CA2236451A1 - Assay system and method for conducting assays - Google Patents

Abstract

The invention relates to an assay system for conducting elevated temperature reactions in a fluid-tight manner within a reaction chamber, the assay system comprising: (a) a first assembly comprising the reaction chamber, and (b) a second assembly for temperature control, wherein the second assembly can be positioned adjacent to the reaction chamber. More particularly, the invention relates to an assay system comprising (a) a reaction chamber having a cover formed of a deformable material and (b) a mechanism for rapidly adjusting the temperature of the reaction chamber.

Description

W O 97/16561 1 PCT~US96/17116 ASSAY ~Y~'l'~ AND METHOD FOR CONDUCTING ASSAYS

The invention was made with U.S. Government support under Contract No. 70NANB5H1037. The U.S. Government has certain rights in this invention.
The invention relates to the field of sri~ntific chemic~ql assays and, in particular, to a system and method for conducting such assays.
S~ientific assays useful in criminal forensics and me(lic~ nl)stics, for e~r~mple, have incre~qingly involved biochemical procedures, such as the polymerase chain reaction ("PCR") which has proven to be very valuable for analysis of trace amounts of DN~ In particular, the PCR
assay has provided a powerful method of assaying for the presence of either ~fin~l segments of ml~ ic acids or segments that are highly homologous to such defined segments. The method can be used to assay body fluids for the presence of nucleic acid specific for particular pathogens, such as the mycobacterium ç~llqing Lyme ~i.qe,q.qe, the HIV virus or any other pathogenic microbe. The microbe tli~nofitic assay functions by ~ in~, to a sample that may contain a target segment of nucleic acid from the microbe's g~.non~e, two "primers" (i.e., relatively short nllrl~ic acid segrn~ntq or nucleic acid ~n~lo~) that specifically bind to (i.e., "hybridize" with) the target segment of nucleic acid. The first primer binds to a first strand of the two-stranded target nllrl~ . acid segment and, when hybridized, can prime the enzymatic reprorl~ ti-n of a copy of the second strand of the target nucleic acid segment in a direction a.bi~ ly tl~.qi~n~tetl as the downstream direction. The second primer binds to the second strand of the target nucleic acid segment at a position downstream from the first primer hybrirli7~tion site and can prime the enzymatic reproduction of a copy of the first strand of the target nucleic acid segment in the upstream direction. (In the case where the sample is made up of single-stranded target nucleic acids, the second primer will hybridize with the theoretical secon-l strand determined with the Watson-Crick base-pairing rules.) To the sample are added the monomer building blocks of nucleic acid and an enzyme that specifically catalyzes nucleic acid reproduction from a single ~ strand of n~ ic acid to which a short primer is bound. The enzyme ispreferably highly resistant to destruction by high temperatures. The sample is he~te-l to a DNA m~lting temperature to separate the two strands of the sample nucleic acid and then cooled to a replicslt.icn -CA 022364~1 1998-04-30 W O 97/16561 2 PCT~US96/17116 temperature. The rep1ic~tion temperature allows the primers to specifically bind to the separated strands and allows the reproductive enzyme to operate. After this cycle, the reaction mix contains two sets of the two stranded nucleic acid segment for each target nucleic acid segment that was ~ in~lly present. ~e~qting and repliç~tion temperature cycles are repeated until sufficient amounts of the nucleic acid segment are created through this expon~nti~1 reproduction method. For instance, after 20 cycles the segment has been ~mp1ifie(1 as much as 22~-fold, or roughly l,OOO,OOO-fold. The PCR process is diagr~mme-l in Figure 9.
11~ rr~ere are several problems associated with aut~m~ting the PC~
re~ction First, the degree of ~mplifiç~tic n achieved by the assay creates a large risk of cont~min~tion from inadvertently miYed samples or oligonucleotides bound to laboratory equipment. Thus far, this risk has been dealt with in commercial or m~nll~l procedures by con(l~ n~ the re~ction.~ in "clean" facilities that are ~ e-.. ely expensive to construct and m~int~in For automation, this risk imp1ie~ that all the reagents n~l~l and the reaction chamber for the ~mp1ifiç~tion should be cont~ine-l in a disposable platform in which the sample can be inserted in a controlled, one-time operation and that sample preparation steps should be . . ,i ~ e-l and, to the extent possible, conducted within a disposable platform.
Second, the high temperatures needed to "melt" the nucleic acid so that the two strands separate imply that the reaction chamber must be well-sealed against vapor loss, even while allowing the insertion and removal of various reagent fluids.
Third, the re~r*on~ should be conducted in relatively small volumes, generally volumes of no more than about 1OO ~il, to conserve expensive reagents and ..~i..i...i~e the amount of sample, which could be a precious sample fluid or tissue that must be conserved to allow for other types of testing or only available in a small amount.
The invention provides a solution to the ~iffic111ti~.~ presented by current methods by providing a small-scale, disposable device that has effective, fluid-tight valves for controlling the insertion into and evacuation out of the reaction chamber of fluids.
SUMMARY OF THE INVENTION
The invention is an assay system for conducting elevated temperature reactions in a fluid-tight manner within a reaction chamber, where the assay system comprises a first assembly comprising the W O 97/16561 3 PCTnUS96/17116 reaction chamber, and a second assembly for temperature control, wherein the ~econd assembly can be positioned adjacent to the reActi-n chamber.
The invention is also a method of conducting a PCR reaction comprising introducing a q~mpl~ into a first re~rti-~n chamber of the assay system of the invention and transferring from one or more fLuid ch~mhers to the first reaction chamber solutions cfnt~ining reagents necessary for conductin~ the PCR re~ction BRIEF DESCRIPTION OF THE DRAWING
The te~rhings of the i~ elltion can be readily understood by 0 c-n.qirl~o.r~n~ the ~ol7Ow~ng deta~lied description in conjunction with the accompanying drawing, in which:
Figure lA displays a top view of the bottom tray of a carousel of the invention.
Figure lB displays a cut-away, perspective view of the bottom tray illustrated in Figure lA, the cut-away is along axis A-B shown in Figure lA.
Figure 2A shows a top view of a reaction chamber disk.
Figure 2B shows a cut-away side view of the rç~cfion chamber disk of Figure 2A.
Figures 2C and 2D show an enlarged view of the sidewall of a re~ctic rl chamber disk.
Figure 3 shows a top view of another bottom tray of a carousel.
Figure 4A illustrates an apparatus for forcing fluids into or out of a reaction chamber.
Figures 4B and 4C illustrate the operation of the apparatus of Figure 4A.
Figure 5 shows a reAr~il n chamber disk that inrh~ .q three reAction chambers.
Figures 6A, 6B and 6C show a merh~ni.~m for rapidly he:~tin~ or cooling the reaction chamber.
Figures 7A and 7B show another merh~ni~m for rapidly he~tin~ or cooling the reaction chamber.
Figures 8A and 8B display an assay cassette used in h~ mple 1.
Figure 9 srhçm~t.ically diagrams the first two cycles of a PCR
re~ction Figure 1O shows an example of a m~gnet useful in providing stirring for the reaction chambers.
To facilitate understqntling, i-l~ntir~l .e~e~ ce numerals have been CA 022364~1 1998-04-30 W O 97/16561 4 PCT~US96/17116 used, where possible, to tleqign~te i-l~ntic~1 elements that are common to the Figures.
DEFINITIONS
The following terms shall have the me~nin q set forth below:
~ "~nnf~.~1ing temperature" means about 5~C below the lowest Tm for one of the primers used in a PCR reaction and the target n~ leic acid seFrn~nt PCR protocols often use an annealing temperature less than the replic~t.ion temperature to accelerate the rate at which the primers bind to (i.e., hybridize with) the sample nl~r.lei~ acid; this temperature is typically between about 4$"C and about 7~~C, of~ten about ~5~C.
~ "DNA strand separation temperature" means the temperature used in a PCR protocol to separate the complementary strands of nucleic acid that may be present in a sample; this temperature is typically between about 92~C and about 97~C, preferably about 94~C.
~ "elevated pressure" means a pressure more than ~mhient atmospheric pressure.
~ "first assembly" means an assembly that in~ q.q at least one reaction chamber, at least one fluid .q~.h~qnge r.h~nne1, and at least one f~uid ~r.h~nge port; wherein the first assembly can form fluid-tight connection with a suitable third assembly.
~ "fluid-tight" means the characteristic of a space that ret~i~s the mass of an aqueous fluid filling the space and heated to a temperature of 90~C to 100~C for one hour; a seal between two materials is fluid-tight if the seal is subst.~nt.i~lly no more permeable to water than the most permeable material.
~ "nucleic acid m~1ting temperature" or "Tm" means the transition temperature for two-stranded duplex of n1~ ic acid at which the eq11i1ihri~
shifts from favoring the base-paired duplex to favoring the separation of the two strands.
~ "re~-.ti~ n cassette" means a disposable unit comprising a first assembly and a third ~.qsP.mh1y.
~ "reduced pressure" means a pressure that is less than ~mhient atmospheric pressure.
~ "repli~t.ion temperature" means the temperature used in PCR to allow the nucleic acid reproductive enzyme to reproduce the complementary strand of a nucleic acid to which a primer is bound (i.e., hyh~rli7:erl); this temperature is typically between about 69~C and about 78~C, preferably CA 022364~1 1998-04-30 W O 97/16561 5 PCT~US96/17116 about 72~C, when using a heat stable polymerase such as Taq polymerase.
~ "second assembly" means an assembly for controlling temperature that includes a heat source or a cooling sink or both; the second assembly is also lefe.led to herein as an "~ ry block," and can include a fluid impeller.
~ "subs~nt.i~lly llniform temperature" means a temperature that varies by no more than about +/- 0.3~C.
~ "target nucleic acid segment" means a segment of nllrl~;c acid that is sought to be i(l~ntifie-l or measured in a sample of nll~ ic acid, such as a sequence int~n~le-1, if present, to be ~mplifi~ in a PCR re~ction; the target se~mf~nt is typically part of a much larger nucleic acid molecule found in the sample.
~ "&d assembly" means an assembly that includes one or more fluid chambers, one or more fiuid ~rh~n~e rh~nnel.c and ports, and which can form a fluid-tight comhin~tion with a sllit~hle first assembly.
DETAILED DESCRIPTION
The invention is an assay system for co~ rt;ng biorh.omir~l reactions in a fluid-tight m~nn~r within a reaction chamber. The assay system is not limit~tl by any particular geometry or configuration of its component parts; preferably, the assay system includes circular, square, or rectangular assemblies, or comhin~tions thereof, such that separate components that respectively comprise a reaction chamber ("a first assembly"), a heat source or a cooling sink or both ("a second assembly"), and a plurality of fiuid chambers ("a third assembly") can move with respect to each other by sliding or translocating, as a~o~l;ate. The first and third ~.c~mhlies may each have an adjoining edge or surface that has fluid ~ h~nge ports that can . eve. ~ibly connect to provide fluid commllnic~tion between the assemblies as a result of the sliding of one assembly with respect to the other along the adjoining edge or surface.
Such sliding occurs in a linear or circular m~nn~r depending on the configuration of the assemblies that is used; if a circular configuration is used, such sliding is alternatively referred to as rotating. The first and second or first and third assemblies or both may also each have an adjoining surface or surfaces that can reversibly be placed in contact with one another to provide temperature c~ntrol, or fluid impelling to or from fluid or re~ctioI chambers, or both, as a result of the translocation of the second assembly to contact or separate from the first or third or both assemblies.

CA 022364~1 1998-04-30 W O 97/16561 6 PCT~US96/17116 A circular embodiment has been drawn and presented in the accompanying Figures. In Figures lA and lB, the bottom tray 110 of a carousel 100 is shown that has 18 fiuid chambers 120A-Q. Each chAmher 120 has a fluid ~r.h~nge port 121 that is or can be in communication with fluid ~rh~nge channels. In the carousel 100, the sidewalls 122 of the chambers are molded together with the base 111 of the carousel 100, which base 111 forms the bottom 123 of each chamber 120A-Q. Each fluid ~rrhflnge port 121A-Q exits at the smooth inner ring surface 140 formed by the carousel 100. Not illustrated is the top cover 130 of the carousel 100 I i~ that i~ïts on top of bottom tray lla. The cover 130 wili generally cover the fiuid chambers, and the junction between the top of the sidewalls 122 and the cover 130 will generally form a fluid-tight seal. Also illustrated is position notch 101, which serves to interact with the merh~ni.c:m controlling the assay system to identify the rot~t.i-~n~l position of the 1 5 carousel 100.
Figures 2A and 2B show the superstructure of a reaction rh~mher disk 200, which is a design that can fit with any system geometry provided that a circular slot is provided for insertion of the reaction chamber disk.
For example, the disk 200 illustrated in Figures 2A and 2B can be used in conjunction with the carousel 100. Alternatively, the re~ction chamber disk could be used in conjunction with a rectangular assembly that comprised a plurality of fluid ch~mbers and inr.lllrle-l a suitable circular slot for insertion of the reaction chamber disk. The disk 200, comprised of one or more reaction chambers, is one embotlim~nt. of a first assembly of the assay system. Alternatively, the first assembly can be any other suitable shape that provides ability to effect or break communication via fluid P.~r.h~n~e rhs~nn~l.s inr.l~ d in the first assembly with those in a third assembly that provides reagents and waste disposal from or to f~uid chambers.
The first assembly, embodied as disk 200, inr.lll-les a structural ring 210, a bevel edge 220 and a spacer ring 230 that ~l~fines the width of the reaction chamber. First fiuid ~rh~n~e channel 231 and second fluid f~r.h~n~e r.h~nnf~.l 232 are located on opposite sides of disk 200. In Figure 2C, the sidewalls 211 of ring 210 has first and second notches 212 and 213.
First and second locking rings 214 and 215, shown in cross-section, lock into place within ring 210 by clipping into notches 212 and 213, respectively.
Membrane 241 fits between first locking ring 214 and first notch 212, _ CA 022364~1 1998-04-30 thereby creating an upper cover 251 for reaction chamber 250. Membrane 242 Rimil~rly fits between second locking ring 215 and second notch 213 to form lower cover 252. Smooth outer surface 216 fits snugly against inner ring surface 140 of the carousel 100 to form a fluid-tight seal, thus forming a snug jlmrtinn A "snug junction" is one that is fluid-tight. In Figure 2D, the disk 200 further inc~ s a gasket 260 that serves to insulate spacer ring 230 and ring 210 from the elevated temperatures of the chamber 250.
Figure 3 illustrates another embo~im~nt of tray 110. In ~ lit;~n to the features i-l~.ntifie~l above, the carousel 100 has contlllitq 125, having 1 0 first opening 126 and second opening 127. Conduits are also referred to herein as f~uid axch~n~e ch~nn~l.q. Second opening 127 is ~ ne~l to facilitate a union with a source of a gas or liquid (not illustrated). Fluid chamber 120-H1 is connected to the outer rim of the carousel by valved portal 124.
1 5 Figures 4A, 4B and 4C illustrate a mech~niRm having upper ~llxili~ry block 300A and lower ~llxili~ry block 300B for forcing fluids into the chamber 250, wherein the assay system has any sllit~hle geometry and structure 300 is an embodiment of a fluid impeller. Upper block 300A
is honeycombed with passageways in-]ll-ling upper gas inlet/outlet 310A, Z0 upper m~nif~lkl 320A and a plurality of upper pressurized l h~nn~lR 321A.
Upper channels 321A exit adjacent to the surface of upper membrane 241.
The corresponding structures of the symmetrical lower block 300B are correspon~lingly numbered using the suffix "B" instead of "A". In Figure 4B, gas pressure has been applied through upper gas inlet/outlet 310A and lower gas inlet/outlet 310B, so that gas exiting upper and lower pressurized channels 321A and 321B forces upper and lower memhranes 241 and 242 together, thereby forcing fluid from chamber 250, thus providing ns~ti~n ofthe afor~mf~ntione(l fluid impeller embo-lim~nt In Figure 4C, a vacuum applied to gas inlet/outlets 310A and 310B creates suction at upper and lower channels 321A and 321B, c~llRing the upper and lower membranes 241 and 242 to adhere to blocks 300A and 300B, respectively, thereby pulling upper and lower m~mhranes 241 and 242 apart and partially evacuating the invention. The partial vacuum in chamber 250 - helps draw fluid into the hlv~:lllion through one of first or second fiuid ~th~nge channels 231 or 232.
In Figure 5, the reaction chamber disk 200 in( lll-l~R three chambers 250A-250C, which can be any suitable shape or vol~lme, each with its own CA 022364~1 1998-04-30 W O 97/16561 8 PCT~US96/17116 set of first fluid ~ h~n~e ~h~nnel~ 231A-231C and second f~uid ~rh~n~e channels 232A-232C.
Figure 6A shows one embo-liment of a second assembly which is ~;mil~r to the ~ ry block illustrated in Figures 4A-4C, except that it inr.llltles additional features. The second assembly 300A is honeycombed with upper conduit 330A. Upper conduit 330A has an upper inlet 33~A and an upper outlet 332A. Upper portion 301A of upper block 300A is fabricated of a heat-inslll~*n~ material, while upper section 302A is fabricated of a heat-conductive material. Upper electrical heaters 340A
1 ~1 are posltloned adl~acent to the chamber 25(~. Generally, a duplicate lower second assembly 300B of the illustrated upper second assembly 300A is positioned beneath the chamber 250.
Figure 6B shows a schematic of the accessory support devices for the upper second assembly 300A of Figure 6A. Chilled water is propelled 1 5 through upper and lower conduits 330A and 330B from pump and water cooler console 350. Pump and water cooler console 350 further incllltle.c fluid valves operating under the control of controller 400. Electrical current is supplied to upper and lower heaters 340A and 340B by power supply 360, which is controlled by controller 400. Controller 400 receives input from upper and lower thermal sensors 370A and 370B.
Figure 6C shows another embodiment of a second assembly 300A
.~imilz~r to that of Figure 6A, except that an upper ç~ten~ion 303A is added to the outer face of the upper z~ln~ y block 300A. The upper f~.xten~ion 303A is generally fabricated of the same material as upper section 302A.
The upper portion 301A can favorably be fabricated of the same material as upper section 302A. First and second upper thermal insulator se~rner~t..~
322A and 323A are interposed between upper portion 301A and upper section 302A. First and second upper thermal sensors 304A and 306A are located in upper section 302A and upper ~ten-~ion 303A, respectively, and are connected to controller 400 by first and second upper leads 305A and 307A, respectively.
In Figure 7A, an upper second assembly 500A in~ a set of paired first and second upper thermoelectric blocks 511A and 612A, wbile lower second assembly 500B has a set of paired first and second lower thermoelectric blocks 511B and 512B. First upper and lower thermoelectric blocks 511A and 511B are made of p-type semiconductor mflt~ri~l, while second upper and second lower thermoelectric blocks 512A

CA 022364~1 1998-04-30 and 512B are made of n-type semiconductor material. The blocks 511 and 512 are electrically connected in series by upper and lower connectors 513A and 613B to form thermoelectric heat pumps. Upper and lower gas inlet/outlets 510A and 510B are connected to upper and lower m~nifoltls 520A and 520B, respectively, formed by the space between the upper and lower thermoelectric blocks 501A and 501B. ~ nifol~ 520A and 520B are connected, respectively, to an upper plurality of passageways 521~A or a lower plurality or passageways 521B. The outer portions of upper and lower blocks 500A and 500B are upper and lower heat sinks 504A and 1 0 504B, respectively. First upper air-tight collar 506A, second upper air-tight collar 507A, first lower air-tight collar 506B and second lower air-tight collar 507B help form manifolds 520A and 520B. Upper and lower thermal sensors 570A and 570B are connectable to a controller or a moni~ring device by upper and lower leads 571A and 571B, respectively. Figure 7B
illustrates the surface of a ceramic end-plate 502 that would face the chamber 250. Figure 7B illustrates one of the three rlimen.~ l aspects of second assemblies 500A and 500B not apparent in Figure 7A; another attribute of the second assemblies not apparent in Figure 7A is that the thermoelectric blocks will typically be arrayed in three tlim~?n~ions rather than two, as illustrated.
Figure 9 shows a schematic of a polymerase chain re~cti~ n Tine 901 represents a first strand of target DNA having, from right to left, 5' to 3' orient~ticn Tine 902 represents the complementary strand of target DNA having 3' to 5' orientation. Primers 903 are complçmerlt~ry to first strand 901 at position I. Primers 904 are compl~m~n~sry to second strand 902 at position J. A temperature stable DNA polymerase (e.g. Taq polymerase) and the nucleotide triphosphates used by the polymerase to replicate DNA are present in the ~ Lule. As a first step, the temperature is raised to a strand separation temperature to separate strands 901 and 902. Then, in a second step, the temperature is lowered to a replication temperature where the abundant primers bind to the first and second strands 901 and 902, respectively, at positions I and J, respectively, and the polymerase builds onto the bound first and second primers 903 and 904 ~ to construct replicas of portions of first strand 902 and second strand 901, respectively. After two cycles, the portion of DNA between the po~ition~ I
and J has been replicated four-fold.
The chamber 250 can have any sllit~hl~ shape. For a circular CA 022364~1 1998-04-30 W O 97/16561 lo PCTAUS96/17116 shape, the af~ entioned third assembly preferably is in the shape of a carousel, i.e., a circular conveyor or depositor of reagents or waste products, respectively. An alternative configuration is a rectangular volume, wherein the third assembly can slide relative to fiuid ~hAn~e ports on a first assembly which includes a reaction chamber. The third assembly can be constructed of a nllmber of materials in~ ing without limitation optical grade glass, silicon-based materials or plastic. In some cases, it may be necessary to surface treat the material, for instance with chloromethylsilane or dichlororlimethyl~qil~ne~ to ..~ e the sites on the 111 m~teri~l that bind to biological molecules such as proteins or nucleic acids.
A highly favored material is polyethylene (PE), particularly high density grade PE, which is favorably tolerant of high temperatures. When constructed of plastic, the third assembly is preferably formed by injection mol~ling Generally, the cover 130 of a third assembly is formed separately from bottom tray 110. The cover 130 is adhered to the tray 110 by, for instance, pressing these parts together while applying heat until the two materials fuse, and then rapidly cooling while contimlin~ to press the parts together until the fused junction s~ ifi~.~. The seals between the sidewalls 122 ofthe chambers 120 and the cover 130 are generally fluid-tight. The cover 130 is formed either of a deformable polymeric material~which is stretchable and elastic, or of a more rigid m~t~riF~l, such as glass. If a more rigid material is used it preferably has a coefficient of e~p~n~ion comparable to that of the material f-rming the tray 110. H~wev~.-, this ~imil~rity of thermal ~p~n.~ion is not critical in this conte~t because the carousel is removed from the portions of the assay system that are subjected to cycling between two or more temperatures.
Where the cover 130 is made up of a deformable material, the fluid impeller for impelling fluid to flow from chamber 120 to chamber 250 can be a mech~nical plunger with substantially as much cross-sectional area as will allow the plunger to fit within the walls of the chamber 120 to drive the fLuid out through the fluid ~ h~n~e port and into a properly aligned reaction chamber. Where the carousel 100 is rotatable, it can be rotated to position the chamber 120 under such a plunger when the assay (or other reaction) being conducted in the assay system calls for the fluid in the chamber 120 to be transferred into the chamber 250. .~imil~rly, in a rectangular system, the first and third assemblies slide relative to each other to align fluid W O 97/16561 11 PCTrUS96/17116 f~x(~hAnFe rh~nnel.q that connect reagent-cont~qinin~ third chambers to reaction chambers. This rotation or sliding is calibrated to align the fluid f~x( h:~n~e port in commllnicAti~n with the fluid chamber with either the fluid ~x~hAn~e port in communication with the first or second fluid ~xlhAn~e ~hAnnçl, 231 or 232, respectively, regarding the carousel embodiment) of a re~ction chamber. In some embodiments, when the IhAnne~l 231 or the ~hAnn~l 232 is align~(l with a fluid ~ hAn~e port 121, the other ~.h~nnel (231 or 232) is Ali~n~ll with another port 121; this dual Alignment. allows fluid to be inserted into the disk 200 through one port 121, while the gas or 0 f~uid previously present in the disk 21)0 is forced out through the other port 121. In other embo~im~ntq the disk 200 is evacuated prior to the insertion of a fluid, which f~cilit~tes the subsequent fluid insertion.
Another means of imp~lling a fluid to flow from chamber 120 to chamber 250 requires ~ nin~ an ~lixili~ry block (such as upper AllxiliAry block 300A of Figure 4A) over chamber 120 having a deformable cover.
The AllxiliAry block includes a plurality of gas vents that are used to create a pressure against the cover 130 to deform it duwllw~ls to move the fluid out of the fluid chamber. The spacing between the block 300A and the infl~xihle portion of the cover 130 adhered to the sidewalls 122 of a chAmber 120A-120Q are sufficiently narrow to allow enough pressure to be applied to the fluid so that it will move out of the chamber 120 through the port 121. A mechAniqm of this type is illustrated in Figure 4A. This type of mech~ni.qm can be operated in reverse by applying a vacuum to the vents so that the cover 130 will seek to adhere to the ~llxiliAry block surface. As the AllxiliAry block, together with the adhered cover 130 is drawn away from the chamber 120, a partial vacuum is created in the chamber, cAll.qing fluid to be drawn into the chamber.
In another embo-liment7 the fluid chambers have valved portals 124 conn~cted to the outer edge of the carousel (see Figure 3A). The carousel 100 can be operated at a right angle to the orient~ti(n illustrated, so that the chamber 120 from which a liquid is to be evacuated can be ~lign~tl above the chamber 250. Gas can be a-lmitted through portal 124 to the chamber 120 to force fluid to evacuate the chamber 120 through the ~x~hAnge port 121 located at the bottom of the chamber 120. One way to assure that gas is not admitted into the chamber 250 is to design the chAmber 250 with less volume than the chamber 120, thereby cAllqing the chamber 250 to fill before all the liquid is drained from the chamber 120. A

CA 022364~1 1998-04-30 W O 97/16561 12 PCT~US96/17116 gas with low solubility in aqueous solutions, such as hPlillm, will generally be preferred in this context.
The valve of portal 124 can be a check valve allowing the ~mi~Rion of fluids but blocking O~W~LL d flows of fluids from chamber 120.
Alternatively, the valve can be an electromagnetically actuated valve connected to contacts on the outer surface of the carousel. These contacts can be aligned with other electrical contacts that provide the power to actuate the valve.
Figure 3 illustrates an alternative embo-lim~nt of the bottom tray 11~ 110 where the ~uid chambers 120A1, 120B1 et seq. comprise a plurality ofvariously sized chambers of round cross-sectional shape. The cover 130 can be replaced by plungers that are per~n~nently ~lign~ with the chambers 120A1 etc. If the carousel 100 is designe-l to rotate, the plungers are fixed to a superstructure that rotates with the carousel 100. To increase the disposability of all components that contact assay fluid, the plungers, and even the superstructure, are preferably fabricated out of an inexpensive, easily molded material such as a plastic. In a ~l~re~,ed embo-lim~nt to facilitate the formation of a tight seal, a material "C" is used to construct the plunger and a material "D" is used to construct the walls of the fluid chamber, wherein materials C and D have Rockwell hardness values that are equal to or greater than R50 to insure that the assemblies will not deform in use. Equally important is use of a m~teri~l with a low permeability to water such as polyethylene. In a l l~relled embodiment, C is a R50 polyethylene, while D is a R110 polycarbonate.
Alternatively, fluid can be drawn out of or into the chambers 120A1 etc. of Figure 3 by the same me~h~ni~m~ described above.
In one embo~iment, the carousel 100 has chambers 120 on the top half and additional chambers 120 on a bottom half (not illustrated). In this embodiment, the assay system can have two means for imp.ollin~ a fluid to ~0 leave a chamber 120: one ~ nerl with the upper part of the carousel and the other aligned with the lower half.
Where the assay system is designed to ~ccommodate more than one chamber 250, such as disk 200, there will p,erelably be at least about nine chambers 120 per first assembly, more ~fe~ably at least about fifteen (15) chambers 120 per first assembly, yet more ~ ably at least about twenty (20) per first assembly.
In ~A~lit;~n to the fluid chambers 120, in some embo-liment,s there CA 022364~l l998-04-30 W O 97/16561 13 PCTnJS96/17116 will be one or more conduits 125 through the third assembly, each c- n~ it having a first opening 126 at the inner ring surface 140 capable of ~ nin~
with a first or second fluid ~h~n~e ~h~nnel (231 or 232, respectively). The conduits will have a second opening 127 capable of forming a union with a source of a gas or liquid, such as, without limit~tion, a wash fLuid or an inertgas.
Typically, the ~lh~n~e ports 121, conduits 125 and portals 124 are t-h~qnn~l~ or openings of diameter between about 125 micrometers (~Im) and about 1250,Um, preferably about 500,Um. Typically, the carousel 1OO has a diameter between about 8 cm and about 1O cm and a depth between about 6 millimeters (mm) and about 8 mm. Other configurations of the third assembly occupy similar volume, having, for ~ mple, a length of between about 7 cm and 9 cm for a square configuration and a depth of between about 5 mm and about 8 mm.
The first assembly comprises one or more reaction chambers and can be constructed of the same materials as those set forth for the third assembly. In one embodiment, the disk 200is made up of a structural ring 210 with a first and a second ~ h~n~e rh~nn~l (231 and 232, respectively), as illustrated in Figures 2A and 2B. The ring 210 fits snugly within the inner ring surface 140 of the carousel 1OO, thus rendering the assay system fluid tight upon assembly.
In one embo-lime~n~ the chamber 250 is formed by stretching two thin, deformable membranes 241 and 242 across the structural ring as illustrated in Figures 2C and 2D. Preferably, the membranes 241 and 242 are formed of a deformable polymeric film that is stretchable and flf~ihl~, such as polyethylene, polyvinylidene fiuoride or polyethylene/polyethylene terepth~l~te bi-layer. Suitable films are av~ hle, for instance from R~p~k Corporation of Minn~rolis, MN or E.I. duPont de Nemours and Co., Wilmington, DE. Preferably, the m Pm hranes 241 and 242 are resistant to temperatures as high as about 120~C. Preferably, the membranes 241 and 242 are between about 25 and about 150 ,Um in thi-kness, more preferably, between about 50 and 1OO ,llm. The thinne.~.~ of the m~mhranes f~ ilit~tes rapid heat ~ h~nge between the re~ctiQn chamber ~ and an ~ c~nt h~tin~ or cooling device. Preferably, the total volume of each chamber 250 is between about 5 ~11 and about 200 ,ul, more preferably, between about 20 ,ul and about 1OO ~1l. Preferably, the chamber 250 has a thi~.kness (i.e., distance between covers 251 and 252) of about 1 mm or less.
When the chamber 250 has a deformable upper cover 251 or lower cover 262 (or both), forces can be applied to these covers in the same ways described above for the chambers 120 to push fluids into or out of the reaction chamber. A ~.er~lled means of doing this is illustrated in Figures 4A, 4B and 4C. Note, how~ve~-, that while the illustrations show both the upper cover 251 and cover 252 being deformed to move fluid into or out of a reaction chamber, one of these covers can be infl~xihle and fluid can be moved into or out of such a reaction chamber using only the deform~tion of 1 0 the one stretchable cover.
The chamber 250 can be rl~.ciFn~rl so that when a fluid ~ch~nFe rh~nnel 231 or 232 is ~ n~rl with the fluid ~xrh~nge port 121 of a given chamber 120, the other _uid ~rh~nge rhzlnn~l is sealed (i.e., not ~
with a _uid ç~h~nFe port). In this po.qi~ion, fluid can be forced from the 1 5 chamber 250 into the ~lignf~ fluid chamber 120, for instance, using the merh~ni.~m illustrated in Figure 4B. Fluid can then be drawn into the chamber 250 by ~qliFninF an ~r(h~n~e ~h~nnel 231 or 232 with another chamber 120 and forcing the fluid in the chamber 120 into the ch~mher 250. The fiuid can be drawn into the chamber 250 using positive pressure applied via mech~ni~m.~ associated with the carousel 100, or with negative pressure such as that applied through the me~h~ni~m illustrated in Figure 4C.
To increase the assay variables that can be accommodated on a single assay system, the first assembly, such as a reaction chamber disk 200, preferably includes more than one chamber 250, ~lef~ldbly at least three, more preferably at least five. A disk 200 with three reaction chambers 250A-C~ is illustrated in Figure 5. Note that the illustrated disk 200 is designed so that each chamber 250 can interact with its own set of chambers 120, with each such set of chambers 120 fiitll~te~ within a separate 120~ arc of the reaction cassette. In a ~lefe~l~d embo~liment the covers 251 or 252 of the chambers 250 of a disk 200 are fl~ih]e membranes 241 and 242.
When ll.cing a multi-reaction chamber system in conjunction with merh~ni.~m~ that push fluid into or out of the reaction ch~mbers, some of the me~hAni~m~ for doing so that are described above may need morlific~ti- n in ways that will be apparent to those of or&a~ skill.
H~3wev~r, the task of mzlking these morlifir~ti(m~ is made easier because CA 022364~1 1998-04-30 W O 97/16~61 15 PCT~US96/17116 typically fluid will be inserted or evacuated from all of the re~ct;o~
chambers at the same time. Because of this concurrent operation of the reaction chambers, the pressure control meçh~ni.~m of evacuating or f~lling reaction chambers that is illustrated in Figures 4A - 4C should not require modification beyond, perhaps, altering the distribution of the f~uid rh~nnal.~
321A and 321B to better align the passageways with the reaction chambers.
When the first assembly cont~in~ mlllt.iple re~ctior- ch~mbers, such as a re~ctiorl chamber disk as illustrated in Figure 5, the fiuid chambers 11~ suitable for use with, for instance, the first chamber 250A has a r.h~nnel 231A at a first position on outer surface 216, while a second chamber 260B
has a fluid ~lh~nge rh~nne] at a second position on outer surface 216.
~;t.i~)n~l chambers 250 have ~.~rhzln~e rh~nnel.~ at other distinct posit.i( n.~. Each chamber 120 suitable for use with a given chamber 250 1 5 has a port 121 positioned at a position on inner surface 140 a~lo~l;ate for ning the port 121 with the channel 231 of the corresponding chamber 250.
Typically, the r.h~nnel.~ 231 and 232 have a diameter between about 125 ~Im and 1250 ,um, preferably about 500 ~Im. Typically, the disk 200 has a di~meter between about 7.5 cm and about 10 cm, and a depth that a~ x;...~tely corresponds with the depth of the carousel 100; first ass~mhlies used in the corlt~.~t of other configurations of the assay system occupy .~imil~r volumes as was analogously discussed above with respect to the third assembly.
Important variables in det~l.. i.. i.. g the quality of the seal between the first and third ass~.mhlies, such as the surface 140 of the carousel 100 and outer surface 216 of the disk 200, are the smoothness of and the fit between these two surfaces. When using a plastic, one way to achieve a sufficiently smooth surface is to form the surfaces by injection mol~ling The injection mol~ing methods described by U.S. Precision Lens (Cinrinn~t.i, OH) or Matrix Inc. (Providence, RI) are particularly suitable for dimensional repro~llr.ihility of s_all assemblies.
In a ~lererled embodiment, to f~rilit~te the formation of a tight seal, - a material "E" is used to construct surface 140 and a material "F" to construct the outer surface 216 of the disk 200, wherein materials E and F
have Rockwell hardness values as given previously. In a preferred embodiment, E is a R50 polyethylene, while F is a R110 polycarbonate.

The third or the first assembly, such as the carousel 100 or the disk 200, respectively, will generally be ~tt~hed to a motorized, mechanical means of sliding or rot~tion Such sliding or rot~tion~l devices are well-known in the ~n~ine~ring arts. Preferably, the means of sliding or rotation is sl7ffi~ nt.1y precise to reproducibly align the first l~h~nn~l 231 or the second channel 232 with any given fiuid ~ h~nge port 121 when the controller 400 selects such an ~ nment as a~lol~.iate for a given part of an assay protocol. For instance, the position notch 101 of the carousel embodiment illustrated in Figure lA can be used to precisely fit the 1 0 carousel within an outer ring that has a uniform set of gear teeth on its outer edge. The gears, in turn, interact with a stepper motor operating under the direction of the controller 400.
Typically, when one of the disks 200 or the carousel 100 is attached to a motorized means of rotation, the other will be f~xed in place during the operation of the assay system. This locking into place re~ s the ~lignment. variables and improves the repro~ ihility of ~lignm~nt. between the carousel 100 and the disk 200.
Numerous means of rotating or sliding one of the third or first assembly, e.g., the carousel or the reaction chamber disk, will be apparent to those of ordinary skill in the me-h~nic~l arts.
The upper or lower second assembly 300A or 300B (or, of course upper and lower second assemblies 500A or 500A) can cont~in a plurality of upper or lower pressurized fluid ~ h~nn~qlR 321A or 321B. The fluid within these ~h~nn~.lR is typically a gas. Gas of higher than atmospheric pressure can be applied to the rh~nnel.~ 321A or 321B from, for instance, a pressurized gas canister or a pump applied to upper or lower gas inlet/outlets 310A or 310B. A vacuum, usually a partial vacuum, can be applied to the channels 321A or 321B using, for instance, a vacuum pump.
Numerous meçh~ni.~m.~ for controlling the pressure of the pressurized fluid ~ h~nn~lR will be reco~ni~e~l by those of ordinary skill in the engineering arts.
As part of a meçh~ni~m for creating a vacuum in an adjacent reaction chamber 250, the block 300A or 300B can have a means for drawing the block 300A or 300B away from the chamber 250, thereby drawing an adherent, stretchable cover 251 or 252 away from the reaction chamber. Such means of drawing the ~ ry block away will typically be a me-h~niç~l or electromechanical device. Such devices are well-known to those of ordinary skill in the engineering arts.

CA 022364~1 1998-04-30 W O 97/16561 17 PCT~US96/17116 In a preferred embo-lim~nt, either, (1) at least one reAction chamber has a transparent external wall that is generally an upper cover or lower cover (or two external walls are transparent), such as in structures 250, 251 and 252 in Figure 2B, or (2) at least one fluid chamber has a transparent external wall that is generally the top cover or bottom of the chamber (or two external walls are transparent). The assay system in this embodiment preferably in~ es a light source capable of directing light to the transparent cover or bottom and a detection device for ~letecting (a) the light reflected from an illllmin~t~-l chamber (such as 250 or 120 in Figure 1 0 8A or 8B, for example), (b) the light transmitted through an illllmin~t~l chamber (260 or 120), or (c) the light emiR~iorl.~ Pm~n~ting from an ~itetl molecule in a chamber (250 or 120). An external wall is "transparent" if it is at least about 80% transparent at a wavelength useful for detec~;ng biologi~l molecules.
1 5 The detection device can incorporate optical fibers. With fiber optics, the size of the detection system that is adjacent to the assay system is ...i.,i,..i7:~-1 This size minimi7~tion facilitates incorporating the dete-ctior~
system together with the temperature control device (incorporated in the second assembly) and rotational me~h~ni~m into the assay system. A
particularly ~ler~lled light source is a solid state laser. The size of these light sources also f~.ilit~tes incorporating a number of ~ ry assqmhlies about the assay cassette. When PCR is contlll-teA in an assay system that incorporates current technology solid state lasers, the method used to detect amplifiied nucleic acid uses a dye that absorbs light at a wavelength higher than about 600 manometers (nm) to in~i~te the presence of ~mplified n~ ic acid, as described below. Examples of such dyes in~
Cy6TM conjugated reagents (Jackson ImmunoResearch Labs, Inc., West Grove, PA), which reagents absorb in the 600-650 nm range and emit a fluorescent signal in the 630-750 nm range, allophycocyanin and allophycocyanin-conjugated reagents (Sigma Chemic~l Co., St. Louis, MO), and C-phycocyanin and C-phycocyanin-conjugated reagents (Sigma Chemic~l Co., St. Louis, MO). The relatively high wavelengths described above avoid much of the background fluorescence associated with - oligonll-leotides, plastics and other components of the assay system. A
~.~relled solid state laser source is a Laser Max, Inc. (Rochester, NY) Model LAS-200-635.5.
~Sign~ from the detection device will typically be input into the CA 022364~1 1998-04-30 W O 97/16561 18 PCT~US96/17116 controller 400, where they can be used to rl~termin~ whether an assay should continue or to generate an assay report.
The speed with which the temperature of the chamber 250 is increased or decreased is important for optimi7~ing various enzymatic-based assays, including PCR-based assays. During the temperature cycling important for PCR, it is important to operate at a relatively lower temperature where the nucleic acid sample is enzym~tic~lly reproduced and at a higher temperature where the nucleic acid sample is melte-l to separate the two strands of the nucleic acid. During the period when the 1~ assay apparatus cycles between the two preselected temperatures believed to be appropriate for a given nucleic acid ~mplific~tion, various unwanted chemistries can be expected to occur. For instance, as the temperature increases from the lower temperature, the replication enzyme can be expected to continue to function, although not necessarily with the appropriate accuracy of replic~tion achieved at the prescribed lower temperature. At the higher temperature set point, this unwanted enzymic activity is inhihite-l by the high temperature. Thus, it is important to rapidly change the reaction temperature between the two operating temperature plateaus.
One me- h~ni.~m by which the temperature can rapidly be changed in the reaction chamber is illustrated in Figures 6A and 6B. Although this illustration is provided with respect to a circularly-configured assay system, the same heat source and cooling sink çlement~ can be used in the context of the assay system of any suitable configuration, as would be understood by those skilled in the art. Assllme that the chamber 250 is operating at lower plateau temperature "G". Under these con~ition.~, cooling water does not flow through upper and lower conduits 330A or 330B.
The temperature is m~int~in~(l by intermittently operating upper and lower heaters 340A and 340B when the temperature in the chamber 250 lowers bene~t.h a temperature of G minus X (where X is a temperature di~el~lllial). At a pre-programmed time, the temperature is raised to higher pl~te~ll temperature "H" by activating heaters 340A and 340B until a temperature is rç~-~he-l that will lead to a temperature qt~hili7~t.ion at temperature H. Water flow through conduits 330A and 330B can be activated to minimi7e temperature overshoots if needed, which action tends to increase the cooling sink efficiency of the second assembly.
Temperature H is m~int~ine-l by intermittently operating heaters 340A

CA 022364~1 1998-04-30 W O 97/16S61 19 PCT~US96/17116 and 340B when the temperature of the chamber 250 lowers bçn~t.h a temperature of H minus Y (where Y is a temperature difrel elltial). To cycle back to temperature G, the controller activates the pump 351 (not illus,trated) of console 350 to cause cooling water to flow through con~ it~
330A and 330B of the second assembly. In another embo~im~nt the cooling sink effect is provided by a suitable material of adequate heat h~nge capacity and sufficient mass thereof that is level;jibly brought into co~t~ct with the reaction chamber in need of being cooled.
The heaters 340A and 340B are generally thin layers of conductive 1 0 material that is separated from the heat conductive upper and lower section 302A and 302B of blocks 300A and 300B by a thin electrical inslll~ti- n layer. The blocks are referred to herein as second assemhlies of the assay system, which assemblies comprise a heat source and a cooling sink. Alternatively, or in A-l~litic~n, such second assemblies can comprise a l 5 mer~h~ni.~m for pushing or pulling a deformable cover on a fluid or reaction chamber. The insulation layer is formed, for example, by direct deposition onto the substrate. For ~ mple, silicon nitride can be deposited from the gas phase or ~ mimlm oxide can be deposited using a ]iquid carrier. The c-~n~ *ng layer forming heaters 340A and 340B are, for ~mple, deposited by vacuum evaporation (e.g, for a nichrome conducting layer) or by deposition from the vapor (e.g., for an intlillm tin oxide conducting layer).Alternately, pre-formed heater sheets are cemented to the substrate, for instance using an epoxy cement or the adhesive recommenfle~ by the vendors. Appropriate heaters can be obtained from Elmwood Sensors Inc.
(Pawtucket, RI) or from Omega ~n~ineering Inc. (Stamford, CT).
In some embo.lim~ntq, the thermal contact between the heaters 340A and 340B and the blocks 300A and 300B, respectively, will be sllffiri~nt so that the temperature of block 300A or 300B can be expected to closely a~loxi~ te the temperature of the ~ cçnt. chamber 250.
Acc~,ldillgly, temperature monitoring means can be mounted to the block 300A or 300B. In other embodiments, typically those where pre-fabricated heaters are mounted onto the auxiliary blocks, other methods of measuring reaction chamber temperature may be required. One such method is - illustrated in Figure 6C, which shows an upper ~ n.~ioTI 303A on the outer face of the block 300A. The ~t~n~i- n 303A is generally fabricated of the same material as upper section 302A. Upper second thermal sensor 306A
provides more direct evidence of the temperature in chamber 250, while CA 022364~l l998-04-30 W O 97/16561 20 PCT~US96/17116 upper first ther_al sensor 304A provides rli~Fnostic temperature information.
To m~imi~e the thermal transfer between the heaters and the chamber 250, the covers 261 and 252 of the chamber 250 are preferably constructed of a fl~nhle material that can conform to the surface of the heater 340A or 340B placed against such a flexible cover.
The non-conductive upper and lower portions 301A and 301B of blocks 300A and 300B are fabricated from a n~nf~on~luctive material such as, without limit~tion, nylon or polycarbonate. The conductive upper and 110 lower sections 302A and 302B of blocks 300A and 300B are fabricated from a material such as, without limitation, ~ minum or copper.
Figure 7A shows alternate heater and cooling devices within upper and lower ~lnrili~ry blocks 500A and 500B. The illustrated blocks 500A
and 500B function to narrow or expand the chamber 250 just as do the 1 5 blocks 300A and 300B of Figures 4A - 4C. The elements such as 511A and 512B are made of a suitable material to provide a strong thermoelectric effect at their junction. ~e~ting is achieved by applying voltage of the proper polarity to upper first and second leads 508A and 509A and to lower leads 508B and 509B. Cooling is achieved by l~V~ lg the polarity of the voltage. An important variable in the operation of these he~ting and cooling devices is temperature unil'()~ily. To increase temperature uniformity, upper and lower first end-plates 502A and 502B are preferably constructed of a material of high thermal conductivity, such as sintered beryllia. Other suitable materials in~ (le, without li...il~lion, ceramics c~ntSlining ~lllminllm rlef~dbly, the thermal conductivity of end-plates 502A and 502B is at least about 0.2 watt/cm~~ , more preferably at least about 2 watt/cm~l/K~l. The upper and lower temperature sensors 570A and 570B can be, without limitation, thermocouples or resistive sensors. The sensors 570A and 570B can, for example, be deposited on the end-plates 502A and 502B as thin films or they can be in the form of thin wires embedded into holes in the end-plates 502A and 502B.
Using these he~ting and cooling devices, inrllllling the device described in the immediately preceding paragraph, ch~mber 250 temperatures between about -20~C and about 100~C can be m~
The higher temperature may require a sllhsi~ ry heater to give a constant temperature bias to the blocks 504A and 504B. The temperature in a reaction chamber ~ef~ably can jump from about 25~C to about 75~C in CA 022364~1 1998-04-30 about 10 seconds, more preferably in about 5 seconds, and more ~lare. ably yet in about 3 seconds. The reciprocal cooling step is preferably achieved in about 10 seconds, more preferably in about 5 seconds, and more preferably yet, in about 3 seconds. Preferably, after a cooling or he~tinF step, the variation in temperature in the reaction chamber is no more than about 1~C, more preferably no more than about 0.5~C, yet more preferably, no more than about 0.1~C.
In one ~lere~led embodiment, when the disk 200 includes more than one re~ction chamber 250, each such chamber 250 will have at least one 1 0 heAtinF and cooling device made up of thermoelectric blocks 501 (such asthe h~AtinF and cooling device described in the paragraph immediately above) capable of being Ali~ne-l with a side of the reaction chamber. More preferably, each chamber 250 will have a heAtin~ and cooling device on each of two opposing sides. In another preferred embo~im~nt7 the cross-1 5 section~l area of the end-plate 502A or 502B subst~ntiAlly matches the largest cross-sectional area of the chamber 250 to which it is int~n~l~ to transfer heat.
The principles of temperature cycling for chambers 250 he~te(l and cooled with blocks 500A and 500B are the same as those ollt.linetl above for the blocks 300A and 300B of Figures 6A, 6B and 6C.
In another embo-lim~nt7 the reAction chamber is he~tetl and cooled by p~inF a heated or cooled fluid, preferably a gas, either ~li. e~;Lly over oneor more surfaces of the chamber 250 or through a heat f~h~nFe apparatus that can be position~d adjacent to one or more surfaces of the reaction chamber. The apparatus illustrated in Figures 6A and 6B can be mo-lifi~rl to operate pursuant to this embo-liment by (a) removing (or not using) the heaters 340A and 340B and (b) adding a heater for he~tinF the fluid. The system preferably has two fluid m~n~Fement systems, one for a hotter fluid and another for a cooler fiuid, together with the valuing required to inject the hotter or cooler fluid into the tubing le~-linF to the chamber 250as appropriate for m~int~ininF a given temperature in the reaction - chamber. Particularly where the he~tinF and cooling fiuid is a gas, the temperature of the gas soon after it has passed by the reaction chamber - will provide a useful in~ tion of the temperature of the re~ction chamber.
The uniformity of the reactions conducted in the chambers 250 can be increased with par~mA~netic beads AFit~te~ by a rotating m~gnetic field. Such par~m~gnetic beads are available from several sources W O 97/16561 22 PCT~US96/17116 in-]ll-ling Bang Laboratories (Carmel, IN) for beads l~rking conjugated biomolecules, Dynal (Lake Success, NY) for beads conjugated to various antibodies (for instance, antibodies that bind to the CD2 cell-surface receptor) and CPG (T.incoln Park, NJ) for beads with a glass matrix and a variety of surface bonded organics. For appliç~tior-.~ where it is anticipated that the beads will be washed into and out of reaction chambers, each bead will ~ r~Lably have a diameter of less than about 26 ,Um, more preferably, less than about 12.5 ,Um, which ~ met~r farilit~tes entry and exit through the ~ h~nnel.~ by which material is inserted or evacuated from the ~ h~mher 2~0. For appliç~tion.~ where the beads are ~n~i~ip~terl to remain in the chamber 250, in one embo-liment, the di~meter is preferably sllffi~i~ntly large to preclude their entry into these ~h~nnel~. The entrances to such channels within the chamber 250 are preferably positioned or ~e.cign~l so as to ~--i llilll i7e the chance that a channel will be blocked by a bead that settles over the channel's entryway. In another embodiment, the beads are locked in place using magnetic fields.
To generate sufficient movement ~mong the beads, it has been determined that the magnet used should ~lefelably generate a sllffi~i~nt m~netiC field gradient within the chamber 250. Such m~gnets can be constructed by forming sharp edges on highly m~gnetic permf-n~nt m~gnetq, such as those formed of rare earths, such as the neodymium-iron-boron class of permanent magnets. Such a permPn~nt m~gnet is av~ hle from, for ~ mple, ~mllntl S-içnt.ific (Barrington, NJ). Sharp edges of tlimen~ n~ suitable for a particular reaction chamber are, for example, formed by abrasive grinding of the magnetic material. An eY~mple of such a shaped m~ net 600is shown in Figure 10, where the N pole of the m~gn~t has a roof-shape. To m~rimi~e the field gradient acting on the par~m~g-etic beads, the peak 601 of the m~gnet 600is placed adjacent to the reaction chamber or other structure in which the beads are located.
The beads are stirred by rotating the magnet 600 about its N to S axis.
The par~qm~netic beads are held in place by leaving the peak 601 adjacent to the beads. By sliding the magnet with its peak 601 adjacent to the beads, the beads are impelled to move with the magnet.
The sharp-edged magnets ~çcl rihed above are effective in ~Ah~ring the par~m~netic beads in one place and in moving beads located, for instance, in a ~s~pilklry or in a reaction chamber, from one location to another. Such magnets thus can help retain the par~m~netic beads in CA 022364~1 1998-04-30 WO 97/16561 23 PCTrUS96/17116 one place, for instance when a fluid in the ch~mber 250 or a chamber 120 is being removed from that chamber but it is desirable to leave the beads in the chamber.
Various cell hin~ing beads (e.g., beads having bound antibodies specific for a certain subset of cells) can be used to adhere selected cells from a population of cells. The beads can be locked in place, for instance magnetically if the beads are par~m~gnetiC~ while non-a&erent cells and fluids are washed away. Thus, cell-hintling beads can be used to concentrate small sub-populations of cells.
110 ~n the PCE~ re~cti-n, mi~nng can be important in the preparatory steps prior to the ~mplification reaction. Hc wev~ during the subsequent temperature cycling, mechanical mixing can be omitted. Thus, in some embo-lim~ntq, the rotatable magnet that induces the mi~nng movement of the beads can be placed adjacent to the chamber 250 immerli~t~ly after the various reagents are introduced. The reagents can then be mi~ed, and the m~gnet withdrawn to f~ilit~te the pl~cement of other me~h~ni.qm.~, such as he~tin~ and cooling devices, adjacent to the ch~mber 250.
The controller 400 will typically be a microprocessor. H~ wev~r, it can also be a simpler device comprised of timers, switches, sol.qn~ and the like. The important feature of controller 400 is that it directs the movement of the carousel 100 or disk 200, the activation of the means for impelling a fluid, and the he~ting and cooling device according to a pre-set or progr~mm~hle schedule that results in the operation of an assay protocol, such as one of the protocols outlined below. Preferably, the controller receives input in(lic~ting the temperature of the reaction chambers of the assay cassette and is capable of adjusting its control ~ign~l~ in response to this input.
Often an important variable in PCR reactions is the amount of interfering cellular debris, including cellular ~h~.mic~ , present in the sample to be assayed. Ideally, only highly purified nucleic acid is used as the sample subjected to a PCR ~mplific~tion However, such purification is - not practical with the small amounts of tissue or fluid available for a nostic assay. Further, given the sen~i~ivily of the assay to coT-t~min~tion by envhol....ent~l sources of nucleic acid, a nucleic acid purific~ti- n step can increase the likelih~lod of getting a false positive result.
In some areas of diagnostic or forensic PCR this concern about interference by c~ r debris has been eased somewhat by im~l vvelllents in the CA 022364~1 1998-04-30 W O 97/16561 24 PCT~US96/17116 characterization of PCR reaction conditions, such that often much cruder nucleic acid samples can be used without adverse effect. See Rolfs et al., PCR: Clinical Dia~znostics and Research, Springer Lab, 1992 (particularly Chapter 4 et seq.). See, also, the literature available with such commercial products as GeneReleaserTM (BioVentures, Inc., Murphreesboro, TN), Pall LeukosorbTM media (Pall, East Eills, NY) and DynbeadsTM DNA DirectTM
(Dynal, Lake Success, NY). On PCR procedures, see generally, Ausubel et al., Current Protocols in Molecular Biolo~, Jo_n Wiley & Sons, New York (AUSUBEL I) and PCR: A Practical Approach, IRL Press, 1991 1 0 (AUSUBEL II). Nonethale.ss, it is desirable to have the c~p~hility of atleast removing the call~ r debris associated with the cell membranes of the cells that may be present in the sample. Such a technique for use in conjunction with the assay system is described below. Such a cleanup step can be applied when needed to achieve the needed level of sensitivity or accuracy, or omitted if not needed.
It is also important to conduct parallel control PCR reactions when con~ ng PCR. One important type of control omits sample from the reaction or uses a sample previously characterized as negative. Another important type of control introduces a known amount of a purified n~ laic acid that is known to cont~in the sequence or sequences that the PCR
re.qction is rl~ netl to amplify. These types of controls can be accompli~h~d on multiple assay system units or cassettes, or, more preferably, in separate reaction chambers on the same first assembly, such as the disk 200.
Another control technique used in PCR is to design the PCR reaction so that it will amplify multiple nucleic acid se~ ents, each in~ tive of a disease or a genetic circumstance. The different se~mants can be ~mI~lifie~l in m~ ;rle reactions or in the s~me reaction chamber. If ~mrlifi~r~ in the same chamber, experience in the field has in(li~s~tafl that hin~ing competition between the various primers necessitates P~t,~n~1in~ the time, in each amplification cycle, spent at the replication temperature.
One tool for removing cellular debris from a sample involves first hin~ling the cells in the sample to a bead that has attached thereto an antibody specific for a cell surface m~ cllle found on the cells. Beads that bind to the CD2 molecule found on white blood cells or to E.coli bacteria (such as the 0157E strain) are available from Dynal (Lake Success, NY).
An ever-growing f~mily of cell-surface molecules found on m~m~n~ n cells, CA 022364~1 1998-04-30 W O 97/16561 25 PCTrUS96/17116 bacterial cells, viruses and parasites has been characterized and antibodies against the majority of these molecules have been developed. See, e.g, ~lh~.~ion Molecules, C.D. Wegner, ed., ~(lemic Press, New York, 1994.
Many of these antibodies are available for use in fabric~ting other types of cell-affinity beads (for instance, from Sigma Chemi~l Co., St. Louis, MO).
The cells can be adhered to the beads and lysed to release their nl7-1~ic acid content. The lysis fiuid together with the rf~le~e~ n~ ic acid can be moved to a separate compartment for further proce.~ing, leaving behind the beads and their adherent c~ r debris.
The lysis fluid used to release nucleic acid from the sample cells can also interfere with the PCR reaction. Thus, in some protocols it is important to bind the ml~lçic acid to a substrate so that the lysis fiuid can be washed away. One such support is provided by beads that bind to DNA, such as glass beads that bind to DNA by ionic and other interaction forces.
Suitable beads, with surfaces ~.hemic~lly treated to m~imi~e the number of interaction sites, are available from, for example, BioRad (Hercules, CA).
Par~m~gnetic beads with a number of DNA hinrling surfaces, such as nitroc~ e or nylon-coated surfaces are anticipated to be useful in operating the invention. In some embodiments, it is desirable for the beads to be par~m~netic so that they can be manipulated using m~netic forces.
Par~m~n~tic glass beads are manufactured by Dynal (Lake Success, NY) or CPG (T incoln Park, NJ). Once the nucleic acid is bound to the beads, the lysis fluid can be washed from the beads. The nll~leit acid can be ~mplifie-l with the beads present.
Z5 The lysis fluid used to release nucleic acid from the cells in a sample typically includes a detergent, ~. ef~lably nonionic, and a buffer, usually the buffer used in the PCR amplific~fiQn reaction. The pH of the lysis fluid is preferably about pH 8 to about pH 8.6. Suitable detergents in~
without limit~ti(m Sarkosyl and Nonidet P-40. Other assemblies can in~ lç salts, in~ tling MgC12, ~hel~tors and proteases such as proteinase K Proteinase K can be inactivated by he~tin~, for instance, to about 100~C
for about 10 minllte~ Dep~n~ling on the composition of the lysis buffer, it can be more or less important to wash the lysis buffer away from the - nucleic acid prior to conducting the amplification assay.
The ~mplific~tion buffer used to support the amplifi-~tion re~tior~
will typically include the four deoxynucleotide triphosphates (NTPs) (e.g, at a concçntration of from about 0.2 mM each), a buffer (e.g., Tris-HCl, 10 CA 022364~1 1998-04-30 W O 97/16561 26 PCT~US96/17116 mM), potassium chloride (e.g., 50 mM) and mAgnç.qium chloride (e.g., 1 to 10 mM, usually optimized for a given PCR assay scheme). The pH is typically about pH 8.4. Other components, such as gelatin (e.g, 0.1 mg/ml), can be added. The individual primers are typically present in the reaction at a conc~ntration of about 0.5 ~lM. The amount of sample nllrl~ic acid n~e~
varies with the type of nllr.l~ir acid and the nllmber of target nllrl~ic acid segmentq in the nucleic acid sample. For g~nr-mic DNA, where each cell in the sample has about 2 copies of target nllrl~ic acid, a concentration of about 10 ,ug/ml is desirable.
11~1 E'or .qimpiirity, the polymerase used in the procedure is a heat-resistant DNA polymerase such as Taq polymerase, recnmhinAnt Taq polymerase, 1~ DNA polymerase and Tli DNA polymerase (all from Promega Corp., Madison, VVI). Heat stability allows the PCR reaction to proceed from cycle to cycle without the need for adding A-l~litir~nAl polymerase during the course of the reaction process to replace polymerase that is ill~velaiibly ~nAtllred when the reaction vessel is brought to a DNA
strand separation temperature. Preferably, the DNA polymerase used has the increased accuracy associated with the presence of a proofreading, 3' to 5' exonuclease activity, such as the proofreading activity of the Tli DNA
polymerase.
Blood provides one of the more convenient samples for (liA~nnstic or genetic PCR testing. For most genetic testing, from about 10 to about 50 ,ul of blood is sufficient to provide enough sample DNA to allow for PCR
amplifi~ti~ n of specific target segments. For fetal cell analysis, howt,v~
as much as about 20 mls, which may cont~in as few as about 400 fetal cells, can be required. Such large sample volumes require concentration, for instance, using the methods described above. For testing for microbial diseases, the concentration of target nucleic acid in the sample can be quite low (e.g., no more than about 2 fg to about 5 fg per bacterial genome).
Thus, when using the assay system to test for such microbes, conc-~nt~ation methods may again be required.
To specifically amplify RNA, it is necessary to first synthesize cDNA strands from the RNA in the sample using a reverse transcriptase (such as AMV reverse transcriptase available from Promega Corp., M~li.qon, ~7VI). Methods for c~n~ *ing a PCR re~ct;on from an RNA
sample are described, for example, in AUSUBEL I and AUSUBEL II. To prepare RNA for this purpose, a facile procedure uses a lysis buffer CA 022364~1 1998-04-30 W O 97/16561 27 PCT~US96/17116 conf~inin~ detergent (such as 0.5% Nonidet P-40), buffer (e.g., pH 8.3) and suitable salts that has been, immediately prior to use, mixed 1:1000 with a 1:10 diethylpyrocarbonate solution in e-th~ncl After sample cells have been lysed with this solution, a supernate cont~ining RNA is separated away from a pellet of nuclei by centrifugation. Primer, which is generally the same as one of the primers used in the subsequent PCR cycling re$lctiorl, is ~nne~led to the RNA by he~ting, e.g to 65~C and subsequently re~ ing the temperature to, generally, about 37~C. The reverse transcriptase, nucleotide triphosphates and suitable buffer (if not already present) are then added to initi~te cDNA synt.he.~ . Generally, a small volume of cDNA synthesis solution is added to a solution c-nt~ining the buffer, DNA polymerase, nucleotide triphosphates and primers n~erl~l for the PCR amplification. The temperature cycling program is then initiated.
These advantages also subst~nt.i~lly apply to con~ ting hybri~ t.ion procedures. The ability of the valves of the assay system to accommodate elevated temperatures allows the system to be used in hybri~ *on protocols. While hybri~ tion reactions are not as sensitive to cont~min~t.ion as PCR re~ctior~ these reactions are nonethele.~.~ very sensitive to c-)nt~min~tinn, the risk of which is substantially re~ e l with the disposable system.
Procedures for conducting hybri~ tion~ are well known in the art.
See, for example, AUSUBEL I and Sambrook et al., Molecular Clonin~: A
Laboratory M~nll~l, 2nd ed., Cold Spring Harbor Press, 1989. In these procedures, one of (a) a sample source of nucleic acid cont~ining a target sequence or (b) a probe nucleic acid is bound to solid support and, after this bin-lin~, the r~m~ining hin~ling sites on the support are inactivated. Then, the other species of nucleic acid, which has bound to it a detect,~hle reporter molecule, is added under a~ riate hybri~ tion con-lition~ After w~.~hing, the amount of reporter molecule bound to (i.e. hybridized with) the nucleic acid on the solid support is measured.
For instance, a hybridization can be cl ntlllcte(l in a reaction chamber ~ in the assay system, where the reaction chamber contains a piece of locellulose mf~.mhrane (or another membrane that binds nll~ l~ic acid) to - which RNA has been bound (for instance, by electrophoretic or ç~pilklry blotting from a separation gel, followed by baking). A Northern prehybri~ tinn solution can then be introduced into the re~ction chamber from one of the fluid ch~mbers. (The recipes for Northern prehybri~ tion CA 022364~1 1998-04-30 solution (p. A1-40), Northern hybridization solution (p. A1-39), SSC (p. A1-53, 20X recipe) and Denhart's solution (p. A1-14, 100X recipe) of AUSUBEL I are incorporated herein by reference to more fully ~ mplify the hybridization methods that can be conducted in the assay system; note that the salmon sperm DNA recited in two of these recipes, which DNA
serves as a competitor to reduce nonspecific hybri-li7Ati~n~, is typically sheared prior to use.) The membrane and prehybri~i7~tion solution are incubated overnight at a temperature between about 37~C and about 42~C, depending on the melting temperature for the interaction between target sequence and the probe sequence. Note that these inrllh~tioI~
temperatures are in the range that is generally appropriate given the presence of 50% form~mi~le in the prehybri-1i7~tion and hybri~1i7~tioIl solutions; for hybri-li7.~t.i-n.~ conducted without form~mi-l~, incllhslt.ion temperatures are typically higher, such as about 55~C or about 70~C. The mf~mhrane is then exposed to Northern hybri-li7~tion solution c-nt~ining melted probe and incubated overnight at the same temperature used in the prehybri(li7~tion Following hybrilli7~tion~ the hybri(li7~ti-n solution is pushed out of the reaction chamber, the reaction chamber is brought to about 25~C and a first wash solution (lX SSC, 0.1% w/v sodium dodecyl sulfate) is introduced. After 15 minutes, the wash is repeated. After an ihon~l 15 minute wash, a third and final wash is c-n-1llcted using 0.25X
SSC, 0.1% W/V sodium dodecyl sulfate.
This hybridization method is exemplary only. Numerous other hybri~ tion methods can be conducted in the assay system, inrllltling those described in the following sections AUSUBEL I which are incorporated herein by reference: Unit 2.9,pp. 2-24 to 2-30 and the recipes of Appendix 1 lerelled to therein; Unit 6.3, pp. 6-6 to 6-7 and the recipes of Appendix 1 referred to therein; and Unit 13.12, p. 13-44 and the recipes of Appendix 1 ~erelled to therein.
The elevated temperatures required for hybri~ tion reactions can be h~n~lled in an automated apparatus. For instance, hybridizations can be conducted at a temperatures a~ )x i ~ tely ~l~.fin~ll by the mf~.ltin~
temperature (Tm). Tm values for any hybri~ tion probe can be calculated using commercially available software such as OligoTM v4.0 from National Bios~ienc~, Inc. Plymouth, MN.
In immllno~q~y procedures, which are conventionally known to the art, the antibody-antigen hin~ling re~ction.~ are generally conllllcted at room CA 022364~1 1998-04-30 temperature or at a reduced temperature, such as about 4~C. After the hin~ing reactions, positive results are generally in~ te-l by an enzymic reaction, such as that metli~ted by the enzyme ~lk~line phosphatase, which enzyme re~ctior is generally conducted at a temperature between about 20~C and about 40~C. The assay allows these assays to be ~11tom:lted in a system that allows fast and reliable temperature reg~ tion in the temperature range between about 0~C and about 40~C.
Typically, modern antibody-based screening procedures use a solid support to which an "antigen" (which is simply a substance that when injected in a s11it~hle form into a suitable ~nim~1 causes the ~nim~1 to manufacture antibodies specific for the antigen) or an antibody has been attached. Alternatively, the antigen is found on the surface of a cell, such as a bacteria or eukaryotic cell, and the cell substitutes for a solid support.
In one assay (indirect ELISA), the antigen is bound to the support and a sample prospectively c-nt~ining a first antibody specific for the antigen and produced by a first ~nim~1 species is incubated with the bound antigen. After a~lo~liate washing steps, a second antibody from a second ~3nim~1 species, which antibody is specific for antibodies of the first species and is attached to a detectable moiety (such as ~lk~linf~ phosphatase), is inc11h~te-l with the support. If the sample collt~ined the first antibody, the second antibody will bind and be detectable using the detectable moiety.
For instance, if the detectable moiety is ~lk~lin~ phosphatase, detection can be conducted by adding a chemical, p-nitrophenyl phosphate, that is converted into a blue substance by the action of the phosphatase enzyme.
This assay can, for instance, be used to test blood for the presence of antibodies to the AIDS virus.
In another assay (direct competitive ELISA) that uses a support with bound antigen, a sample that prospectively cont~in.~ antigen is inc11hs~ted vwith the support together with a 1imiting amount of an antibody specific for the antigen, which antibody has an attached detectable moiety.
Due to competition between the s--llltion phase antigen and the support-bound antigen, the more antigen in the sample, the less antibody that is bound to the support-bound antigen and the weaker the signal produced by the ~letect~hle moiety.
Another assay (antibody-sandwich ELISA) uses a first antibody specific for an antigen, which antibody is bound to the support. A sample that prospectively contains the antigen is then incubated with the support.

CA 022364~1 1998-04-30 W O 97/16561 30 PCT~US96/17116 Following this, a second antibody that binds to a second part of the antigen, and which has an attached detectable moiety, is incubated with the support. If the sample cont~in~d the antigen, the antigen will bind to the support and then bind to the detectable second antibody. This is the basis for a home pregnancy test, where the antigen is the pregn~nr.y-associated hormone chorionic gonadotropin.
In another assay (double antibody-sandwich ELISA) that uses a support with bound antibody, a sample that prospectively cont~in~ a first antibody from a first species is incubated with a support that has bound to 1~11 it a second antibody from a second species that is specific for antibodies of the first species. The antigen for the first antibody is then incubated with the support. Finally, a third antibody specific for a portion of thQ antigen not bound by the first antibody is incllhAte-l with the support. The third antibody has an Att~f~hed detectable moiety. If the sample c-ntAine-l the first antibody, the detectable third antibody will bind to the support.
These and other immunoassays are described in Units 11.1 and 11.2 of AUSUBEL I pp. 11-1 to 11-17, which text and the recipes of Appendix 1 cited therein, are incorporated herein by reference.
The following ~rAmples further illustrate the i.lv~ltion but, of course, should not be construed as in any way limiting~ its scope.
Ample 1 - Heat Source/Coolin~ Sink Operation The performance of a heater device and cooling device used with respect to the illustrated circular configuration of the assay system, and depicted in Figure 6A, has been simulated using a heat transfer sim~ tion computer program using a finite element a~.Jx;lllAtion to the heat flow equation. The simulation was conl1llcted with the following assumptions:
the thickne~.s of the chamber 250 was 0.5 mm, the upper and lower covers were 0.1 _m thick and the ins~ tion between the heater and the ~ ry block was 0.025 mm thick. The ~imlllAtion determined that with appropriate commercially available materials, a jump from 25~C to 75~C
can be achieved within 3.2 seconds, where, after 3.2 seconds, the temperature in the reaction chamber is substantially uniform. The reciprocal cooling step can be achieved within about 3 seconds, resulting in a substantially uniform temperature in the reaction chamber.
~.~mple 2 - PCR Reaction A PCR assay is conducted using the reaction cassette illustrated in Figures 8A and 8B, where the reActiQ~ chamber disk has first through third CA 022364~1 1998-04-30 W O 97/16561 31 PCT~US96/17116 reaction chambers 250A-250C of equal volume and a fourth reaction chamber 250D of volume 8% greater than the combined volume of the first and third reaction chambers (250A and 250C). The carousel has seventeen fluid chambers 120A2 through 120R2. The reActiQrl cassette has a r~pill~ry tubing structure 150 used to introduce sample into the chamber 250D. The capillary tubing structure has an inlet 151 through which sample is introduced into the capillary system. The assay system has upper and lower ~ ry blocks, the lower of which can be drawn sufficiently away from the reaction chamber disk to allow a rotatable i 1~ m~gnet to be posltioned under the reaction chamber clls~ The assay system has a first fluid impeller for impel~ing a fluid to move from a fluid chamber (120A2 to 120R2) to a reaction chamber (250A to 250D) and a second fluid impeller for emptying the reaction chambers 250A-250D, both such impellers found in the upper ~ ry block. Steps 1 through 12, 1 5 ollt~in~rl below, are conducted with the lower ~ ry block drawn away from the reaction chamber disk and with a rotatable magnet pn.ciiti~n~ll under the disk. The rç~ction protocol is as follows:
1. Capillary tubing structure 150 is ~ n~l with chamber 250D of the assay cassette and a blood sample in the capillary tubing 150 is drawn into the chamber 250D using air pressure. (The czlpill~ry tubing is filled with blood by ~ligning the outlet of the çiqpill~ry tubing structure with a vent (not shown) located adjacent to fiuid ~ch~n~e ~h~nnel 231D on the reaction ch~mber disk, and filling the c~pill~ry structure by c~pill~ry action through inlet 151.) The chamber 250D is pre-loaded with a supply of par~m~gn~tic, white blood cell-hin~ing beads having a diameter of 50-100 ,um (Dynal, Lake Success, NY). The chamber 250D is m~int~in~d at a temperature of 2-4~C. The magnet is rotated to agitate the beads, thereby providing stirring 2. After 45 minutes co-incubation of the beads and the blood, the carousel of the cassette is rotated until a first fluid chamber 120A2 is n~l with a fluid ~ h~nge ~hiqnnel 231D. The excess fluid from the ~ sample, along with unbound cells are pushed into the chamber 120A2. The beads are m~gn~tically restrained from leaving the chamber 250D.
~ 3. At this point, the carousel is rotated to align the chamber 250D with a second fluid chamber 120B2 cont~ining a washing solution composed of ~mplific~tion buffer (40 mM NaCl, 20 mM Tris-HCl, pH 8.3, 5 mM
MgSO4). The washing solution is drawn into the chamber 250D.

4. The carousel is rotated to align the chamber 250D with the ch~mher 120A2, into which the wash solution is pushed, again leaving the beads magnetically restrained in the chamber 250D.
5. The carousel is rotated to align chamber 250D with a tllird fluid chamber 120C2, from which a lysis solution (amplific~ti- n buffer supplemented with 0.5~o v/w Tween 20 (Sigma Chemic~l Co., St. Louis, MO) and 100 ~pl proteinase K) is drawn. To lock the lysis solution in place, the carousel is rotated to close the fluid ~x~ hAn~e ~h~nn~l 231D. The temperature of the fourth 250D is now mtqint~inetl at 56~C. Stirring is 1 0 achieved by rotating the adjacent magnet to ~git~te the cell-hin(lin~ beads.
6. After 45 minutes, the carousel is rotated to align the chamber 250D
with a fourth fiuid chamber 120D2, into which the lysis solution, which c-nt~in.q nucleic acid extracted from the cells of the s~mple, is pushed.
Again, the cell-hin-lin~ beads remain in the chamber 250D. The volume of 1 5 lysis solution is 8% greater than the comhin~d volume of the first and third reaction chambers 250A and 250C (exclusive of the volume occupied by nucleic acid-hintlin~ beads).
7. The chambers 250A and 250C are pre-loaded with par~m~netic, nucleic acid-hin(ling beads and are evacuated of fluid. The carousel is serially rotated to align the fourth fiuid chamber 120D2 with a fluid n~e l~h~nnel 231A and then with fluid ~ h~n~e ~h~nnel 232C, and chambers 250A and 250C are filled with the nucleic acid-cont~ining lysis solution from the fourth fluid chamber 120D2. The beads in the chamber 250C are pre-loaded with a purified DNA the includes the amplification sequence in an amount sllffi~ ient to generate a positive result. The chamber 250A is for the experimental ~mplification. The chamber 250C
provides the positive control. The chamber 250B co~t~in~ only the nucleic acid-hinflin~ beads and a pre-loaded volume of lysis solution; it serves as the negative control.
8. The carousel is now rotated to close the chambers 250A-250C. The chambers 250A-250C are m~int~ine-l at a temperature consistent with the binrlin~ material on the beads. For example, for Dynal Dynabeads M-450 Pan T (CDL) bin-ling beads the ~ lL~3 temperature is 2-4~C.
9. After 15 minutes, which is sufficient time to allow the DNA in the lysis s~ ltion to bind the DNA-binding beads, the carousel is rotated to align the chambers 250A-250C with the fifth (120E2), tenth (120J2) and fifteenth (120P2) fluid chambers, respectively, and the lysis buffer is -W O 97/16561 33 PCTnJS96/17116 pushed out of the chambers 250A-250C. DNA-hin-ling beads are m~n~tically restrained from exiting.
10. The carousel is rotated to align the chambers 250A-250C with the sixth (120F2), eleventh (120K2) and.~i~te~nth (120Q2) fluid chambers, respectively, and wash sollltion (described above) is i~ oduced into the chambers 250A-250C. The DNA-binding beads are ~it~ted m~gnetically.
11. After 10 minutes, the carousel is rotated to align the chambers 250A-260C with the fifth (120E2), tenth (120J2) and fifteenth (120P2) fluid chambers, respectively, and the wash solution is pushed out of the l 0 reaction chambers.
12. The carousel is rotated to align the chambers 250A-250C with the seventh (120G2), twelfth (120L2) and seventeenth (120R2) fluid chambers, respectively, from which is drawn an amplification sollltion cont~inin~
amplification buffer (supplemented with 0.01% w/v gelatin, 0.1% v/v Triton 1 5 X-100 (Sigma Ch~mic~l Co., St. Louis, MO)), the needed nucleotide triphosphates, a~o~.iate primers for amplifying the target sequence (0.5 lM) and Taq polymerase (available from Promega Corp., M~-li.qon, VVI).
13. The carousel is rotated to close the chambers 250A-250C and the lower ry block is positioned under the reaction chamber disk to provide more precise temperature control. The controller then initi~tes a temperature program modeled on the protocol described by Wu et al., Proc.
Natl. Acad. Sci. USA, 86, 2752-2760 (1989). The program first heats the chambers 250A-250C to a temperature of 55~C and m~int~in~s that temperature for 2 minutes. Next the controller cycles the temperature Z5 between a replication temperature of 72~C (m~int~in-orl for 3 minlltes) and a DNA melting temperature of 94~C (m~int~in~d for 1 minute). After the replication temperature incllh~tion has been contllll ted 25 times, the material in the chambers 250A-250C is analyzed for the presence of the proper ~mplified sequence.
The eight (120H2), ninth (120I2), thirteenth (120M2) and fourteenth (120N2) f~uid chambers are not used in this protocol and are available for ~ post-reaction manip~ tion~. Note that the sequence by which the carousel is rotated is ~ ned so that no portion of the inner ring surface ~ 140 that has been in contact with the nucleic acid-c~nt~ining m~t~
rotates by the fluid ~ h~n~e ~h~qnn.?l~ for the reaction chamber in which the negative control reaction is con~ cted. This prec~lltion is desirable in view of the sen~i~ivil,y of the PCR reaction to c-~nt~min~tion. Fluid W O 97/16561 34 PCT~US96/17116 ~h~nge ch~nn~l 231D is situated so that when it is ~lign~rl with any fiuid ~rth~n~e port 121-A2 to 121-R2, none of f~uid ~ h~n~e ~-h~nn~l~ 23LA-231C and 232C is ~lign~rl This lack of co-~liFnment between these two groups of reaction chambers prevents inadvertent mi~in~ of fluids.
F'l~mple 3 - PCR Amplification Recently it has been shown that par~m~netic DNA-hin-ling beads can be used directly in the cell lysis stage to bind the DNA rele~e~ from the lysed cells (e.g., Dynabeads(~3) DNA DirectTM, available from Dynal, Lake Success, NY). Acco~ dillgly, the protocol of F'.~mrle 2 can be 11~1 c:ignific~ntly simpli~ied. In this ~mple, each re~cti- n chamber on thereaction chamber disk has a separate and independent set of fluid chambers and, if appropriate, a separate c~pill~ry tubing structure. For simplicity, the protocol outlined below focuses on one reaction chamber and its associated fluid chamber and c~pill~ry tubing structure. The protocol is 1 5 as follows:
1. Capillary tubing structure 160 is ~lign~l with chamber 250 of the assay cassette and a blood sample in the s~rill~ry tubing 150 is drawn into the fourth chamber 250 using air pressure. (The c~rill~ry tubing is filled with blood by ~ligning the outlet of the capillary tubing structure with a vent [not shown] located adjacent to fiuid ~ hAn~e fh~nn~l 231 on the re~ctior- ch~mber disk, and filling the c~pill~ry structure through inlet 151.) The blood sample only fills half the volume of the fourth re~ction chamber.
The chamber 250 is pre-loaded with a supply of par~m~gnetic, DNA-bin(lin~ beads having a diameter of 50-100 ~m (Dynal, Lake Success, NY) and a volume of lysis solution equal to the volllme of the sample is drawn.
The lysis solution is a 2X solution of ~mplification buffer suppl~m~nte-l with 1.0% v/w Tween 20 (Sigma Chemical Co., St. Louis, MO). (The lysis solllt.ion can be substituted with the solution provided by Dynal.) To lock the lysis solution in place, the carousel is rotated to close the ~-h~nnel 231.
The temperature of the chamber 250 is now maintained at 56~C.
2. After 45 minutes, the carousel is rotated to align the chamber 250 with first fluid chamber 120A, into which the lysis s~llltion~ which contains the cPlllll~r residue of the sample, is pushed. The DNA-bin-ling beads, to which the cellular DNA is bound, remain in the chamber 250.

3. The carousel is rotated to align the chamber 250 with a second fluid chamber 120B, and wash solution composed of amplification bu~er (40 mM
NaCl, 20 mM Tris-HCl, pH 8.3, 5 mM MgSO4) is introduced into the W O 97/lC561 35 PCT~US96/17116 chamber 250. The carousel is then rotated to close the chamber 250. The DNA-bin~ling beads are ~it~ted magnetically.

4. After 10 minutes, the carousel is rotated to align the chamber 250 with the chamber 120B and the wash solution is pushed out of the reaction ch~mhers.

5. Using a third fluid chamber 120C, steps 4 and 5 are repeated.

6. The carousel is then rotated to align the chamber 250 with a fourth fluid chamber 120D, from which is drawn an amplific~tic-n s~ t;~n cont~ining amplification buffer (suppl~m~nted with 0.01% w/v gelatin, 0.1%
i t31 v/v ~l~ton X-~ igma C~hemical ~o., ~3t. Louis, ~C))), the n~e~l~
nucleotide triphosphates, a~p~o~.iate primers for amplii~ying the target sequence (0.5 ~lM) and Taq polymerase (available from Promega Corp., Madison, VVI).

7. The carousel is rotated to close the chamber 250 and the lower ~ ry block is po~iti~ned under the reaction chamber disk to provide more precise temperature control. The controller then ini~i~teq a temperature progr~m modeled on the protocol described by Wu et al., Proc.
Natl. Acad. Sci. USA,86, 2752-2760 (1989). The progr~m that first heats the chamber 250 to a temperature of 55~C and m~qint~in.q that temperature for 2 minutes. Next the controller cycles the temperature bet~,veen an replication temperature of 72~C (m~int~ined for 3 minutes) and a DNA strand separation temperature of 94~C (m~int~inefl for 1 minllt,e).
After the replication temperature incubation has been conducted 25 times, the material in the chamber 250 is analyzed for the presence of the proper amplified sequence.
VVhile this invention has been described with an emp~qi.q upon a preferred embo~iment, it will be obvious to those of ordinary skill in the art that variations in the ~le~lred composition and method may be used and that it is int~n-l~l that the invention may be pr~c~ice-l otherwise than as specifically described herein. Accoldillgly, this invention inr~ q all mo(1ifi~ti--n.s encompassed witllin the spirit and scope of the invention as ~ defined by the following ~ imq The invention provides an economical, elevated temperature ~ microreactor with effective valves suitable for use in conducting various forensic and ~ n~stic biochemical assays, such as, but not limit~l to, PCR assays. The microreactor is also suitably adapted for conducting ,qlltom~te-l assays even when high vapor pressure is not a particular CA 022364~1 1998-04-30 concern.
The invention is also a system for conducting elevated temperature reAction.q in a fluid-tight mAnner within a reaction chamber comprising: (a) a first assembly comprising the reaction chamber, and (b) a second assembly for temperature control, wherein the second assembly can be positioned A-ljAc~nt to the reaction chamber. The first assembly comprises a plurality of reArti--n chambers, each having a fluid P~rhAn~e rh~nn~l, and preferably comprises at least three of such reA(~tion chambers. The second assembly comprises a heat source, a cooling sink, or both and preferably a 11~ fLuid imp~ll~r.
The system can also include a third assembly that can be .qli(leAhly positioned in contact with the first assembly and c~-nt~inq a plurality of fluid ch~mbers, each fluid chamber having a fluid ~q~rhAn~e port, a plurality of which fluid f~rhAnge ports can be aligned with the first fluid e~rhAn~e rhAnn~l by .qli~le~hly positioning the third assembly relative to the first assembly and a first fluid imp~ ?r for impelling a fiuid to flow from a fiuid chamber having a fluid ~rhAnge port which can be Ali~n~1 with the first fiuid e~rrhAnge rhAnn~l into the reaction chamber of the Align~rl first fluid f~rrhAn~e rhAnnel; and in some embodiments, a second fluid impeller for impelling a fluid to leave the reAction chamber.
The first assembly fits within the third assembly, which cl mhinAt.ion is fluid-tight, wherein the first and third assemblies can slide relative to each other, such that fiuid does not flow through the first fiuid ~rhAn~e rhAnn~l when the first fluid ~rhAn~e rhF~nnel is not ~ n~l with a fluid ~rhAnge rh~nnel in the third assembly. Further, the fluid-tight seal between the first assembly and the third assembly is effective in ret~3inin~
a fiuid in the reaction chamber when the temperature of the fluid is at about 90~C to about 100~C.
Another embotlim~nt includes at least one reAction or fiuid chamber that has an external transparent wall. The invention also comprises a light source capable of directing light to the external transparent wall of the reaction or fiuid chamber. Such an embodiment inrlll-les a light detection device capable of detecting (a) the light reflected from an illllminAt,erl reActio~ or fluid chamber having the external transparent wall, (b) the light transmitted through an illllminAted reaction or fluid chamber having the external transparent wall, or (c) the light emi.qqinnq ~mAnAting from an excited molecule in a reaction or fluid chamber having the external CA 022364~1 1998-04-30 transparent wall.
Another embodim~ntinrludes a slider for sliding the third assembly or the first assembly and a processor for controlling: (a) the sliding of the third~Rsemhly or the first assembly, and (b) a first fluid impeller for impelling fluid to flow from a fluid chamber to a reaction chamber. The slider effects linear or circular movement between the first and third ass~mhlies.
The invention also can inrlll~e a temperature m-nit~ng device for mc.nitoring the temperature of the re~ctiQn chamber. Other embo-liment~
inrlll-le a processor for receiving a signal from the temperature monitoring 1~1 device and controlling the heat source. 1~e cooling sink used in the context of the present invention can also be under the control of the processor. The cooling sink comprises conduits cont~ining a circlllAtin~ fluid.
Another embodiment includes a perm~nent magnet that can be po~itioned adjacent to the reaction chamber, a rotator for lota~ g the perm~n~nt m~n~t, a slider for sliding the first or third assembly, and a processor for controlling: (a) the sliding of the first assembly or the third assembly, (b) the first fluid impeller for impelling fluid to flow from a fluid chamber to a reaction chamber, (c) the heat source, and (d) the rotation of the m~gnet.
Another embodiment relates to a method of c n~lllrting a PCR
reaction comprising introducing a sample into a first re~ction chamber of the assay system ~lescrihed herein and transferring from one or more fluid chambers to the first reaction chamber solutions cont~ining reagents neces.sslry for con~lllcting the PCR reaction. Preferably, the second assembly of the assay system raises the temperature from about 25~C to about 75~C within about 5 seconds; the second assembly also preferably lowers the temperature from about 75~C to about 50~C within about 5 seconds.
The i~lve~llion includes a merh~ni.~m for rapidly adjusting the 3;0 temperature of a reaction chamber, the assay system comprising: (i) a first assembly comprising a reaction ch~mber having a first and a second cover and a thickness ~l~fin~l by the distance between the first and second covers, (ii) a second assembly comprising a heat source, a cooling sink, and ~ a plurality of pressurized fiuid rh~nnel.~ that open on the surface of the second assembly, and (iii) a pressure controller for controlling the ~luid pressure within the pressurized fiuid r.h~nnel.c; wherein the second ~s~mhly can be positioned adjacent to the reaction chamber such that the CA 022364~1 1998-04-30 W O 97/16561 38 PCT~US96/17116 pressurized fluid channels are in contact with a cover of the re~ction chamber. The heat source included in the second assembly preferably comprises at least one electrical heater attached to the second assembly.
The cooling sink in~ A~l in the second assembly preferably comprises a conduit for water or other fluid integral to the second assembly and a ~luid impeller for ~ ing fluid to flow through the conrlllit, Preferably, the first and third assemblies are used in c- mhin~ion with a second assembly, which comprises (i) first and second end plates, wherein the first end plate can be positioned adjacent to the reaction ï 1~1 chamber and (i~) a plurality of paired p-type and n-type semiconductor blocks positioned between the first and second end-plates and electrically connected in series to form a thermoelectric heat pump. The heat pump pumps heat from the first end plate to the second end plate when a voltage with a first polarity is applied across the serially connected semiconductor blocks and from the second end to the first end plate when a voltage of the polarity opposite the first polarity is applied across the serially connected semiconductor blocks. Preferably, the re~ction chamber thickness is about 1 mm or less.
A preferred embodiment is used when a fiuid in the pressurized fluid Z0 ~h~nnel~q has elevated pressure and the second assembly is po.qit~on~ladjacent to the reaction chamber, pressure is applied to the adjacent surface of the reaction chamber. This use is preferably facilitated when the first cover is formed of a deformable material and wherein, when the second ~.q,s~mhly is positioned adjacent to the first cover, gaseous pressure applied against the first cover via the plurality of adjacent p~.qRt geways is effective to deform the first cover so as to narrow the width of the re~ctior chamber. Thus, when the second assembly is po.qitione~l ~rlj~c~nt to the first cover and when a f~uid in the pressurized fiuid ~-h~nnelq of the second assembly has reduced pressure, the first cover adheres to the second assembly. The invention also preferably comprises a translocator for moving the second assembly away from the first assembly, wherein when the first cover is adhered to the second assembly, the second assembly can be moved away from the first ~ mhly by moving the second assembly away from the first assembly.
A preferred emborlim~nt comprises (a) a re~cti~ n chamber having a cover formed of a deformable material and (b) a me-hAniqm for rapidly adjusting the temperature of the reaction chamber, which mel.h~niqm W O 97/16561 39 PCT~US96/17116 comprises: (i) first and second end plates, wherein the first end plate can be positioned in cont~ct with the reaction chamber cover; and (ii) a plurality of paired p-type and n-type semiconductor blocks po~itil ne-l between the ffrst and second end-plates and electrically connected in series to form a thermoelectric heat pump; wherein the heat pump pumps heat from the first end plate to the second end plate when a current with a first polarity is applied across the serially connected semiconductor blocks and from the second end to the first end plate when a current of the polaLrity opposite the first polarity is applied across the serially connected semicontl~ tor blocks.

Claims (11)

WE CLAIM:

1. An assay system for conducting elevated temperature reactions in a fluid-tight manner within a reaction chamber, the assay system comprising: (a) a first assembly comprising the reaction chamber, and (b) a second assembly for temperature control, wherein the second assembly can be positioned adjacent to the reaction chamber.

2. The system of claim 1, further comprising a third assembly that can be slideably positioned in contact with the first assembly and contains a plurality of fluid chambers, each fluid chamber having a fluid exchange port, a plurality of which fluid exchange ports can be aligned with the first fluid exchange channel by slideably positioning the third assembly relative to the first assembly.

3. The system of claim 2, further comprising a first fluid impeller for impelling a fluid to flow from a fluid chamber having a fluid exchange port which can be aligned with the first fluid exchange channel into the reaction chamber of the aligned first fluid exchange channel.

4. The system of claim 3, further comprising a second fluid impeller for impelling a fluid to leave the reaction chamber and wherein the first assembly fits within the third assembly in a fluid-tight, slideable manner such that fluid does not flow through the first fluid exchange channel when the first fluid exchange channel is not aligned with a fluid exchange channel in the third assembly.

5. The system of claim 2, further comprising a slider for sliding the third assembly or the first assembly and a processor for controlling: (a) the sliding of the third assembly or the first assembly, and (b) a first fluid impeller for impelling fluid to flow from a fluid chamber to a reaction chamber.

6. A method of conducting a PCR reaction comprising introducing a sample into a first reaction chamber of the assay system of claim 1 and transferring from one or more fluid chambers to the first reaction chamber solutions containing reagents necessary for conducting the PCR reaction.

7. The method of claim 6, wherein the second assembly raises the temperature from about 25°C to about 75°C within about 5 seconds.

8. The method of claim 6, wherein the second assembly lowers the temperature from about 75°C to about 50°C within about 5 seconds.

9. An assay system having a mechanism for rapidly adjusting the temperature of a reaction chamber, the assay system comprising: (a) a first assembly comprising a reaction chamber having a first and a second cover and a thickness defined by the distance between the first and second covers, (b) a second assembly comprising a heat source, a cooling sink, and a plurality of pressurized fluid channels that open on the surface of the second component, and (c) a pressure controller for controlling the fluid pressure within the pressurized fluid channels; wherein the second assembly can be positioned adjacent to the reaction chamber such that the pressurized fluid channels are in contact with a cover of the reaction chamber.

10. The system of claim 9, wherein the heat source comprises at least one electrical heater attached to the second assembly and the cooling sink comprises a conduit for water or other fluid integral to the second assembly and a fluid impeller for causing fluid to flow through the conduit.

11. An assay system comprising: (a) a reaction chamber having a cover formed of a deformable material and (b) a mechanism for rapidly adjusting the temperature of the reaction chamber, which mechanism comprises: (i) first and second end plates, wherein the first end plate can be positioned in contact with the reaction chamber cover; and (ii) a plurality of paired p-type and n-type semiconductor blocks positioned between the first and second end-plates and electrically connected in series to form a thermoelectric heat pump;
wherein the heat pump pumps heat from the first end plate to the second end plate when a current with a first polarity is applied across the serially connected semiconductor blocks and from the second end to the first end plate when a current of the polarity opposite the first polarity is applied across the serially connected semiconductor blocks.