CA2172210A1

CA2172210A1 - System for nucleic acid based diagnostic assay

Info

Publication number: CA2172210A1
Application number: CA002172210A
Authority: CA
Inventors: Allen S. Reichler; David J. Antol; Michael L. Lamos; Peter A. Bourdelle; Scott D. Hildebrand
Original assignee: Becton Dickinson and Co
Current assignee: Becton Dickinson and Co
Priority date: 1995-03-24
Filing date: 1996-03-20
Publication date: 1996-09-25
Also published as: JPH08266267A; EP0733905A2; US5578270A; EP0733905A3

Abstract

An automated system is provided for carrying out nucleic acid based assays on a plurality of liquid samples with little or no intervention by a human operator. The system includes at least one reaction station adapted to hold and apply controlled amounts of heat to a plurality of reaction devices in which theliquid samples are receivable. Each of the reaction devices includes a sample area for receiving a liquid sample, a reaction area into which the liquid sampleis movable to carry out a nucleic acid decontamination or amplification reactionon the sample, and a pneumatic port for allowing air to be aspirated from and dispensed into the reaction device to move the sample between the sample area and the reaction area. A robotically controlled aspiration and dispensing head is adapted to move into contact with the pneumatic ports of the reaction devices, and to aspirate air from and dispense air into the pneumatic ports of the reaction devices in order to move the liquid samples between the sample and reaction areas of the reaction devices. A programmable control system is provided for causing the robotically controlled aspiration and dispensing head to move into contact with the pneumatic ports of the reaction devices, and to aspirate air from and dispense air into the reaction devices in order to cause the desired movement of the liquid samples within the reaction devices. The robotically controlled aspiration and dispensing head is also adapted to transfer the liquid samples toand from the reaction devices using disposable pipettes, and to introduce liquidreagents into the reacted samples after the samples have been transferred from the reaction devices to separate assay devices. A robotically controlled wash head is also provided to aspirate and dispense wash fluids and reagent fluids from the assay devices.

Description

rATE~rr SYSTEM FOR NUCLEIC ACID BASED
DIAGNOSTIC ASSAY
Cross-Referenoe to Related Applications r~l~t~ subject matter is disclosed in a copending patent application of Allen S. Reichler et al, filed on even date he~..;lh and entitled "Nucleic Acid Amplification Method and Apparatus" (Attorney's File 2573-P1), and in a copending U.S. patent application of Michael L Iamos et al, filed on even date herewith and entitled "Pipette Tip" (Attorney's File P-31g3), both of said applications being expressly incorporated herein by reference.

Field of the Invention The present invention relates to an automated system for carrying out reactions on a pluralib of liquid s~mp~es~ and particularly relates to an automated system in which loL ~ slly controlled fluid aspirating and dispensing heads executing programrned movements are used to carry out nucleic acid based diagnostic assays on a pluralib of liquid biological samples with little or no ntion by a human operator.

Background of the Invention In the clinical diagnosis of ~i.~&tory bacterial ~l-e-~-r such as tuberculosis, a sample of sputum or other body fluid obtained l~om the paffent is cultured in an agar growth medium to test for the presenoe of the particular bacterium of interest. Unfortunately, this is a relatively time-consuming prooess, generally requiring several days to produoe a definitive result. During this time, a patient suspected of having tuberculosis, for eYqmple, must be i~ t~ to prevent fLrlher spread of the disease.
The advent of DNA probes, which can identify a specific bacterium by testing for the presence of a unique DNA sequenoe in the s~mple obtained from the patient, has greatly increased the speed and reliability of clinical diagnostic 2 1 722 ~ O

tes~i~. A test for the tuberculosis ~ rDb~terium, for example, can be completed within a matter of hours using DNA pnDbe technology. This allows treatment to begin more quicldy and avoids the need for long patient isolation times.
In the use of DNA pnDbes for clinical diagnostic purposes, a nucleic acid amplification reaction is carried out in order to multiply the target nucleic acid into millions of copies or amplicons. Examples of nucleic acid amplification reactions which can be used include strand displaoement amplification (SDA) and polymerase chain reaction (PCR). Unfortunately, nucleic acid amplificaffon reactions can become contaminated with the amplicons produoed by previous amplificaffon reacffons. The contaminating amplicons can, in turn, contaminate new samples entering the lab, leading to false posiffve diagnoses.
Decontamination techniques have been developed in which contaminaffng amplicons produoed by previous amplification reacffons are ~zed and destnDyed. By carrying out a decont~mirnt~ ~n reaction prior to amplificaffon, the possibility that a contaminaffng ampliaDn will be ~nized as a target nucleic acid is greatly decreased. IIowever, because decontamination and amplificaffon reagents are often not con~p~tihle with each o~her and require their own reaction condiffons, they must often be carried out in epa~ale containers. In transferring the sample fnDm one container to another, recontaminaffon can occur.
In order to minimize contaminaffon pnDblems, separate areas of a clinical diagnostic laboratory are often l~ee.~_d for sample preparaffon, amplificaffon/
decontaminaffon and assay (detection). Although this is an eff~t;~ safeguard, it is very labor-intensive and offsets some of the advantages offered by DNA probe techniques. Automaffon of all or part of the prooedure would be desirable, but this is difficult to achieve when many processing steps are involved and the potential for cross-contamination b~ e.. samples is great.
In the aforementioned copending patent application of Allen S. Reichler et al entitled "Nucleic Acid Amplification Method and Apparatus", a disposable, - 3 - rATENT

single-use apparatus or module is described which allows decontamination and amplification of a liquid bic~ nl sample to be ~liC G~ out within the confines of a single container. In general, the disclosed apparatus includes a sample area for receiving a liquid biological sample, at least one reacffon area in fluid communicaffon with the sample area, a pneumaffc area in pneumatic communication with the reacffon area and the sample area, and a pneumatic port in the pneumatic area for allowing connection of the apparatus or module to a pneumatic aspiration/dispensing means. Operation of the pneumatic aspiration/
dispensing means causes the liquid L ~ ol sample to flow between the sample area and the reaction area, and between different zones in the reaction area, ina controlled manner. Reagents necessary for the decontamination and ampliScation reactions are affiYed to separate, discrete locations within the reaction area, and are contacted by the liquid biological sample at different times under the control of the pneumaffc aspiration/dispensing means.
It is an object of the present invention to provide an automated system for carrying out reactions on a plurality of liquid samples using ~ p~l^qble~ single-use modules of the general type described ab~ove.
It is another object of the invention to pl~..d* an au~m~ted system for carrying out reactions on a pluralib of liquid samples, particularly nucleic acid based diagnosffc assays, with little or no inter-~ention by a human operator.
It is further object of the invenffon to ~o.-d~ an automated system for carrying out reactions on a plurality of liquid samples, particularly nucleic acid based diagnostic assays, while minimizing the potenffal for cross-contaminaffon between different samples.
It is a still further object of the invenffon to provide improved methods for carrying out reactions on a plurality of liquid samples, particularly nucleic acid based diagnostic assays, which methods can be carried out using the ~Y~mpl~ry apparatus disclosed and claimed herein.

P 33Cl Summary of the Invention In accordanoe with one aspect of the preænt invention, an automated system for carrying out reactions on a pluralib of liquid samples comprises a reaction station adapted to hold a plurality of reactions devioes in which the liquid ~smpl~e~ are receivable. Each of the reaction devioes includes a sample area for receiving a liquid sam~lc, a reaction area into which the liquid sampleis movable to carry out a reaction on the sample, and a pneumatic port for allowing air to be a~l-~at~d from and ~licpP ced into the reaction devioe to move the liquid ssmrle between the sample area and the reaction area. A robotically controlled aspiration and di~pen~ing head is adapted to move into contact with the p"o--mst:c ports of the reaction devioes, and to aspirate air from and dispenæ
air into the pneumatic ports of the reaction devioes in order to move the liquidsample in the reaction devioes L~h ~e~ the sample and reaction areas of the reaction devioes. A ~.o6~ 1e control devioe is provided for causing the robotically controlled aspiraffon and dispensing head to move into contact with the pne~mst:c ports of the reaction devioes, and to hs~Udle air from and dispP~eair into the reaction devioes in order the move the liquid samples between the sample areas and the reaction areas of the reacffon devioes.
In another aspect of the ~l~s_.ll invention, a reaction station for use in an automsted system for carrying out reaction on a plurality of liquid samples comprises a fLlced heaffng platen for heating the liquid samples, and a removable tray posiffonable on the heaffng platen. The removable tray is adapted to hold a plurality of reaction devioes in which the liquid samples are reoeivable. A
locating devioe is ~u.idcd for locating the removable tray at a pl~el~nined position on the heaffng platen.
In a further aspect of the present invention, an assembly for use in an g l-lmst~ system for carrying out reacffons on a plurality of liquid samples comprises a plurality of reaction devioes in which the Uquid samples are receivable, and a tray adapted to hold the pluraUty of reaction devioes. The s rAT~r r33c reaction devices ha~e svbst~ lly flat bottom surfaoes through which heat can be applied to the liquid samples. The tray is formed with shaped slots or caviffes for reoeiving each of the reaction devices in a p~det~.~ined position and orientation, and with cut-out portions for allowing the substantially flat bottom surfaoes of the reaction devioes to make direct contact with a heating platen.
In a still further aspect of the present invention, an assay devioe for use in an automated system for carrying out reactions on a liquid sample comprises a plurality of connected wells for reoeiving portions of the liquid sample. Eachof the wells has an open top for rl~ifflrlg a portion of the liquid sample, a substantially nat bottom surfaoe for making contact with a heating platen, and interior walls coated with a ~isgr~ s'ic reagent.
The present invention is also directed to methods for carrying out reactions on liquid samples, which . ~ may be implemented using the exemplary apparatus disclosed and claimed herein.

Brief Description of the D~&~.mgs The various objects, advantages and novel features of the invention will be more readily apprehended from the follow~ng detailed description when read in co~unction with the appended ula~.~gi.~ in which:
Fig. 1 is a ~ view of the principal components of an automated system for carrying out nucleic acid based diagnostic assays in accordanoe with a p,~f~ d embodiment of the ~l~se~l invention;
Fig. 2 is a ~ . view of the cabinet or enclosure in which the assays are performed, with the doors of the apparatus shown in an open position to illustrate oertain internal d-t~
Fig. 3 is a detailed ~~ view of the cabinet interior, illustrating the stations p~.idcd in the system and the robotic arms which perform various programmed functions at these stations;

~ 1 7~ 3 U

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Fig. 4 is an exploded ~.li._ view similar to that of Fig. 3, illustrating the manner in which the components of oertain stations are removable, Figs. 5A - SC are detailed views illustrating the manner in which a di~posoble pipette tip is picked up and ejected by one of the robotic arms;
Figs. 6A and 6B are detailed views illustrating the manner in which a pneumatic aspiration and dispensing pipette is picked up and ~ ed by the robotic arm of Figs. 5A - 5C;
Figs. 7A and n are d t~;l ~7 ~ ;.~.li.~ and side views of a wash head that is plo.;ded on a second robotic arm for dispensing and aspirating wash fluid;
Figs. 8A and 8B are d ~oi~- d top plan and sectional views, l~,~.ti._l~, of one of the removable trays shown in Figs. 3 and 4;
Figs. 9A and 9B are ~d~Pi'- d top plan and sectional views similar to those of Figs. 8A and 8B, with a reaction devioe and an assay devioe shown in the removable tray;
Figs. 10A and lOB are detPil-d top plan and sectional views similar to those of Figs. 9A and 9B, with the removable tray shown in position at one of the reaction stations of Figs. 2 - 4;
Figs. 11A and 11B are a ~ views illustrating two possible ents of the assay devioes;
Fig. 12 is an enlarged cross se.lional view of one of the reaction d .i~
illustrating the plaoement of the decontamination and amplification reagents in the ~ n area;
Fig. 13 is a cross se.1ional view of the lo ver portion of the cabinet shown in Figs. 1 - 4, illustrating the arrangement used for cooling the reaction devioe heating pls~n~ during intervals when these heating platens are deacti~vated;
Figs. 14A - 14T are sequence views illustrating the plv~med sequenoe of movements executsd by the two robotic arms of Fig. 3 during an automated nucleic acid assay;

Fig. 15 is a block diagram of the principal iiuidic and pne~mstic components of the automated assay ~ ,t~;
Fig. 16 is a block diagram of the principal electrical components of the automated assay ~;,te~, and Fig. 17 is a aow chart illustrating the sequence of operations carried out by the computer shown in the block diagram of Fig. 16.
Fig. 18 shows the aspiration and ~ ellsillg head carried by the robotic arm of Fig. 3.
Throughout the drawings, like reference numerals will be understood to refer to like parts and components.

Detailed Descl;~lion of the Preferred Embodiments Fig. 1 illustrates an automated nucleic acid based diagnostic assay system 20 constructed in accordanoe with a p~f~.,~ embodiment of the present invention. The system includes a cabinet or enclosure 22 which houses the principal components of the system and the liquid samples to be assa~ed. At the front of the cabinet 22 is a bottom-hinged door 24 which affords access to the cabinet interior, a slide-out drawer 26 which pro~ides access to the system computer, and a side-hinged clear plastic door 28 which allows access to the containers and syringes used for dispensing f~uids. A rear-hinged top door 29 isalso provided to improve the o~.alol s access to the interior of the cabinet 22.The doors 24, 28 and 29 and the drawer 26 are shown in their closed positions inFig. 1. The cabinet 22 has dimensions suitable for pl~ ent on a laboratory counter or tabletop 30, as shown, for convenient access by laboratory personnel.Waste fluids that are produced by the system 20 are pumped into a waste bottle 32 by means of a flexible waste tube 34 that is coupled to a fitting (not shown)on the left side of the cabinet 22. The system computer (not shown) housed within the slide-out drawer 26 is conr~ected to a keyboard 36 (with an i ~
mouse or trackball 40), a numeric keypad 37, a video display unit 38, and a printer 42. These components are provided to allow laboratory personnel to program and initialize the system 20, to select among various system options, and 2 1 722 ~ O
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r~

to monitor the status of the system during automated operation. Also connected to the system computer is a luminometer 43 that performs a chemiluminescent detection step at the conclusion of the automated assay.
Fig. 2 is a detailed pe~ view of the cabinet 22, with the slide-out drawer 26 and doors 24, 28 and 29 shown in the open position. IIoused within the slide-out drawer 26 behind a removable panel 45 is the system computer 44, which is preferably a Model MS-32 computer manufactured by Advanoe Modular Solutions of Acton, Massachusetts. The system computer 44 includes a floppy disk drive 47 which may be used for installing software u~s At the left-hand side of the cabinet 22, the area behind the transparent door 28 houses a first fluid supply bottle 46 for containing a system fluid (typically consisting of water with preservatives) and a second fluid supply bottle 48 for containing a stringency wash solution. The tubes 50 and 52 allow fluids to be drawn from the bottles 46 and 48, res~ , by means of automatically controlled syringe pumps or diluters 54 and 56 - 60. Fluid valves 62 and 641 through 643 (the latter shown behind a cover plate 64), also controlled automatically by the system 20, allow the syringes 54 and 56 - 60 to withdraw nuids from the supply bottles 46 and 48, andto dispense these fluids at p~ d e l~nined locations within the p~ces .;ng area 66 of the cabinet 22 during the automated assay procedure.
With continued referenoe to Fig. 2, the upper door 29 is held in its open or elevated position by means of a holding devioe 68 that is Uh~ by the frame of the cabinet 22. The front door 24, which interlits with the upper door 29 andcabinet opening 70 in a light-tight ~ m~hell manner, is held securely in its horizontal open position by means of stops (not shown) and is slidable by a short distance into a slot 72 lo~ d at the bottom forward edge of the cabinet 22. In this orientation, the nat interior surfaoe 74 of the door 24 p~.i~- a convenientwork surface for laboratory personnel during fi~tDllDtion and removal of comrorents from the p.ocessing area of the cabinet 22. The light-tight fit between the doors 24 and 29 and the cabinet opening 70, which is made possible P~361 by complementary labyrinthine seals formed around their peripheries, allows for the possibility of carlying out chemiluminescent detection within the cabinet 22rather than in a separate luminometer 43.
The components located in the p.o~s;.lng area 66 of the cabinet 22 are shovvn in Figs. 3 and 4. In general, the processing area 66 is defined by a flatlocator plate 76 which is mounted on the deck or base plate 77 of the cabinet 22.
Various stations are provided on the locator plate 76, or on the deck M within cut-out areas of the locator plate 76, for the components ~ d for carrying out the desired nucleic acid based assay. Included among the stations are four identical reaction stations 78, 80, 82 and 84, where the principal p~o~s~.ng steps are carried out on the liquid biological samples to be ass.,~eL Each reaction station l~oe;~_s a removable tray 86 which holds a plurality of reaction d . ~ e -88 and a comsponding plurality of assay de~ices 90. In the preferred embodiment, twel~e reaction devioes 88 and Iwel~e assay ~ . ~ es 90 are ~.;~
by each tray 86. The reaction devices 88 and assay ~d .;~er 90 are heated 1~om the bottom by elongated heating platens 92 and 94, ~ el~, which are installed in the deck or base plate 77. The trays 86 are formed with cut-out portions 96 and 98 for allowing direct contact~etween the bottoms of the de~ioes88 and 90 and their comsponding heating platens 92 and 94. In addition, the top surfaoes of the reaction de~ices 88 are heated by an upper heating platen 100 which is carried by a pivotable arm 102. The arms 102 are carried by a hinges 104 l~-n~ed at the rear of the reaction stations 78 - 84, and are locked in the downward position by means of pivotable U-shaped ~lsmps 106 lcr~t~d at the fon~ard end of the reaction stations 78 - 84. The pivotable arms 102 serve the dual purpose of bringing the upper heating platens 100 firmly into contact with the upper surfaoes of the reaction de~ioes 88, and locking the trays 86 into position on the deck or base plate 76. For illu:".,.li~ purposes, the third reaction station 82 in Fig. 3 is shown with the pivotable arm 102 in the open position and - 10- rATENT
r-336l the tray 86 removed, and both the arms 102 and trays 86 have been deleted for all but the first reaction station 78 in Fig. 4.
Immediately to the left of the first reaction station 78 in Figs. 3 and 4, a sample tube station 108 is pfo.ided The sample tube station includes a remo~able metal rack 110 compri~ing three spaced-apart metal plates 112, 114 and 116. The two upper plates 112 and 114 are formed with rows of aligned apertures 118 which receive and locate a plurality of sample tubes 120. The bottom plate 116 does not include apertures and serves as a base for supporting the sample tubes 120. The metal plates 112, 114 and 116 are held in a parallel, s~- ~e ~ ~prrt relationship by means of metal spaoers 122. The sample tube rack 110 is removable as a whole from the reaction area 66 of the cabinet 22, as indicated in the exploded view of Fig. 4. The locator plate 76 includes a pair of upstanding metal pins 124 which engage corresponding apertures (not shown) in the bottom plate 116 of the sample rack 110 in order to locate the rack 110 in ade l ~rmined position on the deck 76. In practice, the rack 110 will ordinarily be remo~ed from the cabinet n for filling before the start of a nucleic acid assay, and then placed in the position defined by the pins 124 after sample tubes 120 containing the liquid bilsl~ l samples to be~assayed have been placed into the apertures 118. Only a few sample tubes 120 have been shown in Figs. 3 and 4 for clarity, although it will be understood that the rack 10 can accommodate as manysample tubes 120 as there are apertures 118 in each of the plates 112 and 114.
The number of sample tubes 120 used during any given assay will, of course, depend upon the number of liquid biological samples to be &SSa~O~
~ tffl rearwardly of the sample tube staffon 108 in Figs. 3 and 4 is a ~i~pos~lP pipette tip station 126. The ~i~pos~ble pipette tip station 126 includes a rack 127 consisting of a pair of parallel, 3r ~ ~ e d ap~rt metal plates 128 and 130 which are formed with aligned rows of a~ s 132 for receiving and locating a plurality of disposable pipette tips 134. The plates 128 and 130 are held apart by metal ~ .s 136, and the rack 127 as a whole is supported on the locator - 11- PATENr plate 76 by means of six metal supports 138. A metal locating fixture 140 is afffxed to the locator 76 and contains holes 142 for .~oe;~; two of the six metal supports 138 of the rack 127 (Sp~~ olly~ the left-hand and middle supports adjoining the rear of the locator plate 76). In this way, the rack 127 is l~te~lat a known position on the le~t~r plate 76, and the same is true of the individual ~icposnl~le pipette tips 134. As irdi-u~ed in Fig. 4, the .li~ nble pipette tip rack 127 is remo~abk from the deck 76 to make it easier to replenish the supply of d r~; ~nble pipette tips 134. Although only a few sl s~ ~s~ble pipette tips 134 are illustrated in Figs. 3 and 4 for clarity, it will be understood that a large number of ~licrQso~le pipette tips (typically 192 or approximately 4 per sample in the illustrated emho~liment) will ordinarily be provided in the rack 127.
As will be described in some detail hereinafter, the ~ispos~ble pipette tips 134 are used by the system 20 to aspirate and dispense both the liquid biological samples themsel~es and ~arious reagents that are used during the nucleic acid assay. For this purpose, the dicpos~ble pipette tips 134 may be of the conventional type, consisting of au~l~...ble polypropylene with a maximum volume of 300 microliters (I.L). Howe~rer, in order to prevent sample and reagent fluids from being drawn into the robotic aspi~ation and dispensing system (to bedescribed shortly) with which the ~icpo~ble pipette tips 134 are used, each tip 134 is modiffed by providing a plug or insert of filter material (not shown) near its upper end. The fflter material allows air to pass for pneumatic aspiration and dispensing purposes, but blocks the passage of sample and reagent fluids. The filter material is described in detail in the aforementioned copending patent application of Michael L. Lamos et al, entitled "Pipette Tip" (Attorney's File P-3193), which is inco~3~ted herein by referenoe.
Dicpo~ol~le pipette tips are conventionally sold in plastic boxes with cavities or apertures for holding the tips in a rectangular array. If desired, aconventional plastic box of this type may be used in plaoe of the metal rack 127shown in Figs. 3 and 4. An ~Y~mple of a ~ sor le pipette tip box of the type r-336l conte~nr'^t~ is ~li~losed in U.S. Patent No. 4,577,760, to Rainin et al, which is incorporated herein by referenoe.
To the left of the sample tube station 108 and dicposoble pipette ffp station 126 in Figs. 3 and 4 is a pipette tip disposal station 142. The pipette tip lI;C~J~1 station 142 comprises a rectangular box 144 which is supported on the deck or base plate 77 in a shallow cut-out area 146 of the lc-otr- plate 76. The rectangular box 144 is closed on all sides, exoept for a slot or opening 148 which occupies the top right-hand area of the box and extends from front to rear. The slot or opening 148 allows used pipette tips 134 to be dropped or ejE ~ted into the box 144 by a robotic aspiration and dispensing arm, as will be described shortly.
In the preferred ~ iment, the box 144 has an internal volume sufficient to contain appr~Yi~nDt~ly 384 ~licpo~qble pipette tips. When the box 144 has reached its maximum capacity, it can be removed and emptied by laboratory personnel as shown in Fig. 4. For con~enienoe in grasping the box 144 during remo~al, foam spaoers 145 are ~lO. ~ed o the left side of the box 144 to s~l..te the box 144 from the adjaoent interior wall (not shown) of the cabinet 22, and an elongated groo ~e 147 serving as a finger grip is formed along the lower right-hand side of the box 144.
With conffnued referenoe to Figs. 3 and 4, the ~-r~on area 66 of the cabinet 22 also includes a purging staffon 150, a docking staffon 152 for pneumatic aspiraffon and dispensing pipettes, and a reagent station 154. The purging staffon 150 includes a llce st~nding wash cup 156, which is generally rectangular in shape with a cavib or depression 158 in its upper surfaoe to provide a auid reoeptacle. The wash cup 156 ~ auids which are discharged during ~.;Qdic purging of the robotic arms used in the reaction area 66 of the cabinet 22, and may be .~ d from the deck 76 for cleaning as shown in Fig.
4. The docking station 152 includes a metal bracket 160 which is affixed to the deck 76 at a location behind the purging station 150. The bracket 160 has a fon~ardly-extending horizontal lip or flange 161 which is formed with a pair of U-s~a~l notches or cut-outs 162 for ~-cohly carrying a pair of pneumatic aspiration and dispensing pipettes lC4. The pneumatic aspiration and dispensing pipettes 164 are used to induoe sample fluid movement v~ithin the reaction devioes 88 at the reaction stations 78 - 84, as will be ~es~ihed below. The reagent station 154 includes a machined plastic holder 166 which is ~ in a shallow cut-out area 168 of the locator plate 76. The holder 166 is generally ~ _dge-f~oped, v~ith a flat bottom surfaoe 170 and an inclined upper surfaoe 172in which rows of cavities 174, 17C and 178 are formed for holding open reagent bottles 179, 180, 181 and 182 and the caps 184 which have been removed from these b Ott~f S. The reagent bottle cavities 174 and 176 are all cylindricaa in shape, with the uppermost cavity 174 in each row being larger in diameter than the remaining cavities 176 in order to hold a larger reagent bottle 182. The ca~ities 178 for holding the reagent bottle caps 184 are all of the same size, and are semicylindrical in configuration so that the caps 184 are held on their sides asshown. In the specific type of nucleic acid assay to be described below, only four liquid reagents (and henoe four reagent bottles) are required. ~Iowever, the t bottle holder 166 is pre&rably formed with a number of spare reagent bottle and cap cavities, as shown, so that thl~ system 20 can be used for other types of assays in which larger numbers of reagents are required. Alternatively,the spare reagent bottle positions allow the system 20 to be used for carrying out different types of assays on different samples (or groups of samples) at the same time. The reagent bottle holder lC6 is removable from the reaction area 66 of the cabinet 22, as illustrated in Fig. 4, for storage, replenishment and cleaning.
Apertures (not shown) formed on the bottom surfaoe 170 of the holder 166 are engaged by IQ -nting pins 188 affixed to the deck 77 in the cut-out area 168 of the locator plate 76. In this way, both the holder 166 and the individual reagent bottles 179 - 182 are held at p~det~rmined positions within the r~h~n area 66.
If desired, the machined plastic reagent bottle holder 166 may be replaced with a sheet metal rack having rectangular cut-outs, and the reagent bottles 179 - 182 may be packaged as a single unit that is ~ d in one of the r~ngular cut-outs.
In order to transfer the liquid samples and reagents between different containers and locations during the nucleic acid assay, the system 20 includes apair of pro~.--.--cble, independently movable robotic arms 190 and 192 which are movable in three dimensions above the various stations 78- 84, 108, 126, 142, 150, 152 and 1S4 in Fig. 3. The left-hand arm 190 is referred to as the hydropneumatic aspiration and dispensing arm, and the right-hand arm 192 is r. f~ d to as the wash arm. E:xoept for the pneumatic aspiration and dispensing head 216 affixed to the lower end of the arm 190 and the wash head 194 affixed to the lower end of the arm 192, the robotic arms 190 and 192 are conventionaL
A suitable robotic system including the two robotic arms 190 and 192, a fluid aspiration and dispensing system for the arms, and a p~mm-ble control system for controlling the arm movements and fluid aspiraffon and dispensing funcffons, is the TECAN Model RSP 9652 auto---;ed pipetting instrument manufactured by TECAN AG of EIombrechffkon, ~ ;~ nd. Both of the arms 190 and 192 are supported from the rear by means of a horizontal track 196, which allows each arm to be moved independently in the x direcffon (i e., in thedirection parallel to the rear edge of the locator plate 76) by stepper motors under microprocessor control. Each arm 190 and 192 is canfflevered outwardly from the track 196 toward the forward edge of the locator plate 76. The hydropneumatic aspiraffon and dispensing arm 190 includes an elongated metal enclosure 198, open at the bottom, which houses a y - z stepper motor dri~e system for a vertical guide rod 200 and hollow gear rack 202. A slot 204 in the enclosure 198 p~.ides clearanoe for the movement of the guide rod 200 and gear rack 202 in they direction (i.e., toward or away f~om the front edge of the locator plate 76). The guide rod 200 and gear rack 202 are also movable vertically through the slot 204 (i.e., toward or away from the surface of the deck 76) to provide the z-direction movement of the arm 190. The wash arm 192 is generally - 15 2 1 7 2 2 1 0 PATE~

s;~ilDr in construction, and includes an elongated metal enclosure 206, a hollowguide rod 208, a gear rack 210 and a slot 212 for allowing movement of the guiderod 208 and gear rack 210 in the y and z direcffons.
The arm 190 carries out hydropneumaffc aspiraffon and dispensing funcffons, and is fitted with an aspiraffon and dispensing head 21C. The aspiration and dispensing head 216 terminates in a tapered metal tip 218 which is used either for aspiraffng or dispensing controlled amounts of air, or for dispensing system fluid. During aspiration and dispensing operaffons, the metal ffp 218 carries either a ~liqposPhle pipette ffp L34 or one of the pneumatic aspiraffon and dispensing pipettes 164. A flexible tube 221 passes through the hollow gear rack 202 to allow aspiraffon and dispensing to be carried out through the metal ffp 218. The wash arm 192 is fitted with a wash head 194 for a purposeto be described shortly, and a plurality of flexible tubes 214 pass through the hollow guide tube 208 to carly wash fluids to and ~om the wash head 194.
Sinoe the roboffc arms 190 and 192 are, with the exoepffon of the s~ifi~
components mentioned previously, part of a commercially available apparatus, their construction and operation need not be described in detaiL In general, however, the functions of the hydropneumaffc~aspiration and dispensing arm 190 may be summarized as follows: (a) picking up and ejecting liqposoble pipette tips 134 and pneumatic as~Alion and dispensing pipettes 164, (b) liquid level ~t ~ion, (c) controlled st~..;e movement along the ~; y and z axes, and (d) aspiraffon and dispensing of air and liquids.
Picking up a ~licpo~oble pipette tip L34 is accomplished by controlling the arm 190 to place the metal ffp 218 verffcally above one of the (lic~oble pipettetips L34 in the rack 127, and then lowering the head 216 by a p~ e~P~ined number of steps s~ d to be below the point at which a ~lisr ~ -oble pipette ffp 134 is e.lgaged. Engagement of the pipette ffp L34 displaoes a slidable plasffc ejector sleeve 228 (best seen in Figs. 5A - SC) located just above the metal ffp218. By then retTacting the head 216 to its home posiffon, which is deffned by an 2 ~ ~

r-336l electrical contact attached to the upper end of the ejector sleeve 228, the system can determine whether a ffp L34 was engaged by comparing the number of steps travelled upwardly and downwardly, which will differ by an amount corresponding to the ejecffon sleeve displaoement. After use, the ~lis~soble pipette ffps L34 are ejected from the head 216 by the ejector sleeve 228 and are allowed to drop intothe slot 148 of the box 144 at the pipette tip ~licpnqol staffon 142. The pipette tip pickup and ejecffon funcffons will be described in more detail hereinaiter, as will the manner in which the pneumaffc aspiraffon and dispensing pipettes 164 are picked up and ~ t d The liquid ~ ~n function is carried out by selecting an x - y locaffon at which liquid is to be d~ hd (e.g., the locaffon of a sample tube 120 or a reagent bottle 179 - 182), and then sensing the presence of liquid beginning at a defined posiffon along the z axis by discharging air through the metal ffp 218 until the air flow is interrupted by occlusion of the tip. Liquid detecffon is carried out only with a ~ispos~ble pipette ffp 134 attached to the nozzle 218.
After the first liquid detecffon, which occludes the ffp 134 with liquid, subsequent reagent le~el ænsing with the same ~ poc~ble ffp 134 can be ~;ed out empirically by calculating the liquid le~el baæd upon the dimensions of the reagent bottle and the amount of reagent removed. In lieu of the ~irfl_.. methodof liquid level detecffon, a technique baæd on variaffons in the electrical capacitanoe of the metal tip 218 may also be uæd; this capability is also plo.ide d in the above-referenoed TECAN :~5t- -Movement of the hydropneumatic aspiraffon and ~ ng head 216 inthe x, y and z direcffons is accomplished by stepping motors (not shown) which are operated under microprocessor control. A software environment known as "Integrator" has been ~ . la~ E d by Tecan for this purpose, and is described inthm doc.~ . rtc entitled 5000/8000 Series Integrator Software Manual ~Version 7.40, July 1991), Command .S~ ry (Version 2.0, October 23, 198g), and DITI
Option Manual (Document No. 390 542, Version 1.1, October 1992), all of which are incorporated by referenoe herein. In the preferred embodiment of the presentinvention, sof~ commands designed for an OS-2 operating system are used and generate outputs which emulate "Integrator" soflware commands and user interfaoes.
Aspiration and dispensing of air and liquids thmugh the metal tip 218 is achieved by means of the nuid supply bottle 46, syringe pump 54 and fluid valve 62 of Fig. 2. The tubing in the system is primed with system fluid, which can either be dispensed di~e.~ or used as a hydraulic fluid medium for aspirating or dispensing measured amounts of air thmugh the metal tip 218. The syringe pump 54 is driven automatically by a stepping motor under microprocessor control, and the valve C2 is also controlled automatically by a solenoid to either fill the syringe 54 from the supply bottle 4C or to aspirate or dispense air or liquid through the metal ffp 218, depending upon the position of the ~alve 62.
As noted above, the wash arm 192 is generally similar in construction to the hydropneumatic aspiration and dispensing arm 190, exoept that the wash head 194 is installed in plaoe of the hydropneumaffc aspiration and dispensing head 216. The funcffons of the wash arm 192 are as follows: (a) controlled stepwise movement along the x, y and z axes, ~) dispensing of wash fluid into the assay devioes 90, and (c) aspiration of wash fluid and reagents from the assay devioes 90.
Movement of the wash head 192 along the x, y and z axes is carried out by ~t~pl~ motors under microp~ocesscr control, in the same manner as the hydropneumatic aspiration and dispensing arm 19Q Software commands control the speed and position of the wash head 194 at each moment during the operating cycle, with the movements of the wash arm 192 being coordinated and synchronized with those of the hydropneumaffc aspiration and dispensing arm lgO.
Dispen~ing of vrash fluid into the assay devioes 90 is carried out automatically by drawing wash fluid from the supply bottle 48 of Fig. 2 and - 18- PATE~IT

dis~n~ g it through nozzles at the wash head 194. As wiU be ~ in more detail hereinafter, the wash head 194 has three separate nozzles for dispensing wash fluid, with one nozzle being aligned with each weU of a given assay devioe 9Q There is a separate syringe pump 56, 58 and 60 in Fig. 2 for each of the washhead dispensing nozzles, and the fluid control valves 64-1 through G43 of Fig. 2connect the syringes either to the supply bottle 48 (to fill the syIinges) or to the dispensing nozzles at the wash head 194. As in the case of the hydropneumatic aspiraffon and dispensing head 216, the syringes S6 - 60 which supply the dispensing nozzles of the wash head 194 are controUed automaffcally by stepper motors to deliver pr~:te~,~ ined amounts of wash fluid at controUed rates. The fluid control valves 641 through 64-3 are also controUed automaffcaUy, with the position of the valves being the same for each of the three syringes 56 - 60 at any given time.
Aspiraffon of wash fluid and liquid reagents from the weUs of the assay ~ .ic~- 90 is carried out by providing the wash head 194 with a second set of nozzles which are used only for aspiraffon. These nozzles are connected by flexible tubes to pumps 222, visible in Fig. 3, which are switched on and o~
avtoroti~lly at the appropriate ffmes under~ computer controL
Additional features of the reacffon area 66 of the cabinet 22 will be evident from Fig. 3, in which a rear panel has been removed from the reaction area 66 to iUustrate cQmponents that are not visible in Fig. 2. A circuit board 224 mounted vertically on a rear waU of the cabinet 22 carries ~ o~ drivers for the heating platens 92, 94 and 100 at the reaction stations 78 - 84. The circuitboard 224 is connected by wires (not shown) to the electrical heating elements and to platinum RTD (resistanoe temperature devioe) temperature sensors located in the heating platens 92, 94 and 100. A temperature controller (not shown) controls the duty cycle of the electrical power provided to the heating elements, so that the temperature at each of the platens 92, 94 and 100 can be preciseb regulated. In the preferred embodiment, separate temperature feed~

~1 72210 - 19- r~T~NT
r 3361 loops are provided for the assay devioe heaffng platens 94 at each of the reaction staffons 78 - 84, but the lower and upper reacffon devioe heating platens 92 and100 at each reacffon station are controlled by a common feedback loop. A
suitable mulffple-loop t.~ye.Alure controller for use in the present invention is the ANAFAZE BCLS Loop System, which is a~ailable from ANAFA~E, Inc. of Watsonville, California. Alternaffvely, a mulffple-loop controller d~ hffl in a commonly-assigned, copending U.S. patent application of Gene A. Benton, Serial No. 08/177,829, filed on January 5, 1994 and incorporated herein by referenoe, may be used. The heating platens 92, 94 and 100 are convenffonal resistanoe heaffng laminates, app.~ At~ly 1116 inch in thickness, and are available commercially from Watlow of St. Louis, Missouri. Thermal fuses (not shown) are provided to protect the heating platens 92, 94 and 100 against overheaffng. Alsovisible in Fig. 3 are four cooling fans 226 which are installed on an elevated shelf at the rear of the cabinet 22, immediately behind the rear edge of the locator plate 7C. As will be described in more detail hereinafter, these fans draw air ~om a plenum l---ted below the reacffon staffons 78 - 84 in order to hasten the cooling of the reacffon devioe heating platens 92 at each reacffon staffon afterpower is removed from these platens.
As shown in the exploded view of Fig. 4, many of the components of the reacffon area 66 are removable from the locator plate 76 or deck 77 by the operator. These include the trays 86, the sample tube rack 110, the dicpQsol le pipette tip rack 127, the pipette ffp di~posol container 144, the wash cup 156 and the reagent bottle holder 166. This is advantageous not only in f... ~ .\g the introduction and removal of samples and expendable supplies by laboratory personnel, as described earlier, but also in allowing the locator plate 76 or deck 77 to be cleaned. If desired, the design of the cabinet 22 may be modi~ied by eliminating the locator plate 76 and placing all locating devioes lih~ on the deck 77, thereby providing a smoother surfaoe to facilitate routine ~ ning and containment of spilled liquids.

- 20 - PATI~NT

Figs. SA through SC illustrate the manner in which a dis~oble pipette tip 134 is picked up and ejected by the hydropneumatic aspiration and dispensinghead 216. In Fig. 5A, the lower portion of the pneumatic aspiration and dispensing head 21C is shown without a ~is~soble pipette tip in plaoe. The metaltip 218 extends downwardly by a short distanoe beyond the lower edge of a s~ lc plastic ejector sleeve 228, and carries a small-diameter tube or nozzle 219 through which air or system fluid is aspirated or dispensed. In order to pick upa ~ os- I)le pipette tip L34, the head 216 is lowered in order to foroe the nozzle 218 (which is slightly conical and l~.eled at its lower end as shown) into frictional engagement with the opening or lumen at the upper end of the ~ po^oble tip 134. This is possible sinoe the d;~los~l)le pipette L34 is held against downward movement by the rack 127 of Fig. 3. With the head 216 and ~i~posoble pipette tip L34 thus joined, the combined structure can be used for aspirating and dispensing liquids (i.e., liquid biological samples and reagents) by drawing or discharging precisely co..l~.~le~ amounts of air through the nozzle 219. This is done by controlling the syringe pump 54 of Fig. 2 to displaoe a cu,l~s~onding amount of system fluid in the tube connecting the syringe 54 and nozzle 219, while maintaining a volume of a~ (rather than system fluid) at the end of the tube which adjoins the nozzle 219. When it is desired to eject the p~rol~1~ pipette tip L34 into the di~posol cont. iner 144 of Figs. 3 and 4, the Ctic arm 190 of fig. 3 is moved to the upper limit of its z-direction travel, causing the upper end of the sleeve 228 (not shown in Figs. 5A - 5C) to strike afixed stop or obstruction. This has the effect of displacing the sleeve 288 in adownward direction, against a spring force, as illustrated in Fig. SC. This causes the di~posoble tip 134 to separate from the nozzle 218 and to fall by gravity into the slot 148 of the di~posol container 144. When the robotic arm 190 again moves downwardly from the upper limit of its z-direction travel, the upper end of the sleeve 228 separates from the stop and the lower end of the sleeve returns to the position shown in Fig. SA. The tip ejection function using the slidable - 21 - PATENlr ejector sleeve 228 is a standard feature of the TECAN system referred to previously, but the illustrated metal ffp 218 and nozzle 219 represent modifications made to the TECAN system for the purposes of the present invention. These modifications will be de-^r-h~ in more detail hereinafter.
Figs. 6A and 6B illustrate the manner in which the head 216 picks up and es one of the two pneumatic as~ilAIion and dispensing pipettes 164 of Fig.
3. The construction of the pneumatic aspiration and dispensing pipettes 164 is disclosed in more detail in the aforementioned copending U.S. patent applicationof Allen S. Reichler et al, filed on e~en date herewith and entitled nNucleic Acid Amplification Method and Apparatus" (Attorney's file 2573-rl), which is incorporated herein by referenoe. Briefly, the pneumatic aspiration and dispensing pipette 64 includes a rigid, generally cylindrical plastic portion 230 which is attached at its lower end to a resilient ffp 234 made of silicone rubber or the like. The resilient tip 234 is formed with a hole (not shown) on its lower faoe which communicates with the lumen 236 of the plasffc portion 230, and is adapted to be brought into contact with a pneumaffc port on each of the reacffondevioes 88 of Fig. 4 in order to control the movement of a liquid sample within the reacffon devioe. When the pneumaffc aspiraffon and dispensing pipette 164 is not in use, it is held on the bracket 160 of Figs. 3 and 4 by ~irtue of the engagement between a reslli l d or narrowed area 238 of the plasffc portion 230 and one of the U-shaped notches or cut-outs 162 in the upper horizontal lip or flange 161 of the bracket 160. When it is desired to use the pipette 164 to car~y out pne~nsti^ aspiraffon or dispensing on one of the reacffon de.; -- 88, the roboffc arm 190 is controlled to bring the metal ffp 218 on the pneumaffc aspiraffon and dispensing head 216 into alignment with the lumen 236 of the pipette 164 as shown in Fig. 6A. The head 216 is then moved downwardly to bring the ffp 218 into fricffonal c~ge~ent with the lumen 236, thereby coupling the head 216 with the pipette 164. This is followed by a he .20~1 mo.~ nt of the head 216 in they direcffon to ~ erlgage the pipette 164 from the cut-out 162, - 22 - rATENT
r 3361 and then by an upward ,,.... ~nt in the z direction to clear the bracket lC0. The resulting positions of the head 216, pipette 164 and bracket 160 are illu;,~ in Fig. 6B. At this point, the pipette 164 can be moved into contact with the pneumatic port of one of the reaction devioes 88 by appropriate movements of therobotic arm 190, and used to aspirate air from or dispense air into the reactiondevioe by auton-off~olly controlling the syringe pump 54 of Fig. 2 in the mannerdescribed previously. When it is desired to return the pipette 164 to the bracket 160, the roboffc arm 190 is controlled to maneuver the n~ d or restricted area 238 of the pipette 164 into horizontal alignment with one of the notches 162 in the bracket 160. A further horizontal movement of the head 21C in the y direcffon brings the pipette 164 into engagement with the notch 162 of the bracket 160, and a subsequent upward movement of the head 216 in the z direction separates the nozzle 218 from the lumen 236 of the pipette 164. This returns thecomponents to the posiffons shown in Fig. 6A, whereupon the pneumaffc aspiraffon and dispensing head 216 is free to perform other funcffons.
As shown in Figs. 3 and 4, the bracket 160 preferably holds two pneumatic aspiraffon and dispensing pipettes 164. This p.~. ~s redundancy in case one of the pipettes 164 becomes dislodged from the~bracket 160 and cannot be picked up by the robotic arm 190. The roboffc arm 190 is capable of detecting whether a pipette lC4 has been engaged, in the same way as described previously in connecffon with the disposable pipette tips 134. If the first pipette 164 cannot be engaged, the control system is programmed to move to the locaffon of the second pipette 164 and to engage that pipette as a backup.
Figs. 7A and 7B are enlarged views of the wash head 194 that is carried by the wash`arm 192 of Fig. 3. The flexible tubes 214 of Fig. 3 have not been shown in Figs. 7A and 7B for clarity. The wash head 194 comprises a solid, generally rectangular body 240 of plasffc material, such as polyvinylchloride (PVC), with a rear extension 242 that permits attachment to the rack 210 of the wash arm 192 by means of sn a,cl lul ~ 244. ~Ioles are formed through the main - 23 - rATENT
p 33Cl portion of the plsstic body 240 to tightly receive two sets of rigid metal tubes or conduits 246 and 248. The conduits 246 extend vertically through the plastic body 240, while the conduits 248 extend at a compound angle of applo ;-- ~t~ 1~
10 from the vertical when viewed from the end of the plastic body 240 in Fig. 7B
and approximately 41 from the vertical when viewed from the front of the plastic body in Fig. 7A. The conduits 246 are used for dispensing wash fluid into the wells of the assay devioes 90, while the conduits 248 are used to aspirate reagents and wash nuid from the wells of the assay devioes 90. The diameter of the aspiration conduits 248 is larger than the diameter of the dispensing conduits 246, and the aspiration conduits 248 extend slightly lower than the dispensing conduits 246 as illustrated in Fig. 7B. The lowermost ends of both sets of conduits 246 and 248 are ~ d or narrowed, as shown, to form nozzles.
I~fe~..bly, an adhesive is used to bond the conduits 246 and 248 to the holes inthe plastic body 240.
As will be described in more detail in connection with Figs. 14R and 14S, the wash head 194 is l~.~.cd by the wash arm 192 of Fig. 3 so that the lower (nozzle) ends of the conduits 246 and 248 are ~ d in the wells of the assay devices 90, with each well receiving the lower ends of a l~s~.~ pair of conduits246 and 248 simultaneously. To this end, the spacing between the lower ends of each pair of conduits 246 and 248 is such that both conduits are receivable within the di~meter of an assay device well. Depending upon the function being carried out by the wash head 194, fluid is either ~lic~--sed from the conduits 246 or aspirated into the conduits 248 at any given time. Although not illu;.l~_t d in Figs. 7A and 7B, the nexible tubes 214 of Fig. 3 are attached to the upper ends of the conduits 246 and 248, in the area adjoining the upper surfaoe of the plastic body 240. One set of flexible tubes delivers wash nuid to the dispensing conduits 246 from the supply bottle 48 and syringes 56 - 60, and the other set of flexible tubes couples the aspiration co~duils 248 to the pumps 222 and waste bottle 32 - 24 - rATE~T

of Fig. 1. The length and flexibility of the flexible tubes 214 is sufficient to allow for the desired range of movement of the wash arm 192 of Fig. 3.
Figs. 8A and 8B are top plan and side sectional views, res~li~ , of one of the removable trays 8C shown in Figs. 3 and 4. The purpose of the tray 86 is to hold a plurality of reaction devioes 88 and assay devioes 90 for convenient handling by laboratory personnel, and to locate these devioes at p,~dele~ined positions at the reaction stations 78 - 84. To this end, the tray 86 is formed with two opposed rows of slots or cavities 250 and 252 which are shaped to reoeive the ends of the ~ CR- r devioes 88, and with two opposed rows of slots or cavities 254 and 256 which are shaped to receive the ends of the assay devices 90. In the illustrated ~ ment~ the tray 86 accommodates twelve reaction devioes 88 and twelve assay devioes 90, with each assay devioe 90 l~ce;~d at a posiRon adjaoentto a comsponding one of the reaction devioes 88. Cut-out portions 258 and 260 are formed in the bottom of the tray for allowing the bottom surfaoes of the reactions devioes 88 and assay devioes 90 to make direct contact with the ,. s~tive heaRng platens 92 and 94 of Figs. 3 and 4. The tray 86 is preferably made from a suitable heat-re~i~t~rt plasRc material, such as Delrin or Ultem 1000.
Figs. 9A and 9B are plan and sectional views siilar to Figs. 8A and 8B, but with one reaction devioe 88 and its corresponding assay device 90 shown in position within the tray 8C. It will be understood that, although only one reaction devioe 88 and one assay devioe 90 are shown in Figs. 9A and 9B, the tray 86 willnormally be filled with as many reacffon d .i~es 88 and assay devioes 90 as there are samples to be assayed at the corresponding reacffon staffon 78 - 84. Each reacffon device 88 includes a sample tower 262 through which liquid biological samples are introduced, an elongPted rectangular body portion 264 through which the sample is moved during the decontamination and amplification reactions, and a pneumatic tower 266 through which pneumatic aspiration and dispensing is carried out in order to move the sample v,~ithin the body portion 264. The reaction devioe 88 has a substantially flat boV-~n surfaoe 268, a portion of which (corresponding to the locations of the decontamination and amplification areas within the body portion 264) is ~ eA through the cut-out 2S8 at the bottom of the tray 86.
The assay devioe 90 comprises three connected microwells 270, 272 and 274 which are generally cylindrical in configuration, with their side walls tapering slightly inward from top to bottom to produoe a frusto-conical shape. The interior walls of the sample wells are coated with a dried capture ~,cnl (typically biotinilated BSA/Streptavidin) for use during the nucleic acid assay.The microweUs 270, 272 and 274 are connected to each other by means of a generally planar, horizontal flange 276 extending between and parallel to the open tops of the m c~o.~lls, and by vertical webs 278 which are formed h~h._~n adjaoent wells imn~e 'i~t ~Iy below the flange 276. Each of the micl~,.. _lls 270, 272 or 274 has a substantially flat bottom surfaoe 280, 282 or 284. As illu~h_l ~ inFig. 8B, the slots or ca~ilies 254 and 256 of the tray 86 which support the assay devioes 90 are formed with L~ ly-facing ledges 286 and 288 which are engaged by a notch or step 290 that is formed around the perimeter of the assay dence 90. As a result of this arrangemenS the alssay devioe 90 is supported at a predetermined vertical positions within the tray 86 and are held against downward movemenS This position is such that the nat bottom surfaoes 280 -284 of the microwells 270 - 274 extend slightly below the bottom surfaoe 292 of the tray 86, as shown in Fig. 9B.
Figs. 10A and 10B are plan and sectional views similar to Figs. 9A and 9B, showing the tray ~6 in place at one of the reacffon staffons 78 - 84 of Figs. 3 and 4. The pivotable arm 102 is shown in the closed posiffon, with the U-shaped clamp 106 of Figs. 3 and 4 serving to lock the arm 102 in this posiffon and to compress the body portion 264 of the reaction device 88 l~t~.~e.. the heaffng platens 92 and 110. This provides efficient heat transfer from the heaffng platens 92 and 110 to the liquid biological sample contained within the reacffon device - 21 72~1G

r~

88. As shown in Fig. 10B, the interior of the arm 102 is substantially hollow (exoept for stiffening ribs that are not visible in the drawing); this pl-,.~e-thermal insulation between the platen 100 and the top surfaoe of the arm 102 andthus protects the operator against exposure to high temperatures.
The flat bottom surfaoes 280 - 284 of the microwells 270 - 274 of the assay devioe 90 are brought into contact with the upper surfaoe of the heating platen 94 when the tray 8C is installed at the reaction station. As this occurs, the assay devioe 90 is lifted slightly in the tray 86, causing the peripheral notch or step 290 to separate from the ledges 286 and 288. By allowing the assay devioe to "float"in the tray 86 in this manner, good thermal contact between the bottom surfaoes 280 - 284 of the assay devioes 90 and the upper surfaoe of the heating platen 94is assured.
As illustrated in Fig. 10A, the tray 8C is held in a p~ d ~f ~ined position and orientation at the reaction station by means of three locating devioes 294, 296 and 298. The l~t;~ devioe 294 is a plate-like structure that afflxes the rear hinge 104 of the arm 102 to the deck 76, as shown in Fig. 4, and the ~r~ ng devioe 296 is a similar plate-like structure that affixes the U-shaped clamp 106to the deck 76. The locating devioes 294 and 296 make contact with opposite endsof the tray 86 in order to properly locate the tray at the reaction station. Thethird locating devioe 298 is in the form of a diagonal block which is affixed to the deck 76, and which makes contact with the forward left-hand corner of the tray 86. As illustrated, the forward left-hand corner of the tray 86 is angled or b~.. Ie~
in such a manner as to conform to the angle of the block 298. In this way, the tray 86 has only one possible or=nt~1ion at the reaction station and cannot inadvertently be installed inco .. cll~. This insures that the reaction de~ices 88 and assay devices 90 proper make contact with their ~s~ c heating platens 92 and 94.
Figs. 11A and 11B are a ~ c.ti-~ views illu~ li g two alternative embodiments of the assay devices 90. In the embodiment of Fig. 11A, the assay r~

devioes 90 are manufactured in strips 300 of four, with each assay devioe 90 being connected to the next by means of a narrow web or tab 302 extending from the upper nange 276. The webs or tabs are formed on alternating sides of the strip 300 from one assay devioe 90 to the next. The assay ~ es 90 are preferably made of a molded plastic material, such as polystyrene, with the tabs 302 integrally formed using the same materiaL The individual assay devices 90 are easily separated from each other by bending or twisting the strip 300 to break the tabs 302. In the embodiment of Fig. 11B, the assay devioes 90 are formed indi~idually, rather than in strips; this produoes more uniform edges around theassay devioes 90 sinoe the tabs 302 are no longer required. In both emho~liments, the assay d .-~ es are formed with thin bottom walls (preferably about 0.022 inch in thickness) to promote efflcient heating of the liquid samples, and are preferably white in color with a high pigment content to enhanoe light collection and reduoe cross ~PII~ during the chemiluminesoent detection step. The horizontal nange 276 and webs 278 which connects the three microwells 270 - 274 of each assay devioe 90 are advantageous in that they resist bowing of the assay devioe and thus maintain the nat bottoms 280 - 284 of the microwells in a parallel, coplanar relationship, so that proper contact~with the heating platen 94 can be achieved.
Fig. 12 is a cross-sectional view illustrating the internal details of one of the reaction ~ 88. The reaction devioe 88 is disclosed in more detail in the aforementioned copending patent application of Allen S. Reichler et al, entitled"Nucleic Acid Amplification Method and Apparatusn, which is incorporated herein by referenoe. The sample tower 262 of the reaction de~ioe 88 is provided with a sample port 304 through which a liquid biological sample (not shown) is inll~ d ~ed. The sample passes through an a~.l~ 306 at the bottom of the ~smp~e tower, and is l~oe;~d in a sample area 308 in the form of a liquid bolus.The pne~lm~tic tower 266 at the opposite end of the reaction de~ioe 88 includes a l~cl-m~tic port 310 through which pneumatic aspiration and dicpP~;ng is - 28 - rATENT

carried out in order to move the liquid bolus horizontaUy within the reaction devioe 88. InitiaUy, air is aspirated through the pneumatic port 310 to cause the liquid sample to move from the sample area 308 to the decontamination zone 312 of the reaction area 314, where the sample makes contact with dried decontamination reagents 31h After a suitable incubation ffme, during which heat is applied to the reaction area 314 by the heating platens 92 and 100 of Fig.
10B, further aspiration through the pneumatic port 310 causes the liquid sample to move from the decontamination zone 312 to the amplification zone 318. In the amplification zone 318, the liquid sDmp'e contacts dried amplifi~Dffon reagents 320 and undergoes a nucleic amplification reaction. A suitable incubation periodis pl~ d for the amplification reaction, and heat is applied to the reaction area 314 by the heating platens 92 and 100 during this interval. The heat is then in.l~as~d for a short period of time to provide a heat spike that terminates theamplificaffon reacffon. FoUowing compleffon of the amplificaffon reaction, air is dispensed into the pneumaffc port 310 to cause the liquid sample to move fromthe amplifi-Dt;on zone 318 through the decontaminaffon zone 312 and back to the sample area 308. The liquid sample is then withdrawn from the reaction de ioe 88 by inserffng a pipette 134 (not shown) throi~gh the sample port 304 and orifice 306.
Fig. 13 is a partial cross-secffonal view through the deck 76 of Fig. 3, iUu~LAlil.g a cooling arrangement for the reaction devioe heating platens 92 and94. For clarity, the components which are normaUy mounted on the deck 76 have been removed in Fig. 13, and the portions of the cabinet 22 above the level of the deck 76 have also been removed. Beneath the forward edge of the cabinet 22, an air inlet 322 communicates with a bamed plenum chamber 324 l~Dted below the deck 76. At the rear of the cabinet 22, the plenum chamber 324 communicates with an a~. lurc 326 on which one of the fans 226 of Fig. 3 is mounted. The fan 226 draws air from the plenum chamber 324 through the aperture 326, and exhausts the air through an air outlet 328 located at the rear of the cabinet 22.

- 29 - rATENT

In this way, a continuous circulation of air is maintained in the plenum chamber324. At the forward end of the plenum chamber 324, immediately above and behind the air inlet 322, a cut-out is provided for recei~ing one of the heatingplatens 92 of Figs. 3 and 4. Similar cut-outs are plo.i~ed for the heating platens 92 of the remaining reaction staffons. When power is removed from the heating platens 92 foUowing the heat spike l~f~ d to earlier, the circulation of air in the plenum .~1 gn her 324 provides a cooling effect which aUows the heating platens 92 to reach ambient temperature more quickly. In this way, more rapid temperature transitions can be obtained in the reaction r~ es 88. Bafnes 331 divide the plenum ~h~her 324 into channels extending from front to rear beneath the deck 76, in order to confine the cooling air flow to the l~~.ff~
devioe heating platens 92 and to isolate the air flow from the assay de~ioe heating platens 94, the latter operating at lower temperatures and not requiring an air nOw for cooling.
Figs. 14A - 14T are a series of sequenoe views iUustrating the pl~al lmed series of mo~ements carried out by the robotic arms 190 and 192 during the course of a nucleic acid assay. Prior to the start of the assay, the trays 86 are loaded with the desired number of loe~h~ ~ d~vioes 88 and assay devioes 90, withboth bpes of devioes being equal in number and loaded at adjaoent ~s;t~ in the tray 86. I~f~ ~Ably, the trays 86 are sequentially loaded (without empty slots) and are fuUy loaded exoept for the last tray used, which may have unoccupied slots depending upon the total number of samples and controls to be ass..~d.
The trays 86 are filled from front to back beginning with the first reaction staffon 78 and ending with the last l~- ct ~n station 84. FiUed reagent bottles 179 - 182 are placed into the left-hand row of cavities in the ~..1 holder 166, with theircaps removed and placed in the Plj~nt cavities 178. The three smaller reagent bottles 179 - 181 contain hybridization reagents of different specificity which are used during the nucleic acid assay, and the larger reagent bottle 182 contains achemiluminescent reagent such as Lumi-Phos 530 (a tr~len~rk of Lumigen Inc.

r~

Soutl-ff-ld Michigan). The disposable pipette rack 127 is also installed with a supply of ~licposoble pipette ffps 134 in plaoe. Preferably, the rack 127 is completely fflled with ~licpossble pipette tips 134 to insure that an adequate supply of tips will be available. Finally, the sample tube rack 110 is installed and is plo.id~d with sample tubes 120 containing the liquid biological samples to beassayed. The first som. le tube 120 occupies the right front aperture 118 of thesample rack 110, and subsequent sample tubes 120 are loaded from ~ont to back Before commencing the assay, the fluid supply bottles 46 and 48 of Fig. 2 are checked to insure that there is an adequate supply of system fluid and wash solution.
The assay is commenced by sending an appropriate command to the system computer through the k~bc-rd 36 of Fig. 1. As the first step in the process, the robotic arms 190 and 192 move from their home positions (shown in Fig. 3) to positions above the wash cup 156 as shown in Fig. 14A. Air is thenpurged from the pneumatic aspiration and dispensing head 216 by dispensing a small amount of system fluid from the nozzle 218, and the dispensing nozzles 246of the wash head 194 are purged in a similar manner. Eluids discharged by the hydropneumatic aspiration and ~icpenC-ing ~head 216 and wash head 194 are collected by the wash cup 156 and are then aspirated by the aspiration nozzles 248 of the wash head 194.
In Fig. 14B, the mbotic arm 190 moves the hydmpneumatic aspiration and dispensing head 216 to a position above the licposoble pipette tip rack 127 andpicks up one of the ~icpvcoh~e pipette ffps 134. The first tip 134 to be picked up is at the rear left-hand corner of the rack 127, and subsequent tips are picked up fmm back to front.
In Fig.14C, the robotic arm 190 maneuvers the hydmpneumatic aspiration and ~lic~ncing head 216 (now car~ing a ~ po~qhle pipette tip L34) to a position above the lowermost reagent bottle 179. The ~licpos~ble pipette tip 134 is then lowered into the reagent bottle 179 while the hydropneumatic aspiration and - 31 - rATENT

dispensing head 216 is operated in the liquid d~ ion mode. This allows the reagent level in the bottle 179 to be d eI e ,1 ~ d so that a warning can be produced on the video monitor 38 of Fig. 1 in the event that an insufficient quantity of reagent remains for the desired number of assays.
In Fig. 14D, the robotic arm 190 moves the hydropneumatic aspiraffon and n~ing head 216 to a position above the pipette tip di~posDI station 142, and the used pipette tip 134 is e~ected into the slot 148 of the box 144. This completes the liquid level check for the first reagent bottle 179. The robotic arm 190 then maneuvers the hydropneumatic aspiration and ~ ing head 216 to pick up a new pipette tip 134 from the rack 127, and to check the reagent level in the next reagent bottle 180 in the holder 166. The new pipette tip 134 is then discarded, and the y~ocess is l ~At~d for the remaining two r~e~t bottles 181 and 182. By using a new diSF~ le pipette tip 134 for each of the reagent bottles179 - 182, cross-contamination among different reagents is avoided.
In Fig. 14E, the h~dlop~ &tiC aspiration and dispensing head 216 is shown after having picked up a new ~ pos~le pipette tip L34 from the station 126. The head 216 has now been moved to a ~osition above the first sample tube 120, and the pipette tip L34 is lowered into the sample tube 120 until the liquid -sample is det~ te~ (as before, this is done by o~l..ling the head 216 in the liquid d e t ,t on mode). The liquid sample preferably has a minimum volume of about 250 I~L; of this, about 55 ~L is drawn up into the pipette tip 134 by aspirating air into the nozzle 218 of the head 216. To prevent a droplet from forming at the bottom of the pipette tip L34 after the tip is ~e~o._d from the sample tube 120,a transport air gap of about 10 IlL is maintained l~t~._e~ the tip opening and the bottom level of the liquid sample held in the tip. In general, it will be desirable to maintain a transport air gap in the pipette tip 134 for all liquid transfers that are carried out by the hydropneumatic aspiration and dispensing head 214.
In Fig. 14F, the hydropneumatic aspiration and dispensing head 216 has been moved by the robotic arm 190 to a position above the sample port 304 of the - 32 - PATEN~
r-33cl first reaction device 88. The head 216 is then lowered to move the pipette ffp 134 into the sample area 308 of the reacffon device 88, and the liquid biological sample is discharged into the sample area 308 by dispensing air from the head 216. When this operaffon is completed, the pipette ffp 134 is withdrawn and ejected into the box 144 of Figs. 3 and 4, and a new pipette ffp is picked up from the rack 127. The procedure illustrated in figs. 14E and 14F is then ~ le~l inorder to transfer the liquid sample from the next sample tube 120 to the next reacffon devioe 88. This sequenoe is ~ ted for each of the sample tubes 120 and reaction devioes 88, using a new ~i ir ~sohle pipette tip 134 each ffme. When all of the liquid samples have been transferred, the head 216 is moved to the docking station 1~2 to pick up one of the pneumaffc aspiraffon and dispensing pipettes 164 using the prooedure shown in Figs. 6A and 6B.
In Fig. 14G, the head 216 (now carrying a pneumatic aspiraffon and dispensing pipette 164 with its resilient ffp 234) has been moved to a position above the pneumaffc port 310 of the first reacffon devioe 88. The head 216 is then lowered to bring the resilient ffp 234 of the pipette 164 into engagement with the pneumatic port 310 of the reaction devioe 88, and a sufflcient amount of airis aspirated from the reacffon devioe to cause ~he liquid sample to move from the sample area 308 to the decontaminaffon zone 312 of the reaction area 314. This procedure is ~~ d for each of the reaction devioes at the reacffon station 78, and for the reacffon devioes 88 at the remaining reaction staffons 80 - 84, using the same pneumatic aspiration and dispensing pipette 164. Use of the same pipette 164 does not give rise to cross-contaminaffon p~ m~, sinoe the pipette 164 does not make contact with the liquid biological samples within the reacffondevioes 88.
After the pipette 164 is removed from the pneumaffc port 310 of the last reaction devioe 88, and with the liquid biological s~mpl~^ now l~oei~d in the decont~mi~tion zones 312 of the reaction devioes 88, the heating ,~!~te~-~ 92 and 100 of that reaction station are e~erl;ized to heat the liquid s~mples to a P-~361 temperature of 41 C. This le.~ ture is maintained for an incubation period of 50 minutes, during which the decontDmirD~ion reaction occurs. This is illustrated in Fig. 14E~. The S0-minute incubaffon ~. clldr (and all subsequent decontamination, ampliP -nt on and assay steps) are staggel~ d among the reaction stations 78 - 84, with a 16-minute inter~al (the longest period of time required to carlg out any operation at a given reaction station) from one to the next. This insures that the chemical reactions occurring in all of the reaction devioes andassay ~ are of the same duraffon, regardless of the reacffon staffon involved.
Following completion of the ~d~ntDminaffon ~ n at a gi~en reaction station, the robotic arm 190 returns the head 216, with the pneumaffc aspiraffonand dispensing pipette 164 and resilient ffp 234" to a posiffon abo~e the pneumaffc port 310 of the first reaction devioe 88. The resilient ffp 234 of thepipette 164 is then brought into contact with the pneumaffc port 310, as shown in Fig. 14I, and a controlled amount of air is aspirated from the reacffon devioe 88 to cause the liquid biological sample to move from the decontaminaffon zone 312 to the amplificaffon zone 318. This prooedure is reF~t~ for all of the remaining reaction devioes 88 at the reaction staffon, using the same pipette 164.
When the liquid biological samples in all bf the reacffon devioes 88 at the reacffon staffon ha~e been transferred to the amplificaffon zones, an incubaffonperiod of 120 minutes begins, during which the amplificaffon reacffon occurs in the Dmplificaffon zones 318 of the reaction ~.ices 88. This is illustrated in Fig.
14J. At the conclusion of the 120-minute incubaffon period, the amplificaffon reacffon is stopped by operaffng the heaffng platens 92 and 100 to raise the temperature of the samples to 80 C for five minutes. After the fi~e minute heatspike, the fans 226 of Fig. 3 are turned on to cool the heaffng platens 92 and the platen temperature is reduced to 41 C.
In Fig. 14K, the resilient ffp 234 of the pneumatic aspiraffon and dispensing pipette 164 has again been brought into contact with the pneumatic port 310 of the first reaction device 88 at one of the reaction stations 78 - 84. A

- 34 - rATENT
P~361 controlled amount of air is dispensed through the pipette 164 by the head 216 tocause the liquid biological sample within the reaction devioe 88 to mo~e from the amplification zone 318 of the reaction area 314 back to the sample area 308.
This procedure is ,~t~d for each of the remaining reaction devioes 88 at the reaction station. The state of each reaction devioe 88 at this point is illush _te d in Fig. 14L.
After the liquid biological samples have been returned to the sample areas 308 of the reaction ~ e s 88 at the reaction station, at the pneumatic aspiration and ~ pPn~ing pipette 164 is returned to the docking station 152 of Figs. 3 and 4 using the proce.l~ shown in Figs. 6A and 6B. The hydropneumatic aspiration and dispensing head 216 is then moved by the robotic arm 190 to a position above the wash cup 156, as illustrated in Fig. 14M, and a small amount of systemnuid is discharged into the wash cup to purge air from the nozzle 218.
In Fig. 14N, the nozzle 218 has been moved to a position above the first well (ie the well closest to the row of ~q~n ~ es 88) of the first assay devioe 90 at one of the reaction stations 78 - 84. A quantity of system fluid isdischarged into the well for subsequent mixing with the amplified sample, typically 30 I~L ~co.~d &om the origina~ 55 ~L, from the comsponding reaction devioe 88. For an amplified sample volume of ap~ n~ely 30 ~L, the volume of system fluid discharged into the first well is app~ tPIy 60 Each microwell of the assay devioe 90 has a capacity of about 400 ,uL.
Following the discharge of system fluid into the first well of each assay devioe 90 of the reaction station, the robotic arm 190 moves the hydropneumatic aspiration and dispensing head 216 to a positi~ above the ~i~po~ le pipette tube rack 127 and picks up a new ~ po~s~)le pipette ffp 134. With the tffp in place, the head 216 is moved to a position above the sample port 304 of the first reaction device 88, and is then lowered to move the pipette ffp 134 into the sample area 308 of the reaction device as illustrated in Fig. 140. The liquid sample is then aspirated from the reaction device 88 into the disposable pipette r-33cl tip L34. Because the sample will be tran~ r t~d over a short distanoe (to the adjaoent assay devioe 90) and will not pass over any other samples, a transport air gap need not be maintained at the bottom of the pipette tip during this transfer.
In Fig. 14P, the robotic arm 190 has moved the hydropneumatic aspiration and dispensing head 216 (with the pipette tip 134 containing the liquid aspirated from the first reaction devioe 88) to a position above the first microwell of the first assay devioe 90. The pipette 134 is then lowered into the microwell and the 30 ~L of amplified sample is ~liCr--~d into the 60 ~.L of system fluid. Then, 60~L of the mixture is aspirated into the pipette 134, and the pipette is elevatedabove the remaining fluid in the microwell. A 30 ~L volume of air is aspirated into the pipette ffp 134, the 30 ~.L of air and 60 ~L of fluid are dispensed into the microwell, and the pipette tip 134 is again l~ d into the microwell to begin a second aspiration. This prooess is then l~nt~l to insure complete mixing. At this point, the head 216 aspirates 60 ,uL of the mixture and dispenses 30 ~L of the mixed sample into each of the two remaining mic~u. _lls of the first assay devioe 90, leaving 30 I~L in the first microwell. The pipette tip 134 is then ejected into the box 144 at the pipette tip ~;cpo^~l sta~ion 142, a new tip is obtained from the ~liC~Cnl-l~ pipette tip station 126, and the sample aspiration, mixing and dispensing prooedure is re~ e d for the next reaction devioe 88 and assay devioe90. This procedure is ~ ~1 for each of the remaining reaction devioes 88 and assay devioes 90 of the reaction staffon, using a new disposable pipette ffp 134each time, until all of the ~ liquid samples have been removed from the reaction devioes 88 and transferred to the microwells of the comsponding assay devioes 90.
After the liquid samples at the last reaction staffon 84 have been tr~r~f. I ~d to the assay devioes 90, the hydropneumatic aspiraffon and dispensing head 216 ejects the last used pipette ffp L34 at the tip disposal station 142 and picks up a new tip from the disposable pipette tip station 126. With the new tip - 36 - PATEN~
r-33cl 134 in place, the head 216is carried by the roboffc arm 190 to the reagent staffon 154, where a quDnffty of a first hybridizaffon reagent is aspirated into the pipette ffp from the lowermost reagent bottle 179. The first hybridizaffon reagent is a signature reagent which detects whether any nucleic acid amplificaffon has occurred in the sample. The position of the hydropneumaffc aspiraffon and dispensing head 216 at this point is the same as indicated in Fig. 14C. The robotic arm 190 then returns the head 216 to the first reacffon staffon 78 and dispenses the reagent into the first (innermost) microwell of the first assay device 90. This procedure is repoot~ (using the same ~licposoble ffp L34) for the firstmicrowell of each of the remaining assay devices 90, with the head 216 returningto the reagent bottle 179 each ffme that a microwell is filled. After the first microwells of all of the assay devices 90 have been filled with the first hybridizaffon reagent, the hydropne~mD~ic aspiraffon and dispensing head 216 ejects the used pipette ffp 134 in the box 144 and obtains a new ffp L34 S om the pipette ffp station 126. With the new tip 134 attached, the head 216 aspirates asecond hybridization reagent from the next reagent bottle 180 and transfers the reagent to the second (middle) microwell of the first assay devioe 90. The second hybridization reagent is a genus reagent whic~h detects whether a mycobacterial DNA sequenoe has been amplii;ed. Again, using the same llicr~Dble pipette ffp 134, this prooedure is ~ l in order to dispense the second hybridizaffon reagent from the second reagent container 180 to the second microwell of each of the remaining assay devices 90 at the reacffon staffon. After ejecffng the used pipette ffp 134 and picking up a new pipette ffp, the prooedure is repe-D~ed once again in order to dispense the third hybridization reagent from the third reagent container 181 into the third (outermost) microwell of each assay devioe 90. The third h~ i7~ion reagent is a species reagent that detects any tubercle bacillus DNA s~q-.moes in the ~mplified sample.
At this point, the diluted liquid samples in the first, second and third microwells of each assay device 90 at the reaction station contain the first, second r~

and third hyb~ i7st ~n reagents, r.s~ . An incubation period of 50 minutes then commenoes, during which the heating platen 94 lccst~d beneath the assay devioes 90 is controUed to raise the temperature of the liquid samples to 33 C After the incubation period, the heating platen 94 is deactivated and a wash step is carried out by the wash head 194 to remove the liquid samples and reagents from the microweUs of the assay devioes 90, leaving only the reacted material which is bound to the inside waUs of the assay device weUs. Prior to the wash step, the wash head 194 is moved by the robotic arm 192 to a position abovethe wash cup 156, as shown in Fig. 14Q. Wash fluid is then dispensed from the dispensing no_zles 246 of the wash head 194 in order to purge the dispensing no~les of air. The robotic ar n 192 then moves the wash head 194 to a position above the first assay device 90, and the pumps 222 of Fig. 3 are turned on. The robotic arm 192 then slowly lowers the nozzles 246 and 248 into the microweUs of the assay device, as shown in Fig. 14R. In this position, the ends of the aspiration nozzles 248 of the wash head 194 are very close to the bottom surfaoes of the microweUs, and due to their inclination are directed toward the peripheryof the microweUs. The sample and reagent fluids are then aspirated from the microweUs of the first assay devioe 90. By ~lowly moving the wash head 214 downward while aspirating the sample and reagent fluids, actual wetting of the aspiration nozzles 248 by these fluids (and consequent cross-contaminaffon between samples) is &v~ ~e d No~le wetting is also avoided by maintaining a relatively high aspiraffon rate through the nozzles 248, sinoe the resulffng high-velocity airflow around the nozzles prevents the aspirated fluids from touching the nozzle sur~ces ~ .tl~.
After aspirating the csmple and reagent fluids, the wash head 194 is moved slightly upward and toward the center of the microweUs in order to separate the aspirating no~les 246 from the bottom of the microweUs of the assay devioe 90.
The wash head 194 is then raised to a di~ ing height, as shown in Fig. 14S, and wash fluid is dispensed into the microwells of the assay devioe 90 from the - 38 - rATENT

dispensing nozzles 246. The wash head 194 is then moved to each of the remaining assay devioes 90 at the reaction station and ~ts the same operations. Wash fluid is dicpenced into and aspirated from the microwells of the assay h.-~es 90 two more times, with the wash head 214 moving betveen the positions shown in Figs. 14R and 14S during each assay devioe washing operation.After the last aspiration cycle, the microwells of the assay devioe 90 are substantially empty exoept for the amplicons bound to the walls of the microwells.
The washing prooedure is carl ~ out on each of the assay ~ es 90 at the reaction station, with each of the aspiration/dispensing gcles occurring at all of the assay d~ e- 90 in sucoession to ~ o.;d~ a soak time for the wash fluid between suc~s ,i~ gcles.
When the washing of all of the assay devioes 90 at a given assay station is complete, the wash head 194 is returned to the home position (shown in Fig. 3) by the robotic arm 192. The robotic arm 190 then causes the hydropneumatic aspiration and dispensing head 216 to move to a position above the wash cup lS6 and dispense a small amount of system fluid in order to purge air from the nozzle 218. The hydropneumatic aspirating and dispensing head 216 then moves to a position above the first microwell of the Lst assay devioe 90, and dispenses a small amount of system fluid into the well. The position of the head 216 at this point is the same as illustrated in Fig. 14N. The head 216 then p~ooeeds to dispense system fluid into each of the remaining wells of the first assay devioe 90, and into the wells of all of the remaining assay ~ e s 90 at the reaction station.
With a small amount of system fluid now present in the microwells of all of the assay devioes 90 at the reaction station, the hydropneumatic aspiration and dicp~n~ing head 216 picks up a new pipette tip f~om the pipette tip staffon 126 and moves to a position above the chemiluminesoent ~ t bottle 182 at the l~genl station 154. Using the pipette tip 134, the head 216 then draws a quantity of chemiluminescent reagent from the fourth reagent bottle 182, as illustrated in Fig. 14T. The head 216 then returns to the first assay device 90 and dispenses 217221û

r-3~6l an equal amount of the chemiluminesoent reagent into the first, second and thirdmicrowells of the assay devioe 90. This procedure is ~ d for each of the remaining assay devioes 90 at the reaction station with the head 216 returning to the reagent bottle 182 after each assay devioe 90 is filled. The pipette tip 134 is replaoed each time that 12 microwells (i e., 4 assay devioes 90) have been filled, to prevent bubbles from forming as a result of accumulated residual liquid in the pipette tip. The wells of all of the assay devioes 90 at the reaction station now contain the chemiluminesoent reagent mL~ed with the system fluid dispensed previously, and the heating platen 94 is now operated to incubate the chemiluminesoent reagent in the assay c~ es at 37 C for 30 minutes.
When the ro~ego.hg sequenoe of operations has been completed for each of the reaction stations 78- 84, the automated portion of the nucleic acid assayis complete. The trays 86 are now be removed from the reaction area 66 of the cabinet 22, and plaoed in the luminometer 43 of Fig. 1. The function of the luminometer 43 is to detect lumincsce..cc within each microwell of each assay devioe 90, as will occur when the chemiluminesoent reagent reacts with the hybridized amplified material which has become bound to the interior walls of the assay devioes 90. Such luminesoenoe i~dicates that a target nucleic acid sequenoe has been ~d:le~ed. The luminometer is pre&rably a Model ML 2200 luminometer manufactured by l~ynatech Laboratories of Chantilly, Virginia.
Fig. 15 is a schematic diagram of the principal pneumatic and fluidic components of the system 20, illustrating the manner in which they are interconnected. The supply bottle 46 containing system fluid is coupled by meansof the nexible tube 50 to one port of the control valve 62, which is in turn connected to the ~r;~t,e pump 54. The second port of the valve 62 is connected to the hydropneumatic aspiration and dispensing head 216 by means of a tube 332. Depending upon the position of the valve 62, the syringe 54 is connected either to the system fluid supply bottle 46 (to fill the ~l~ge) or to the hydrcl cumatic aspiration and dispensing head 216 (to dispense or aspirate air, 2172~10 40- rATENT
r-or to dispense system fluid). The wash fluid supply bottle 48 is connected bymeans of the tube 52 to a three-way coupling or m~ "~ld 338, the outputs of which are connected to the ganged control valves 641, 642 and 643 via tubes 340, 342 and 344, r~ . The control valves 641, 642 and C43 are coupled to the ~es~ syringes 56, 58 and 60 and to corresponding output tubes 346, 348 and 350, l~ r~ . Depending upon the position of the control ~alves 641 through 643, the ~l. ges 56 - G0 either draw fluid from the wash nuid supply bottle 48 (to fill the syringes) or dispense the wash fluid to the wash head 194through the tubes 34G - 350. The tubes 346 - 350 are coupled to the wash head dispensing nozzles 246 of Figs. 7A and 7B and additional tubes 351, 352 and 353 couple the wash head aspiration nozzles 248 of Figs. 7A and n to the pumps 222 and waste bottle 32 through a th,~c~ coupling or manifold 354.
Fig. 16 is a block diagram illustrating the principal electrical components of the system 20. The system computer 44 is connected to the keyboard 36, numeric keypad 37, monitor 38, and printer 42 of Fig. 1, and also to the floppy disk drive 47 and luminometer 47. The floppy disk drive 3C2 allows control programs to be loaded into the computer 44 (including software updates), and also allows assay results to be stored on flopp~ disks. The luminometer 43, which s the assay devices 90 after they are removed from the cabinet 22 of the system 20, is also connected to the computer 44 (via a ærial card) so that the final results of the assay can be logged automatically. An uninterruptible powersupply (UPS) 360 ~ power to the system components and has a logic connection to the computer 44 to allow for orderly system shut-down in the eventof a power failure.
The computer 44 controls the functions of the system 20 through a system controller 366. The system controller 366 is connected to the syringe pumps 54 -60 of Figs. 2 and 15, to the robotic arms 190 and 192 of Fig. 3, and to atemperature control circuit 368 which regulates the temperatures of the heating p'-ten~ 92, 94 and 100 and switches the &ns 226 on and off. The system - 41 - rATENT
r~

controller 366 is also connected by means of an input/output board 370 and an rr~ess ~.~ board 372 to various other components of the system 20, including thecontrol ~alves 62 and 64-1 through 643, pumps 222, and interlocks for the doors 24 and 29 and pivotable arms 102. These components are represented c~ vely by the block 374 in Fig. 16.
Fig. 17 is a flow chart which summarizes the operations carried out by the system computer 44 of Fig. 16 in executing the motions illustrated in Figs. 14A -14T at each of the reaction stations 78 - 84. Following start-up, an initioli70tion proc~d. l ~ is carried out in block 376 to allow the operator to specify the desired values of certain system parameters. These include transport air gap volume, aspiration and dispensing volumes and speeds, incubation times, and number of samples and controls. Following initioli7o~ion~ the computer proceeds to block 378 and purges the wash head 194 and hydropneumatic aspiration and dispensing head 216 of air. The levels of the four liquid reagents are then checked in blocks 380 and 382, and any inadequate l~&ge..l levels are brought to the attention of the operator in block 384 by producing an output on the video display monitor 338.
If the reagent levels are found to be adequate, the system prooeeds to block 386and transfers the liquid biological samples l~rom the sample tubes 120 to the reaction devioes 88 using the hydropneumatic aspiration and dispensing head 216 and the ~i~po-vqble pipette 134. When this is complete, the system prooeeds to block 388 and uses one of the two pneumatic aspiration and dispensing pipettes 164 to move the samples to the decontamination zones 312 of the reaction d~ . c 88. This is ff'~ d by the an incubation period in block 390, during which decontaminaffon takes place. In block 392, the pneumatic aspiration and ~ispen~ing pipette 164 is used once again to move the liquid samples to the amplification zones 318 of the reactions devices 88, and this is followed by a further incubq1;~n period and heat spike in block 394. When ampl;fi~tion is complete, the liquid samples are moved back to the sample areas 308 of the reaction devices 88 as indicated in block 396. With the pipette 164 restored to the -- 42 - rAl~r r~

d~r~ng station 152, the hydropneumatic aspiration and dispensing head 216 is purged in block 398, and system fluid is then ll;c~r~ into the first well of each assay devioe 90 in block 400. In block 402, the ~ ted liquid samples are tron~ d from the reaction devioes 88 to the assay de~ioes 90 using the icpo^oble pipette tips 134, and are mnYed with system fluid in the manner described previously. In block 4Q4, the three hybridization reagents are dispensed sequentially from the reagent bottles 179 - 181 into the corresponding wells of the assay devioes 90, and this is followed by an incubation period in block 406 and by washing and aspiration of the assay devioes 90 in block 408. In blocks 410 and 412, system iiuid and chemiluminescent reagents are llispP~c~l into the assay devioes 90. This is followed by an incubation period in block 414. After incubation, the ass. y devioes are manually ll~...sr~ d to the luminometer 43 ofFigs. 1 and 16. In block 41C, the output of the luminometer 43 (~ _nting the final results of the assay) are read by the computer 44 and are displayed to theuser via the monitor 38 and printer 42. The assay procedure is now complete, and the subsequent assays may be C&l~;~ out by re-initializing the system in themanner described previously.
A number of modifications may be ma~de to the automated assay system 20, in addition to those already described. With referenoe to Figs. 3 and 4, onepossible modification comprises a rearrangement of the reaction area 66 to oe-te the pipette ffp dicposol staffon 142 from the posiffon shown to a new posiffon on the right side of the reagent staffon 154. This may be preferable inthat it Q~O.-~S ~ separaffon l~h.oen the hydropneumaffc aspiraffon and dispencing head 216 and the sample tube rack 110 when the head 216 is ejecRng a used pipette ffp 134, thereby lessening the chanoes of cross-cont minaffon dueto airborne droplet generation by the ejected tip. The reagent bottle holder 166may be reduoed in size (e.g., by eli~nin~ing the cap caviffes 178) in order to accommodate the new location of the pipette tip disposal staRon 142.

r-336l As another modification, the hydropneumatic aspiration and dispensing head 216 may be modified so that a ~lisposoble pipette tip 134 and a pneumaffc aspiration and dispensing pipette 164 can be carried by the head 216 at the sametime. In this modification, the ~ic~osoble pipette tip L34 and pneumatic Ds~&lion and dispensing pipette 164 are preferably se~l~d from each other by a distance comsponding to the distanoe between the sample tower 262 and pneumatic tower 266 of a reaction devioe 88. This aUows a disposable pipette ffp1~4 to be introduced into the sample tower 262 at the same time as the resilienttip 234 of the pneumatic aspiration and dispensing pipette 164 is brought into contact with the pneumatic tower 266. Appropriate changes may also be made in the fluidic aspiration and dispensing system of Fig. 15 to aUow the ~1;C~OSD~I~
pipette tip 134 and pneumatic aspiration and dispensing pipette 164 to be operated independently of each other.
Fig. 18 illustrates the details of the hydropneumaffc aspiraffon and dispensing head 216 that is carried by the roboffc arm 190 of Fig. 3. The construction shown represents a modificaffon of the basic TECAN design, and is the embodiment preferred for use in the present invenffon. The metal ffp 218 is an ~Yten~ on of an elongated metal cylinder i20 which is threadably engaged at its upper end 422 with a hollow rod 424. A length of h~cd^~i- tubing 426 passes through an axial bore 430 in the metal cylinder 420, and p~ojects throughthe ffp 218 to form the aspiraffon and dispensing nozzle 219 referred to previously. A nange 428 is formed near the upper end of the tube 426 to hold thetube in place with re~ to the metal cylinder 420. The flexible tube 332 of Fig.
15 is attached to the upper end of the tube 426 to provide hydropneumaffc aspiration and dispensing through the nozzle 219. The tube 426 fits loosely within the bore 430, and the annular space between the outside of the tube 426 and the inside of the bore 430 forms an air passage for the liquid detecffon function of the TECAN system. At its bottom end, the air passage te~ninates in an annular outlet (not visible in Fig. 18) which surrounds the nozzle 219 at the - 44 - rATENT
P ;3361 bottom faoe of the tip 218. At its upper end, the air flow ~r~s~ terminates in a lateral bore 432 formed near the upper end of the metal cylinder 420. The lateral bore 432 communicates with the hollow interior of the tube 424, in whichan air flow is maintained by the liquid detection system (not shown) of the TECAN unit.
With continued reference to Fig. 18, it will be observed that the metal cylinder 420 and hollow tube 424 are both ~ce;~d in the slidable ejector sleeve 228 described previously. At its upper end, the ejector sleeve 228 is expanded to form a partially cylindrical structure 434 whose upper end 436 is displaced downwardly when the robotic arm 190 is moved to the upper limit of its travel in the z direction. An electrically cond~ .g strip 438 is attached by means of a screw 440 to the interior surface of the cylindrical structure W and terminates in a U-shaped contact 442 which fits over the upper edge of the cylindrical structure 434 as shown. As the robotic arm 190 approaches its uppermost position, the contact 442 is brought into contact with a conductive, spring-lr - ~ e d plunger 444. Ap~ e electrical circuitry (not shown) detects ~le~~ol continuity between the contact 442 and plunger 444 to determine that the roboticarm is ner its uppermost posiffon. Furthell upward travel of the robotic arm will cause the upper edge of the cylindrical structure 434 to be brought in contact with a fixed abutment 446 in which the plunger is mounted, thereby displacing the cylindrical structure 434 and ejector sleeve 228 downwardly to eject a di~poso IE pipette tip L34 in the manner described previously.
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof, as numerous alternatives to the devioes and methods described which incD~ nte the present invention will be apparent to those skilled in the art. The invention is accordingly defined by the following ~loim~, with equivalents of the claims to be included therein.

Claims

1. An automated system for carrying out reactions on a plurality of liquid samples, comprising:
a reaction station adapted to hold a plurality of reaction devices in which said liquid samples are receivable, each of said reaction devices including a sample area for receiving a liquid sample, a reaction area into which said liquid sample is movable to carry out a reaction on said sample, and a pneumatic port for allowing air to be aspirated from and dispensed into said reaction device tomove said liquid sample between said sample area and said reaction area;
a robotically controlled aspiration and dispensing head adapted to move into contact with the pneumatic ports of said reaction devices, and to aspirate air from and dispense air into said pneumatic ports in order to move the liquid samples in said reaction devices between said sample areas and said reaction areas; and a programmable control device for causing said robotically controlled aspiration and dispensing head to move into contact with the pneumatic ports of said reaction devices, and to withdraw air from and dispense air into said reaction devices in order to move said liquid samples between said sample areas and said reaction areas.

2. An automated system as claimed in claim 1, wherein said robotically controlled aspiration and dispensing head is adapted to be brought into contact with the pneumatic port of only one of said reaction devices at a time, and wherein said programmable control device causes said robotically controlled aspiration and dispensing head to move into contact with each of said reaction devices in sequence.

3. An automated system as claimed in claim 1, wherein said robotically controlled aspiration and dispensing head carries a detachable pipette for engaging the pneumatic ports of said reaction devices, and wherein said system further comprises a docking station for holding said detachable pipette when said pipette is not attached to said robotically controlled aspiration and dispensinghead.

4. An automated system as claimed in claim 3, wherein said programmable control device causes said robotically controlled aspiration and dispensing headto pick up said detachable pipette from said docking station before moving into contact with the pneumatic ports of said reaction devices, and to return said detachable pipette to said docking station after withdrawing air from and dispensing air into said reaction devices.

5. An automated system as claimed in claim 4, wherein said docking station includes a bracket with which said detachable pipette is engageable by means of a generally horizontal motion of said robotically controlled aspiration and dispensing head, said pipette being detachable from said aspiration and dispensing head by means of a generally upward motion of said aspiration and dispensing head while said pipette is engaged with said bracket.

6. An automated system as claimed in claim 1, wherein said programmable control device causes said robotically controlled aspiration and dispensing headto move said liquid samples from said sample areas to said reaction areas, to allow said liquid samples to remain in said reaction areas for predetermined intervals, and to return said liquid samples to said sample areas after said predetermined intervals have elapsed.

7. An automated system as claimed in claim 6, wherein said predetermined intervals are equal for all of said reaction devices.

8. An automated system as claimed in claim 1, further comprising:
a disposable pipette tip station adapted to hold a plurality of disposable pipette tips which are individually attachable to said robotically controlled aspiration and dispensing head; and a sample receptacle station adapted to hold a plurality of sample receptacles in which said liquid samples are initially provided;
wherein said programmable control device causes said robotically controlled aspiration and dispensing head to pick up disposable pipette tips from said disposable pipette tip station, to move to said sample receptacle station and aspirate liquid samples into said disposable pipette tips from said sample receptacles, and to move to said reaction station and dispense said liquid samples into said reaction devices.

9. An automated system as claimed in claim 8, wherein said robotically controlled aspiration and dispensing head is adapted to pick up only one of saiddisposable pipette tips at a time, and wherein said programmable control device causes said robotically controlled aspiration and dispensing head to move to said disposable pipette tip station and to pick up a new diposable pipette tip beforeaspirating a liquid sample from each of said sample receptacles.

10. An automated system as claimed in claim 1, further comprising:
a disposable pipette tip station adapted to hold a plurality of disposable pipette tips which are individually attachable to said robotically controlled aspiration and dispensing head; and a reagent station adapted to hold a plurality of reagent containers which contain liquid reagents to be added to said liquid samples;

wherein said programmable control device causes said robotically controlled aspiration and dispensing head to pick up disposable pipette tips from said disposable pipette tip station, to move to said reagent station and aspirate liquid reagents into said disposable pipette tips from said reagent containers, and to move to said reaction station and dispense said liquid reagents into said liquid samples.

11. An automated system as claimed in claim 10, wherein said robotically controlled aspiration and dispensing head is adapted to pick up only one of saiddisposable pipette tips at a time, and wherein said programmable control device causes said robotically controlled aspiration and dispensing head to move to said disposable pipette tip station and pick up a new disposable pipette tip before aspirating a liquid reagent from each of said reagent containers.

12. An automated system as claimed in claim 10, wherein said reaction station is adapted to hold a plurality of assay devices to which said liquid samples aretransferred after reactions are carried out on said samples in said reaction devices, and wherein the dispensing of said liquid reagents into said liquid samples is carried out by dispensing said reagents into said assay devices.

13. An automated system as claimed in claim 12, wherein prior to aspiration and dispensing of said liquid reagents into said assay device, said programmablecontrol device causes said robotically controlled aspiration and dispensing headto pick up disposable pipette tips from said disposable pipette tip station, to move to said reaction station and aspirate reacted liquid samples from said reaction devices, and to dispense said reacted liquid samples into said assay devices.

14. An automated system as claimed in claim 13, wherein said robotically controlled aspiration and dispensing head is adapted to pick up only one of said disposable pipette tips at a time, and wherein said programmable control device causes said robotically controlled aspiration and dispensing head to move to said disposable pipette tip station and pick up a new disposable pipette tip before aspirating a reacted liquid sample from each of said reaction devices.

15. An automated system as claimed in claim 12, wherein each of said assay devices includes a plurality of separate portions, each of said portions being adapted to receive part of the same liquid sample, and wherein the dispensing ofsaid reagents into each of said assay devices is carried out by dispensing a different one of said reagents into each portion of said assay device.

16. An automated system as claimed in claim 12, further comprising a robotically controlled wash head adapted to dispense a wash fluid into said assay devices and to aspirate fluids from said assay devices, said robotically controlled wash head being controlled by said programmable control device.

17. An automated system as claimed in claim 16, wherein each of said assay devices includes a plurality of separate portions, each of said portions being adapted to receive part of the same liquid sample, and wherein said robotically controlled wash head includes separate aspiration/dispensing nozzles for washingall of said portions simultaneously.

18. An automated system as claimed in claim 12, wherein each of said assay devices is held adjacent to a corresponding one of said reaction devices at saidreaction station, with the number of assay devices and reaction devices being equal.

19. An automated system as claimed in claim 18, wherein said assay devices and said reaction devices are carried by a tray which is removable from said automated system.

20. An automated system as claimed in claim 1, wherein said reaction station includes a heating platen for heating said plurality of reaction devices.

21. An automated system as claimed in claim 1, wherein said reaction station is one of a plurality of reaction stations in said automated system, and whereinsaid programmable control device causes said robotically controlled aspiration and dispensing head to carry out substantially the same functions at each of said reaction stations.

22. A reaction station for use in an automated system for carrying out reactions on a plurality of liquid samples, comprising:
a fixed heating platen for heating said liquid samples;
a removable tray positionable on said heating platen, said removable tray being adapted to hold a plurality of reaction devices in which said liquid samples are receivable; and a locating device for locating said removable tray at a predetermined position on said heating platen.

23. A reaction station as claimed in claim 22, wherein said removable tray is formed with cut-out portions to allow said reaction devices to make direct contact with said heating platen.

24. A reaction station as claimed in claim 22, wherein said removable tray is formed with shaped slots or cavities for receiving each of said reaction devices in a predetermined position and orientation.

25. A reaction station as claimed in claim 22, wherein said removable tray is adapted to hold a plurality of assay devices equal in number to said reaction devices, with each of assay devices being held adjacent to a corresponding one of said reaction devices.

26. A reaction station as claimed in claim 22, wherein said fixed heating platen is adapted to heat first sides of said reaction devices, and further comprising a second heating platen for heating second sides of said reaction devices located opposite to said first sides.

27. A reaction station as claimed in claim 26, wherein said second heating platen is movable into and out of contact with said second sides of said reaction devices to allow said removable tray to be positioned on and removed from said fixed heating platen.

28. A reaction station as claimed in claim 27, wherein said second heating platen is carried by a pivoting arm for movement into and out of contact with said reaction devices.

29. A reaction station as claimed in claim 28, further comprising a locking device for locking said pivoting arm into contact with said reaction devices andfor compressing said reaction devices between said second heating platen and said fixed heating platen.

30. An assembly for use in an automated system for carrying out reactions on a plurality of liquid samples, comprising:
a plurality of reaction devices in which said liquid samples are receivable, said reaction devices having substantially flat bottom surfaces through which heat can be applied to said liquid samples; and a tray adapted to hold said plurality of reaction device, said tray being formed with shaped slots or cavities for receiving each of said reaction devices in a predetermined position and orientation, and with cut-out portions for allowingthe substantially flat bottom surfaces of said reaction devices to make direct contract with a heating platen.

31. An assembly as claimed in claim 30, further comprising a plurality of assay devices equal in number to said reaction devices to which said liquid samples are transferrable after reactions are carried out on said samples in said reaction devices, each of said assay devices being held in said tray at a position adjacent to a corresponding one of said reaction devices.

32. An assembly as claimed in claim 31, wherein said assay devices have substantially flat bottom surfaces through which heat can be applied to said liquid samples, and wherein said tray is formed with cut-out portions for allowing the substantially flat bottom surfaces of said assay devices to make direct contact with a heating platen.

33. An assembly as claimed in claim 32, wherein said tray is formed with shaped slots or cavities for receiving each of said assay devices in a predetermined position and orientation, each of said slots or cavities being configured to support said assay devices in said tray at vertical positions suchthat the substantially flat bottom surfaces of said assay devices extend below the bottom surface of said tray.

34. An assembly as claimed in claim 33, wherein the slots or cavities in said tray which support said assay devices are formed with upwardly-facing ledges forsupporting said assay devices at said vertical positions, and wherein said assaydevices are formed with notched or stepped side surfaces for engaging said ledges, whereby said assay devices are held against downward movement in said tray but are free to move upwardly upon contact between the substantially flat bottom surfaces of said assay devices and a heating platen.

35. An assay device for use in an automated system for carrying out reactions on a liquid sample, said assay device comprising a plurality of connected wells for receiving portions of said liquid sample, each of said wells having an open top for admitting a portion of said liquid sample, a substantially flat bottom surface for making contact with a heating platen, and interior walls coated with a reagent.

36. An assay device as claimed in claim 35, wherein each of said wells is substantially cylindrical in configuration.

37. An assay device as claimed in claim 36, wherein said wells are connected to each other by means of a generally planar flange extending between the open tops of said wells.

38. An assay device as claimed in claim 37, wherein at least three of said wellsare connected together by said flange to form said assay device.