U.S. patent application number 12/634661 was filed with the patent office on 2010-06-17 for automated parallel capillary electrophoresis system with hydrodynamic sample injection.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Thomas E. Kane, Qingbo Li, Changsheng Liu.
Application Number | 20100147692 12/634661 |
Document ID | / |
Family ID | 23532811 |
Filed Date | 2010-06-17 |
United States Patent
Application |
20100147692 |
Kind Code |
A1 |
Liu; Changsheng ; et
al. |
June 17, 2010 |
Automated Parallel Capillary Electrophoresis System with
Hydrodynamic Sample Injection
Abstract
An automated capillary zone electrophoretic system is disclosed.
The system employs a capillary cartridge having a plurality of
capillary tubes. The cartridge has a first array of capillary ends
projecting from one side of a plate. The first array of capillary
ends is spaced apart in substantially the same manner as the wells
of a microtitre tray of standard size. This allows one to
simultaneously perform capillary electrophoresis on samples present
in each of the wells of the tray. The system includes a stacked,
dual carrousel arrangement to eliminate cross-contamination
resulting from reuse of the same buffer tray on consecutive
executions from electrophoresis. The system also has a container
connected to the detection end of the capillaries. The container is
provided with valving which facilitate cleaning the capillaries,
loading buffer into the capillaries, introducing samples to be
electrophoresced into the capillaries, and performing capillary
zone electrophoresis on the thus introduced samples.
Inventors: |
Liu; Changsheng; (State
College, PA) ; Kane; Thomas E.; (State College,
PA) ; Li; Qingbo; (State College, PA) |
Correspondence
Address: |
LIFE TECHNOLOGIES CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
23532811 |
Appl. No.: |
12/634661 |
Filed: |
December 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12290087 |
Oct 27, 2008 |
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12634661 |
|
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|
11204773 |
Aug 15, 2005 |
7459070 |
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12290087 |
|
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|
10011977 |
Dec 11, 2001 |
6953521 |
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11204773 |
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09388125 |
Aug 31, 1999 |
6352633 |
|
|
10011977 |
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Current U.S.
Class: |
204/603 |
Current CPC
Class: |
Y10T 436/2575 20150115;
G01N 27/44782 20130101; C07K 1/26 20130101; G01N 27/44704 20130101;
G01N 27/44743 20130101 |
Class at
Publication: |
204/603 |
International
Class: |
G01N 27/00 20060101
G01N027/00 |
Claims
1. A capillary zone electrophoresis subsystem configured to
cooperate with a light source and a light detector to detect
migrating samples, the subsystem comprising: a fluid container; a
plurality of capillary tubes, each capillary tube having a first
end and a second end, the first ends being arranged to receive
samples thereinto and each of the second ends terminating in the
fluid container; a power supply configured to apply a voltage
across the capillary tubes; a pump operably connected to the fluid
container, the pump configured to introduce a liquid into the
container when the pump is in an operating mode; a vacuum device
connected to the fluid container via a vacuum conduit entering the
fluid container at a level higher than a level of each second end
of the capillary tubes, the vacuum device configured to cause a
negative pressure in the container, when the pump is not in the
operating mode and the container is sealed; a gas release valve
connected to the container and configured to vent a gas in the
container when the gas release valve is opened; and a drain valve
connected to the container and configured to drain a liquid in the
container, when the drain valve is open.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 12/290,087, filed Oct. 27, 2008, which is a
Continuation of U.S. Ser. No. 11/204,773, filed Aug. 15, 2005, now
U.S. Pat. No. 7,459,070, which is Continuation to U.S. patent
application Ser. No. 10/011,977, filed Dec. 11, 2001, now U.S. Pat.
No. 6,953,521, which is a Continuation of U.S. patent application
Ser. No. 09/388,125, filed Aug. 31, 1999, now U.S. Pat. No.
6,352,633, all of which are incorporated by reference in their
entirety herein.
INTRODUCTION
[0002] The present teachings relate to an automated apparatus for
performing multiplexed Capillary Electrophoresis. The present
teachings are especially useful in an automated Capillary Zone
Electrophoresis (CZE) system for loading samples into a plurality
of capillaries from wells of commercially available, microtitre
trays of standard size.
[0003] The contents of commonly-owned U.S. patent application Ser.
No. 09/105,988, which issued as U.S. Pat. No. 6,027,627 and also
was published as WO 99/00664, are incorporated by reference to the
extent necessary to understand the present teachings. This
reference discloses an automated apparatus for capillary
electrophoresis.
[0004] FIG. 1 illustrates a prior art automated electrophoretic
apparatus discussed in the above-referenced patent application for
capillary electrophoresis. The apparatus includes a light source
452, a processor/controller 404, a dual carrousel arrangement
having an upper carrousel 601 and a lower carrousel 602 which are
aligned and spaced apart along a common axis and operated by a
rotor 604, a DC motor 605 having a movable member 603 to move a
tray 214 placed on one of the carrousels along a common axis toward
or away from an array of capillary ends belonging to a capillary
cartridge 300, a detector 408 for detecting, at a window region 130
of the capillaries, the fluorescence emitted by samples migrating
along the capillaries, and a computer monitor 406 to view the
results of the migration. An electrophoretic medium, such as a gel,
can be introduced into the capillaries via a conduit 606 in
preparation for an electrophoretic run.
[0005] FIG. 2 illustrates a prior art plumbing system in accordance
with the above-identified reference, for performing capillary
electrophoresis using the device of FIG. 1. In particular, FIG. 2
shows the integration of a gel syringe 804 and an HPLC wash solvent
system 807 into the solvent/gel delivery module. A solvent manifold
850 connects three inlets from the feeder tubes 806 of the solvent
containers 801, 802, 803 to an outlet. Feeder tubes 806 from the
solvent containers 801, 802, 803 are connected to the inlets of the
solvent manifold 850 by tubing 860. The controller 404 pictured in
FIG. 1 controls the solvent manifold 850 to select solvent from one
of the three solvent containers 801, 802, 803. The inlet of the
HPLC pump 807 is connected to the outlet of the solvent manifold
850 by tubing 861 and the outlet of the HPLC pump 807 is connected
to an inlet of a valve manifold 851 by tubing 862.
[0006] The valve manifold 851 connects two inlets and an outlet.
One inlet of the valve manifold 851 is connected to the gel syringe
804 by tubing 863 and the other inlet of the valve manifold 851 is
connected to the outlet of the HPLC pump 807. The outlet of the
valve manifold 851 is connected to the solvent/gel input port 606
by tubing 864. The controller 404 pictured in FIG. 11 causes the
valve manifold 851 to select either the inlet connected to the gel
syringe 804 or the inlet connected to the HPLC pump 807. In this
manner, gel and solvents are delivered to the capillary cartridge
909 in preparation for capillary gel electrophoresis of samples in
microtitre tray 852.
[0007] In some embodiments, the tubing connecting the feeder tubes
806 of the solvent containers 801, 802, 803 to the inlets of the
solvent manifold 850 is standard Teflon tubing with a diameter of
1/8 inches. The tubing 861 connecting the outlet of the solvent
manifold 850 to the inlet of the HPLC pump 807 is PEEK tubing with
a diameter of 1/16 inches. The tubing 861 connecting the outlet of
the solvent manifold 850 to the inlet of the HPLC pump 807, the
tubing 862 connecting the outlet of the HPLC pump 807 to an inlet
of the valve manifold 851, the tubing 863 connecting the gel
syringe 804 to an inlet of the valve manifold 851 and the tubing
864 connecting the outlet of the valve manifold 851 to the
solvent/gel input port are PEEK tubing with a diameter of 1/16
inches.
[0008] FIG. 3 illustrates an embodiment of capillary cartridge 1180
in accordance with the above-identified application. In this
embodiment, the capillary tubes run from their first ends 1188
disposed in an electrode/capillary array 1181. The capillary tubes
then run inside multilumen tubing 1183. The multilumen tubing is
taught in detail in U.S. Pat. No. 6,063,251, which is incorporated
by reference herein. The multilumen tubing 1183 is held firmly in
place by tubing holders 1185. The capillary tubes, without the
protection of the multilumen tubing, pass through an optical
detection region 1187. Beyond the optical detection region 1187,
the capillary tubes have a common termination and are bundled
together and cemented into a high pressure T-shaped fitting 1182
made from electrically conductive material, which, during
electrophoresis, is connected to electrical ground.
[0009] The tubing holders 1185 and the T-fitting 1182 are fixed to
a cartridge base 1186. The cartridge base 1186 is made from
polycarbonate plastic for its dielectric characteristic. The base
1186 in turn is removably attached to a shuttle 1179 which includes
a set of rail couplings 1184 protruding from its bottom. These rail
couplings 1184 are arranged so that they fit on to a railing system
(not shown in FIG. 18) of the apparatus in FIG. 1. The railing
system allows the shuttle 1184 to move between an in position and
out position. The base 1186 is detached from the shuttle 1179 so
that the cartridge 1180 is disposed (or cleaned) and a new (or
cleaned) capillary cartridge is attached when the shuttle 1179 is
in its out position. The combination of the railing system and the
shuttle 1179 allows the newly attached capillary cartridge to be
repeatedly located at the same position as that of the disposed
capillary cartridge in relation to a camera and a laser (not shown
in FIG. 3) when the shuttle 1179 is in its in position. In a
preferred embodiment, the shuttle 1179 extends the length of the
base 1186 with an opening to accommodate the electrode/capillary
array 1181; the shuttle 1179 is attached to the base 1186 by a
plurality of removable fasteners 1178.
[0010] The prior art plumbing system of FIG. 2 and T-fitting of
FIG. 3 are best suited for capillary gel electrophoresis. In
capillary gel electrophoresis, the gel is fairly viscous, on the
order of 50,000 centi-poise. This requires a system which can
create pressure sufficient to load gel into the capillaries in
preparation for a capillary electrophoresis run, and sufficient to
expel the gel from the capillaries during reconditioning.
[0011] In contrast to the gels that are used in capillary gel
electrophoresis, buffers are used to load the capillaries in
capillary zone electrophoresis (CZE). These buffers have a
viscosity on the order of that of water, i.e., about 1 centi-poise.
While the low viscosity of buffers has the advantage of not needing
high pressure to load and unload the electrophoretic medium, CZE
with buffers does have the disadvantage of capillary siphoning.
Capillary siphoning is characterized by the buffer solution at one
end of the capillaries being completely drawn into the capillaries,
thereby depleting the buffer at that one end. Like siphoning of any
tubing, this problem occurs when the two ends of the capillaries
terminate at different heights. The obvious solution to this
problem is to ensure that opposite ends of the capillaries are
maintained at the same level. This, however, is less than an ideal
solution.
SUMMARY
[0012] The present teachings are directed to an automated parallel
capillary zone electrophoresis (CZE) system. The CZE system of the
present teachings is realized by modifying the prior art capillary
gel electrophoresis (CGE) system of the above-referenced prior art.
More particularly, the present teachings are principally realized
by modifying the plumbing at the ends of the capillaries towards
which samples in the capillaries migrate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The skilled artisan will understand that the drawings
described below are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0014] FIG. 1 is a side view of a prior art automated capillary
electrophoresis system suitable for capillary gel
electrophoresis;
[0015] FIG. 2 illustrates a prior art plumbing system for the
electrophoresis system of FIG. 1;
[0016] FIG. 3 is a side view of a prior art capillary cartridge for
use with the electrophoresis system of FIGS. 1 and 2; and
[0017] FIG. 4a shows an embodiment of the present teachings for
performing capillary zone electrophoresis;
[0018] FIG. 4b shows a sequence of valve settings for the
embodiment of FIG. 4a;
[0019] FIG. 5 shows an embodiment of a system in accordance with
the present teachings;
[0020] FIGS. 6a & 6b show two versions of an embodiment of a
system in accordance with the present teachings;
[0021] FIG. 7 shows intensity images comprising fluorescence data
from experimental samples in 96 capillaries simultaneously
migrating; and
[0022] FIGS. 8a, 8b & 8c show intensity plots for experimental
samples migrating in three of the 96 capillaries.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0023] The contents of commonly-owned, aforementioned U.S. patent
application Ser. No. 09/105,988, which issued as U.S. Pat. No.
6,027,627 and also was published as WO 99/00664, are incorporated
by reference to the extent necessary to understand the present
teachings.
[0024] FIG. 4a shows a buffer cell 100 connected to a capillary
cartridge 102 via a pressure fitting 104 not unlike that shown in
FIG. 3. Indeed, capillary cartridge 102 is similar in structure to
the capillary cartridge 1180 of FIG. 3, except that capillary
cartridge 102 does not include the T-fitting 1182. In the present
teachings, the buffer cell 100 and its associated hardware shown in
FIG. 4a replace the prior art T-fitting 1182 of FIG. 3 and some of
the prior art plumbing system seen in FIG. 2.
[0025] The buffer cell 100 has an interior cavity 106 which
preferably is sealed from the exterior, except for openings
discussed below. In some embodiments, the cell is formed from an
acrylic plastic, which is an electrical insulating material. Inner
walls of the cell are shaped and sized to provide an interior
cavity 106 into which a buffer or other liquid 112 may be
introduced. In some embodiments, the container has a capacity of
about 100 ml, by volume.
[0026] A high voltage electrode 110 connected to a power supply
(not shown) is in contact with the liquid 112 in the cell 100 for
the purpose of applying a predetermined potential to the liquid in
the container, and thereby also to the first, cell ends 107 of the
capillaries which are in communication with the liquid 112. During
CZE, the high voltage electrode 110 is held at ground, while a
non-zero voltage is applied to the second, sample ends 108 of the
capillaries, with the polarity of the voltage being determined by
the charge-type of the samples being separated. The magnitude of
the applied voltage is on the order of 10-15 kV, not unlike that
used in capillary gel electrophoresis.
[0027] A plurality of conduits communicates with the cavity 106 via
corresponding valves. In some embodiments, the valves are solenoid
valves or the like, which can be controlled by computer, much as
discussed in the above-identified U.S. patent application Ser. No.
09/105,888. In FIG. 4a, each of the five conduits connected to the
cell 100, whether it is an inlet or an outlet, or serves as both,
is shown to have a separate valve. It is understood, however, that
one or more of these valves may be internal to equipment connected
to the corresponding conduit, rather than being a discrete
valve.
[0028] Drain outlet 114 and drain valve 116 allow a liquid in the
cavity 106 to exit the cell 100 into a waste container (not shown).
Air conduit 118 and gas (air) release valve 120 provide a path from
the interior of the cavity 106 to the atmosphere when air valve
release 120 is open. Pump inlet 122 and pump valve 124 provide a
path for buffers, solvents and other liquids in containers, such as
those indicated by 801, 802 and 803, to enter the cell 100 via one
or more manifolds 850, under assistance of an HPLC pump 807, or the
like. Pressure conduit 126 and pressure valve 128 connect a syringe
130 or other pressure applicator to the cavity 106 at a point above
the level of liquid 112 therein. Finally, overflow outlet 132 and
overflow valve 134 cooperate to provide a passage from the interior
of the cavity 106 to a waste container, so as to ensure that the
cell 100 does not overfill. While the various valves 116, 120, 124,
128 and 134 are shown to be distinct devices, it should be kept in
mind that one or more of these valves may be an integral part of
another device. For instance, pump valve 124 may be integrally
formed as part of HPLC pump 807, and pressure valve 128 may be
replaced by precisely controlling the syringe's piston 136 by a
stepper motor, or the like, under the direction of a
controller.
[0029] FIG. 4a depicts the valve positions for performing steps
associated with preparing and conducting electrophoresis on the
samples in the capillary tubes of the capillary cartridge 102.
[0030] When the cell 100 is to be drained, the pressure valve 128
and the pump valve 124 are closed, and the drain valve 116 and at
least one, if not both, of the air valve 120 and the overflow valve
134 are opened. This allows the liquid in the cell to drain via
drain conduit 114.
[0031] Once the cell 100 has been completely drained, it may be
partially filled with a liquid. For this, the drain valve 116 and
the pressure valve 128 are closed, and the pump valve 124 and at
least one, if not both, of the air valve 120 and overflow valve 134
open. The pump 807 is then operated to introduce a selected one of
the liquids in containers 801, 802, 803 into the cell 100. Because
the pump introduces liquid into the reservoir and, because at least
one of the air valve 120 and the overflow valve 132 is open, the
liquid is not forced into the capillaries. However, the pump is
controlled to turn off when the liquid reaches a predetermined
level within the cell.
[0032] When the capillaries are to be cleaned, a cleaning solution,
or the like, present in one or more of the containers 801, 802,
803, is forced into the cell 100, into the cell ends 107 of the
capillary tubes, and out the sample ends 108 of the capillary
tubes. For this, only the pump valve 124 is open while all the
other valves are closed. Under such conditions, when the HPLC pump
807 operates, it forces liquid into the cell 106, increasing the
pressure therein. The increased pressure forces the cleaning
solution into the cell ends 107, through the capillary tubes and
out the sample ends 108. Once cleaning solution has been forced
through, the pump valve may be closed, and the cell 100 drained, as
discussed above.
[0033] After cleaning, the cell can be filled with buffer to a
predetermined level by selecting the appropriate container 801,
802, 803 with the manifold 850, and operating the pump 807 with the
drain valve 116 and the pressure valve 128 closed, and the pump
valve 124 and at least one, if not both, of the air valve 120 and
overflow valve 134 open. The predetermined level of buffer should
exceed the level of the bundle of capillary cell ends 107.
[0034] Once the level of the buffer has exceeded the level of the
capillary cell ends 107, buffer may be loaded into the capillaries.
For this, only the pump valve 124 is left open, and all other
valves are closed. The buffer enters the capillary cell ends 107,
thereby forcing any material within the capillary tubes out the
capillary sample ends 108 into a waste container (not shown), and
loading the capillary tubes with buffer. At this point, the cell
100 is filled with buffer to just below the level of the overflow
conduit 132, yet above the level of the capillary cell ends. In
some embodiments, the overflow conduit 132 is at about the 60% fill
level and so the cell 100, having a capacity of 100 ml, contains
approximately 60 ml of buffer.
[0035] It should be evident that filling the capillaries with
buffer is similar to the procedure for cleaning the capillaries,
except that buffer, rather than a cleaning solvent, is used. As
discussed above, this is controlled by operating the manifold 850
connected to the containers 801, 802 and 803 holding buffers,
cleaning solutions and other liquids. It should be noted, however,
that buffer itself can be used to clean the capillaries
[0036] To introduce a sample into the sample ends 108 of the
capillaries, the sample ends 108 are first dipped into wells of a
microtitre tray of standard size, such as those having a
rectangular array of 8 rows of 12 wells, or those having 16 rows of
24 wells. The wells contain the samples to be electrophoresced.
[0037] The samples can be introduced into the sample ends 108 of
the capillaries in one of two ways. One way is electro-kinetic
injection wherein a voltage differential is applied between the
sample ends and the cell ends of the capillaries so as to cause a
portion of the sample to enter the sample ends. During
electro-kinetic injection, the air valve 120 is kept open to keep
the reservoir 100 at atmospheric pressure, equilibrated with the
cell ends 107 of the capillary. By applying a high voltage
differential, the electro-osmotic flow causes sample to enter the
capillary sample ends 108. Once the sample has been introduced into
the sample ends from the wells of the microtitre tray, the sample
tray is replaced by a buffer tray and electrophoretic separation
can take place in the capillaries under high voltage.
[0038] A second way in which to load samples into the sample ends
108 of the capillaries is by hydrodynamic injection. First, air
valve 120 is opened and all other valves are closed to equilibrate
both ends of the capillaries with atmospheric pressure. After
equilibration, the air valve 120 is also closed, and so no valves
are left open. At this point, the plunger 136 of the syringe 130 is
pulled back by a predetermined volume. This causes the air above
the liquid level in the cell to expand into a slightly greater
volume and thereby create a vacuum, or negative pressure. At this
point, the pressure valve 128 is opened, thereby applying this
negative pressure to the surface of the buffer 112 in the cell 100.
Due to the negative pressure, a small amount of sample (or other
substance in each of the wells of the microtitre tray) is sucked in
at each of the capillary sample ends. However, because air expands
to fill the volume, there is a slight time lag between opening the
pressure valve 128 and the uptake of sample. After the sample is
allowed to enter due to the negative pressure for a predetermined
period of time, typically on the order of a few seconds, the air
valve 120 is opened, thereby stopping the injection process.
Experiments have shown that hydrodynamic injection produces more
reproducible results, and more even sample injection into the
capillaries. This is because the volume into which the air expands
does not immediately cause an instantaneous, corresponding intake
of sample at the capillary sample ends, when the pressure valve 126
is opened. Instead, a fairly even uptake into each of the capillary
sample results.
[0039] The pulling volume of the syringe controls the degree of
negative pressure or vacuum. In some embodiments, the plunger is
pulled back by an amount sufficient to displace about 2 ml. In a
100 ml container having 60 ml of buffer therein, there is about 40
ml of air. When the plunger is pulled back by 2 ml, a negative
pressure (relative to atmospheric) of 2.0 ml/40.0 ml=0.05 atm (or
about 0.7 psi) is generated. Assuming a syringe precision of 0.1 ml
and a container volume of 100 ml, the precision of the negative
pressure can be controlled to about 0.001 atm.
[0040] Once the sample has been introduced into the capillary
sample ends, the sample tray is preferably replaced by a buffer
tray in preparation for electrophoresis. Replacing the sample trays
with buffer trays helps ensure that excess sample is not taken into
the capillary tubes, and also ensures that both ends of the
capillary tubes are inserted into buffer. Using a device in
accordance with the present teachings, electrophoresis can take
place in either a static mode, or a dynamic mode.
[0041] In the static mode, the pump 807 is not operational and only
the air valve 120, or the overflow valve 134, or both, are open,
with the remaining valves closed. Under these conditions, the
buffer in the cell 112 is substantially stagnant during
electrophoresis.
[0042] In the dynamic mode, the pressure valve 128 is closed, and
all other valves are open, and the pump is operational, with buffer
continuously being pumped into the cell through the pump inlet 122
and exiting the cell via drain outlet 114. This ensures that fresh
buffer bathes the capillary cell ends during electrophoresis while
older buffer drains from the cell. Samples which have completed
migrating from the sample end all the way to the cell end are also
drained through drain outlet 114 and drain valve 116. At the same
time, since air conduit 118 and air valve 120 are open, the
atmospheric pressure at both ends of the capillaries is equalized,
thereby counteracting the siphoning effect, especially when the
capillary ends are at the same height.
[0043] The dynamic mode, in which there is continuous flushing of
the cell 100, provides several advantages. First, continuously
providing fresh buffer solution to the capillary cell ends removes
charge depletion during electrophoresis. Charge depletion happens
when anion and cation layers build up around the electrode, thereby
resulting in a voltage drop between these layers which, in turn,
reduces the voltage drop across the capillary tubes for
separation.
[0044] Flowing buffer helps retard the formation of such layers so
that sample separation is more reproducible from run to run.
[0045] A second advantage to constant flushing is that it assists
in removing fluids and contaminants introduced into the cell by
electro-osmotic flow (EOF) during electrophoresis. EOF is a
continuous pumping process which brings small amounts of
sample-laden buffer into the cell. This can cause a change in
buffer conductivity during electrophoresis. Constant flushing helps
mitigate the problem of a solute-imbalance. Sensors and feedback
control systems connected to the pump and to the pump and drain
valves can ensure that the liquid level in the cell is maintained
at a predetermined level.
[0046] A third advantage to continuous flushing is that it reduces
the time spent cleaning the capillary tubes between runs. Because
fresh buffer is constantly being introduced into the cell in the
dynamic mode, one need not spend as much time rinsing out the cell,
upon conclusion of each run.
[0047] A fourth advantage to continuous flushing is that it removes
air bubbles which otherwise collect around the capillary cell ends
107 during electrophoresis. Such removal is believed to be brought
about by the buffer flowing past this area.
[0048] In one example of continuous flushing using capillaries with
an inner diameter of 50 .mu.m, a voltage differential of 10 kV
across the capillary ends and borate buffer at a pH of 10.5, EOF
speed is about 12 cm/min. This causes the liquid volume of the cell
to increase at the rate of about 53 .mu.l/min. If a drain is
provided, the buffer must be replenished, as needed. In some
embodiments, only about 1 ml/min of fresh buffer is introduced into
the cell while the drain valve is opened during
electrophoresis.
[0049] Despite the above-stated advantages, it should be kept in
mind that continuous flushing, though preferable, is not an
absolute requirement in the present teachings. Indeed, the primary
requirements for carrying out CZE in accordance with the present
teachings are that a cell be provided, the cell having a liquid
therein with the capillary cell ends terminating in said liquid,
and that some mechanism be provided for creating a vacuum, or
suction effect, at the capillary cell ends so as to draw samples
into capillary sample ends.
[0050] FIG. 5 presents another embodiment in accordance with the
present teachings. In the embodiment of FIG. 5, a sealed, or at
least sealable, cell 100 partially filled with a liquid 112 is
provided. The capillary cell ends 107 terminate in this liquid 112.
An air syringe 130 and an HPLC pump 807 are also provided. When the
syringe plunger 136 is pulled in the direction shown by the arrow
A1, sample is introduced into the capillary sample ends 108, as
depicted by arrow A2. As discussed above with reference to FIG. 4a,
conduits for drain, air release and overflow may also be provided.
To clean the cell in this embodiment, one simply restrains the
syringe plunger and runs the pump to flush out the liquid in the
cell and in the capillary tubes via the capillary second ends.
[0051] FIG. 6a presents yet another embodiment in accordance with
the present teachings. In this embodiment, which is similar to
embodiment of FIG. 5, the entire cell and the syringe are filled
with liquid and no air (or other gas) is used. Unlike air, liquid
is incompressible, and so there is neither a time delay nor a
variation in volume, between pulling the syringe plunger and the
introduction of samples into the capillary sample ends. This means
that the syringe must be much more precisely controlled in the
embodiment of FIG. 6a than in the embodiment of FIG. 5. For this, a
micro-syringe operated by high-precision stepper motors, or the
like, is used to ensure that only a small quantity of sample, about
0.1 .mu.l or so, per capillary, is introduced into each of the
capillary second ends. To clean the cell and the capillary tubes in
the embodiment of FIG. 6a, one may either push on the syringe
plunger or run the pump; either one forces buffer into the cell and
out through the capillary sample ends.
[0052] FIG. 6a presents still another embodiment in accordance with
the present teachings. In this embodiment, the syringe is replaced
by a narrow-diameter drain outlet 140 controlled by a valve 142
situated at a vertical position lower than that of the capillary
sample ends 108. In this embodiment, gravity is used to cause a
negative pressure. With the pump off, when the valve 142 is opened,
liquid drains through the conduit 140 as indicated by arrow A3.
This siphons liquid into the capillary sample ends, as indicated by
arrow A4.
[0053] In the embodiments of FIGS. 5, 6a and 6b, discrete valves
between the pump and the cell are not shown; it is understood,
however, that such valves may be integral with the pump. Similarly,
no such valves are shown between the syringe and the cell. As
explained above, the syringe plunger may be restrained and
controlled by a motor so as to exert sufficient force in the
appropriate direction, as dictated by a microprocessor or other
controller. Also, with regard to the embodiments of FIGS. 6a and
6b, it is noted that since only a very minute quantity of liquid is
introduced from the capillary tubes into the cell, there is no
appreciable increase in pressure within the cell, which is
substantially able to accommodate the added amount.
EXPERIMENTAL EXAMPLE
[0054] In an experimental set-up, capillary zone electrophoresis
was carried out simultaneously in 96 capillaries using a device
substantially arranged as shown in FIG. 4a. About 60 ml of buffer
was introduced into a 100 ml cell. The buffer used was a 10 mM
borate solution in de-ionized water, adjusted to a pH 10.5 with
NaOH. The viscosity of the buffer was almost the same as that of
water.
[0055] Ninety-six capillaries, each having a length of about 50 cm,
and ID of 50 .mu.m and a 150 .mu.m OD, available from Polymicro
Technology of Phoenix, Ariz. were used. A window region was burned
into each capillary using a hot wire at a point approximately 10 cm
from one end of the capillaries, thereby providing an effective
migration distance of about 40 cm from the sample end to the window
region at which sample detection would take place. The capillaries
were arranged substantially parallel to one another in a
ribbon-like arrangement.
[0056] More specifically, for most of their length from the sample
ends to the window, the capillaries were spaced apart from one
another by about 150 .mu.m and, at the window region, were spaced
apart by about 300 .mu.m. Beyond the window region, the cell ends
of the 96 capillaries were bound together as a bundle with Torr
Seal, available from Varian Vacuum Products of Lexington, Mass.
This bundle was connected to the cell shown in FIG. 4a with a
Swagelock fitting, with the capillaries being in communication with
the buffer. Meanwhile, the sample ends of the capillaries formed a
two-dimensional array with a spacing corresponding to that of the
wells of an 8.times.12 microtitre tray of standard size.
[0057] A 3 .mu.l sample was introduced into each of the wells of an
8.times.12 microtitre tray. The sample comprised a protein cluster
separated from among a multitude of such clusters in a protein
mixture extracted from bacteria. The proteins were labeled with
fluorescein dye, which has its absorption maximum at 495 nm. The
sample ends of the capillaries were inserted into corresponding
wells of the microtitre tray, in contact with the sample therein.
Samples in each of the 96 wells were then hydrodynamically injected
into the sample ends of the capillaries. This was performed by
creating a vacuum by pulling on the syringe plunger to displace a 3
ml volume with all valves closed, and holding the plunger in place.
At this point, the pressure valve was opened, thereby causing a
negative pressure at the air-buffer interface on the surface of the
buffer in the cell. The pressure valve was opened for about 20
seconds, permitting sufficient time for sample to be sucked into
each of the capillary sample ends. At this point, the air valve was
opened to alleviate the negative pressure and stop further
hydrodynamic injection of sample.
[0058] Next, the microtitre tray containing samples was replaced
with a microtitre tray containing buffer, in preparation for
electrophoresis. A voltage differential of 10 kV was applied for
about 10 minutes across the 50 cm-long capillaries, thereby
providing an electric field of 200 v/cm and causing the samples to
migrate under electro-osmotic flow, along with the buffer. An
all-line Argon-ion laser, available from Spectra-Physics of
Mountain View, Calif., and having an emissions peak not far from
495 nm, was used to illuminate the capillaries substantially at
right angles thereto at the window region during electrophoresis. A
CCD camera, available from PixelView of Beaverton, Oreg., was used
to detect the fluorescence of the samples as they passed through
the window region of the capillaries. The camera was set up
substantially as disclosed in co-owned issued U.S. Pat. No.
5,998,796, also published as WO 99/32877.
[0059] FIG. 7 shows the fluorescence intensities at 530.+-.8 nm, as
a function of time, of the samples in the 96 capillaries. In FIG.
7, the abscissa (x-axis) represents the capillary number while the
ordinate (y-axis) represents time. Darker spots represent higher
fluorescence intensity; thus, the darker the spot, the higher the
intensity.
[0060] FIGS. 8a, 8b and 8c show plots of relative intensities for
edge and center capillaries (capillary nos. 1, 48 and 96) in the
array, as a function of time. In FIG. 8, the abscissa (x-axis)
represents time, while the ordinate (y-axis) represents the
intensity. As seen in FIG. 8, the intensity contours are
substantially the same, exhibiting similar peaks from each
capillary, albeit at slightly different migration times for each
capillary.
[0061] As seen in this experimental example, CZE can be used to
separate proteins in a buffer having a predetermined pH. For
example, CZE can be used for human growth hormone separation, Ca++
binding protein separation, and recombinant human erythroprotein
protein separation, among others. The separation mechanism in CZE
is based on the ratio of the net charge to the size of the
proteins. The net charge can be of either polarity, depending on
the buffer pH and the protein's structure. Electro-osmotic flow of
the buffer in the capillaries sweeps neutral molecules, as well as
charged proteins, toward the detection window. The buffer
preferably has a viscosity about the same as that of water.
[0062] The present teachings may also be used in other capillary
electrophoresis settings in which the separation media has low
viscosity, on the order of 1-150, and more preferably on the order
of 1-50, centipoise. At these viscosities, the separation media can
be pumped into the capillaries under pressure without damage to the
capillaries or other components of the system, and the samples
injected hydrodynamically. A number of these other approaches and
applications are now discussed.
[0063] Sodium Dodecyl Sulfate (SDS)-type Capillary Gel (CGE)/NGE
(Non-Gel) Electrophoresis. In this approach, the proteins are bound
with the surfactant SDS to form negatively charged aggregates. A
polymer-based sieving matrix, such as polyethylene oxide (PEO),
preferably kept at a low pH to extend the lifetime of the
capillaries, is used as the separation medium. Applications for
this include, for example, peptide mapping, molecular weight
estimation, protein quantization and protein stability analysis. In
some cases, CGE with a low viscosity separation media, such as
polyvinylpyrrolidone (PVP), which has a viscosity of 1-25
centipoise when in a weight percentage of 0.1-5%, can be used for
DNA separation, as reported in Gao & Yeung, Anal. Chem., 1998,
v. 70, pp. 1382-1388.
[0064] Capillary Iso-Electric Focusing (CIEF), in which the
proteins are separated according to their unique iso-electric
points in a separation medium having a viscosity similar to that of
water, may also be performed using the device and method of the
present teachings.
[0065] Affinity Capillary Electrophoresis (ACE), in which proteins
are separated on the basis of specific bonding to other molecules
in a separation medium having a viscosity of about 5-50 centipoise,
may also be performed using the device and method of the present
teachings.
[0066] Micellular Electrokinetic Capillary Chromatography (MEKC),
in which compounds are separated based on their hydro-phobicity in
a separation medium having a viscosity of about 5-50 centipoise,
may also be performed using the device and method of the present
teachings. Such an approach is especially useful in separating
non-charged species.
[0067] Capillary Isotachphoresis (CITP), which is used for
in-capillary protein pre-concentration, immediately preceding CZE,
may be performed using the device and method of the present
teachings.
[0068] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described in any way.
[0069] While the present teachings are described in conjunction
with various embodiments, it is not intended that the present
teachings be limited to such embodiments. On the contrary, the
present teachings encompass various alternatives, modifications,
and equivalents, as will be appreciated by those of skill in the
art.
* * * * *