Method and apparatus for the delivery of samples to a chemical sensor array (2025)

U.S. patent number 7,022,517 [Application Number 09/616,731] was granted by the patent office on 2006-04-04 for method and apparatus for the delivery of samples to a chemical sensor array.This patent grant is currently assigned to Board of Regents, The University of Texas System. Invention is credited to Eric V. Anslyn, Damon V. Borich, John T. McDevitt, Dean P. Neikirk, Jason B. Shear.

United States Patent	7,022,517
McDevitt , et al.	April 4, 2006

Method and apparatus for the delivery of samples to a chemicalsensor array

Abstract

A system for the rapid characterization of multi-analyte fluids,in one embodiment, includes a light source, a sensor array, and adetector. The sensor array is formed from a supporting member intowhich a plurality of cavities may be formed. A series of chemicallysensitive particles are, in one embodiment positioned within thecavities. The particles may be configured to produce a signal whena receptor coupled to the particle interacts with the analyte.Using pattern recognition techniques, the analytes within amulti-analyte fluid may be characterized.

Inventors:	McDevitt; John T. (Austin,TX), Anslyn; Eric V. (Austin, TX), Shear; Jason B.(Austin, TX), Neikirk; Dean P. (Austin, TX), Borich;Damon V. (Austin, TX)
Assignee:	Board of Regents, The University ofTexas System (Austin, TX)
FamilyID:	36101916
Appl.No.:	09/616,731
Filed:	July 14, 2000

Related U.S. Patent Documents


ApplicationNumber	Filing Date	Patent Number	Issue Date
60144436	Jul 16, 1999
60144435	Jul 16, 1999
60144126	Jul 16, 1999

Current U.S.Class:	435/288.5;435/287.1; 435/287.3; 435/288.2; 435/288.4; 436/518; 436/524;436/531
Current CPCClass:	G01N21/05(20130101); G01N 21/6428(20130101); G01N21/6452(20130101); G01N 21/6454(20130101); G01N21/6486(20130101); G01N 2021/0346(20130101); G01N2021/056(20130101)
Current InternationalClass:	C12M1/34(20060101); G01N 33/543(20060101)
Field ofSearch:	;422/68.1,81-82.09;435/286.5-7.1,288.4-7,808 ;436/518,524,531,56,164,172,805

References Cited [Referenced By]

U.S. Patent Documents


3696932	October 1972	Rosenberg
3701433	October 1972	Krakauer et al.
3709868	January 1973	Spector
3843696	October 1974	Wagner et al.
3856469	December 1974	Schneider et al.
3876504	April 1975	Koffler
3954623	May 1976	Hammer et al.
3964974	June 1976	Banauch et al.
4036946	July 1977	Kleinerman
4050898	September 1977	Goffe et al.
4069017	January 1978	Wu et al.
4115277	September 1978	Swank
4189382	February 1980	Zine, Jr.
4200613	April 1980	Alfrey et al.
4245041	January 1981	Denney
4246107	January 1981	Takenaka et al.
4294817	October 1981	Burgett
4344743	August 1982	Bessman et al.
4360611	November 1982	Wakimoto et al.
4378429	March 1983	Modrovich
4477575	October 1984	Vogel et al.
4493815	January 1985	Fernwood et al.
4567149	January 1986	Sell et al.
4596657	June 1986	Wisdom
4623461	November 1986	Hossom et al.
4661445	April 1987	Saxinger et al.
4672028	June 1987	Olson
4681742	July 1987	Johnson et al.
4683195	July 1987	Mullis et al.
4683202	July 1987	Mullis
4753776	June 1988	Hillman et al.
4795698	January 1989	Owen et al.
4810378	March 1989	Carmen et al.
4813277	March 1989	Miller et al.
4828386	May 1989	Matkovich et al.
4855225	August 1989	Fung et al.
4874499	October 1989	Smith et al.
4908112	March 1990	Pace
4925800	May 1990	Kovacs
4938742	July 1990	Smits
4965188	October 1990	Mullis et al.
4988618	January 1991	Li et al.
4997577	March 1991	Stewart
5053197	October 1991	Bowen
5071076	December 1991	Chagnon et al.
5108933	April 1992	Liberti et al.
5126276	June 1992	Fish et al.
5130238	July 1992	Malek et al.
5137833	August 1992	Russell
5143853	September 1992	Walt
5147606	September 1992	Charlton et al.
5156810	October 1992	Ribi
5156972	October 1992	Issachar
5162863	November 1992	Ito
5168044	December 1992	Joyce et al.
5182366	January 1993	Huebner et al.
5188934	February 1993	Menchen et al.
5209904	May 1993	Forney et al.
5211850	May 1993	Shettigar et al.
5219763	June 1993	Van Hoegaerden
5223393	June 1993	Khanna et al.
5235028	August 1993	Barany et al.
5240640	August 1993	Siiman et al.
5244636	September 1993	Walt et al.
5244813	September 1993	Walt et al.
5248742	September 1993	McGarry et al.
5250264	October 1993	Walt et al.
5252294	October 1993	Kroy et al.
5252494	October 1993	Walt et al.
5262127	November 1993	Wise et al.
5278303	January 1994	Krepinsky et al.
5288214	February 1994	Fukuda et al.
5307144	April 1994	Hiroshi et al.
5366860	November 1994	Bergot et al.
5374530	December 1994	Nuzzolo et al.
5382512	January 1995	Smethers et al.
5385709	January 1995	Wise et al.
5391272	February 1995	ODaly et al.
5405784	April 1995	Van Hoegaerden
5480723	January 1996	Klainer et al.
5491097	February 1996	Ribi et al.
5499909	March 1996	Yamada et al.
5503985	April 1996	Cathey et al.
5512490	April 1996	Walt et al.
5518887	May 1996	Parsons et al.
5541057	July 1996	Bogart et al.
5547682	August 1996	Chagnon et al.
5550373	August 1996	Cole et al.
5563042	October 1996	Phillips et al.
5567627	October 1996	Lehnen
5583054	December 1996	Ito et al.
5587128	December 1996	Wilding et al.
5597531	January 1997	Liberti et al.
5611676	March 1997	Ooumi et al.
5616698	April 1997	Krepinsky et al.
5616790	April 1997	Arnold et al.
5631130	May 1997	Leckie et al.
5632876	May 1997	Zanzucchi et al.
5637469	June 1997	Wilding et al.
5654497	August 1997	Hoffheins et al.
5677195	October 1997	Winkler et al.
5677196	October 1997	Herron et al.
5690807	November 1997	Clark, Jr. et al.
5698089	December 1997	Lewis et al.
5698271	December 1997	Liberti et al.
5700897	December 1997	Klainer et al.
5705018	January 1998	Hartley
5707502	January 1998	McCaffrey et al.
5714122	February 1998	Bretscher et al.
5747349	May 1998	Van Den Engh et al.
5748091	May 1998	Kim
5755942	May 1998	Zanzucchi et al.
5759015	June 1998	Van Lintel et al.
5770370	June 1998	Kumar
5773307	June 1998	Colin et al.
5779907	July 1998	Yu
5788814	August 1998	Sun et al.
5797898	August 1998	Santini, Jr. et al.
5814524	September 1998	Walt et al.
5827748	October 1998	Golden
5834318	November 1998	Buettner
5837552	November 1998	Cotton et al.
5840256	November 1998	Demers et al.
5843655	December 1998	McGall
5843767	December 1998	Beattie
5846396	December 1998	Zanzucchi et al.
5846708	December 1998	Hollis et al.
5854141	December 1998	Cronin et al.
5854684	December 1998	Stabile et al.
5856174	January 1999	Lipshutz et al.
5858804	January 1999	Zanzucchi et al.
5863957	January 1999	Li et al.
5866099	February 1999	Owen et al.
5866430	February 1999	Grow
5869241	February 1999	Edwards et al.
5872170	February 1999	Mine et al.
5872623	February 1999	Stabile
5876605	March 1999	Kitajima et al.
5891656	April 1999	Zarling et al.
5914042	June 1999	Ball et al.
5922617	July 1999	Wang et al.
5942443	August 1999	Parce et al.
5945334	August 1999	Besemer et al.
5965590	October 1999	Rossignol
5965695	October 1999	Simon et al.
5980704	November 1999	Cherukuri et al.
5981297	November 1999	Baselt
5985120	November 1999	Cholli et al.
5992820	November 1999	Fare et al.
6008031	December 1999	Modrich et al.
6013440	January 2000	Lipshutz et al.
6023540	February 2000	Walt et al.
6037137	March 2000	Komoriya et al.
6045579	April 2000	Hochshuler et al.
6048732	April 2000	Anslyn et al.
6063581	May 2000	Sundrehagen
6074616	June 2000	Buechler et al.
6083761	July 2000	Kedar et al.
6083763	July 2000	Balch
6103479	August 2000	Taylor
6127139	October 2000	Te Koppele et al.
6133047	October 2000	Elaissari et al.
6151973	November 2000	Geysen et al.
6168948	January 2001	Anderson et al.
6171489	January 2001	Ballard et al.
6171780	January 2001	Pham et al.
6210910	April 2001	Walt et al.
6245296	June 2001	Ligler et al.
6254830	July 2001	Pivarnik et al.
6288220	September 2001	Kambara et al.
6039889	October 2001	Cutler et al.
6331441	December 2001	Balch et al.
6355431	March 2002	Chee et al.
6355491	March 2002	Zhou et al.
6413786	July 2002	Hansen et al.
6482593	November 2002	Walt et al.
6485690	November 2002	Pfost et al.
6514415	February 2003	Hatch et al.
6680206	January 2004	McDevitt et al.
2001/0002984	June 2001	Vetter

Foreign Patent Documents


339 623	Nov 1989	EP
0 518 557	Dec 1992	EP
439182	Apr 1996	EP
2 300 258	Oct 1996	GB
2 315 131	Jan 1998	GB
90/01069	Feb 1990	WO
92/0880	Jan 1992	WO
94/19690	Sep 1994	WO
97/35189	Sep 1997	WO
98/17383	Apr 1998	WO
98/25701	Jun 1998	WO
98/40726	Sep 1998	WO
98/53300	Nov 1998	WO
99/17139	Apr 1999	WO
99/18434	Apr 1999	WO
99/67024	Dec 1999	WO
00/04372	Jan 2000	WO
00/20117	Apr 2000	WO
00/4372	Jul 2000	WO

Other References

Stanley, "UT scientists engineer a tiny arbiter of taste," AustinAmerican State man, Jul. 26, 1998. cited by other .
"Biosensors respond with colored light," Science News, vol. 152,Nov. 15, 1997, 9. 317. cited by other .
Ricco et al, "Surface Acoustic Wave Chemical Sensor Arrays: NewChemically Sensitive Interfaces Combined with Novel ClusterAnalysis to Detect Volatile Organic Compounds and Mixtures,"Accounts of Chemical Research, vol. 31, No. 5, 1998, pp. 289-296.cited by other .
Grate et al., "Hydrogen Bond Acidic Polymers for Surface AcousticWave Vapor Sensors and Arrays," Analytical Chemistry, vol. 71, No.,5, Mar. 1, 1999, pp. 1033-1040. cited by other .
Johnson et al., "Identification of Multiple Analytes Using andOptical Sensor Array and Pattern Recognition Neural Networks,"Analytical Chemistry, vol. 69, No. 22, Nov. 15, 1997, pp.4641-4648. cited by other .
Healey et al. "Fast Temporal Response Fiber-Optic Chemical SensorsBased on the Photodeposition of Micrometer-Scale Polymer Arrays,"Analytical Chemistry, vol. 69, No. 11, Jun. 1, 1997, pp. 2213-2216.cited by other .
White et al., "Rapid Analyte Recognition in a Device Based onOptical Sensors and the Offactory System," Analytical Chemistry,vol. 68, No. 13, Jul. 1, 1996, pp. 2191-2202. cited by other .
Potyrailo et al., "Optical Time-of Flight Chemical Detection:Absorption-Modulated Fluorescence for Spatially Resolved AnalyteMapping in a Bidirectional Distributed Fiber-Optic Sensor."Analytical Chemistry, vol. 70, No. 16, Aug. 15, 1998, pp.3407-3412. cited by other .
Holz et al., "Intelligent Polymerized Crystalline Colloidal Arrays:Novel Chemical Sensor Materials," Analytical Chemistry, vol. 70,No. 4, Feb. 15, 1998, pp. 780-791. cited by other .
Lavigne et al., "Solution-Based Analysis of Multiple Analytes by aSensor Array: Toward the Development of an Electronic Tongue," J.Am. Chem. Soc., vol. 120, No. 25, Jul. 1, 1998, pp. 6429-6430.cited by other .
Han et al., "Fabrication and characterization of a Fabry-Perotbased chemical sensor," Microelectronics Research Center andDepartment of Electrical and Computer Engineering, Feb. 7, 1997.cited by other .
Han et al, "Deflection behavior of Fabry-Perot pressure sensorshaving planar and corrugated diaphragms," Microelectronics ResearchCenter and Department of Electrical and Computer Engineering, Feb.4, 1997. cited by other .
Patent Abstracts of Japan, Publication No. 10332593, PublicationDate Dec. 18, 1998. cited by other .
International Search Report, Application No. PCT/US99/16162, mailedNov. 26, 1999. cited by other .
Adler, M; Nicholson, J.D.; Hackley, B.E., Jr. "Efficacy of a novelmetalloprotease inhibitor on dotalinum neurotoxin B activity" FEBSLett., 1998, 429, 234-238. cited by other .
James, T.D.; Sandanayake, K.R.A.S.; Iguchi, R.; Shinkai, S.J. Am.Chem. Soc 1995, 117, 8982-8987. cited by other .
Murukami, H.; Nagasaki, T.; Hamachi, I.; Shinkai, S. TetrahedronLett., 1993, 34, 6273, 6276. cited by other .
Shinkai, S.; Tsukagohsi, K.; Ishikawa, Y.; Kunitake, T.J. Chem.Soc. Chem. Commun. 1991, 1039-1041. cited by other .
Kondo, K.; Shiomi, Y.; Saisho, M.; Harada, T.; Shinkai, S.Tetrahedron. 1992, 48, 8239-8252. cited by other .
Shiomi, Y.; Saisho, M.; Tsukagoshi, K.; Shinkai, S.J. Chem. Soc.Perkin Trans I 1993, 2111-2117. cited by other .
Deng, G.; James, T.D.; Shinkai, S.J. Am. Chem. Soc. 1994, 116,4567-4572. cited by other .
James, T.D.; Harada, T.; Shinkai, S.J. Chem. Soc. Chem. Commun.1993, 857-860. cited by other .
James T.D.; Murata, K.; Harada, T.; Ueda, K.; Shinkai, S.Chem.Lett. 1994, 273-276. cited by other .
Ludwig, R.; Harada, T.; Ueda, K.; James, T.D.; Shinkai, S.J., Chem.Soc. Perkin Trans 1, 1994, 697-702. cited by other .
Sandanayake, K.R.A.S.; Shinkai, S.J. Chem. Soc., Chem Commun. 1994,1083-1084. cited by other .
Nagasaki, T.; Shinmori, H.; Shinkai, S. Tetrahedron Lett. 1994, 35,2201-2204. cited by other .
Murakami, H.; Nagasaki, T.; Hamachi, I.; Shinkai, S., J. Chem. Soc.Perkin Trans 2. 1994, 975-981. cited by other .
Nakashima, K.; Shinkai, S,.Chem. Lett. 1994, 1267-1270. cited byother .
Sandanayake, K.R.A.S.; Nakashima, K.; Shinkai, S.J., Chem. Soc.,Chem. Commun. 1994, 1621-1622. cited by other .
James, T.D.; Sandanayake, K.R.A.S.; Shinkai, S.,J. Chem. Soc.,Chem. Comun. 1994, 477-478. cited by other .
James T.D.; Sandanayake, K.R.A.S.; Shinkai, S., Angew. Chem., Int.Ed. Eng. 1994, 33, 2207-2209. cited by other .
James T.D.; Sandanayake, K.R.A.S.; Shinkai, S. Nature, 1995, 374,345-347. cited by other .
Forster, Th. Transfer Mechanisms of Electronic Excitation;,Discuss. Faraday Soc., 1959,27, 7-17. cited by other .
Khanna, P.L., Ullman, E.F. "4`,5`--Dimethoxyl-6-carboxyfluorescein; A novel dipolediple coupledfluorescence energy transfer acceptor useful for fluorescenceimmunoassays", Anal. Biochem. 1980, 108, 156-161. cited by other.
Morrison, L.E. "Time resolved Detection of Energy Transfer: Theoryand Application to Immunoassays", Anal. Biochem. 1988, 174,101-120. cited by other .
Schmidt, J.J.; Stafford, R.G.; Bostian, K.A.; "Type A botulinumneurotoxin proteolytic activity; development of competitiveinhibitors and implicatins for substrate specificity at theS.sub.1'binding subsite", FEBS Lett., 1998, 435, 61-64. cited byother .
Shone, C.C.; Roberts, A.K., "Peptide substrate specificity andproperties of the zincendopetidase activity of botulinum type Bneurotoxin", Eur. J. Biochem., 1994, 225, 263-270. cited by other.
Soleihac, J.-M.; Cornille, F.; Martin, L.; Lenoir, C.;Fournie-Zaluski, M.-C.; Roques, B.P. A sensitive and rapidfluorescence-based assay for determination of tetnus toxinpeptidase activity; Anal. Biochem., 1996, 241, 120-127. cited byother .
Stimpson, D.I.; Hoijer, J.V.; Hsieh, WT.; Jou, C.; Gordon, J.;Theriault, t.; Gamble, R.; Baldeschwieler, J.D.; Proc. Natl. Acad.Sci. USA 1995, 92, 6379-6383. cited by other .
Niikura, K.; Metzger, A.; Anslyn, E.V., "A Sensing Ensemble withSelectivity for Iositol Triphosphate", J. Am. Chem. Soc., 1998,120, 8533-8534. cited by other .
Kaiser, E.; Colescott, R.L.; Bossinger, C.D.; Cook, P.I.; "ColorTest for Detection of Free Terminal Amino Groups in the Solid-PhaseSynthesis of Peptides", Anal. Biochem., 1970, 34, 595-598. cited byother .
Hamasaki, K.; Ikeda, H.; Nakamura, A.; Ueno, A.; Toda, F.: Suzuki,I.; Osa, T. "Fluorescent Sensors of Molecular Recognition. ModifiedCyclodextrins Capable of Exhibiting Guest-Responsive TwistedIntramolecular Charge Transfer Fluorescence", J. Am. Chem. Soc.,1993, 115, 5035-5040. And reference 5 therein. cited by other .
Youil R; Kemper, B; Cotton, RGH, "Detection of 81 of 81 Known MouseBeta-Globin Promoter Mutations with T4 Endonuclease-VII-The EMCMethod", Genomics, 1996, 32, 431-5. cited by other .
Pabo and Sauer, 1992, Annu. Rev. Biochem, 61, 1053-1095. cited byother .
Harrison, 1991, Nature, 353, 715-719. cited by other .
Klug A., Gene 1993, 135-83-92. cited by other .
Hsu, I.C.; Yang, Q.P.; Kahng, M.W.; Xu, J.F.; "Detection of DNAPoint Mutations with DNA Mismatch Repaire Enzymes", Carcinogenesis,1994, 15, 1657-1662. cited by other .
Saiki, et al., Science, 1985, 230, 1350-1354. cited by other .
Mullis, et al., Cold Springs Harbor Symp. Quant. Biol., 1986, 51,263-273. cited by other .
Mullis K.B.; and Faloona, F.A., Methods Enzymol., 1987, 155,335-350. cited by other .
Barany, F. PCR Methods and Applications, 1991, 1, 5-16. cited byother .
Wu D.Y.; Wallace, R.B., Genomics, 1989, 4:560-569. cited by other.
Barany, F., Proc. Natl. Acad. Sci. USA, 1991, 88, 189-193. cited byother .
Kwoh, et al., Proc. Natl. Acad. Sci. USA, 1989, 86,1173-1177. citedby other .
Guatelli, et al., Proc. Natl. Acad, Sci., 1990, USA, 87, 1874-1878.cited by other .
Goldrick, M.M.; Kimball, G.R.; Liu, Q.: Martin, L.A.; Sommer, S.S.:Tseng, J.Y.H.; "Nirca.TM.-A Rapid Robust Method for Screening forUnknown Point Mutations", BioTechniques, 1996, 21, 106-112. citedby other .
Cho et al., "An Unnatural Biopolymer," Science, 1993, 261, p.1303-1305. cited by other .
Lauritzen et al., "Peptide Dot Immunoassay and Iimmunoblotting:Electroblotting from Aluminum Thin-layer Chromatography Plates andIsoelectric Focusing Gels to Activated Nitrocellulose,"Electrophoresis, 1993, 14, p: 852-859. cited by other .
Schutz et al., "Direct Observation of Ligand Colocalization onIndividual Receptor Molecules," Biophysical Journal, 1998, 74.cited by other .
Internationall Search Report for PCT/US99/16162 mailed Nov. 26,1999. cited by other .
International Search Report for PCT/US00/19302 mailed Feb. 22,2001. cited by other .
International Search Report for PCT/US00/19351 mailed Feb. 22,2001. cited by other .
International Search Report for PCT/US00/19350 mailed Feb. 22,2001. cited by other .
International Search Report for PCT/US01/03141 mailed Oct. 19,2001. cited by other .
International Search Report for PCT/US01/03139 mailed May 7, 2001.cited by other .
International Search Report for PCT/US01/03240 May 7, 2001. citedby other .
International Search Report for PCT/US01/03241 mailed May 7, 2001.cited by other .
International Search Report for PCT/US02/3277 mailed Feb. 13, 2003.cited by other .
International Search Report for PCT/US02/3275 mailed May 7, 2003.cited by other .
International Search Report for PCT/US03/12951 mailed Oct. 14,2003. cited by other .
International Search Report for PCT/US03/23131 mailed Dec. 12,2003. cited by other .
Dickinson et al., "Convergent, Self-Encoded Bead Sensor Arrays inthe Design of an Artificial Nose", Anal. Chem, Jun. 1, 1999, vol.71, No. 11, pp. 2192-2198. cited by other .
Michael et al., "Making Sensors out of Disarry: Optical SensorMicroarray", SPIE, 1998, vol. 3270, pp. 34-41. cited byother.

Primary Examiner: Le; Long V.
Assistant Examiner: Yang; Nelson
Attorney, Agent or Firm: Meyertons, Hood, Kivlin, Kowert& Goetzel, P.C. Meyertons; Eric B.

Government Interests

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Research leading to this invention was federally supported, inpart, by grant No. 1R01GM57306-01 entitled "The Development of anElectronic Tongue" from the National Institute of Health and theU.S. Government has certain rights to this invention.

Parent Case Text

PRIORITY CLAIM

This application claims priority to U.S. Provisional ApplicationNo. 60/144,436 entitled "DETECTION SYSTEM BASED ON AN ANALYTEREACTIVE PARTICLE," filed Jul. 16, 1999, U.S. ProvisionalApplication No. 60/144,435 entitled "GENERAL SIGNALING PROTOCOLSFOR CHEMICAL RECEPTORS IN IMMOBILIZED MATRICES," filed Jul. 16,1999, and U.S. Provisional Application No. 60/144,126 entitled"METHOD AND APPARATUS FOR THE DELIVERY OF SAMPLES TO A CHEMICALSENSOR ARRAY," filed Jul. 16, 1999.

Claims

What is claimed is:

1. A system for detecting an analyte in a fluid comprising: a lightsource; a sensor array, the sensor array comprising a supportingmember comprising at least one cavity formed within the supportingmember; a particle, the particle positioned in the cavity, whereinthe particle produces a signal when the particle interacts with ananalyte during use; a vacuum apparatus at least partiallyincorporated into the supporting member, wherein the vacuumapparatus is coupled to the cavity, and wherein the vacuumapparatus produces a vacuum in the cavity such that the producedvacuum pulls fluid through the cavity during use, and wherein thevacuum apparatus comprises a vacuum chamber, and wherein the vacuumchamber comprises a breakable barrier positioned between thechamber and the cavity, and wherein the chamber applies a vacuum tothe cavity when the breakable barrier is punctured; and a detector,the detector being configured to detect the signal produced by theinteraction of the analyte with the particle during use; whereinthe light source and detector are positioned such that light passesfrom the light source, to the particle, and onto the detectorduring use.

2. The system of claim 1, wherein the system comprises a pluralityof particles positioned within a plurality of cavities, and whereinat least a first part of the plurality of particles is adapted todetect at least one analyte, and wherein the analyte that isdetected by the portion of the plurality of particles is notdetected by second part of the plurality of particles.

3. The system of claim 1, wherein the system comprises a pluralityof particles positioned in the cavity.

4. The system of claim 1, wherein the light source comprises alight emitting diode.

5. The system of claim 1, wherein the light source comprises awhite light source.

6. The system of claim 1, wherein the sensor array furthercomprises a bottom layer and a top cover layer, wherein the bottomlayer is positioned below a bottom surface of the supportingmember, and wherein the top cover layer is positioned above theupper surface of the supporting member, and wherein the bottomlayer and the top cover layer are positioned such that the particleis substantially contained within the cavity by the bottom layerand the top cover layer.

7. The system of claim 1, wherein the bottom layer and the topcover layer are substantially transparent to light produced by thelight source.

8. The system of claim 1, wherein the sensor array furthercomprises a bottom layer and a top cover layer, wherein the bottomlayer is coupled to a bottom surface of the supporting member, andwherein the top cover layer is coupled to a top surface of thesupporting member; and wherein both the bottom layer and the topcover layer are coupled to the supporting member such that theparticle is substantially contained within the cavity by bottomlayer and the top cover layer.

9. The system of claim 8, wherein the bottom layer and the topcover layer are substantially transparent to light produced by thelight source.

10. The system of claim 1, wherein the sensor array furthercomprises a bottom layer coupled to the supporting member, andwherein the supporting member comprises silicon, and wherein thebottom layer comprises silicon nitride.

11. The system of claim 1, further comprising a conduit coupled tothe sensor array, wherein the conduit is configured to conduct thefluid sample to and away from the sensor array.

12. The system of claim 1, wherein the supporting member is formedfrom a plastic material, and wherein the sensor array furthercomprises a top cover layer, the top cover layer being coupled tothe supporting member such that the particle is substantiallycontained within the cavity, and wherein the top cover layercomprises one or more openings that allow the fluid to pass throughthe top cover layer to the particle, and wherein both thesupporting member and the top cover layer are substantiallytransparent to light produced by the light source.

13. The system of claim 1, wherein the cavities are configured toallow the fluid to pass through the supporting member duringuse.

14. The system of claim 13, wherein the cavity is configured tosubstantially contain the particle.

15. The system of claim 13, further comprising a cover layercoupled to the supporting member and a bottom layer coupled to thesupporting member, wherein the cover layer and the bottom layer areremovable.

16. The system of claim 13, further comprising a cover layercoupled to the supporting member and a bottom layer coupled to thesupporting member, wherein the cover layer and the bottom layer areremovable, and wherein the cover layer and the bottom layer includeopenings that are substantially aligned with the cavities duringuse.

17. The system of claim 13, further comprising a cover layercoupled to the supporting member and a bottom layer coupled to thesupporting member, wherein the bottom layer is coupled to a bottomsurface of the supporting member and wherein the cover layer isremovable, and wherein the cover layer and the bottom layer includeopenings that are substantially aligned with the cavities duringuse.

18. The system of claim 13, further comprising a cover layercoupled to the supporting member and a bottom layer coupled to thesupporting member, wherein an opening is formed in the cover layersubstantially aligned with the cavity, and wherein an opening isformed in the bottom layer substantially aligned with thecavity.

19. The system of claim 13, wherein the cavity is substantiallytapered such that the width of the cavity narrows in a directionfrom a top surface of the supporting member toward a bottom surfaceof the supporting member, and wherein a minimum width of the cavityis substantially less than a width of the particle.

20. The system of claim 13, wherein a width of a bottom portion ofthe cavity is substantially less than a width of a top portion ofthe cavity, and wherein the width of the bottom portion of thecavity is substantially less than a width of the particle.

21. The system of claim 13, further comprising a cover layercoupled to the supporting member and a bottom layer coupled to thesupporting member, wherein the particle is positioned on the bottomlayer, and wherein an opening is formed in the cover layersubstantially aligned with the cavity.

22. The system of claim 13, wherein the supporting member comprisesa dry film photoresist material.

23. The system of claim 13, wherein the supporting member comprisesa plurality of layers of a dry film photoresist material.

24. The system of claim 13, wherein an inner surface of the cavityis coated with a reflective material.

25. The system of claim 1, wherein the detector comprises acharge-coupled device.

26. The system of claim 1, wherein the detector comprises anultraviolet detector.

27. The system of claim 1, wherein the detector comprises afluorescence detector.

28. The system of claim 1, wherein the detector comprises asemiconductor based photodetector, and wherein the detector iscoupled to the sensor array.

29. The system of claim 1, wherein the particle ranges from about0.05 micron to about 500 microns.

30. The system of claim 1, wherein a volume of the particle changeswhen contacted with the fluid.

31. The system of claim 1, wherein the vacuum apparatus comprises avacuum pump.

32. The system of claim 1, wherein the particle comprises areceptor molecule coupled to a polymeric resin.

33. The system of claim 1, wherein the polymeric resin comprisespolystyrene-polyethylene glycol-divinyl benzene.

34. The system of claim 33, wherein the receptor molecule producesthe signal in response to the pH of the fluid.

35. The system of claim 33, wherein the analyte comprises a metalion, and wherein the receptor produces the signal in response tothe presence of the metal ion.

36. The system of claim 33, wherein the analyte comprises acarbohydrate, and wherein the receptor produces a signal inresponse to the presence of a carbohydrate.

37. The system of claim 33, wherein the particle further comprisesa first indicator and a second indicator, the first and secondindicators being coupled to the receptor, wherein the interactionof the receptor with the analyte causes the first and secondindicators to interact such that the signal is produced.

38. The system of claim 33, wherein the particle further comprisesan indicator, wherein the indicator is associated with the receptorsuch that in the presence of the analyte the indicator is displacedfrom the receptor to produce the signal.

39. The system of claim 33, wherein the receptor comprises apolynucleotide.

40. The system of claim 33, wherein the receptor comprises apeptide.

41. The system of claim 33, wherein the receptor comprises anenzyme.

42. The system of claim 33, wherein the receptor comprises asynthetic receptor.

43. The system of claim 33, wherein the receptor comprises anunnatural biopolymer.

44. The system of claim 33, wherein the receptor comprises anantibody.

45. The system of claim 33, wherein the receptor comprises anantigen.

46. The system of claim 33, wherein the analyte comprises phosphatefunctional groups, and wherein the particle produces the signal inthe presence of the phosphate functional groups.

47. The system of claim 1, wherein the analyte comprises bacteria,and wherein the particle produces the signal in the presence of thebacteria.

48. The system of claim 1, wherein the system comprises a pluralityof particles positioned within a plurality of cavities, and whereinthe plurality of particles produce a detectable pattern in thepresence of the analyte.

49. The system of claim 1, further comprising a filter coupled tothe conduit and the sensor array, wherein the fluid passes throughthe filter before reaching the sensor array.

50. The system of claim 49, wherein the fluid is a blood sample,and wherein the filter comprises a membrane for the removal ofparticulates.

51. The system of claim 49, wherein the fluid is a blood sample,and wherein the filter comprises a membrane for removal of whiteand red blood cells from the blood.

52. The system of claim 1 further comprising a reagent deliveryreservoir coupled to the sensor array via a conduit, wherein thefluid passes through the reagent delivery reservoir before enteringthe cavity, and wherein reagents enter the fluid as the fluidpasses through the reagent delivery reservoir during use.

53. The system of claim 52, wherein the reagent delivery reservoircomprises an indicator.

54. A system for detecting an analyte in a fluid comprising: asensor array, the sensor array comprising a supporting membercomprising at least one cavity formed within the supporting member;a particle, the particle positioned within the cavity, wherein theparticle produces a signal when the particle interacts with ananalyte during use; a vacuum apparatus at least partiallyincorporated into the supporting member, wherein the vacuumapparatus is coupled to the cavity, and wherein the vacuumapparatus produces a vacuum in the cavity such that the producedvacuum pulls fluid through the cavity during use, and wherein thevacuum apparatus comprises a vacuum chamber, and wherein the vacuumchamber comprises a breakable barrier positioned between thechamber and the cavity, and wherein the chamber applies a vacuum tothe cavity when the breakable barrier is punctured; and a detector,the detector being configured to detect the signal produced by theinteraction of the analyte with the particle during use.

55. The system of claim 54, wherein the system comprises aplurality of particles positioned in a plurality of cavities, andwherein at least a first part of the plurality of particles isadapted to detect at least one analyte, and wherein the analytethat is detected by the portion of the plurality of particles isnot detected by second part of the plurality of particles.

56. The system of claim 54, wherein the system comprises aplurality of particles positioned in the cavity.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and device for thedetection of analytes in a fluid. More particularly, the inventionrelates to the development of a sensor array system capable ofdiscriminating mixtures of analytes, toxins, and/or bacteria inmedical, food/beverage, and environmental solutions.

2. Brief Description of the Related Art

The development of smart sensors capable of discriminatingdifferent analytes, toxins, and bacteria has become increasinglyimportant for clinical, environmental, health and safety, remotesensing, military, food/beverage and chemical processingapplications. Although many sensors capable of high sensitivity andhigh selectivity detection have been fashioned for single analytedetection, only in a few selected cases have array sensors beenprepared which display solution phase multi-analyte detectioncapabilities. The advantages of such array systems are theirutility for the analysis of multiple analytes and their ability tobe "trained" to respond to new stimuli. Such on site adaptiveanalysis capabilities afforded by the array structures make theirutilization promising for a variety of future applications. Arraybased sensors displaying the capacity to sense and identify complexvapors have been demonstrated recently using a number of distincttransduction schemes. For example, functional sensors based onSurface Acoustic Wave (SAW), tin oxide (SnO.sub.2) sensors,conductive organic polymers, and carbon black/polymer compositeshave been fashioned. The use of tin oxide sensors, for example, isdescribed in U.S. Pat. No. 5,654,497 to Hoffheins et al. Thesesensors display the capacity to identify and discriminate between avariety of organic vapors by virtue of small site-to-sitedifferences in response characteristics. Pattern recognition of theoverall fingerprint response for the array serves as the basis foran olfaction-like detection of the vapor phase analyte species.Indeed, several commercial "electronic noses" have been developedrecently. Most of the well established sensing elements are basedon SnO.sub.2 arrays which have been derivatized so as to yieldchemically distinct response properties. Arrays based on SAWcrystals yield extremely sensitive responses to vapor, however,engineering challenges have prevented the creation of large SAWarrays having multiple sensor sites. To our knowledge, the largestSAW device reported to date possesses only 12 sensor elements.Additionally, limited chemical diversity and the lack ofunderstanding of the molecular features of such systems makes theirexpansion into more complex analysis difficult.

Other structures have been developed that are capable ofidentifying and discriminating volatile organic molecules. Onestructure involves a series of conductive polymer layers depositedonto metal contacting layers. When these sensors are exposed tovolatile reagents, some of the volatile reagents adsorb into thepolymer layers, leading to small changes in the electricalresistance of these layers. It is the small differences in thebehavior of the various sites that allows for a discrimination,identification, and quantification of the vapors. The detectionprocess takes only a few seconds, and sensitivities ofpart-per-billion can be achieved with this relatively simpleapproach. This "electronic nose" system is described in U.S. Pat.No. 5,698,089 to Lewis et al. which is incorporated herein byreference as if set forth herein.

Although the above described electronic nose provides an impressivecapability for monitoring volatile reagents, the system possesses anumber of undesirable characteristics that warrant the developmentof alternative sensor array systems. For example, the electronicnose can be used only for the identification of volatile reagents.For many environmental, military, medical, and commercialapplications, the identification and quantification of analytespresent in liquid or solid-phase samples is necessary. Moreover,the electronic nose systems are expensive (e.g., the Aromascansystem costs about $50,000/unit) and bulky (.gtoreq.1 ft.sup.3).Furthermore, the functional elements for the currently availableelectronic nose are composed of conductive polymer systems whichpossess little chemical selectivity for many of the analytes whichare of interest to the military and civilian communities.

One of the most commonly employed sensing techniques has exploitedcolloidal polymer microspheres for latex agglutination tests (LATs)in clinical analysis. Commercially available LATs for more than 60analytes are used routinely for the detection of infectiousdiseases, illegal drugs, and early pregnancy tests. The vastmajority of these types of sensors operate on the principle ofagglutination of latex particles (polymer microspheres) whichoccurs when the antibody-derivatized microspheres becomeeffectively "cross-linked" by a foreign antigen resulting in theattachment to, or the inability to pass through a filter. Thedye-doped microspheres are then detected colorimetrically uponremoval of the antigen carrying solution. However, the LATs lackthe ability to be utilized for multiple, real time analytedetection schemes as the nature of the response intrinsicallydepends on a cooperative effect of the entire collection ofmicrospheres.

Similar to the electronic nose, array sensors that have shown greatanalytical promise are those based on the "DNA on a chip"technology. These devices possess a high density of DNAhybridization sites that are affixed in a two-dimensional patternon a planar substrate. To generate nucleotide sequence information,a pattern is created from unknown DNA fragments binding to varioushybridization sites. Both radiochemical and optical methods haveprovided excellent detection limits for analysis of limitedquantities of DNA. (Stimpson, D. I.; Hoijer, J. V.; Hsieh, W.; Jou,C.; Gardon, J.; Theriault, T.; Gamble, R.; Baldeschwieler, J. D.Proc. Natl. Acad. Sci. USA 1995, 92, 6379). Although quitepromising for the detection of DNA fragments, these arrays aregenerally not designed for non-DNA molecules, and accordingly showvery little sensitivity to smaller organic molecules. Many of thetarget molecules of interest to civilian and military communities,however, do not possess DNA components. Thus, the need for aflexible, non-DNA based sensor is still desired. Moreover, while anumber of prototype DNA chips containing up to a few thousanddifferent nucleic acid probes have been described, the existingtechnologies tend to be difficult to expand to a practical size. Asa result, DNA chips may be prohibitively expensive for practicaluses.

Systems for analyzing fluid samples using an array formed ofheterogeneous, semi-selective thin films which function as sensingreceptor units are described in U.S. Pat. Nos. 6,023,540;5,814,524; 5,700,897; 5,512,490; 5,480,723; 5,252,494; 5,250,264;5,244,813; 5,244,636; and 5,143,853 which are incorporated hereinby reference as if set forth herein. These systems appears todescribe the use of covalently attached polymeric "cones" which aregrown via photopolymerization onto the distal face of fiber opticbundles. These sensor probes appear to be designed with the goal ofobtaining unique, continuous, and reproducible responses from smalllocalized regions of dye-doped polymer. The polymer appears toserve as a solid support for indicator molecules that provideinformation about test solutions through changes in opticalproperties. These polymer supported sensors have been used for thedetection of analytes such as pH, metals, and specific biologicalentities. Methods for manufacturing large numbers of reproduciblesensors, however, has yet to be developed. Moreover, no methods foracquisitions of data streams in a simultaneous manner arecommercially available with this system. Optical alignment issuesmay also be problematic for these systems.

A method of rapid sample analysis for use in the diagnosticmicrobiology field is also desirable. The techniques now used forrapid microbiology diagnostics detect either antigens or nucleicacids. Rapid antigen testing is based on the use of antibodies torecognize either the single cell organism or the presence ofinfected cell material. Inherent to this approach is the need toobtain and characterize the binding of the antibody to uniquestructures on the organism being tested. Since the identificationand isolation of the appropriate antibodies is time consuming,these techniques are limited to a single agent per testing moduleand there is no opportunity to evaluate the amount of agentpresent.

Most antibody methods are relatively insensitive and require thepresence of 10.sup.5 to 10.sup.7 organisms. The response time ofantibody-antigen reactions in diagnostic tests of this type rangesfrom 10 to 120 minutes, depending on the method of detection. Thefastest methods are generally agglutination reactions, but thesemethods are less sensitive due to difficulties in visualinterpretation of the reactions. Approaches with slower reactiontimes include antigen recognition by antibody conjugated to eitheran enzyme or chromophore. These test types tend to be moresensitive, especially when spectrophotometric methods are used todetermine if an antigen-antibody reaction has occurred. Thesedetection schemes do not, however, appear to allow the simultaneousdetection of multiple analytes on a single detector platform.

The alternative to antigen detection is the detection of nucleicacids. An approach for diagnostic testing with nucleic acids useshybridization to target unique regions of the target organism.These techniques require fewer organisms (10.sup.3 to 10.sup.5),but require about five hours to complete. As with antibody-antigenreactions this approach has not been developed for the simultaneousdetection of multiple analytes.

The most recent improvement in the detection of microorganisms hasbeen the use of nucleic acid amplification. Nucleic acidamplification tests have been developed that generate bothqualitative and quantitative data. However, the current limitationsof these testing methods are related to delays caused by specimenpreparation, amplification, and detection. Currently, the standardassays require about five hours to complete. The ability tocomplete much faster detection for a variety of microorganismswould be of tremendous importance to military intelligence,national safety, medical, environmental, and food areas.

It is therefore desirable that new sensors capable ofdiscriminating different analytes, toxins, and bacteria bedeveloped for medical/clinical diagnostic, environmental, healthand safety, remote sensing, military, food/beverage, and chemicalprocessing applications. It is further desired that the sensingsystem be adaptable to the simultaneous detection of a variety ofanalytes to improve throughput during various chemical andbiological analytical procedures.

SUMMARY OF THE INVENTION

Herein we describe a system and method for the analysis of a fluidcontaining one or more analytes. The system may be used for eitherliquid or gaseous fluids. The system, in some embodiments, maygenerate patterns that are diagnostic for both the individualanalytes and mixtures of the analytes. The system in someembodiments, is made of a plurality of chemically sensitiveparticles, formed in an ordered array, capable of simultaneouslydetecting many different kinds of analytes rapidly. An aspect ofthe system is that the array may be formed using a microfabricationprocess, thus allowing the system to be manufactured in aninexpensive manner.

In an embodiment of a system for detecting analytes, the system, insome embodiments, includes a light source, a sensor array, and adetector. The sensor array, in some embodiments, is formed of asupporting member which is configured to hold a variety ofchemically sensitive particles (herein referred to as "particles")in an ordered array. The particles are, in some embodiments,elements which will create a detectable signal in the presence ofan analyte. The particles may produce optical (e.g., absorbance orreflectance) or fluorescence/phosphorescent signals upon exposureto an analyte. Examples of particles include, but are not limitedto functionalized polymeric beads, agarous beads, dextrose beads,polyacrylamide beads, control pore glass beads, metal oxidesparticles (e.g., silicon dioxide (SiO.sub.2) or aluminum oxides(Al.sub.2O.sub.3)), polymer thin films, metal quantum particles(e.g., silver, gold, platinum, etc.), and semiconductor quantumparticles (e.g., Si, Ge, GaAs, etc.). A detector (e.g., acharge-coupled device "CCD") in one embodiment is positioned belowthe sensor array to allow for the data acquisition. In anotherembodiment, the detector may be positioned above the sensor arrayto allow for data acquisition from reflectance of the light off ofthe particles.

Light originating from the light source may pass through the sensorarray and out through the bottom side of the sensor array. Lightmodulated by the particles may pass through the sensor array andonto the proximally spaced detector. Evaluation of the opticalchanges may be completed by visual inspection or by use of a CCDdetector by itself or in combination with an optical microscope. Amicroprocessor may be coupled to the CCD detector or themicroscope. A fluid delivery system may be coupled to thesupporting member of the sensor array. The fluid delivery system,in some embodiments, is configured to introduce samples into andout of the sensor array.

In an embodiment, the optical detector may be integrated within thebottom of the supporting member, rather than using a separatedetecting device. The optical detectors may be coupled to amicroprocessor to allow evaluation of fluids without the use ofseparate detecting components. Additionally, a fluid deliverysystem may also be incorporated into the supporting member.Integration of detectors and a fluid delivery system into thesupporting member may allow the formation of a compact and portableanalyte sensing system.

A high sensitivity CCD array may be used to measure changes inoptical characteristics which occur upon binding of thebiological/chemical agents. The CCD arrays may be interfaced withfilters, light sources, fluid delivery and micromachined particlereceptacles, so as to create a functional sensor array. Dataacquisition and handling may be performed with existing CCDtechnology. CCD detectors may be configured to measure white light,ultraviolet light or fluorescence. Other detectors such asphotomultiplier tubes, charge induction devices, photo diodes,photodiode arrays, and microchannel plates may also be used.

In one embodiment, a naturally occurring or synthetic receptor isbound to a polymeric bead in order to create the particle. Theparticle, in some embodiments, is capable of both binding theanalyte(s) of interest and creating a detectable signal. In someembodiments, the particle will create an optical signal when boundto an analyte of interest.

In an embodiment, a large number of chemical/biological agents ofinterest to the military and civilian communities may be sensedreadily by the described array sensors. Bacteria may also bedetected using a similar system. To detect, sense, and identifyintact bacteria, the cell surface of one bacteria may bedifferentiated from other bacteria, or genomic material may bedetected using oligonucleic receptors. One method of accomplishingthis differentiation is to target cell surface oligosaccharides(i.e., sugar residues). The use of synthetic receptors which arespecific for oligosaccharides may be used to determine the presenceof specific bacteria by analyzing for cell surfaceoligosaccharides.

In one embodiment, a receptor may be coupled to a polymeric resin.The receptor may undergo a chemical reaction in the presence of ananalyte such that a signal is produced. Indicators may be coupledto the receptor or the polymeric bead. The chemical reaction of theanalyte with the receptor may cause a change in the localmicroenvironment of the indicator to alter the spectroscopicproperties of the indicator. This signal may be produced using avariety of signalling protocols. Such protocols may includeabsorbance, fluorescence resonance energy transfer, and/orfluorescence quenching. Receptor-analyte combination may include,but are not limited to, peptides-proteases,polynucleotides-nucleases, and oligosaccharides-oligosaccharidecleaving agents.

In one embodiment, a receptor and an indicator may be coupled to apolymeric resin. The receptor may undergo a conformational changein the presence of an analyte such that a change in the localmicroenvironment of the indicator occurs. This change may alter thespectroscopic properties of the indicator. The interaction of thereceptor with the indicator may be produce a variety of differentsignals depending on the signalling protocol used. Such protocolsmay include absorbance, fluorescence resonance energy transfer,and/or fluorescence quenching.

In an embodiment, the sensor array system includes an array ofparticles. The particles may include a receptor molecule coupled toa polymeric bead. The receptors, in some embodiments, are chosenfor interacting with analytes. This interaction may take the formof a binding/association of the receptors with the analytes. Thesupporting member may be made of any material capable of supportingthe particles, while allowing the passage of the appropriatewavelengths of light. The supporting member may include a pluralityof cavities. The cavities may be formed such that at least oneparticle is substantially contained within the cavity. A vacuum maybe coupled to the cavities. The vacuum may be applied to the entiresensor array. Alternatively, a vacuum apparatus may be coupled tothe cavities to provide a vacuum to the cavities. A vacuumapparatus is any device capable of creating a pressure differentialto cause fluid movement. The vacuum apparatus may apply a pullingforce to any fluids within the cavity. The vacuum apparatus maypull the fluid through the cavity. Examples of vacuum apparatussinclude pre-sealed vacuum chamber, vacuum pumps, vacuum lines, oraspirator-type pumps.

BRIEF DESCRIPTION OF THE DRAWINGS

The above brief description as well as further objects, featuresand advantages of the methods and apparatus of the presentinvention will be more fully appreciated by reference to thefollowing detailed description of presently preferred butnonetheless illustrative embodiments in accordance with the presentinvention when taken in conjunction with the accompanying drawingsin which:

FIG. 1 depicts a schematic of an analyte detection system;

FIG. 2 depicts a particle disposed in a cavity;

FIG. 3 depicts a sensor array;

FIGS. 4A F depict the formation of a Fabry-Perot cavity on the backof a sensor array;

FIG. 5 depicts the chemical constituents of a particle;

FIG. 6 depicts the chemical formulas of some receptorcompounds;

FIG. 7 depicts a plot of the absorbance of green light vs.concentration of calcium (Ca.sup.+2) for a particle which includesan o-cresolphthalein complexone receptor;

FIG. 8 depicts a schematic view of the transfer of energy from afirst indicator to a second indicator in the presence of ananalyte;

FIG. 9 depicts a schematic of the interaction of a sugar moleculewith a boronic acid based receptor.

FIG. 10 depicts various synthetic receptors;

FIG. 11 depicts a synthetic pathway for the synthesis ofpolythioureas;

FIG. 12 depicts a synthetic pathway for the synthesis ofpolyguanidiniums;

FIG. 13 depicts a synthetic pathway for the synthesis of diaminesfrom amino acids;

FIG. 14 depicts fluorescent diamino monomers;

FIG. 15 depicts a plot of counts/sec. (i.e., intensity) vs. time asthe pH of a solution surrounding a particle coupled too-cresolphthalein is cycled from acidic to basic conditions;

FIG. 16 depicts the color responses of a variety of sensingparticles to solutions of Ca.sup.+2 and various pH levels;

FIG. 17 depicts an analyte detection system which includes a sensorarray disposed within a chamber;

FIG. 18 depicts an integrated analyte detection system;

FIG. 19 depicts a cross-sectional view of a cavity covered by amesh cover;

FIG. 20 depicts a top view of a cavity covered by a mesh cover;FIGS. 21A G depict a cross-sectional view of a series of processingsteps for the formation of a sensor array which includes aremovable top and bottom cover;

FIGS. 22A G depict a cross-sectional view of a series of processingsteps for the formation of a sensor array which includes aremovable top and a stationary bottom cover;

FIGS. 23A G depict a cross-sectional view of a series of processingsteps for the formation of a sensor array which includes aremovable top;

FIGS. 24A D depict a cross-sectional view of a series of processingsteps for the formation of a silicon based sensor array whichincludes a top and bottom cover with openings aligned with thecavity;

FIGS. 25A D depict a cross-sectional view of a series of processingsteps for the formation of a photoresist based sensor array whichincludes a top and bottom cover with openings aligned with thecavity;

FIGS. 26A E depict a cross-sectional view of a series of processingsteps for the formation of a plastic based sensor array whichincludes a top and bottom cover with openings aligned with thecavity;

FIGS. 27A D depict a cross-sectional view of a series of processingsteps for the formation of a silicon based sensor array whichincludes a top cover with openings aligned with the cavity and atapered cavity;

FIGS. 28A E depict a cross-sectional view of a series of processingsteps for the formation of a photoresist based sensor array whichincludes a top cover with openings aligned with the cavity and atapered cavity;

FIGS. 29A E depict a cross-sectional view of a series of processingsteps for the formation of a photoresist based sensor array whichincludes a top cover with openings aligned with the cavity and abottom cover;

FIGS. 30A D depict a cross-sectional view of a series of processingsteps for the formation of a plastic based sensor array whichincludes a top cover with openings aligned with the cavity and abottom cover;

FIG. 31 depicts a cross-sectional view of a schematic of amicropump;

FIG. 32 depicts a top view of an electrohydrodynamic pump;

FIG. 33 depicts a cross-sectional view of a sensor array whichincludes a micropump;

FIG. 34 depicts a cross-sectional view of a sensor array whichincludes a micropump and channels which are coupled to thecavities;

FIG. 35 depicts a cross-sectional view of a sensor array whichincludes multiple micropumps each micropump being coupled to acavity;

FIG. 36 depicts a top view of a sensor array which includesmultiple electrohydrodynamic pumps;

FIG. 37 depicts a cross-sectional view of a sensor array whichincludes a system for delivering a reagent from a reagent particleto a sensing cavity;

FIG. 38 depicts a cross-sectional view of a sensor array whichincludes a vacuum chamber;

FIG. 39 depicts a cross-sectional view of a sensor array whichincludes a vacuum chamber, a filter, and a reagent reservoir.

FIG. 40 depicts a general scheme for the testing of an antibodyanalyte;

FIG. 41 depicts general scheme for the detection of antibodieswhich uses a sensor array composed of four individual beads;

FIG. 42 depicts a sensor array which includes a vacuum chamber, asensor array chamber, and a sampling device;

FIG. 43 depicts a flow path of a fluid stream through a sensorarray from the top toward the bottom of the sensor array;

FIG. 44 depicts a flow path of a fluid stream through a sensorarray from the bottom toward the top of the sensor array;

FIGS. 45A C depict the disruption of neuromuscular communication bya toxin;

FIG. 45D depicts the attachment of differentially protected lysineto a bead;

FIG. 46 depicts a system for measuring the absorbance or emissionof a sensing particle;

FIG. 47 depicts receptors 3 6;

FIG. 48 depicts pH indicators which may be coupled to aparticle;

FIG. 49 depicts a device for the analysis of IP.sub.3 in cells;

FIG. 50 depicts the structure of Indo-1 and compound 2 and theemission spectra of Indo-1 and compound 2 in the presence of Ca(II)and Ce(III), respectively;

FIG. 51 depicts a scheme wherein binding of citrate to a receptorfrees up the Indo-1 for Ca(II) binding;

FIG. 52 depicts the change in FRET between coumarin and5-carboxyfluorescein on resin beads as a function of thesolvent;

FIG. 53 depicts a scheme wherein a signal of apo-7 to citrate istriggered by Cu(II) binding;

FIG. 54 depicts the response of receptor 3 and 5-carboxyfluorosceinon a resin bead to the addition of citrate;

FIGS. 55A I depict various sensing protocols forreceptor-indicator-polymeric resin particles;

FIG. 56 depicts a peptide trimer receptor and a pair of fluorescentindicators coupled to a polymeric resin;

FIG. 57 depicts a synthetic scheme for anchoring dansyl and dapoxylindicators to 6% agarose glyoxalated resin beads;

FIG. 58 depicts the RGB epifluorescence of 6 in EtOH with varyingratio buffer concentrations;

FIG. 59 depicts indicators and polymeric beads used forfluorescence studies;

FIG. 60 depicts Emission spectra of derivatized dapoxyl dyes invarious solvents;

FIG. 61 depicts a general structure of a chemically sensitiveparticle that includes a receptor and multiple indicators coupledto a polymeric resin;

FIGS. 62A D depict various sensing protocols forreceptor-indicator-polymeric resin particles in which a cleavagereaction occurs;

FIG. 63 depicts a plot of the fluorescence signal of a chemicallysensitive particle in the presence of trypsin.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Herein we describe a system and method for the simultaneousanalysis of a fluid containing multiple analytes. The system may beused for either liquid or gaseous fluids. The system may generatepatterns that are diagnostic for both individual analytes andmixtures of the analytes. The system, in some embodiments, is madeof a combination of chemically sensitive particles, formed in anordered array, capable of simultaneously detecting many differentkinds of analytes rapidly. An aspect of the system is that thearray may be formed using a microfabrication process, thus allowingthe system to be manufactured in an inexpensive manner.

System for Analysis of Analytes

Shown in FIG. 1 is an embodiment of a system for detecting analytesin a fluid. The system, in some embodiments, includes a lightsource 110, a sensor array 120 and a detector 130. The light source110 may be a white light source or light emitting diodes (LED). Inone embodiment, light source 110 may be a blue light emitting diode(LED) for use in systems relying on changes in fluorescencesignals. For colorimetric (e.g., absorbance) based systems, a whitelight source may be used. The sensor array 120, in someembodiments, is formed of a supporting member which is configuredto hold a variety of particles 124. A detecting device 130 (e.g., acharge-coupled device "CCD") may be positioned below the sensorarray to allow for data acquisition. In another embodiment, thedetecting device 130 may be positioned above the sensor array.

Light originating from the light source 110, in some embodiments,passes through the sensor array 120 and out through the bottom sideof the sensor array. The supporting member and the particlestogether, in some embodiments, provide an assembly whose opticalproperties are well matched for spectral analyses. Thus, lightmodulated by the particles may pass through the sensor array andonto the proximally spaced detector 130. Evaluation of the opticalchanges may be completed by visual inspection (e.g., with amicroscope) or by use of a microprocessor 140 coupled to thedetector. For fluorescence measurements, a filter 135 may be placedbetween supporting member 120 and detector 130 to remove theexcitation wavelength. A fluid delivery system 160 may be coupledto the supporting member. The fluid delivery system 160 may beconfigured to introduce samples into and out of the sensorarray.

In an embodiment, the sensor array system includes an array ofparticles. Upon the surface and within the interior region of theparticles are, in some embodiments, located a variety of receptorsfor interacting with analytes. The supporting member, in someembodiments, is used to localize these particles as well as toserve as a microenvironment in which the chemical assays can beperformed. For the chemical/biological agent sensor arrays, theparticles used for analysis are about 0.05 500 microns in diameter,and may actually change size (e.g., swell or shrink) when thechemical environment changes. Typically, these changes occur whenthe array system is exposed to the fluid stream which includes theanalytes. For example, a fluid stream which comprises a non-polarsolvent, may cause non-polar particles to change in volume when theparticles are exposed to the solvent. To accommodate these changes,it is preferred that the supporting member consist of an array ofcavities which serve as micro test-tubes.

The supporting member may be made of any material capable ofsupporting the particles, while allowing the passage of theappropriate wavelength of light. The supporting member is also madeof a material substantially impervious to the fluid in which theanalyte is present. A variety of materials may be used includingplastics, glass, silicon based materials (e.g., silicon, silicondioxide, silicon nitride, etc.) and metals. In one embodiment, thesupporting member includes a plurality of cavities. The cavitiesmay be formed such that at least one particle is substantiallycontained within the cavity. Alternatively, a plurality ofparticles may be contained within a single cavity.

In an embodiment, the supporting member may consist of a strip ofplastic which is substantially transparent to the wavelength oflight necessary for detection. A series of cavities may be formedwithin the strip. The cavities may be configured to hold at leastone particle. The particles may be contained within the strip by atransparent cover which is configured to allow passage of theanalyte containing fluid into the cavities.

In another embodiment, the supporting member may be formed using asilicon wafer as depicted in FIG. 2. The silicon wafer 210 mayinclude a substantially transparent layer 220 formed on the bottomsurface of the wafer. The cavities 230, in one embodiment, areformed by an anisotropic etch process of the silicon wafer. In oneembodiment, anisotropic etching of the silicon wafer isaccomplished using a wet hydroxide etch. Photolithographictechniques may be used to define the locations of the cavities. Thecavities may be formed such that the sidewalls of the cavities aresubstantially tapered at an angle of between about 50 to 60degrees. Formation of such angled cavities may be accomplished bywet anisotropic etching of <100> silicon. The term"<100> silicon" refers to the crystal orientation of thesilicon wafer. Other types of silicon, (e.g., <110> and<111> silicon) may lead to steeper angled sidewalls. Forexample, <111> silicon may lead to sidewalls formed at about90 degrees. The angled sides of the cavities in some embodiments,serve as "mirror layers" which may improve the light collectionefficiency of the cavities. The etch process may be controlled sothat the formed cavities extend through the silicon wafer to theupper surface of transparent layer 220. While depicted aspyramidal, the cavities may be formed in a number of shapesincluding but not limited to, spherical, oval, cubic, orrectangular. An advantage to using a silicon wafer for the supportmember, is that the silicon material is substantially opaque to thelight produced from the light source. Thus, the light may beinhibited from passing from one cavity to adjacent cavities. Inthis manner, light from one cavity may be inhibited frominfluencing the spectroscopic changes produced in an adjacentcavity.

The silicon wafer, in some embodiments, has an area ofapproximately 1 cm.sup.2 to about 100 cm.sup.2 and includes about10.sup.1 to about 10.sup.6 cavities. In an embodiment, about 100cavities are formed in a ten by ten matrix. The center to centerdistance between the cavities, in some embodiments, is about 500microns. Each of the cavities may include at least oneparticle.

The transparent layer 220 may serve as a window, allowing light ofa variety of wavelengths to pass through the cavities 230 and tothe detector. Additionally, the transparent layer may serve as aplatform onto which the individual particles 235 may be positioned.The transparent layer may be formed of silicon dioxide (SiO.sub.2),silicon nitride (Si.sub.3N.sub.4) or silicon dioxide/siliconnitride multi-layer stacks. The transparent layer, in someembodiments, is deposited onto the silicon wafer prior to theformation of the cavities.

The cavities 230 may be sized to substantially contain a particle235. The cavities are, in some embodiments, larger than a particle.The cavities are, in some embodiments, sized to allow facileplacement and removal of the particle within the cavities. Thecavity may be substantially larger than the particle, thus allowingthe particle to swell during use. For example, a particle may havea size as depicted in FIG. 2 by particle 235. During use, contactwith a fluid (e.g., a solvent) may cause the particle to swell, forexample, to a size depicted as circle 236. In some embodiments, thecavity is sized to allow such swelling of the particle during use.A particle may be positioned at the bottom of a cavity using, e.g.,a micromanipulator. After a particle has been placed within thecavity, a transparent cover plate 240 may be placed on top of thesupporting member to keep the particle in place.

When forming an array which includes a plurality of particles, theparticles may be placed in the array in an ordered fashion usingthe micromanipulator. In this manner, a ordered array having apredefined configuration of particles may be formed. Alternatively,the particles may be randomly placed within the cavities. The arraymay subsequently undergo a calibration test to determine theidentity of the particle at any specified location in thesupporting member.

The transparent cover plate 240, in some embodiments, is coupled tothe upper surface of the silicon wafer 220 such that the particlesare inhibited from becoming dislodged from the cavity. Thetransparent cover plate, in some embodiments, is positioned a fixeddistance above the silicon wafer, as depicted in FIG. 2, to keepthe particle in place, while allowing the entrance of fluids intothe cavities. The transparent cover plate, in some embodiments, ispositioned at a distance above the substrate which is substantiallyless than a width of the particle. The transparent cover plate maybe made of any material which is substantially transparent to thewavelength of light being utilized by the detector. The transparentcover plate may be made of plastic, glass, quartz, or silicondioxide/silicon nitride.

In one embodiment, the transparent cover plate 240, is a thin sheetof glass (e.g., a microscope slide cover slip). The slide may bepositioned a fixed distance above the silicon wafer. Supportstructures 241 (See FIG. 2) may be placed upon the silicon wafer210 to position the transparent cover plate 240. The supportstructures may be formed from a polymer or a silicon basedmaterial. In another embodiment, a polymeric substrate is coupledto the silicon wafer to form the support structures 241 for thetransparent cover plate 240. In an embodiment, a plastic materialwith an adhesive backing (e.g., cellophane tape) is positioned onthe silicon wafer 210. After the support structures 241 are placedon the wafer the transparent cover plate 240 is placed upon thesupport structures. The support structures inhibit the transparentcover sheet from contacting the silicon wafer 200. In this manner,a channel is formed between the silicon wafer and the transparentcover plate which allow the fluid to pass into the cavity, whileinhibiting displacement of the particle by the fluid.

In another embodiment, the transparent cover plate 240 may befastened to the upper surface of the silicon wafer, as depicted inFIG. 3. In this embodiment, the fluid may be inhibited fromentering the cavities 230 by the transparent cover plate 240. Toallow passage of the fluid into the cavities, a number of channels250 may be formed in the silicon wafer. The channels, in oneembodiment, are oriented to allow passage of the fluid intosubstantially all of the cavities. When contacted with the fluid,the particles may swell to a size which may plug the channels. Toprevent this plugging, the channels may be formed near the upperportion of the cavities, as depicted in FIG. 3. The channels, inone embodiment, are formed using standard photolithographic maskingto define the regions where the trenches are to be formed, followedby the use of standard etching techniques. A depth of the cavitymay be such that the particle resides substantially below theposition of the channel. In this way, the plugging of the channelsdue to swelling of the particle may be prevented.

The inner surfaces of the cavities may be coated with a material toaid the positioning of the particles within the cavities. In oneembodiment, a thin layer of gold or silver may be used to line theinner surface of the cavities. The gold or silver layer may act asan anchoring surface to anchor particles (e.g., via alkylthiolbonding). In addition, the gold or silver layer may also increasethe reflectivity of the inner surface of the cavities. Theincreased reflectance of the surface may enhance the analytedetection sensitivity of the system. Alternatively, polymer layersand self-assembled monolayers formed upon the inner surface of thecavities may be used to control the particle adhesion interactions.Additional chemical anchoring methods may be used for siliconsurfaces such as those based on siloxane type reagents, which maybe attached to Si--OH functionalities. Similarly, monomeric andpolymeric reagents attached to an interior region of the cavitiescan be used to alter the local wetting characteristics of thecavities. This type of methodology can be used to anchor theparticles as well as to alter the fluid delivery characteristics ofthe cavity. Furthermore, amplification of the signals for theanalytes may be accomplished with this type of strategy by causingpreconcentration of appropriate analytes in the appropriate type ofchemical environment.

In another embodiment, the optical detector may be integratedwithin the bottom transparent layer 220 of the supporting member,rather than using a separate detecting device. The opticaldetectors may be formed using a semiconductor-based photodetector255. The optical detectors may be coupled to a microprocessor toallow evaluation of fluids without the use of separate detectingcomponents. Additionally, the fluid delivery system may also beincorporated into the supporting member. Micro-pumps andmicro-valves may also be incorporated into the silicon wafer to aidpassage of the fluid through the cavities. Integration of detectorsand a fluid delivery system into the supporting member may allowthe formation of a compact and portable analyte sensing system.Optical filters may also be integrated into the bottom membrane ofthe cavities. These filters may prevent short wavelength excitationfrom producing "false" signals in the optical detection system(e.g., a CCD detector array) during fluorescence measurements.

A sensing cavity may be formed on the bottom surface of the supportsubstrate. An example of a sensing cavity that may be used is aFabry-Perot type cavity. Fabry-Perot cavity-based sensors may beused to detect changes in optical path length induced by either achange in the refractive index or a change in physical length ofthe cavity. Using micromachining techniques, Fabry-Perot sensorsmay be formed on the bottom surface of the cavity.

FIGS. 4A F depict a sequence of processing steps for the formationof a cavity and a planar top diaphragm Fabry-Perot sensor on thebottom surface of a silicon based supporting member. A sacrificialbarrier layer 262a/b is deposited upon both sides of a siliconsupporting member 260. The silicon supporting member 260 may be adouble-side polished silicon wafer having a thickness ranging fromabout 100 .mu.m to about 500 .mu.m, preferably from about 200 .mu.mto about 400 .mu.m, and more preferably of about 300 .mu.m. Thebarrier layer 262a/b may be composed of silicon dioxide, siliconnitride, or silicon oxynitride. In one embodiment, the barrierlayer 262a/b is composed of a stack of dielectric materials. Asdepicted in FIG. 4A, the barrier layer 262a/b is composed of astack of dielectric materials which includes a silicon nitridelayer 271a/b and a silicon dioxide layer 272a/b. Both layers may bedeposited using a low pressure chemical vapor deposition ("LPCVD")process. Silicon nitride may be deposited using an LPCVD reactor byreaction of ammonia (NH.sub.3) and dichlorosilane(SiCl.sub.2H.sub.2) at a gas flow rate of about 3.5:1, atemperature of about 800.degree. C., and a pressure of about 220mTorr. The silicon nitride layer 271a/b is deposited to a thicknessin the range from about 100 .ANG. to about 500 .ANG., preferablyfrom 200 .ANG. to about 400 .ANG., and more preferably of about 300.ANG.. Silicon dioxide is may be deposited using an LPCVD reactorby reaction of silane (SiH.sub.4) and oxygen (O.sub.2) at a gasflow rate of about 3:4, a temperature of about 450.degree. C., anda pressure of about 110 mTorr. The silicon dioxide layer 272a/b isdeposited to a thickness in the range from about 3000 .ANG. toabout 7000 .ANG., preferably from 4000 .ANG. to about 6000 .ANG.,and more preferably of about 5000 .ANG.. The front face silicondioxide layer 272a, in one embodiment, acts as the main barrierlayer. The underlying silicon nitride layer 271a acts as anintermediate barrier layer to inhibit overetching of the mainbarrier layer during subsequent KOH wet anisotropic etchingsteps.

A bottom diaphragm layer 264a/b is deposited upon the barrier layer262a/b on both sides of the supporting member 260. The bottomdiaphragm layer 264a/b may be composed of silicon nitride, silicondioxide, or silicon oxynitride. In one embodiment, the bottomdiaphragm layer 264a/b is composed of a stack of dielectricmaterials. As depicted in FIG. 4A, the bottom diaphragm layer264a/b is composed of a stack of dielectric materials whichincludes a pair of silicon nitride layers 273a/b and 275a/bsurrounding a silicon dioxide layer 274a/b. All of the layers maybe deposited using an LPCVD process. The silicon nitride layers273a/b and 275a/b have a thickness in the range from about 500.ANG. to about 1000 .ANG., preferably from 700 .ANG. to about 800.ANG., and more preferably of about 750 .ANG.. The silicon dioxidelayer 274a/b has a thickness in the range from about 3000 .ANG. toabout 7000 .ANG., preferably from 4000 .ANG. to about 6000 .ANG.,and more preferably of about 4500 .ANG..

A cavity which will hold the particle may now be formed in thesupporting member 260. The bottom diaphragm layer 264b and thebarrier layer 262b formed on the back side 261 of the siliconsupporting member 260 are patterned and etched using standardphotolithographic techniques. In one embodiment, the layers aresubjected to a plasma etch process. The plasma etching of silicondioxide and silicon nitride may be performed using a mixture ofcarbontetrafluoride (CF.sub.4) and oxygen (O.sub.2). The patternedback side layers 262b and 264b may be used as a mask foranisotropic etching of the silicon supporting member 260. Thesilicon supporting member 260, in one embodiment, isanisotropically etched with a 40% potassium hydroxide ("KOH")solution at 80.degree. C. to form the cavity. The etch is stoppedwhen the front side silicon nitride layer 271a is reached, asdepicted in FIG. 4B. The silicon nitride layer 271a inhibitsetching of the main barrier layer 272a during this etch process.The cavity 267 may be formed extending through the supportingmember 260. After formation of the cavity, the remaining portionsof the back side barrier layer 262b and the diaphragm layer 264bmay be removed.

Etch windows 266 are formed through the bottom diaphragm layer 264aon the front side of the wafer. A masking layer (not shown) isformed over the bottom diaphragm layer 264a and patterned usingstandard photolithographic techniques. Using the masking layer,etch windows 266 may be formed using a plasma etch. The plasmaetching of silicon dioxide and silicon nitride may be performedusing a mixture of carbontetrafluoride (CF.sub.4) and oxygen(O.sub.2). The etching is continued through the bottom diaphragmlayer 264a and partially into the barrier layer 262a. In oneembodiment, the etching is stopped at approximately half thethickness of the barrier layer 262a. Thus, when the barrier layer262a is subsequently removed the etch windows 266 will extendthrough the bottom diaphragm layer 264a, communicating with thecavity 267. By stopping the etching at a midpoint of the barrierlayer, voids or discontinuities may be reduced since the bottomdiaphragm is still continuous due to the remaining barrierlayer.

After the etch windows 266 are formed, a sacrificial spacer layer268a/b is deposited upon the bottom diaphragm layer 264a and withincavity 267, as depicted in FIG. 4C. The spacer layer may be formedfrom LPCVD polysilicon. In one embodiment, the front side depositedspacer layer 268a will also at least partially fill the etchwindows 266. Polysilicon may be deposited using an LPCVD reactorusing silane (SiH.sub.4) at a temperature of about 650.degree. C.The spacer layer 268a/b is deposited to a thickness in the rangefrom about 4000 .ANG. to about 10,000 .ANG., preferably from 6000.ANG. to about 8000 .ANG., and more preferably of about 7000 .ANG..The preferred thickness of the spacer layer 268a is dependent onthe desired thickness of the internal air cavity of the Fabry-Perotdetector. For example, if a Fabry-Perot detector which is toinclude a 7000 .ANG. air cavity between the top and bottomdiaphragm layer is desired, a spacer layer having a thickness ofabout 7000 .ANG. would be formed. After the spacer layer has beendeposited, a masking layer for etching the spacer layer 268a (notshown) is used to define the etch regions of the spacer layer 268a.The etching may be performed using a composition of nitric acid(HNO.sub.3), water, and hydrogen fluoride (HF) in a ratio of25:13:1, respectively, by volume. The lateral size of thesubsequently formed cavity is determined by the masking patternused to define the etch regions of the spacer layer 268a.

After the spacer layer 268a has been etched, the top diaphragmlayer 270a/b is formed. The top diaphragm 270a/b, in oneembodiment, is deposited upon the spacer layer 268a/b on both sidesof the supporting member. The top diaphragm 270a/b may be composedof silicon nitride, silicon dioxide, or silicon oxynitride. In oneembodiment, the top diaphragm 270a/b is composed of a stack ofdielectric materials. As depicted in FIG. 4C, the top diaphragm270a/b is composed of a stack of dielectric materials whichincludes a pair of silicon nitride layers 283a/b and 285a/bsurrounding a silicon dioxide layer 284a/b. All of the layers maybe deposited using an LPCVD process. The silicon nitride layers283a/b and 285a/b have a thickness in the range from about 1000.ANG. to about 2000 .ANG., preferably from 1200 .ANG. to about 1700.ANG., and more preferably of about 1500 .ANG.. The silicon dioxidelayer 284a/b has a thickness in the range from about 5000 .ANG. toabout 15,500 .ANG., preferably from 7500 .ANG. to about 12,000.ANG., and more preferably of about 10,500 .ANG..

After depositing the top diaphragm 270a/b, all of the layersstacked on the bottom face of the supporting member (e.g., layers268b, 283b, 284b, and 285b) are removed by multiple wet and plasmaetching steps, as depicted in FIG. 4D. After these layers areremoved, the now exposed portions of the barrier layer 262a arealso removed. This exposes the spacer layer 268a which is presentin the etch windows 266. The spacer layer 268 may be removed frombetween the top diaphragm 270a and the bottom diaphragm 264a by awet etch using a KOH solution, as depicted in FIG. 4D. Removal ofthe spacer material 268a, forms a cavity 286 between the topdiaphragm layer 270a and the bottom diaphragm layer 264a. Afterremoval of the spacer material, the cavity 286 may be washed usingdeionized water, followed by isopropyl alcohol to clean out anyremaining etching solution.

The cavity 286 of the Fabry-Perot sensor may be filled with asensing substrate 290, as depicted in FIG. 4E. To coat the cavity286 with a sensing substrate 290, the sensing substrate may bedissolved in a solvent. A solution of the sensing substrate isapplied to the supporting member 260. The solution is believed torapidly enter the cavity 286 through the etched windows 266 in thebottom diaphragm 264a, aided in part by capillary action. As thesolvent evaporates, a thin film of the sensing substrate 290 coatsthe inner walls of the cavity 286, as well as the outer surface ofthe bottom diaphragm 264a. By repeated treatment of the supportingmember with the solution of the sensing substrate, the thickness ofthe sensing substrate may be varied.

In one embodiment, the sensing substrate 290 ispoly(3-dodecylthiophene) whose optical properties change inresponse to changes in oxidation states. The sensing substratepoly(3-dodecylthiophene) may be dissolved in a solvent such aschloroform or xylene. In one embodiment, a concentration of about0.1 g of poly(3-dodecylthiophene)/mL is used. Application of thesolution of poly(3-dodecylthiophene) to the supporting membercauses a thin film of poly(3-dodecylthiophene) to be formed on theinner surface of the cavity.

In some instances, the sensing substrate, when deposited within acavity of a Fabry-Perot type detector, may cause stress in the topdiaphragm of the detector. It is believed that when a sensingpolymer coats a planar top diaphragm, extra residual stress on thetop diaphragm causes the diaphragm to become deflected toward thebottom diaphragm. If the deflection becomes to severe, stickingbetween the top and bottom diaphragms may occur. In one embodiment,this stress may be relieved by the use of supporting members 292formed within the cavity 286, as depicted in FIG. 4F. Thesupporting members 292 may be formed without any extra processingsteps to the above described process flow. The formation ofsupporting members may be accomplished by deliberately leaving aportion of the spacer layer within the cavity. This may beaccomplished by underetching the spacer layer (e.g., terminatingthe etch process before the entire etch process is finished). Theremaining spacer will behave as a support member to reduce thedeflection of the top diaphragm member. The size and shape of thesupport members may be adjusted by altering the etch time of thespacer layer, or adjusting the shape of the etch windows 266.

In another embodiment, a high sensitivity CCD array may be used tomeasure changes in optical characteristics which occur upon bindingof the biological/chemical agents. The CCD arrays may be interfacedwith filters, light sources, fluid delivery and micromachinedparticle receptacles, so as to create a functional sensor array.Data acquisition and handling may be performed with existing CCDtechnology. Data streams (e.g., red, green, blue for colorimetricassays; gray intensity for fluorescence assays) may be transferredfrom the CCD to a computer via a data acquisition board. CurrentCCDs may allow for read-out rates of 10.sup.5 pixels per second.Thus, the entire array of particles may be evaluated hundreds oftimes per second allowing for studies of the dynamics of thevarious host-guest interaction rates as well as the analyte/polymerdiffusional characteristics. Evaluation of this data may offer amethod of identifying and quantifying the chemical/biologicalcomposition of the test samples. CCD detectors may be configured tomeasure white light, ultraviolet light or fluorescence. Otherdetectors such as photomultiplier tubes, charge induction devices,photodiode, photodiode arrays, and microchannel plates may also beused. It should be understood that while the detector is depictedas being positioned under the supporting member, the detector mayalso be positioned above the supporting member. It should also beunderstood that the detector typically includes a sensing elementfor detecting the spectroscopic events and a component fordisplaying the detected events. The display component may bephysically separated from the sensing element. The sensing elementmay be positioned above or below the sensor array while the displaycomponent is positioned close to a user.

In one embodiment, a CCD detector may be used to record colorchanges of the chemical sensitive particles during analysis. Asdepicted in FIG. 1, a CCD detector 130 may be placed beneath thesupporting member 120. The light transmitted through the cavitiesis captured and analyzed by the CCD detector. In one embodiment,the light is broken down into three color components, red, greenand blue. To simplify the data, each color is recorded using 8 bitsof data. Thus, the data for each of the colors will appear as avalue between 0 and 255. The color of each chemical sensitiveelement may be represented as a red, blue and green value. Forexample, a blank particle (i.e., a particle which does not includea receptor) will typically appear white. For example, when brokendown into the red, green and blue components, it is found that atypical blank particle exhibits a red value of about 253, a greenvalue of about 250, and a blue value of about 222. This signifiesthat a blank particle does not significantly absorb red, green orblue light. When a particle with a receptor is scanned, theparticle may exhibit a color change, due to absorbance by thereceptor. For example, it was found that when a particle whichincludes a 5-carboxyfluorescein receptor is subjected to whitelight, the particle shows a strong absorbance of blue light. TheCCD detector values for the 5-carboxyfluorescein particle exhibitsa red value of about 254, a green value of about 218, and a bluevalue of about 57. The decrease in transmittance of blue light isbelieved to be due to the absorbance of blue light by the5-carboxyfluorescein. In this manner, the color changes of aparticle may be quantitatively characterized. An advantage of usinga CCD detector to monitor the color changes is that color changeswhich may not be noticeable to the human eye may now bedetected.

The support array may be configured to allow a variety of detectionmodes to be practiced. In one embodiment, a light source is used togenerate light which is directed toward the particles. Theparticles may absorb a portion of the light as the lightilluminates the particles. The light then reaches the detector,reduced in intensity by the absorbance of the particles. Thedetector may be configure to measure the reduction in lightintensity (i.e., the absorbance) due to the particles. In anotherembodiment, the detector may be placed above the supporting member.The detector may be configured to measure the amount of lightreflected off of the particles. The absorbance of light by theparticles is manifested by a reduction in the amount of light beingreflected from the cavity. The light source in either embodimentmay be a white light source or a fluorescent light source.

Chemically Sensitive Particles

A particle, in some embodiments, possess both the ability to bindthe analyte of interest and to create a modulated signal. Theparticle may include receptor molecules which posses the ability tobind the analyte of interest and to create a modulated signal.Alternatively, the particle may include receptor molecules andindicators. The receptor molecule may posses the ability to bind toan analyte of interest. Upon binding the analyte of interest, thereceptor molecule may cause the indicator molecule to produce themodulated signal. The receptor molecules may be naturally occurringor synthetic receptors formed by rational design or combinatorialmethods. Some examples of natural receptors include, but are notlimited to, DNA, RNA, proteins, enzymes, oligopeptides, antigens,and antibodies. Either natural or synthetic receptors may be chosenfor their ability to bind to the analyte molecules in a specificmanner. The forces which drive association/recognition betweenmolecules include the hydrophobic effect, anion-cation attraction,and hydrogen bonding. The relative strengths of these forces dependupon factors such as the solvent dielectric properties, the shapeof the host molecule, and how it complements the guest. Uponhost-guest association, attractive interactions occur and themolecules stick together. The most widely used analogy for thischemical interaction is that of a "lock and key". The fit of thekey molecule (the guest) into the lock (the host) is a molecularrecognition event.

A naturally occurring or synthetic receptor may be bound to apolymeric resin in order to create the particle. The polymericresin may be made from a variety of polymers including, but notlimited to, agarous, dextrose, acrylamide, control pore glassbeads, polystyrene-polyethylene glycol resin, polystyrene-divinylbenzene resin, formylpolystyrene resin, trityl-polystyrene resin,acetyl polystyrene resin, chloroacetyl polystyrene resin,aminomethyl polystyrene-divinylbenzene resin, carboxypolystyreneresin, chloromethylated polystyrene-divinylbenzene resin,hydroxymethyl polystyrene-divinylbenzene resin, 2-chlorotritylchloride polystyrene resin, 4-benzyloxy-2'4'-dimethoxybenzhydrolresin (Rink Acid resin), triphenyl methanol polystyrene resin,diphenylmethanol resin, benzhydrol resin, succinimidyl carbonateresin, p-nitrophenyl carbonate resin, imidazole carbonate resin,polyacrylamide resin,4-sulfamylbenzoyl-4'-methylbenzhydrylamine-resin (Safety-catchresin), 2-amino-2-(2'-nitrophenyl) propionic acid-aminomethyl resin(ANP Resin), p-benzyloxybenzyl alcohol-divinylbenzene resin (Wangresin), p-methylbenzhydrylamine-divinylbenzene resin (MBHA resin),Fmoc-2,4-dimethoxy-4'-(carboxymethyloxy)-benzhydrylamine linked toresin (Knorr resin), 4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (Rink resin),4-hydroxymethyl-benzoyl-4'-methylbenzhydrylamine resin (HMBA-MBHAResin), p-nitrobenzophenone oxime resin (Kaiser oxime resin), andamino-2,4-dimethoxy-4'-(carboxymethyloxy)-benzhydrylamine handlelinked to 2-chlorotrityl resin (Knorr-2-chlorotrityl resin). In oneembodiment, the material used to form the polymeric resin iscompatible with the solvent in which the analyte is dissolved. Forexample, polystyrene-divinyl benzene resin will swell withinnon-polar solvents, but does not significantly swell within polarsolvents. Thus, polystyrene-divinyl benzene resin may be used forthe analysis of analytes within non-polar solvents. Alternatively,polystyrene-polyethylene glycol resin will swell with polarsolvents such as water. Polystyrene-polyethylene glycol resin maybe useful for the analysis of aqueous fluids.

In one embodiment, a polystyrene-polyethylene glycol-divinylbenzene material is used to form the polymeric resin. Thepolystyrene-polyethylene glycol-divinyl benzene resin is formedfrom a mixture of polystyrene 375, divinyl benzene 380 andpolystyrene-polyethylene glycol 385, see FIG. 5. The polyethyleneglycol portion of the polystyrene-polyethylene glycol 385, in oneembodiment, may be terminated with an amine. The amine serves as achemical handle to anchor both receptors and indicator dyes. Otherchemical functional groups may be positioned at the terminal end ofthe polyethylene glycol to allow appropriate coupling of thepolymeric resin to the receptor molecules or indicators.

The chemically sensitive particle, in one embodiment, is capable ofboth binding the analyte(s) of interest and creating a detectablesignal. In one embodiment, the particle will create an opticalsignal when bound to an analyte of interest. The use of such apolymeric bound receptors offers advantages both in terms of costand configurability. Instead of having to synthesize or attach areceptor directly to a supporting member, the polymeric boundreceptors may be synthesized en masse and distributed to multipledifferent supporting members. This allows the cost of the sensorarray, a major hurdle to the development of mass-producedenvironmental probes and medical diagnostics, to be reduced.Additionally, sensor arrays which incorporate polymeric boundreceptors may be reconfigured much more quickly than array systemsin which the receptor is attached directly tot he supportingmember. For example, if a new variant of a pathogen or a pathogenthat contains a genetically engineered protein is a threat, then anew sensor array system may be readily created to detect thesemodified analytes by simply adding new sensor elements (e.g.,polymeric bound receptors) to a previously formed supportingmember.

In one embodiment, a receptor, which is sensitive to changes in thepH of a fluid sample is bound to a polymeric resin to create aparticle. That is, the receptor is sensitive to the concentrationof hydrogen cations (H.sup.+). The receptor in this case istypically sensitive to the concentration of H.sup.+ in a fluidsolution. The analyte of interest may therefore be H.sup.+. Thereare many types of molecules which undergo a color change when thepH of the fluid is changed. For example, many types of dyes undergosignificant color changes as the pH of the fluid medium is altered.Examples of receptors which may be used to monitor the pH of afluid sample include 5-carboxyfluorescein and alizarin complexone,depicted in FIG. 6. Each of these receptors undergoes significantcolor changes as the pH of the fluid is altered.5-carboxyfluorescein undergoes a change from yellow to orange asthe pH of the fluid is increased. Alizarin complexone undergoes twocolor changes, first from yellow to red, then from red to blue asthe pH of the fluid increases. By monitoring the change in colorcaused by dyes attached to a polymeric particle, the pH of asolution may be qualitatively and, with the use of a detector(e.g., a CCD detector), quantitatively monitored.

In another embodiment, a receptor which is sensitive to presence ofmetal cations is bound to a polymeric particle to create aparticle. The receptor in this case is typically sensitive to theconcentration of one or more metal cations present in a fluidsolution. In general, colored molecules which will bind cations maybe used to determine the presence of a metal cation in a fluidsolution. Examples of receptors which may be used to monitor thepresence of cations in a fluid sample include alizarin complexoneand o-cresolphthalein complexone, see FIG. 6. Each of thesereceptors undergoes significant color changes as the concentrationof a specific metal ion in the fluid is altered. Alizarincomplexone is particularly sensitive to lanthanum ions. In theabsence of lanthanum, alizarin complexone will exhibit a yellowcolor. As the concentration of lanthanum is increased, alizarincomplexone will change to a red color. o-Cresolphthalein complexoneis particularly sensitive to calcium ions. In the absence ofcalcium, o-cresolphthalein complexone is colorless. As theconcentration of calcium is increased, o-cresolphthalein complexonewill change to a blue color. By monitoring the change in color ofmetal cation sensitive receptors attached to a polymeric particle,the presence of a specific metal ion may be qualitatively and, withthe use of a detector (e.g., a CCD detector), quantitativelymonitored.

Referring to FIG. 7, a graph of the absorbance of green light vs.concentration of calcium (Ca.sup.+2) is depicted for a particlewhich includes an o-cresolphthalein complexone receptor. As theconcentration of calcium is increased, the absorbance of greenlight increases in a linear manner up to a concentration of about0.0006 M. A concentration of 0.0006 M is the solubility limit ofcalcium in the fluid, thus no significant change in absorbance isnoted after this point. The linear relationship betweenconcentration and absorbance allows the concentration of calcium tobe determined by measuring the absorbance of the fluid sample.

In one embodiment, a detectable signal may be caused by thealtering of the physical properties of an indicator ligand bound tothe receptor or the polymeric resin. In one embodiment, twodifferent indicators are attached to a receptor or the polymericresin. When an analyte is captured by the receptor, the physicaldistance between the two indicators may be altered such that achange in the spectroscopic properties of the indicators isproduced. A variety of fluorescent and phosphorescent indicatorsmay be used for this sensing scheme. This process, known as Forsterenergy transfer, is extremely sensitive to small changes in thedistance between the indicator molecules.

For example, a first fluorescent indicator 320 (e.g., a fluoresceinderivative) and a second fluorescent indictor 330 (e.g., arhodamine derivative) may be attached to a receptor 300, asdepicted in FIG. 8. When no analyte is present short wavelengthexcitation 310 may excite the first fluorescent indicator 320,which fluoresces as indicated by 312. The short wavelengthexcitation, however, may cause little or no fluorescence of thesecond fluorescent indicator 330.

After binding of analyte 350 to the receptor, a structural changein the receptor molecule may bring the first and second fluorescentindicators closer to each other. This change in intermoleculardistance may allow the excited first indicator 320 to transfer aportion of its fluorescent energy 325 to the second fluorescentindicator 330. This transfer in energy may be measured by either adrop in energy of the fluorescence of the first indicator molecule320, or the detection of increased fluorescence 314 by the secondindicator molecule 330.

Alternatively, the first and second fluorescent indicators mayinitially be positioned such that short wavelength excitation, maycause fluorescence of both the first and second fluorescentindicators, as described above. After binding of analyte 350 to thereceptor, a structural change in the receptor molecule may causethe first and second fluorescent indicators to move further apart.This change in intermolecular distance may inhibit the transfer offluorescent energy from the first indicator 320 to the secondfluorescent indicator 330. This change in the transfer of energymay be measured by either a drop in energy of the fluorescence ofthe second indicator molecule 330, or the detection of increasedfluorescence by the first indicator molecule 320.

In another embodiment, an indicator ligand may be preloaded ontothe receptor. An analyte may then displace the indicator ligand toproduce a change in the spectroscopic properties of the particles.In this case, the initial background absorbance is relatively largeand decreases when the analyte is present. The indicator ligand, inone embodiment, has a variety of spectroscopic properties which maybe measured. These spectroscopic properties include, but are notlimited to, ultraviolet absorption, visible absorption, infraredabsorption, fluorescence, and magnetic resonance. In oneembodiment, the indicator is a dye having either a strongfluorescence, a strong ultraviolet absorption, a strong visibleabsorption, or a combination of these physical properties. Examplesof indicators include, but are not limited to, carboxyfluorescein,ethidium bromide, 7-dimethylamino-4-methylcoumarin,7-diethylamino-4-methylcoumarin, eosin, erythrosin, fluorescein,Oregon Green 488, pyrene, Rhodamine Red, tetramethylrhodamine,Texas Red, Methyl Violet, Crystal Violet, Ethyl Violet, Malachitegreen, Methyl Green, Alizarin Red S, Methyl Red, Neutral Red,o-cresolsulfonephthalein, o-cresolphthalein, phenolphthalein,Acridine Orange, B-naphthol, coumarin, and a-naphthionic acid. Whenthe indicator is mixed with the receptor, the receptor andindicator interact with each other such that the above mentionedspectroscopic properties of the indicator, as well as otherspectroscopic properties may be altered. The nature of thisinteraction may be a binding interaction, wherein the indicator andreceptor are attracted to each other with a sufficient force toallow the newly formed receptor-indicator complex to function as asingle unit. The binding of the indicator and receptor to eachother may take the form of a covalent bond, an ionic bond, ahydrogen bond, a van der Waals interaction, or a combination ofthese bonds.

The indicator may be chosen such that the binding strength of theindicator to the receptor is less than the binding strength of theanalyte to the receptor. Thus, in the presence of an analyte, thebinding of the indicator with the receptor may be disrupted,releasing the indicator from the receptor. When released, thephysical properties of the indicator may be altered from those itexhibited when bound to the receptor. The indicator may revert backto its original structure, thus regaining its original physicalproperties. For example, if a fluorescent indicator is attached toa particle that includes a receptor, the fluorescence of theparticle may be strong before treatment with an analyte containingfluid. When the analyte interacts with the particle, thefluorescent indicator may be released. Release of the indicator maycause a decrease in the fluorescence of the particle, since theparticle now has less indicator molecules associated with it.

An example of this type of system is illustrated by the use of aboronic acid substituted resin 505 as a particle. Prior to testing,the boronic acid substituted resin 505 is treated with a sugar 510which is tagged with an indicator (e.g., resorufin) as depicted inFIG. 9. The sugar 510 binds to the boronic acid receptor 500imparting a color change to the boronic substituted resin 505(yellow for the resorufin tagged sugar). When the boronic acidresin 505 is treated with a fluid sample which includes a sugar520, the tagged sugar 510 may be displaced, causing a decrease inthe amount of color produced by the boronic acid substituted resin505. This decrease may be qualitatively or, with the use of adetector (e.g., a CCD detector), quantitatively monitored.

In another embodiment, a designed synthetic receptor may be used.In one embodiment, a polycarboxylic acid receptor may be attachedto a polymeric resin. The polycarboxylic receptors are discussed inU.S. patent application Ser. No. 08/950,712 which is incorporatedherein by reference.

In an embodiment, the analyte molecules in the fluid may bepretreated with an indicator ligand. Pretreatment may involvecovalent attachment of an indicator ligand to the analyte molecule.After the indicator has been attached to the analyte, the fluid maybe passed over the sensing particles. Interaction of the receptorson the sensing particles with the analytes may remove the analytesfrom the solution. Since the analytes include an indicator, thespectroscopic properties of the indicator may be passed onto theparticle. By analyzing the physical properties of the sensingparticles after passage of an analyte stream, the presence andconcentration of an analyte may be determined.

For example, the analytes within a fluid may be derivatized with afluorescent tag before introducing the stream to the particles. Asanalyte molecules are adsorbed by the particles, the fluorescenceof the particles may increase. The presence of a fluorescent signalmay be used to determine the presence of a specific analyte.Additionally, the strength of the fluorescence may be used todetermine the amount of analyte within the stream.

Receptors

A variety of natural and synthetic receptors may be used. Thesynthetic receptors may come from a variety of classes including,but not limited to, polynucleotides (e.g., aptamers), peptides(e.g., enzymes and antibodies), synthetic receptors, polymericunnatural biopolymers (e.g., polythioureas, polyguanidiniums), andimprinted polymers, some of which are generally depicted in FIG.10. Natural based synthetic receptors include receptors which arestructurally similar to naturally occurring molecules.Polynucleotides are relatively small fragments of DNA which may bederived by sequentially building the DNA sequence. Peptides may besynthesized from amino acids. Unnatural biopolymers are chemicalstructure which are based on natural biopolymers, but which arebuilt from unnatural linking units. Unnatural biopolymers such aspolythioureas and polyguanidiniums may be synthesized from diamines(i.e., compounds which include at least two amine functionalgroups). These molecules are structurally similar to naturallyoccurring receptors, (e.g., peptides). Some diamines may, in turn,be synthesized from amino acids. The use of amino acids as thebuilding blocks for these compounds allow a wide variety ofmolecular recognition units to be devised. For example, the twentynatural amino acids have side chains that possess hydrophobicresidues, cationic and anionic residues, as well as hydrogenbonding groups. These side chains may provide a good chemical matchto bind a large number of targets, from small molecules to largeoligosaccharides. Amino acid based peptides, polythioureas, andpolyguanidiniums are depicted in FIG. 10.

Techniques for the building of DNA fragments and polypeptidefragments on a polymer particle are well known. Techniques for theimmobilization of naturally occurring antibodies and enzymes on apolymeric resin are also well known. The synthesis of polythioureasupon a resin particle may be accomplished by the synthetic pathwaydepicted in FIG. 11. The procedure may begin by deprotection of theterminal tBoc protecting group on an amino acid coupled to apolymeric particle. Removal of the protecting group is followed bycoupling of the rigid spacer 410 to the resulting amine 405 usingdiisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole hydrate(HOBT). The spacer group may inhibit formation of a thiazolone byreaction of the first amino acids with subsequently formedthioureas. After the spacer group is coupled to the amino acid,another tBoc deprotection is performed to remove the spacerprotecting group, giving the amine 415. At this point, monomer maybe added incrementally to the growing chain, each time followed bya tBoc deprotection. The addition of a derivative of the diamine420 (e.g., an isothiocyanate) to amine 415 gives the mono-thiourea425. The addition of a second thiourea substituent is alsodepicted. After the addition of the desired number of monomers, asolution of benzylisothiocyanate or acetic anhydride may be addedto cap any remaining amines on the growing oligomers. Between 1 to20 thioureas groups may be formed to produce a syntheticpolythiourea receptor.

The synthesis of polyguanidiniums may be accomplished as depictedin FIG. 12. In order to incorporate these guanidinium groups intothe receptor, the coupling of a thiourea with a terminal amine inthe presence of Mukaiyama's reagent may be utilized. The couplingof the first thiourea diamine 430 with an amino group of apolymeric particle gives the mono-guanidinium 434. Coupling of theresulting mono-guanidinium with a second thiourea diamine 436 givesa di-guanidinium 438. Further coupling may create a tri-guanidinium440. Between 1 to 20 guanidinium groups may be formed to produce asynthetic polyguanidinium receptor.

The above described methods for making polythioureas andpolyguanidiniums are based on the incorporation of diamines (i.e.,molecules which include at least two amine functional groups) intothe oligomeric receptor. The method may be general for any compoundhaving at least two amino groups. In one embodiment, the diaminemay be derived from amino acids. A method for forming diamines fromamino acids is shown in FIG. 13. Treatment of a protected aminoacid 450 with borane-THF reduces the carboxylic acid portion of theamino acid to the primary alcohol 452. The primary alcohol istreated with phthalimide under Mitsunobu conditions(PPh.sub.3/DEAD). The resulting compound 454 is treated withaqueous methylamine to form the desired monoprotected diamine 456.The process may be accomplished such that the enantiomeric purityof the starting amino acid is maintained. Any natural or syntheticamino acid may be used in the above described method.

The three coupling strategies used to form the respectivefunctional groups may be completely compatible with each other. Thecapability to mix linking groups (amides, thioureas, andguanidiniums) as well as the side chains (hydrophobic, cationic,anionic, and hydrogen bonding) may allow the creation of adiversity in the oligomers that is beyond the diversity ofreceptors typically found with natural biological receptors. Thus,we may produce ultra-sensitive and ultra-selective receptors whichexhibit interactions for specific toxins, bacteria, andenvironmental chemicals. Additionally, these synthetic schemes maybe used to build combinatorial libraries of particles for use inthe sensor array.

In an embodiment, the indicator ligand may be incorporated intosynthetic receptors during the synthesis of the receptors. Theligand may be incorporated into a monomeric unit, such as adiamine, that is used during the synthesis of the receptor. In thismanner, the indicator may be covalently attached to the receptor ina controlled position. By placing the indicator within the receptorduring the synthesis of the receptor, the positioning of theindicator ligand within the receptor may be controlled. Thiscontrol may be difficult to achieve after synthesis of the receptoris completed.

In one embodiment, a fluorescent group may be incorporated into adiamine monomer for use in the synthetic sequences. Examples ofmonomeric units which may be used for the synthesis of a receptorare depicted in FIG. 14. The depicted monomers include fluorescentindicator groups. After synthesis, the interaction of the receptorwith the analyte may induce changes in the spectroscopic propertiesof the molecule. Typically, hydrogen bonding or ionic substituentson the fluorescent monomer involved in analyte binding have thecapacity to change the electron density and/or rigidity of thefluorescent ring system, thereby causing observable changes in thespectroscopic properties of the indicator. For fluorescentindicators such changes may be exhibited as changes in thefluorescence quantum yield, maximum excitation wavelength, and/ormaximum emission wavelength. This approach does not require thedissociation of a preloaded fluorescent ligand, which may belimited in response time by k(.sub.off)). While fluorescent ligandsare shown here, it is to be understood that a variety of otherligand may be used including colorimetric ligands.

In another embodiment, two fluorescent monomers for signaling maybe used for the synthesis of the receptor. For example, compound470 (a derivative of fluorescein) and compound 475 (a derivative ofrhodamine), depicted in FIG. 14, may both be incorporated into asynthetic receptor. Compound 470 contains a commoncolorimetric/fluorescent probe that will, in some embodiments, sendout a modulated signal upon analyte binding. The modulation may bedue to resonance energy transfer to compound 475. When an analytebinds to the receptor, structural changes in the receptor may alterthe distance between monomeric units 470 and 475. It is well knownthat excitation of fluorescein can result in emission fromrhodamine when these molecules are oriented correctly. Theefficiency of resonance energy transfer from monomers 470 to 475will depend strongly upon the presence of analyte binding; thus,measurement of rhodamine fluorescence intensity (at a substantiallylonger wavelength than fluorescein fluorescence) may serve as anindicator of analyte binding. To greatly improve the likelihood ofa modulatory fluorescein-rhodamine interaction, multiple rhodaminetags may be attached at different sites along a receptor moleculewithout substantially increasing background rhodamine fluorescence(only rhodamine very close to fluorescein will yield appreciablesignal). This methodology may be applied to a number of alternatefluorescent pairs.

In an embodiment, a large number of chemical/biological agents ofinterest to the military and civilian communities may be sensedreadily by the described array sensors including both small andmedium size molecules. For example, it is known that nerve gasestypically produce phosphate structures upon hydrolysis in water.The presence of molecules which contain phosphate functional groupsmay be detected using polyguanidiniums. Nerve gases which havecontaminated water sources may be detected by the use of thepolyguanidinium receptors described above.

In order to identify, sense, and quantitate the presence of variousbacteria using the proposed micro-machined sensor, two strategiesmay be used. First, small molecule recognition and detection may beexploited. Since each bacteria possesses a unique and distinctiveconcentration of the various cellular molecules, such as DNA,proteins, metabolites, and sugars, the fingerprint (i.e., theconcentration and types of DNA, proteins, metabolites, and sugars)of each organism is expected to be unique. Hence, the analytesobtained from whole bacteria or broken down bacteria may be used todetermine the presence of specific bacteria. A series of receptorsspecific for DNA molecules, proteins, metabolites, and sugars maybe incorporated into an array. A solution containing bacteria, ormore preferably broken down bacteria, may be passed over the arrayof particles. The individual cellular components of the bacteriamay interact in a different manner with each of the particles. Thisinteraction will provide a pattern within the array which may beunique for the individual bacteria. In this manner, the presence ofbacteria within a fluid may be determined.

In another embodiment, bacteria may be detected as whole entities,as found in ground water, aerosols, or blood. To detect, sense, andidentify intact bacteria, the cell surface of one bacteria may bedifferentiated from other bacteria. One method of accomplishingthis differentiation is to target cell surface oligosaccharides(i.e. sugar residues). Each bacterial class (gram negative, grampositive, etc.) displays a different oligosaccharide on their cellsurfaces.

The oligosaccharide, which is the code that is read by other cellsgiving an identification of the cell, is part of the cell--cellrecognition and communication process. The use of syntheticreceptors which are specific for oligosaccharides may be used todetermine the presence of specific bacteria by analyzing for thecell surface oligosaccharides.

In another embodiment, the sensor array may be used to optimizewhich receptor molecules should be used for a specific analyte. Anarray of receptors may be placed within the cavities of thesupporting member and a stream containing an analyte may be passedover the array. The reaction of each portion of the sensing arrayto the known analyte may be analyzed and the optimal receptordetermined by determining which particle, and therefore whichreceptor, exhibits the strongest reaction toward the analyte. Inthis manner, a large number of potential receptors may be rapidlyscanned. The optimal receptor may then be incorporated into asystem used for the detection of the specific analyte in a mixtureof analytes.

It should be emphasized that although some particles may bepurposefully designed to bind to important species (biologicalagents, toxins, nerve gasses, etc.), most structures will possessnonspecific receptor groups. One of the advantages associated withthe proposed sensor array is the capacity to standardize each arrayof particles via exposure to various analytes, followed by storageof the patterns which arise from interaction of the analytes withthe particles. Therefore, there may not be a need to know theidentity of the actual receptor on each particle. Only thecharacteristic pattern for each array of particles is important. Infact, for many applications it may be less time consuming to placethe various particles into their respective holders without takingprecautions to characterize the location associated with thespecific particles. When used in this manner, each individualsensor array may require standardization for the type of analyte tobe studied.

On-site calibration for new or unknown toxins may also be possiblewith this type of array. Upon complexation of an analyte, the localmicroenvironment of each indicator may change, resulting in amodulation of the light absorption and/or emission properties. Theuse of standard pattern recognition algorithms completed on acomputer platform may serves as the intelligence factor for theanalysis. The "fingerprint" like response evoked from thesimultaneous interactions occurring at multiple sites within thesubstrate may be used to identify the species present in unknownsamples.

The above described sensor array system offers a number of distinctadvantages over exiting technologies. One advantage is that "realtime" detection of analytes may be performed. Another advantage isthat the simultaneous detection of multiple analytes may berealized. Yet another advantage is that the sensor array systemallows the use of synthetic reagents as well as biologicallyproduced reagents. Synthetic reagents typically have superiorsensitivity and specificity toward analytes when compared to thebiological reagents. Yet another advantage is that the sensor arraysystem may be readily modified by simply changing the particleswhich are placed within the sensor array. This interchangabilitymay also reduce production costs.

EXAMPLES

1. The Determination of pH Using a Chemically SensitiveParticle.

Shown in FIG. 15 is the magnitude of the optical signal transmittedthrough a single polymer particle derivatized witho-cresolphthalein. Here, a filter is used to focus the analysis onthose wavelengths which the dye absorbs most strongly (i.e., about550 nm). Data is provided for the particle as the pH is cycledbetween acid and basic environments. In acidic media (i.e., attimes of 100 150 seconds and 180 210 seconds), the particle isclear and the system yields large signals (up to greater than300,000 counts) at the optical detector. Between times of 0 100 and150 180 seconds, the solution was made basic. Upon raising the pH(i.e., making the solution more basic), the particle turns purplein color and the transmitted green light is greatly diminished.Large signal reductions are recorded under such circumstances. Theevolution of the signal changes show that the response time isquite rapid, on the order of 10 seconds. Furthermore, the behavioris highly reproducible.

2. The Simultaneous Detection of Ca.sup.+2, Ce.sup.+3, and pH by aSensor Array System.

The synthesis of four different particles was accomplished bycoupling a variety of indictor ligands to a polyethyleneglycol-polystyrene ("PEG-PS") resin particle. The PEG-PS resinparticles were obtained from Novabiochem Corp., La Jolla, Calif.The particles have an average diameter of about 130 .mu.m when dryand about 250 .mu.m when wet. The indicator ligands of fluorescein,o-cresolphthalein complexone, and alizarin complexone were eachattached to PEG-PS resin particles using a dicyclohexylcarbodiimide(DCC) coupling between a terminal resin bound amine and acarboxylic acid on the indicator ligand.

These synthetic receptors, localized on the PEG-PS resin to createsensing particles, were positioned within micromachined wellsformed in silicon/silicon nitride wafers, thus confining theparticles to individually addressable positions on a multicomponentchip. These wells were sized to hold the particles in both swollenand unswollen states. Rapid introduction of the test fluids can beaccomplished using these structures while allowingspectrophotometric assays to probe for the presence of analytes.For the identification and quantification of analyte species,changes in the light absorption and light emission properties ofthe immobilized resin particles can be exploited, although onlyidentification based upon absorption properties are discussed here.Upon exposure to analytes, color changes for the particles werefound to be 90% complete within one minute of exposure, althoughtypically only seconds were required. To make the analysis of thecolorimetric changes efficient, rapid, and sensitive, acharge-coupled-device (CCD) was directly interfaced with the sensorarray. Thus, data streams composed of red, green, and blue (RGB)light intensities were acquired and processed for each of theindividual particle elements. The red, blue, and green responses ofthe particles to various solutions are graphically depicted in FIG.16.

The true power of the described bead sensor array occurs whensimultaneous evaluation of multiple chemically distinct beadstructures is completed. A demonstration of the capacity of fivedifferent beads is provided in FIG. 16. In this case, blank,alizarin, o-cresol phthalein, fluorescein, and alizarin-Ce3+complex derivatized beads serve as a matrix for subtledifferentiation of chemical environments. The blank bead is simplya polystyrene sphere with no chemical derivatization. The beadderivatized with o-cresolphthalein responds to Ca+2 at pHs valuesaround 10.0. The binding of calcium is noted from the large greencolor attenuation noted for this dye while exposed to the cation.Similarly, the fluorescein derivatized bead acts as a pH sensor. AtpHs below 7.4 it is light yellow, but at higher pHs it turns darkorange. Interesting, the alizarin complexone plays three distinctroles. First, it acts as a proton sensor yielding a yellow color atpHs below 4.5, orange is noted at pHs between 4.5 and 11.5, and atpHs above 11.5 a blue hue is observed. Second, it functions as asensor for lanthanum ions at lower pHs by turning yellow to orange.Third, the combination of both fluoride and lanthanum ions resultsin yellow/orange coloration.

The analysis of solutions containing various amount of Ca.sup.+2 orF.sup.- at various pH levels was performed using alizarincomplexone, o-cresolphthalein complexone, 5-carboxy fluorescein,and alizarin-Ce.sup.3+ complex. A blank particle in which theterminal amines of a PEG-PS resin particle have been acylated wasalso used. In this example, the presence of Ca.sup.+2 (0.1 MCa(NO.sub.3).sub.2) was analyzed under conditions of varying pH.The pH was varied to values of 2, 7, and 12, all buffered by amixture of 0.04 M phosphate, 0.04 M acetate, and 0.04 M borate. TheRGB patterns for each sensor element in all environments weremeasured. The bead derivatized with o-cresolphthalein responds toCa.sup.+2 at pH values around 12. Similarly, the 5-carboxyfluorescein derivatized bead acts as a pH sensor. At pHs below 7.4it is light yellow, but at higher pHs it turns dark orange.Interesting, the alizarin complexone plays three distinct roles.First, it acts as a proton sensor yielding a yellow color at pHsbelow 4.5, orange is noted at pHs between 4.5 and 11.5, and at pHsabove 11.5 a blue hue is observed. Second, it functions as a sensorfor lanthanum ions at lower pHs by turning yellow to orange. Third,the combination of both fluoride and lanthanum ions results inyellow/orange coloration.

This example demonstrates a number of important factors related tothe design, testing, and functionality of micromachined arraysensors for solution analyses. First, derivatization of polymerparticles with both colorimetric and fluorescent dyes wascompleted. These structures were shown to respond to pH andCa.sup.2+. Second, response times well under 1 minute were found.Third, micromachined arrays suitable both for confinement ofparticles, as well as optical characterization of the particles,have been prepared. Fourth, integration of the test bed arrays withcommercially available CCD detectors has been accomplished.Finally, simultaneous detection of several analytes in a mixturewas made possible by analysis of the RGB color patterns created bythe sensor array.

3. The Detection of Sugar Molecules Using a Boronic Acid BasedReceptor.

A series of receptors were prepared with functionalities thatassociate strongly with sugar molecules, as depicted in FIG. 9. Inthis case, a boronic acid sugar receptor 500 was utilized todemonstrate the functionality of a new type of sensing scheme inwhich competitive displacement of a resorufin derivatized galactosesugar molecule was used to assess the presence (or lack thereof) ofother sugar molecules. The boronic acid receptor 500 was formed viaa substitution reaction of a benzylic bromide. The boronic acidreceptor was attached to a polyethylene glycol-polystyrene("PEG-PS") resin particle at the "R" position. Initially, theboronic acid derivatized particle was loaded with resorufinderivatized galactose 510. Upon exposure of the particle to asolution containing glucose 520, the resorufin derivatizedgalactose molecules 510 are displaced from the particle receptorsites. Visual inspection of the optical photographs taken beforeand after exposure to the sugar solution show that the boronsubstituted resin is capable of sequestering sugar molecules froman aqueous solution. Moreover, the subsequent exposure of thecolored particles to a solution of a non-tagged sugar (e.g.,glucose) leads to a displacement of the bound colored sugarreporter molecule. Displacement of this molecule leads to a changein the color of the particle. The sugar sensor turns from darkorange to yellow in solutions containing glucose. The particleswere also tested in conditions of varying pH. It was noted that thecolor of the particles changes from dark orange to yellow as the pHis varied from low pH to high pH.

Further Improvements

1. System Improvements

Shown in FIG. 17 is an embodiment of a system for detectinganalytes in a fluid. In one embodiment, the system includes a lightsource 512, a sensor array 522, a chamber 550 for supporting thesensor array and a detector 530. The sensor array 522 may include asupporting member which is configured to hold a variety ofparticles. In one embodiment, light originating from the lightsource 512 passes through the sensor array 522 and out through thebottom side of the sensor array. Light modulated by the particlesmay be detected by a proximally spaced detector 530. While depictedas being positioned below the sensor array, it should be understoodthat the detector may be positioned above the sensor array forreflectance measurements. Evaluation of the optical changes may becompleted by visual inspection (e.g., by eye, or with the aid of amicroscope) or by use of a microprocessor 540 coupled to thedetector.

In this embodiment, the sensor array 522 is positioned within achamber 550. The chamber 550, may be configured to allow a fluidstream to pass through the chamber such that the fluid streaminteracts with the sensor array 522. The chamber may be constructedof glass (e.g, borosilicate glass or quartz) or a plastic materialwhich is transparent to a portion of the light from the lightsource. If a plastic material is used, the plastic material shouldalso be substantially unreactive toward the fluid. Examples ofplastic materials which may be used to form the chamber include,but are not limited to, acrylic resins, polycarbonates, polyesterresins, polyethylenes, polyimides, polyvinyl polymers (e.g.,polyvinyl chloride, polyvinyl acetate, polyvinyl dichloride,polyvinyl fluoride, etc.), polystyrenes, polypropylenes,polytetrafluoroethylenes, and polyurethanes. An example of such achamber is a Sykes-Moore chamber, which is commercially availablefrom Bellco Glass, Inc., in New Jersey. Chamber 550, in oneembodiment, includes a fluid inlet port 552 and a fluid outlet port554. The fluid inlet 552 and outlet 554 ports are configured toallow a fluid stream to pass into the interior 556 of the chamberduring use. The inlet and outlet ports may be configured to allowfacile placement of a conduit for transferring the fluid to thechamber. In one embodiment, the ports may be hollow conduits. Thehollow conduits may be configured to have an outer diameter whichis substantially equal to the inner diameter of a tube fortransferring the fluid to or away from the chamber. For example, ifa plastic or rubber tube is used for the transfer of the fluid, theinternal diameter of the plastic tube is substantially equal to theouter diameter of the inlet and outlet ports.

In another embodiment, the inlet and outlet ports may be Luer lockstyle connectors. Preferably, the inlet and outlet ports are femaleLuer lock connectors. The use of female Luer lock connectors willallow the fluid to be introduced via a syringe. Typically, syringesinclude a male Luer lock connector at the dispensing end of thesyringe. For the introduction of liquid samples, the use of Luerlock connectors may allow samples to be transferred directly from asyringe to the chamber 550. Luer lock connectors may also allowplastic or rubber tubing to be connected to the chamber using Luerlock tubing connectors.

The chamber may be configured to allow the passage of a fluidsample to be substantially confined to the interior 556 of thechamber. By confining the fluid to a small interior volume, theamount of fluid required for an analysis may be minimized. Theinterior volume may be specifically modified for the desiredapplication. For example, for the analysis of small volumes offluid samples, the chamber may be designed to have a small interiorchamber, thus reducing the amount of fluid needed to fill thechamber. For larger samples, a larger interior chamber may be used.Larger chambers may allow a faster throughput of the fluid duringuse.

In another embodiment, depicted in FIG. 18, a system for detectinganalytes in a fluid includes a light source 512, a sensor array522, a chamber 550 for supporting the sensor array and a detector530, all enclosed within a detection system enclosure 560. Asdescribed above, the sensor array 522 is preferably formed of asupporting member which is configured to hold a variety ofparticles. Thus, in a single enclosure, all of the components of ananalyte detection system are included.

The formation of an analyte detection system in a single enclosuremay allow the formation of a portable detection system. Forexample, a small controller 570 may be coupled to the analytedetection system. The controller 570 may be configured to interactwith the detector and display the results from the analysis. In oneembodiment, the controller includes a display device 572 fordisplaying information to a user. The controller may also includeinput devices 574 (e.g., buttons) to allow the user to control theoperation of the analyte detection system. For example, thecontroller may control the operation of the light source 512 andthe operation of the detector 530.

The detection system enclosure 560, may be interchangeable with thecontroller. Coupling members 576 and 578 may be used to remove thedetection system enclosure 560 from the controller 570. A seconddetection system enclosure may be readily coupled to the controllerusing coupling members 576 and 578. In this manner, a variety ofdifferent types of analytes may be detecting using a variety ofdifferent detection system enclosures. Each of the detection systemenclosures may include different sensor arrays mounted within theirchambers. Instead of having to exchange the sensor array fordifferent types of analysis, the entire detection system enclosuremay be exchanged. This may prove advantageous, when a variety ofdetection schemes are used. For example a first detection systemenclosure may be configured for white light applications. The firstdetection system enclosure may include a white light source, asensor that includes particles that produce a visible lightresponse in the presence of an analyte, and a detector sensitive towhite light. A second detection system enclosure may be configuredfor fluorescent applications, including a fluorescent light source,a sensor array which includes particles which produce a fluorescentresponse on the presence of an analyte, and a fluorescent detector.The second detection system enclosure may also include othercomponents necessary for producing a proper detection system. Forexample, the second detection system may also include a filter forpreventing short wavelength excitation from producing "false"signals in the optical detection system during fluorescencemeasurements. A user need only select the proper detection systemenclosure for the detection of the desired analyte. Since eachdetection system enclosure includes many of the requiredcomponents, a user does not have to make light source selections,sensor array selections or detector arrangement selections toproduce a viable detection system.

In another embodiment, the individual components of the system maybe interchangeable. The system may include coupling members 573 and575 that allow the light source and the detector, respectively, tobe removed from the chamber 550. This may allow a more modulardesign of the system. For example, an analysis may be firstperformed with a white light source to give data corresponding toan absorbance/reflectance analysis. After this analysis isperformed the light source may be changed to a ultraviolet lightsource to allow ultraviolet analysis of the particles. Since theparticles have already been treated with the fluid, the analysismay be preformed without further treatment of the particles with afluid. In this manner a variety of tests may be performed using asingle sensor array.

In one embodiment, the supporting member is made of any materialcapable of supporting the particles, while allowing the passage ofthe appropriate wavelength of light. The supporting member may alsobe made of a material substantially impervious to the fluid inwhich the analyte is present. A variety of materials may be usedincluding plastics (e.g., photoresist materials, acrylic polymers,carbonate polymers, etc.), glass, silicon based materials (e.g.,silicon, silicon dioxide, silicon nitride, etc.) and metals. In oneembodiment, the supporting member includes a plurality of cavities.The cavities are preferably formed such that at least one particleis substantially contained within the cavity. Alternatively, aplurality of particles may be contained within a single cavity.

In some embodiments, it will be necessary to pass liquids over thesensor array. The dynamic motion of liquids across the sensor arraymay lead to displacement of the particles from the cavities. Inanother embodiment, the particles are preferably held withincavities formed in a supporting member by the use of a transmissionelectron microscope ("TEM") grid. As depicted in FIG. 19, a cavity580 is formed in a supporting member 582. After placement of aparticle 584 within the cavity, a TEM grid 586 may be placed atopthe supporting member 582 and secured into position. TEM grids andadhesives for securing TEM grids to a support are commerciallyavailable from Ted Pella, Inc., Redding, Calif. The TEM grid 586may be made from a number of materials including, but not limitedto, copper, nickel, gold, silver, aluminum, molybdenum, titanium,nylon, beryllium, carbon, and beryllium-copper. The mesh structureof the TEM grid may allow solution access as well as optical accessto the particles that are placed in the cavities. FIG. 20 furtherdepicts a top view of a sensor array with a TEM grid 586 formedupon the upper surface of the supporting member 582. The TEM grid586 may be placed on the upper surface of the supporting member,trapping particles 584 within the cavities 580. As depicted, theopenings 588 in the TEM grid 586 may be sized to hold the particles584 within the cavities 580, while allowing fluid and opticalaccess to cavities 580.

In another embodiment, a sensor array includes a supporting memberconfigured to support the particles, while allowing the passage ofthe appropriate wavelength of light to the particle. The supportingmember, in one embodiment, includes a plurality of cavities. Thecavities may be formed such that at least one particle issubstantially contained within the cavity. The supporting membermay be configured to substantially inhibit the displacement of theparticles from the cavities during use. The supporting member mayalso be configured to allow the passage of the fluid throughcavities, e.g., the fluid may flow from the top surface of thesupporting member, past the particle, and out the bottom surface ofthe supporting member. This may increase the contact time betweenthe particle and the fluid.

FIGS. 21A G depict a sequence of processing steps for the formationof a silicon based supporting member which includes a removable topcover and bottom cover. The removable top cover may be configuredto allow fluids to pass through the top cover and into the cavity.The removable bottom cover may also be configured to allow thefluid to pass through the bottom cover and out of the cavity. Asdepicted in FIG. 21A, a series of layers may be deposited upon bothsides of a silicon substrate 610. First removable layers 612 may bedeposited upon the silicon substrate. The removable layers 612 maybe silicon dioxide, silicon nitride, or photoresist material. Inone embodiment, a layer of silicon dioxide 612 is deposited uponboth surfaces of the silicon substrate 610. Upon these removablelayers, covers 614 may be formed. In one embodiment, covers 614 areformed from a material that differs from the material used to formthe removable layers 612 and which is substantially transparent tothe light source of a detection system. For example, if theremovable layers 612 are formed from silicon dioxide, the cover maybe formed from silicon nitride. Second removable layers 616 may beformed upon the covers 614. Second removable layers 616 may beformed from a material that differs from the material used to formthe covers 614. Second removable layers 616 may be formed from amaterial similar to the material used to form the first removablelayers 612. In one embodiment, first and second removable layers612 and 616 are formed from silicon dioxide and covers 614 areformed from silicon nitride. The layers are patterned and etchedusing standard photolithographic techniques. In one embodiment, theremaining portions of the layers are substantially aligned in theposition where the cavities are to be formed in the siliconsubstrate 610.

After the layers have been etched, spacer structures may be formedon the sidewalls of the first removable layers 612, the covers 614,and the second removable layers 616, as depicted in FIG. 21B. Thespacer structures may be formed from the same material used to formthe second removable layers 616. In one embodiment, depositing aspacer layer of the appropriate material and subjecting thematerial to an anisotropic etch may form the spacer structures. Ananisotropic etch, such as a plasma etch, employs both physical andchemical removal mechanisms. Ions are typically bombarded at anangle substantially perpendicular to the semiconductor substrateupper surface. This causes substantially horizontal surfaces to beremoved faster than substantially vertical surfaces. During thisetching procedure the spacer layers are preferably removed suchthat the only regions of the spacer layers that remain may be thoseregions near substantially vertical surfaces, e.g., spacerstructures 618.

After formation of the spacer structures 618, cover supportstructures 620, depicted in FIG. 21C, may be formed. The coversupport structures may be initially formed by depositing a supportstructure layer upon the second removable layer 616 and spacerstructures 618. The support structure layer is then patterned andetched, using standard photolithography, to form the supportstructures 620. In one embodiment, the support structures areformed from a material that differs from the removable layersmaterial. In one embodiment, the removable layers may be formedfrom silicon dioxide while the support structures and covers may beformed from silicon nitride.

Turning to FIG. 21 D, the second removable layers 616 and an upperportion of the spacer structures 618 are preferably removed using awet etch process. Removal of the second removable layers leaves thetop surface of the covers 614 exposed. This allows the covers to bepatterned and etched such that openings 622 are formed extendingthrough the covers. These openings 622 may be formed in the covers614 to allow the passage of fluid through the cover layers. In oneembodiment, the openings 622 are formed to allow fluid to passthrough, while inhibiting displacement of the particles from thesubsequently formed cavities.

After the openings 622 have been formed, the remainder of the firstremovable layers 612 and the remainder of the spacer structures 618may be removed using a wet etch. The removal of the removablelayers and the spacer structures creates "floating" covers 614, asdepicted in FIG. 21E. The covers 614 may be held in proximity tothe silicon substrate 610 by the support structures 620. The covers614 may now be removed by sliding the covers away from the supportstructures 620. In this manner removable covers 614 may beformed.

After the covers 614 are removed, cavities 640 may be formed in thesilicon substrate 610, as depicted in FIG. 21F. The cavities 640may be formed by, initially patterning and etching a photoresistmaterial 641 to form a masking layer. After the photoresistmaterial 641 is patterned, the cavities 640 may be etched into thesilicon substrate 610 using a hydroxide etch, as describedpreviously.

After the cavities 640 are formed, the photoresist material may beremoved and particles 642 may be placed within the cavities, asdepicted in FIG. 21G. The particles 642, may be inhibited frombeing displaced from the cavity 640 by placing covers 614 back ontothe upper and lower faces of the silicon substrate 610.

In another embodiment, a sensor array may be formed using asupporting member, a removable cover, and a secured bottom layer.FIGS. 22 A G depict a series of processing steps for the formationof a silicon based supporting member which includes a removable topcover and a secured bottom layer. The removable top cover ispreferably configured to allow fluids to pass through the top coverand into the cavity. As depicted in FIG. 22A, a series of layersmay be deposited upon both sides of a silicon substrate 610. Afirst removable layer 612 may be deposited upon the upper face 611of the silicon substrate 610. The removable layer 612 may besilicon dioxide, silicon nitride, or photoresist material. In oneembodiment, a layer of silicon dioxide 612 is deposited upon thesilicon substrate 610. A cover 614 may be formed upon the removablelayer 612 of the silicon substrate 610. In one embodiment, thecover 614 is formed from a material that differs from the materialused to form the removable layer 612 and is substantiallytransparent to the light source of a detection system. For example,if the removable layer 612 is formed from silicon dioxide, thecover layer 614 may be formed from silicon nitride. In oneembodiment, a bottom layer 615 is formed on the bottom surface 613of the silicon substrate 610. In one embodiment, the bottom layer615 is formed from a material that is substantially transparent tothe light source of a detection system. A second removable layer616 may be formed upon the cover 614. Second removable layer 616may be formed from a material that differs from the material usedto form the cover layer 614. Second removable layer 616 may beformed from a material similar to the material used to form thefirst removable layer 612. In one embodiment, first and secondremovable layers 612 and 616 are formed from silicon dioxide andcover 614 is formed from silicon nitride. The layers formed on theupper surface 611 of the silicon substrate may be patterned andetched using standard photolithographic techniques. In oneembodiment, the remaining portions of the layers formed on theupper surface are substantially aligned in the position where thecavities are to be formed in the silicon substrate 610.

After the layers have been etched, spacer structures may be formedon the side walls of the first removable layer 612, the cover 614,and the second removable layer 616, as depicted in FIG. 22B. Thespacer structures may be formed from the same material used to formthe second removable layer 616. In one embodiment, the spacerstructures may be formed by depositing a spacer layer of theappropriate material and subjecting the spacer layer to ananisotropic etch. During this etching procedure the spacer layer ispreferably removed such that the only regions of the spacer layerwhich remain may be those regions near substantially verticalsurfaces, e.g., spacer structures 618.

After formation of the spacer structures 618, cover supportstructures 620, depicted in FIG. 22C, may be formed upon theremovable layer 616 and the spacer structures 618. The coversupport structures 620 may be formed by depositing a supportstructure layer upon the second removable layer 616 and spacerstructures 618. The support structure layer is then patterned andetched, using standard photolithography, to form the supportstructures 620. In one embodiment, the support structures areformed from a material that differs from the removable layermaterials. In one embodiment, the removable layers may be formedfrom silicon dioxide while the support structures and cover may beformed from silicon nitride.

Turning to FIG. 22 D, the second removable layer 616 and an upperportion of the spacer structures 618 may be removed using a wetetch process. Removal of the second removable layer leaves the topsurface of the cover 614 exposed. This allows the cover 614 to bepatterned and etched such that openings 622 are formed extendingthrough the cover 614. These openings 622 may be formed in thecover 614 to allow the passage of fluid through the cover. In oneembodiment, the openings 622 are formed to allow fluid to passthrough, while inhibiting displacement of the particle from acavity. The bottom layer 615 may also be similarly patterned andetched such that openings 623 may be formed extending thorough thebottom layer 615.

After the openings 622 and 623 are formed, the first removablelayer 612 and the remainder of the spacer structures 618 may beremoved using a wet etch. The removal of the removable layers andthe spacer structures creates a "floating" cover 614, as depictedin FIG. 22E. The cover 614 may be held in proximity to the siliconsubstrate 610 by the support structures 620. The cover 614 may nowbe removed by sliding the cover 614 away from the supportstructures 620. In this manner a removable cover 614 may beformed.

After the cover 614 is removed, cavities 640 may be formed in thesilicon substrate 610, as depicted in FIG. 22F. The cavities 640may be formed by, initially patterning and etching a photoresistmaterial 641 to form a masking layer. After the photoresistmaterial 614 is patterned, the cavities 640 may be etched into thesilicon substrate 610 using a hydroxide etch, as describedpreviously.

After the cavities 640 are formed, the photoresist material may beremoved and particles 642 may be placed within the cavities, asdepicted in FIG. 22G. The particles 642, may be inhibited frombeing displaced from the cavity 640 by placing cover 614 back ontothe upper face 611 of the silicon substrate 610. The bottom layer615 may also aid in inhibiting the particle 642 from beingdisplaced from the cavity 640. Openings 622 in cover 614 andopenings 623 in bottom layer 615 may allow fluid to pass throughthe cavity during use.

In another embodiment, a sensor array may be formed using asupporting member and a removable cover. FIGS. 23A G depict aseries of processing steps for the formation of a silicon basedsupporting member which includes a removable cover. The removablecover is preferably configured to allow fluids to pass through thecover and into the cavity. As depicted in FIG. 23A, a series oflayers may be deposited upon the upper surface 611 of a siliconsubstrate 610. A first removable layer 612 may be deposited uponthe upper face 611 of the silicon substrate 610. The removablelayer 612 may be silicon dioxide, silicon nitride, or photoresistmaterial. In one embodiment, a layer of silicon dioxide 612 isdeposited upon the silicon substrate 610. A cover 614 may be formedupon the removable layer 612. In one embodiment, the cover isformed from a material which differs from the material used to formthe removable layer 612 and which is substantially transparent tothe light source of a detection system. For example, if theremovable layer 612 is formed from silicon dioxide, the cover 614may be formed from silicon nitride. A second removable layer 616may be formed upon the cover 614. Second removable layer 616 may beformed from a material that differs from the material used to formthe cover 614. Second removable layer 616 may be formed from amaterial similar to the material used to form the first removablelayer 612. In one embodiment, first and second removable layers 612and 616 are formed from silicon dioxide and cover 614 is formedfrom silicon nitride. The layers formed on the upper surface 611 ofthe silicon substrate may be patterned and etched using standardphotolithographic techniques. In one embodiment, the remainingportions of the layers formed on the upper surface aresubstantially aligned in the position where the cavities are to beformed in the silicon substrate 610.

After the layers have been etched, spacer structures 618 may beformed on the side walls of the first removable layer 612, thecover layer 614, and the second removable layer 616, as depicted inFIG. 23B. The spacer structures 618 may be formed from the samematerial used to form the second removable layer 616. In oneembodiment, the spacers may be formed by depositing a spacer layerof the appropriate material upon the second removable layer andsubjecting the material to an anisotropic etch. During this etchingprocedure the spacer layer is preferably removed such that the onlyregions of the spacer layer which remain may be those regions nearsubstantially vertical surfaces, e.g., spacer structures 618.

After formation of the spacer structures 618, cover supportstructures 620, depicted in FIG. 23C, may be formed upon theremovable layer 616 and the spacer structures 618. The coversupport structure may be formed by initially depositing a supportstructure layer upon the second removable layer 616 and spacerstructures 618. The support structure layer is then patterned andetched, using standard photolithography, to form the supportstructures 620. In one embodiment, the support structures 620 areformed from a material that differs from the removable layermaterials. In one embodiment, the removable layers may be formedfrom silicon dioxide while the support structure and cover layermay be formed from silicon nitride.

Turning to FIG. 23D, the second removable layer 616 and an upperportion of the spacer structures 618 may be removed using a wetetch process. Removal of the second removable layer leaves the topsurface of the cover 614 exposed. This allows the cover 614 to bepatterned and etched such that openings 622 are formed extendingthrough the cover 614. These openings 622 may be formed in thecover 614 to allow the passage of fluid through the cover 614.

After the openings 622 are formed, the remainder of the firstremovable layer 612 and the remainder of the spacer structures 618may be removed using a wet etch. The removal of the removablelayers and the spacer structures creates a "floating" cover 614, asdepicted in FIG. 23E. The cover 614 is preferably held in proximityto the silicon substrate 610 by the support structures 620. Thecover 614 may now be removed by sliding the cover 614 away from thesupport structures 620. In this manner a removable cover 614 may beformed.

After the cover 614 is removed, cavities 640 may be formed in thesilicon substrate 610, as depicted in FIG. 23F. The cavities 640may be formed by initially depositing and patterning a photoresistmaterial 641 upon the silicon support 610. After the photoresistmaterial 614 is patterned, the cavities 640 may be etched into thesilicon substrate 610 using a hydroxide etch, as describedpreviously. The etching of the cavities may be accomplished suchthat a bottom width of the cavity 643 is less than a width of aparticle 642. In one embodiment, the width of the bottom of thecavity may be controlled by varying the etch time. Typically,longer etching times result in a larger opening at the bottom ofthe cavity. By forming a cavity in this manner, a particle placedin the cavity may be too large to pass through the bottom of thecavity. Thus, a supporting member that does not include a bottomlayer may be formed. An advantage of this process is that theprocessing steps may be reduced making production simpler.

After the cavities 640 are formed, the photoresist material may beremoved and particles 642 may be placed within the cavities, asdepicted in FIG. 23G. The particles 642, may be inhibited frombeing displaced from the cavity 640 by placing cover 614 back ontothe upper face 611 of the silicon substrate 610. The narrow bottomportion of the cavity may also aid in inhibiting the particle 642from being displaced from the cavity 640.

FIGS. 24A-d depict a sequence of processing steps for the formationof a silicon based supporting member which includes a top partialcover and a bottom partial cover. The top partial cover and bottompartial covers are, in one embodiment, configured to allow fluidsto pass into the cavity and out through the bottom of the cavity.As depicted in FIG. 24A, a bottom layer 712 may be deposited ontothe bottom surface of a silicon substrate 710. The bottom layer 712may be silicon dioxide, silicon nitride, or photoresist material.In one embodiment, a layer of silicon nitride 712 is deposited uponthe silicon substrate 710. In one embodiment, openings 714 areformed through the bottom layer as depicted in FIG. 24A. Openings714, in one embodiment, are substantially aligned with the positionof the cavities to be subsequently formed. The openings 714 mayhave a width that is substantially less than a width of a particle.Thus a particle will be inhibited from passing through the openings714.

Cavities 716 may be formed in the silicon substrate 710, asdepicted in FIG. 24B. The cavities 716 may be formed by initiallydepositing and patterning a photoresist layer upon the siliconsubstrate 710. After the photoresist material is patterned,cavities 716 may be etched into the silicon substrate 710 using anumber of etching techniques, including wet and plasma etches. Thewidth of the cavities 716 is preferably greater than the width of aparticle, thus allowing a particle to be placed within each of thecavities. The cavities 716, in one embodiment, are preferablyformed such that the cavities are substantially aligned over theopenings 714 formed in the bottom layer.

After the cavities have been formed, particles 718 may be insertedinto the cavities 716, as depicted in FIG. 24C. The etched bottomlayer 712 may serve as a support for the particles 718. Thus theparticles 718 may be inhibited from being displaced from thecavities by the bottom layer 712. The openings 714 in the bottomlayer 712 may allow fluid to pass through the bottom layer duringuse.

After the particles are placed in the cavities, a top layer 720 maybe placed upon the upper surface 717 of the silicon substrate. Inone embodiment, the top layer 720 is formed from a material issubstantially transparent to the light source of a detectionsystem. The top layer may be formed from silicon nitride, silicondioxide or photoresist material. In one embodiment, a sheet ofphotoresist material is used. After the top layer 620 is formed,openings 719 may be formed in the top layer to allow the passage ofthe fluid into the cavities. If the top layer 720 is composed ofphotoresist material, after depositing the photoresist materialacross the upper surface of the silicon substrate, the openings maybe initially formed by exposing the photoresist material to theappropriate wavelength and pattern of light. If the top layer iscompose of silicon dioxide or silicon nitride the top layer 720 maybe developed by forming a photoresist layer upon the top layer,developing the photoresist, and using the photoresist to etch theunderlying top layer.

Similar sensor arrays may be produced using materials other thansilicon for the supporting member. For example, as depicted inFIGS. 25 A D, the supporting member may be composed of photoresistmaterial. In one embodiment, sheets of photoresist film may be usedto form the supporting member. Photoresist film sheets arecommercially available from E.I. du Pont de Nemours and Company,Wilmington, DE under the commercial name RISTON. The sheets come ina variety of sizes, the most common having a thickness ranging fromabout 1 mil. (25 .mu.m) to about 2 mil. (50 .mu.m).

In an embodiment, a first photoresist layer 722 is developed andetched such that openings 724 are formed. The openings may beformed proximate the location of the subsequently formed cavities.Preferably, the openings have a width that is substantially smallerthan a width of the particle. The openings may inhibit displacementof the particle from a cavity. After the first photoresist layer720 is patterned and etched, a main layer 726 is formed upon thebottom layer. The main layer 720 is preferably formed from aphotoresist film that has a thickness substantially greater than atypical width of a particle. Thus, if the particles have a width ofabout 30 .mu.m, a main layer may be composed of a 50 .mu.mphotoresist material. Alternatively, the photoresist layer may becomposed of a multitude of photoresist layers placed upon eachother until the desired thickness is achieved, as will be depictedin later embodiments.

The main photoresist layer may be patterned and etched to form thecavities 728, as depicted in FIG. 25B. The cavities, in oneembodiment, are substantially aligned above the previously formedopenings 724. Cavities 728, in one embodiment, have a width whichis greater than a width of a particle.

For many types of analysis, the photoresist material issubstantially transparent to the light source used. Thus, asopposed to a silicon supporting member, the photoresist materialused for the main supporting layer may be substantially transparentto the light used by the light source. In some circumstances, thetransparent nature of the supporting member may allow light fromthe cavity to migrate, through the supporting member, into a secondcavity. This leakage of light from one cavity to the next may leadto detection problems. For example, if a first particle in a firstcavity produces a fluorescent signal in response to an analyte,this signal may be transmitted through the supporting member anddetected in a proximate cavity. This may lead to inaccuratereadings for the proximately spaced cavities, especially if aparticularly strong signal is produced by the interaction of theparticle with an analyte.

To reduce the occurrence of this "cross-talk", a substantiallyreflective layer 730 may be formed along the inner surface of thecavity. In one embodiment, the reflective layer 730 is composed ofa metal layer which is formed on the upper surface of the mainlayer and the inner surface of the cavity. The metal layer may bedeposited using chemical vapor deposition or other known techniquesfor depositing thin metal layers. The presence of a reflectivelayer may inhibit "cross-talk" between the cavities.

After the cavities 728 have been formed, particles 718 may beinserted into the cavities 728, as depicted in FIG. 25C. The firstphotoresist layer 722 may serve as a support for the particles 718.The particles may be inhibited from being displaced from thecavities by the first photoresist layer 722. The openings 724 inthe first photoresist layer 722 may allow fluid to pass through thebottom layer during use.

After the particles 728 are placed in the cavities 728, a topphotoresist layer 732 may be placed upon the upper surface of thesilicon substrate. After the cover layer is formed, openings 734may be formed in the cover layer to allow the passage of the fluidinto the cavities.

In another embodiment, the supporting member may be formed from aplastic substrate, as depicted in FIGS. 26A D. In one embodiment,the plastic substrate is composed of a material which issubstantially resistant to the fluid which includes the analyte.Examples of plastic materials which may be used to form the plasticsubstrate include, but are not limited to, acrylic resins,polycarbonates, polyester resins, polyethylenes, polyimides,polyvinyl polymers (e.g., polyvinyl chloride, polyvinyl acetate,polyvinyl dichloride, polyvinyl fluoride, etc.), polystyrenes,polypropylenes, polytetrafluoroethylenes, and polyurethanes. Theplastic substrate may be substantially transparent or substantiallyopaque to the light produced by the light source. After obtaining asuitable plastic material 740, a series of cavities 742 may beformed in the plastic material. The cavities 740 may be formed bydrilling (either mechanically or with a laser), transfer molding(e.g., forming the cavities when the plastic material is formedusing appropriately shaped molds), or using a punching apparatus topunch cavities into the plastic material. In one embodiment, thecavities 740 are formed such that a lower portion 743 of thecavities is substantially narrower than an upper portion 744 of thecavities. The lower portion 743 of the cavities may have a widthsubstantially less than a width of a particle. The lower portion743 of the cavities 740 may inhibit the displacement of a particlefrom the cavity 740. While depicted as rectangular, with a narrowerrectangular opening at the bottom, it should be understood that thecavity may be formed in a number of shapes including but notlimited to pyramidal, triangular, trapezoidal, and oval shapes. Anexample of a pyramidal cavity which is tapered such that theparticle is inhibited from being displaced from the cavity isdepicted in FIG. 25D.

After the cavities 742 are formed, particles 718 may be insertedinto the cavities 742, as depicted in FIG. 26B. The lower portion743 of the cavities may serve as a support for the particles 718.The particles 718 may be inhibited from being displaced from thecavities 742 by the lower portion 743 of the cavity. After theparticles are placed in the cavities 740, a cover 744 may be placedupon the upper surface 745 of the plastic substrate 740, asdepicted in FIG. 26C.

In one embodiment, the cover is formed from a film of photoresistmaterial. After the cover 744 is placed on the plastic substrate740, openings 739 may be formed in the cover layer to allow thepassage of the fluid into the cavities.

In some circumstances a substantially transparent plastic materialmay be used. As described above, the use of a transparentsupporting member may lead to "cross-talk" between the cavities. Toreduce the occurrence of this "cross-talk", a substantiallyreflective layer 748 may be formed on the inner surface 746 of thecavity, as depicted in FIG. 26E. In one embodiment, the reflectivelayer 748 is composed of a metal layer which is formed on the innersurface of the cavities 742. The metal layer may be deposited usingchemical vapor deposition or other techniques for depositing thinmetal layers. The presence of a reflective layer may inhibitcross-talk between the cavities.

In another embodiment, a silicon based supporting member for asensing particle may be formed without a bottom layer. In thisembodiment, the cavity may be tapered to inhibit the passage of theparticle from the cavity, through the bottom of the supportingmember. FIG. 27A D, depicts the formation of a supporting memberfrom a silicon substrate. In this embodiment, a photoresist layer750 is formed upon an upper surface of a silicon substrate 752, asdepicted in FIG. 27A. The photoresist layer 750 may be patternedand developed such that the regions of the silicon substrate inwhich the cavities will be formed are exposed.

Cavities 754 may now be formed, as depicted in FIG. 27B, bysubjecting the silicon substrate to an anisotropic etch. In oneembodiment, a potassium hydroxide etch is used to produced taperedcavities. The etching may be controlled such that the width of thebottom of the cavities 750 is less than a width of the particle.After the cavities have been etched, a particle 756 may be insertedinto the cavities 754 as depicted in FIG. 27C. The particle 756 maybe inhibited from passing out of the cavities 754 by the narrowerbottom portion of the cavities. After the particle is positionedwithin the cavities 754, a cover 758 may be formed upon the siliconsubstrate 752, as depicted in FIG. 27D. The cover may be formed ofany material substantially transparent to the light produced by thelight source used for analysis. Openings 759 may be formed in thecover 758 to allow the fluid to pass into the cavity from the topface of the supporting member 752. The openings 759 in the coverand the opening at the bottom of the cavities 754 together mayallow fluid to pass through the cavity during use.

In another embodiment, a supporting member for a sensing particlemay be formed from a plurality of layers of a photoresist material.In this embodiment, the cavity may be tapered to inhibit thepassage of the particle from the cavity, through the bottom of thesupporting member. FIGS. 28A E depict the formation of a supportingmember from a plurality of photoresist layers. In an embodiment, afirst photoresist layer 760 is developed and etched to form aseries of openings 762 which are positioned at the bottom ofsubsequently formed cavities, as depicted in FIG. 28A. As depictedin FIG. 28B, a second layer of photoresist material 764 may beformed upon the first photoresist layer 760. The second photoresistlayer may be developed and etched to form openings substantiallyaligned with the openings of the first photoresist layer 760. Theopenings formed in the second photoresist layer 764, in oneembodiment, are substantially larger than the layers formed in thefirst photoresist layer 760. In this manner, a tapered cavity maybe formed while using multiple photoresist layers.

As depicted in FIG. 28C, additional layers of photoresist material766 and 768 may be formed upon the second photoresist layer 764.The openings of the additional photoresist layers 766 and 768 maybe progressively larger as each layer is added to the stack. Inthis manner, a tapered cavity may be formed. Additional layers ofphotoresist material may be added until the desired thickness ofthe supporting member is obtained. The thickness of the supportingmember, in one embodiment, is greater than a width of a particle.For example, if a layer of photoresist material has a thickness ofabout 25 .mu.m and a particle has a width of about 100 .mu.m, asupporting member may be formed from four or more layers ofphotoresist material. While depicted as pyramidal, the cavity maybe formed in a number of different shapes, including but notlimited to, rectangular, circular, oval, triangular, andtrapezoidal. Any of these shapes may be obtained by appropriatepatterning and etching of the photoresist layers as they areformed.

In some instances, the photoresist material may be substantiallytransparent to the light produced by the light source. As describedabove, the use of a transparent supporting member may lead to"cross-talk" between the cavities. To reduce the occurrence of this"cross-talk", a substantially reflective layer 770 may be formedalong the inner surface of the cavities 762, as depicted in FIG.28D. In one embodiment, the reflective layer is composed of a metallayer which is formed on the inner surface of the cavities 762. Themetal layer may be deposited using chemical vapor deposition orother techniques for depositing thin metal layers. The presence ofa reflective layer may inhibit "cross-talk" between thecavities.

After the cavities 762 are formed, particles 772 may be insertedinto the cavities 762, as depicted in FIG. 28D. The narrow portionsof the cavities 762 may serve as a support for the particles 772.The particles 772 may be inhibited from being displaced from thecavities 762 by the lower portion of the cavities. After theparticles 772 are placed in the cavities 762, a cover 774 may beplaced upon the upper surface of the top layer 776 of thesupporting member, as depicted in FIG. 28E. In one embodiment, thecover 774 is also formed from a film of photoresist material. Afterthe cover layer is formed, openings 778 may be formed in the cover774 to allow the passage of the fluid into the cavities.

In another embodiment, a supporting member for a sensing particlemay be formed from photoresist material which includes a particlesupport layer. FIGS. 29A E depict the formation of a supportingmember from a series of photoresist layers. In an embodiment, afirst photoresist layer 780 is developed and etched to form aseries of openings 782 which may become part of subsequently formedcavities. In another embodiment, a cavity having the appropriatedepth may be formed by forming multiple layers of a photoresistmaterial, as described previously. As depicted in FIG. 29B, asecond photoresist layer 784 may be formed upon the firstphotoresist layer 780. The second photoresist layer 784 may bepatterned to form openings substantially aligned with the openingsof the first photoresist layer 782. The openings formed in thesecond photoresist layer 784 may be substantially equal in size tothe previously formed openings. Alternatively, the openings may bevariable in size to form different shaped cavities.

For reasons described above, a substantially reflective layer 786may be formed along the inner surface of the cavities 782 and theupper surface of the second photoresist layer 784, as depicted inFIG. 29C. In one embodiment, the reflective layer is composed of ametal layer. The metal layer may be deposited using chemical vapordeposition or other techniques for depositing thin metal layers.The presence of a reflective layer may inhibit "cross-talk" betweenthe cavities.

After the metal layer is deposited, a particle support layer 788may be formed on the bottom surface of the first photoresist layer780, as depicted in FIG. 29D. The particle support layer 788 may beformed from photoresist material, silicon dioxide, silicon nitride,glass or a substantially transparent plastic material. The particlesupport layer 788 may serve as a support for the particles placedin the cavities 782. The particle support layer, in one embodiment,is formed from a material that is substantially transparent to thelight produced by the light source.

After the particle supporting layer 788 is formed, particles 785may be inserted into the cavities 782, as depicted in FIG. 29E. Theparticle support layer 788 may serve as a support for theparticles. Thus the particles 785 may be inhibited from beingdisplaced from the cavities by the particle support layer 788.After the particles 785 are placed in the cavities 782, a cover 787may be placed upon the upper surface of the second photoresistlayer 784, as depicted in FIG. 29E. In one embodiment, the cover isalso formed from a film of photoresist material. After the cover isformed, openings 789 may be formed in the cover 787 to allow thepassage of the fluid into the cavities. In this embodiment, thefluid is inhibited from flowing through the supporting member.Instead, the fluid may flow into and out of the cavities via theopenings 789 formed in the cover 787.

A similar supporting member may be formed from a plastic material,as depicted in FIGS. 30A D. The plastic material may besubstantially resistant to the fluid which includes the analyte.The plastic material may be substantially transparent orsubstantially opaque to the light produced by the light source.After obtaining a suitable plastic substrate 790, a series ofcavities 792 may be formed in the plastic substrate 790. Thecavities may be formed by drilling (either mechanically or with alaser), transfer molding (e.g., forming the cavities when theplastic substrate is formed using appropriately shaped molds), orusing a punching machine to form the cavities. In one embodiment,the cavities extend through a portion of the plastic substrate,terminating proximate the bottom of the plastic substrate, withoutpassing through the plastic substrate. After the cavities 792 areformed, particles 795 may be inserted into the cavities 792, asdepicted in FIG. 30B. The bottom of the cavity may serve as asupport for the particles 795. After the particles are placed inthe cavities, a cover 794 may be placed upon the upper surface ofthe plastic substrate 790, as depicted in FIG. 30C. In oneembodiment, the cover may be formed from a film of photoresistmaterial. After the cover 794 is formed, openings 796 may be formedin the cover to allow the passage of the fluid into the cavities.While depicted as rectangular, is should be understood that thecavities may be formed in a variety of different shapes, includingtriangular, pyramidal, pentagonal, polygonal, oval, or circular. Itshould also be understood that cavities having a variety ofdifferent shapes may be formed into the same plastic substrate, asdepicted in FIG. 30D.

In one embodiment, a series of channels may be formed in thesupporting member interconnecting some of the cavities, as depictedin FIG. 3. Pumps and valves may also be incorporated into thesupporting member to aid passage of the fluid through the cavities.A schematic figure of a diaphragm pump 800 is depicted in FIG. 31.Diaphragm pumps, in general, include a cavity 810, a flexiblediaphragm 812, an inlet valve 814, and an outlet valve 816. Theflexible diaphragm 812, during use, is deflected as shown by arrows818 to create a pumping force. As the diaphragm is deflected towardthe cavity 810 it may cause the inlet valve 814 to close, theoutlet valve 816 to open and any liquid which is in the cavity 810will be forced toward the outlet 816. As the diaphragm moves awayfrom the cavity 810, the outlet valve 816 may be pulled to a closedposition, and the inlet valve 814 may be opened, allowingadditional fluid to enter the cavity 810. In this manner a pump maybe used to pump fluid through the cavities. It should be understoodthat the pump depicted in FIG. 31 is a generalized version of adiaphragm based pump. Actual diaphragm pumps may have differentshapes or may have inlet and outlet valves which are separate fromthe pumping device.

In one embodiment, the diaphragm 810 may be made from apiezoelectric material. This material will contract or expand whenan appropriate voltage is applied to the diaphragm. Pumps using apiezoelectric diaphragms are described in U.S. Pat. Nos. 4,344,743,4,938,742, 5,611,676, 5,705,018, and 5,759,015, all of which areincorporated herein by reference. In other embodiments, thediaphragm may be activated using a pneumatic system. In thesesystems, an air system may be coupled to the diaphragm such thatchanges in air density about the diaphragm, induced by thepneumatic system, may cause the diaphragm to move toward and awayfrom the cavity. A pneumatically controlled pump is described inU.S. Pat. No. 5,499,909 which is incorporated herein by reference.The diaphragm may also be controlled using a heat activatedmaterial. The diaphragm may be formed from a temperature sensitivematerial. In one embodiment, the diaphragm may be formed from amaterial which is configured to expand and contract in response totemperature changes. A pump system which relies on temperatureactivated diaphragm is described in U.S. Pat. No. 5,288,214 whichis incorporated herein by reference.

In another embodiment, an electrode pump system may be used. FIG.32 depicts a typical electrode based system. A series of electrodes820 may be arranged along a channel 822 which may lead to a cavity824 which includes a particle 826. By varying the voltage in theelectrodes 820a current flow may be induced in the fluid within thechannel 822. Examples of electrode based systems include, but arenot limited to, electroosmosis systems, electrohydrodynamicsystems, and combinations of electroosmosis and electrohydrodynamicsystems.

Electrohydrodynamic pumping of fluids is known and may be appliedto small capillary channels. In an electrohydrodynamic systemelectrodes are typically placed in contact with the fluid when avoltage is applied. The applied voltage may cause a transfer incharge either by transfer or removal of an electron to or from thefluid. This electron transfer typically induces liquid flow in thedirection from the charging electrode to the oppositely chargedelectrode. Electrohydrodynamic pumps may be used for pumping fluidssuch as organic solvents.

Electroosmosis, is a process which involves applying a voltage to afluid in a small space, such as a capillary channel, to cause thefluid to flow. The surfaces of many solids, including quartz, glassand the like, become variously charged, negatively or positively,in the presence of ionic materials, such as for example salts,acids or bases. The charged surfaces will attract oppositelycharged (positive or negative) counterions in aqueous solutions.The application of a voltage to such a solution results in amigration of the counterions to the oppositely charged electrode,and moves the bulk of the fluid as well. The volume flow rate isproportional to the current, and the volume flow generated in thefluid is also proportional to the applied voltage. Anelectroosmosis pump system is described in U.S. Pat. No. 4,908,112which is incorporated herein by reference.

In another embodiment, a combination of electroosmosis pumps andelectrohydrodynamic pumps may be used. Wire electrodes may beinserted into the walls of a channel at preselected intervals toform alternating electroosmosis and electrohydrodynamic devices.Because electroosmosis and electrohydrodynamic pumps are bothpresent, a plurality of different solutions, both polar andnon-polar, may be pump along a single channel. Alternatively, aplurality of different solutions may be passed along a plurality ofdifferent channels connected to a cavity. A system which includes acombination of electroosmosis pumps and electrohydrodynamic pumpsis described in U.S. Pat. No. 5,632,876 which is incorporatedherein by reference.

In an embodiment, a pump may be incorporated into a sensor arraysystem, as depicted in FIG. 32. A sensor array 830 includes atleast one cavity 832 in which a particle 834 may be placed. Thecavity 832 may be configured to allow fluid to pass through thecavity during use. A pump 836 may be incorporated onto a portion ofthe supporting member 838. A channel 831 may be formed in thesupporting member 838 coupling the pump 836 to the cavity 832. Thechannel 831 may be configured to allow the fluid to pass from thepump 836 to the cavity 832. The pump 836 may be positioned awayfrom the cavity 832 to allow light to be directed through thecavity during use. The supporting member 838 and the pump 836 maybe formed from a silicon substrate, a plastic material, orphotoresist material. The pump 836 may be configured to pump fluidto the cavity via the channel, as depicted by the arrows in FIG.32. When the fluid reaches the cavity 832, the fluid may flow pastthe particle 834 and out through the bottom of the cavity. Anadvantage of using pumps is that better flow through the channelsmay be achieved. Typically, the channels and cavities may have asmall volume. The small volume of the cavity and channel tends toinhibit flow of the fluid through the cavity. By incorporating apump, the flow of fluid to the cavity and through the cavity may beincreased, allowing more rapid testing of the fluid sample. While adiaphragm based pump system is depicted in FIG. 33, it should beunderstood that electrode based pumping systems may also beincorporated into the sensor array to produce fluid flows.

In another embodiment, a pump may be coupled to a supporting memberfor analyzing analytes in a fluid stream, as depicted in FIG. 34. Achannel 842 may couple a pump 846 to multiple cavities 844 formedin a supporting member 840. The cavities 842 may include sensingparticles 848. The pump may be configured to create a flow of thefluid through the channel 842 to the cavities 848. In oneembodiment, the cavities may inhibit the flow of the fluid throughthe cavities 844. The fluid may flow into the cavities 844 and pastthe particle 848 to create a flow of fluid through the sensor arraysystem. In this manner a single pump may be used to pass the fluidto multiple cavities. While a diaphragm pump system is depicted inFIG. 33, it should be understood that electrode pumping systems mayalso be incorporated into the supporting member to create similarfluid flows.

In another embodiment, multiple pumps may be coupled to asupporting member of a sensor array system. In one embodiment, thepumps may be coupled in series with each other to pump fluid toeach of the cavities. As depicted in FIG. 35, a first pump 852 anda second pump 854 may be coupled to a supporting member 850. Thefirst pump 852 may be coupled to a first cavity 856. The first pumpmay be configured to transfer fluid to the first cavity 856 duringuse. The cavity 856 may be configured to allow the fluid to passthrough the cavity to a first cavity outlet channel 858. A secondpump 854 may also be coupled to the supporting member 850. Thesecond pump 854 may be coupled to a second cavity 860 and the firstcavity outlet channel 858. The second pump 854 may be configured totransfer fluid from the first cavity outlet channel 858 to thesecond cavity 860. The pumps may be synchronized such that a steadyflow of fluid through the cavities is obtained. Additional pumpsmay be coupled to the second cavity outlet channel 862 such thatthe fluid may be pumped to additional cavities. In one embodiment,each of the cavities in the supporting member is coupled to a pumpconfigured to pump the fluid stream to the cavity.

In another embodiment, multiple electrode based pumps may beincorporated herein into the sensor array system. The pumps may beformed along the channels which couple the cavities. As depicted inFIG. 36, a plurality of cavities 870 may be formed in a supportingmember 872 of a sensor array. Channels 874 may also be formed inthe supporting member 872 interconnecting the cavities 870 witheach other. An inlet channel 876 and an outlet channel 877, whichallow the fluid to pass into and out of the sensor array,respectively, may also be formed. A series of electrodes 878 may bepositioned over the channels 874, 876, and 877. The electrodes maybe used to form an electroosmosis pumping system or anelectrohydrodynamic pumping system. The electrodes may be coupledto a controller 880 which may apply the appropriate voltage to theappropriate electrodes to produce a flow of the fluid through thechannels. The pumps may be synchronized such that a steady flow offluid through the cavities is obtained. The electrodes may bepositioned between the cavities such that the electrodes do notsignificantly interfere with the application of light to thecavities.

In some instances it may be necessary to add a reagent to aparticle before, during or after an analysis process. Reagents mayinclude receptor molecules or indicator molecules. Typically, suchreagents may be added by passing a fluid stream which includes thereagent over the sensor array. In an embodiment, the reagent may beincorporated herein into the sensor array system which includes twoparticles. In this embodiment, a sensor array system 900 mayinclude two particles 910 and 920 for each sensing position of thesensor array, as depicted in FIG. 37. The first particle 910 may bepositioned in a first cavity 912. The second particle 920 may bepositioned in a second cavity 922. In one embodiment, the secondcavity is coupled to the first cavity via a channel 930. The secondparticle includes a reagent which is at least partially removablefrom the second particle 920. The reagent may also be configured tomodify the first particle 910, when the reagent is contacted withthe first particle, such that the first particle will produce asignal when the first particle interacts with an analyte duringuse. The reagent may be added to the first cavity before, during orafter a fluid analysis. The reagent is preferably coupled to thesecond particle 920. The a portion of the reagent coupled to thesecond particle may be decoupled from the particle by passing adecoupling solution past the second particle. The decouplingsolution may include a decoupling agent which will cause at least aportion of the reagent to be at released by the particle. Areservoir 940 may be formed on the sensor array to hold thedecoupling solution.

A first pump 950 and a second pump 960 may also be coupled to thesupporting member 915. The first pump 950 may be configured to pumpfluid from a fluid inlet 952 to the first cavity 912 via channel930. The fluid inlet 952 is the location where the fluid, whichincludes the analyte, is introduced into the sensor array system. Asecond pump 950 may be coupled to the reservoir 940 and the secondcavity 922. The second pump 960 may be used to transfer thedecoupling solution from the reservoir to the second cavity 922.The decoupling solution may pass through the second cavity 922 andinto first cavity 912. Thus, as the reagent is removed the secondparticle it may be transferred to the first cavity912, where thereagent may interact with the first particle 910. The reservoir maybe refilled by removing the reservoir outlet 942, and addingadditional fluid to the reservoir 940. While diaphragm based pumpsystems are depicted in FIG. 37, it should be understood thatelectrode based pumping systems may also be incorporated hereininto the sensor array to produce fluid flows.

The use of such a system is described by way of example. In someinstances it may be desirable to add a reagent to the firstparticle prior to passing the fluid which includes the analyte tothe first particle. The reagent may be coupled to the secondparticle and placed in the sensor array prior to use, typicallyduring construction of the array. A decoupling solution may beadded to the reservoir before use. A controller 970 may also becoupled to the system to allow automatic operation of the pumps.The controller 970 may be configured to initiate the analysissequence by activating the second pump 960, causing the decouplingsolution to flow from the reservoir 940 to the second cavity 922.As the fluid passes through the second cavity 922, the decouplingsolution may cause at least some of the reagent molecules to bereleased from the second particle 920. The decoupling solution maybe passed out of the second cavity 922 and into the first cavity912. As the solution passes through the first cavity, some of thereagent molecules may be captured by the first particle 910. Aftera sufficient number of molecules have been captured by the firstparticle 910, flow of fluid thorough the second cavity 922 may bestopped. During this initialization of the system, the flow offluid through the first pump may be inhibited.

After the system is initialized, the second pump may be stopped andthe fluid may be introduced to the first cavity. The first pump maybe used to transfer the fluid to the first cavity. The second pumpmay remain off, thus inhibiting flow of fluid from the reservoir tothe first cavity. It should be understood that the reagent solutionmay be added to the first cavity while the fluid is added to thefirst cavity. In this embodiment, both the first and second pumpsmay be operated substantially simultaneously.

Alternatively, the reagent may be added after an analysis. In someinstances, a particle may interact with an analyte such that achange in the receptors attached to the first particle occurs. Thischange may not, however produce a detectable signal. The reagentattached to the second bead may be used to produce a detectablesignal when it interacts with the first particle, if a specificanalyte is present. In this embodiment, the fluid is introducedinto the cavity first.

After the analyte has been given time to react with the particle,the reagent may be added to the first cavity. The interaction ofthe reagent with the particle may produce a detectable signal. Forexample, an indicator reagent may react with a particle which hasbeen exposed to an analyte to produce a color change on theparticle. Particle which have not been exposed to the analyte mayremain unchanged or show a different color change.

As shown in FIG. 1, a system for detecting analytes in a fluid mayinclude a light source 110, a sensor array 120 and a detector 130.The sensor array 120 is preferably formed of a supporting memberwhich is configured to hold a variety of particles 124 in anordered array. A high sensitivity CCD array may be used to measurechanges in optical characteristics which occur upon binding of thebiological/chemical agents. Data acquisition and handling ispreferably performed with existing CCD technology. As describedabove, colorimetric analysis may be performed using a white lightsource and a color CCD detector. However, color CCD detectors aretypically more expensive than gray scale CCD detectors.

In one embodiment, a gray scale CCD detector may be used to detectcolorimetric changes. In one embodiment, a gray scale detector maybe disposed below a sensor array to measure the intensity of lightbeing transmitted through the sensor array. A series of lights(e.g., light emitting diodes) may be arranged above the sensorarray. In one embodiment, groups of three LED lights may bearranged above each of the cavities of the array. Each of thesegroups of LED lights may include a red, blue and a green light.Each of the lights may be operated individually such that one ofthe lights may be on while the other two lights are off. In orderto provide color information while using a gray scale detector,each of the lights is sequentially turned on and the gray scaledetector is used to measure the intensity of the light passingthrough the sensor array. After information from each of the lightsis collected, the information may be processed to derive theabsorption changes of the particle.

In one embodiment, the data collected by the gray scale detectormay be recorded using 8 bits of data. Thus, the data will appear asa value between 0 and 255. The color of each chemical sensitiveelement may be represented as a red, blue and green value. Forexample, a blank particle (i.e., a particle which does not includea receptor) will typically appear white. When each of the LEDlights (red, blue and green) are operated the CCD detector willrecord a value corresponding to the amount of light transmittedthrough the cavity. The intensity of the light may be compared to ablank particle, to determine the absorbance of a particle withrespect to the LED light which is used. Thus, the red, green andblue components may be recorded individually without the use of acolor CCD detector. In one embodiment, it is found that a blankparticle exhibits an absorbance of about 253 when illuminated witha red LED, a value of about 250 when illuminated by a green LED,and a value of about 222 when illuminated with a blue LED. Thissignifies that a blank particle does not significantly absorb red,green or blue light. When a particle with a receptor is scanned,the particle may exhibit a color change, due to absorbance by thereceptor. For example, it was found that when a particle whichincludes a 5-carboxyfluorescein receptor is subjected to whitelight, the particle shows a strong absorbance of blue light. When ared LED is used to illuminate the particle, the gray scale CCDdetector may detect a value of about 254. When the green LED isused, the gray scale detector may detect a value of about 218. Whena blue LED light is used, a gray scale detector may detect a valueof about 57. The decrease in transmittance of blue light isbelieved to be due to the absorbance of blue light by the5-carboxyfluorescein. In this manner the color changes of aparticle may be quantitatively characterized using a gray scaledetector.

As described above, after the cavities are formed in the supportingmember, a particle may be positioned at the bottom of a cavityusing a micromanipulator. This allows the location of a particularparticle to be precisely controlled during the production of thearray. The use of a micromanipulator may, however, be impracticalfor production of sensor array systems. An alternate method ofplacing the particles into the cavities may involve the use of asilk screen like process. A series of masking materials may beplaced on the upper surface of the sensor array prior to fillingthe cavities. The masking materials may be composed of glass, metalor plastic materials. A collection of particles may be placed uponthe upper surface of the masking materials and the particles may bemoved across the surface. When a cavity is encountered, a particlemay drop into the cavity if the cavity is unmasked. Thus particlesof known composition are placed in only the unmasked regions. Afterthe unmasked cavities are filled, the masking pattern may bealtered and a second type of particles may be spread across thesurface. Preferably, the masking material will mask the cavitiesthat have already been filled with particle.

The masking material may also mask other non-filled cavities. Thistechnique may be repeated until all of the cavities are filled.After filling the cavities, a cover may be placed on the supportmember, as described above, to inhibit the displacement and mixingof the particles. An advantage of such a process is that it may bemore amenable to industrial production of supporting members.

2. Further System Improvements

One challenge in a chemical sensor system is keeping dead volume toa minimum. This is especially problematic when an interface to theoutside world is required (e.g., a tubing connection). In manycases the "dead volume" associated with the delivery of the sampleto the reaction site in a "lab-on-a-chip" may far exceed the actualamount of reagent required for the reaction. Filtration is alsofrequently necessary to prevent small flow channels in the sensorarrays from plugging. Here the filter can be made an integral partof the sensor package.

In an embodiment, a system for detecting an analyte in a fluidincludes a conduit coupled to a sensor array and a vacuum chambercoupled to the conduit. FIG. 38 depicts a system in which a fluidstream (E) passes through a conduit (D), onto a sensor array (G),and into a vacuum apparatus (F). The vacuum apparatus (F) may becoupled to the conduit (D) downstream from the sensor array (G). Avacuum apparatus is herein defined to be any system capable ofcreating or maintaining a volume at a pressure below atmospheric.Examples of vacuum apparatus include vacuum chambers. Vacuumchamber, in one embodiment, may be sealed tubes from which aportion of the air has been evacuated, creating a vacuum within thetube. A commonly used example of such a sealed tube is a"vacutainer" system commercially available from Becton Dickinson.Alternatively, a vacuum chamber which is sealed by a movable pistonmay also be used to generate a vacuum. For example, a syringe maybe coupled to the conduit. Movement of the piston (i.e., theplunger) away from the chamber will create a partial vacuum withinthe chamber. Alternatively, the vacuum apparatus may be a vacuumpump or vacuum line. Vacuum pumps may include direct drive pumps,oil pumps, aspirator pumps or micropumps. Micropumps that may beincorporated into a sensor array system have been previouslydescribed.

As opposed to previously described methods, in which a pump as usedto force a fluid stream through a sensor array, the use of a vacuumapparatus allows the fluid to be pulled through the sensor array.Referring to FIG. 39, the vacuum apparatus (F) is coupled todownstream from a sensor array. When coupled to the conduit (D),the vacuum apparatus may exert a suction force on the fluid stream,forcing a portion of the stream to pass over, and in someinstances, through the sensor array. In some embodiments, the fluidmay continue to pass through the conduit, after passing the sensorarray, and into the vacuum apparatus. In an embodiment where thevacuum apparatus is a pre-evacuated tube, the fluid flow willcontinue until the air within the tube is at a pressuresubstantially equivalent to the atmospheric pressure. The vacuumapparatus may include a penetrable wall (H). The penetrable wallforms a seal inhibiting air from entering the vacuum apparatus.When the wall is broken or punctured, air from outside of thesystem will begin to enter the vacuum apparatus. In one embodiment,the conduit includes a penetrating member, (e.g., a syringeneedle), which allows the penetrable wall to be pierced. Piercingthe penetrable wall causes air and fluid inside the conduit to bepulled through the conduit into the vacuum apparatus until thepressure between the vacuum apparatus and the conduit isequalized.

The sensor array system may also include a filter (B) coupled tothe conduit (D) as depicted in FIG. 39. The filter (B) may bepositioned along the conduit, upstream from the sensor array.Filter (B) may be a porous filter which includes a membrane forremoving components from the fluid stream. In one embodiment, thefilter may include a membrane for removal of particulates above aminimum size. The size of the particulates removed will depend onthe porosity of the membrane as is known in the art. Alternatively,the filter may be configured to remove unwanted components of afluid stream. For example, if the fluid stream is a blood sample,the filter may be configured to remove red and white blood cellsfrom the stream, while leaving in the blood stream blood plasma andother components therein.

The sensor array may also include a reagent delivery reservoir (C).The reagent delivery system is preferably coupled to the conduitupstream from the sensor array. The reagent delivery reservoir maybe formed from a porous material which includes a reagent ofinterest. As the fluid passes through this reservoir, a portion ofthe reagent within the regent delivery reservoir passes into thefluid stream. The fluid reservoir may include a porous polymer orfilter paper on which the reagent is stored. Examples of reagentswhich may be stored within the reagent delivery reservoir include,but are not limited to, visualization agents (e.g., dye orfluorophores), co-factors, buffers, acids, bases, oxidants, andreductants.

The sensor array may also include a fluid sampling device (A)coupled to the conduit (D). The fluid sampling device is configuredto transfer a fluid sample from outside the sensor array to theconduit. A number of fluid sampling devices may be used including,but not limited to a syringe needle, a tubing connector, acapillary tube, or a syringe adapter.

The sensor array may also include a micropump or a microvalvesystem, coupled to the conduit to further aid in the transfer offluid through the conduit. Micropumps and valves have beenpreviously described. In one embodiment, a micro-valve or micropumpmay be used to keep a fluid sample or a reagent solution separatedfrom the sensor array. Typically, these microvalves and micropumpsinclude a thin flexible diaphragm. The diaphragm may be moved to anopen position, in one embodiment, by applying a vacuum to theoutside of the diaphragm. In this way, a vacuum apparatus coupledto the sensor array may be used to open a remote microvalve orpump.

In another embodiment, a microvalve may be used to control theapplication of a vacuum to the system. For example, a microvalvemay be positioned adjacent to the vacuum apparatus. The activationof the microvalve may allow the vacuum apparatus to communicatewith the conduit or sensor array. The microvalve may be remotelyactivated at controlled times and for controlled intervals.

In one embodiment, a sensor array system, such as depicted in FIG.39, may be used for analysis of blood samples. A micropuncturedevice (A) is used to extract a small amount of blood from thepatient, e.g., through a finger prick. The blood may be drawnthrough a porous filter that serves to remove the undesirableparticulate matter. For the analysis of antibodies or antigens inwhole blood, the filtering agent may be chosen to remove both thewhite and red blood cells, while leaving in the fluid stream bloodplasma and all of the components therein. Methods of filteringblood cells from whole blood are taught, for example, in U.S. Pat.Nos. 5,914,042; 5,876,605, and 5,211,850 which are incorporated byreference. The filtered blood may also be passed through a reagentdelivery reservoir that may consist of a porous layer that isimpregnated with the reagent(s) of interest. In many cases, avisualization agent will be included in this layer so that thepresence of the analytes of interest in the chip can be resolved.The treated fluid may be passed above the electronic tongue chipthrough a capillary layer, down through the various sensingparticles and through the chip onto the bottom capillary layer.After exiting the central region, the excess fluid flows into thevacuum apparatus. This excess fluid may serve as a source of samplefor future measurements should more detailed analyses be warranted.A "hard copy" of the sample is thus created to back up theelectronic data recorded for the specimen.

Other examples of testing procedures for bodily fluids aredescribed in the following U.S. Pat. Nos. 4,596,657, 4,189,382,4,115,277, 3,954,623, 4,753,776, 4,623,461, 4,069,017, 5,053,197,5,503,985, 3,696,932, 3,701,433, 4,036,946, 5,858,804, 4,050,898,4,477,575, 4,810,378, 5,147,606, 4,246,107, and 4,997,577 all ofwhich are incorporated by reference.

This generally described sampling method may also be used foreither antibody or antigen testing of bodily fluids. A generalscheme for the testing of antibodies is depicted in FIG. 40. FIG.40A depicts a polymer bead having a protein coating that can berecognized in a specific manner by a complimentary antibody. Threeantibodies (within the dashed rectangle) are shown to be present ina fluid phase that bathes the polymer bead. Turning to FIG. 40B,the complimentary antibody binds to the bead while the other twoantibodies remain in the fluid phase. A large increase in thecomplimentary antibody concentration is noted at this bead. In FIG.40C a visualization agent such as protein A (within the dashedrectangle) is added to the fluid phase. The visualization agent ischosen because it possesses either a strong absorbance property orit exhibits fluorescence characteristics that can be used toidentify the species of interest via optical measurements. ProteinA is an example of a reagent that associates with the common regionof most antibodies. Chemical derivatization of the visualizationagent with dyes, quantum particles or fluorophores is used to evokethe desired optical characteristics. After binding to thebead-localized antibodies, as depicted in FIG. 40D, thevisualization agent reveals the presence of the complimentaryantibodies at the specific polymer bead sites.

FIG. 41 depicts another general scheme for the detection ofantibodies which uses a sensor array composed of four individualbeads. Each of the four beads is coated with a different antigen(i.e. a protein coating). As depicted in FIG. 41A, the beads arewashed with a fluid sample which includes four antibodies. Each ofthe four antibodies binds to its complimentary antigen coating, asdepicted in FIG. 41B. A visualization agent may be introduced intothe chamber, as depicted in FIG. 41C. The visualization agent, inone embodiment, may bind to the antibodies, as depicted in FIG.41D. The presence of the labeled antibodies is assayed by opticalmeans (absorbance, reflectance, fluorescence). Because the locationof the antigen coatings is known ahead of time, thechemical/biochemical composition of the fluid phase can bedetermined from the pattern of optical signals recorded at eachsite.

In an alternative methodology, not depicted, the antibodies in thesample may be exposed to the visualization agent prior to theirintroduction into the chip array. This may render the visualizationstep depicted in 41C unnecessary.

FIG. 42 depicts a system for detecting an analyte in a fluidstream. The system includes a vacuum apparatus, a chamber in whicha sensor array may be disposed, and an inlet system for introducingthe sample into the chamber. In this embodiment, the inlet systemis depicted as a micro-puncture device. The chamber holding thesensor array may be a Sikes-Moore chamber, as previously described.The vacuum apparatus is a standard "vacutainer" type vacuum tube.The micro puncture device includes a Luer-lock attachment which canreceive a syringe needle. Between the micro-puncture device and thechamber a syringe filter may be placed to filter the sample as thesample enters the chamber. Alternatively, a reagent may be placedwithin the filter. The reagent may be carried into the chamber viathe fluid as the fluid passes through the filter.

As has been previously described, a sensor array may be configuredto allow the fluid sample to pass through the sensor array duringuse. The fluid delivery to the sensor array may be accomplished byhaving the fluid enter the top of the chip through the showncapillary (A), as depicted in FIG. 43. The fluid flow traverses thechip and exits from the bottom capillary (B). Between the top andbottom capillaries, the fluid is passed by the bead. Here the fluidcontaining analytes have an opportunity to encounter the receptorsites. The presence of such analytes may be identified usingoptical means. The light pathway is shown here (D). In the forwardflow direction, the beads are typically forced towards the bottomof the pit. Under these circumstances, the bead placement is idealfor optical measurements.

In another embodiment, the fluid flow may go from the bottom of thesensor array toward the top of the sensor array, as depicted inFIG. 44. The fluid exits from the top of the chip through the showncapillary (A). The fluid flow traverses the chip and enters fromthe bottom capillary (B). Between the top and bottom capillaries,the fluid can avoid the bead somewhat by taking an indirect pathway(C). The presence of analytes is identified using optical means asbefore. Unfortunately, only a portion of the light passes throughthe bead. In the reverse flow direction, the beads can be dislodgedaway from the analysis beam by the upwards pressure of the fluid,as shown in FIG. 44. Under these circumstances, some of the lightmay traverse the chip and enter the detector (not shown) withoutpassing through the sensor bead (Path E).

In any microfluidic chemical sensing system there may be a need to"store" the chemically sensitive elements in an "inert"environment. Typically, the particles may be at least partiallysurrounded by an inert fluid such as an inert, non reactive gas, anon-reactive solvent, or a liquid buffer solution. Alternatively,the particles may be maintained under a vacuum. Before exposure ofthe particles to the analyte, the inert environment may need to beremoved to allow proper testing of the sample. In one embodiment, asystem may include a fluid transfer system for the removal of aninert fluid prior to the introduction of the sample with minimumdead volume.

In one embodiment, a pumping system may be used to pull the inertfluid through from one side (by any pumping action, such as thatprovided by a vacuum downstream from the array). The inert fluidmay be efficiently removed while the beads remain within the sensorarray. Additionally, the analyte sample may be drawn toward thesensor array as the inert fluid is removed from the sensor array. Apocket of air may separate the analyte sample from the inert fluidas the sample move through the conduit. Alternatively, the samplemay be pumped from "upstream" using a micropump. Note that a vacuumdownstream can produce a maximum of one atmosphere of headpressure, while an upstream pump could in principle produce anarbitrarily high head pressure. This can effect the fluid transportrates through the system, but for small volume microfluidicsystems, even with low flow coefficients, one atmosphere of headpressure should provide acceptable transfer rates for manyapplications.

In another embodiment, the vacuum apparatus may be formed directlyinto a micromachined array. The vacuum apparatus may be configuredto transmit fluid to and from a single cavity or a plurality ofcavities. In one embodiment, a separate vacuum apparatus may becoupled to each of the cavities.

3. Chemical Improvements

The development of smart sensors capable of discrimination ofdifferent analytes, toxins, and bacteria has become increasinglyimportant for environmental, health and safety, remote sensing,military, and chemical processing applications. Although manysensors capable of high sensitivity and high selectivity detectionhave been fashioned for single analyte detection, only in a fewselected cases have array sensors been prepared which displaymulti-analyte detection capabilities. The obvious advantages ofsuch array systems are their utility for the analysis of multipleanalytes and their ability to be "trained" to respond to newstimuli. Such on site adaptive analysis capabilities afforded bythe array structures makes their utilization promising for avariety of future applications.

Single and multiple analyte sensors both typically rely on changesin optical signals. These sensors typically make use of anindicator that undergoes a perturbation upon analyte binding. Theindicator may be a chromophore or a fluorophore. A fluorophore is amolecule that absorbs light at a characteristic wavelength and thenre-emits the light most typically at a characteristically differentwavelength. Fluorophores include, but are not limited to rhodamineand rhodamine derivatives, fluorescein and fluorescein derivatives,coumarins and chelators with the lanthanide ion series. Theemission spectra, absorption spectra and chemical composition ofmany fluorophores may be found, e.g., in the "Handbook ofFluorescent Probes and Research Chemicals", R. P. Haugland, ed.which is incorporated herein by reference. A chromophore is amolecule which absorbs light at a characteristic wavelength, butdoes not re-emit light.

As previously described, the receptor itself may incorporate theindicator. The binding of the analyte to the receptor may directlylead to a modulation of the properties of the indicator. Such anapproach typically requires a covalent attachment or strongnon-covalent binding of the indicator onto or as part of thereceptor, leading to additional covalent architecture. Each andevery receptor may need a designed signaling protocol that istypically unique to that receptor. General protocols for designingin a signal modulation that is versatile and general for most anyreceptor would be desirable.

In one embodiment, a general method for the creation of opticalsignal modulations for most any receptor that is coupled to animmobilized matrix has been developed. Immobilized matricesinclude, but are not limited to, resins, beads, and polymersurfaces. By immobilization of the receptor to the matrix, thereceptor is held within a structure that can be chemicallymodified, allowing one to tune and to create an environment aroundthe receptor that is sensitive to analyte binding. Coupling of theindicator to an immobilization matrix may make it sensitive tomicroenvironment changes which foster signal modulation of theindicator upon analyte binding. Further, by coupling the indicatorto an immobilization matrix, the matrix itself becomes thesignaling unit, not requiring a specific new signaling protocol foreach and every receptor immobilized on the matrix.

In an embodiment, a receptor for a particular analyte or class ofanalytes may be designed and created with the chemical handlesappropriate for immobilization on and/or in the matrix. A number ofsuch receptors have been described above. The receptors can be, butare not limited to, antibodies, aptamers, organic receptors,combinatorial libraries, enzymes, and imprinted polymers.

Signaling indicator molecules may be created or purchased whichhave appropriate chemical handles for immobilization on and/or inthe immobilization matrix. The indicators may possess chromophoresor fluorophores that are sensitive to their microenvironment. Thischromophore or fluorophore may be sensitive to microenvironmentchanges that include, but are not limited to, a sensitivity tolocal pH, solvatophobic or solvatophilic properties, ionicstrength, dielectric, ion pairing, and/or hydrogen bonding. Commonindicators, dyes, quantum particles, and semi-conductor particles,are all examples of possible probe molecules. The probe moleculesmay have epitopes similar to the analyte, so that a strong or weakassociation of the probe molecules with the receptor may occur.Alternatively, the probe molecules may be sensitive to a change intheir microenvironment that results from one of the affects listedin item above.

Binding of the analyte may do one of the following things,resulting in a signal modulation: 1) displace a probe molecule fromthe binding site of the receptor, 2) alter the local pH, 3) changethe local dielectric properties, 4) alter the features of thesolvent, 5) change the fluorescence quantum yield of individualdyes, 6) alter the rate/efficiency of fluorescence resonance energytransfer (FRET) between donor-acceptor fluorophore pairs, or 7)change the hydrogen bonding or ion pairing near the probe.

In an alternative embodiment, two or more indicators may beattached to the matrix. Binding between the receptor and analytecauses a change in the communication between the indicators, againvia either displacement of one or more indicators, or changes inthe microenvironment around one or more indicators. Thecommunication between the indicators may be, but is not limited to,fluorescence resonance energy transfer, quenching phenomenon,and/or direct binding.

In an embodiment, a particle for detecting an analyte may becomposed of a polymeric resin. A receptor and an indicator may becoupled to the polymeric resin. The indicator and the receptor maybe positioned on the polymeric resin such that the indicatorproduces a signal in when the analyte interacts with the receptor.The signal may be a change in absorbance (for chromophoricindicators) or a change in fluorescence (for fluorophoricindicators).

A variety of receptors may be used, in one embodiment, the receptormay be a polynucleotide, a peptide, an oligosaccharide, an enzyme,a peptide mimetic, or a synthetic receptor.

In one embodiment, the receptor may be a polynucleotide coupled toa polymeric resin. For the detection of analytes, thepolynucleotide may be a double stranded deoxyribonucleic acid,single stranded deoxyribonucleic acid, or a ribonucleic acid.Methods for synthesizing and/or attaching a polynucleotide to apolymeric resin are described, for example, in U.S. Pat. No.5,843,655 which is incorporated herein by reference."Polynucleotides" are herein defined as chains of nucleotides. Thenucleotides are linked to each other by phosphodiester bonds."Deoxyribonucleic acid" is composed of deoxyribonucleotideresidues, while "Ribonucleic acid" is composed of ribonucleotideresidues.

In another embodiment, the receptor may be a peptide coupled to apolymeric resin. "Peptides" are herein defined as chains of aminoacids whose .alpha.-carbons are linked through peptide bonds formedby a condensation reaction between the a carboxyl group of oneamino acid and the amino group of another amino acid. Peptides isintended to include proteins. Methods for synthesizing and/orattaching a protein or peptides to a polymeric resin are described,for example, in U.S. Pat. Nos. 5,235,028 and 5,182,366 which isincorporated herein by reference.

Alternatively, peptide mimetics may be used as the receptor.Peptides and proteins are sequences of amide linked amino acidbuilding blocks. A variety of peptide mimetics may be formed byreplacing or modifying the amide bond. In one embodiment, the amidebond may be replaced by alkene bonds. In another embodiment, theamide may be replaced by a sulphonamide bond. In another embodimentthe amino acid sidechain may be placed on the nitrogen atom, suchcompounds are commonly known as peptoids. Peptides may also beformed from non-natural D-stereo-isomers of amino acids. Methodsfor synthesizing and/or attaching a peptide mimetic to a polymericresin is described, for example, in U.S. Pat. No. 5,965,695 whichis incorporated herein by reference.

In another embodiment, the receptor may include an oligosaccharidecoupled to a polymeric resin. An "oligosaccharide" is an oligomercomposed of two or more monosaccharides, typically joined togethervia ether linkages. Methods for synthesizing and/or attachingoligosaccharides to a polymeric resin are described, for example,in U.S. Pat. Nos. 5,278,303 and 5,616,698 which are incorporatedherein by reference.

In another embodiment, polynucleotides, peptides and/oroligosaccharides may be coupled to base unit to form a receptor. Inone embodiment, the base unit may have the general structure:(R.sup.1).sub.n--X--(R.sup.2).sub.m wherein X comprises carbocyclicsystems or C.sub.1 C.sub.10 alkanes, n is an integer of at least 1,m is an integer of at least 1; and wherein each of R.sup.1independently represents--(CH.sub.2).sub.y--NR.sup.3--C(NR.sup.4)--NR.sup.5,--(CH.sub.2).sub.y--NR.sup.6R.sup.7, --(CH.sub.2).sub.y--NH--Y,--(CH.sub.2).sub.y--O--Z; where y is an integer of at least 1;where R.sup.3, R.sup.4, and R.sup.5 independently representhydrogen, alkyl, aryl, alkyl carbonyl of 1 to 10 carbon atoms, oralkoxy carbonyl of 1 to 10 carbon atoms, or R.sup.4 and R.sup.5together represent a cycloalkyl group; where R.sup.6 representshydrogen, alkyl, aryl, alkyl carbonyl of 1 to 10 carbon atoms, oralkoxy carbonyl of 1 to 10 carbon atoms; where R.sup.7 representsalkyl, aryl, alkyl carbonyl of 1 to 10 carbon atoms, or alkoxycarbonyl of 1 to 10 carbon atoms; where R.sup.6 and R.sup.7together represent a cycloalkyl group; where Y is a peptide, orhydrogen and where Z is a polynucleotide, an oligosaccharide orhydrogen; and wherein each of R.sup.2 independently representshydrogen, alkyl, alkenyl, alkynyl, phenyl, phenylalkyl, arylalkyl,aryl, or together with another R.sup.2 group represent acarbocyclic ring. The use of a base unit such as described abovemay aid in the placement and orientation of the side groups tocreate a more effective receptor.

The receptor and indicators may be coupled to the polymeric resinby a linker group. A variety of linker groups may be used. The term"linker", as used herein, refers to a molecule that may be used tolink a receptor to an indicator; a receptor to a polymeric resin oranother linker, or an indicator to a polymeric resin or anotherlinker. A linker is a hetero or homobifunctional molecule thatincludes two reactive sites capable of forming a covalent linkagewith a receptor, indicator, other linker or polymeric resin.Suitable linkers are well known to those of skill in the art andinclude, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers.Particularly preferred linkers are capable of forming covalentbonds to amino groups, carboxyl groups, or sulfhydryl groups orhydroxyl groups. Amino-binding linkers include reactive groups suchas carboxyl groups, isocyanates, isothiocyanates, esters,haloalkyls, and the like. Carboxyl-binding linkers are capable offorming include reactive groups such as various amines, hydroxylsand the like. Sulfhydryl-binding linkers include reactive groupssuch as sulfhydryl groups, acrylates, isothiocyanates, isocyanatesand the like. Hydroxyl binding groups include reactive groups suchas carboxyl groups, isocyanates, isothiocyanates, esters,haloalkyls, and the like. The use of some such linkers is describedin U.S. Pat. No. 6,037,137 which is incorporated herein byreference.

A number of combinations for the coupling of an indicator and areceptor to a polymeric resin have been devised. These combinationsare schematically depicted in FIG. 55. In one embodiment, depictedin FIG. 55A, a receptor (R) may be coupled to a polymeric resin.The receptor may be directly formed on the polymeric resin, or becoupled to the polymeric resin via a linker. An indicator (I) mayalso be coupled to the polymeric resin. The indicator may bedirectly coupled to the polymeric resin or coupled to the polymericresin by a linker. In some embodiments, the linker coupling theindicator to the polymeric resin is of sufficient length to allowthe indicator to interact with the receptor in the absence of ananalyte.

In another embodiment, depicted in FIG. 55B, a receptor (R) may becoupled to a polymeric resin. The receptor may be directly formedon the polymeric resin, or be coupled to the polymeric resin via alinker. An indicator (B) may also be coupled to the polymericresin. The indicator may be directly coupled to the polymeric resinor coupled to the polymeric resin by a linker. In some embodiments,the linker coupling the indicator to the polymeric resin is ofsufficient length to allow the indicator to interact with thereceptor in the absence of an analyte. An additional indicator (C)may also be coupled to the polymeric resin. The additionalindicator may be directly coupled to the polymeric resin or coupledto the polymeric resin by a linker. In some embodiments, theadditional indicator is coupled to the polymeric resin, such thatthe additional indicator is proximate the receptor during use.

In another embodiment, depicted in FIG. 55C, a receptor (R) may becoupled to a polymeric resin. The receptor may be directly formedon the polymeric resin, or be coupled to the polymeric resin via alinker. An indicator (I) may be coupled to the receptor. Theindicator may be directly coupled to the receptor or coupled to thereceptor by a linker. In some embodiments, the linker coupling theindicator to the polymeric resin is of sufficient length to allowthe indicator to interact with the receptor in the absence of ananalyte, as depicted in FIG. 55E.

In another embodiment, depicted in FIG. 55D, a receptor (R) may becoupled to a polymeric resin. The receptor may be directly formedon the polymeric resin, or be coupled to the polymeric resin via alinker. An indicator (B) may be coupled to the receptor. Theindicator may be directly coupled to the receptor or coupled to thereceptor by a linker. In some embodiments, the linker coupling theindicator to the polymeric resin is of sufficient length to allowthe indicator to interact with the receptor in the absence of ananalyte, as depicted in FIG. 55F. An additional indicator (C) mayalso be coupled to the receptor. The additional indicator may bedirectly coupled to the receptor or coupled to the receptor by alinker.

In another embodiment, depicted in FIG. 55G, a receptor (R) may becoupled to a polymeric resin. The receptor may be directly formedon the polymeric resin, or be coupled to the polymeric resin via alinker. An indicator (B) may be coupled to the polymeric resin. Theindicator may be directly coupled to the polymeric resin or coupledto the polymeric resin by a linker. In some embodiments, the linkercoupling the indicator to the polymeric resin is of sufficientlength to allow the indicator to interact with the receptor in theabsence of an analyte. An additional indicator (C) may also becoupled to the receptor. The additional indicator may be directlycoupled to the receptor or coupled to the receptor by a linker.

In another embodiment, depicted in FIG. 55H, a receptor (R) may becoupled to a polymeric resin by a first linker. An indicator (I)may be coupled to the first linker. The indicator may be directlycoupled to the first linker or coupled to the first linker by asecond linker. In some embodiments, the second linker coupling theindicator to the polymeric resin is of sufficient length to allowthe indicator to interact with the receptor in the absence of ananalyte.

In another embodiment, depicted in FIG. 55I, a receptor (R) may becoupled to a polymeric resin by a first linker. An indicator (B)may be coupled to the first linker. The indicator may be directlycoupled to the first linker or coupled to the first linker by asecond linker. In some embodiments, the second linker coupling theindicator to the first linker is of sufficient length to allow theindicator to interact with the receptor in the absence of ananalyte. An additional indicator (C) may be coupled to thereceptor. The additional indicator may be directly coupled to thereceptor or coupled to the receptor by a linker.

These various combinations of receptors, indicators, linkers andpolymeric resins may be used in a variety of different signallingprotocols. Analyte-receptor interactions may be transduced intosignals through one of several mechanisms. In one approach, thereceptor site may be preloaded with an indicator, which can bedisplaced in a competition with analyte ligand. In this case, theresultant signal is observed as a decrease in a signal produced bythe indicator. This indicator may be a fluorophore or achromophore. In the case of a fluorophore indicator, the presenceof an analyte may be determined by a decrease in the fluorescenceof the particle. In the case of a chromophore indicator, thepresence of an analyte may be determined by a decrease in theabsorbance of the particle.

A second approach that has the potential to provide bettersensitivity and response kinetics is the use of an indicator as amonomer in the combinatorial sequences (such as either structureshown in FIG. 14), and to select for receptors in which theindicator functions in the binding of ligand. Hydrogen bonding orionic substituents on the indicator involved in analyte binding mayhave the capacity to change the electron density and/or rigidity ofthe indicator, thereby changing observable spectroscopic propertiessuch as fluorescence quantum yield, maximum excitation wavelength,maximum emission wavelength, and/or absorbance. This approach maynot require the dissociation of a preloaded fluorescent ligand(limited in response time by koff), and may modulate the signalfrom essentially zero without analyte to large levels in thepresence of analyte.

In one embodiment, the microenvironment at the surface and interiorof the resin beads may be conveniently monitored using spectroscopywhen simple pH sensitive dyes or solvachromic dyes are imbedded inthe beads. As a guest binds, the local pH and dielectric constantsof the beads change, and the dyes respond in a predictable fashion.The binding of large analytes with high charge and hydrophobicsurfaces, such as DNA, proteins, and steroids, should induce largechanges in local microenvironment, thus leading to large andreproducible spectral changes. This means that most any receptorcan be attached to a resin bead that already has a dye attached,and that the bead becomes a sensor for the particular analyte.

In one embodiment, a receptor that may be covalently coupled to anindicator. The binding of the analyte may perturb the localmicroenvironment around the receptor leading to a modulation of theabsorbance or fluorescence properties of the sensor.

In one embodiment, receptors may be used immediately in a sensingmode simply by attaching the receptors to a bead that is alreadyderivatized with a dye sensitive to its microenvironment. This isoffers an advantage over other signalling methods because thesignaling protocol becomes routine and does not have to beengineered; only the receptors need to be engineered. The abilityto use several different dyes with the same receptor, and theability to have more than one dye on each bead allows flexibilityin the design of a sensing particle.

Changes in the local pH, local dielectric, or ionic strength, neara fluorophore may result in a signal. A high positive charge in amicroenvironment leads to an increased pH since hydronium migratesaway from the positive region. Conversely, local negative chargedecreases the microenvironment pH. Both changes result in adifference in the protonation state of pH sensitive indicatorspresent in that microenvironment. Many common chromophores andfluorophores are pH sensitive. The interior of the bead may beacting much like the interior of a cell, where the indicatorsshould be sensitive to local pH.

The third optical transduction scheme involves fluorescence energytransfer. In this approach, two fluorescent monomers for signalingmay be mixed into a combinatorial split synthesis. Examples ofthese monomers are depicted in FIG. 14. Compound 470 (a derivativeof fluorescein) contains a common colorimetric/fluorescent probethat may be mixed into the oligomers as the reagent that will sendout a modulated signal upon analyte binding. The modulation may bedue to resonance energy transfer to monomer 475 (a derivative ofrhodamine). When an analyte binds to the receptor, structuralchanges in the receptor will alter the distance between themonomers (schematically depicted in FIG. 8, 320 corresponds tomonomer 470 and 330 corresponds to monomer 475). It is well knownthat excitation of fluorescein may result in emission fromrhodamine when these molecules are oriented correctly. Theefficiency of resonance energy transfer from fluorescein torhodamine will depend strongly upon the presence of analytebinding; thus measurement of rhodamine fluorescence intensity (at asubstantially longer wavelength than fluorescein fluorescence) willserve as a indicator of analyte binding. To greatly improve thelikelihood of a modulatory fluorescein-rhodamine interaction,multiple rhodamine tags can be attached at different sites along acombinatorial chain without substantially increasing backgroundrhodamine fluorescence (only rhodamine very close to fluoresceinwill yield appreciable signal). In one embodiment, depicted in FIG.8, when no ligand is present, short wavelength excitation light(blue light) excites the fluorophore 320, which fluoresces (greenlight). After binding of analyte ligand to the receptor, astructural change in the receptor molecule brings fluorophore 320and fluorophore 330 in proximity, allowing excited-statefluorophore 320 to transfer its energy to fluorophore 330. Thisprocess, fluorescence resonance energy transfer, is extremelysensitive to small changes in the distance between dye molecules(e.g., efficiency.about.[distance].sup.6).

In another embodiment, photoinduced electron transfer (PET) may beused to analyze the local microenvironment around the receptor. Themethods generally includes a fluorescent dye and a fluorescencequencher. A fluorescence quencher is a molecule that absorbs theemitted radiation from a fluorescent molecule. The fluorescent dye,in its excited state, will typically absorbs light at acharacteristic wavelength and then re-emit the light at acharacteristically different wavelength. The emitted light,however, may be reduced by electron transfer with the fluorescentquncher, which results in quenching of the fluorescence. Therefore,if the presence of an analyte perturbs the quenching properties ofthe fluorescence quencher, a modulation of the fluorescent dye maybe observed.

The above described signalling methods may be incorporated into avariety of receptor-indicator-polymeric resin systems. Turning toFIG. 55A, an indicator (I) and receptor (R) may be coupled to apolymeric resin. In the absence of an analyte, the indicator mayproduce a signal in accordance with the local microenvironment. Thesignal may be an absorbance at a specific wavelength or afluorescence. When the receptor interacts with an analyte, thelocal microenvironment may be altered such that the produced signalis altered. In one embodiment, depicted in FIG. 55A, the indicatormay partially bind to the receptor in the absence of an analyte.When the analyte is present the indicator may be displaced from thereceptor by the analyte. The local microenvironment for theindicator therefore changes from an environment where the indicatoris binding with the receptor, to an environment where the indicatoris no longer bound to the receptor. Such a change in environmentmay induce a change in the absorbance or fluorescence of theindicator.

In another embodiment, depicted in Turning to FIG. 55C, anindicator (I) may be coupled to a receptor (R). The receptor may becoupled to a polymeric resin. In the absence of an analyte, theindicator may produce a signal in accordance with the localmicroenvironment. The signal may be an absorbance at a specificwavelength or a fluorescence. When the receptor interacts with ananalyte, the local microenvironment may be altered such that theproduced signal is altered. In contrast to the case depicted inFIG. 55A, the change in local microenvironment may be due to aconformation change of the receptor due to the biding of theanalyte. Such a change in environment may induce a change in theabsorbance or fluorescence of the indicator.

In another embodiment, depicted in FIG. 55E, an indicator (I) maybe coupled to a receptor by a linker. The linker may have asufficient length to allow the indicator to bind to the receptor inthe absence of an analyte. The receptor (R) may be coupled to apolymeric resin. In the absence of an analyte, the indicator mayproduce a signal in accordance with the local microenvironment. Asdepicted in FIG. 55E, the indicator may partially bind to thereceptor in the absence of an analyte. When the analyte is presentthe indicator may be displaced from the receptor by the analyte.The local microenvironment for the indicator therefore changes froman environment where the indicator is binding with the receptor, toan environment where the indicator is no longer bound to thereceptor. Such a change in environment may induce a change in theabsorbance or fluorescence of the indicator.

In another embodiment, depicted in FIG. 55H, a receptor (R) may becoupled to a polymeric resin by a first linker. An indicator may becoupled to the first linker. In the absence of an analyte, theindicator may produce a signal in accordance with the localmicroenvironment. The signal may be an absorbance at a specificwavelength or a fluorescence. When the receptor interacts with ananalyte, the local microenvironment may be altered such that theproduced signal is altered. In one embodiment, as depicted in FIG.55H, the indicator may partially bind to the receptor in theabsence of an analyte. When the analyte is present the indicatormay be displaced from the receptor by the analyte. The localmicroenvironment for the indicator therefore changes from anenvironment where the indicator is binding with the receptor, to anenvironment where the indicator is no longer bound to the receptor.Such a change in environment may induce a change in the absorbanceor fluorescence of the indicator.

In another embodiment, the use of fluorescence resonance energytransfer or photoinduced electron transfer may be used to detectthe presence of an analyte. Both of these methodologies involve theuse of two fluorescent molecules. Turning to FIG. 55B, a firstfluorescent indicator (B) may be coupled to receptor (R). Receptor(R) may be coupled to a polymeric resin. A second fluorescentindicator (C) may also be coupled to the polymeric resin. In theabsence of an analyte, the first and second fluorescent indicatorsmay be positioned such that fluorescence energy transfer may occur.In one embodiment, excitation of the first fluorescent indicatormay result in emission from the second fluorescent indicator whenthese molecules are oriented correctly. Alternatively, either thefirst or second fluorescent indicator may be a fluorescencequencher. When the two indicators are properly aligned, theexcitation of the fluorescent indicators may result in very littleemission due to quenching of the emitted light by the fluorescencequencher. In both cases, the receptor and indicators may bepositioned such that fluorescent energy transfer may occur in theabsence of an analyte. When the analyte is presence the orientationof the two indicators may be altered such that the fluorescenceenergy transfer between the two indicators is altered. In oneembodiment, the presence of an analyte may cause the indicators tomove further apart. This has an effect of reducing the fluorescentenergy transfer. If the two indicators interact to produce anemission signal in the absence of an analyte, the presence of theanalyte may cause a decrease in the emission signal. Alternatively,if one the indicators is a fluorescence quencher, the presence ofan analyte may disrupt the quenching and the fluorescent emissionfrom the other indicator may increase. It should be understood thatthese effects will reverse if the presence of an analyte causes theindicators to move closer to each other.

In another embodiment, depicted in FIG. 55D, a first fluorescentindicator (B) may be coupled to receptor (R). A second fluorescentindicator (C) may also be coupled to the receptor. Receptor (R) maybe coupled to a polymeric resin. In the absence of an analyte, thefirst and second fluorescent indicators may be positioned such thatfluorescence energy transfer may occur. In one embodiment,excitation of the first fluorescent indicator may result inemission from the second fluorescent indicator when these moleculesare oriented correctly. Alternatively, either the first or secondfluorescent indicator may be a fluorescence quencher. When the twoindicators are properly aligned, the excitation of the fluorescentindicators may result in very little emission due to quenching ofthe emitted light by the fluorescence quencher. In both cases, thereceptor and indicators may be positioned such that fluorescentenergy transfer may occur in the absence of an analyte. When theanalyte is presence the orientation of the two indicators may bealtered such that the fluorescence energy transfer between the twoindicators is altered. In one embodiment, depicted in FIG. 55D, thepresence of an analyte may cause the indicators to move furtherapart. This has an effect of reducing the fluorescent energytransfer. If the two indicators interact to produce an emissionsignal in the absence of an analyte, the presence of the analytemay cause a decrease in the emission signal. Alternatively, if onethe indicators is a fluorescence quencher, the presence of ananalyte may disrupt the quenching and the fluorescent emission fromthe other indicator may increase. It should be understood thatthese effects will reverse if the presence of an analyte causes theindicators to move closer to each other.

In a similar embodiment to FIG. 55D, the first fluorescentindicator (B) and second fluorescent indicator (C) may be bothcoupled to receptor (R), as depicted in FIG. 55F. Receptor (R) maybe coupled to a polymeric resin. First fluorescent indicator (B)may be coupled to receptor (R) by a linker group. The linker groupmay allow the first indicator to bind the receptor, as depicted inFIG. 55F. In the absence of an analyte, the first and secondfluorescent indicators may be positioned such that fluorescenceenergy transfer may occur. When the analyte is presence, the firstindicator may be displaced from the receptor, causing thefluorescence energy transfer between the two indicators to bealtered.

In another embodiment, depicted in FIG. 55G, a first fluorescentindicator (B) may be coupled to a polymeric resin. Receptor (R) mayalso be coupled to a polymeric resin. A second fluorescentindicator (C) may be coupled to the receptor (R). In the absence ofan analyte, the first and second fluorescent indicators may bepositioned such that fluorescence energy transfer may occur. In oneembodiment, excitation of the first fluorescent indicator mayresult in emission from the second fluorescent indicator when thesemolecules are oriented correctly. Alternatively, either the firstor second fluorescent indicator may be a fluorescence quencher.When the two indicators are properly aligned, the excitation of thefluorescent indicators may result in very little emission due toquenching of the emitted light by the fluorescence quencher. Inboth cases, the receptor and indicators may be positioned such thatfluorescent energy transfer may occur in the absence of an analyte.When the analyte is presence the orientation of the two indicatorsmay be altered such that the fluorescence energy transfer betweenthe two indicators is altered. In one embodiment, the presence ofan analyte may cause the indicators to move further apart. This hasan effect of reducing the fluorescent energy transfer. If the twoindicators interact to produce an emission signal in the absence ofan analyte, the presence of the analyte may cause a decrease in theemission signal. Alternatively, if one the indicators is afluorescence quencher, the presence of an analyte may disrupt thequenching and the fluorescent emission from the other indicator mayincrease. It should be understood that these effects will reverseif the presence of an analyte causes the indicators to move closerto each other.

In another embodiment, depicted in FIG. 55I, a receptor (R) may becoupled to a polymeric resin by a first linker. A first fluorescentindicator (B) may be coupled to the first linker. A secondfluorescent indicator (C) may be coupled to the receptor (R). Inthe absence of an analyte, the first and second fluorescentindicators may be positioned such that fluorescence energy transfermay occur. In one embodiment, excitation of the first fluorescentindicator may result in emission from the second fluorescentindicator when these molecules are oriented correctly.Alternatively, either the first or second fluorescent indicator maybe a fluorescence quencher. When the two indicators are properlyaligned, the excitation of the fluorescent indicators may result invery little emission due to quenching of the emitted light by thefluorescence quencher. In both cases, the receptor and indicatorsmay be positioned such that fluorescent energy transfer may occurin the absence of an analyte. When the analyte is presence theorientation of the two indicators may be altered such that thefluorescence energy transfer between the two indicators is altered.In one embodiment, the presence of an analyte may cause theindicators to move further apart. This has an effect of reducingthe fluorescent energy transfer. If the two indicators interact toproduce an emission signal in the absence of an analyte, thepresence of the analyte may cause a decrease in the emissionsignal. Alternatively, if one the indicators is a fluorescencequencher, the presence of an analyte may disrupt the quenching andthe fluorescent emission from the other indicator may increase. Itshould be understood that these effects will reverse if thepresence of an analyte causes the indicators to move closer to eachother.

In one embodiment, polystyrene/polyethylene glycol resin beads maybe used as a polymeric resin since they are highly water permeable,and give fast response times to penetration by analytes. The beadsmay be obtained in sizes ranging from 5 microns to 250 microns.Analysis with a confocal microscope reveals that these beads aresegregated into polystyrene and polyethylene glycol microdomains,at about a 1 to 1 ratio. Using the volume of the beads and thereported loading of 300 pmol/bead, we can calculate an averagedistance of 35 .ANG. between terminal sites. This distance is wellwithin the Forester radii for the fluorescent dyes that we areproposing to use in our fluorescence resonance energy transfer("FRET") based signaling approaches. This distance is alsoreasonable for communication between binding events andmicroenvironment changes around the fluorophores.

The derivatization of the beads with our receptors and with theindicators may be accomplished by coupling carboxylic acids andamines using EDC and HOBT. Typically, the efficiency of couplingsare greater that 90% using quantitative ninhydrin tests. (SeeNiikura, K.; Metzger, A.; and Anslyn, E. V. "A Sensing Ensemblewith Selectivity for lositol Trisphosphate", J. Am. Chem. Soc.1998, 120,0000, which is incorporated herein by reference). Thelevel of derivatization of the beads is sufficient to allow theloading of a high enough level of indicators and receptors to yieldsuccessful assays. However, an even higher level of loading may beadvantageous since it would increase the multi-valency effect forbinding analytes within the interior of the beads. We may increasethe loading level two fold and ensure that two amines are close inproximity by attaching an equivalent of lysine to the beads (seeFIG. 45D). The amines may be kept in proximity so that binding ofan analyte to the receptor will influence the environment of aproximal indicator.

Even though a completely random attachment of indicator and areceptor lead to an effective sensing particle, it may be better torationally place the indicator and receptor in proximity. In oneembodiment, lysine that has different protecting groups on the twodifferent amines may be used, allowing the sequential attachment ofan indicator and a receptor. If needed, additional rounds ofderivatization of the beads with lysine may increase the loading bypowers of two, similar to the synthesis of the first fewgenerations of dendrimers.

In contrast, too high a loading of fluorophores will lead toself-quenching, and the emission signals may actually decrease withhigher loadings. If self quenching occurs for fluorophores on thecommercially available beads, we may incrementally cap the terminalamines thereby giving incrementally lower loading of theindicators.

Moreover, there should be an optimum ratio of receptors toindicators. The optimum ratio is defined as the ratio of indicatorto receptor to give the highest response level. Too few indicatorscompared to receptors may lead to little change in spectroscopysince there will be many receptors that are not in proximity toindicators. Too many indicators relative to receptors may also leadto little change in spectroscopy since many of the indicators willnot be near receptors, and hence a large number of the indicatorswill not experience a change in microenvironment. Through iterativetesting, the optimum ratio may be determined for any receptorindicator system.

This iterative sequence will be discussed in detail for a particledesigned to signal the presence of an analyte in a fluid. Thesequence begins with the synthesis of several beads with differentloadings of the receptor. The loading of any receptor may bequantitated using the ninhydrin test. (The ninhydrin test isdescribed in detail in Kaiser, E.; Colescott, R. L.; Bossinger, C.D.; Cook, P. I. "Color Test for Detection of Free Terminal AminoGroups in the Solid-Phase Synthesis of Peptides", Anal. Biochem.1970, 34, 595 598 which is incorporated herein by reference). Thenumber of free amines on the bead is measured prior to and afterderivatization with the receptor, the difference of which gives theloading. Next, the beads undergo a similar analysis with varyinglevels of molecular probes. The indicator loading may bequantitated by taking the absorption spectra of the beads. In thismanner, the absolute loading level and the ratio between thereceptor and indicators may be adjusted. Creating calibrationcurves for the analyte using the different beads will allow theoptimum ratios to be determined.

The indicator loading may be quantitated by taking the absorptionspectra of a monolayer of the beads using our sandwich technique(See FIG. 46D). The sandwich technique involves measuring thespectroscopy of single monolayers of the beads. The beads may besandwiched between two cover slips and gently rubbed together untila monolayer of the beads is formed. One cover slip is removed, andmesh with dimensions on the order of the beads is then place overthe beads, and the cover slip replaced. This sandwich is thenplaced within a cuvette, and the absorbance or emission spectra arerecorded. Alternatively, an sensor array system, as describedabove, may be used to analyze the interaction of the beads with theanalyte.

A variety of receptors may be coupled to the polymeric beads. Manyof these receptors have been previously described. Other receptorsare shown in FIG. 47.

As described generally above, an ensemble may be formed by asynthetic receptor and a probe molecule, either mixed together insolution or bound together on a resin bead. The modulation of thespectroscopic properties of the probe molecule results fromperturbation of the microenvironment of the probe due tointeraction of the receptor with the analyte; often a simple pHeffect. The use of a probe molecule coupled to a common polymericsupport may produce systems that give color changes upon analytebinding. A large number of dyes are commercially available, many ofwhich may be attached to the bead via a simple EDC/HOBT coupling(FIG. 48 shows some examples of indicators). These indicators aresensitive to pH, and also respond to ionic strength and solventproperties. When contacted with an analyte, the receptor interactswith the analyte such that microenvironment of the polymeric resinmay become significantly changed. This change in themicroenvironment may induce a color change in the probe molecule.This may lead to an overall change in the appearance of theparticle indicating the presence of the analyte.

Since many indicators are sensitive to pH and local ionic strength,index of refraction, and/or metal binding, lowering the localdielectric constant near the indicators may modulate the activityof the indicators such that they are more responsive. A highpositive charge in a microenvironment leads to an increased pHsince hydronium ions migrate away from the positive region.Conversely, local negative charge decreases the microenvironmentpH. Both changes result in a difference on the protonation state ofa pH sensitive indicator present in that microenvironment. Thealtering of the local dielectric environment may be produced byattaching molecules of differing dielectric constants to the beadproximate to the probe molecules. Examples of molecules which maybe used to alter the local dielectric environment include, but arenot limited to, planar aromatics, long chain fatty acids, andoligomeric tracts of phenylalanine, tyrosine, and tryptophan.Differing percentages of these compounds may be attached to thepolymeric bead to alter the local dielectric constant.

Competition assays may also be used to produce a signal to indicatethe presence of an analyte. The high specificity of antibodiesmakes them the current tools of choice for the sensing andquantitation of structurally complex molecules in a mixture ofanalytes. These assays rely on a competition approach in which theanalyte is tagged and bound to the antibody. Addition of theuntagged analyte results in a release of the tagged analytes andspectroscopic modulation is monitored. Surprisingly, althoughcompetition assays have been routinely used to determine bindingconstants with synthetic receptors, very little work has been doneexploiting competition methods for the development of sensors basedupon synthetic receptors. Yet, all the ways in which themicroenvironment of the chromophore can be altered, as describedabove, may be amenable to the competition approach. Those that havebeen developed using synthetic receptors are mostly centered uponthe use of cyclodextrins. (See e.g., Hamasaki, K.; Ikeda, H.;Nakamura, A.; Ueno, A.; Toda, F.; Suzuki, I.; Osa, T. "FluorescentSensors of Molecular Recognition. Modified Cyclodextrins Capable ofExhibiting Guest-Responsive Twisted Intramolecular Charge TransferFluorescence" J. Am. Chem. Soc. 1993, 115, 5035, and reference (5)therein, which are incorporated herein by reference) A series ofparent and derivatized cyclodextrins have been combined withchromophores that are responsive to the hydrophobicity of theirmicroenvironment to produce a sensor system. Displacement of thechromophores from the cyclodextrin cavity by binding of a guestleads to a diagnostic spectroscopy change.

This competitive approach has been used successfully, in oneembodiment, for the detection of carbohydrates such asinositol-1,4,5-triphosphate (IP.sub.3). In one embodiment, asynthetic receptor 5 may be paired with an optical signalingmolecule 5-carboxyfluorescein, to quantitate IP.sub.3 at nMconcentrations. A competition assay employing an ensemble of5-carboxyfluorescein and receptor 5 was used to measure bindingconstants. The addition of receptor 5 to 5-carboxyfluoresceinresulted in a red shift of the absorption of 5-carboxyfluorescein.Monitoring the absorption at 502 nm, followed by analysis of thedata using the Benesi-Hildebrand method, gave affinity constants of2.2.times.10.sup.4 M.sup.-1 for 5-carboxyfluorescein binding toreceptor 5. Addition of IP.sub.3 to a solution of the complexesformed between 5 and 5-carboxyfluorescein resulted in displacementof 5-carboxyfluorescein and a subsequent blue shift.

In order to enhance the affinity of receptor 5 for IP.sub.3,similar assays were repeated in methanol, and with 2% of thesurfactant Triton-X. In methanol and the detergent solutions,5-carboxyfluorescein prefers a cyclized form in which the2-carboxylate has undergone an intramolecular conjugate addition tothe quinoid structure. This form of 5-carboxyfluorescein iscolorless and nonfluorescent. Upon addition of receptor 5 theyellow color reappears as does the fluorescence. The positivecharacter of the receptor induces a ring opening to give thecolored/fluorescent form of 5-carboxyfluorescein. Using theBenesi-Hildebrand method applied to absorption data a bindingconstant of 1.2.times.10 M.sup.-1 was found for receptor 5 and5-carboxyfluorescein. As anticipated based upon the differences inthe spectroscopy of 5-carboxyfluorescein when it is bound toreceptor 5 or free in solution, addition of IP.sub.3 to a solutionof receptor 5 and 5-carboxyfluorescein resulted in a decrease ofabsorbance and fluorescence due to release of 5-carboxyfluoresceininto the methanol solution. Binding constants of 1.0.times.10.sup.8M.sup.8 and 1.2.times.10.sup.7 M.sup.-1 for IP.sub.3 and receptor 5were found for methanol and the surfactant solutionrespectively.

Since fluorescence spectroscopy is a much more sensitive techniquethan UV/visible spectroscopy, and the use of methanol gavesignificantly stronger binding between receptor 5 and5-carboxyfluorescein, as well as between receptor 5 and IP.sub.3,the monitoring of fluorescence was found to be the method of choicefor sensing nM concentrations of IP.sub.3. We find that theaddition of IP.sub.3 to an ensemble of receptor 5 and5-carboxyfluorescein in water may detect and quantitate IP.sub.3 ata concentration as low as 1 mM. Importantly, in methanol a 10 nMIP.sub.3 concentration was easily detected. A detection level inthe nM range is appropriate for the development of an assay usingmethanol or surfactant as an eluent and capillary electrophoresisto sample and fractionate cellular components.

We have shown that receptor 5 binds IP.sub.3 quite selectively overother similarly charged species present in cells. Polyanions withcharges higher than IP.sub.3, such as IP.sub.4, IP.sub.5, andoligonucleotides, however, are expected to bind with higheraffinities. In order to fractionate the cellular components duringsignal transduction, and specifically monitor IP.sub.3, acombination of a chemically sensitive particle and capillaryelectrophoresis (CE) may be used. As has been described above, asensor array may include a well in which the particle is placed,along with a groove in which the capillary will reside. Thecapillary will terminate directly into the interior of the bead(See FIG. 49). Illumination from above and CCD analysis from belowmay be used to analyze the particle. Samples as small as 100femtoliters may be introduced into an electrophoresis capillary foranalysis. Using high sensitivity multiphoton-excited fluorescenceas few as .about.50,000 molecules of various precursors/metabolitesof the neurotransmitter, serotonin may be detected. Cytosolicsamples may be collected and fractionated in micron-diametercapillary electrophoresis channels. At the capillary outlet,components may migrate from the channel individually, and will bedirected onto a bead that houses immobilized receptor 5 and thedyes appropriate for our various signaling strategies. Receptorbinding of IP.sub.3 or IP.sub.4 will elicit modulations in theemission and/or absorption properties.

Dramatic spectroscopy changes accompany the chelation of metals toligands that have chromophores. In fact, mostcolorimetric/fluorescent sensors for metals rely upon such astrategy. Binding of the metal to the inner sphere of the ligandleads to ligand/metal charge transfer bands in the absorbancespectra, and changes in the HOMO-LUMO gap that leads tofluorescence modulations.

In one embodiment, the binding of an analyte may be coupled withthe binding of a metal to a chromophoric ligand, such that themetal may be used to trigger the response of the sensor for theanalyte. The compound known as Indo-1 (see FIG. 50 for thestructure and emission properties) is a highly fluorescentindicator that undergoes a large wavelength shift upon exposure toCa(II). Further, compound 2 binds Ce(III) and the resulting complexis fluorescent. In one embodiment, the binding of Ca(II) or Ce(III)to these sensors may be altered by the addition of an analyte ofinterest. By altering the binding of these metals to a receptor asignal may be generated indicating the presence of thereceptor.

In one embodiment, fluorescent indicators that have been used tomonitor Ca(II) and Ce(III) levels in other applications may beapplied to a polymeric supported system. Using the Ca(II) sensorIndo-1 as an example, the strategy is shown in FIG. 51. Indo-1binds Ca(II) at nM concentrations (see FIG. 50). Attachment ofIndo-1 and one of our guanidinium/amine based receptors 3 6 to aresin bead (derivatized with lysine as depicted in FIG. 45D) maylead to intramolecular interactions between the carboxylates ofIndo-1 and the guanidiniums/ammoniums of a receptor. Thecoordination of the carboxylates of Indo-1 may result in adecreased affinity for Ca(II). However, there should be cooperativebinding of Ca(II) and our analytes. Once one of the anionicanalytes is bound to its respective receptor, it will competitivelydisplace the carboxylates of Indo-1 leading to increased Ca(II)binding, which in turn will result in a fluorescence modulation.Similarly, binding of Ca(II) to Indo-1 leaves the guanidiniums ofthe receptors free to bind citrate. The assays will likely be mostsensitive at concentrations of the analytes and Ca(II) near theirdissociation constants, where neither receptor is saturated andsmall changes in the extent of binding lead to large changes influorescence.

We also may switch the role of the metal and the ligand. Indo-1 isfluorescent with and without the Ca(II). However, compound 2 is notfluorescent until Ce(III) binds to it. Thus, a similar assay thatrelies upon a change of microenvironment in the interior of thebead depending upon the presence or absence of the analyte shouldperturb the binding of Ce(III) to compound 2. In this case, arepulsive interaction is predicted for the binding of Ce(III) whenthe positive charges of the guanidinium based receptors are notneutralized by binding to the anionic analytes.

In one embodiment, an indicator may be coupled to a bead andfurther may be bound to a receptor that is also coupled to thebead. Displacement of the indicator by an analyte will lead tosignal modulation. Such a system may also take advantage offluorescent resonance energy transfer to produce a signal in thepresence of an analyte. Fluorescence resonance energy transfer is atechnique that can be used to shift the wavelength of emission fromone position to another in a fluorescence spectra. In this mannerit creates a much more sensitive assay since one can monitorintensity at two wavelengths. The method involves the radiationlesstransfer of excitation energy from one fluorophore to another. Thetransfer occurs via coupling of the oscillating dipoles of thedonor with the transition dipole of the acceptor. The efficiency ofthe transfer is described by equations first derived by Forester.They involve a distance factor (R), orientation factor (k), solventindex of refraction (N), and spectral overlap (J).

In order to incorporate fluorescence resonance energy transfer intoa particle a receptor and two different indicators may beincorporated onto a polymeric bead. In the absence of an analytethe fluorescence resonance energy transfer may occur giving rise toa detectable signal. When an analyte interacts with a receptor, thespacing between the indicators may be altered. Altering thisspacing may cause a change in the fluorescence resonance energytransfer, and thus, a change in the intensity or wavelength of thesignal produced. The fluorescence resonance energy transferefficiency is proportional to the distance (R) between the twoindicators by 1/R.sup.6. Thus slight changes in the distancebetween the two indicators may induce significant changes in thefluorescence resonance energy transfer.

In one embodiment, various levels of coumarin and fluorescein maybe loaded onto resin beads so as to achieve gradiations in FRETlevels from zero to 100%. FIG. 52 shows a 70/30 ratio of emissionfrom 5-carboxyfluorescein and coumarin upon excitation of coumarinonly in water. However, other solvents give dramatically differentextents of FRET. This shows that the changes in the interior of thebeads does lead to a spectroscopic response. This data also showsthat differential association of the various solvents and5-carboxyfluorescein on resin beads as a function of solvents. Thisbehavior is evoked from the solvent association with the polymeritself, in the absence of purposefully added receptors. We may alsoadd receptors which exhibit strong/selective association withstrategic analytes. Such receptors may induce a modulation in theratio of FRET upon analyte binding, within the microenvironment ofthe polystyrene/polyethylene glycol matrices.

In order to incorporate a wavelength shift into a fluorescenceassays, receptors 3 6 may be coupled to thecourmarin/5-carboxyfluorescein beads discussed above. When5-carboxyfluorescein is bound to the various receptors and coumarinis excited, the emission will be primarily form coumarin since thefluorescein will be bound to the receptors. Upon displacement ofthe 5-carboxyfluorescein by the analytes, emission should shiftmore toward 5-carboxyfluorescein since it will be released to thebead environment which possesses coumarin. This will give us awavelength shift in the fluorescence which is inherently moresensitive than the modulation of intensity at a signalwavelength.

There should be large changes in the distance between indicators(R) on the resin beads. When the 5-carboxyfluorescein is bound, thedonor/acceptor pair should be farther than when displacement takesplace; the FRET efficiency scales as 1/R.sup.6. The coumarin may becoupled to the beads via a floppy linker, allowing it to adopt manyconformations with respect to a bound 5-carboxyfluorescein. Hence,it is highly unlikely that the transition dipoles of the donor andacceptor will be rigorously orthogonal.

In one embodiment, a receptor for polycarboxylic acids and anappropriate probe molecule may be coupled to a polymeric resin toform a particle for the detection of polycarboxylic acid molecules.Receptors for polycarboxylic acids, as well as methods for theiruse in the detection of polycarboxylic acids, have been describedin U.S. Pat. No. 6,045,579 which is incorporated herein byreference. This system involves, in one embodiment, the use of areceptor 3 which was found to be selective for the recognition of atricarboxylic acid (e.g., citrate) in water over dicarboxylates,monocarboxylates, phosphates, sugars, and simple salts. Thereceptor includes guanidinium groups for hydrogen bonding andcharge pairing with the tricarboxylic acid.

An assay for citrate has employed an ensemble of5-carboxyfluorescein and 3. The binding between 3 and5-carboxyfluorescein resulted in a lowering of the phenol pK.sub.aof 5-carboxyfluorescein, due to the positive microenvironmentpresented by 3. This shift in pK.sub.a (local pH) caused the phenolmoiety to be in a higher state of protonation when5-carboxyfluorescein was free in solution. The absorbance orfluorescence of 5-carboxyfluorescein decreases with higherprotonation of the phenol. The intensity of absorbance increaseswith addition of host 3 to 5-carboxyfluorescein, and as predictedthe intensity decreases upon addition of citrate to the ensemble of3 and 5-carboxyfluorescein. The same effect was seen in thefluorescence spectrum (.lamda.max=525 nm).

In an embodiment, a metal may be used to trigger the response of achromophore to the presence of an analyte. For example, compound 7binds Cu(II) with a binding constant of 4.9.times.10.sup.5 M.sup.-1(See FIG. 53). Addition of 1 eq. of Cu(II) increases the bindingconstant of citrate to compound 7 by a factor of at least 5.Importantly, the addition of citrate increases the binding ofCu(II) to the receptor by a factor of at least 10. Therefore thecitrate and Cu(II) enhance each other's binding in a cooperativemanner. Further, the emission spectra of compound 7 is quitesensitive to the addition of citrate when Cu(II) is present, buthas no response to the addition of citrate in the absence ofCu(II). Thus the binding of a "trigger" may be perturbed with ananalyte of interest, and the perturbation of the binding of thetrigger may be used to spectroscopically monitor the binding of theanalyte. The triggering of the sensing event by an added entity issimilar to the requirement for enzymes in saliva to degrade foodparticulants into tastants recognizable by the receptors onmammalian taste buds.

In one embodiment, citrate receptor 3 may be immobilized on apolystyrene/polyethylene glycol bead, where on the same bead mayalso be attached a fluorescent probe molecule (FIG. 54). Solutionsof citrate at different concentrations may be added to the beads,and the fluorescence spectra of the monolayer recorded. We findexactly the same fluorescence response toward citrate for theensemble of receptor 3 and 5-carboxyfluorescein on the beads as insolution. Apparently, a similar microenvironment change to modulatethe spectroscopy of 5-carboxyfluorescein occurs in the beads,although both 5-carboxyfluorescein and receptor 3 are just randomlyplaced throughout the bead.

Additional sensor system include sensors for tartrate andtetracycline Compound 4 binds tartrate in buffered water (pH 7.4)with a binding constant of approximately 10.sup.5 M.sup.-1. Thebinding is slow on the NMR time scale, since we can observe boththe bound and free receptor and tartrate. This binding issurprisingly strong for pure water. It must reflect goodcooperativity between the host's boronic acid moiety and the twoguanidinium groups for the recognition of the guest's vicinal dioland two carboxylates respectively. Compound 6 may act as amolecular receptor for tetracyclin. The compound has beensynthesized, and by variable temperature NMR it has been found tobe in a bowl conformation. Its binding properties with severalindicators have been explored (most bind with affinities near10.sup.4M.sup.-1). More importantly, the binding of tetracyclin hasalso been explored, and our preliminary results suggests that thebinding constant in water is above 10.sup.3 M.sup.-1.

In another embodiment, a sensing particle may include an oligomerof amino acids with positively charged side chains such as thelysine trimer, depicted in FIG. 56, designed to act as the anionreceptor, and an attached FRET pair for signaling. Sensing ofdifferent anions may be accomplished by optically monitoringintensity changes in the signal of the FRET pair as the analyteinteracts with the oligomer.

Upon introduction of an anionic species to 1, the analyte may bindto the trimer, disturbing the trimer-fluorescein interaction,thereby, altering the fluorescein's ability to participate in theenergy transfer mechanism. Using a monolayer of resin in aconventional fluorometer, the ratio of D:A emission for the FRETpair attached to TG-NH.sub.2 resin is sensitive to differentsolvents as well as to the ionic strength of the solution.Epifluorescence studies may be performed to test the solventdependence, ionic strength, and binding effects of different anionson the FRET TG-NH.sub.2 resins. The images of the FRET TG-NH.sub.2resins within a sensor array, taken by a charged coupled device(CCD) may result in three output channels of red, green, and bluelight intensities. The RGB light intensities will allow forcomparison of the results obtained using a conventionalfluorometer.

The signal transduction of 1 may be studied using a standardfluorometer and within the array platform using epifluorescencemicroscopy. The RGB analysis may be used to characterize therelative changes in emission of the FRET pair. Other resin-boundsensors may be synthesized by varying the amino acid subunitswithin the oligomers and the length of the peptide chains.

In another embodiment, solvatochromic dyes may be covalently linkedto a receptor unit tethered to a resin bead that is capable ofbinding to small organic guests. In one example, dansyl and dapoxylmay act as sensitive probes of their microenvironment. Whenselecting a dye for use, characteristics such as high extinctioncoefficients, high fluorescence quantum yields, and large Stoke'sshifts should be considered. Dapoxyl and dansyl were anchored to 6%agarose resin beads, in an effort to enhance the signaling responseof these resin bound fluorophores in various solvent systems.Agarose beads are crosslinked galactose polymers that are morehydrophilic than the polystyrene-polyethylene glycol resins. Theattachment of these solvatochromic dyes to the agarose resin beadsis outlined in FIG. 57.

The dapoxyl labeled resin (6) was formed by reductively aminatingglyoxalated agarose resin with mono (Fmoc)-butyldiaminehydrochloride salt using sodium borohydride as the reducing agent.The base labile protecting group, Fmoc, was removed from 3 withdilute base, and the solvatochromic dye was anchored to 4 through areaction to form a sulfonamide bond resulting in 6. The tetheringof dansyl to agarose resin was performed similarly.

Analysis of the agarose resins derivatized with dansyl and dapoxylwas attempted several times using a monolayer sample cell in aconventional fluorometer. However, satisfactory emission spectra of5 and 6 in different solvent systems were not obtained due to thefragile nature of the agarose resin which placed restrictions onthe manufacturing of the monolayer sample cell.

Significant signal enhancement of 5 and 6 was seen when the solventsystem was changed from a 50 mM phosphate buffer (pH=7.0) toethanol (EtOH), methanol (MeOH), and acetonitrile (CH.sub.3CN). Theemission of 6 increased three fold in EtOH and five fold inCH.sub.3CN when compared to the emission of 6 in a buffer. Theagarose-dansyl resin, 5, demonstrated similar trends in response todifferent solvents; however, the intensities were smaller than for6. For instance, the emission of 5 in EtOH for the red channel was61% smaller in intensity units compared to 6 (2200 vs. 5800arbitrary intensity units). This observation has been attributed tothe lower quantum yield of fluorescence and the smaller extinctioncoefficient of dansyl to that of dapoxyl. From these initialstudies, the average fluorescence intensity of the three beads oftype 6 in EtOH across the red channel was 5800.+-.300 arbitraryintensity counts with a percent standard deviation of 5.0%. Also,before changing to a new solvent, the agarose beads were flushedwith the buffer for 5 minutes in order to return the agarose-dyeresin to a "zero" point of reference. The background variance ofthe fluorescence intensity of 6 when exposed to each of the bufferwashes between each solvent system was 5.0% and 4.0% in the red andgreen channels, respectively.

The response of 5 and 6 to varying ratios of two different solventswas also studied. As seen in FIG. 58, a detectable decrease in theemission of 6 is observed as the percent of the 50 mM phosphatebuffer (pH=7) is increased in ethanol. The fluorescence intensityof 6 decreased by three fold from its original value in 100% EtOHto 100% buffer. There was an incremental decrease in thefluorescence emission intensities of 6 in both the red and greenchannels. Once again, 5 demonstrated similar trends in response tothe varying ratios of mixed solvent systems; however, theintensities were smaller than 6.

In another example, each dye was derivatized with benzyl amine (24) for studies in solution phase and anchored to resin (5 7) forstudies using the sandwich method and epi-fluorescence. The dyesand corresponding resins are depicted in FIG. 59.

Fluorescence studies have been performed for each dye in solutionphase and attached to resin. FIG. 60 illustrates an example of theemission changes in 4 (part A.) and 7 (part B.) that result fromexposure to different solvent systems. The quantum yield of 4diminished in more polar protic media (i.e. ethanol); whereas, thequantum yield of 4 increased in more hydrophobic environments (i.e.cyclohexane). Also, the Stoke's shift of each probe changedsignificantly between nonpolar and polar media. For example, theStoke's shift of 4 (.lamda..sub.em-.lamda..sub.abs) in 1:1 mixtureof methanol and 1.0 M aqueous phosphate buffer was 221 nm, but theStoke's shift of 4 was 80 nm in cyclohexane. 7 displayed similartrends, but the Stoke's shift from solvent to solvent was not asdramatic. The optical properties of 5 7 only varied slightly whencompared to their homogeneous analogs.

Of the three fluorophores, the solvatochromic properties ofcoumarin were not as dramatic when compared to dansyl and dapoxyl.6 and 7 displayed the largest Stoke's shifts. The emissionwavelength for 5 7 red shifted when placed in more polar solvents.However, when 6 was placed in water, the Stoke's shift was the sameas in when placed in cyclohexane as seen in FIG. 60. This trend wasobserved with each fluoresently labeled resin, and may be explainedby the fact that these probes are hydrophobic and that they mayactually reside within the hydrophobic core of the PEG-PS resinwhen submerged in water.

In another example a selective chemosensor for ATP was found. Abead with a polyethylene-glycol base was attached via guanidiniumto two long polypeptide arms that were known to interact with theadenine group of ATP, as depicted in FIG. 61. The tripeptide armscontained two flourophore attachment sites for 5-carboxyfluorescein(fluorescein), and an attachment site for7-diethylaminocoumarin-3-carboxylic acid (coumarin) located on theterminal end of the lysine that was attached to the core structure.The fluorophores act as receptors for the desired analyte. Thefluorophores also act as indicators to signal changes in theenvironment before and after the addition of analytes.

Fluorescently labeled N-methylanthraniloyl-ATP were chosen toscreen for ATP receptors. Sequences of amino acids were linked astripeptides and equilibrated with a buffer. The resin wastransferred to a microscope slide and illuminated with UV light.The results yielded 6 sequences with active beads that displayedfluorescent activity, and 3 sequences with inactive beads wherethere was no detectable fluorescent activity.

Three of the 6 active beads, and 1 of the 3 inactive beads werearbitrarily chosen to react with ATP (Sequences below in bold).When the fluorescein and coumarin were excited there was nodetectable difference in the FRET upon addition of ATP. This may bedue to there being an average distance between the fluorophoreswithin the beads which does not significantly change upon bindingATP. However, all but one active bead (Thr-Val-Asp) exhibited afluorescence modulation upon excitation of fluorescein. The lack ofresponse from an active bead shows that screening against aderivatized analyte (MANT-ATP in this case) will not guarantee thatthe active beads are successful sensors when synthesized withattached fluorophores. Either this active bead binds the MANTprotion of MANT-ATP or there is no significant microenvironmentchange around the fluorophores of the Thr-Val-Asp receptor uponbinding ATP.

TABLE-US-00001 Active Beads Inactive Beads His-Ala-Asp His-Phe-GlyGlu-Pro-Thr Ser-Ala-Asp Thr-Val-Asp Trp-Asn-Glu Met-Thr-HisAsp-Ala-Asp Ser-Tyr-Ser

A large spectral response upon addition of ATP was observed withthe Ser-Tyr-Ser sequence in the active bead. The increase influorescein emission is possibly due to a higher local pH aroundthe fluorescein upon binding of ATP. Further studies were performedwith the Ser-Tyr-Ser sequence and analytes, AMP, and GTP, which arestructurally similar to ATP. This peptidic library member exhibitedvery high detection selectivity for ATP over these structurallysimilar potentially competing analytes. The lack of response to AMPsuggests the necessity for triphosphates to bind strongly to theguanidinium entities of the receptor, while the lack of response toGTP indicates the specificity for nucleotide bases imparted by thetripeptide arms. The combination of serine and tyrosine suggests.pi.-stacking between the phenol of tyr and adenine and hydrogenbonding interactions between the serine OH and/or the ribose oradenine. These studies have demonstrated that the union of a provencore with combinatorial methods, followed by the attachment offluorophores, can create resin bound chemosensors with excellentselectivity.

As described above, a particle, in some embodiments, possesses boththe ability to interact with the analyte of interest and to createa modulated signal. In one embodiment, the particle may includereceptor molecules which undergo a chemical change in the presenceof the analyte of interest. This chemical change may cause amodulation in the signal produced by the particle. Chemical changesmay include chemical reactions between the analyte and thereceptor. Receptors may include biopolymers or organic molecules.Such chemical reactions may include, but are not limited to,cleavage reactions, oxidations, reductions, addition reactions,substitution reactions, elimination reactions, and radicalreactions.

In one embodiment, the mode of action of the analyte on specificbiopolymers may be taken advantage of to produce an analytedetection system. As used herein biopolymers refers to natural andunnatural: peptides, proteins, polynucleotides, andoligosaccharides. In some instances, analytes, such as toxins andenzymes, will react with biopolymer such that cleavage of thebiopolymer occurs. In one embodiment, this cleavage of thebiopolymer may be used to produce a detectable signal. A particlemay include a biopolymer and an indicator coupled to thebiopolymer. In the presence of the analyte the biopolymer may becleaved such that the portion of the biopolymer which includes theindicator may be cleaved from the particle. The signal producedfrom the indicator is then displaced from the particle. The signalof the bead will therefore change thus indicating the presence of aspecific analyte.

Proteases represent a number of families of proteolytic enzymesthat catalytically hydrolyze peptide bonds. Principal groups ofproteases include metalloproteases, serine porteases, cysteineproteases and aspartic proteases. Proteases, in particular serineproteases, are involved in a number of physiological processes suchas blood coagulation, fertilization, inflammation, hormoneproduction, the immune response and fibrinolysis.

Numerous disease states are caused by and may be characterized byalterations in the activity of specific proteases and theirinhibitors. For example emphysema, arthritis, thrombosis, cancermetastasis and some forms of hemophilia result from the lack ofregulation of serine protease activities. In case of viralinfection, the presence of viral proteases have been identified ininfected cells. Such viral proteases include, for example, HIVprotease associated with AIDS and NS3 protease associated withHepatitis C. Proteases have also been implicated in cancermetastasis. For example, the increased presence of the proteaseurokinase has been correlated with an increased ability tometastasize in many cancers.

In one embodiment, the presence of a protease may be detected bythe use of a biopolymer coupled to a polymeric resin. For thedetection of proteases, the biopolymer may be a protein or peptide.Methods for synthesizing and/or attaching a protein or peptides toa polymeric resin are described, for example, in U.S. Pat. No.5,235,028 which is incorporated herein by reference. "Proteins" and"peptides" are herein defined as chains of amino acids whose.alpha.-carbons are linked through peptide bonds formed by acondensation reaction between the a carboxyl group of one aminoacid and the amino group of another amino acid. Peptides alsoinclude peptide mimetics such as amino acids joined by an ether asopposed to an amide bond.

The term "protease binding site" as used herein refers to an aminoacid sequence that may be recognized and cleaved by a protease. Theprotease binding site contains a peptide bond that is hydrolyzed bythe protease and the amino acid residues joined by this peptidebond are said to form the cleavage site. The protease binding siteand conformation determining regions form a contiguous amino acidsequence. The protease binding site may be an amino acid sequencethat is recognized and cleaved by a particular protease. It is wellknown that various proteases may cleave peptide bonds adjacent toparticular amino acids. Thus, for example, trypsin cleaves peptidebonds following basic amino acids such as arginine and lysine andchymotrypsin cleaves peptide bonds following large hydrophobicamino acid residues such as tryptophan, phenylalanine, tyrosine andleucine. The serine protease elastase cleaves peptide bondsfollowing small hydrophobic residues such as alanine. A particularprotease, however, may not cleave every bond in a protein that hasthe correct adjacent amino acid. Rather, the proteases may bespecific to particular amino acid sequences which serve as proteasebinding sites for each particular protease. Any amino acid sequencethat comprises a protease binding site and may be recognized andcleaved by a protease is a suitable protease receptor. Knownprotease binding sites and peptide inhibitors of proteases possesamino acid sequences that are recognized by the specific proteasethey are cleaved by or that they inhibit. Thus known substrate andinhibitor sequences provide the basic sequences suitable for use asa protease receptor. A number of protease substrates and inhibitorsequences suitable for use as protease binding sites are describedin U.S. Pat. No. 6,037,137 which is incorporated herein byreference. One of skill will appreciate that the proteasesubstrates listed in U.S. Pat. No. 6,037,137 is not a complete listand that other protease substrates or inhibitor sequences may beused.

Proteases (e.g., botulinum and tetanus toxins) cleave peptide bondsat specific sequence sites on the proteins that "dock"neurotransmitter secretory vesicles to their cellular release sites(FIGS. 45A, 45B). When one or more of these proteins is degraded inthis fashion, secretion is blocked and paralysis results (FIG.45C). It is known that relatively low molecular weight peptides(.about.15 35 amino acids) based on the normal protein substratesof the botulinum toxins can be rapidly cleaved in solution by atoxin in a manner similar to the full-length protein. Suchexperiments have been described by Schmidt, J. J.; Stafford, R. G.;Bostian, K. A. "Type A botulinum neurotoxin proteolytic activity:development of competitive inhibitors and implications forsubstrate specificity at the S.sub.1'binding subsite" FEBS Lett.,1998, 435, 61 64 and Shone, C. C.; Roberts, A. K. "Peptidesubstrate specificity and properties of the zinc-endopeptidaseactivity of botulinum type B neurotoxin" Eur. J. Biochem., 1994,225, 263 270, both of which are incorporated herein by reference asif set forth herein. It has also been demonstrated that thesepeptide substrates can retain high levels of activity for bothbotulinum and tetanus toxins even when chemically modified by aminoacid substitutions and fluorescence labeling (See also Soleihac,J.-M.; Cornille, F.; Martin, L.; Lenoir, C.; Fournie-Zaluski,M.-C.; Roques, B. P. "A sensitive and rapid fluorescence-basedassay for determination of tetanus toxin peptidase activity" Anal.Biochem., 1996, 241, 120 127 and Adler, M.; Nicholson, J. D.;Hackley, B. E., Jr. "Efficacy of a novel metalloprotease inhibitoron botulinum neurotoxin B activity" FEBS Lett., 1998, 429, 234 238both of which are incorporated herein by reference).

For newly discovered proteases, or proteases of which the proteaserecognition sequence is not known, a suitable amino acid sequencefor use as the protease binding site may be determinedexperimentally. The synthesis of libraries of peptides and the useof these libraries to determine a protease binding sequence for aparticular protease is described in U.S. Pat. No. 5,834,318 whichis incorporated herein by reference. Generally, combinatoriallibraries composed of between about 2 to about 20 amino acids maybe synthesized. These libraries may be used to screen for aninteraction with the protease. Analysis of the sequences that bindto the protease may be used to determine potential bindingsequences for use as a receptor for the protease.

The interaction of the receptor with a protease may be indicated byan indicator molecule coupled to the receptor or the polymericresin. In one embodiment, the indicator may be a chromophore or afluorophore. A fluorophore is a molecule that absorbs light at acharacteristic wavelength and then re-emits the light mosttypically at a characteristic different wavelength. Fluorophoresinclude, but are not limited to rhodamine and rhodaminederivatives, fluorescein and fluorescein derivatives, coumarins andchelators with the lanthanide ion series. A chromophore is amolecule which absorbs light at a characteristic wavelength, butdoes not re-emit light.

In one embodiment, a peptide containing the cleavage sequence isimmobilized through a covalent or strong non-covalent bond to anaddressable site on a sensor array. In one embodiment, this may beaccomplished by coupling the peptide to a polymeric resin, asdescribed above. The polymeric resin may be positioned in a cavityof a sensor array, such as the sensor arrays described above. Insome embodiments, different peptides containing different cleavagesequences for the various proteases may be immobilized at differentarray positions. A sample containing one or more proteases may beapplied to the array, and peptide cleavage may occur at specificarray addresses, depending on the presence of particular proteases.Alternatively, different peptides containing different cleavagesequences may be coupled to a single polymeric bead. In thismanner, a single bead may be used to analyze multipleproteases.

A variety of signaling mechanisms for the above described cleavagereactions may be used. In an embodiment, a fluorescent dye and afluorescence quencher may be coupled to the biopolymer on oppositesides of the cleavage site. The fluorescent dye and thefluorescence quencher may be positioned within the Forster energytransfer radius. The Forster energy transfer radius is defined asthe maximum distance between two molecules in which at least aportion of the fluorescence energy emitted from one of themolecules is quenched by the other molecule. Forster energytransfer has been described above. Before cleavage, little or nofluorescence may be generated by virtue of the molecular quencher.After cleavage, the dye and quencher are no longer maintained inproximity of one another, and fluorescence may be detected (FIG.62A). The use of fluorescence quenching is described in U.S. Pat.No. 6,037,137 which is incorporated herein by reference. Furtherexamples of this energy transfer are described in the followingpapers, all of which are incorporated herein by reference: James,T. D.; Samandumara, K. R. A.; Iguchi, R.; Shinkai, S. J. Am. Chem.Soc. 1995, 117, 8982. Murukami, H.; Nagasaki, T.; Hamachi, I.;Shinkai, S. Tetrahedron Lett., 34, 6273. Shinkai, S.; Tsukagohsi,K.; Ishikawa, Y.; Kunitake, T. J. Chem. Soc. Chem. Commun. 1991,1039. Kondo, K.; Shiomi, Y.; Saisho, M.; Harada, T.; Shinkai, S.Tetrahedron. 1992,48, 8239. Shiomi, Y.; Kondo, K.; Saisho, M.;Harada, T.; Tsukagoshi, K.; Shinkai, S. Supramol. Chem. 1993, 2,11. Shiomi, Y.; Saisho, M.; Tsukagoshi, K.; Shinkai, S. J. Chem.Soc. Perkin Trans 11993, 2111. Deng, G.; James, T. D.; Shinkai, S.J. Am. Chem. Soc. 1994, 116, 4567. James, T. D.; Harada, T.;Shinkai, S. J. Chem. Soc. Chem. Commun. 1993, 857. James, T. D.;Murata, K.; Harada, T.; Ueda, K.; Shinkai, S. Chem. Lett. 1994,273. Ludwig, R.; Harada, T.; Ueda, K.; James, T. D.; Shinkai, S. J.Chem. Soc. Perkin Trans 2. 1994, 4, 497. Sandanayake, K. R. A. S.;Shinkai, S. J. Chem. Soc., Chem. Commun. 1994, 1083. Nagasaki, T.;Shinmori, H.; Shinkai, S. Tetrahedron Lett. 1994, 2201. Murakami,H.; Nagasaki, T.; Hamachi, I.; Shinkai, S. J. Chem. Soc. PerkinTrans 2. 1994, 975. Nakashima, K.; Shinkai, S. Chem. Lett. 1994,1267. Sandanayake, K. R. A. S.; Nakashima, K.; Shinkai, S. J. Chem.Soc. 1994, 1621. James, T. D.; Sandanayake, K. R. A. S.; Shinkai,S. J. Chem. Soc., Chem. Commun. 1994,477. James, T. D.;Sandanayake, K. R. A. S.; Angew. Chem., Int. Ed. Eng. 1994, 33,2207. James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Nature,1995, 374, 345.

The fluorophores may be linked to the peptide receptor by any of anumber of means well known to those of skill in the art. In anembodiment, the fluorophore may be linked directly from a reactivesite on the fluorophore to a reactive group on the peptide such asa terminal amino or carboxyl group, or to a reactive group on anamino acid side chain such as a sulfur, an amino, a hydroxyl, or acarboxyl moiety. Many fluorophores normally contain suitablereactive sites. Alternatively, the fluorophores may be derivatizedto provide reactive sites for linkage to another molecule.Fluorophores derivatized with functional groups for coupling to asecond molecule are commercially available from a variety ofmanufacturers. The derivatization may be by a simple substitutionof a group on the fluorophore itself, or may be by conjugation to alinker. Various linkers are well known to those of skill in the artand are discussed below.

The fluorogenic protease indicators may be linked to a solidsupport directly through the fluorophores or through the peptidebackbone comprising the indicator. In embodiments where theindicator is linked to the solid support through the peptidebackbone, the peptide backbone may comprise an additional peptidespacer. The spacer may be present at either the amino or carboxylterminus of the peptide backbone and may vary from about 1 to about50 amino acids, preferably from 1 to about 20 and more preferablyfrom 1 to about 10 amino acids in length. The amino acidcomposition of the peptide spacer is not critical as the spacerjust serves to separate the active components of the molecule fromthe substrate thereby preventing undesired interactions. However,the amino acid composition of the spacer may be selected to provideamino acids (e.g. a cysteine or a lysine) having side chains towhich a linker or the solid support itself, is easily coupled.Alternatively the linker or the solid support itself may beattached to the amino terminus of or the carboxyl terminus.

In an embodiment, the peptide spacer may be joined to the solidsupport by a linker. The term "linker", as used herein, refers to amolecule that may be used to link a peptide to another molecule,(e.g. a solid support, fluorophore, etc.). A linker is a hetero orhomobifunctional molecule that provides a first reactive sitecapable of forming a covalent linkage with the peptide and a secondreactive site capable of forming a covalent linkage with a reactivegroup on the solid support. Linkers as use din these embodimentsare the same as the previously described linkers.

In an embodiment, a first fluorescent dye and a second fluorescentdye may be coupled to the biopolymer on opposite sides of thecleavage site. Before cleavage, a FRET (fluorescence resonanceenergy transfer) signal may be observed as a long wavelengthemission. After cleavage, the change in the relative positions ofthe two dyes may cause a loss of the FRET signal and an increase influorescence from the shorter-wavelength dye (FIG. 62B). Examplesof solution phase FRET have been described in Forster, Th."Transfer Mechanisms of Electronic Excitation:, Discuss. FaradaySoc., 1959, 27, 7; Khanna, P. L., Ullman, E. F."4',5'-Dimethoxy-6-carboxyfluorescein: A novel dipole--dipolecoupled fluorescence energy transfer acceptor useful forfluorescence immunoassays", Anal. Biochem. 1980, 108, 156; andMorrison, L. E. "Time resolved Detection of Energy Transfer: Theoryand Application to Immunoassays", Anal. Biochem. 1998, 174, 101,all of which are incorporated herein by reference.

In another embodiment, a single fluorescent dye may be coupled tothe peptide on the opposite side of the cleavage site to thepolymeric resin. Before cleavage, the dye is fluorescent, but isspatially confined to the attachment site. After cleavage, thepeptide fragment containing the dye may diffuse from the attachmentsite (e.g., to positions elsewhere in the cavity) where it may bemeasured with a spatially sensitive detection approach, such asconfocal microscopy (FIG. 62C). Alternatively, the solution in thecavities may be flushed from the system. A reduction in thefluorescence of the particle would indicate the presence of theanalyte (e.g., a protease).

In another embodiment, a single indicator (e.g., a chromophore or afluorophore) may be coupled to the peptide receptor on the side ofthe cleavage site that remains on the polymeric resin or to thepolymeric resin at a location proximate to the receptor. Beforecleavage the indicator may produce a signal that reflects themicroevironment determined by the interaction of the receptor withthe indicator. Hydrogen bonding or ionic substituents on theindicator involved in analyte binding have the capacity to changethe electron density and/or rigidity of the indicator, therebychanging observable spectroscopic properties such as fluorescencequantum yield, maximum excitation wavelength, or maximum emissionwavelength for fluorophores or absorption spectra for chromophores.When the peptide receptor is cleaved, the local pH and dielectricconstants of the beads change, and the indicator may respond in apredictable fashion. An advantage to this approach is that it doesnot require the dissociation of a preloaded fluorescent ligand(limited in response time by koff). Furthermore, several differentindicators may be used with the same receptor. Different beads mayhave the same receptors but different indicators, allowing formultiple testing for the presence of proteases. Alternatively, asingle polymeric resin may include multiple dyes along with asingle receptor. The interaction of each of these dyes with thereceptor may be monitored to determine the presence of theanalyte.

Nucleases represent a number of families of enzymes thatcatalytically hydrolyze the phosphodiester bonds of nucleic acids.Nucleases may be classified according to the nucleic acid that theyare specific for. Ribonucleases ("RNases") are specific forribonucleic acids while deoxyribonucleases ("DNases") are specificfor deoxyribonucleic acids. Some enzymes will hydrolyze bothribonucleic acids and deoxyribonucleic acids. Nucleases may also beclassified according to their point of attack upon the nucleicacid. Nucleases that attack the polymer at either the 3' terminusor the 5' terminus are known as exonucleases. Nucleases that attackthe nucleic acid within the chain are called endonucleases.

Restriction enzymes recognize short polynucleotide sequences andcleave double-stranded nucleic acids at specific sites within oradjacent to these sequences. Approximately 3,000 restrictionenzymes, recognizing over 230 different nucleic acid sequences, areknown. They have been found mostly in bacteria, but have also beenisolated from viruses, archaea and eukaryotes. Because many ofthese restriction enzymes are only found in a particular organism,nucleic acids may be used as a receptor to determine if aparticular organism is present in a sample by analyzing forrestriction enzymes. Restriction endonucleases specifically bind tonucleic acids only at a specific recognition sequence that variesamong restriction endonucleases. Since restriction enzymes only cutnucleic acids in the vicinity of the recognition sequence, areceptor may be designed that includes the recognition sequence forthe nuclease being investigated.

Most nucleases bind to and act on double stranded deoxyribonucleicacid ("DNA"). Restriction endonucleases are typically symmetricaldimers. Each monomeric unit binds to one strand of DNA andrecognizes the first half the DNA recognition sequence. Eachmonomer also typically cuts one strand of DNA. Together, the dimerrecognizes a palindromic DNA sequence and cuts both strands of DNAsymmetrically about the central point in the palindromic sequence.Typically, each monomer of the restriction endonucleases needs atleast two specific nucleotides that it recognizes, though in a fewcases a restriction endonuclease monomer only needs to bind to onespecific nucleotide and two others with less specificity. Thismeans that restriction endonucleases may recognize a sequence of 4nucleotides at a minimum, and generally recognize sequences thatcontain an even number of nucleotides (since the same sites arerecognized by each monomer. Restriction endonucleases are knownthat recognize 4, 6, or 8 nucleotides, with only a few 8-cuttersknown. Some restriction endonucleases bind to recognition sequencesthat have an odd number of nucleotides (typically this is 5 or 7)with the central nucleotide specifically recognized or with some orstrict specificity for a central base pair. The origin and sequencespecificity of hundreds of restriction endonucleases are known andcan be found from catalogs available from New England Biolabs,Boston, Mass.; Life Technologies, Rockville, Md.; PromegaScientific, Madison, Wis., Rouche Molecular Biochemicals,Indianapolis, Ind.

In one embodiment, the presence of a nuclease may be detected bythe use of a polynucleotide coupled to a polymeric resin. For thedetection of nucleases, the polynucleotide may be a double strandeddeoxyribonucleic acid or a ribonucleic acid. Methods forsynthesizing and/or attaching a polynucleotide to a polymeric resinare described, for example, in U.S. Pat. No. 5,843,655 which isincorporated herein by reference. "Polynucleotides" are hereindefined as chains of nucleotides. The nucleotides are linked toeach other by phosphodiester bonds. "Deoxyribonucleic acid" iscomposed of deoxyribonucleotide residues, while "Ribonucleic acid"is composed of ribonucleotide residues.

The term "nuclease binding site" as used herein refers to apolynucleotide sequence that may be recognized and cleaved by anuclease. The nuclease binding site contains a phosphodiester bondthat is cleaved by the nuclease and the polynucleotide residuesjoined by this phosphodiester bond are said to form the cleavagesite.

For newly discovered nucleases, or nucleases of which the nucleaserecognition sequence is not known, a suitable polynucleotidesequence for use as the nuclease binding site may be determinedexperimentally. Generally, combinatorial libraries ofpolynucleotides composed of between about 2 to about 20 nucleotidesmay be synthesized. The synthesis of such libraries is described,for example, in U.S. Pat. No. 5,843,655 which is incorporatedherein by reference. These libraries may be used to screen for aninteraction with the nuclease. Analysis of the sequences that bindto the nuclease may be used to determine potential bindingsequences for use as a receptor for the nuclease.

The interaction of the receptor with a nuclease may be indicated byan indicator molecule coupled to the receptor or the polymericresin. In one embodiment, the indicator may be a chromophore or afluorophore.

In one embodiment, a polynucleotide containing the nuclease bindingsequence is immobilized through a covalent or strong non-covalentbond to an addressable site on a sensor array. In one embodiment,this may be accomplished by coupling or synthesizing thepolynucleotide on a polymeric resin, as described above. Thepolymeric resin may be positioned in a cavity of a sensor array,such as the sensor arrays described above. In some embodiments,different polynucleotides containing different cleavage sequencesfor the various nucleases may be immobilized at different arraypositions. A sample containing one or more nucleases may be appliedto the array, and polynucleotide cleavage may occur at specificarray addresses, depending on the presence of particular nucleases.Alternatively, different polynucleotides containing differentcleavage sequences may be coupled to a single polymeric bead. Inthis manner, a single bead may be used to analyze multiplenucleases.

A variety of signaling mechanisms for the above described cleavagereactions may be used. In an embodiment, a fluorescent dye and afluorescence quencher may be coupled to the polynucleotide onopposite sides of the cleavage site. The fluorescent dye and thefluorescence quencher may be positioned within the Forster energytransfer radius. Before cleavage, little or no fluorescence may begenerated by virtue of the molecular quencher. After cleavage, thedye and quencher are no longer maintained in proximity of oneanother, and fluorescence may be detected (FIG. 62A).

The fluorophores may be linked to the polynucleotide receptor byany of a number of means well known to those of skill in the art.Examples of methods of attaching fluorophores and dyes topolynucleotides are described in U.S. Pat. Nos. 4,855,225;5,188,934, and 5,366,860 all of which are incorporated herein byreference.

In another embodiment, a first fluorescent dye and a secondfluorescent dye may be coupled to the polynucleotide receptor onopposite sides of the cleavage site. Before cleavage, a FRET(fluorescence resonance energy transfer) signal may be observed asa long wavelength emission. After cleavage, the change in therelative positions of the two dyes may cause a loss of the FRETsignal and an increase in fluorescence from the shorter-wavelengthdye (FIG. 62B).

In another embodiment, a single fluorescent dye may be coupled tothe polynucleotide receptor on the opposite side of the cleavagesite to the polymeric resin. Before cleavage, the dye isfluorescent, but is spatially confined to the attachment site.After cleavage, the nucleic acid fragment containing the dye maydiffuse from the attachment site (e.g., to positions elsewhere inthe cavity) where it may be measured with a spatially sensitivedetection approach, such as confocal microscopy (FIG. 62C).Alternatively, the solution in the cavities may be flushed from thesystem. A reduction in the fluorescence of the particle wouldindicate the presence of the analyte (e.g., a nuclease).

In another embodiment, depicted in FIG. 62D, a single indicator(e.g., a chromophore or a fluorophore) may be coupled to thepolynucleotide receptor on the side of the cleavage site thatremains on the polymeric resin or to the polymeric resin at alocation proximate to the polynucleotide receptor. Before cleavagethe indicator may produce a signal that reflects themicroevironment determined by the interaction of the receptor withthe indicator. Hydrogen bonding or ionic substituents on theindicator involved in analyte binding have the capacity to changethe electron density and/or rigidity of the indicator, therebychanging observable spectroscopic properties such as fluorescencequantum yield, maximum excitation wavelength, or maximum emissionwavelength for fluorophores or absorption spectra for chromophores.When the polynucleotide receptor is cleaved, the local pH anddielectric constants of the beads change, and the indicator mayrespond in a predictable fashion. An advantage to this approach isthat it does not require the dissociation of a preloadedfluorescent ligand (limited in response time by k.sub.off).Furthermore, several different indicators may be used with the samereceptor. Different beads may have the same receptors but differentindicators, allowing for multiple testing for the presence ofnucleases. Alternatively, a single polymeric resin may includemultiple dyes along with a single receptor. The interaction of eachof these dyes with the receptor may be monitored to determine thepresence of the analyte.

In another embodiment, polynucleotide receptors may be used todetermine the presence of other types of analytes. It someinstances, polynucleotide receptors will bind to small organicmolecules. These small organic molecules may disrupt the action ofnucleases upon the polynucleotide receptor. Typically, the smallmolecules will occupy the preferred binding site of the nuclease,inhibiting the action of the nuclease on the polynucleotide. Thusthe presence of a small organic molecule, which is known to bind toa specific polynucleotide, may be detected by the observation ofreduced nuclease activity at the specific polynucleotide. Ananalogous methodology may be applied to a peptide-proteasereaction.

In another embodiment, oligosaccharides may also be used todetermine the presence of analytes. In a system similar to thosedescribed above for peptides and polynucleotides, oligosaccharidesmay be coupled to a polymeric resin. In the presence ofoligosaccharide cleaving agents (e.g., enzymes such as amylase, anenzyme that cleaves a long saccharide polymer and disaccharidecleaving enzymes such as invertase, .beta.-galactosidase, andlactase, to name a few) the oligosaccharide may be cleaved. Thecleavage of the oligosaccharide may be used to generate a signal.Methods for synthesizing and/or attaching oligosaccharides to apolymeric resin are described, for example, in U.S. Pat. Nos.5,278,303 and 5,616,698 which are incorporated herein byreference.

In another embodiment, an analyte may cause a change to abiopolymer, but not necessarily cleavage of the biopolymer, whenthe analyte interacts with the biopolymer. The induced change maycause a detectable signal to be generated. Typically, the bindingor association ability of an indicator molecule with a biopolymeris dependent upon the structure of the biopolymer. If the structureof the biopolymer is altered, the association of an indicatormolecule may be significantly altered. Such a change may beaccompanied by a change in the signal produced by the indicator.For biopolymers many different types of enzymes may induce avariety of structural changes to the biopolymer which may alter thebinding site of an associated indicator molecule. Such changes mayoccur without cleavage of the biopolymer.

Alternatively, an indicator and a biopolymer may be coupled to apolymeric bead. The biopolymer may undergo a chemical reaction inthe presence of an analyte. This chemical reaction may also inducea change in the chemical structure of the indicator. The change inthe chemical structure of the indicator may lead to a detectablechange in the optical properties of the particle, signaling thepresence of the analyte.

In one example, NAD and glucose may be coupled to a polymeric bead.This system may be used to detect the presence of an carbohydratemodifying enzyme. For example, the system may be used to detect thepresence of glucose dehydrogenase. In the presence of glucosedehydrogenase, glucose may be consumed, and in the process wouldconvert the coupled NAD into NADH. NADH has both different UVabsorbance and different fluorescence properties from NAD. Thesedifferences may be used to signal the presence of glucosedehydrogenase in a fluid sample. Many other types of enzymes may bedetected in a similar manner.

In an example, the protease trypsin was analyzed using animmobilized "sacrificial receptor" that is cleaved by trypsin, anevent that results in modulation of a fluorescence signal. In anembodiment of a protease assay, a peptide that may be cleavedbetween two amino acids by the enzyme trypsin was immobilized. Thisimmobilization was accomplished by first conjugating manystreptavidin molecules to aldehyde-activated 6% agarose beads usinga reductive amination procedure. A biotin chemical group attachedto the amino-terminus of the peptide was strongly bound by theimmobilized streptavidin molecules, thereby immobilizing thepeptide chains. A fluorescein group was attached to thecarboxyl-terminus of the peptide, thereby making the bead highlyfluorescent. Importantly, the immobilized peptide contains acleavage site recognized by trypsin between the biotin attachmentsite and the fluorescein, so that exposure of the bead to trypsinanalyte causes release of fluorescent peptide fragments from thebead. This release may be visualized either as a decrease in thefluorescence at the bead, or by an increase in the fluorescence ofthe surrounding solution (see FIG. 63).

Further modifications and alternative embodiments of variousaspects of the invention will be apparent to those skilled in theart in view of this description. Accordingly, this description isto be construed as illustrative only and is for the purpose ofteaching those skilled in the art the general manner of carryingout the invention. It is to be understood that the forms of theinvention shown and described herein are to be taken as thepresently preferred embodiments. Elements and materials may besubstituted for those illustrated and described herein, parts andprocesses may be reversed, and certain features of the inventionmay be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description ofthe invention. Changes may be made in the elements described hereinwithout departing from the spirit and scope of the invention asdescribed in the following claims.

* * * * *