AU2002306207B2 - Apparatus for performing assays at reaction sites - Google Patents

Description

AUSTRALIA

Patents Act 1990 ALEXION PHARMACEUTICALS, INC.

COMPLETE SPECIFICATION STANDARD PATENT

DIVISIONAL

Invention Title: Apparatus for performing assays at reaction sites The following statement is a full description of this invention including the best method of performing it known to us:ja 2B 1 P APP ARATUS;FOR' PERFORMINGASSAYS AT REACTION

SITES

C:t b2 U OF T-HEINVENTION S-1. Field of the Invention 2 1X The invention generally relates to' apparatus'for performing assays, such .as chemical assaysand biochemical reactiobis, or the'like, at reaction sitesbh a siisirate.

Sin paicular.the invention relates to apparatu's fdr perfoiffiing assiy, siuch sh'mica assaysand!biocheflcal reactioisby. delivering'a selected aliquiot br seleted 'ailiquots to a reactioni,site or sites -on a substrate that inainclude >plurality' of' layirs of semiconductor material rk. Description of the Relate&~rt Until the relatively recent advent of combinatorial chemistry and'geiietic research spawnedthe need'for high-throughput ahalyzing and screening techniques, researchers perfdrmed such assaysusing vials, tubes, and beakers. Howe&er; with ever more.substances available via synthesis or via combinatorial techniques'fdr testing, the need has arisen to test the possible role of thousands-or- even nillions f substances, in comparable numbers of possible reactions.,. Miiiiatiizition has been identified as a promising'pathto more efficient, g less expensive$ chemical, and in partidular,' drug, analysis and screening. Discussions of various aspects of such analysis and screening techniques are found in J.D. Devlin, ed., High hrounhut Screeiinr: The Discovery of Bioactive Substances (Marcel Dekker, Inc., New York, 1997); which is incorporated herein by reference to more fully describe the state of the art to which the present invention pertains.

Miniaturization apparatus may be broadly classified into at least two categories. A first category involves the placement of chemical substances in mall amounts in sites formed on glass or a similar substrate. Micro-chemistry includes processes carried out in small volumes, e, between nanoliter and microliter aliquots, whereby reaction times may be shortened significantly over those conducted in reaction vessels holding on the order a fraction of a milliliter, as currently achievable by a lab technician working "by hand." In addition to microchenical testing, levels of gene expression may be tested on a large scale.

An example of this first category is the development of microplate technology in which a glass substrate may include site densities of about 10,000 sites.

This technology may include the use of complex micro-robotics or the adaptation of inkjet technology to apply chemical and biochemical substances to chosen sites on the substrates. Frequently, at least one of the reactants in a chemical assay to be performed is chemically linked to or otherwise immobilized at the reaction site. This is done, so that fluids may be added to and removed from the reaction site without removing at least one intermediate or end product of the reaction, which intermediate or end product(s) is (are) to be retained at the reaction site, so that the outcome of the chemical assay may thereby be detected.

Orchid Biocomputer ("Orchid") of Princeton, New Jersey, USA, has indicated that it plans to create a credit-card sized glass chip with 10,368 sites. See M.

Leach, Update: Discovey on a Credit Card?, DRUG DISCOVERY TODAY, 253-4 (Vol.

2, No. 7 (July 1997). For example, each site may cover an area of 00;m 2 and may contain less than I .l in volume. The chip is a glass sandwich formed from individual chip layers, which then may be glued together to form tubes to move substances between sites. Such tubes are formed in this device by cutting, etching, trenches or grooves in a first layer and then sandwiching the trenches under a second layer.

A second category of miniaturization apparatus employs silicon in some fUnctional, electrical or mechanical, modality as the substrate, and chemicals then are tested on the substrate. In some cases, micro-robotics or micro-chemistry, or both, may be employed with such substrates. For example, Orchid's chip may employ microfluidic pumps, electronic pumps having no moving pans, to move substances between sites. Nanogen, Inc. also has developed a microelectronic device for handling low-dilutions of charged molecules. However, unlike Orchid, which may use 3 electrokinetic pressure .pumping,-heNanogRen-device .employs electrophoresis:as a motive agent to. analyze chemical .reactiops actingover.the. surface .of the 'silicon substrate at about twenty-five reaction sites. Electrokinetic pre§spuwiunpirl hias. been described as a combination of electroporesis and-electro-smosis!T Other research has addressed-developig,productswhich-lemployin-place silicon substrates or devices for chemical testing4hat ipcludeeither electrical oik;micromechanical technlogies, or, both.. for exampe,: Syntejqninc.has.developed a process which simultaneously measures the expression of thousands'of genes using.microscopic cDNA portions placed on a substrate, ,Synteni-alsothas developed.a companion 'reader that uses two-color fluorescence hybridizationdetectioP; Genometrix; Inc: also employs a fluorescence analysis technique that appars sinilarnin :concept to the .Synteni's process, but carries out the reactionson miniature,scale;ri:e., onafilm that:eventuilly fits over the surface ofa reaction reader.. Such a readeris manufactured from'a silicon A J~ '1 ''ttI chip or wafer modified to function as a potodetector, such as a dharge-couipled device

(CCD).

Fluorescence generated. on,,the film produces a:.photocurrent,"which provides an electrical charge to a CCP.site, andwhich subsequently may be gated out hF'i "hi. for analysisip a manner analogous to the function of.a CCD detectorarray in a digital camera. Thus, known digitizing technology may,be cobined, with the placexhent of an arrays ofchemicals on the surface of a plastic film. The plastic reaction. arrayfilm may be fitted over the surface of silicon chip or wafer that acts.as the reader and when ultraviolet light is flooded over the film surface,.fluorescenceis elicited from.the chemical reaction sites. Each reaction site on the film is. aligned with an analyzing site on the reader, and, therefore, a coordinate on the reader corresponds to a reaction site in the chemical array.

Nevertheless, previous attempts to achieve high-throughput analyzing and screening techniques for chemical reactions have required complex operations using combinations of films and substrates or complex robotics for the precise placement of fluids carrying chemical compositions, or both. Such complex systems are-subject to failure due to the failure of any system component. Further, such complex systems, especially those including robotics, are expensive to manufacture and maintain.

SUMMARY OF THE INVETIOT-TN Thus, a need has arisen for an efficient, simple to operate, and relatively low cost apparatus for performing a high throughput of chemical assays and biological reactions at reaction sites on a substrate.

A further need has arisen for apparatus which allows high-throughput analyzing and screening techniques for chemical assays of biochemical reactions within an aliquot or between aliquots to be performed at discrete reaction sites, Further, it is a feature of the invention that the delivery of aliquot(s) may be accurately and automatically controlled and monitored, by a rotatable substrate and a movable fluid dispenser. It is a technical advantage of the invention that etch geometry may be used to form reaction sites which may have added advantages in that they may reduce evaporation and aid in the retention of a portion of the fluid.

Yet a further need has arisen for easily assembled and simply and accurately controlled apparatus for delivering an aliquot or aliquots to reaction sites in order to perform chemical or biochemical testing or both. It is a feature of the invention that the apparatus achieves a high degree of accuracy in the delivery of fluids to reaction sites. It is a technical advantage of the apparatus that it may employ prepackaged engines or motors, such as linear and rotary stepper motors, to move and position at least one fluid dispenser outlet over a reaction site. Such stepper motors provide a high degree of accuracy and repeatability of movement. Such stepper motors also permit the use of an integrated control system with electronic damping and an integrated indexing system. Moreover, the control systems for such stepper motors may readily be 2S customized to provide for variable speed and continuous speed operation.

Still a further need has arisen for an apparatus which aligns at least one fluid dispenser outlet with at least on reaction site without the use of complex robotics.

It is a technical advantage that the linear stepper motor(s) move(s) the fluid dispenser outlet(s) in one dimension along at least one rail, and that the rotary stepper motor(s) rtts)the substrateabound, an axis. -It is a fuirther4echnical advantage of th~iasb of )naandroayse -or.I athey,mAyte ]ess .expnsv to afature, .maintain, andxreplac ;ta3 nperocts izn(m' Yet another need ha.aie for wapparatus-having amunilti-fhrni ibri head comprising.at least one fluid Aispepe~{keieig~fti~rfud -tob~ d-i'l .oneof~ ap uralitygfqfyacton sites andat- east -one. readout _devi6-EtL ,,'%Jft .The readout devjce( s) mayste~ puaiyxffi~in nldn ,monitorng the progresp~f as~ays, scanning thexreaction. site(s) to- determiife tHFlsults of assays,loca ngareaction 2 ,it9 or, sites~by readinga-.Iocatiiig. mark;:' an-d-uidingdP~east one ese ueto~a, recinste~or-.stes-by means 'of' a trkdkingg~mark?-iIi'is a technical advantage of the multi-function head that the operation and constiuciidi dfthe ~~apratus is simplifie by_3lhegombination'of~multiplC ftmctidns.oF a single movable head. It is a ftirther technical-advantagesfthemulti-fiiunction head-.that'a:- inglett 6 t6ol ma yposition both theat, lea$ ~oneifluid -dispensir aidmte'at Ieas-foii?&kabout device, jhereby, eliminating alignment differen~ces between-these components %I f-is tet .aoerechnical advantage~ fhs mul4ti-function rhead that; rapidly!'or instantahfid &usly occurring assays m X h,1e 4 n 9 tpredc, imrnedi ately .after,-initiation, 'and "monitored' ufitil completion. .It.Jis still,,anpther techical advantage -of thr apparatus~'that- a micrqpO5itionrsc,,5sl3y,tpree-aiszflicrOp~OSitioneT, -may be. controlled- to 'make adjustments, L.g 2 ,O jLtrenta153 range.of less thanwabou l5itm withan accuratty of about one micron, aoqng Cartein. axes in the position of the at Ileastv one fluid dispenser outlet andthe readoutdevice.-j, iIt)* The invention is an apparatus for performing-a plurality of assays, such as a plurality of chemical assays or a plurality of biochemical reactions comprising an axially -rotatable substrate including a plurality of radially-arrayed reaction sites.

7 -Other assays include cellular assays.. as well as physical and biophysical assays 4

M.

chemiluminescence luminescence, dielectric field strength, resistivity, impedance, circular dichroism, refractivity, surface plasmon resonance, optical- absorbance, magnetic resonance, and the like. Assay components may include, for example, synthetic organic compounds (R.jL, compounds of less than 1001000 molecular weight, preferably compounds of less than 10,000 molecular weight, more preferably compounds of less than 1,000 molecular weight) proteins (egenzymes, amnyloid proteins, receptors, cytokines, and antibodies) peptides, oligopeptid-es, nucleic acids (including modified synthetic derivatives thereof, DNA, RNA oligonucleotide and monomeric nucleotides, nucleosides, modified synthetic variants thereof, and the like) cells (abacterial cells; yeast or other flumgal cells; unicellular organisms such as protozoans; animal cells including insect, avian, and mammalian cells; and plant cells) cell membranes and other cellular components, buffers, salts, ions such as metal ions, lipids, carbohydrates, vitamrins, extracellular matrixes or components thereof, as well as blood serum, or other bodily fluids.

The substrate may be manufactured from glass, ceramics, semiconductor materials, plastics, composites, and combinations thereof Semiconductor materials are solid crystalline materials whose electrical conductivity is intermediate between that of a conductor and an insulator, ranging from 1 05 mhos and 10"4 mho per meter and is usually strongly temperature dependant. Semiconductor materials may include silicon, germanium, and gray tin. For example, the substrate may include a plurality of layers of semiconductor material which may partially or completely cover the surface of the substrate. Alternatively, the plurality of layers may lie beneath the surface of the substrate and extend for a portion or for the entire area of the substrate.

The apparatus ifurther comprises means for rotating and controlling the rotation of the substrate and at least one fluid dispenser for conveying at least one fluid to at least one of the reaction sites. The means for rotating may comprise a engine, such as an air driven turbine, or a motor. Each of such fluid dispensers includes a fluid dispenser outlet. In addition, the apparatus includes means for identify~ing the at least one reaction site, and means for aligning the at least one fluid dispenser outlet with the at least one reaction site.

In particular, the apparatus may comprise at least one multi-function head, such as a dual function head, including at least one fluid dispenser for conveying at least one fluid to at least one of the reaction sites and at least:6ne:readout device:. The readout.device mayjncludemeans for :locating.a.reaction site, suchtas the iheans -for identifying a location.mark, and for monitoring the chemical or'biochemical reactions at the reaction sites. .Each of such fluid dispensersdincludesaifluid dispenser outlet. 'Thus, the fluidics and locating and monitoring, functions ofthe apparafus may be'combihed in a multi-finction.head. *4 The operation of,stepper motors is.known-inthe artt For.examnile, such motors are used in computer disk drives., Generally; astepper motor rotates in short, essentially uniform regular movements. The stepped movements are;obtained iby means T P

I

of electromagnetc ,controls..t Although-the:.apparatusr-maysrinclude: arotary -stepper Itd I t 1 t i f n J.L motor, the means for rotating also may rotate the substrate i.atan: adjustable' or substantially continuous, speed,.or both, and-may control the rotationiof the substrate by adjusting the speed and adirection-of rotation. .Further the means .for rotatiii is controllable to rotate the substrate ata speed, such that a:portnion:ofthe at.least'one fluid 'T;7 cii fj3 115a c: ia, tt:jlr is.removable from the at least one.reaction site -by a.centnifugalbforce igenerated by:the rotation of the substrate. Moreover, at,least one -channel may join the at leastrone reaction site.to at least one other reaction site. The meansfor rotating further maylbe controllable to rotate the substrate at a speed, such that-the at leastone fliidis: diiwn .from the at least one reactionsite,through the at least one channel to the atleast'one other reaction site by a.centrifigal force.generated-by.the rotation'of the substrates ,J i ''a The fluid(s) delivered to-the reaction,site may comprise at least'a first amount of at least one fluid aliquot ;and.at least.a, second amountrof at least 'one separating fluied,.., a solvent, oil, air, immiscible fluid ,or the like. For.example,r the first amount of at least one. fluid aliquotmay b.subSantialyIidentical-to the second amount of at least on separating fluid. In another embodiment, however, the first amounts of the at least one fluid aliquot may be substantially identical-to each-other while the second amounts of the at leastvone separately fluid are of a different amount and are substantially identical to each other.. For example, an oil or air may be a preferred separating fluid for water-based aliquots. Further, the at least one fluid dispenser may include one or more pumps, suction devices, and timing devices for controlling the pump(s) or the suction device(s), or both. The pump(s) may include conduits and valves, whereby the pump(s) may alternately draw at least one of the first amount, Lg,, in a range of about 0.0001 to 5 ul, and preferable about 3 to 5 ul, of the at least one fluid aliquot and at least one of the second amount of the at least one separating fluid into the dispenser tube and delivers an alternating stream of the at least one aliquot and the at least one separating fluid to the at least one fluid dispenser outlet under a controlled pressure differential relative to the ambient pressure surrounding the fluid dispenser outlet(s). The timing device(s) then may control the operation of the suction device(s), such that the suction device(s) may draw off the stream from the fluid dispenser outlet(s).

Specifically, the timing device(s) may measure a flow rate of the stream through the dispenser tube and deactivate and subsequently reactivate the suction device(s), such that at least one first amount of the at least one aliquot is delivered to the reaction site. The suction device(s) may create a suction pressure less than the ambient pressure surrounding the dispenser outlet(s), a vacuum sufficient to remove fluid from the dispenser outlet(s). Alternatively, a plurality of suction devices may create different degrees of pressure differential across the orifices of such suction devices, different levels of vacuum, with respect to the ambient pressure surrounding the dispenser outlet(s). In still another alternative, a library of tubes may be provided, each tube having a predetermined amount of a chemical or solution for use in performing a chemical assay or causing biological reaction. A desired amount of the chemical solution may then be drawn or pumped from the tube and deposited at a reaction site or reaction sites. The unused portion of the chemical or solution may be discarded or recovered for recycling or reuse, or the tube also may be discarded or refilled, sealed, and reused.

Other means for dispensing or removing fluids at reaction sites also may be used in accordance with the invention. See, L, D.W. Brandt, Multiplexed Nanoliter Transfers for High Throughput Drug Screening Using the BIOMEK 2000 and the High Densi, Replicating Tool, J. BIOMOLECULAR SCREENING 2:111-116 (1997); which is incorporated herein by, reference to more fully describe the -state of the&'irt -t vo hich' thEprent invention pertains. El.f

.MI.

;7T a J he, dispenser .outlet(s):may -berombvabl9 inotihtedhii aiilwkh transects the substrate and is oriented:substantially:parallel tota'sirface of he sbsfrate, si s suspended over the substrate, andJafiistniotor .may bitised tb'r'tite the substrate..The means for.aligning comprises;a second motor-forjpositionihg hihit least one fluid;dispqnseroutlet along the rail. Moreover,.as noted abovet firs motor iay be acrotary stepper motor rand the-second,motor may* 'be a 'lineastippe motor-fi8 n addition, the means for aligning-may comprise a:corriputer (including micoprocessor or other electronic device) which receives, processes, and preserits datandfii .wUhi6h stores a startlocation on the substrate's surface for thidipene outi The computer and additional funmtionally linked electronics including; for-example, asigfial gEnefratbr such as an .electromagnetic energys source and a icalibrating 'sensdr;' ich' 1 ian electromagnetic energy sensor, may provide i mboement sighals tothe fist indec'nd motor. Thus, the computer and the additional electronics'generatesignalkto'alighe ie d espnseroutlet over the reaction site. Alternatively, the fluid dispee dutlet(s)hay bemounted pn a pivotable, arm.which-may be rotated through an arc across the-sirfate of, .over, the rotating substrate. In this erhbodiment, thelsciond 6ibtdr-ihay alI&be a rotary steppr motor.

C.

In addition, the-apparatus may position'the multi-functionhead byineans .of a two step process. First, the apparatus may direct the head to.the general vicinity of a selected reaction site. Second, the multi-function head may utilize the rmeans for identifying to interrogate or read the locating marks to identify the selected reaction site and to align the dispenser with-that reaction site.

The means for identifying may-include at least one sensor. This sensor may be positioned in the same manner as the fluid dispenser outlet, it may be joined to a linear. stepper motor which is mounted on a rail above the substrate. Preferably, the sensor is incorporated into the head. This at least.one sensor may receive a signal emanating from the substrate, or the at least one sensor may transmit an -interrogating signal and receive a locating signal in response. Further, the at least one sensor may read at least one locating mark, an indexing mark, a tracking mark, a bar code, or combinations thereof, on the substrate's surface. Examples of the locating mark are discussed below. Se Fig. 7. However, as noted above the locating mark may consist of an indexing mark, which identifies the particular reaction site, and at least one tracking mark which helps the means for aligning to guide the multi-function head and its associated fluid dispenser(s) and readout device(s) over the reaction site. In particular, the tracking mark may be recognized and help guide the head to the reaction site by its size or shape or by its physical relationship to, distance from or direction to the reaction site.

In another embodiment of the invention, the apparatus for performing a plurality of assays again comprises an axially rotatable substrate including a plurality of radially, arrayed reaction sites; means for rotating the substrate; and at least one multifiumction head including at least one fluid dispenser for conveying at least one fluid to at least one of the reaction sites and at least one readout device. The at least one fluid dispenser also may include at least one fluid dispenser outlet. The apparatus also may include means for identifying the at least one reaction site and means for aligning the at least one multi-fuinction head, such that the at least one fluid dispenser outlet is aligned with the at least one reaction site. The means for rotating may be controllable to rotate the substrate at a speed, such that a portion of the at least one fluid is removable from the at least one reaction site by a centrifugal force generated by the rotation of the substrate.

In stil another embodiment of the invention, the apparatus for performing a plurality of assays comprises an axially rotatable substrate including a plurality of radial ly-ar-rayed reaction sites; means for rotating the substrate; at least one fluid dispenser for conveying at least one fluid to at least one dispersion point, preferably located on the substrate; and means for identifying at least one of the reaction sites.

Further, the apparatus, and preferably the substrate, may include at least one channel joining the at least one dispersion point to the at least one reaction site. Alternatively, this embodiment may include at least one multi-function head including'at least one fluid dispenser for;convgyyngat least.ne fluid to at least idne dispersion point and-at least one readout device. The means for rotating may be controllable to rotate saidsuibstrite at a.speed, such that.the.at least one fluidis conveyed from the at-least one disp&fsion point to the at leastone reaction site by a centrifgal force generated by.the rotation of the i su b s trat e 7 U 0 .1 W7 t t *Q 'I :ri In yet another embodiment of the invention, the apparatus for perforriiing a lurality of assays compses an axially rotatable substrate including a plurality of radialy-arrayed reaction sites;,at.leastone fluid dispenserifor convyeying at least one;fluid to the substrate; means for identifying at least one of the reaction sites;.and meansfor rotating the substrate., For exmple, the at. least.one fluid,dispenserj.may,convey at least one fluid to the substrate througha spindle -aroundwhich the substrate,rotates:, tjThe .tL;z c' *"LilsF;, t~ U t~ *means for rotating is con t rollable to rotate the substrate at a speed,such that the at least one fluid is drawn across the reaction sites X4? centrifugal force generated:bythe rotation of the substrate. It2O .W NW J k- f. T .1 Other features and technical advantages wjll be apparent.o persns skilled in the relevant art in view of the following detaileddescription and accompanying drawings. .tt" WI 3. t 0r$ K BRIEF DESCRIPTION OF THE DRAWINGS For a more complete understanding of the present, invention and the U\ aii.B'.technical advantages thereof, reference now is made to the following description,taken in conjunction with the accompanying drawings,, in which like reference numerals represent like referenced parts, wherein: Fig. 1 depicts a perspective view of a first embodiment of the apparatus of the present invention;, Fig.. 2A depicts a perspective view ofa fluid dispenser with a fluid dispenser outlet mounted on a linear stepper motor, and Figs. 2B and 2C depict two embodiments of a delivery mechanism for controlling the delivery of fluids through the fluid dispenser Ot. to h t s outlet to the reaction sites; Fig. 3 depicts a perspective view of a second embodiment of the apparatus of the present invention including a fluid dispenser mounted on a pivotable arm and a calibrating device; Fig. 4A is a schematic view of another embodiment of the fluid dispenser, Fig.

4B is an enlarged view of a fluid inlet tube 20' illustrating a fluid including alternating amounts of aliquot and of a separating fluid, and Fig. 4C is an enlarged view of an aliquot in a fluid inlet tube Fig. 5 depicts a perspective view of a third embodiment of the apparatus of the present invention including a multi-function head comprising a fluid dispenser and a readout device.

Fig. 6 is a bottom view of readout device depicting the configuration of the readout device including the arrangement of readout and tracking optical fibers.

Fig. 7 depicts a locating mark combining tracking and indexing marks for guiding the multi-function head to the reaction site.

Fig. SA depicts a perspective view of a reaction site on a substrate, and Fig. 8B depicts an overhead view of the reaction site of Fig. SA; Fig. 9A depicts a perspective view of a substrate, and Figs. 9B-9D depict overhead views of solid angle sectors of the substrate containing embodiments of reaction sites; Fig. 10 depicts an overhead view of a pair of reactions sites joined by a channel and a microfluidic device; Fig. 11 is a graph of the laser detection scanning of a droplet of Rhodamine 610 solution deposited on a silicon substrate; Fig. 12A is a graph of the forward laser size scanning of a droplet of Rhodamine 610 solution deposited on a silicon substrate, and Fig. 12B is a graph of the reverse laser size scanning of a droplet of Rhodamine 610 solution deposited on a silicon substrate; Fig. 13 is a graph of the showing the detectable volume of a droplet of Rhodamine 610 solution deposited on a silicon substrate and the effects of evaporation on the droplet volume over time; .13 i -Yr- Fig. 14 is a graph showing the ability of the laser readout device:to read a 3-bit code, and r M; 711- Fig. 15 is a schematicdia: gr Odepicting:theapparatus.rw, c'x et0 S I> DETAILED DESCRIPTION OF PREFERRED EMBOD1MENTSz- ap.

4 Referring to ig. -aperspective viewtof a first embodimentof the apparatus of the present invention is depicted.. An apparatus l:fordelivering flibiditoa reaction site,includes a substrate 10 such as a silicon wafer,:mounted: on a platformi. 2, which includes a rotary stepper motor.(not shown), such as a Zeta 5751-10 Motor arid whichinclues a otar r, a a Zeta,4 Rotary Driver manufacturedby ,arker Compumotor:Coinpanyiof Rdhriert Park, Califomia, U.S.A. The rotary stepper. motor.rotates:substrateil 0inithediredtions T~F t~ife. t. .,.bisects,.substratel% .7A of arrow A. A rail 14 is suspended above and transects,:L,.bisects,.substratelO.

A

linear stepper motor 16, such as .a.,L20, StepeMotor;:manufactured byParker v 4 0Swppr votor, Compumnotor Company of Rohnert Park, California, U.S.A.;is mounted oh railhl4% i kh that linear stepper motor 16 is movable-in one dimension in the;directibns of arrdw B along rail 14 over.the surface of substrate 10. n:i SThe rota stepper motor. is controllable. to rotate substrate 10 at a plurality of variable and continuous speeds in eitherdirection of arrow:A.. For example, an AT6200 Controller, manufactured by Parker.Compumotopr. Company of Rohnet 1.

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Park, California, may be used to control the.operation of-the, rotary stepper motor. Further, the rotary stepper motor.my beoperated to rotate -substratet0 ri7f1

I

continuously in a manner similar to that of the disk in a compact,disc player, so that a centrifugal force is generated on substrate 10, or incrementally, so tatsubstrate be moved a less than one revolution with respect to the positionof linear stepper motor 16.

Substrate 10, platform 12, rail 14, and linear stepper motor 16 may be enclosed within a container 18. Container 18 allows the atmosphere surrounding the reaction sites to be strictly monitored and controlled during testing. Container 18 may be airtight to prevent dust and moisture from settling on and effecting the operation of linear stepper motor 16 or the rotary stepper motor and to permit the maintenance of positive Or negative pressure within container 1 8. Moreover, dust and Moisture may adversely effect the chemi~cal or biochemical reactions, or both, occurring at the reaction sites and alter the outcomes of chemical and biochemical tests. For example, container 18 may be equipped with a humidity and temperature sensor 19, so that the level of and changes in humidity and temperature may be detected. When low or high relative humidity or temperature, or both, is (are) detected, assaying operations may be terminated, or the atmospheric conditions within container I 8 may be corrected.

Changes in humidity or temperature, or both, may cause evaporation of all or a portion of the deposited fluid or dilution of the fluid with condensation. In addition, the container environment may be controlled by establishing a vacuum, increasing air pressure, regulating environmental temperature, or establishing a predetermined container atmosphere, such as a nitrogen atmosphere, an oxygen-rich atmosphere, a nobel gas (inert) atmosphere, or a combination thereof Further, because of the importance of delivering precise amounts of fluid to small reaction sites, container 18 also serves to reduce or eliminate air disturbances at or near the surface of substrate Fig. 2A depicts a perspective view of a fluid dispenser 2, which may include liear stepper motor 16 and which may be mounted on rail 14. Referring to Fig.

2A, a fluid stream comprising a serialization of measured amounts of aliquot(s) and measured amounts of separating fluid(s) is delivered to fluid dispenser 2 through a fluid input/return tube 20. Fluid dispenser 2 includes a dispenser platform 15 that Is suspended below linear stepper motor 16 by four dispenser platform supports .17.

Components relating to the aligning of a dispenser outlet 21 over a reaction site on substrate 10 also may be mounted and stabilized on platform 15. Preferably, the length of fluid dispenser outlet 21 is minimized in order to reduce or eliminate dead space.

From fluid input/return tube 20, the fluid is received by a fluid input tube and delivered to fluid dispenser outlet 2 1. Fluid, which is not delivered to dispenser outlet 21, is returned to fluid input/return tube 20 through a fluid return tube Further, fluid that is delivered to dispenser outlet 21, but which is not delivered to substrate 10 may be withdrawn through suction return tube 22. A suction device (not fshown) draws undelivered fluid from-. di sjensersout1~t 2Th 'J h .,ressure differential acrossjhe-orifice dtsuctiohi retum'tube'22 ,-LichthtiJih' ressure in suction, returntub>%4 2 is-tessltnntfle' amolent.preS~usu Urr'i t djn-gtri'' e a acumsfficient to remove fluid exirin' iph& u .,Liter stepp~prjpotorJ 6:,receive'slmovemensighfrt Iiomaniiil Iopter(no ow) throughpa computer conne~tion;(hot Ishowh)' sitiilii-rl&ting 4AWvie2,Se' an-epmrnc timing, devicem fay ineasur6 flo~w. ate&an'drdete-r-miti- the amount of fluid delivered from dispenser outlet;2-11shy nieaxwn'lS)bW~l 4 Rs pitionedadicto senser outlet. Fornexarnple,.floW'§sr 2 a dtc the.rintrruiptionor.,diminlution -of an electrorhignet ic, energyba iI'isef 6tbi~h intensity lgt: beam., directe4~across the orilk~e df dise n ser ou tlet 2 Uh.- I6Wse'ns s ts2 ,,beonnected toj inng. deie23by a timingtconnectiofl lint 24i 'F~t in nig di overa tin s Ig nal1 :retuiirn I lie 2 4,A fa i ~arthtfi6sfeirihe to .a comptt (not ,-,ho~ri)_to.,activate -anddeactivate 'h6 sUcti6An T&ic'e(Hbt shop nt~ threy rcp l. the- amount, of fluid -delivered to F ubstrate1 oIbthr6tigh dispenser..outlet 21 I. I 'V

C

n addition. a fluddispenser 2.may; inclu dea aidetection-mechiti, 7 these, comp9qentsmay. M~mnearulti-fuincti on, head; to-d&rinine \vhether-iiid:_o what extent a chemical reaction has occurred at-a reaction'.site-: A. light source -(not hcyn lghtofudisns2thcugh a' light source input ~27 to a light input optical fiber Light inpyt opticalfiber 27W' is~fixed adjadentfto ,dispenser outlet 21,.so thathigh intensity fighr-may. be directed'onito at'least onetfeatioin site of substrate 10. The amount and natuire of the ight,,reflected from the: reacti o 'site ma~y9ndicate the occurrence and progress of the reaction. Such' reflecttd light'may be c ,ollected. by aligh tqrceiving qpticaJ -fiber ?28, as.eceivipg end of -which alsormay be positioned adjacent to dispenser outlet 21. Further, Ilightfrom-light input opiical fiber 2'may stimulate fluorescence at the at least one reaction site;.and such fluorescence may by detected by light receiving optical fiber,28. Light received by light riceiving optical fiber,28. is conducted to a photomultip'lier tube 29 that includes a band pass.filter and generates an electrical signal describing the nature and amount of the light reflected from the at least one reaction site. Such a photomultiplier tube may also be a photodiode array. Photo-diode arrays are known light detecting elements of a charge coupled device (CCD). Alternatively, a source of electromagnetic energy may be supplied to detection mechanism and directed onto at least one reaction site of substrate 10. Again, the amount. and nature of the reflected electromagnetic energy may indicate the occurrence and progress of the reaction. This signal then is returned to a computer (not shown) for analysis over signal transfer line 120.

Additionally, at least one sensor may be mounted on fluid dispenser 2 in 1 0 order to identifiy reaction sites or to aid in determining a start location for dispenser outlet 21 wi'th respect to the surface of substrate 10, or both. In particular, the at least one sensor may receive a signal emanating from an emitter positioned on substrate (sEee Fig. 6C, described below), or the at least one sensor may transmit an interrogating signal to substrate 10, g. to a transponder positioned adjacent to a reaction site or 1 5 group of reaction sites, and then receive a locating signal in response. Further, the a: least one sensor may read, ije, scan, at least one locating mark, an indexing mark or a bar code, on the surface of substrate 10. Such a locating mark also may include a tracking mark to guide Fluid dispenser 2 to a reaction site. Bar codes, micro-scale bar codes, and bar code readers are known in the ant.

Figs. 2B and 2C depict two embodiments of a delivery mechanism 200 and 200', respectively, for controlling the delivery of fluids through a dispenser outlet to the reaction sites. Referring to Fig. 2B, delivery mechanism 200 includes a portion of fluid input tube 20' and a portion of fluid return tube 20" forming a U-shaped connection. Arrows F depict the flow path of the serialized fluid through the U-shaped connection. Dispenser outlet 21 extends substantially perpendicular to the fluid flow path as fluid passes from fluid input tube 20' into fluid return tube 20". A first electromechanical controller 202, such as a solenoid, controls a return side valve 204, and first controller 202 may receive control signals 206 from timing device 23. Preferably, first controller 202 and return side valve 204 are positioned sufficiently distant from 17 dispenser outlet 21, such that vibrations or. mpovement, or both, caused by the'tope tion of first controller 202 orreturn.side valve 204, or both, :do:not.effect the aligri itbf ~±yio "-aimentb dispenser outlet 21 over-at leastone reaction site on substrate 10:i, 4 nrtt t Fluid may be supplied to.fluid input tube.20.'.unider-a pressure'differethia determined relative to the ambient air pressure within-ontainer 18K Nevertheles$,ihis myil 34 -NW f7-L r pressure is sufficient to force.the.majorjty of thefluid,in fluid .inutt6hbe 20' to $-ass directly across dispenser outlet orifice,208.an into-fluid-return tube 20Z",and aily fldid entering dispenser outlet.21 may been collected by-the suction device (not'shon) :3 -nri,r~4ifl 05~3! It'~ through suction return tube 22. However.. when return,sidewalve 204is :clos&dfl6d from fluid input tube 20',flows direct lyinto dispenservoutleq21.7 Moreover, because dispenser outlet 21 may narrow toward a dispenser- outlet tip 210,the flow:velbditycof fluid leaving dispenser outlet 21 maybeeater than the flowyelocity vwithin fliid in ut o.Y For example, dispenser outlet orifice 10,:may have aidiameter'6f1es than about 10 pm and preferaby, less than.about.4 gm At awflow.rate'ofabdut2 gl/sec., a pressure differential in a range ofKaboutQ0l, 1 to 2000 psi is creatd across dispenser outlet orifice 210 and preferably, a positive pressure ina ranger to 500 psi. If a suction device is used to draw off portionsof the flow ofserialized fluid from dispenser outlet orifice 210, a similar, but jinerse, pressuredifferential is created .4 across the orifice of suction returnmtube,22 adjacent to-dispensercoutlet orifice 210sl Referring to Fig. 2C,-delivery mechanism 200' also includes a portion of fluid input tube 20' and a portion of fluid returntube 20".forming a U-shaped connection. Arrows F again depict the flow path of the serialized fluid through the'Ushaped connection. Dispenser outlet 21 :extends substantially perpendicular -to the fluid I flow path as fluid passes from fluid input tube 20' into fluid return tube 20". First controller 202 controls return side valve 204, and, first solenoid.206 again may receive control signals 206 from timing device 23. Further, a second electro-mechanical controller 212, such as a solenoid, controls an input side valve 214, and second controller 212 also may receive control signals 206 from timing device- 23. In addition, delivery mechanism 200' includes a four-way connection 216 having dispenser outlet orifice 208 providing access to dispenser outlet 21 and a dispenser fluid pump orifice 218 providing access to dispenser fluids (not shown) provided by a dispenser fluid pump 220. The dispenser fluids may be the same as the separating fluid(s) in the serialized fluid. Preferably, first and second controllers 202 and 212, return side valve 204, input side valve 214, dispenser fluid pump 220 are positioned sufficiently distant from dispenser outlet 2 1, such that vibrations or movement, or both, caused by the operation of any or all of these components does (do) not effect the alignment of dispenser outlet 21 over at least one reaction site on substrate As noted above, fluid may be supplied to fluid input tube 20' under a small pressure differential, t.g& less than about 10 psi, and preferably less than about psi, deterrntned relative to the ambient pressure within container 18. Nevertheless, this pressure is sufficient to force the majority of the fluid in fluid input tube 20' to pass directly across dispenser outlet connecti on 208 and dispenser fluid pump connection 218 and into fluid return tube 20". However, when return side valve 204 and input side valve 214 are closed and a dispenser fluid, such as a separating fluid is pumped into.

dispenser fluid pump orifice 218 by dispenser fluid pump 220, fluid from fluid input tube flows directly into dispenser outlet 2 1. Moreover, because dispenser outlet 21 may narrow toward dispenser outlet orifice 2 10, the flow velocity of fluid leaving dispenser outlet 21 may. be greater than the flow velocity within fluid input tube Essentially, delivery mechanisms 200 and 200' redirect the flow of fluid in fluid input tube 20' into dispenser outlet 21. In delivery mechanism 200, control of the amount of fluid and the number and type of aliquot(s) supplied to the various reaction sites on substrate 10 may be accomplished by the control of return side valve 204. Similarly, in delivery mechanism 200', control of the amount of fluid and the number and type of aliquot(s) supplied to the various reaction sites on substrate 10 may be accomplished by the control of return side valve 204 and input side valve 214, as well as dispenser fluid pump 220. Alternatively, or in addition to the fluid dispensing control accomplished by controlling valves 204 and 214 and pump 220, the suction device (not :19 shownl) may be used to daw.off fluid* through suctioni.returM" tibe '22 Ahd t~dj controlling the amount 2 of fluid dispqrnsed from dispenser- outlet 21 that-is-delii-eradcftb substrate ri~l~ ~Deivern. echanism. 200and200 t .may -comprise'of.chiiinels and micromechanical devices'formed, within a blockof mnteri al, subch;a semiconductor material. For exampltgrqqyesoQ,;renches mayfbe~etcfiedifitb a'blbtdk of semiconductor material and imicro-mechanical,.devices, such aswvalves 204 .and 2l'4 may -be formed integrally,.,wi h'h-grooves,-ortrdnches.c An-sec~o'dt-bldck2. 16f semiconductor material, may ,betJ oined to,,the ~etched :block-to. coV&r theg'ro'bV'esZor treches fouiimtube-like channels.-.6Ar Alternatively, jthe. fluid 1 dispene-may ploy.. ink-jet technology' td providJe measured amout another embodiment, a fluid dispenser rnay:qJec a micro-drcipletrdamn of te-aiasf one fluid from the dispensr ou~tlet, and an'eiectrostatic,.accelerator -and deflecior-fliy' direct the micro,-droplet stream ao at, least .one, ofthe reaction,"sites. Moreover, a fluid dispenser may include a micro-fluidic deyice.employing *an'zoscillating. .solenoid--for pumping fluid from a capillar tub,,or apiezoelpctric device having:a piezoelectrid-ttibe Z_ i -1 O J 4 to dispense measured aliquots separated by air,, from -capillary:-tube,, orthe'like: Because the aliquots are separated~bymar, 2 excessiveedilutjpn, of the aliquots~is Avoided.

-Such devices also may includle the nioJei: uanutJ r ~m fluid, dispenser, which is manufactured by.~ipDot, Inc. of Irvine, Califomnia,-U.S.A. .This device emplcdys an inkdot-type fluid deiiyery system. -This device mayzachieve flow ratesmupito 50, gI/sec of a fluid having a viscosity in aqrange of I to 20 centipqier-Thisrangeof flowi-ates, 44 z C'~f 1 r however, may be extended dependdent on fluid rheology., Moreover,. thisidevice may, deliver lines of fluid with volumes as low as 250 ni/cm 2 and line widths -in a range of 0!25 to 5 mm. The specifications were determined by dispenising,deionized- water with surf'actant added. This device also may deliver droplets, of fluid with volumes as low as 4 ni/droplet and dropl~et dianieters in a range of 0.25 to 5 mm. -Achievable droplet volumes and diameters are dependent upon fluid, and -fluid membrane characteristics.

Biolet Quanti3OOOTm fluid dispenser may achieve a flow repeatability of less than 1% cumulative volume variations for delivered lines and of less than 5% variation between drops.

The BioJet Quanti3000Tm fluid dispenser carries a swept volume of less than 40,p1 and is supplied by a feeding tube carrying 5,p11cm. In addition, this fluid dispenser may be equipped with a filter the fluid reagents before delivery to a reaction site. Such a filter may provide for removal of particulates with a diameter of less than microns, Moreover, the fluid dispenser may be equipped with means for de-aeraxing the fluid flow. These devices may employ an oscillating solenoid, for example, a solenoid oscillating at a rate of about 100 Hz, for pumping fluid from a capillary tube or a piezoelectric tube that is excited by frequencies up -to about 1000 Hz which surrounds a capillary tube, respectively. Such devices may achieve flow rates of less than about to 500,pllsec., and preferably, of less than about 200,p1/sec., the orifices of the capillary tubes of these devices may have a diameter in a range of about 50 to 175 pm.

Fig. 3 depicts a perspective view of a second embodiment of the apparatus of the present invention. An apparatus 3 for delivering fluid to a reaction site includes substrate 10, such as a silicon wafer, mounted on platform 12, which includes a first rotary stepper motor (not shown), such as a Zeta 57-5 1- 10 Motor and a Zeta 4 Rotary Driver. The first rotary stepper motor rotates substrate 10 in the directions of arrow A. A second rotary stepper motor 3 0, such as a Zeta 5 7-5 1 -10 Motor or a Zeta 4 Rotary Driver, is mounted on platform 12, and a pivot arm 32 is mounted on second rotary stepper motor 30. A fluid dispenser 34 is mounted on pivot arm 32, and pivot arm 32 is of sufficiently length, such that fluid dispenser 34 may be rotated through an arc in two dimensions by the rotation of second rotary stepper motor 30 in the directions of arrow B to reach the entire surface of rotating substrate The first rotary stepper motor is Controllable to rotate substrate 10 at a variety of speeds in either direction of arrow A. For example, an AT6200 Controller may be used to control the operation of the first rotary stepper motor. Further, the first rotary stepper motor may be operated continuously, so that a centrifugal force is :21 generated on substrate 10, or incrementally, so that substrate 10 may be moved less than one revolution and stopped at a.new, orientation with, respect-to,the position pf~fluid 4~4~JL T;flfL-aX .i Lb 2 1 dispesr 34. For ample, pivot arm 32.may beppere td in a manner similar to'thafof a tracking arm of a hard disk magnetic computer, memory r A-firstQtary stepper:.motor iiL~S4 r a may rotate,substrate 10 while fluid dispenser 34is positioned oversubstrate- I 0by the rotar motion of rotating second, rotar y st pperot 3.tpivot aim 32. eSi Substrate 10, platform 12, second stepper motor 30, pivot arm -32;,iand fluid dispenser 3may be enclosed within container.l Container 18 allows the .'Zic~LL 13)liiri4 m.A:;I.f~rtIga S .i.

atmosphere surrounding the reaction sites to-bestrictlx monitored and controlled during testig. Container 18-may be airtighto prevent dustand.moisturefrom settlingop and effecting the operation of the rotary stepper motors -and to permit thejnaintenance of positive ornegatepressure within container- Moreover, dust and moisturey adversely effect the chemical and biochemical reactions occurring at the reaction sites and alter the outcomes of chemical and biochemical tests. .For example,container(1.8 may be equipped with a humidity and temperature sensor 19 tso. that levels-ofgand changes in humidity and temperature may detected When low pr high relative, humidity as. lt :u a :st or temperature, or both, is (are) deteted, assay ing operaons raybe terminatedor the J. 1J !m -j '15aton.m y atmospheric conditions within container 18 may be corrected. Further, because of the importance of delivering precise amounts of fluid to small reaction sites, container 18 ,1 p -iZ .4 -sts also serves to reduce or eliminate air disturbances atorynear the surface of In addition, substrate 10 may be equipped witha pluralityof calibrating holes 36. Such calibrating holes 6 may be employed in combination,with Ein. place of the locating marks described above. For example, substrate ,1 .mayinclude. four calibrating holes 36 positiqned around the outer circumference of substrate 10 at intervals. Anelectromagnetic energy sensor (not shown) may be.aligned beneath each of calibrating holes 36, such that when an electromagnetic energy squrce (not shown), which may be mounted on fluid dispenser 34, is directly over one of calibrating holes.36 electromagnetic energy generated by the electromagnetic energy source is.detected by the corresponding electromagnetic energy sensor. This combination of the electromagnetic energy source with corresponding electromagnetic energy sensors may be used to locate fluid dispenser 34 over substrate 10 and to calibrate the aligning means of fluid dispenser 34. Further, each reaction site may be positioned a precise number of stepper motor "steps" from each other and from at least one of calibrating holes 36.

Therefore, once fluid dispenser 34 locates and identifies one of calibrating holes 36, fluid dispenser 34 may be moved quickly and precisely between reaction sites and over the surface of substrate Fig. 4A is a schematic view of another embodiment of the fluid dispenser.

A chemical or biological sample or samples are drawn from a fluid serializer 40, which accesses an aliquot fluid source and a separating fluid source (not shown). For example, in one embodiment of serializer 40, a pump may alternately draw amounts of an aliquot fluid or fluids from an aliquot fluid source through an aliquot fluid conduit and amounts of a separating fluid, such as a solvent, oil, air, immiscible fluid, a noble gas, or the like, from a separating fluid source through a separating fluid conduit, so that a serialized fluid stream, ije., a fluid stream including alternating amounts of an aliquot or aliquots and amounts of separating fluid. Alterniatively, serialized samples may be prepared as part of a serialized library, which are preformatted and stored for later use in the apparatus.

in another embodiment, a pressure control device 42, including an air pump and an air pressure gage, may create a vacuum within container 18, whereby amounts of aliquot fluid or fluids and amounts of separating fluid may be drawn into dispenser input tube 20'. For example, a valve may be used to alternatively place dispenser input tube 20' in communication with a source or sources of aliquot fluid or fluids and a source of a separating fluid. The vacuum in container 18 then may cause amounts of aliquot fluid or fluids and separating fluid to be drawn into dispenser input tube 20'. Alternatively, amounts of aliquot fluid or fluids and amounts of separating fluid may be formed as droplets, and an open end of dispenser input tube 20' may be positioned to draw selected droplets into dispenser input tube V23 $tc !t0r&~h 1 .Fig._4.B. nnlarged, view 'ofdigpefsef'i 1l tiuie ~2"iI srating a serialized fluid stream including* alternth am ts -of 'bhofiwo- ,separatirng.fluid, such as an immiscible fluid or solt'bnt! A's6lvent n6-ay be chobsen as a separating fluid? so.that aiquptjresidue iserernovedby: the.soIV6ti iiis'i4lid fluid passes through djspenseryjput tube 2 -20''oup-,i ?Pl_ "2ti n1.

2Refenrig again to~F 4A, a- timing- detice 23 iiicl~dei a flow sensor to determine the rate of flow ofjhe serializedhfluid, so'ithAtacti~ating nd deactivating signamaybeset to suction devc 4vaaiigsga 6n~t~~6 Fr'ime theamunt o aiqu :r~spa.ijpg fluid;may- include' a cbm ffdtf qF sensor 25. Fluid drawn from disppenser-,outlet -21liby sucti &i'die t"fr the apparatus via.,utinreturn tube22. sAs shown intFig$'4A, -thiEfluiiddi~ponrii"'iet 2 1, as 1 well as the fluid dispenser and thesubsttat& Th'.ay be'endlosdd Wi thin Eco'iitain& 1'8.

.fPressure, control device42, may be-used:lto- maintain t lT' pd~itiV&-'6f."'gaiv resr 'within container8 1 y: 1 YiI"t'F L-:ri 27 jRfrnx Fig13, an enlargedinofAlidiltubQ'sov" illustratinig a fluid 400including alternatingtamountsof'aliquot afi&o 6f 4 Mepaiiirig' flidr pecifically,,!luicMQ00 comprises eaaigfud42 li l~~b 4;ha second fluid -aliquot,406,. Second-fluid aliquot'406 iiicludi~s a"Idiili6f flo' "'s"hdr ,conoetfl0L8For-exanpJlecqmponents 408 'may bernagnexic beads; auid'skich 66as mybe detected asghey~ipassthrough fluid inlet tube 20!-iW clos'e'p-rhfmity to an coil or other magetic sensor. -If-the diameter- of fluidlinlet.tubd 20', the order, of ehali~ifin, and the amounts of aliqqot and. separating fluid are'known'.xhe flo w rate of thialze fluid stream may be determined. Further, the identity -of each :amount rdfaliqbot or sep tjg fluid may be determined from a signal generated by flow sens6r- conp~f'i 408 in flow sensoix?4, by varying the strength of the magnetic field geiiera&1 by the magnetc, beads. pr-.h9 gensityLof the magnetic bead in an Aliquot or 'a separating fluid, or both. Nevertheless, other types of-sensor. components -may generate: detectable signal~s such as fluorescence, radiation, electrical charge, or the like:- For example, each fluid aliquot may have a volume in the range of about-3 to 5 ul, and preferably,' in 'a range of about 4 to 5 Al. However, the amount delivered to any particular reaction site may be in a range of about 50 nanoliters (nW) to, and still more preferably, in a range of about 4 to 5nl. In view of these volumes, timing device 23 may deactivate and reactivate suction device 44 or energize and de-energize first controller 202 or controllers 202 and 212 to deliver a measured amount of aliquot to a particular reaction site.

In order to control the amount of a fluid aliquot to be delivered to a reaction site, the frictional effects of contact between the aliquot and the inner surface of dispenser input tube 20' also are considered. Referring to Fig. 4C, a furnher enlargement of a portion of dispenser input tube 20'is shown. As first fluid aliquot 404 is pumped through dispenser input tube 20', friction between first aliquot 404 and the walls of dispenser input tube 20' causes the outer leading and trailing edges of the aliquot to mix with the separating fluid 402 and to create mixing zones 414. In order to ensure that pure aliquot portions are delivered to the reaction sites on substrate delivered portions of first fluid aliquot 404 are drawn from within aliquot boundaries 416. A leading edge 410 and a trailing edge 412 of first fluid aliquot 404 are formed in the direction of fluid flow. Aliquot boundaries 416 identify the portion of the aliquot unaffected by frictional forces and by mixing with the separating fluid 402. The location of aliquot boundaries 416 depends in part on the pressure at which fluid 400 is pumped, the composition of first fluid aliquot 404, and the material of dispenser input tube Referring to Fig. 5, a third embodiment of the apparatus I may include a multi- function head 4 including at least one fluid dispenser 50 for conveying at least one fluid form a fluid line 51 to at least one of the reaction sites and a readout device Readout device 5 may include means for locating a reaction site, such as the means for reading a locating mark at a reaction site and for monitoring the chemical assays or biochemical reactions at the reaction sites. Each of such fluid dispensers 50' includes a fluid dispenser outlet (not shown). Thus, the fluidics and detection functions of the apparatus may be combined in a multi-function head 4.

In Fig. 5, a perspective view of a third embodiment of the present invention, apparatus F' includes a substrate 10 mounted on a rotary chuck 110 including -a So i I rotary stepper motor (not..shoivn) m~yY 6_td~tisu6strate107in eithr d0,irection.o anrowA4J. A-rail: Wissuspended ab6ve ad -ftiM'ic5td- .biSdctS z ubste1 4 inear~stepper-motor. 1:'ismounted on hdiil4 'h di iei~f inir-ter motr,'is, movable in- one dimensionifi the -directionsocfaifroiB alonigral4'oe S hesurfa~ce of substrate,1Q. ~Because pegtr e used a fi tidWh~iay be ovd Aqiacu1.qr reaction'site and stopped'andd:&ai th4tite 'unil ij co9"lte or. for.,a.predetermined.period -of timi' a'ii& sufficient t6'Illow r deetino asa hIsJI-' f

I

z)iu~-~~st4J,.4 i isjno~unted o? a. supporting! arm '137>-Siippoftii irih 13"iay'be jnounte ona aoqpigotori(not' showni), -such that mfu I ti~~tAi t a raised or lowered withrespecttpisdbstrate I 0?k' z: Apazaus1 may.,include-subtantalS uy hae 5'tfi 1 5 il iihy ;.,e~sspende4 by .four. platformsj suppots* 17W frornt'Iineaf-stiepp"r motor 1 A !r ppe/edotupr 115 is. ,poitioned'.within.th&I 6p~nI'dino rd,-hi~ed pafr-1..Fluid depe 50..inciLding'fiid 'supply lin'e-5 inc1u. Anthr e. opticaj fiber .rcpals52 and 54a and -54W-and'ih~ir as 6iit $ii_[a fibers: read fiber: 5Anditracking, fibers 55a and S5bt'are.motf-n teldo n'sb'jpkddYlS :C3 1 6#9hogh fluid dispenser 50, and -readout d'evi&:'-5 titiitliallyji'o'iii~oi'.'d h'oughthe interaption of the movement of substrate -10 by tihe-rotai y~iepp6r WotoribYot shownr) and the movement. of, u-shaped :platform 15' by ,liieErstepper' mi&or"16', adjustzynts to the position of fluid dispenserSO5 andreidout device 5S may biiaddty three-,Ns. micropositioner 56.§ ipne/edi upr 115 ni o~fiunt1 tee-axis micropositioner 56, which may in turn be mounted on u-shaped platfonY Ts, micropositioner 56 may. mqo\vg dispenser/readout support] 115 along three-axes to adjust the position of the fluid dispenser outlet (not shown) and optical, fiberS3-ind and 55b. 7j The readout device 5 may be combined with a tracking system, iich as in a (Compact Disc) CD pick-up head, tg_. Sony KSS367IA Optical Pickup manufactuied by Sony Corporation of Tokyo, Japan, or the readout device 5 may be phy'iically separate from the tracking system. In known optical pickup heads, such as those found in CD players, light emitted from a laser is split into three beams, a central and two flanking beams, and is directed onto the surface of the CD via a turning mirror and a lens. The central beam impinges upon the CD surface in order to read the binary coded information and to provide a feedback signal for focusing the central beam on the CD surface while the two flanking beams provide feedback signals for tracking the pickup head over the CD surface. The light reflected from the CD surface is received through the lens, is deflected by the turning mirror through the beam splitter, and impinges upon a photodiode array. Feedback signals then are generated by virtue of the geometry of the photodiode array. The operation of such feedback control systems is described in more detail in 0. Thomas and W. Ophey, "Optical Recording," PHYSICS WORLD, 36-41 (Dec. 1990), the disclosure of which is incorporated herein by reference.

The track pitch of a CD is about 1 .6 microns and the width of the encoding pits on the CD surface is about 0.5 microns, The flanking beams described above, may strike the surface of the CD surface at a separation of about 2. 1 microns apart, the CD track pitch plus the pit width. While a tracking system substantially similar to the CD tracking system described above may be suitable for the present invention, certain modifications may be necessary depending upon the precise configurations of the multi-function head and the substrate. In the present invention, for example, it may be necessary to alter the angle of separation of the flanking beams, so that they strike the CD surface at a wider or narrower separation to accommodate different sizes of reaction sites. This may be accomplished by altering or replacing the lens, as appropriate.

As noted above, the CD tracking system may employ a laser diode, which may emit primarily infrared light. The laser diode found in CD players typically emits a fraction of a milliwatt. Due to the nature of the assays performed with the present invention, the laser diode may be replaced with a fiber optic connector allowing a source of electromagnetic energy of a predetermined power and wavelength to be optically coupled to the apparatus. Moreover, a diffraction grating (not shown) may be placed 27 in the path of the reflected teims, WheA suichdiffradted 6&inis impijnge upon alinear :photbdiode array,:a spectiometermaybetreaited."Such aspectromeer may'beused to detect .the~progress oroccur-rence dfia'biologiciVoi cmiariiitarection site.

t v -In:afr eferred:eitodiment; the bptics- ~adlthie'li~iipU or t tbD S r~~ig~yt9 my~e'oifedoialit taar exitati source and ~as a -means;for:mnonitoring~and-rd'ading~the ,assay trlt oanihr~i~~ii nadto -takn-the reaction. sites-Referring to the'fdre* bi nk-de§*crion h bt~t~C -trackng -system is altered -toaincrease the sepataion b'&ti'~ethetii6e rojei&Piight bas sch! that the two -oiitbranosi'beaffis istraddle th e'll it a~readti6d iite t-fi6 la~er diode ofithe'.CD,,rackingmsyst~m~rhay-thefl .be-feplaced:,itti a fb&i o60ticti"cnnedtor to couple light in ffrm any type of laser. Finally, the photodiodes in the photodiode array .of.the CD tracking -systemn may bi6 sdlectively replaCed;4accordiritg- 6iheir sensitivity~to ,~ielgth anqlw light-levels that are expetted' tobeeved frdrnth rea~ctio s'ites.

Altrnaixeysome pr.:alb of the- photodiodes raybe -rtpla& idt: a pront _photoqppltiplier~tube. i 1 g~ ~As an alterniati~e: toathis CD rracking'systeit.?eiddtit" devi&& 5 may ,comprise three,. optical,,fibers, :such, -as athreetflffy, (5 0) 'micron "fib ers;- warii~d 'ifca .tI"nglaT~Onfigation ih the,'muliiiftin~ti on head 4---Maepiafedi-in ig.~ frst .optical .fiberyreceptacle 52 1receives a: first fibter 53. -First opricabfiber 53 tMiy~b& -slt ~g.jyifurcated, suchkthat first fiber 53 may ftnciontd eivlaserlighttliAbsar sufaeand .to -receive reflecteddlight from .the. substrate: surface: Cohsequtbdiy, flh'st optical fiber 5.3. enables the apparatus to detect-chemical and -biochfmidaI chantgs at the reaction sites ad to -read. any indexing marks. The laser orrlight source is- elkctd'bas d on the type of assay(s) to be performed and is.coupled'-to first fiber 53 -by- an optical filber launcher, such 'as a 1(7210 Optical Fiber.Launcher, 'manufactured by Thor Labs, mc, of Newton, New jersey, *U.S.-An ,For example;, first fiber -53--may be coupled. to -a photjnuliir tb(& a photodiode array) or a spectrometer. -Second and thifd optical fiber receptacles 54a and 54b, receive a second fiber 55a and 55b, respectively.

These two fibers 55a and 55b trail first fiber 53 and may be coupled to individual photodiodes to receive reflected electromagnetic energy and generate tracking signals.

Referring to Fig. 6, a bottom view of readout device 5 is depicted. This figure shows the arrangement of readout fiber 53 and tracking fibers 55a and 55b, with respect to the fluid dispenser outlet 60. In this alternative tracking system, an antireflective coating is applied in specific geometric configurations at precise locations along the substrate, such as that depicted in Fig. 7. Electromagnetic energy may be transmitted down the first or read fiber 53 and the second and third or tracking fibers and 55b or down the read fiber exclusively. The modulation of the intensity of the reflected light by the anti-reflective coating may enable the indexing and tracking signals to be read.

Referring to Fig. 7, a locating mark 70 may comprise both a start bit 72, an indexing mark 74, a dispense/stop bit 76, and a tracking bit 78. Start bit 72 instructs the readout device to begin reading the locating mark. Indexing mark 74 identifies the particular reaction site and may consist of one or more submarks, such as submarks 7 4a and 74b. Dispense/stop bit 76 may instruct multi-function head 4 to pause at theat position or to initiate the dispensing of an aliquot or aliquots for perform a chemical assay or to cause a biochemical reaction. Tracking mark 78 then guides the dispenser outlet (not shown) over a reaction site 80. As may be seen from a comparison of Figs.

6 and 7, if multi-function head 4 is properly centered over locating mark 70, tracking fibers 55a and 55b receive an equal amount of reflected electromagnetic energy from the substrate surface. By analyzing a differential signal, shifts by multi-function head 4 or misalignment of the multi-function head 5 to the right or left may be detected, and these offsets may be corrected. For example, as depicted in Fig. 5, fluid dispenser 50 and redout device 5 comprising first, second and third fiber receptacles 52, 54a and 54b, respectively, may be mounted together on u-shaped platform 15' movably affixed to a three-axis micropositioner 56. Micropositioner 56 may permit further adjustments, eg,, in a range of less than about 15mm with an accuracy of about one micron, in the position of fluid dispenser 50 and first, second and third fiber receptacles 52, 54a and 54b in three 2- @9I dimensions to improve the alignment of fluid dispgnseroutlet -60 or the read'and t tacking optical fibers or to adjust the focus of the. electromagnetic energy, teflected *6~ffi the substrate surface. In. addition, to determinigg.the. occurrence hndtprogress of the .reaion at the reaction.site, yead fiber. 53 may scan the iifdexinglmark. Reid fiber-53 also may function as a proxiritysensogpo correct for nfidvement bythe muiti-functibh hea 4 wa frm~otward the, substrate. This -my be.accorhplished.by monitdtitg th6 head 4r away Lro4.q n. ,.tt ~N light intensity of the reflected light in are-as in.whichttheanti- reflective coatirig'iskabstnt! SRough calibration, may ,be pecessilry,,-and as axresult,.the entire multi- L0 function head and feedback~pstoii~ytr may-be mounted on~a XYZ~translatidn stage that has a range~of motion significantly,,greatg ;thanjthat of th&~efebc positioning system that is mounted to the. XYZ *trans] ation,stage!Yeedback actuat ion may be used to make fine adjustments to the -positjpning of the fluid:dispeitand the readout device and maybe provided by vareyo eni.phspizeeti.blocks; voice coils, worm drives, and the like. T~he a paraitus, maybemodified~to providddi6' such finre adjustments by the addition of a secn he-xsmcooiird'ewe-h ~~?i;-':t}Irreaou~s p.nmvf !ta cnhtre iscoioneire tenter first mi crop ositioner and the dispenser/readouy t11- Ppf:Tiscfd.icdoiife has. a smaller range of~movernent ada higher- dpgrge.1of accurac~ygthan the Lfirst microposltloner,, arneof adjustment of less,-tharn- about- LQ00 -micronramnd an accuracy of about 0. 1 mi cron t v.

The shield structure also ma~y ppn se-a, cap: formed eover the geometric cavity of each reaction site. -Such caps may includea apenin sufficiently large to allow ivv 4 u U Iop.nIlg a stream of fluid to be directed therethrougb, but. sufficiently small, so as~to*retainnhe' fudwtithcaiy and reduce or-elimint evprt~nof the fluid from-Ithe cavity.

For example, streams containi'ng nanoliter.,volumes. of reagents, cleaning fluids~buffers; and the like, may be directed, through the cap, openipg. 1 &J4rg9 volume droplet, gj, a dtope t having avolume in a range of about 9.1 !o 0.5 uI,, is then: dispensed onto the c ap opening. If the droplet has sufficient surface tension and.is. dispensed -at a~low velocity, it will seal the cap opening without entering the cavity. This sealing. droplet may acts as a removable cover that may be removed by centiftigal force caused by the rotation of the substrate.

In addition, the sealing droplet may be electrically gazed. By changing the electical potential of the droplet through the use of appropriate electrodes and voltages, the wetting capabilities of the droplet may be altered to increase or decrease its adherence to the cap. Moreover, electronic components may be placed in the cavity to control the electrical gating of the covering drops.

Referring to Figs. SA and 8B, a reaction site 80 is depicted as a geomeernc cut or etched cavity formed in the surface of substrate 10. Reaction sites may be formed by chemical etching or ion etching, or the like. Moreover, the shape of reaction site 80 may depend in part on the crystalline structure of substrate Although the reaction site depicted in Figs. 8A and 8B is shown to have a simple rectangular shape, it has a locating mark 82 and is equipped with a shield structure 84.

Shield structure 84 aids in the reduction of evaporation losses from reaction site 80, the retention of fluid within reaction site 80, and the prevention fluid fr-om covering locating mark 82. Surface micromachining, especially on semiconductor materials, is known in the art. Techniques for rnicromachining are discussed in K. Gabriel, Enmineein Micro)scopic M-achines, SciEwnIc AMERICANj 150-53 -(Sept. 1995), which is incorporated herein by reference. Substrate 10 may include at least about 20,000 reaction sites, and preferably, at least about 100,000 reaction sites.

For example, a radial pattern comprising substantially square wells with edges with a length of about 1 10 microns may be formed in an oxide layer grown on a silicon wafer. A photo resist, such as a Microposit 18 10 photoresist, manufactured by Shipley Company, Marlborough Massachusetts, may be spun onto the oxide layer on the surface of the silicon wafer. The photo resist may then be exposed through a mask to produce-the appropriate pattern, and then developed and allowed to dry. The water then maybe placed in a hydrofluoric acid bath to remove the exposed oxide layer, and the wafer may be placed in a potassium oxide bath to etch the uncovered silicon.

Acetone may be used to remove the residual photo resist, and a second hydrofluoric icid ,,bath -may, be~ used -to! 'remove,-r.he -,-eann .'oxide. 'This rpfoIUf6U;-pJ-ou'-e-s anstojalpqhd el.wt the geometric, shape dfat iiverf d p~inid,;!-) Solid support substrates (not shown) may be'formed"Wthiffr e~tibit sif -Teetucms provide-a-point. at, which biochiefficil',probes-may b affixed tothe reactionsites 8O;jhe~prpkes may be usedt:bind-.pgriculaffargetsuch~s-p-lymierls,C yoyf ul4eo9tesiPA RA rNA; ahd,,anti bod'i, of 'antibod fagments -or mitrsmereof Ankadd$ipfl, th-e probes may~bitnd-,airg~ts; guch. as'individual nucko tides .andnuloies such- as, adenosine,,, gianosine;!- dytosinae,--!thyrnMidine,' urdc6i-vhor combinations .thereof, 1 and. moleclar .structure~s-sucht.ai eniymeslpiroteihi'- I-~1sfi chromosomes and chromatids, and cellular structures, such asnito 66hidria,zribbsohie-s, t adthe like.. M ovqr, -micyorgantsmrrissuchfds pr~karygpticib-r."eukaryotic cells,including. mnammallan ceilsi;bateiafya$ an&rtoo iruses, p hagest an combinations thereof, rny e,bpup4 to theprobest T hese-tuctures,:may. hfomdypotolithbgraphicaechdi~uso ae~i _.naybF9incorporated into substrate 10 o. form:n. thesol id support structures.ns For ~,example- suitable, materials, which, may provide points -for di rectprobe~atachment, may include electrometal materials, such as goldj niobium oxide?, iridium, oxide, .platinurn, titanium, zinc, and other metals. Solid support substtattS. and probes- may'be usedto .ensure that parnicuar targets are retaine4 injpanj94uar reaction sites,for. testing. The use of such stucures and probes~is describedi inUS. Patet.No.,5,532,12B to Egg~rs eal.,'1 which is inoprtdhri yyeference.

.Locating mas 82,are discussed -in- detail zwth.respect to-Fig..7and may serve several fiunctions., Y'rsthey allow the apparatusio determhine its starting: position.

Second, thy allow, thea4pparatus,to acpcirately,.4!liver fluidto avpredetermined -reaction ,,site bysirply,scanning the locat~ing mafrsunil-te-desired -site is found., -Third, although ,the precision achieal .th. seelotr anaethaparatus to,track its movemntsacross, the surface ,of substra,_9 ,by.A formn of dead-reckoning-t-the apparatus may detec positioning errors by comparing theappaais's position 'bydeadreckoning to that of a known 1 calibration point or reaction site. Fourth, because positioning errors may be determidned, the apparatus may re-calibrate it self by comparing its calculated position to its known position with respect to the calibration points or reaction sites of substrate Ref~rring to Fig. 9A, a perspective view of substrate 10 having a spindle 90 is depicted. Spindle 90 may support substrate 10 and pen-nit the rotary stepper motor to engage substrate 10. Further, spindle 90 may permit electric connections between electronic and electromechanical elements, uicroelectromechanical systems, positioned on substrate 10 and power and control sources separate from substrate Figs. 9B-9D depict overhead views of solid angle sectors of substrate 10 exemplary of embodiments of substrate Referring to Fig. 9B1, a solid angle sector 92 is shown, which includes a plurality of reaction sites 80 aligned radially from spindle 60. Each of reaction sites has locating mark 82, which is depicted as comprising two bars. The use of this identifying mark is intended to be merely exemplary, and other identifiying marks, such as indexing marks, bar codes, number codes, color codes, or the like, may also be employed. Further, an identifying mark may consist of a combination of characters or markings, or both, and the position of the mark with respect to the center of substrate or with respect to sp indle Referring to Fig. 9C, a solid angle sector 92' is shown, which includes a plurality of circular reaction sites 80' and electronic or electro-mechanical elements 93, which are integral with, adjacent to, or connected to reaction sites 8O'. Examples of such electronic elements include transponders for receiving and responding to an interrogation signal; heating coils for raising the temperature at a reaction site, temperature sensors to measure or monitor temperature or temperature changes at a reaction site; electric field generating elements for altering the electro-magnetic field at a reaction site, gZ,, to produce electrophoresis or to denature substances; photo-sensing elements for detecting light emissions caused by chemical or biochemical reactions at a reaction site or for detecting changes in the amount of l ight passing through substances as a result of a chemical or biochemical interaction;, chemicay sensitive gates, e~.

1.33 ChemnFETS: ionsensitiv gaeIjSFETS; 'or (a-cbhibinatioitfferof -tfurher, a serpentine resistor~imay be,used~to h'etproteiniuntil d~ntiatdedI'.',' I L k1fl 4

T

,:,Qprally,;.chemically:-.s~nstivejte~i 1 ioi estv-gates' are transistors ,,whose gatesw have 'been-re iacec,!wiith- hby 'arfn :perrneable .rembrane9t Cemically, and ion sensit~eigaxis'iybe usdtd det-ect changes in pH as well, as ~chagsi chemical- and ion Qcompositi6nYJ-,).Flrtir; sub~tr&E -10 hckd JnterdigAreoarraysfIDAs) h An IDA may ,comprisdiwo arrays '6ffefii6af elec'tr des ,that, re pWaced so that,,the arrays intermesh ,b'utdo notFcontitd~ e f~hdP~ dl& icai -reactiqpspccurrig tween. the -arraysc-arid- generating t eedt-ricdl'!cha ges 6'aiise' a resultant current'tp 'flow in,the,-arraysa-7heresuiltant curretrfloWIW]"ay bd rfbiiii6fe'ato indirectly,,;rnonitpr. the. progress, sQfcherncl atin0 th reatio sie0leto 1 ,iechanical, elements 93, mayproduice. vibiition at-the, reaction §ite tt-stii dt-hix-a _,plurait3yof sub stancesto, facilitateo or acceletate chemi~i 6r,,bi chelmical intetictioiis.

:.;wmi addition substrate:10 ztfiy'ihclude elements, electrod~' >ltha acte,.aterthe electrostatic-qharge on;substrate-l10 in: local aieas.c*'Suchan 6ftatiic chag ma ttat repiAicrovolumes'of fluid. C6nsequentlyfP-tudfhel6ctrodb may eue-to ,aid in-deliverp pcieanmounts of the-at least one fluid to the i ag't6die reaction site. With a plurality,.6T.controllkd.'electrbd~s; an'eleirostaticLfdid"t6hsigti~g ojj ey 'm~y bgenerated across the Enhtire surface'of substrate 10, thus, creating a plurality ot"virtuaf', reaqtion.sites. -Generally- these electrostatic fields ffay b e used to create vertical and, hoizontal containentflelds.yiThese virtual reaction- site s may ,reduce crosspwotaminatiomtletween sites..acThe surface -charge.. also miy be manpultedby oaxng r dpositing a material havingia desirdc' g ntlh~rfc 2 oswbsftate:10.. Thistechnique may be used in .conjunction.-with, the elements described above.. J R eaction. sites.,80' mayr-be joined to elements 93 by-a reacxion'-site connection 64. As demonstrated by electronic or electro-mechanical elements ,93 described above, connections 64 may transfer information coakeming the reaction site or may serve to transfer energy to alterithe chemical or biochemical interactions at the reaction site, or both. In addition, elements 93 may be connected to each other, interconnected, by axia connections 95 or by radial connections 96, or by a combination of these connections. Further, elements 93 may be interconnected radially through spindle 90, whereby signals may be transmitted to elements 93 from a computer (not shown) or other signal source and data from the reaction sites may be sent for storage and analysis to a computer or other data storage and analysis device or displayed on a monitor or printer or the like. Alternatively, elements may be interconnected via a device for combining two or more signals, a multiplexer (not shown). For example, by using a plurality of multiplexers with between about 10 to 100 connections per multiplexer, all of the reaction sites on the substrate may be interconnected.

Referring to Fig. 9D, a solid angle sector 920" is. shown, which includes a plurality of circular reaction sites 80', a dispersion point 97, and a plurality of substantially radial channels 98, which channels 98 join dispersion point 97 to reaction sites 80'. In operation, dispenser outlet 21 may be aligned by rotary stepper motor 12 and linear stepper motor 16 with dispersion point 67. An amount of fluid then may be delivered to dispersion point 97. The rotary stepper motor may be used to rotate substrate 10 at sufficient speed to create a centriffugal force to draw a portion of the fluid from dispersion point 97 through channels 98 to reaction sites As noted above, Fig. 9D depicts an embodiment using channels 98 and the centrifugal force generated by the rotation of substrate 10. However, it is not necessary to use such channels to transfer fluids to various reaction sites. If sufficient fluid is placed in a dispersion point 97 or in a plurality of dispersion points 97, the centrifugal force created by a rotating substrate 10 transfers a fluid across the entire surface of substrate 10 and to all reaction sites 80'. The amount of fluid required depends upon the size of the substrate, the number of reaction sites, the amount of fluid required at each reaction site, and the placement of the dispersion point or dispersion points.

Fig. 10 depicts an overhead view of a pair of reactions sites, reaction sites 80, joined by a channel 100 and a microfluidic device 102, such as a microfluidic puip..or~valve.J Staged, :hemical ;and bitochemical' t~eth,~ tinAaoSi b mc piliedby providing a fluid-or fluids to .a~reaction-,site'-foa'fir-&§ita&g -ftkh and 'th& the product or products -of such first stage,testingni' be thifferrecd by nicans ofdeices 102 through channelsA 10, to anotherreactionmsiteobr sit~frsuosie'queit testing stages.

Channels 100 may be formed or etched or otherwise ctt.nt6.th'c7su't i6o~sist 1 0.

Alternatively,.channels 4 lOOQmay kej ormed as-.tubes bo7t6hdiitV'rassing beneath the surface of., substrat 1-t ent mlicl! trnfe *tuis or th'& pioducts of ,hmcland biochemical'-reactions between reaction ~sitesS The invention may be more. fuill'I--und~r-f&6-id~b '6ikdrto f-h foil owing examples, and test results.,,whi ch: ateinfend-dPt 63 be pfrly, exeffiolaiy of the invention and its operation,and-,usests Jc Fr f FT- T's O'atl W ~~i ~sk w t :rc'~~:Examples and Test Rtsjults--( t>In~a first. exajnpe,,theaiquotstwould b6 deii'Wt-ed' Ida disptri'ser ouitlet via a dispe n~er input~tub,. A~iquotsand separatiti9 fli e. -ni~i mxisdible fluid or a sont au. s pa-r throug ,te gispensetrinppt -tube, as'a serifized fldi £A gaps would be formed between~eachaiqiuots, And each7xamouht:ff 6f9~~-~mg fh'id&' 'The Serialized fluid then wudbdeirdlth dispenser'outlet i iiiur'differziial relative to the ambientpesr withincontainer l-8'j ,ap9 E0 S- -py~tS*~ ~~2g~7&6~ii~eressuremn a-range of about 0.01 to 2000 psi,-and preferably, a positive pressureir i -age o 0 t-~'ihaTik .about 59.0 psi, such that the airxgapsdo not compiess signifid ahtlgKThe moeme6hCf the aliquots may be monitored using a photo-,diode~and a photo-trangniifte;--ikhich se6.n.s e the change in the refractive index as~the air~gaps pass..the s-e'sor ',"The time between sensing and dispensigol becluaebased on the".sptedd d-f thi fluiid fldow.rb The fluid foqi my Pq dispensed continuougly, 'Ho-w-e-r~a suioni return tube would be positioned at the dispenser outlet, .such ithat thersucttin'etiirn tube, dr&Ws away the separating fjUid..portiop of the fihid flow and leadingtidtaiingpi-tons of the aliquot. See.Fig. 4C. The. portion of fluid-that is-not- drawn away; wodid'ble timed8 to flow straight from the,,dispenser.,outlet onto- the surface of siliconiafer, Ti-e suction device' which is connected to suction returnitube, would b&-A:.highivacuu~m system that is controlled via high-power solenoids located at a sufficient distance from the dispenser outlet, so that the forces do not cause shaking of the dispenser outlet in the vicinity of the silicon substrate. Thus, a continuous Stream of fluid may be applied to cover everything, reaction sites and non-reaction sites, or individual aliquots may be delivered to individual reaction sites.

Once the reactions have occurred 1 the reaction results may be read. This would be accomplished by using a fiber optic tube that mounted adjacent to the dispenser outlet. The entire substrate surface then would be exposed to ultra-violet light. Reaction sites in which a blocking reaction occurred would show no fluorescence due to a fluorophor carried in the aliquot(s), conjugated with proteins contained in an aliquot. Other reaction sites would emit fluorescence. Preferably, however, the inverse situation would be employed, in which a researcher would determine the occurrence of a reaction by the presence, rather than absence, of the fluorescence.

An identifying mark, indexing marks and bar codes, may be placed 1 5 adjacent to each reaction site, whereby a identifying mark reader would detect and read the mark by changes in its reflectance. Either ultra-violet or non-ultraviolet light may be used to determine the occurrence of a reaction, as well as to determine or verify the identity of a reaction site. Generally, reaction sites may be located via dead-reckoning.

However, a dead-reckoning system would require periodic calibration. A feedback system, such as one utilizing identifying marks, may be used to confirmn the accuracy of and, if necessary, re-calibrate a dead-reckoning system.

The following tests were conducted using samples consisting of Rhodarnine 610 perchiorate dissolved in butanol at a concentration of about 2E-4 molar (hereinafter "Rhodamine solution"). Referring to the apparatus as depicted in Figs. and 6, a green He-Ne laser, lasing at about 543.5 nm, was used to transmit light along a first portion of a bifiurcated optical fiber, L& optical fiber 53. The comfmon end of this bifurcated fiber was oriented orthogonally to a silicon wafer (the substrate) mounted on a rotary chuck, such as rotary chuck 1 10 depicted in Fig. 5. The bifurcated optical fiber allows about 53 microwatts of laser light to impinge upon droplets of Rhodane ,t37 solution dispensed by a.BioJet Quanti300'TM fuid dispenser. The'portion 'of tf6lA'e light reflected from the subsiyate 'surface is, gathered, via -the'. cotfmor h'd "of the ,bifiircated3 fiber and. transmnitted .along. a seconr4,portionwof the biftircaitd UlpticAlfdier.

The signa received by. the second .porti on of the fiberis passed thrbugh- a -hig h p ass. filter order to remove noise and feedback associated with the laserfatta cuioffkwaV'enh of about 565 nrn This filtered signal was then transmitted to a photomultiplterFttibe, (PMT) Lg., a Hamnamatsu PMT, Model tNo;.5784-O .rnanufactutil by Hamamatsu Corporation, of Bridgeaer.New Jersey, :S4, for analysis:;t Tet. t_ eatr~ I0 ,..,Thep this ft.!sisl) 'to .detenniriwh ther the'tadbut devices of the type ntended for- use in embofiiments,,ofshis apparatus couldratiially detect the small volumes of a Rhodarme solution. dispensed: .frdiii the fluid: dispeinsers suitable for use in the embodimentsydiscussed above and td,,determnineth6 sigiial ~ak and the signal-to-noise ratio (SNR).lThe test~i inoled the steps'ofrtdi-pensing a droplet of Rhodarnine solutipn. having-.A volume,of aboqvt 10:4 nkonto Ea silicon -waferw-and stepping .the readout device-across- the, droplet.. atla. rate of abdut .1 'mm/sec;iWhile sa mpling the output of the PM a_ 't-j&1t~f bu, W Referin toFig._ 1 1, the 10.4ni~droplet is-cleArly detectabl&, ?kOeak output of about 3.5 V with a noise level, of about. 250 m~j..were measured. Tbus,'a SNR of about 14.1l was calculated using available fluid -dispensing and- readouti-devicesrtA.sinfit porin f th ,tsignal!nois could~haye b~eneliminated'by using a baindpass filter, centered on a wavelength, of. about 580nm ie 'the- emission peak' of the at9.Qte _4k ifnstead of a ,highpass filter. ,Additionally, because the'light exiting the fiber was not collimatedj.firther. signal degradati on was experienced.-., a' 4 The approximate 'diameter, of the- droplet -on the' -silicon wafer:, is determinable from the sampling data to be 4 about .650 microns. However, a I 0.4' nI sphere of fluid iscalculated to, have,.diameter of about 270 microns. Surface energy interactions between the droplet and the silicon 'wafer may-cause the -droplet bDf Rhodarnune solution tospra ouan ot hufce. Such spreading occurred in part 1~ ou nLot h uf.

because the droplets .were placed on a flat surface of the silicon wafer, rather than into an etched well. As a result, the effective path length Of light within the sample decreased, which weakened the resulting signal. Using appropriate geometry, ite, wells, and appropriate surface coatings, it seems likely that the path length could be increased by at least a factor or two.

Test 2.

The purpose of this second test was to create an intensity/thickness profile of a 10.4 nil droplet, to confirm the lower bounds Of the dynamic range available for sensing, using available readout devices, and to determine source of hysteresis in the intensity signal. The test involved the steps of: dispensing a droplet of Rhodamine solution having a volume of about 10.4 n] onto a silicon wafer and stepping the readout device forwards and backwards across the droplet at a rate of about 0.5 mm/see, while sampling the output of the PMT at a sampling rate of about 500 Hz.

The intensity plot of a droplet is primarily a function of two variables; the excitation path length, which is essentially the thickness of the drop, and the focusing effect created as light of the passes through the droplet. Because the tested droplets were relatively flat, the majority of the modulation of the intensity signal was primarily attributable to the thickness modulation across the droplet, rath'er than to any focusing effects. Consequently, the forward scan data depicted in Fig. 12A indicates that the excitation path length of the droplet (which is proportional to the thickness of the droplet) is greatest in the center of the droplet and least at the periphery of the droplet.

This result was as expected. The left edge of the intensity profile indicated that a measurable signal was generated across a distance of about 85 microns. However, the right edge of the profile indicated that across a-sinilar distance, the signal generated was about 1.5 times that of the left edge. This difference indicated that smaller areas of the droplet may be resolved if the path length were increased.

There was a distinct asymmetry in the forward scan intensity curve.

Referring to Fig. 12B, the reverse scan intensity curve indicated that the source of hysteresis was the droplet itself The reverse intensity profile was a mirror image of the 1 .forward intensityp rofle. ;-hjs suggested that the droplet sitting -on hsesilid-oivafei Wvas "ushed" slightly 'to the right. The cause of thisoffset -may'bek~iie td .the'fluid dyiiiinhcs the fluid disper eetraic efets, or a combinatior'.thtef Mroer h .about. oe.half-volt. ofipsign~al lost on* the, reverse 'intensitymcurve -was diue-7o' ihe .pevapor t f~h4e droplet-iurinjgetp~ rxJi~' wo~ i 2l 1~C ,The~jpup§q of this ,third- test .was t'b.shoW 'phlfacrc ofth feedba-tepperjngtrs,9d iyp rtjake~airoughi uihtitati& agsesiher& o-;f $,iiim 0 4e~g~al~xp9rn,: CbjtsM involved the: steps -of'di spenisirfg'in -Ie d6 kl6ii, vo,.,lume of about 10 4. in ,ajcnowp .locationmaaid -,fflovirjg :the;r'eadodrd16;ievi by did to moTnitoring the outp~i f hPM atdThifga&o *about,1OqO 4 H#z untilQ1he4?nopet exaporated.;r:5 :1w tSt ~S n t 'Fi 413; the iccuricyobfth opea iofi d 1 5Fdf the stepper motor is evidenced by the intensity plot. The peak value of about 1 3.25 'Vots is simi]aLito the~peak~vl4esobtained-in Test-lI&TheicanningtA'r?6piet. This indicates that the.,steipper motor~fld.motor controls, Are capab le 6f. pdsiiihgnif-iilifiii&ion head; substantiafiy ,qt te 9jter of atoplet:. Fulrther,: as discii'ssad abov th6eheeie *micropostn mTay be usedgjo make adjustments;! .±gh adjastmetsiria'raijebff lss Ibu~~rwta 1.-curajcyof about onetmicronAif Any ofth'-eCirtisiaK~e rto,,the position of the-,nulti-finction. head,'-once thex 'head !hd's..brin poiii~ned s hevolurneof the 4roplet decreases dtie to evaporation'tth61inteisity signal will decrease thereby tracking ,the iloss-of droplet ,volurnert, At' soiii critical volume. whicbwi 1 @dpekn 'A i'l3 at ij the initial:droplet olume, the tomnpositi on of the dropl, ,~anothe disposition of the. droplet_; -on. the~flat -substrate-surface oriri a geoetrc ell an te eapration process accderates ,significantly: 7Assumidng-that volume is linearly related to intensiw and that the drop still has a volume of 10 4 -nl Whien iniialy dtecedbyex trapolation, a:volt signal may. be;correlated to a. volum&efabu intaly deetd by of-t-; 3 ni. Consequently, a volume of about 3 n]i may be a lower limit for the detection of a droplet using the equipment confi guration of this test.

Nevertheless, other configurations may permit detection of still smaller volumes. These configurations may include modifications to the readout device'a transm-rission and analysis of the interrogating light transmission, such as increasing the path length of the laser detection beam, using a bandpass filter to reduce or eliminate noise, collimating the light exiting the first portion of the biffurcated optical fiber, increasing the power of the excitation source, g h ae, n obntin hro In addition, the structure of the substrate may be modified to improve detection capabilities, such as forming wells that are geometrically designed to reflect light back into the second portion of the bifurcated optical fiber. incorporating photodiodes at the base of each well, and the like. Finally, detection capabilities may be improved by systemic changes such as the reduction of coupling losses throughout the apparatus.

Test 4.

The purpose of this fourth test was to demonstrate that a binary code could be successfully placed on the substrate and read by the readout device. The test involved the steps of depositing a 3-bit code that included a start and a stop bit on a silicon wafer at an annulus and subsequently, stepping the rotary stepper motor, such that the circumference of the annulus to be scanned passes under the bifurcated optical fiber. The resulting intensity signal was sampled at ]1000 Hz. The binary code was formed from 0. 1 microliter droplets of Rhodainine solution with the presence of a droplet indicating a one and the absence of a droplet indicating a zero. Each of the start and stop bits were indicated with a single droplet.

Referring to Fig. 14, the 3-bit code was successfully laid down and read back. The variation in pulse amplitude and duration were primarily due to evaporation of the droplets over the duration of the readout procedure. Generally, bits read later in the readout procedure generated lower signal intensity magnitudes. This indicates that a significant amount of evaporation occurred during the test. Nevertheless, the bit code remained readable even at the reduced droplet volumes.

v:_ssC: .Referring tpo Fig. 15, aniecond exaipole6srbn the nea oth compon~ents .of an ,e ikocigen: .otithe:ap-ats~aig' mutfnto11 d is decie ~In thisembkoqien,.overaU cqontrol ofihe6pe"ti6noft'h;-a5rS ay~b m gnrl:uppsed -comnpiter.35e02csu6i avr~?c~o cmrsn a Pentium~' II microprocessor, manufactured' by Intel Corporatin'k Sant C ltara, 43 CaiforiaJ.S.,;ir ite2 ike;,apable 16f 1bptratinfg:at as abu 30 Mt ~.Moreover, ~computer 15 S'sjpreferably ,equipped withfriditAb6"s'oftwar'A to create a .Coding~ environment: ft,.acs~igpr~ea--onpfiff ia ~~software. includes LabVIEW~software;'PatsN o.-7,7 66.70-03'; j- k'dthe'ab 6 AEW& P ID Control Toolkcit software, Part No i ill ilfim National Instruments, oa,-f tut..TxsU. S7AW 2 J2I. CoinpqitervJqj,9Lis, linked t& a-imotorl'toniir6llEr". l-i 'g9 Controller, manufactured by ParkergompumiotornCom'paui abf'Rdhief Paikfl,"Nifriia, U~s.A. .Motor~coritroller, 151 relaysjinstructiohnsiftoi.co'MputierilS0to a linear stepper motor 152a, a L20 Ste pprMotor, mranuficxiredby'rker. ofnptu~i6ibf Cdiijkny of.Rohnert Park,, California, U. .,.ad.4rtr..tpe'fitI51,7 -51-10 Motor, and, wa Zeta. 41,Rotary Dniver,inianufadtured by Park&- CoiUWht6tor Company of Rohneft,ParI, 9alfopia,-U.S.At,-via motoi.!coiitd '0 i'P9K 'lB iiW>. In addition, comnputer 150.mayibe linked to aiiiiLilti~fuhdti6ii c85fftrblt,4 gaDAQ.Board ModelNo.AT1l96 zAnufatur yN atibfan.fsruments, Icof.Austin, Texas, U.S. rA micropositioner, 155; Mg2;a Low-Profile Three-Axis Micropositioner,, Part, N~o. CR4000; manufactjredby,-the';Daedal -Divi~iohn of Pirker C ompumotor Com pofRohnert Park -California;-U. ,?rn4be operated dire6ily to~ mak Toiinn ijj~ppt in alrangezofless-thain about'l 5mmnwith-ri Atdrabcy ,pou n cron,*alog pae~a~ s in.,the: position of a fluid disjiensero ui-Renda a readout device. NMufti-function control 1-53 provides operating insiructib-is recived from computer, 150 lto at least a fluid dispenser 1-54, L. a Biolet Quanti3 OO'~h fluid dispenser, manufactured by BioDot, Inc. of Irvin,-jCalifonia, U.S.A.,and -alaser 156.

Laser 156 delivers a beam of light to the surface of a substrate 10, via a first bindpass filter 157. Laser 156 and first bandpass filter 157 are selected according to the assay to be accomplished. Further, light reflected from the substrate's surface then may pass through a second bandpass filter 158 to a photomultiplier tube 159, a H-amamatsu PMT, Mode! No. 5784-0 1, manufactured by Hamamatsu Corporation, of Bridgewater, New Jersey,

U.S.A.

In this embodiment, fluid dispenser 154, micropositioner 155, and the readout device are combined on a multi-function head. Light from laser 156 is transmitted to the readout device over a fiber optic cable and from the end of the fiber optic cable is projected onto the surface of substrate 10. A suitable fiber optic cable for transmitting the light from laser 156 is a bifurcated cable, Bifurcated 50 1 um Fiber Optic Cable, Part No. 83F50 U[V[VIS, manufactured by Ocean Optics, Inc. of Dunedin, Florida, U.S.A. Such a cable allows a single cable to deliver laser light to the surface of substrate 10 and return it to photomultiplier tube 159.

As discussed above with respect to Figs. 5 and 6, tracking fibe rs may be 1 5 used to help align and guide the multi-function head to the reaction site. Such tracking fibers may be designed only to receive reflected light from the surface of substrate which was generated by laser 156 and supplied by the bifurcated cable described above.

Alternatively, the tracking fibers may also comprise bifurcated fiber optic cables and may provide their own light for tracking purposes. The light supplied to the tracking fibers may be generated by laser 156 or by a separate laser or lasers.

In one embodiment, each of the tracking fibers may comprise bifurcated cables and may supply light generated by laser 15 6 to the surface of substrate 10. Prior to the projection of the light from these tracking fibers onto the surface of substrate the light transmitted by each fiber may pass through a separate bandpass filter, so that the light delivered by each tracing fiber is distinguishable over the light delivered by the other tracking fiber and over that delivered by the read fiber. As described above, with respect to the read fiber, in this embodiment, the reflected light gathered by the tracking fibers may also be filter again before it is returned to a photomultiplier or photo-diode array for analysis.

43 Other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and these examples be considered as exemplary only. While the invention has been described in connection with preferred embodiments, it will be understood by those skilled in the art that other variations and modifications of these preferred embodiments described above can be made without departing from the scope of the invention.

Claims (40)

1. 2. The apparatus of claim 1, wherein said means for rotating is adapted to rotate and stop said substrate at an adjustable speed and controls the rotation of said substrate by adjusting said speed, acceleration, and a direction of rotation.
2. 3. The apparatus of claim 1, wherein said means for rotating is controllable to rotate said substrate at speeds, such that a portion of said at least one fluid removable from said at least one reaction site by a centrifugal force generated by the rotation of said substrate.
3. 4. The apparatus of claim 1, further comprising at least one channel joining said at least one reaction site to at least one other reaction site; wherein said means for rotating is controllable to rotate said substrate at speeds, such that said at least one fluid flows from said at least one reaction site to said at least one other reaction site by a centrifugal force generated by the rotation of said substrate. The apparatus of claim 4, further comprising microfluidic devices adapted to regulate the flow of said at least one fluid through said at least one channel between said reaction sites.
4. 6. The apparatus of claim 1, wherein said at least one head including at least one fluid dispenser is movably mounted on at least one rail, said at least one rail being oriented substantially parallel to a surface of said substrate; and said means for aligning comprises a linear stepper motor for positioning said at least one fluid dispenser along said at least one rail such that said at least one fluid dispenser outlet is directed toward said rotated substrate.
5. 7. The apparatus of claim 6, wherein said rail transects said substrate.
6. 8. The apparatus of claim 6, wherein said means for rotating comprises a rotary stepper motor.
7. 9. The apparatus of claim 6, wherein said at least one multi-function head is mounted on said linear stepper motor.
8. 10. The apparatus of claim 6, wherein said means for identifying includes at least one sensor mounted on said at least one multi-function head.
9. 11. The apparatus of claim 10, wherein said at least one sensor receives a signal emanating from said substrate.
10. 12. The apparatus of claim 10, wherein said at least one sensor transmits an interrogating signal and receives a locating signal.
11. 13. The apparatus of claim 10, wherein said at least one sensor reads at least one locating mark on said substrate's surface.
12. 14. The apparatus of claim 10, wherein said at least one sensor reads at least one tracking mark on said substrate's surface. The apparatus of claim 6, wherein said means for aligning comprises a computer having a memory for storing a start location on said substrate's surface for said at least one head and said computer provides movement signals to said rotary stepper motor and linear stepper motor, whereby said motors align said at least one multi-function head, such that said at least one fluid dispenser is aligned over said at least one reaction site.
13. 16. The apparatus of claim 1, wherein said at least one head comprises a chemical reaction detection mechanism including an electromagnetic energy source, whereby electromagnetic energy is directed into said at least one reaction site, and an electromagnetic energy receiver, whereby electromagnetic energy generated in said at least one reaction site is received and analyzed to detect a chemical reaction or the products thereof
14. 17. The apparatus of claim 15, wherein said electromagnetic energy source includes an optic fiber coupled to a light source, whereby light generated by said light source is directed into said at least one reaction site, and wherein said electromagnetic energy receiver includes a optic fiber functionally coupled to a photomultiplier having a bandpass filter, whereby an electrical signal is generated and transmitted to a-computer to detect said chemical reaction. S18 The apparatus of claim 1, wherein said at least one head comprises a chemical reaction detection mechanism including an electromagnetic energy receiver, whereby electromagnetic emissions caused by chemical or biological reactions at said at least one reaction site are channeled to an electromagnetic energy detector for detection.
15. 19. The apparatus of claim 1, wherein said at least one reaction site is a geometric cavity formed into said substrate. The apparatus of claim 14, wherein said at least one reaction site is a geometric cavity formed in said substrate, said geometric cavity having a plurality of surfaces which are oriented, such that said electromagnetic energy is reflected within said cavity.
16. 21. The apparatus of claim 15, wherein said at least one reaction site is a geometric cavity formed in said substrate, said geometric cavity having a plurality of surfaces which are oriented, such that said electromagnetic energy is reflected within said cavity to increase an electromagnetic energy path length.
17. 22. The apparatus of claim 20, wherein reflective barriers are formed on a periphery of each of said cavities to prevent electromagnetic interference from adjacent cavities.
18. 23. The apparatus of claim 1, wherein said substrate is a manufactured from a material selected from the group consisting of glass, ceramics, semiconductor materials, plastics, composites, and combinations thereof
19. 24. The apparatus of claim 1, wherein said substrate includes solid support structures formed within said at least one reaction site, which provide a plurality of points at which probes affix to said at least one reaction site. The apparatus of claim 24, wherein said structures are formed from an electrometal material.
20. 26. The apparatus of claim 1, wherein said at least one fluid comprises discrete amounts of at least one fluid aliquot and at least one separating fluid and wherein said dispenser comprises a pump, which includes conduits whereby said pump alternately draws at least one first discrete amount of said at least one fluid aliquot and at least one second discrete amount of said at least one separating fluid into a dispenser tube and delivers a serialized fluid of said at least one fluid aliquot and said at least one separating fluid to said at least one dispenser under controlled pressure; at least one suction device, which is adapted to draw said stream from said at least one dispenser; and at least one timing device for controlling said at least one suction device.
21. 27. The apparatus of claim 26, wherein said at least one timing device measures a flow rate of said stream through said at least one dispenser tube and deactivates and subsequently reactivates said at least one suction device, such that a portion of said at least one first discrete amount of said at least one fluid aliquot is delivered to said at least one reaction site.
22. 28. The apparatus of claim 26, wherein said first discrete amount of said at least one fluid aliquot is substantially identical to said at least one second discrete amount of said at least one separating fluid, wherein said at least one fluid aliquot includes a flow sensor.component, and wherein said at least one timing device includes a flow sensor for detecting said flow sensor component as said flow sensor component passes said sensor.
23. 29. The apparatus of claim 27, wherein said flow sensor component is a plurality of magnetic beads and said flow senor is a magnetic sensor. The apparatus of claim 1, wherein said at least one fluid comprises discrete amounts of at least one fluid aliquot and at least one separating fluid and wherein said at least one fluid dispenser comprises at least one pump, which includes conduits whereby said pump alternately draws at least one first discrete amount of said at least one fluid aliquot and at least one second discrete amount of said at least one separating fluid into a dispenser tube and delivers a serialized fluid of said at least one fluid aliquot and said at least one separating fluid to said at least one dispenser under controlled pressure; a valve mechanism whereby a portion of said serialized fluid is dispensed from said at least one dispenser to said at least one reaction site; said at least one suction device for removing an unwanted portion of said portion of said serialized fluid draws said stream; and at least one timing device for controlling said valve mechanism and said at least one suction device.
24. 31. The apparatus of claim 30, wherein said at least one dispenser extends perpendicular to a direction of fluid flow within said dispenser tube and said valve mechanism comprises a controller and a valve controlled by said controller, said value being positioned downstream from said at least one dispenser and said controller being controlled by said at least one timing device, such that when said valve is closed, said at least one fluid flows to said at least one dispenser outlet.
25. 32. The apparatus of claim 1, wherein said at least one dispenser extends perpendicular to a direction of fluid flow within at least one dispenser tube and a valve mechanism for controlling fluid flow from said at least one dispenser, said valve mechanism comprises a controller and a valve controlled by said controller, said valve being positioned downstream from said at least one dispenser and said controller being controlled by at least one timing device, such that when said valve is closed, said at least one fluid flows to said at least one dispenser outlet.
26. 33. The apparatus of claim 32, wherein said at least one timing device measures a flow rate of said fluid flow through said at least one dispenser tube.
27. 34. The apparatus of claim 32, wherein said valve mechanism comprises a four-way valve in said dispenser tube; a first controller and a first valve controlled by said first controller, said first value being positioned downstream from said four-way connection; and a second controller and a second valve controlled by said second co ntroller, said second valve being positioned upstream from said four-way connection, and wherein said at least one dispenser extends from a first orifice of said four-way connection and a dispenser pump is joined to a second orifice of said four-way connection, such that said first and second controllers are controlled by said at least one timing device and when said first and second valves are closed, a dispenser fluid is pumped through said four-way connection and forces said at least one fluid to said at least one dispenser outlet. The apparatus of claim 1, wherein said at least one fluid dispenser ejects a micro- droplet stream of said at least one fluid from said at least one dispenser and an electrostatic accelerator and deflector directs said micro-droplet stream to said at least one reaction site.
28. 36. The apparatus of claim 1, wherein said means for identifyting includes an light source and a florescence detector and wherein a plurality of said at least one fluid aliquot is delivered to said plurality of reaction sites and said at least one fluid includes a flourophor, whereby florescence occurs at least one of said plurality of reaction sites.
29. 37. The apparatus of claim 1, wherein said substrate comprises a plurality of layers of a semiconductor material and said apparatus further comprises at least one electronic element is formied on said plurality of layers of semiconductor material and joined to said at least one reaction site.
30. 38. The apparatus of claim 37, wherein said at least one electronic element is selected from the group consisting of transponders, heating coils, temperature sensors, electric field generating elements, photosensing elements, electrophoresing elements, denaturing elements, chemically sensitive gates, ion sensitive gates, interdigitated arrays, and combinations thereof
31. 39. The apparatus of claim 37, wherein said, at least one electronic element comprises a plurality of electronic elements and said plurality of elements are interconnected. The apparatus of claim 39, further comprising a spindle around which said IS substrate rotates and wherein at least a pair of said plurality of electronic elements are radially interconnected through said spindle.
32. 41. The apparatus of claim 39, wherein at least a pair of said plurality of electronic elements are axially interconnected,
33. 42. The apparatus of claim 39, further comprising a multiplexor for interconnecting said plurality of electxrnic elements.
34. 43. The apparatus of claim 1, further comprising at least one electro-mechanical element that is formed on said substrate at said at least one reaction site. 44, The apparatus of claim 43, wherein said at least one electro-mechanical element produces a vibration to agitate said at least one fluid at said at least one reaction site. The apparatus of claim 1, wherein said means for rotating comprises a first rotary stepper motor; said at least one dispenser outlet is mounted on a pivot armn, said pivot arm being mounted on a second rotary stepper motor, such that said at least one dispenser outlet is controllably pivotable over said rotatable substrate.
35. 46. The apparatus of clam 1, wherein said substrate and said at least one fluid dispenser are enclosed within an airtight container.
36. 47. The apparatus of claim 1, wherein said at least one reaction site is a geometric cavity formed in said substrate, said geometric cavity having a shield structure to prevent fluid loss from said reaction site.
37. 48. The apparatus of claim 47, further comprising a pressure control device for controlling atmospheric pressure within said container.
38. 49. An apparatus for performing a plurality of assays comprising: an axially rotatable substrate including a plurality of radially-arrayed reaction sites; .means for rotating said substrate; at least one multi-function head including at least one fluid dispenser for conveying at least one fluid to at least one of said reaction sites, including at least one fluid dispenser outlet, and a at least one readout device; means for identifying said at least one reaction site; and means for aligning said at least one miulti-fuinction head, such that said at least one fluid dispenser outlet is aligned with said at least one reaction site; wherein said means for rotating is controllable to rotate said substrate at a speed, such that a portion of said at least one fluid is removable from said at least one reaction site by a centrifugal force generated by the rotation of said substrate. The apparatus of claim 49, further comprising at least one channel joining said at least one reaction site to at least one other reaction site and said at least one fluid is conveyed from said at least one reaction site to said at least one other reaction site by said centrifugal force generated by the rotation of said substrate.
39. 51. An apparatus for performing a plurality of assays comprising: an axially rotatable substrate including a plurality of radially-arrayed reaction sites; means for rotating said substrate; at least one multi-function head including at least one fluid dispenser for conveying at least one fluid to at least one dispersion point, and a readout device; means for identifying at least one of said reaction sites; and at least one channel joining said at least one dispersion point to said at least one reaction site; wherein said means for rotating is controllable to rotate said substrate at a speed, such that said at least one fluid is conveyed from said at least one dispersion point to said at least one reaction site by a centrifugal force generated by the rotation of said substrate.
40. 52. An apparatus for performing a plurality of assays substantially as herein described with reference to any one of the accompanying figures. Dated this twenty-seventh day of November 2002 Alexion Pharmaceuticals, Inc. Patent Attorneys for the Applicant: F B RICE CO