CA2215974A1 - Capillary electrophoresis apparatus and method - Google Patents

Abstract

This invention involves method and apparatus for multiplexing electrophoresis analysis. An array of samples in multi well plates are simultaneously transferred to an array of electrophoresis column where electrophoresis is simultaneously carried out followed by analysis of the columns. The methods and apparatus of this invention are, for example, useful for DNA analysis, including sequencing, and for measuring reactions between specifically binding proteins and their binding partners.

Description

W O 96/29595 PCT~US96/03828 (~APn,T,ARY F,T,F,(~TR(~PT-T()RF~$;T~;; APPARATTT~ ANT~ lV~,TFT~ n pLAl~K~TR()~Jl~ (lF TFTT~', ~VF,NTTON

A. Fi~ l nf th~ TnvPntinn ~ 5 This invention is in the field of se~r~tinn of biomolecules and, in particular, sep~dtions by capillary electrophoresis and the use of the capillary electrophoresis to detect such molecnl~s.

B. R~rkgrmlnll nf th~ Prinr Art Capillary-based se~,,.l;ons are widely used for analysis of a variety of analyte speciçs Numerous :iubteclmiques, all based on electrokinetic-driven s~alions, have been developed. ~rill~ry electrophoresis is one of the more popular of these techniques and can be considered to encompass a number of related sep~r~tion techniques such as capillary zone electrophoresis, capillary gel electrophoresis, capillary i~oelectric focusing, capillary isotachophoresis, andmicellar electrokinetic chromatography. In the context used throughout this application, the phrase "capillary electrophoresis" is used to refer to any and all of the aforementioned electrokinetic separation subtechniques.
Electrophoresis is a se~a dLion process in which molecules with a net charge migrate through a meAillm under the infl~lçnce of an electric field. Traditionally, slab gel electrophoresis has been a widely used tool in the analysis of genetic materials. See, for eY~mple, G. L. Trainor, Anal. Chem., 62, 418-426 (1990).
Capillary electrophoresis has emerged as a powerful separation technique, with applicability to a wide range of molecules from simple atomic ions to large DNA

W O 96/29595 PCTrUS~h'03X2 r.~p,...~,..,~. In particular, capillary electrophoresis has become an attractive ~lt~-n~tive to slab gel electrophoresis for biomolecule analysis, inrllldin~ DNA~luP~ g. See,for~.~"~plç,Y.Babaetal.,TrendsinAnal.Chem.,11,280-287 (1992). This is gen~ lly because the small size of the capillary greatly reduces Joule hlo~ting ~ceoci~t~d with the applied ~ rtric~l potential. Furthermore, capillaryele~ u~ l'c5iS l~UilCS less sample and produces faster and better separations than slab gels.
Currently, sophictie~tecl experiments in ch.-mi~try and biology, particularly molecular biology, involve evaluating large numbers of samples. For eY~mple, DNA sequencing of genes is time con~umin~ and labor intensive. In the mapping of the human genome, a rcseal~her must be able to process a large number of samples on a daily basis. If capillary electrophoresis can be conduct~l and monitored ~imlllt~neously on many capillaries, i.e., multiplexed, the cost and labor for such projects can be ~ignifi~ntly reduced. Attempts have been made to sequence DNA in slab gels with multiple lanes to achieve multiplexing. However, slab gelsare not readily amenable to a high degree of multiplexing and automation.
Diffieulti~ exist in ~ ~ing uniform gels over a large area, m~int~ining gel to gel repr~ çihility and loading sample wells. Furthermore, difficulties arise as a result of the large physical size of the separation medium, the requirements for uniform cooling, large amounts of media, buffer, and samples, and long run times for extended reading of nucleotide sequences. Unless capillary electrophoresis can be highly multiplexed and mllltirle c~rill~ries run in parallel, the advantages of capillary electrophoresis cannot produce substantial improvement in shortening the W 096/29595 PCTrUS96/03828 time needed for sequ~--n~in~ the human genome.
~pill~ry electrophoresis pocc~cces several char~etP~ tics which makes it ~mPn~hle to this applic~tion The substantial reduction of Joule heating per lanemakes the overall cooling and ele~tri~l r~ui-cl.,ents more manageable. The cost S of m~t~-ri~lc per lane is reduced because of the smaller sample sizes. The reduced band ~limencinnC are ideal for e~cit~tion by laser beams, as well as focused broad band sources, and for im~ging onto array ~ie~ectors or discrete spot de~c~;lol~. The concentration of analyte into such small bands results in high sensitivity. The use of electromigration injection, i.e., applying the sample to the capillary by an ele~tri~l field, provides reproducible sample introduction with little band spreading, minim~l sample consumption, and little labor.
Among the techniques used for cietecting target species in capillary electrophoresis, laser-excited fluore.scen~e detection so far has provided the lowest dete~tion limits. Therefore, fluor~s~en~e detection has been used for the detectit n of a variety of analytes, e~c~i~lly macromol~ul~-s, in capillary electrophoresis. For es~mpl~, Zare et al. (U.S. Patent No. 4,675,300) ~ic~ cces a fluoroassay method for the detection of macromolecules such as genetic m~tt~ri~lc and proteins by capillary electrophoresis. Yeung et al. (U.S. Patent No. 5,006,210) presented a system forcapillary zone electrophoresis with indirect laser-induced fluorescence detection of macromolecules, including proteins, amino acids, and genetic materials.
Systems such as these generally involve only one capillary. There have been ~llr~ to imrlt-m~nt the analysis of more than one capillary simultaneously in the electrophoresis system, but the number of capillaries has been quite small. For W 096/29595 ~1l~S96103828 c -, S. T~l~h~chi et aL, ProceeAingc of ~'~pill~ry Electrophoresis Sylnpo~
~r."h.., 1992, referred to a multi-capillary eleiL~u~horesis system in which DNAfi~mPnt ~mrlPs were analyzed by laser irr~ tion causing fluorescence. This method, however, relies on a relatively poor focus (large focal spot) to allow S cuul.l;.. p to only a few c~pill~ri~-c Thus, this method could not be applied to a large nul..be~ of capillaries. This method also results in relatively low intensity and thus poor sensitivity because of the large beam. Furthermore, detection in one capillary can be influPncel by light absorption in the ~ ent capillaries, thus affecting accuracy due to cross-talk between adjacent ç~pill~ri~ps.
Attempts have been made to pelrc,l.-- parallel DNA sequp-ncing runs in a set of up to 24 ç~rill~riPc by providing laser-excited fluorometric ~letectiQn and coupling a confocal illumin~tion geometry to a single laser beam and a single photoml-ltiplier tube. See, for example, X. C. Huang et al., Anal. Chem., 64, 967-972 (1992), andAnal. Chem., 64, 2149-2154 (1992). Also see U.S. Patent No. 5,274,240.
However, observation is done one capillary at a time and the capillary bundle isPd across the Py~it~tir~n/detection region at 20 mm/sec by a mPch~niç~l stage.
There are features inherent in the confocal eYcit~tinn scheme that limit its usefor very large numbers of c~rill~riPc. Re~ ce data acquisition is sequential and not truly parallel, the Illtim~te sequenccing speed is generally determined by the observation time needed per DNA band for an adequate signal-to-noise ratio.
Moveover, the use of a translational stage can become problematic for a large c~rill~ry array. Recalls~P of the need for tr~ncl~tional movement, the amount ofcycling and therefore bending of the capillaries naturally increases with the number W O 96~9595 PCTrUS96/03828 in the array. It has been shown that bending of the c~pill~rie~s can result in loss in the cf~ ;O~ effi~iency~ This is attributed to distortions in the gel and multir~th effects. Sensitive laser l,~ci~d nuu~ "ce dete~tinn also requires careful ~lignmPnt both in eYcit~tion and in light c-lle~ti~-n to provide for efficient coupling with the small inside tli~meter of the capillary and tlicc~ ;on of stray light. The tr~n~l~tion~l movement of the c~pill~ne-s thus has to m~int~in stability to the order of the confocal ~dlllC;l~l (around 25 ~m) or else the cylindrical capillary walls will distort the spatially sPl~octeA image due to mic~lignment of the capillaries in relation to the light source and photodetector. In addition, long capillaries provide slow s~dtion, foul easily, and are difficult to replace.
U.S. Patent No. 5,324,401 to Yeung et al. describes a mllltiple~e,d capillary electrophoresis system where excitation light is introduced through an optical fiber i~.~d into the capillary. In this system the capillaries remain in place, i.e. in the buffer solutions when the capillaries are read.
U.S. Patent No. S,332,480 (Datta et al.) describes a multiple capillary electrophoresis device for continuous batch electrophoresis.
U.S. Patent No. S,277,780 (l~mb~ra) describes a two ~lim~ncinnal capillary electrophoresis a~pdldt~ls for use with a two ~limçrlcion~l array of capillaries for m~curing samples, such as DNA s~mple-c, in an array of test wells.
. 20 U.S. Patent 5,413,686 (Klein and Miller) describes a multi-ch~nntol du~llld~d capillary electrophoresis analyzer in which multiple individual s~dtion c~rill~riP~c are inct~ l in a instnlmen~l analyzer which serves to flush and fill the ~pill~riPs and associated buffer reservoirs from supplies of buffer sit~t~-A within the W O 96/29~9~ PCTrUS96103828 instrument.

U.S. Patent 5,439,578 (Dovichi and Zhang) describes a multiple capillary bioch~omi~l analyzer based on an array of separation capillaries termin~ting in a S sheath flow cuvette. The use of the sheath flow cuvette f~ilit~t~s det~tion of the analyte bands by rerlll~ing the m~gnitutle of scaU~,ed radiation from the ~let~ti- n zone.
U.S. Patent No. 5,338,427 (Shartle et al.) describes a single use capillary cartridge having electri~lly conductive films as electrodes; the system does notprovide for multiplexed sampling, sample handling, and electrophoresis.
U.S. Patent Nos. 5,091,652 (Mathies et al.) and 4,675,300 (Zare et al.) describe means for dPte~ting s~mrles in a capillary.
U.S. Patent 5,372,695 (Demorest) describes a system for delivering reagents to serve a fix capillary scanner system.
Numerous el~ml)les of sample handling for capillary electrophoresis are known. For eY~mplP7 James in U.S. 5,286,652 and C~hricti~ncon in U.S. 5,171,531 are based on ~l~sel.l;ng a single vial of sample to a single separation capillary for a sequential series of analyses.
Goodale in U.S. 5,356,525 describes a device for prçsPnt~tion of a tray of 7 vials of samples to an array of seven capillaries for the sample injection process.
Carson in U.S. 5,120,414 describes injection of a sample contained within a porous membrane onto a single-capillary electrophoresis device. The end of thecapillary must be in in~im~t~ contact with the porous membrane to effect sample CA 0221~974 1997-09-19 W 096/29595 PCTrUS96/03828 introduction into the capillary.
In cQnt~t, the present invention provides short disposable c~rill~ri~s mounted in a frame which is integr~l with a llquid h~ntilin~ system. This system a rapid m--ltipleY~A approach to capillary electrophoresis.
Numerous ~x~mpl~s of multi-well devices with integral membranes are known (e.g. Mann in U.S. 5,043,215, M~tthi.~ in U.S. 4,927,604, Bowers in U.S.
5,108,704, Clark in U.S. 5,219,528). Many of these devices attach to a base unitwhich can be evacuated, drawing samples through the membrane for filtration.
Numerous eY~mplçs of multi-channel met~ring devices such as multi-ch~nnPl pipettes are known. One example is described in a device by Schramm in U.S.
4,925,629, which utilizes an eight-channel pipette to meter samples/reagents to/from multi-well plates. A second example is a 96-channel pipetting device described by Lyman in U.S. 4,626,509. These devices use positive displ~cem~-nt plungers in cc,lle~onding cylinders to draw in and expel liquid in the sampling/metering step.
Finally, Flesher in U.S. 5,213,766 describes a 96-channel device which contains flexible "fingers" which can be deformed out of a common plane; each "finger" can be deflected into a well of a multi-well plate to acquire a small aliquot of sample by one of several mech~nicm~.
The present invention differs in that it provides for simultaneously sampling of an array of ~mples; ~imlllt~n~usly handling the samples and ~ s~ g an array of the samples for capillary electrophoresis; simultaneously transferring the array of presented samples to an array of c~pill~ries; and simultaneously conducting separations in the capillary electrophoresis columns.

CA 0221~974 1997-09-19 .
W O 96r29595 PCTrUS~-'03828 ~ M AR Y ~F l'HF~rNrvF~rrT~ N
The present invention ~ncol"l~.s mPtho~lc and apparatus for .cimlllt~neously transferring ~mrles from an array of sample holders to an array of capillary el~;LI~horesis columns, simlllt~neously conducting electrophoresis, and analyzing S the capillary electrophoresis columns.
The invention encomr~cces a system for multiplexing capillary electrophoresis analysis of mllltiple samples comprising:
a) a means for cimlllt~nrnusly acquiring an array of aliquots of sample from an array of c~mrles in sample containers;
b) a means, in combination with means (a), for simultaneously processing the array of 5~mrles to provide an array of processed samples and presçnting the array of processed samples for capillary electrophoresis;
c) means for simultaneously transferring an array of processed s~mplçs to an array.of capillary electrophoresis columns;
d) means for cimllls~n~usly conducting capillary electrophoresis on the array of the capillary electrophoresis columns from (c); and e) means for.analyzing capillary electrophoresis columns from (d).
The invention also encomp~cces an electrophoresis separation plate comprising:
a) a frame having a first and second end and having an array of electrophoresis capillaries with first and second ends mounted respectively in the first and second end of the frame;

-W O 96/29595 P~~

b) the first end of the frame having a buffer reservoir and an electrode in the buffer reservoir wl.Glein the first end of the c~pill~ries are in liquid cc,,....,~,.lir~tinn with the buffer reservoir;
c) the second end of the frame having a means .for placing the second end of the ç~rill~ri~os in contact with an array of liquid samples or run buffer which is in contact with an array of electrodes and wherein there is fluid communication between the buffer reservoir and the sample or run buffer through the capillaries and electrical commnnir~tic-n between the electrode in the buffer reservoir and the electrodes in the s~mrle~s or run buffer by way of the electrophoresis capillaries.
The invention further encQmr~s an apparatus for processing samples from an array of sample containers compri~ing:
a) a sample h~nrlling plate which defines an array of sample h~n-lling plate wells in liquid co.. ,l.. ir~tion wlth an array of sipper c~rill~ri~s and wherein a porous matrix is interposed between each sipper capillary and the sample h~n-llin~ plate well, wherein the array of sipper r~pill~ri~s will simultaneously fill with samples from an array of sample containers;
. 20 b) a base plate which defines an opening to receive the sample handling plate and defines an inner chamber and means associated with the base plate and sample h~n-lling plate to seal the inner chamber and provide a sealed inner chamber;

-CA 0221~974 1997-09-19 W O 96/2959S PCTrUS96/03828 c) means for p~ . ;7in~ and ev~u~ting the sealed inner chamber to move liquid on either side of the porous matrix to the other side of the porous matrix.
The invention also encomr~csPs, in another embotlimpnt~ a electrophoresis S sep~r~tinn plate comprising a frame having a reservoir at one end and a plurality of sample sites at the other end and having an electrode at each end and a means for mounting capillary electrophoresis columns on the frame so that there is ~lectrit~
co,~ ie~ti-)n beLweell the electrodes and fluid communication between the samplesite and reservoir when there is fluid in the sample site and reservoir. The electrophoresis plate may be transportable so that it can be moved to various locations and stored.
The invention further involves a method for m-lltirlexecl analysis of multiple samples by capillary electrophoresis comprising:
a) providing the samples in an array of sample containers;
b) simultaneously sampling the c~mrlt~c with a sample handling plate having an array of sample h~n-lling plate wells with sipper capillaries;
c) transferring the sample h~n-lling plate to a base plate which provides for simultaneously processing and presentin~ the samples for electrophoresis;
d) cimnlt~nPously transferring pl~~ A sample to an array of capillary electrophoresis columns;
e) simultaneously conducting separation by electrophoresis;
f) analyzing the capillary electrophoresis columns.

CA 0221~974 1997-09-19 W 096/29595 PCT~US96103828 The el~llu~h~ is plate co~ iscs-a frame with at least one reservoir at one end. The electrophoresis plate has a means for mounting a plurality of capillaryde~lul~hol~sis c~ mnc, having first and.second ends, on the frame so that there is fluid col-----u~ tion be~ween the reservoir and the first end of the plurality of S ç~ril1~riPc, when there is fl~id in the reservoir, and a plurality of sample introduction sites near or at the second end of the .-~pill~ri.oF. There is a means to make electrical connection to each end of the frame, with.at least one common electrode in the reservoir in the frame at the first end of the capillaries and at least one electrode in a second common reservoir in the base plate in fluid comm~lni~tion with the plurality of sample sites at the second end of the r~rill~ries There is a plurality of electrodes electrically connected in parallel and positioned in each of the sample intr~clucction sites at the second end of the c~rill~ripc This arrangement of electrodes provides for electrical communication between the electrodes at each end of the frame and the capillaries, when there is fluid in the reservoirs, capillaries and the 1~ sample introduction sites.
The invention includes methods for analyzing samples of substances which are ~.p~r~bl~ by electrophoresis providing an electrophoresis plate having a plurality of electrophoresis columns, simultaneously transferring sample from the multiplesamples to the electrophoresis columns in the plate, simultaneously con~ucting ele~ u~hore~is on the columns in the plate to separate the substance to be analyzed and analyzing the substance(s) to be analyzed which are se.p~r~tP~ in the electrophoresis columns in the electrophoresis plate. The capillary columns may be dynamically analyzed by detecting a separated band as it moves past a stationary 096/29595 PCTrUS96/03828 analysis system, or the columns may be s~nned by a moveable analysis system, or imaged by an array detert--r.
The present invention has many adv~ges over conventional electrophoresis technology, whereas in the prior art the confirmation of proper amplification of nucleic acids by convPnti--n~l ~r~tiQn on gels takes hours, whereas, the system of this invention can do cr~"I~ ion in about 10 min~tPs Conventic-n~l gel separations are manually intensive. The invention only requires sample introduction, with the separation, detection and analysis being automatic. The invention system will p~, rO.... conrl~ ationS in parallel. For example, 96 samples can be processed on a standard gel, but only with col"plo",ises in resolution and sensitivity.
The invention system can resolve a difference of 2-3 base pairs among double stranded DNA fragments of 200-300 bps. If 96 s~mrles are run in parallel on a single standard gel, resolution for the same range of targets would be 5 to 10 fold poorer. Working with ~mplps of unpurified PCR-amplified DNA, the present system found multiple ~mplifiPd targets in several s~mples where the standard gel methods only dPtectPcl a single band.
The present invention system typically requires only 1 to 5 % of the amount of sample used on a standard gel because of the high sensitivity and hence less ,mplifiP~ m~tPri~l is required, reducing reagent cost and/or amplification time.
The capability to add a common reagent to multiple samples, mix and react means that the primary amplification step could be done on the sample handling plate, with an a~~ iately dP~i~nP~ thermocycler, with the benefits of amplifiç~tiQn done in discrete small volumes, in parallel with precise timing, with a minimum of W O 96/29595 PCTrUS96/03828 carry-ovff and cross con~...;n~i;on This invention has adv~ulk~es with regard to ~ n of ~mplified nucleic acids. After amplific~tic-n, the standard methods are patterned after conventional i.n~ ~ys requiring solid phase re~ tionC (on the surface of 96-well plates or onS beads), followed by washes for s~ lion.and subsequent rP~ctirn~ for signal generation. The whole procedure takes several hours, with many steps, and with ,inal sensitivity and precision.
The present invention system can pelÇolln a ~luan~i~ative determination in a much simpler format. The target is amplified with sequence specificity in the standard way; that is, for PCRTM, specificity is derived from the primers and for LCR~M ~ificity is derived from the ligation of adjacent hybridized probes. Then the amplified m~tt~ri~liss~l~aldt~d on columns and the amount of target is measurèd at the anticipated position on the column, based on the size of the target. That is, one qll~ntit~fPc the amount of target, in addition to obtaining a size-based S cO.. r, IllaLion without the need for any solid phase reactions. If ~diti~m~l sequence spe~ifi~ity is required, then an additional hybridization step could be incorporated into the procedure.
Since reactions are in the liquid phase, the invention system provides greater speed, greater specificity and less background biochemical noise and quantitation is achieved in tens of minutes instead of hours.
The present invention system has demonstrated the detection of as little as 8 million DNA molecules (300 to 1300 bps) in the sample and therefore has high sensitivity. ~e~use of the high sensitivity of the present system, less biologi~l CA 0221~974 1997-09-l9 096/29595 PCTrUS96/03~28 ~mrlifis~tion is required. For example, 20 PCRTM cycles o~cl~ling at optimum effiri~ncy will produce 1 million molec~ s, starting from a single target molecule in the c~mple Conventional quasi-q~.~nl;l;.tive methods typically require 30 to 40 cycles to produce enough target for reliable detectil~n. However, the biological gain per cycle decreases as one amplifies at the higher cycle numbers (experts agree that such variability can occur at greater than about 20 cycles). This variability is a primary cause for the lack of assay precision in the conventional methods. The present invention system only requires 2 ,ul of sample, reducing the amount of primers, bases, enzymes, etc. required for the amplification step.
The c~p~hility to add a common reagent to multiple s~mples, mix and react means that the primary ~mplifit~tion step could be done on the sample h~nflling plate, with an a~ opliately decign~l thermo-cycler, with the benefits of amplification done in discrete small volumes, in parallel with precise timing, with a minimum of carry-over and cross contamination.
Many of these advantages are also achieved with regard to conventional binding assays such as ELISA's. For example, qu~ntit~tion can be done in lO's of minutes on 2 ,ul sample volumes. The capability to add a common reagent to multiple samples in, for example, a 96-well or a multi-well plate, mix, and react on the sample handling plate means that a primary reaction step (e.g. displacement of a common ligand to a receptor) can be done in discrete small volumes, in parallel with precise timing, with a minimum of carry-over and cross cont~mination, and without cont~min~tion of the starting m~t~ri~l (e.g. any array or library of compounds).

W 096129595 PCTrUS96/03828 RR~h.F ~F~ R~ N C-F T~F, Fr~lJRF.~
Figure 1 is a ~ e ;live view of the electrophoresis ~dlion plate.
Figure 2 is a cross-s~ctinn~l view of thè se~a-dLion plate through a capillary.
Figure 3a shows an exI)lod~rl sectional view of the well with the well Selectrode and capillary positioned in the well.
Figure 3b shows a sample inJected into the well.
Figure 3c shows- the wdl with buffer and sample in the well.
Figure 3d shows the well when the buffer has diluted the sample.
Figure 4a-c shows a sr~hem~ti~ of how the components of the system interact.
Figure Sa shows a cross-se~tic-n~l view of several sample h~nrlling plate wells.
Figure Sb shows a schematic of liquid flow from sipper capillary to sample h~nflling plate well.
Figure 6 shows flow of sample into sipper capillary.
lS Figure 7a - 7e shows the flow of sample and reagent mixing.
Figure 8 shows sample h~n~ling plate with sample h~n~lling plate well and waste and primary buffer wells.
Figure 9 shows top plan view of a preferred separation plate.
Figure 10 shows a cross-sectional view of the l lefell~d separation plate through a capillary.
Figure 11 shows a schem~tic diagram of the optical system for reading c~rill~n~-s.
Figure 12 shows a-graph of Msp I pBR322 separation.

W O 9612959S PCTrUS9GJ~82B

Figure 13 shows a trace of a .ct~ inn of single str~ndecl DNA fr~gm~ntc ~iffering by one base.
Figure 14 shows sep~r~tion of unknown DNA c~mples derived from PCRTM
~mplifi~ti~ n.
S Figure 15 shows separation of a protein/binding partner complex.
Figure 16 shows a plot of the concentrations of a separation of a ~licpl~e labeled peptide versus concentration of a colnl eLilor.
Figure 17 illustrates the limits of detection of DNA.
Figure 18 shows the efficiency of mixing of two s~mpl~s in the sample h~n-iling plate.
Figure 19 illustrates the reproducibility of simultaneous sipping, sample h~n~iling, electrophoresis and then analysis of DNA samples.
Figure 20a shows a ~refelled embodiment of the separation plate composed of an array of 8 separation capillaries in a frame aiong with an integral bufferreservoir for supplying the s~,p~rAtion ç~rill~Ti.os and associated run buffer wells with run buffer Figure 20b shows a cross-sectional view of a s~ ti~ n plate/sample h~ntlling plate assembly and illustrates the l l~felled embodiment of the geometry and positioning of the run buffer wells on the sample handling plate and the s~al~tion plate.
Figure 21 a and b each illustrate an embodiment of the sipper/porous matrix/sample h~n~llin~ well feature on the sample h~ntlling plate.

CA 0221~974 1997-09-19 W 096/29595 PCT/U~ /03X.

nF.TA n,F.~ nF~RnPT~ N ~ F ll~F rNrVF.NTT~ N
The invention includes as shown in Figures l and 2 an elong~t~ri Cl~llu~ c~iS se~r~tif~n plate 1 which has a plurality of sample wells 2 at one end S and a common buffer reservoir :~ at the other end. A first master electrode ~ is electrically connected to a cell electrode ~ in the sample wells 2. A second master electrode l is in the common buffer reservoir ~. Capillary electrophoresis columns are mounted in the plate 1 so that there is electrical communication between thefirst master electrode S by way of the capillary electrophoresis column ~ when the sample wells 2 and the reservoir 3 are filled with electrically conductive liquid. In operation, current between the master electrodes permits electrophoresis of the sample from the sample well 2 to the reservoir ~.
Figure 3a is a partial sectional view through a sample well 2 showing the well electrode ~ and capillary ele ;llupho.esis column ~. Figure 3b illustrates the injection of a sample 1~ so that there is liquid commlmi~tion between the capillary ~ and cell de~L.udc ~i. The sample is loaded on the capillary via electromigration injection and then the residual sample in well 2 is diluted with buffer 14 before the electrophoresis process takes place as shown in Figure 3d. In an alternative implementation, Figure 3c shows a mech~ni~1 liquid transfer system where about 4 ~Ll of buffer 17 is first added to establish liquid communication between the capillary ~ and cell electrode fi. Then 4 ~l of sample 18 contained within a pipetter tip is placed in contact with the buffer 17 and sample is loaded on the column via electromigration injection.After removing the pipette tip, the well is then filled with buffer 1~ as shown in CA 0221~974 1997-09-19 W 096/29595 PCTrUS9''0~8 Figure 3d and the electrophoresis is concluc.t~
Sample may be.made available for injection from the introcl~lction site to the capillary such that only small aliquots of the primary sample are required at the introcluction sites, 4u~ e injection is possible, and there is a minimllm of carryover from one injection to anoth.er (if the plates are reused). Such sampleintroduction may be accomplished by a variety of means, including:
a) physically moving the c~pill~n~s relati~e to the intr~dllction sites and immersing the tips of the capillaries into the respective sample aliquots po.citinned within the introduction sites. In this embodiment the plurality of sample introduction sites might.be constructed on a separate disposable part, which moves into position for sample introduction, and then is disposed.
b) physically moving the capillaries relative to the introduction sites and bringing the tips of the capillaries to the close proximity of the sample aliquots in the introduction sites.
c) each introduction site is perm~n~.ntly imm~i~tely adjacent to the respective capillary tip.
Sample injection may be accompli.~h~cl, simultaneously.and in parallel, for the plurality of capillaries by a variety of means, including:
a) electroinjection, under the action of an electric field due to a voltage dirrerel-ce applied to the a~ ol"iate electrodes.
b) pressure injection, under the action of a pressure or suction applied to the fluid at one or both ends of the c~pill~ri~-ls.
With regard to electrodes, some or all of the electrodes may be within the CA 0221~974 1997-09-19 W O 96/29595 PCTrUS96/03828 sample h~n-llin~ plate or within the electrophoresis sep~r~ti~ n plate, with eYtern~l connections to power supplies, or some or all of the electrodes might be on a sep~ part (e.g. built into the injection and sel)A.~tion station), such that theelectrodes can be immersed into the ~upliate fluid reservoirs at the time of S injection or separation. The electrodes may also be integral with the s~a~ ion plate. They may be strip metal ele;tludes formed in a stamping process or chemical etching process. The electrodes may be wires or strips either soldered or glued wi~h epoxy and can be made of conrlllctive m~tPri~lc such as platinum, gold, carbon fibers and the like. The electrodes would preferably be an integral part of the s~ dtion plate and could be deposited, coated or plated onto a section of the exterior wall of the capillary near each end of the capillary. Controlled vapor deposition of gold, pl~timlm, or p~lWtillm metal onto the exterior wall of the capillary is one method of forming the electrodes. This technique can be used to produce an electrode layer with a thickness up to several microns. Thicker electrodes could be subsequentlyformed by electrochemically plating gold, p~ ium or platinum onto the thin electrode formed by the vapor deposition process. Electrodes could be integral with the sample h~nrlling plate formed by silk screening process, printing, vapor deposition, electrode-less plating process, etc. Carbon paste, conductive ink, and the like could be used to form the electrode.
Those skilled in the electrophoresis arts will recognize a large number of c~rill~nrs useful for pr~rticing this invention. For exarnple, fused silica is used with an outside coating of polyimide for strengthening, with inside bore ID's from 10 to 200 microns, more typically from 25 to 100 microns, and OD's greater than 200 , =

W O 96/29595 PCT~US96/03828 microns. Tn~.orn~l coating may be used to reduce or reverse the elec~oosnlotic flow (EOF). The ~ t "coating" involves running at a low pH such that some of the silanol negative charge is nPutr~li7Pcl Other co~ting~ inc~ le silylation, polyacrylamide (vinyl-bound), methylcP-li-llose, polyether, polyvinylpyrrolidoneS (PVP), and polyethylene glycol. C~pill~ries made from other m~teri~l~ such as glass, polypropylene, PyrexTM and TeflonTM may also be used. In ~rltlition, capillariesprepared by microfabrication techniques in which a small channel is etched into a planar substrate such as glass, silica, or ceramic couldalso be used.
Conventional buffers include the Good's buffers (HEPES, MOPS, MES, Tricine, etc.), and other organic buffers (Tris, acetate, citrate, and formate),including standard inorganic compounds (phosphate, boMte, etc.). Two preferred buffered systems are:
i) 100 mM sodium phosphate, pH 7.2 ii) 89.5 mM tris-base, 89.5 mM Boric acid, 2 mM ETDA, pH 8.3.
Buffer additives include: methanol, metal ions, urea, surf~t~nt~, and zwitterions interc~l~ting dyes and other labeling reagents. Polymers can be added to create a sieving buffer for the ~lirrt~ tial separation of DNA based on fragment length.
Fy~mples of such polymers are: polyacrylamide (cross-linked or linear), agarose,methylcellulose and derivatives, dextrans, and polyethylene glycol. Inert polymers can be added to the separation buffer to stabilize the separation matrix against factors such as convection mixing.
Alternatively, buffers cont~ining mi~elles could be used for effecting s~al~ion of electrically neutral or hydrophobic analyte species. The miç~lles are CA 0221~974 1997-09-19 W 096/29595 PCTrUS96/03828 formed in the buffer by ~ lition of an a~ u~liate surfactant at a conc~ntr~ti-n eYce~iin~ the critical micelle c~?ncçntration of that d~ gellt. Useful surf~c-t~nt~
include but are not limited to sodium dodecyl sulfate, dodecyltrim~thyl ~mmonillm bromide, etc. Weakly cha,~ed or apolar analytes partition into the micelles to different degrees depending upon their degrée of hydrophobicity and thus can be separated. This subtechnique of capillary electrophoresis is termed micellar electrokinetic chromatography.
Those skilled in the electrophoresis arts will recognize a wide range of useable electric field strengths, for example, fields of 10 to 1000 V/cm are used with 200-600 V/cm being more typical. The upper voltage limit for the commercial systems is 30 kV, with a capillary length of 40-60 cm, giving a maximum field ofabout 600 V/cm. There are reports of very high held strengths (2500 - 5000 V/cm)with short, small bore (10 microns) capillaries micro-machined into an in~ul~ting substrate.
Normal polarity is to have the injection end of the capillary at a positive potential. The electroosmotic flow is normally toward the cathode. Hence, with normal polarity all positive ions and many negative ions will run away from the injection end. Generally, the "end-of-capillary" detector will be near the cathode.
The polarity may be reversed for strongly negative ions so that they run against the electroosmotic flow. For DNA, typically the capillary is coated to reduce EOF, and the injection end of the capillary is m~int~inçd at a negative potential.
Figures 4a-c show the interaction of various parts of the electrophoresis system. Figure 4a shows the sample handling plate ~Zl with an array of sample W 096/29595 PCT~US96/03828 h~n~l1in~ wells 1~ with an'colle:~ollding array of sipp~r c~rill~ries 82. The array of sipper c~ri11~ries is aligned'with wells of a multiwell plate which contain ~mp1es ~~. When the sipper capillaries 82 are in the sample, an- aliquot of sample is rr,. . ed to the sipper capillary by capillary action. The sample h~n(l1ing plate I
S is then moved to base,plate 1~, as shown in Figure 4b. Sample h~n~1ing plate ~Zl and base plate 72 fit togeth'er to form a sealed inner chamber ~ which can be p,ressurized or evacuated through port 7~. In this way, the s~mrlP-c in sipper c~ri11~riPs ~2 can be m~nir~ t~1 and eventually presented in sample h~ntlling wells lg for electrophoresis. Figure 4c shows how the electrophoresis sep~r~tion plate(s) 1~2Q Cf~.ll .illi.-g an array of elec~lu~l1olesis capillaries ln1 are aligned with the sample handling plate wells 1~. A stack of sep~r~tiQn plates 1 0C) is formed to provide an array of separation c~ri11~ries that con~olllls 'to the spacing format of the sample h~nrl!ing wells 74.in sam,ple h~n-l1ing plate 71 or to the spacing format of a multiwell plate that contains s~mp1es ~.
Figure 5a shows a cross-s~ctit)n~1 view through several wells 74 The sample h~n~l1ing plate 11 is assembled from a ~mrling block 7~ which defines the funnelshaped base wells with openings 77. Mixer block ~ has passages 1~ which are aligned with U~llingS 11. The mixer block :Z~ and s~mr1ing block :Z~ are sep~r~tP,A
by a porous matrix ~Q, such' as a membrane. Aligned with mixer block ~Z8 is sipper block ~1 with sipper capillaries ~2. ~a is filled with sample and ~k is not.' The sipper block ~1, mixer-block ~, and ~mrling block 1~ are fastened together so that a channel a-h is defined which is interrupted by the matrix ~Q as shown in Figure 5b.
Sipper c~rill~riP~ can be constructed by any number of means. A major requirement -CA 0221F,974 1997-09-19 WO 96/2959S P~ 5/03828 is that the sipper capillaries must be sl-fficiçntly hydrophilic to draw in several microliters of liquid sample by capillary action. A suitable sipper capillary could be constructed from glass or silica tubing of a~lu~liate dim~ncions. ,Altt~.rn~tively, a suitable sipper capillary could be constructed from a plastic m~tPri~l such as S ~lyt;lllylene, polypropylene, poly~l~onate, polysulfone, etc. In the case of sipper capillaries constructed from plastic materials, however, the inner bore of the plastic sipper capillary must be treated in some way to make the inner walls of the sipper capiilary s~ifflciçntly hydrophilic to draw in the sample. by capillary action.
Appropriate tre~tmt--ntc for ~lt-.nng the normally hydrophobic surface of the plastic sippers and i~ ~ling hydrophilicity to the inner walls include coating the walls with a surfactant or wetting agent, grafting a layer of hydrophilic polymer onto the wall of the hydrophobic sipper capillary or treating the walls of the sipper capillary by plasma etching.
The volume of sample drawn into the sipper capillary is controlled by the length and the bore ~ met~r of the sipper capillary. Typically, the bore diameter will be several hundred microns and the length of the sipper capillary could range up to 10-20 mm. Sipper r~pill~ri~s of these ~limtoncinnc can fill with sample volumes in the 1 to 10 ~L range. The volume of fluid drawn into the sipper capillary can be controlled by carefully dçfining the bore ~ m~ter of the sipper capillary and bydefining the length of the sipper capillary. The latter ~iimçn.cion can be defined by posihoning a stop junction along the inner wall of the sipper capillary. This stop junction could be an abrupt increase in the bore ~ meter of the sipper capillary.
Altematively, using the techniques describe above, only a defined length of the inner -CA 0221~974 1997-09-19 .
W 096/29S95 P~1/~_''03828 wall of the sipper capillary could be made sl~fficiently hydrophilic to draw in aqueous sarnple by capillary action; the boundary between the hydrophilic and hydrophobic cllrf~ s of the inner wall would thus define stop junction.
Matrix ~Q is typically made of a wide variety of porous matrix m~tPri~lc S where, for most applic~tinnc, the porous matrix m~tPri~lc should have little or no affinity for sample. Useful porous matrix m~tPri~lc include membrane m~teri~lc such as regenerated cPllllloce, cellulose acetate, polysulfone, polyvinylidine fluoride, polycarbonate and the like. For DNA samples, a cellulose acetate membrane such as that available from Amicon is useful. For protein s~mI-les, a membrane co.,.~osed of polysulfone such as those available from Amicon or Gelman is useful.
Alternatively, porous matrix ~Q could be a porous cylindric~l or spherical plug of sintered polymer particles. Such porous materials are available from Porex or Interflow and are typically comprised of a bed of small polymeric particles that have been fused together by heat and pressure (cint~ring) to form a porous plug of precle-fined geometry. The bed of particles forms a network of tortuous interstitial pores through which the sample is passed and subsequently mixed.
~PnPr~lly, porous matrix ~Q is an inert matrix that serves to mix the sample and added reagent upon passage through the pores of the matrix. However, porous matrix ~Q might also be ~ecignPd to provide some selective fractionation function to remove unwanted species from the sample. For example, porous matrix ~fl might also serve as a filtering medium to purify the sample of particulate debris prior to injection into the capillary.
In another implemçnt~tion, porous matrix ~Q might be an ultrafiltration CA 02215974 1997-o9-19 W 096/29595 PCTnUS96103828 .,.~.,~1..,..~.~ with a defined molec~ r weight cut-off, such that porous matrix ~n will allow small molecul~r weight col,iponents to pass through porous matrix ~Q. The m~ r weight cut-off of ult~filt~.~tinn me~ es can vary considerably, r~nging typically from approximately 1,000 to upwards of 500,000 Daltons. Thus, an ultrafiltration membrane with a m~-lecul~r weight cut-off of 1,000 daltons wouldallow co,n~nents with molecular weights lower than ~ 1,000 Daltons to pass through while ret~ining co"~ponents with molecular weights greater than 1,000 Daltons. By proper s~l~ticn of the molecl-l~r weight cut-off of the membrane used as porous matrix ~Q, small molecular weight collipolltlll~ of the sample are passed through the membrane, whereas high molecular weight components are retained ibelow the membrane.
Alternatively, porous matrix ~ could be derivatized with some biochemical agent to impart a selective binding capability to matrix ~2; in this instance, porous matrix ~n could also serve to remove high concentrahons of a specific biomaterial or specific class of bio.. ~ lc from the sample prior to injection into the capillary.
Specific examples of the biochemical agent used to derivatize the porous matrix would be protein G (used to remove immunoglobulins) or lectins (used to remove carbohydrates). Altematively, porous matrix ~Q could be derivatized with biotin or streptavidin, thereby pçrmitting ~tt~chm~nt of a variety of a~p~ iated derivatized (biotinylated) species to porous matrix ~Q.
Porous matrix ~Q also serves another illlpol~ll function to prevent loss of the mixed volume of sample/fluid from the sipper during the plessulization/evacuation protocol implemented during the mixing step. The mixed volume of sample/fluid ~ ~ .
CA 02il5974 1997-09-19 WO 96/29595 P~ 1 iL~ 31PX

is slowly passed through the porous matrix during the yle~ I;on phase of one cycle of the mixing step. When the trailing edge mPni~cus of the sample/fluid bolus reaches the face of porous matrix ~Q, flow of the sample/fluid bolus will stop as the yl~ssulization gas reaches the face of porous matrix ~Q. This occurs because of dilr~ ces in surface tension-inrlllce~ forces that origin~tP- when the porous plug is cont~te~ by the pre-c~l-ri7~tion gas versus contact by the sample/fluid solution.
I~lcewise, during the evacuation phase of the mixing cycle when the sample/fluid is passing back through porous matrix ~Q in the opposite direction, the flow of sarnple/fluid will stop when the opposite mPni~cu~ of the sample/fluid bolus contacts the opposite face of porous matrix ~Q. In the absence of porous matrix f~ll in the flow conduit in the sipper capillary, the sipped sample and any added reagents could be ejected from the sipper capillary flow conduit during the pres~llri7~tion or evacuation phase of the mixing cycle.
Figure 6 illustrates the flow of sample ~ from a well of a multiwell plate into sipper capillary ~2. Thus the ends of the array of sipping capillaries ~2 on sample handling plate Zl are dipped into ~mples contained in an array of samples such as a 96 well plate and the samples are metered into the sipping capillaries by capillary action. At this point, the sample handling plate ~Zl with its sipper capillaries filled with samples is placed on base 12 to form a sealed inner chamber ~4, Figure 4b.
Figure 7a - 7e ilh~ lPs the flow of sample in response to ples~lllization and ev~rll~tic n of the inner sealed c-h~mhPr ~4 through port 1~. For example, a positive Ult~ moves the sample from the sipping capillary ~2 to the area below the porous CA 0221~974 1997-09-19 .
W O 96/2959S PCTnUS96/03828 matrix, as shown in la, through the membrane, and into the well 19 above the ,,..~..,1~.~.~ in plate :Zl. R~gent~ 7 can be added to the wells l~L as shown in Figure 7b and the reagent and sample can be mixed as shown in 7c and 7d~by forcing the sample and reagent back and forth through the membrane ~Q in response to S pressu.izatlon and evacuation of the inner sealed chamber fi4. . Finally, the mixed sample fi~ is presented in well 74 for injection into an electrophoresis column as shown in 7e.
Figure 21a shows a pl~felled embodiment of the sipper components of the sample h~n-lling plate. Sample h~n~lling plate Zl is composed of an array of sipper capillaries one capillary ~2, one of which is illustrated. Sipper capillary ~2 in conjunction with porous matrix ~1 and sample h~n~ling plate well l~L constitute a flow channel in which sample 18Q is manipulated. Sipper capillary ~2 is composedof a conduit region 1~ which spontaneously fills with sample 180 when proximal end 1~ is immersed in sample. The conduit region 1~2 must be sufficiently hydrophilic to allow the conduit to ~n~leollsly fill with sample by capillary action.
The distal end of the conduit region 1~2 is le,.--il-~t~ by stop junction 1~ in the conduit bore. Sample fluid will spontaneously fill the conduit 1~ until the fluid level reaches the level of the stop junction 1~. Thus the conduit region defined by the proximal end and the stop junction in conjunction with the bore of the conduit region determine the volume of fluid "sipped" into the sipper capillary. The proximal end 1~ of the sipper capillary ~ is formed with a chamfer 1~1 on the exterior wall to minimi7e the volume of sample that could cling to the exterior wall of the sipper capillary.

W O 96/29595 PCTrUS96/03828 ~Sih-~t~ u~ I inside the enlarged bore region 1 g~ of the sipper capillary as defined by the stop junction is porous matrix ~Q. Porous matrix ~Q is ideally a cylinrlric~1 porous plug formed from sintered polymeric particles. Further upstream of porous matrix X0 is sample h~n-lling plate well l~L, where the sample is ~l~s~nted S for injection into the sep~r~ti~ n capillary.
Figure 21b depicts another embodiment of the sipper components, where the stop junction is replaced with a junction defined by the boundary ~ between s~cti-~nc of hydrophobic 1~1 and hydrophilic 192 wall which form the inner bore of the sipper capillary. The inner wall of 192 is sl~fficiently hydrophilic to draw in sample and fill conduit 1~ with fluid 1 2Q The rise of sample fluid inside the conduit is termin~ted when the advancing fluid level reaches the boundary 14n, where the sample fluid will no longer wet the inside wall of the sipper because of the presence of hydrophobic wall 141 and capillary action stops.
Figure 8 illustrates the sample well 1~ flanked with a well for waste ele ;LIopl~ is buffer 9n from a previous se~aldLion and a well for fresh run buffer ~1 which is deposited from a capillary ln~ during a flushing before injecting c;sellL~d sample from the sample-h~n-lling well into the capillary 1 n~ and then used during the electrophoresis separation. The capillary 1(~ addresses the waste 9n,buffer 91 and sample 1~ positions by moving the separation plate with respect to the sample h~nrlling plate.
Figure 9 shows the electrophoresis sep~r~tion plate 111~ having 8 capillaries lnl mounted on a frame ln2; upper buffer reservoir lQ~ provides buffer to the r~pill~ri~s ln1. Orient~ti~n notch 11~ provides a means for ~ligning the separation W 096/29595 PCTrUS9"0~828 plate for ~ r~-. 1 ;llg sample or reading columns. Electrode 1 n4 and an electrode at the injection end of each of the .se~ n capillary 1n~ provide for elpctr~
communication through the buffer. Figure lO is a cross-section~l view through a se~;.l,.l;on plate.
S As shown, separation pIate lQQ contains buffer in upper reservoir ln~; the buffer in lQ~ is drawn from a source P~t~m~1 to separation plate 1 nn. In a p-ere led embo iimtont as shown in Figure 20a, s~ tion plate on may also be equipped with an integral (or deAir~ted) supply of buffer l~Q on-board the separatio~ plate in a storage vessel 1~1 (or tank) ~tt~h~d to separation plate 1 nn. A portion of the buffer in this integral supply 1~1 can be used to flush the upper buffer wells 1~ and the r~rill~riPs 1nl after each analysis via a set of manifolds 1~4 in the separation plate.
Another manifold 1~ is employed to deliver buffer via nozzles 1~ to run buffer well 21 on the sample h~n-l1ing plate.
In this way, s~dLion plate lQQ becomes a self-contained, limited-use, application-specific device. Sufficiçnt buffer of the ~p~ul~liate composition for a predetermined number of uses can be packaged with the separation plate for user convenience. The a~l~liate buffer can be packaged with the separation plate of capillaries to preclude use of the wrong buffer composition with a specific set of r~ril1~ri.os. Finally, application-spe~r-ific separation plates can be developed, where - 20 the capillary and buffer chemistry are optimized for separation of a particular set of analytes, thereby insuring optimum separation performance from the system.
Figure 20b shows the s~dlion plate 1 nn and sample h~n~lling plate Zl in cross-section and illustrates important features of the buffer well geometries of the W 096/29S95 PCTrUS96/03828 s~p~ ;nn plate 1nn and sample h~nr'1ing plate 1. Buffer is delivered to upper buffer well 1~2 via manifold 1n~. .Se~ t;.-g upper buffer well 1~'~ and manifold1n~ iS an orifice 1 'i9 which controls the flow of buffer into the upper buffer well.
Buffer 1~51 partially fills upper buffer well 1~2, forming a meni~cu~ 1~ at one end S of the buffer well. An overflow basin 1 f~l catches buffer that might flow from upper buffer reservoir 1~'~ and returns expended buffer from 1~'7 to a waste reservoir. One end of the sc~dLion capillary 1n1 is immersed in the buffer in upper buffer wellThe other end of the sep~r~tion capillary 1 nl is immersed in sample handling plate buffer well 91 which is situated on sample h~n-l1in~ plate 71. Likewise, sample h~nrl1ing plate buffer well 91 iS filled with buffer 1~ which forms a meniscus 1~7.
Buffers 1~1 and 1~8 are identical in chemical composition since they were both delivered from buffer supply 1 ',n in buffer tank 1~1 . Ideally, upper buffer well 1~2 and sample h~n-l1ing plate well 91 have idtontic~1 cross-sectional geometries and cc"l,~al~ble if not identical well surface plu~,e"ies, such that buffer 1~1 wets upper buffer well 1~2 and buffer l~i8 wets sample h~n-l1ing plate well 91 to the same extent and forms the same curvature of meniscus in each. In this way, the volumes of buffer fluid in each buffer well experience i~1enti~1 forces and thus the tendency for buffer fluid to flow through the capillary to balance any imb~1~nces in these forces is elilll;o~lr~. Tmb~1~nces in the forces experienced by the buffers in the twû wells could result frûm dirrc;lclll wetting plùl~clLies of the two well structures as evidenced by different contact angles for the buffer in the two wells and this would manifest itself in dirrc~c~t curvatures of the meniscus in the two wells. Likewise the geomPtry of the upper buffer well 1~ and the sample h~n~i1ing plate well 91 should CA 022l5974 l997-09-l9 W 096129S95 PCTrUS96/03828 i~eally be iAentie~l- Also, as shown, upper buffer well ~2 and sample h~ndling plate buffer well ~1 are ~eei~ne~ to permit removal or addition of some fraction of the buffer from the wells by electroosmotic flow during the s~d dtion p.ucess without a change in the height of buffer fluid in either well. The absence of this change in fluid level means that no hydrodynamic flow of buffer fluid will occurduring the separation process; such hydrodynamic flow would result from syphoriing induced by differences in fluid level and the presence of such flow could ~e detrim~nt~l to the separation process.
Those skilled in the arts will r~cog~ that parts such as the sample h~n-lling plate; base plate and frame of the separation plate can be m~ehine l or molded from chemiç~l resistant plastlcs such as polystyrene or the like.
Thus, in opPr~tion, .c~n-. '~ from an array of samples ~ such as a multiwell plate are wicked into an array of sipping c~pill~riee ~2 of the sample handling plate 11. The sample handling plate ~1 is placed on base 12 and the sample is manipulated by pre~lln7ing the ch~mher ~ defined by the sample handling and baseplates and finally moved to the base plate wells 1~ for present~tion to the r~rill~ries in the separation plate ~. However, prior to transferring the sample to the capillary, the capillaries are washed with buffer and primed with buffer. .S~mples are injected into the c~rill~ries and electrophoresis is conducted in the capillaries in the ~p~r~tinn plate. When the electrophoresis is finished, the separation plate may be moved to an analysis station. The over all scheme is shown in Figures 4a-c.
After electrophoresis, the separation plate can be stored or read as shown in Figure 11.

CA 02215974 1997-09-l9 W 096/29595 PcTAus96J~3x2 An il"~ L~IL feature of the present invention is the fleYihility of the system to allow ~mrl~c to be run simultaneously under a variety of sep~r~hnn con~litionc This is poccihlP because each of the sPp~r~ti~n plates in the array of separation plates as shown in Figure 4C iS a se!f-contained unit and each plate can be filled with a dirr~"~ buffer and can be equipped with a different capillary chemistry. Thus, it is possible to form a stack of 12 different separation plates filled with 12 different ~ ti~m buffers and to analyze a set of 8 different samples under 12 dirr~ t setsof con~1itinn.c using the format i~ ctr~trd in Figure 4. This feature is useful for rapid scr~ni,lg of a~ iaLe sep~rAtion conditions when developing a separation method or when analyzing a set of samples for various subsets of components that are best separated under differing separation conditions.
Capillary electrophoresis columns can be anaiyzed in variety of ways inrln~ing the methr~c shown in U.S. Patents 4,675,300,4,274,240 and 5,324,401.
The-sample injection and cP.p~rAti~n are conducted in one location and the plate may be Lldns~l~d to a dirrel~ or~tion for analysis. Figure 11 shows a block diagram of one optical system for reading the capillaries. Power supply :~Q energizes the photomultiplier tube ~1. Power supply 32 energizes a 75 watt Xenon lamp ~.
Light from the lamp ~ is con-lPncPA by focusing lens ~ which passes light to theexcitation filter ~. A dichroic mirror ~ directs excitation light to microscope objective ~1. The separation plate lQQ with capillaries lnl is mounted on a rP~tilinP~r scanner to pass the r~pill~rips over the light from the microscope objective ~7. Fluorescent emission light is collected by objective ~1, passed through a dichroic mirror ~, emission filter ~n, spatial filter 52 before reAching CA 0221~974 1997-09-19 W 096/29595 PCTnUs95/o~

photoml-ltirliPr (PMT) 31. The output signal of PMT ~l is fed to an analog-to-digital converter ~, which in turn is connP~t~P~1 to co",puler 54.
Alternatively, a static d~Ptection system in which a stationary det~Pction pointsome ~ t~nce from the injection end of the capillary is monitored as analyte bands traverse the length of the capillary and pass by the detP~tiQn zone could be used.
This type of d~PtPction could be imrlçment~pd using optical fibers and lenses to deliver the PYrit~tion radiation to the capillary and to collect the fluorescent emission radiation from the detection zone in the capillary. Appropriate multiplexing anddPmllltiI~lexing protocols might be used to sequentially irradiate and monitor a large array of c~rill~ri~s using a single source and a single or a small number of photodeL~;~ . Using this approach, each capillary in the array is sequentially polled to detect any analyte band in the detection zone of that capillary.
Those skilled in this art will recognize that the above liquid h~n-lling system provides for simnlt~neous and ~luanLiL~tive sampling of a large array of s~mpl~Ps by sipping from the 96, 192 or 384-well plates or arrays of microtubes with an array of sipper ~rill~riPc. It provides for mixing sPp~r~t~P aliquots in the ,LI range by cycling the aliquots back and forth through a porous matrix such as a membrane. The invention provides an arra~ of ~lAition and mixing sites for the simultaneous addition and mixing of reagents for achieving either a constant or a gradient of mixed m~tPri~l across the array and for precisely controlling for the simultaneous starting or stopping of reactions.
Use of activated membranes in the base plate provides for selective removal of some col,lpollents of the sample of reaction mixture prior to injection. For W 096/29595 PCT/U~96'0~82 .~Y~mp~ tr~filtr~tinn .~ lbl.li-e may be used for the removal of high molecular weight con~fituent~ or an affinity membrane,.(e.g., protein-A mçmhr~nPs) for theremoval of IgG or lectin-membranes for removal of carbohydrates or membranes with a specific antibody directed against biopharm~euti~l product to remove the S great excess of product for i~ y analysis for process and quality control.

CA 02215974 1997-09-19 ..
W 096/29595 PCTnUS9610382B

EXAMPLE I
This eY~mrl~ s .~r~tion and detection of Msp I pBR322 fr~gm~nt~
under the following con~1ition~.
SEPARATOR
BREADBOARD: SEPARATION PLATE AS SHOWN IN
FIG. 9.
CAPILLARIES
TYPE: 30 MICRON ID FUSEDSILICA
DERIVATIZED WITH 3.5%
LINEAR POLYACRYLAMIDE.
LENGTH: 109MM
WINDOW LOCATION: 10-100 MM; BARE SILICA
CLEANING PROCEDURE FLUSHED WITH WATER, THEN
BUFFER
SAMPLES: Msp I pBR322 DNA
5 uG/ML IN 0.5 X TBE
LOADED 5 IN CYLINDRICAL ~ LTM
WELL FOR INJECTION
DETECTOR: NIKON EPI-FLUORESCENCE
MICROSCOPE
PTI ANALOGUE PM SYSTEM
GAIN: 0.01 ,uA/VOLT
TIME CONSTANT: 50 MSEC
PMT VOLTAGE: 1000V
LAMP XENON
IRIS: Open N.D. FILTERS: None FILTER SET: G2A CUBE (ETBR): (Dich 580 nm, Exc 510-560 nm, Em 590 nm) FOCUS & SLITS: focus on inner bore, set slits +25 ,um on either side of bore diameter DETECTOR POSITION: 45 mm scan of capillary along x axis using detection system illustrated in Figure 11.
OBJECTIVE: 10X
DATA SYSTEM: PE NELSON MODEL 1020 DATA COLL. RATE: 20-40 HZ
BUFFERS
SAMPLE: 0.5 X TE

-CAPILLARY (pre load): STOCK NUCLEOPHORTM BUPFER + 2.5 ,ug/mL ethi~ m bromide END CHAMBERS: At ground: ethi~ium bromide sieving buffer, not plugged (vented to atm pres.); at Sample S end: 2.5 ug/ml ethitlil-m bromide in TBE
electrolyte INJECTION
METHOD: Electrokinetic, capillary c~ccette in horizontal position TIME: 10 sec VOLTAGE: 3.33k~
SAMPLE REMOVAL: Removed TefzelTM cylindrical well; flushed well; refilled with buffer SEPARATION RUN
VOLTAGE: 3.33 kV; run for ~ 90 sec; capillary ç~ette in horizontal position CURRENT: 1.8 ~A measured by hand-held m--ltim~ter POLARITY: Negative at injecti- n end; detector near ground.
BUFFER: Std NucleophorTM buffer + 2.5 ~g/ml EtBr DETECTION: Auto scan from x = 190000 to x = 26000 via Cell Robotics SmartstageTM;
('~pill~ry in focus +/-5 microns across sc~nned length. Focus also intentionally misadjusted 1/2, 1, and 2 turns for scan at 500 ,~m/sec. scans.
Figure 12 illustrates separation of the DNA fr~gm~n~c, E:XAMPLE 2 This eY~mple iliustrates the separation of a series of single-stranded oligonucleotides that differ by the addition of a single base to the previous oligo.
The sampie consists of a pdAlo fragment which was labeled with a fluorophore (FAM) on the 5' end. The S'FAM-pdA,o fragments were then enzym~tie~lly extended with terminal transferase by addition of dATP onto the 3' end of the fragments. This process gave a (~nCci~n distribution of 5'FAM-pdA~ fragments, -W 096~9595 PCTrUS96103828 with X ranging from ~ 20 to 75. Separation of this sample mimics a DNA
seqllen-~ing separation in that single-stranded DNA fr~gm~-ntc that differ by 1 base are se~ A and detect~ by fluorescen~e detection.

S ~pill~ry: 10 cm length 100 ~m id window at 7.5 cm, internally coatedwith linear polyacrylamide, po~ition~i in separation plate illustrated in Figure 1.
Buffer: Solution of 10% W/v linear polyacrylamide in lX TBE (89 mM
Tris, 89 mM borate, 2mM EDTA) plus 7M urea loaded into the capillary via syringe.
Anode/Cathode Reservoir Buffer: lX TBE, 7M urea Injection: 10 second at 2 kV electroinjection at cathode end.
Separation: 2 kV (- 12,uA current) const. potential Sample: 50 nM total DNA; average of 1 nM each fr~gm~ont Detection: Static; fluorçscence; 470 - 490 nm excitation > 520 nm emission using detection scheme illustrated in Figure 11, but with static detection.
Slit: 110 ,um x 20 ,um, positioned 7.5 cm from injection end.
The results are shown in Figure 13.

This example illustrates the separation of some non-standard samples of DNA. The s~mples were obtained by amplification of human genomic DNA
samples via the PCRTM (polymerase chain reaction) process. S~mples were not purified prior to use. Samples were diluted with a predetermined concentration of . 35 a r~lihr~ticn standard which c~nt~in~ PCRTM fr~gm~nt~ of known sizes of 50, 100, 200, 300, 400, 500 . . . 1000 bp. The calibration standards were obtained from Bio-Synthesis, Inc., Louisville, TX. .S~mples were diluted by 25 ~ into the calibration W 096129595 PcTlu~6l?3x28 standard and .sep~t~d under the following conditions:

W O 96/29595 PCT~US96/03828 ill~ry: 9.2 cm length 30~Lm id 4% linear polyacrylamide coated, window at 7.0 cm from r~thode end, po~itionP~ in an electrophoresis illnstr~t~l in Figure 1.
S Buffer: Nu~ l.. ,~ sieving buffer. (Dionex Corp., Sunnyvale, CA) + 2.5 ~Lg/mL ethir1inm bromide; loaded into capillary via syringe.
~tho~le Buffer: lX TBE (89 mM Tris, 89 mM borate, 2 mM EDTA) + 2.5 ,ug/mL ethidium bromide.
Injection: 10 sec. at 3 kV electroinjection at cathode.
Separation: 3 kV constant potential.
Detection: Fycit~tinn - 510-560 nm.
Emission - > 590 nm, using the static detection scheme illustrated in Figure 11.
The ~ ;~d size of this DNA sample was 211 bp. The size as determined from the separation shown in Figure 14 was 210 bp.

DNA Seq~enl~in~
25A 96 multi-well plate with ternrl~tes 1-8 to be sequenced placed in all rows of ~ ,ec~ e lanes. Primers 1-8 are in all rows of respective lanes, and all 4 bases are in all 96 positions.
The above alTay is sipped into the Base Plate. For sequencing, it is desirable to ~im~llt~n~oously sip four samples and mix those samples prior to sequencing. For 30e ~m~ , with reactions involving four separate reactions with color coded primers, place base Plate with cover into instrument station which can control ~r~ssule within the Base Plate, control tt~ dtllre, and automatically add reagents to the top of the , W 096t29S95 PCTrUS96/03828 Base Plate at the various positions.
Sim-llt~nPously add the polymerase to all 96 positions and start the ul~/vacuum cycling to mix at the membranes and to start the chain eYt~Pn~ion re~ction, S With an eight-by ~i~Uel, add fluorescently labeled, chain eYten~iQn Lt~ Jl~i (for all 4 bases, color coded), to all 8 positions of the first row. Chain extension ends in these positions, producing fragments ext~Pn~ling from 10 (allowing for the primer) to 120 bases, for all 8 templ~tPs After a predetermined period of time (m~tc,heA to the polymerization rate, controlled by buffer con-litic nc), the same l~l.. i.. alor mix is added to the second row of eight lanes. Chain extension ends in the second row, producing fragments extending from 80 to 220 bases from the starting point, for all 8 templates. Thestagger and overlap of fragment sizes is determined by the time interval betweenterminator additions, the concentration of polymerase and bases, and the buffer conditions. The stagger of fragments may be obtained by ch~nging the relative concentration of terminators versus bases at the various rows, or by starting all polymPri7~tion reactions simultaneously and adding an enzyme-stopper at various time points).
~c~r1ition~ of ~l...ina~ol~, at predetermined time intervals is continued for all rows.
The double stranded DNA is melted and injected and then s~aldled on the respective capillaries.
~ach separation plate (at a given row) will have on-board sieving buffer CA 02215974 1997-09-l9 W 096/29595 PCTrUS96/03828 optimi7e~ for the fr~gment size range for that row which is nominally a range ofabout 100 bases. Therefore re~uc-in~ the relative size resolution required to obtain single base se~a.dlion.
At the end of the entire ~lucess, one has simultaneously sequenced 8 ~,.. ~ s, each for about 1200 bases, in about 30 minllt~s Those skilled in this also know how to re-prime and continue the process.
The m-otho l~ and a~dldllls of this invention have many advantages for DNA
sequencing. The primary advantages of this sequencing method is that one obtainslong read lengths by co,--b;.~ g continuous (or overlapping) read windows. Hence, within each row of 8 capillaries, one needs single base resolution for a defined read window (e.g. from 490 to 610 bases). Therefore, the sieving buffer for each row of 8 c~pill~ri~-s, on a given electrophoresis plate, can be optimized for the particular read window.
Additional advantages are that the sequencing throughput is increased by 3 to 10 fold over current m~.tho 1c and that small volumes of template and reagents are r~uil~ d. The main reason for the higher throughput is that s~dtion and reading is done simultaneously for a large number of short capillaries.
Finally, though this sequencing method is ideally suited to the devices of this invention, those experienced in the art of sequencing will realize that this method - 20 may be practiced on other devices such as standard gel-based DNA separation systems.

=
CA 0221F,974 1997-09-19 WO 96/29595 PCl/u~ x Method for I'~leill Binding Assay of F,nl~r~ lin An~ c ~pill~ry 31 ~m ID fused silica, 10 cm mounted in a separation plate shown in Figure 1. Wash at begi..l-;n~ and end of day with phosphate buffer and water (5 min).
S M~t.-.n~lc Run buffer was 62.5 mM sodium phosphate, pH 8.5 with 0.01% (BSA)bovine serums albumin. T ~hel (F-l 1, YGGFLTSEK(-fluorescein)SQ (TANA Labs, ~cu~tc-n, TX), CO~ or (YGGFLK - American Peptide Co., Sunnyvale, CA) and F~b. monoclonal antibody fragment (Gramsch Labs Schwabhausen Germany) were diluted in Run Buffer. Antibody and label were mixed at a concentration of 12.5 nM
each; this typically added to competitor withm 5 minutes after mixing. Competitor was diluted to several concentrations. Add 40 ,L~l of Ab/label mixture to 10 ,ul of competitor and incubated 10 minutes before assaying. Mix by pumping action of pipet. These mixtures were stable for several hours at room temperature, if protected from evaporation and were held in the dark. Final concentrations were:lO nM F"b., 10 nM F-ll, and 0, 20, 40 or 200 nM for the colllpelitoi.
Tnj~tinn- The injection area of the separation plate was rinsed with about 1 ml run buffer and all liquid removed. Using a micl~ipet, 4 ,ul of sample was delivered to the area between the capillary end and (sample well) the electrode. Sample was introduced into the capillary via electrokinetic injection for 10 seconds at 2.3 kV at the anode, at the end of which the injection area was flushed with 4 drops of run buffer to remove residual sample.
.~Pr~r~ti~n: Voltage was set at 3 kV.
net~tinn: Laser light source; PMT; gain set at 10-l, 500 msec; detector window at W O 96129595 PCTrU~

6 cm from iniection Argon ion laser, 488 nm eYcit~tion. Fluorescein was ~ete~tçdwith a long pass filter above 520 nm.
An~ly~i~ . Two areas in the fluo,~,scent electrophelogldm measured as shown in Figure 15.
Area i - under the fre label peak.
Area 2 - total fluorescen~e area, including the complex, free label, and fluorescence between the two peaks due to label coming off the Ab during migration.
Area 1/Area 2 is plotted vs. [colll~~LiLol] as shown in Figure 16.

EXAMPLE ~i Proce~lu~ for DNA Detection Limit Breadboard:
('~pill~ry 10.1 cm of 30 ~m id fused silica, covalently coated with polymeric layer of polyacrylamide; window at 6. 6 cm from cathrode end, and positioned in a separation plate as illustrated in Figure 1.

Anode/Capillary Buffer: NucleophorTM sieving buffer (Dionex, Sunnyvale, CA) plus 2.5 ,ug/ml ethidium bromide.

Cathode buffer: lX TBE (89.5 mM Tris, 89.5 mM borate; 2.0 mM
EDTA, pH 8.3) plus 2.5 ,~Lg/ml ethidium bromide.
Detector: as illustrated in Figure 13, run in the static detectic-n mode.
excitation 510-560 nm emission > 590 nm Injection Run: 30 sec at 3.0 kV @ cathode W O 96/29595 PCTfUS96/03828 3.0 kV constant voltage .~mrl~ 0.033 ,ug/ml Haelll digest of 0X 174 DNA (Sigma Ch~mic~l CO., St. Louis, MO) in water Figure 17 shows that 12 picogram or 14 attomoles of 1353 bp DNA can be dete~te~ and that 2-3 picograms or 14 attomoles of 310 bp DNA can be ~etectecl Figure 18 illustrates the efficiency of mixing very small volumes of reagents as delsr-rihed in Figures 7a-e. Thus, 1.9 ,~1 of a dye (xylene cyanol) was mixed with 1.9 ,ul of water and the mixed sample was passed back and forth through the membrane ~n for 0.4, 2.1 or 4.2 minutes.

This eY~mrle d~,-,o~ .c a combined sample sipping, sample pres~nt~ti~ n, sample injection, separation, and sc~nnin~ detection of s~y~ d sample co,ilponents.

~Sirrt?r/Mix~,r Sipper: 2.5 cm lengths of 538 micron id fused silica capillary in a polycarbonate plate Mixing Block: 764 micron x 16 mm PEEK channels in polycarbonate block Top Plate: polycarbonate Membrane: 0.45 Micron pore membrane Volume Speed: 7.30 microliters from array of PCRTM tubes, each with 30 CA 022l5974 l997-09-l9 W 096/29595 PCT~US96/03828 microliters sample Breadboard: six çh~nn~l~ with 6 p~ m wire electrodes at injection end C~ ries Type: 100 micron id fused silica derivatized with 4% linear polyacrylamide coating Length: 109 mm Window Location: 90 mm Total: +/- 45 mm from center point of capillaries ning Procedure: Flushed with water, then sieving buffer solution from syringe Samples: Haelll digest of 0Xl74 DNA from Sigma (~hemir.~l Co. 10 microgram/mL in 0.5% TE buffer.
30 microliters of sample were loaded into each of 6 500 mL
tubes as primary sample array Detector Optical Lamp: 75 watt Xenon Iris: 1/8 opened N.D. Filters: NDl and ND2 out Gain: 0.1 microamp/volt Time Constant: 50 ~illi.c~onds PMT
Voltage: 850 Volts Filter Set: G2A cube (ETBR):(Dichroic 580 nm, Exc. 510-560 nm~ EM. 590 nm) Focus & Slits: Focus on inner bore of capillary; Slits 150 X 20 microns centered over inner bore Objective: 10x Scanner: Cell Robotics SmartstageTM scanner Scan Speed: 1760 Microns/Sec X Scan Range: X =.33000 to X =.36000 microns Buffers W 096/2959S PCTrUS96/03828 '46-.S~mplf~ O.SX TE (5 mM TRIS, pH 7.5, 1 mM EDTA) C~pill~ry: Stock NucleophorTM buffer (Dionex CO1~ ation) + 2.5 micrugl~,./mL eth~ lm bromide Common S Reservoir: Stock Nuclec,phoiT~ buffer (Dionex Co-~o-a~ion) + 2.5 microgram/mL ethidium bromide Array Reservoirs: 1 x TBE (89.5 mM tris base; 89.5 mM boric acid, 2 mM
EDTA, pH 8.3) + 2.5 microgram/mL ethi~lil-m bromide Power Supply: Bertan, modified with timer/controller Polarity: Negative Injection Method: Electrokinetic, from s~mpling plate on horizontal Duration: 15 seconds Voltage: 3.3 kV
Sample Removal: Moved electrophoresis plate to transfer capillaries from sample wells to run buffer wells Separation Run Voltage: 3.3 kV
Duration: 120 seconds Current: 156 microamps total for the 6 capillaries Detection: sequential scan of the 6 individual capillaries at 1770 microns per second Data System: PE/Nelson Model 1020 Input: 10 Volts fulls scale Sampling Rate: 20 Hertz Thus, simultaneously sipping an array of six samples, simultaneously pres.-nting the s~mple~ through the sample handling block, simultaneously electroinjecting the ~lc;s~n~d .c~mpltoc into the capillaries, simultaneously conducting the electrophoresis following by analysis is demonstrated. Figure 14 illustrates the separation and reproduceability of separation of thç system for DNA sequences W 096/29S95 PCTnUS96/03828 having a slze between 72 and 310. base pairs.
The above ~y~mples are int~ndecl to illustrate the present invention and not to limit it in spirit and scope.

Claims (45)

WHAT IS CLAIMED IS:

1. A separation plate comprising:
a) a frame having first and second ends and having an array of separation capillaries with first and second ends mounted in the frame;
b) the first end of the frame having at least one buffer reservoir and at least one electrode in the buffer reservoir, wherein the first ends of the capillaries are in liquid communication with the buffer reservoir; and c) the second end of the frame having means for placing the second ends of the capillaries in contact with an array of samples or run buffer which are in contact with electrodes, and wherein there is liquid communication between the buffer reservoir at the first end of the frame and the array of samplesor the run buffer at the second end of the frame through the separation capillaries and there is electrical communication between the electrode in the buffer reservoir at the first end of the frame and the electrodes in the array of samples or run buffer at the second end of the frame by way of the separation capillaries.

2. A separation plate according to claim 1, wherein the plate contains 8, 12, 16, or 24 separation capillaries spaced apart to match the spacing format in a row or column of 8, 12, 16, or 24 wells in a 96, 192, or 384 multi-well plate.

3. The separation plate of claim 1, wherein electrodes are mounted in the frame near the second end of the capillaries so that when the second end of the capillaries are in the samples or in the run buffer at the second end of the frame there is electrical communication between the electrode in the buffer reservoir and the electrodes near the second end of the capillaries.

4. The separation plate of claim 1, wherein the electrodes in the buffer reservoir at the first end of the separation plate and the electrodes in the samples or run buffer at the second end of the separation plate are plated or coated onto the exterior of the separation capillaries

5. The separation plate of claim 1, further comprising a buffer-filled storage vessel supplying run buffer for replenishing the buffer reservoir at the first end of the separation plate, for flushing and refilling the capillaries, and for supplying run buffer to the second end of the capillaries in the separation plate.

6. An apparatus for processing an array of samples containing two or more components in an array of sample containers comprising:
a) a sample handling plate which defines an array of sample handling plate wells and an array of sipper capillaries having an inner wall, an outer wall, a distal end, a proximal end, an inner bore diameter and an outer bore diameter, wherein each well of the sample handling plate is in liquid communication with at least one sipper capillary and wherein a porous matrix is interposed between each sipper capillary and the corresponding sample handling plate well, and wherein the array of sipper capillaries will simultaneously fill with sample by capillary action from the array of sample containers;
b) a base plate which defines an opening to receive the sample handling plate and which defines an inner chamber with means associated with the base plate and the sample handling plate to seal the inner chamber and to provide a sealed inner chamber; and c) means for pressurizing and evacuating the sealed inner chamber to move liquid on either side of the porous matrix to the other side of the porous matrix.

7. The apparatus of claim 6 wherein the sipper capillaries of the sample handling plate are in an array which conforms to the spacing format of a 96, 192, or 384 multi-well plate.

8. The apparatus of claim 6, wherein the wells of the sample handling plate conform to the spacing format of a 96, 192, or 384 multi-well plate.

9. The apparatus of claim 6 wherein the means for pressurizing and evacuating is programmed to alternately pressurize and evacuate the sealed inner chamber to mix fluids above the porous matrix with sample below the porous matrix.

10. The apparatus of claim 6 wherein there is provided a run buffer well adjacent to the sample handling plate wells.

11. The apparatus of claim 6 wherein the porous matrix is a membrane.

12. The apparatus of claim 6 wherein the porous matrix is a bed of sintered polymeric particles.

13. The apparatus of claim 6, wherein the porous matrix contains a specific binding agent which removes at least which removes at least one component from the sample.

14. The apparatus of claim 6, wherein the porous matrix is a filtration membrane which removes insoluble material from the sample.

15. The apparatus of claim 6, wherein the porous matrix is an ultrafiltration membrane which passes low molecular weight components membrane and retains high molecular weight components.

16. The apparatus of claim 6, wherein each sipper capillary has a stop junction on the inner wall of the sipper, said stop junction being situated a defined distance from the proximal end of the sipper capillary, said stop junction being formed by an abrupt increase of the inner bore diameter of the sipper capillary, and the distance from the proximal end of the sipper capillary to the stop junction effectively defining the length of the sipper capillary that can be filled with sample by capillary action when the proximal end of the sipper capillary is immersed in the sample.

17. The apparatus of claim 6, wherein each sipper capillary has a stop junction on the inner wall of the sipper, said stop junction being situated a defined distance from the proximal end of the sipper capillary, said stop junction being formed by an abrupt change in the wetting properties of the inner bore diameter of the sipper capillary, and the distance from the proximal end of the sipper capillary to the stop junction effectively defining the length of the sipper capillary that can be filled with sample by capillary action when the proximal end of the sipper capillary is immersed in the sample.

18. The apparatus of claim 6, wherein the sipper capillaries are made of plastic with inner walls sufficiently hydrophilic to exhibit capillary action.

19. The apparatus of claim 18, wherein a portion of the inner walls of the sipper capillary are coated with a surfactant.

20. The apparatus of claim 18, wherein a hydrophilic polymer is grafted on to a portion of the inner walls of the sipper capillary.

21. The apparatus of claim 18, wherein a portion of the inner walls of the sipper capillaries is subjected to a plasma etch treatment.

22. A separation plate comprising:

a) a frame having a buffer reservoir at one end and a plurality of sample sites at the other end and having an electrode at each end; and b) a means for mounting separation capillaries on the frame so that there is electrical communication between the electrodes and fluid communication between the sample sites and the buffer reservoir when there is fluid in the sample sites, the separation capillaries, and the buffer reservoir.

23. A system for analysis of an array of samples in an array of sample containers comprising:
a) means for simultaneously transferring at least a portion of each sample in the array of sample containers to a corresponding array of separation capillaries, wherein the separation capillaries are in the separation plate of claim 1;
b) means for simultaneously conducting electrokinetic separations on the array of transferred samples in the separation capillaries; and c) means for analyzing the separations from (b).

24. A system according to claim 23, wherein the array of samples conforms to the spacing format of a 96, 192, or 384 multi-well plate.

25. A system for analysis of an array of samples in an array of sample containers comprising:
a) means for simultaneously acquiring an aliquot of each sample from the array of sample containers to form an aliquot array of samples;
b) means, in combination with means (a), for simultaneously processing the aliquot array of samples to provide an array of processed samples and for presenting the array of processed samples for electrokinetic analysis;
c) means for simultaneously transferring at least a portion of each sample in the array of processed samples to an array of separation capillaries;
d) means for simultaneously conducting an electrokinetic separation on the array of transferred processed samples in the array of separation capillaries from (c); and e) means for analyzing separations from (d).

26. A system according to claim 25, wherein the array of samples conforms to the spacing format of a 96, 192, or 384 multi-well plate.

27. An assembly of the separation plates, consisting of at least two separation plates of claim 1 in which the separation capillaries are arranged to conform to both the row and column spacing format of a 96, 192, or 384 multi-well plate.

28. An assembly of the separation plates of claim 27, wherein at least one different run buffer is used in at least one buffer reservoir.

29. An apparatus comprising:
a) a frame having first and second ends and having an array of separation capillaries with first and second ends mounted in the frame, the first end of the frame having at least one buffer reservoir and at least one electrode in the buffer reservoir, wherein the first ends of the capillaries are in liquid communication with the buffer reservoir and the second end of the frame having means for placing the second ends of the capillaries in contact with an array of samples or run buffer; and b) the apparatus of claim 6, wherein the buffer reservoir of (a) and the sample handling plate well of the apparatus of claim 6 are horizontally disposed vessels with one end of each vessel at least partially closed off and the vessels possess comparable or identical cross-sectional geometries, possess comparable or identical surface contact angles or wetting properties and are positioned such that the buffer reservoir of (a) and the sample handling plate well of the apparatus of claim 6 are coaxial relative to each other.

30. A method for analysis of an array of samples in an array of sample containers comprising;
a) providing an array of samples in an array of sample containers;
b) simultaneously transferring at least a portion of each sample in the array of sample containers to a corresponding array of separation capillaries wherein the separation capillaries are in the separation plate of claim 1;
c) simultaneously conducting electrokinetic separation of the transferred samples;
and d) analyzing the separations of (c).

31. The method of claim 30, wherein the array of samples is nucleic acids of varying length.

32. The method of claim 30, wherein the array of samples is a specifically binding biomolecule, its binding partner or the complex of the two.

33. The method of claim 30, wherein the array of separation capillaries is rinsed for reuse to separate additional samples.

34. A method for analysis of an array of samples in an array of sample containers comprising;
a) providing the array of samples in an array of sample containers;
b) simultaneously acquiring an aliquot of each sample from the array of samples with the sample handling plate of claim 6;
c) transferring the sample handling plate to a base plate which provides for simultaneously processing and then presenting the samples for electrokinetic separation;
d) simultaneously transferring at least a portion of the presented samples to a corresponding array of separation capillaries wherein the separation capillaries are in the separation plate of claim 1;
e) simultaneously conducting electrokinetic separation of the transferred samples;
and f) analyzing the separations of (e).

35. The method of claim 34, wherein the array of samples is nucleic acids of varying length.

36. The method of claim 34, wherein the array of samples is a specifically binding biomolecule, its binding partner or the complex of the two.

37. The method of claim 34, wherein one or more fluids are added to the sample handling plate wells and mixed with the samples.

38. The method of claim 34, wherein the array of separation capillaries is rinsed for reuse to separate additional samples.

39. An apparatus for mixing two volumes of fluid consisting of:
a) two chambers for containing the volumes of fluid separated by an interposed porous matrix and;
b) means for moving the fluid from one side of the porous matrix through the porous matrix to the other side of the porous matrix, thereby mixing the two volumes of fluid.

40. The apparatus of claim 39, wherein the porous matrix is a membrane.

41. The apparatus of claim 39, wherein the porous matrix is a bed of sintered polymeric particles.

42. The apparatus of claim 39, wherein the porous matrix contains a specific binding agent which removes at least which removes at least one component from the sample.

43. A method of mixing two volumes of fluid by passage through a porous matrix having two sides consisting of:
a) positioning a first fluid in a fluid chamber on a first side of a porous matrix;
b) displacing the first fluid to the second side of the porous matrix;
c) positioning a second fluid volume in the fluid chamber on the first side of the porous matrix;
d) displacing the first fluid to the second side of the porous matrix in such a way as to contact the first fluid; and e) passing the contacted fluid volumes through the porous matrix at least one time to mix the first fluid volume with the second fluid volume.

44. The apparatus of claim 43, wherein the porous matrix is a membrane.

45. The apparatus of claim 43, wherein the porous matrix is a bed of sintered polymeric particles.