Microfabricated capillary array electrophoresis device and method

A capillary array electrophoresis (CAE) micro-plate with an array of separation channels connected to an array of sample reservoirs on the plate. The sample reservoirs are organized into one or more sample injectors. One or more waste reservoirs are provided to collect wastes from reservoirs in each of the sample injectors. Additionally, a cathode reservoir is also multiplexed with one or more separation channels. To complete the electrical path, an anode reservoir which is common to some or all separation channels is also provided on the micro-plate. Moreover, the channel layout keeps the distance from the anode to each of the cathodes approximately constant.

BACKGROUND OF THE INVENTION 
This invention relates to electrophoresis generally, and more particularly, 
to an apparatus and method for performing capillary array electrophoresis 
on microfabricated structures. 
In many diagnostic and gene identification procedures such as gene mapping, 
gene sequencing and disease diagnosis, deoxyribonucleic acid (DNA), 
ribonucleic acid (RNA) or proteins are separated according to their 
physical and chemical properties. In addition to DNA, RNA or proteins, 
other small molecule analytes may also need to be separated. 
One electrochemical separation process is known as electrophoresis. In this 
process, molecules are transported in a capillary or a channel which is 
connected to a buffer-filled reservoir. An electric field in the range of 
kilovolts is applied across both ends of the channel to cause the 
molecules to migrate. Samples are typically introduced at a high potential 
end and, under the influence of the electric field, move toward a low 
potential end of the channel. After migrating through the channel, the 
separated samples are detected by a suitable detector. 
Typically, electrophoretic separation of nucleic acids and proteins is 
carried out in a gel separation medium. Although slab gels have played a 
major role in electrophoresis, difficulties exist in preparing uniform 
gels over a large area, in maintaining reproducibility of the different 
gels, in loading sample wells, in uniformly cooling the gels, in using 
large amounts of media, buffers, and samples, and in requiring long run 
times for extended reading of nucleotides. Moreover, slab gels are not 
readily amenable to a high degree of multiplexing and automation. 
Recently, micro-fabricated capillary electrophoresis (CE) devices have 
been used to separate fluorescent dyes and fluorescently labeled amino 
acids. Additionally, DNA restriction fragments, polymerase chain reaction 
(PCR) products, short oligonucleotides and even DNA sequencing fragments 
have been effectively separated with CE devices. Also, integrated 
micro-devices have been developed that can perform polymerase chain 
reaction amplification immediately followed by amplicon sizing, DNA 
restriction/digestion and subsequent size-based separation, and cells 
sorting and membrane lysis of selected cells. However, these 
micro-fabricated devices only perform analysis on one channel at a time. 
For applications such as population screening or DNA sequencing, such a 
single channel observation and analysis results in an unacceptable delay 
for screening many members of a population. 
SUMMARY OF THE INVENTION 
The invention provides a capillary array electrophoresis (CAE) micro-plate. 
The micro-plate has an array of separation channels connected to an array 
of sample reservoirs on the plate. The sample reservoirs are organized 
into one or more sample injectors. A waste reservoir is provided to 
collect wastes from sample reservoirs in each of the sample injectors. 
Additionally, a cathode reservoir is multiplexed with one or more 
separation channels. An anode reservoir which is common to some or all 
separation channels is also provided on the micro-plate. Moreover, the 
distance from the anode to each of the cathodes is kept constant by 
deploying folded channels. The corners on these turns may be right angle 
turns or more preferably, smooth curves to improve electrophoretic 
resolution. 
In one aspect, the reservoir layout on the substrate separates the sample 
reservoirs by a predetermined spacing to facilitate the simultaneous 
loading of multiple samples. 
In another aspect, cathode, anode and injection waste reservoirs are 
combined to reduce the number of holes N in the substrate to about 5/4N 
where N is the number of samples analyzed. 
In another aspect, the separation channels are formed from linear segments. 
In another aspect, the separation channels are formed from curvilinear 
segments, which may include radial segments. 
In yet another aspect, the separation channels span from the perimeter of 
the plate to the central region of the plate. The separation channels may 
span the plate in a linear or a radial fashion. 
In yet another aspect, a CAE micro-plate assembly is formed using a 
micro-plate, a reservoir array layer, and an electrode array. The assembly 
simplifies sample handling, electrode introduction and allows an increased 
volume of buffer to be present in the cathode and anode reservoirs. 
Advantages of the invention include the following. The micro-plate of the 
present invention permits analysis of a large number of samples to be 
performed at once on a small device. Moreover, the micro-plate allows 
samples to be easily loaded while minimizing the risk of contamination. 
Additionally, the micro-plate is easy to electrically address. Further, 
the micro-plate supports a wide variety of formats that can provide higher 
resolution separation and detection of samples, faster separation and 
detection of samples, or separation and detection of more samples. 
Other features and advantages will be apparent from the following 
description and the claims.

DESCRIPTION 
Referring now to FIG. 1, a capillary array electrophoresis (CAE) 
micro-plate 10 is shown. The micro-plate 10 has an array of capillaries or 
separation channels 50 etched thereon. In one embodiment of FIG. 1, 48 
individual separation channels are etched in a 150 micron (.mu.m) periodic 
array. In this embodiment, the separation channels 50 branch out to an 
8.times.12 array of sample reservoirs 101, each of which is spaced a 
predetermined distance apart to facilitate loading with an 8-tipped 
pipetter. In this case, each sample reservoir 101 is spaced in one 
dimension nine millimeters apart from another sample reservoir. The 
separation channels 50 extend by a first predetermined distance from an 
injection region to an anode reservoir 180 and by a second predetermined 
distance from an injector group 100 to a cathode reservoir 120. The first 
predetermined distance may be about 10 centimeters, while the second 
predetermined distance may be about 1.8 centimeters. 
Each of the sample reservoirs 101 belongs to an injector group such as one 
of injector groups 100-116. Additionally, injector groups 100, 102 and 104 
are connected to a cathode reservoir 120. Although the cathode reservoir 
120 is connected to three sample injectors 100, 102 and 104, other cathode 
injectors may be connected to more than three sample injectors. For 
instance, a cathode injector 130 is connected to sample injectors 106, 
108, 110, 112, 114 and 116. 
The anode reservoir 180 is placed in a non-symmetrical manner in this case 
to avoid a conflict with a scanning system. Moreover, the distance for 
paths from the anode reservoir 180 to any one of cathodes 120 or 130 is 
identical for all separation channels. The equal distance is achieved by 
providing folded paths connecting certain sample reservoirs that are close 
to the anode 180 to increase the path length and to achieve a uniform 
distance between the anode reservoir 180 and the cathode reservoirs 120 
and 130 for all sample reservoirs. 
In the embodiment of FIG. 1, the number of holes H in the micro-plate 10 is 
about 5N/4, and more exactly, 5N/4+7, where N is a number of samples. As 
the embodiment of FIG. 1 addresses 96 samples in parallel, 127 holes are 
required to be drilled. This number of holes is close to a theoretical 
minimum number of holes of N+3. The reduction in hole counts is 
advantageous as fewer holes need to be drilled into the micro-plate 10, 
thereby increasing manufacturing efficiency as well as decreasing the 
potential for defects in the production of micro-plates, as caused by 
mechanical stress associated with the drilling process. Another reason for 
multiplexing the cathode, anode and waste reservoirs is to make it more 
feasible to fit 96 separation system on a single substrate. The above 
advantages are also applicable in the event that the holes are formed by a 
molding process or a bonding process in lieu of the drilling process. 
Turning now to FIG. 2, details of the sample injector 100 of FIG. 1 are 
shown. The sample injector 100 has a plurality of sample reservoirs 200, 
204, 220 and 224. Sample reservoirs 200 and 220 contain a first sample, 
while sample reservoirs 204 and 224 contain a second sample. 
The sample injector 100 also has a first separation channel 202 and a 
second separation channel 222. The sample injector 100 thus permits a 
serial analysis of two different samples on each separation channel. The 
first and second separation channels 202 and 222 are connected to a waste 
reservoir 208 by a cross channel 207. The sample injector 100 also has a 
cathode end 210 as well as an anode end 212. The cathode and anode ends 
210 and 212 are at opposite ends of the first separation channel 202. 
Similarly, a second cathode end 214 is connected to a second anode end 216 
by a separation channel 222 that is connected to the waste reservoir 208. 
As illustrated below, by a proper biasing of the anode reservoirs 211 and 
212, cathode reservoirs 200 and 214, sample reservoirs 200, 204, 220, 224, 
and waste reservoir 208, samples may be moved from their respective sample 
reservoirs 200, 204, 220 and 224 through the cross channel to the waste 
reservoir thereby facilitating an insertion into the separation channel. 
Referring now to FIGS. 3A, 3B, 3C and 3D, a process for loading a sample 
from its respective sample reservoir into the cross channel and then 
performing a separation is shown. In FIG. 3A, an injection voltage, 
preferably about 300 volts (3.0 V/cm), is applied between the sample 
reservoir 200 and the injection waste reservoir 208 to draw a sample 
through a channel that passes from the sample reservoir to the waste 
reservoir and crosses the separation channel. 
In FIG. 3B, a separation voltage of about 3700 volts (300 V/cm), for 
example, is applied between the cathode end 210 and the anode end 212. 
This causes the electrophoretic separation of the sample. In addition, a 
back-bias of the potential between the sample reservoir 200 and the 
injection waste reservoir 208 is applied. Preferably, the back biasing 
voltage is about 720 volts. The back-biasing operation clears excess 
samples from the injection cross-channel 213. As illustrated in FIG. 3B, a 
100 .mu.m sample plug is injected and any residual sample is pulled away 
from the injection region to avoid tailing side-effects. 
FIGS. 3C and 3D represents analogous injections from the second sample 
reservoir 204. Although the embodiment of FIGS. 2 and 3A-3D operates on 
two samples, four samples may be injected onto a single capillary without 
any significant cross-contamination. 
The process of etching patterns into a representative micro-plate is 
discussed next. In one microfabricated embodiment, Borofloat glass wafers 
available from Schott Corporation of Yonkers, N.Y. are pre-etched in 49% 
HF for 15 sec and cleaned before deposition of an amorphous silicon 
sacrificial layer of about 1500 .ANG. in a plasma enhanced chemical vapor 
deposition (PECVD) system. The wafers are primed with 
hexamethyldisilazane, spin coated at 5000 rpm with a photoresist such as a 
1818 photoresist available from Shipley Corp. of Marlborough, Mass. The 
photoresist is developed in a 1:1 mixture of Microposit developer 
concentrate available from Shipley and water. The wafers are then 
soft-baked at 90.degree. C. for 30 minutes. The mask pattern is 
transferred to the substrate by exposing the photoresist to ultraviolet 
radiation in a Quintel contact mask aligner. The mask pattern is 
transferred to the amorphous silicon by a CF.sub.4 plasma etch performed 
in the PECVD reactor. The wafers are etched in a 49% HF solution for about 
3 minutes at an etch rate of 7 .mu.m/min to form a final etch depth of 21 
.mu.m and channel width of .about.60 .mu.m at the bonded surface. The 
photoresist is stripped and the remaining amorphous silicon is removed in 
a CF.sub.4 plasma etch. Holes are drilled into the etched plate using a 
1.25 mm diameter diamond-tipped drill bit, available from Crystalite 
Corporation of Westerville, Ohio. The etched and drilled plate is 
thermally bonded to a flat wafer of similar size and type in a 
programmable vacuum furnace. After bonding, the channel surfaces are 
coated using a coating protocol. 
Turning now to FIGS. 4A and 4B, an exploded view and a cross-sectional side 
view of a CAE micro plate are shown. In FIG. 4A, a CAE micro-plate 302 
with etched separation channels 301 and a plurality of reservoirs 303 
formed thereon is provided. A reservoir array layer 304 is mounted above 
the CAE micro-plate 302 to provide additional reservoir space above the 
reservoirs formed on the micro-plate 302. The presence of the reservoir 
array layer 304 increases the volume of buffers in the cathode and anode 
reservoirs and simplifies sample handling and electrode introduction. 
Preferably, the reservoir array layer 304 is a one millimeter thick 
elastomer sheet which makes a watertight seal when it is in contact with 
the glass micro-plate 302. The reservoir array layer 304 may be an 
elastomer such as Sylgard 184, available from Dow Corning of Midland, 
Mich. 
The reservoir array layer 304 is placed onto the micro-plate 302 before the 
channels are filled with a separation medium. Preferably, the separation 
medium is 0.75 percent weight/volume hydroxyethylcellulose (HEC) in a 
1.times. TBE buffer with 1 .mu.M ethidium bromide. Additionally, the 
reservoir array 304 fully isolates the reservoirs from each other. The 
separation channels are pressure filled with a sieving matrix from the 
anode reservoir 180 until all channels have been filled. The anode and 
cathode reservoirs 180 and 120 are then filled with a 10.times. TBE buffer 
to reduce ion depletion during electrophoresis. The sample reservoirs are 
rinsed with deionized water. Samples are then loaded from a micro-titer 
plate using an 8-tipped pipetter. 
After the reservoir array layer 304 is positioned on the micro-plate 302, 
an electrode array 306 is placed above the reservoir array 304. The 
electrode array 306 is fabricated by placing an array of conductors such 
as platinum wires through a printed circuit board. Each conductor is 
adapted to engage a reservoir on the micro-plate 302. Moreover, the wires 
are electrically connected with metal strips on the circuit board to allow 
individual reservoirs of a common type to be electrically addressed in 
parallel. The electrode array 306 also reduces the possibility of buffer 
evaporation. The electrode array 306 in turn is connected to one or more 
computer controlled power supplies. 
As shown in FIG. 4B, the reservoir array layer 304, when used in 
conjunction with the micro-plate 302, enlarges the effective volume of the 
reservoirs originally formed on the micro-plate 302. Moreover, electrodes 
from the electrode array 306 are adapted to probe the reservoirs on the 
micro-plate 302 and the reservoir array layer 306. The solutions are 
placed in the reservoirs by a pipetter 308. 
After assembly, the CAE micro-plate 302 is probed with a galvo-scanner 
system 400, as shown in FIG. 5. The system 400 measures fluorescence using 
a detector at a detection zone of the channels. During the process of 
electrophoresis, as a fluorescent species traverses a detection zone, it 
is excited by an incident laser beam. In a direct fluorescence detection 
system, either the target species is fluorescent, or it is transformed 
into a fluorescent species by tagging it with a fluorophore. The passing 
of the fluorescent species across the detection zone results in a change, 
typically an increase in fluorescence that is detectable by the system 
400. 
Turning now to the analysis system, the galvo-scanner 400 has a 
frequency-doubled YAG laser such as YAG laser available from Uniphase 
Corporation of San Jose, Calif. The YAG laser generates a beam which may 
be a 30 mW, 532 nm beam. The beam generated by the laser 402 travels 
through an excitation filter 404 and is redirected by a mirror 406. From 
the mirror 406, the beam travels through a beam expander 408. After 
expansion, the beam is directed to a dichroic beam splitter 410. The laser 
beam is directed to a galvonometer 420 which directs the beam to a final 
lens assembly 422. In this manner, the beam is focused on a spot of about 
5 .mu.m where it excited flourescence from the molecules in the channels 
and is scanned across the channels at 40 Hz. The resulting flourescence is 
gathered by the final lens and passed through the galvomirror and the 
dichroic beam splitter 410 to an emission filter 412 which operates in the 
range of about 545-620 nm. After passing through the emission filter 412, 
the beam is focused by a lens 414. Next, the beam is directed through a 
pinhole 416 such as a 400 .mu.m pinhole for delivery to a photomultiplier 
(PMT) 418. 
The electrode array 306 is connected to one or more power supplies 428 such 
as a series PS300, available from Stanford Research Systems of Sunnyvale, 
Calif. The power supplies are connected to a computer and software 
controlled to automatically time and switch the appropriate voltages into 
the electrode array 306. The software may be written in a conventional 
computer language, or may be specified in a data acquisition software such 
as LabVIEW, available from National Instruments of Austin, Tex. Data 
corresponding to spatially distinct fluorescent emission may then be 
acquired at about 77 kHz using a 16 bit A/D converter from Burr-Brown 
Corporation of Tucson, Ariz. Logarithmic data compression is then applied 
to generate five linear orders of dynamic measurement range. The data is 
obtained as a 16 bit image, and electropherograms are then generated using 
a suitable software such as IPLab, available from Signal Analytics, 
Vienna, Va., to sum data points across each channel. A detection of all 
lanes with a 0.09 second temporal resolution has been achieved by the 
system 400. 
EXPERIMENTS 
An electrophoretic separation and fluorescence detection of HFE, a marker 
gene for hereditary hemochromatosis, was performed to demonstrate the 
high-throughput analysis of biologically relevant samples using the CAE 
micro-plates of the present invention. HFE is a genetic disorder that 
causes a buildup of iron in tissues resulting over time in disease. The 
buildup primarily affects the liver. Between 0.1 and 0.5% of the Caucasian 
population are homozygous for an HFE C282Y variant responsible for this 
disease. If detected early, treatment can be initiated and long term 
effects avoided. To screen the population for this marker gene, a high 
throughput screening system is needed. 
In this experiment, samples were prepared using PCR amplification and 
digestion to assay the C282Y mutation in the HFE gene. This G A mutation 
at nucleotide 845 creates a Rsa I restriction site in the HFE gene. DNA 
materials were isolated from peripheral blood leukocytes using standard 
methods. A segment of an HFE exon containing the variant site was 
amplified with the following primers: 
HH-E4B: 5'GACCTCTTCAGTGACCACTC3' 
HC282R: 5'CTCAGGCACTCCTCTCAACC3' 
The HC282R primer is a primer discussed in Feder et al., Nature Genet. 13, 
399-408 (1996), hereby incorporated by reference. The HH-E4B primer 
contains a 5' biotin tag. The 25 .mu.l amplification reaction contained 10 
mM Tris-HCl (pH=8.8), 50 mM KCl, 0.75 mM MgCl.sub.2, 0.2 mM dNTPs, 7.5 pM 
of each primer and 1.5 U AmpliTaq DNA, available from Perkin Elmer, 
Branchburg, N.J. The PCR was carried out under three consecutive 
conditions: 5 cycles (95.degree. C. for 1 min, 64.degree. C. for 1 min, 
72.degree. C. for 1 min), 5 cycles (95.degree. C. for 1 min, 60.degree. C. 
for 1 min, 72.degree. C. for 1 min), and 25 cycles (95.degree. C. for 1 
min, 56.degree. C. for 1 min, 72.degree. C. for 1 min). The restriction 
digestion of amplified product was carried out by adding 4 .mu.l of each 
amplified sample to 6 .mu.l buffer containing 2 U Rsa I (Sigma, St. Louis, 
Mo.) and digesting for 90 minutes at 37.degree. C. Samples were dialyzed 
against DI water on a 96 sample dialysis plate, available from Millipore, 
Bedford, Mass. Sample types were initially established by separation of 
restriction fragments on 1% Agarose-3% SeaPlaque gel, available from FMC 
Bioproducts, Rockland, Me, in 0.5.times.TBE. Gels were stained in 0.5 
.mu.g/ml ethidium bromide for 30 minutes and visualized on a UV 
transilluminator, a Spectroline model TR-302, using a 123-bp ladder, 
available from Life Technologies Inc., Gaithersburg, Md., to determine 
fragment sizes. 
FIG. 6A and 6B present images of separations of 96 HFE samples on a CAE 
micro-plate. The 96 samples were separated in two runs of 48 samples, 
corresponding to two injection reservoirs per channel. In this experiment, 
nineteen different samples were dispersed among the 96 sample wells, 
giving a 5-fold redundancy in sample analysis. An original image 500 was 
obtained for the first injection, while an original image 504 was obtained 
for the second injection. Additionally, expanded images 502 and 506, 
corresponding to original images 500 and 504 are shown. The width of the 
electrophoretic image shown is 7.4 mm for 48 lanes and the complete 
analysis of 96 samples was performed in under 8 minutes. The expanded 
images show that the bands are of high intensity and resolution. The image 
exhibits a smile with the right lanes about 20 seconds faster than the 
left. This is caused by a gradient in the electrophoresis voltages caused 
by the placement of the anode to the side of the injection region to 
ensure adequate clearance from the scanning lens. 
FIG. 7 presents the 96 electropherograms obtained from the images in FIGS. 
6A and 6B. All electropherograms have been shifted to align a 167-bp 
doublet in order to compare the separations. The 167-bp fragment appears 
as a doublet due to a partial biotinylation of the HH-E4B primer, as the 
biotinylated form accounts for the slower migrating fragment in the 
doublet. The 167-bp doublet provides a useful reference point for the 
alignment of electropherograms to compare separations and allows an 
accurate genotyping without requiring a sizing ladder. As shown in FIG. 7, 
an average distance between the 111 and 140-bp bands is 7.3 seconds with a 
standard deviation (SD) of 0.8 second and 0.6 second, respectively, for 
the first injection and 6.6 sec with a SD of 1.1 second and 0.5 second, 
respectively, for the second injection. Using a t-test, the typings for 
both injections are determined to be at about a 99.9% confidence level. 
Referring to FIG. 8, a second embodiment of the CAE micro-plate 600 is 
shown. In FIG. 8, the micro-plate 600 is an array of injectors, each of 
which includes waste reservoirs 602 and 608, sample reservoirs 604, 606, 
610 and 612. Each injector unit is connected to one of two cathode 
reservoirs 614 or 616, respectively. Additionally, each injector unit is 
connected to one capillary in an array of capillaries or channels 620. The 
capillaries or channels 620 are connected to an anode 630. In this design, 
96 samples can be analyzed by injecting four samples serially on a single 
capillary. Further, 24 separation capillaries or channels are used to 
analyze the material in 96 sample reservoirs. Moreover, each of the 
injector units has two waste reservoirs. In total, the embodiment of FIG. 
8 has a hole count of 3N/2+3. 
Referring now to FIG. 9, a third embodiment of the CAE micro-plate 650 is 
disclosed. In the CAE micro-plate 650 of FIG. 9, cathode reservoirs 652 
are positioned on a perimeter of the CAE micro-plate 650. Additionally, an 
anode reservoir 660 is positioned in the center of the CAE micro-plate 
650. Separation channels or capillaries may emanate from an outer 
perimeter of the micro-plate 650 toward the center of the micro-plate 650 
in a spiral pattern if longer separation channels are desired. 
Alternatively, if short paths are desired, the separation channels or 
capillaries may simply be a straight line connecting the perimeter of the 
micro-plate 650 to the center 660 of the CAE micro-plate 650. 
Turning now to FIGS. 10 and 11, an injector unit of the CAE micro-plate of 
FIG. 9 and its position on a perimeter of the micro-plate of FIG. 9 are 
illustrated in detail. In FIG. 10, two separation channels or capillaries 
670 and 671 are connected to a common waste reservoir 672 and a common 
cathode reservoir 674. Additionally, the separation channels 670 and 671 
are connected to sample reservoirs 676 and 678. As shown in FIGS. 10 and 
11, the connections between the sample and waste reservoirs may intersect 
in an off-set manner. 
Referring now to FIG. 12, the common anode 660 of FIG. 9 is illustrated in 
detail. As shown in FIG. 11, a plurality of separation channels or 
capillaries 800-810 form a curvilinear pattern, which may be a radial 
pattern, converging on a central region 820. From the central region 820, 
the separation channels or capillaries form a passageway from the 
perimeter of the central region 820 to the common anode reservoir 660 at 
the center of the CAE micro-plate. The center area 820 is the area where a 
rotating scanner may be used for detection purposes. 
Samples may be loaded manually or automatically. Serial injections may be 
used to increase the sample throughput with a predetermined number of 
capillaries. Moreover, while one embodiment of the present invention 
injects two samples per channel, an injection of four samples per channel 
may be used to analyze 192 samples per plate. Further, an increase in the 
number of capillaries on the CAE micro-plate would increase the throughput 
correspondingly without introducing any sample contamination. Moreover, 
the plate may be made of glass or plastic. 
In addition, the scanning detection system may be altered by inverting its 
objective lens and scanning from below. Placing of the optics below the 
plate would permit facile manipulation and introduction of samples. The 
inverted scanning would also avoid spatial conflict with the anode 
reservoir, thereby permitting a central placement of the anode. Moreover, 
an array of PCR reaction chambers may be used with the micro-plate of the 
invention to allow for integrated amplification of low volume samples, 
eliminate sample handling and manual transfer, and reduce cost. 
Furthermore, the present invention contemplates that electronic heaters, 
thermocouples and detection systems may be used with an array of 
microfluidic capillaries to enhance the CAE electrophoresis process. 
While the invention has been shown and described with reference to an 
embodiment thereof, those skilled in the art will understand that the 
above and other changes in form and detail may be made without departing 
from the spirit and scope of the following claims.