U.S. patent application number 09/946074 was filed with the patent office on 2002-03-14 for multi-channel capillary electrophoresis device including sheath-flow cuvette and replaceable capillary array.
This patent application is currently assigned to The Perkin-Elmer Corporation. Invention is credited to Carrillo, Albert L., Demorest, David M., Nordman, Eric S., Shigeura, John, Wunderle, Philip J..
Application Number | 20020029972 09/946074 |
Document ID | / |
Family ID | 22540848 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020029972 |
Kind Code |
A1 |
Nordman, Eric S. ; et
al. |
March 14, 2002 |
Multi-channel capillary electrophoresis device including
sheath-flow cuvette and replaceable capillary array
Abstract
A multi-channel capillary electrophoresis apparatus is
disclosed. The apparatus includes a capillary array assembly
comprising a plurality of capillaries, each capillary having a
capillary outlet, and an outlet support for supporting the
capillary outlets. The apparatus further includes a cuvette
defining a receiving slot, a gap region and a detection zone, where
the receiving, slot is adapted to removably receive the outlet
support, and wherein when the outlet support is inserted into the
receiving slot, the capillary outlets are positioned in the gap
region in proximity to the detection zone, and a flow channel is
formed by the outlet support and the receiving slot such that the
flow channel is in fluid communication with the gap region. In
addition, the apparatus includes a front plumbing block in fluid
communication with the flow channel for supplying a fluid flow
through the gap region sufficient to transport material downstream
the capillary outlets to the detection zone.
Inventors: |
Nordman, Eric S.; (Palo
Alto, CA) ; Shigeura, John; (Fremont, CA) ;
Carrillo, Albert L.; (Redwood City, CA) ; Demorest,
David M.; (Soquel, CA) ; Wunderle, Philip J.;
(El Sobrante, CA) |
Correspondence
Address: |
PATTI SELAN, PATENT ADMINISTRATOR
APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Assignee: |
The Perkin-Elmer
Corporation
|
Family ID: |
22540848 |
Appl. No.: |
09/946074 |
Filed: |
September 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09946074 |
Sep 4, 2001 |
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09805624 |
Mar 13, 2001 |
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09805624 |
Mar 13, 2001 |
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09621071 |
Jul 21, 2000 |
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6231739 |
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09621071 |
Jul 21, 2000 |
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09151928 |
Sep 11, 1998 |
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6162341 |
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Current U.S.
Class: |
204/601 |
Current CPC
Class: |
G01N 2035/00237
20130101; G01N 27/44782 20130101 |
Class at
Publication: |
204/601 |
International
Class: |
G01N 027/447 |
Claims
We claim:
1. A multi-channel capillary electrophoresis apparatus comprising:
a capillary array assembly comprising a plurality of capillaries,
each capillary having a capillary outlet, and an outlet support for
supporting the capillary outlets; a cuvette defining a receiving
slot, a gap region, and a detection zone; where the receiving slot
is adapted to removably receive the outlet support; wherein when
the outlet support is inserted into the receiving slot, the
capillary outlets are positioned in the gap region in proximity to
the detection zone, and a flow channel is formed by the outlet
support and the receiving slot such that the flow channel is in
fluid communication with the gap region; and a front plumbing block
in fluid communication with the flow channel for supplying a fluid
flow through the gap region sufficient to transport material
downstream from the capillary outlets to the detection zone.
2. The apparatus of claim 1 wherein the capillaries are in the form
of discrete capillary tubes.
3. The apparatus of claim 2 wherein the discrete capillary tubes
have a cylindrical cross-section.
4. The apparatus of claim 1 wherein an inner radius of the
capillaries is from 20 to 200 .mu.m.
5. The apparatus of claim 1 wherein the capillary array assembly
comprises between 20 and 200 capillaries.
6. The apparatus of claim 1 further including an inlet support.
7. The apparatus of claim 6 wherein the inlet support comprises a
body, a registration feature, and a plurality of capillary
alignment grooves.
8. The apparatus of claim 7 wherein the capillary alignment grooves
are V-shaped grooves.
9. The apparatus of claim 7 wherein the alignment grooves have a
pitch that is an integral fraction of 9 mm.
10. The apparatus of claim 7 wherein the capillary inlets are
arranged in a multi-tier configuration.
11. The apparatus of claim 7 wherein the capillaries are potted
into the inlet support with a potting material.
12. The apparatus of claim 1 wherein the outlet support comprises a
platform having a support surface.
13. The apparatus of claim 12 wherein the support surface has
capillary alignment grooves located thereon.
14. The apparatus of claim 13 wherein the capillary alignment
grooves are V-shaped grooves.
15. The apparatus of claim 1 wherein the capillary outlets are
arranges in a linear array.
16. The apparatus of claim 12 wherein the platform further
comprises two or more guide rails.
17. The apparatus of claim 16 wherein at least one surface of the
guide rails includes one or more flexure features located
thereon.
18. The apparatus of claim 16 wherein the guide rails include a
chamfer feature.
19. The apparatus of claim 12 wherein the platform is divided into
a first portion and a second portion such that the first and second
portions are connected by the capillaries.
20. The apparatus of claim 1 further including a detector in
optical communication with the detection zone.
21. The apparatus of claim 20 wherein the detector is a
protomultiplier tube, a photodiode or a charged coupled device.
22. The apparatus of claim 1 wherein the detection zone is located
between about 20 .mu.m and 2000 .mu.m from the capillary
outlets.
23. The apparatus of claim 22 wherein the detection zone is located
between about 100 and 500 .mu.m from the capillary outlets.
24. The apparatus of claim 1 wherein walls of the cuvette bounding
the detection zone include a window that is substantially
transparent to light.
25. The apparatus of claim 1 wherein the cuvette is formed from
glass, quartz or fused silica.
26. The apparatus of claim 1 wherein the cuvette is formed from
fused silica.
27. The apparatus of claim 1 further comprising a heater located in
thermal contact with the cuvette.
28. The apparatus of claim 1 wherein the cuvette is mounted in a
clamping block.
29. The apparatus of claim 28 wherein the clamping block comprises:
a first support for contacting a first external surface of the
cuvette; a second support for contacting a second external surface
of the cuvette; and a clamp for urging the first support and the
second support against the first and second external surfaces of
the cuvette.
30. The apparatus of claim 29 wherein the first support and/or the
second support includes a window.
31. The apparatus of claim 1 wherein the front plumbing block
comprises an inlet port, an inlet channel, a front plenum, an
outlet channel, and an outlet port.
32. The apparatus of claim 1 further including a rear plumbing
block in fluid communication with the gap region.
33. The apparatus of claim 32 wherein the rear plumbing block
comprises a rear plenum, a rear plenum exit channel, a waste valve
port, a weir, waste channel, a fill channel, and a fill port.
34. The apparatus of claim 33 wherein the weir has a height such
that there is substantially no pressure drop between the inlets and
outlets end of the capillary tubes.
35. The apparatus of claim 33 further including an electrode
reservoir in fluid communication with the waste valve port and the
weir.
36. The apparatus of claim 35 further including any electrode
located in the electrode reservoir.
37. A multi-channel capillary electrophoresis apparatus of a type
including a capillary array assembly comprising a plurality of
capillaries, each capillary having a capillary outlet, and outlet
support for supporting the capillary outlets, the apparatus
comprising: a cuvette defining a receiving slot, a gap region, and
a detection zone; where the receiving slot is adapted to removably
receive the outlet support, wherein when the outlet support is
inserted into the receiving slot, the capillary outlets are
positioned in the gap region in proximity to the detection zone,
and a flow channel is formed by the outlet support and the
receiving slot such that the flow channel is in fluid communication
with the gap region.
38. A multi-channel capillary electrophoresis apparatus of a type
having a cuvette defining a receiving slot, a gap region, and a
detection zone, where the receiving slot is adapted to removably
receive a capillary array, the apparatus comprising: a capillary
array assembly comprising a plurality of capillaries, each
capillary having a capillary outlet, and an outlet support for
supporting the capillary outlets, wherein when the outlet support
is inserted into the receiving slot, the capillary outlets are
positioned in the gap region in proximity to the detection zone,
and a flow channel is formed by the outlet support and the
receiving slot such that the flow channel is in fluid communication
with the gap region.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/621,071 filed Jul. 21, 2000, which is a divisional of
application Ser. No. 09/151,928 filed Sep. 11, 1998, now U.S. Pat.
No. 6,162,341, all of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention relates to apparatus and methods useful for
biochemical analysis. More specifically, this invention relates to
a highly automated capillary electrophoresis apparatus for the
simultaneous analysis of multiple samples, and methods for using
such apparatus.
BACKGROUND
[0003] Capillary electrophoresis (CE) is a powerful analytical
separation technique that brings speed, quantitation,
reproducibility and automation to the inherently highly resolving
but typically labor intensive methods of electrophoresis (e.g.,
Capillary Electrophoresis Theory and Practice, Grossman and
Colburn, eds., Academic Press (1992)). While early capillary
electrophoresis systems utilized only a single capillary tube,
multi-capillary systems have been developed to provide increased
throughput (e.g., Mathies et al., U.S. Pat. No. 5,247,240; Dovichi
and Zhang, U.S. Pat. No. 5,439,578; kambara, U.S. Pat. No.
5,516,406; Takahashi, et. al., Anal. Chem., 66, 1021-1026 (1994)).
Such multi-capillary CE systems are particularly attractive for use
in large scale DNA sequencing projects.
[0004] However, existing multi-channel capillary electrophoresis
systems have several significant shortcomings that limit their
utility, particularly for applications requiring a high degree of
automation throughput, detection-sensitivity and reliability. For
example, existing systems do not provide for a sheath-flow
detection cuvette wherein a capillary array may be replaced by a
user without extensive disassembly of the cuvette. In addition,
existing systems do not provide for a sheath-flow detection cuvette
wherein fresh separation media and/or capillary wash solutions may
be introduced into outlets of the capillary tubes under high
pressure. Thus, there remains a continuing need for an automated
multi-channel capillary electrophoresis device including these
features.
SUMMARY
[0005] The present invention is directed towards our discovery of a
multi-channel capillary electrophoresis device including a
sheath-flow detection cuvette wherein a capillary array is easily
replaceable by a user, and wherein fresh separation media and/or
capillary wash solutions may be introduced into outlets of the
capillary tubes under high pressure.
[0006] In a first aspect, the invention comprises a multi-channel
capillary electrophoresis apparatus. The apparatus includes a
capillary array assembly comprising a plurality of capillaries,
each capillary having an outlet, and an outlet support for
supporting the capillary outlets. In addition, the apparatus
includes a cuvette defining a receiving slot, a gap region, and a
detection zone, where the receiving slot of the cuvette is adapted
to removably receive the outlet support. When the outlet support is
inserted into the receiving slot, the capillary outlets are
positioned in the gap region in proximity to the detection zone,
and a flow channel is formed by the outlet support and the
receiving slot such that the flow channel is in fluid communication
with the gap region. The apparatus further includes a plumbing
block in fluid communication with the flow channel for supplying a
fluid flow through the gap region sufficient to transport material
downstream from the capillary outlets to the detection zone.
[0007] These and other features and advantages of the present
invention will become better understood with reference to the
following description, drawings, and appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows a persoective view of a preferred capillary
array assembly with an outlet support in the foreground.
[0009] FIG. 2 shows a perspective view of a preferred capillary
array assembly with an inlet support in the foreground.
[0010] FIG. 3 shows a cross-section of a preferred capillary
tube.
[0011] FIG. 4 shows a perspective view of a preferred inlet
support.
[0012] FIG. 5 shows a persoective view of a preferred inlet support
wherein the inlet support is registered with respect to a frame
member.
[0013] FIG. 6 shows a side view of a preferred inlet support.
[0014] FIG. 7 shows a perspective view of a preferred outlet
support.
[0015] FIGS. 8a, 8b and 8c show an expanded perspective view of a
platform portion of a preferred outlet support.
[0016] FIGS. 9a and 9b show a side view of a platform portion of a
preferred outlet support.
[0017] FIG. 10 shows a side view of an alternative platform portion
of a preferred outlet support.
[0018] FIG. 11 shows a perspective view of a preferred cuvette with
a receiving slot in the foreground.
[0019] FIG. 12 shows a side view of a preferred cuvette.
[0020] FIG. 13 shows a perspective view of a preferred cuvette with
a gap region in the foreground.
[0021] FIG. 14 shows a front view of a preferred cuvette located in
a clamping block of the present invention.
[0022] FIG. 15 shows a perspective view of a preferred front
plumbing block.
[0023] FIG. 16 shows a cutaway view of a preferred front plumbing
block and a cuvette located behind the front plumbing block.
[0024] FIG. 17 shows a cross-section of a preferred operation
combination of an outlet support, front plumbing block, and
cuvette.
[0025] FIG. 18 shows a cross-section through the front plumbing
block of FIG. 17.
[0026] FIG. 19 shows a cross-section through the cuvette and
platform portion of the outlet support of FIG. 17.
[0027] FIG. 20 shows a see-through view of a preferred rear
plumbing block.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] Reference will now be made in detail to several preferred
embodiments of the invention, examples of which are illustrated in
the accompanying drawings. While the invention will be described in
conjunction with these preferred embodiments, it will be understood
that they are not intended to limit the invention to these
embodiments. On the contrary, the invention is intended to cover
alternatives, modifications, and equivalents, that may be included
within the invention as defined by the appended claims.
[0029] Generally, the present invention is directed to a
multi-channel capillary electrophoresis device comprising a
capillary array assembly and a cuvette assembly for use in a
sheath-flow detection system wherein the capillary array assembly
is adapted to be removably inserted into the cuvette assembly such
that the capillary array assembly may be easily replaced by a user.
In addltion, the cuvette is adapted to permit a high-pressure flow
of fluid through the cuvette into outlets of the capillary tubes
for filling the capillary tubes with fresh separation medium and/or
wash solutions.
[0030] I. Definitions
[0031] Unless stated otherwise, the following terms and phrases as
used herein are intended to have the following meanings:
[0032] "Sheath-flow detection system" means a detection system
wherein a sample is detected outside of a separation capillary
after being transported from an outlet of such capillary into a
detection zone by a flow of a "sheath fluid" (e.g., Cheng and
Dovichi, Science 242: 5602-564 (1988); Kambara and Takahashi,
Nature 361:565-566 (1993)). The sheath fluid may be any fluid
capable of supporting a sample eluting from the capillary outlet.
Preferred sheath fluids include aqueous buffers, both with and
without polymers dissolved therein. In a particularly preferred
embodiment of the present invention, the sheath fluid is the
separation medium used to effect the electrophoretic separation in
the capillary tubes, e.g., a flowable solution containing an
un-crosslinked polymer.
[0033] "Separation medium" refers to a medium located within the
lumen of a capillary tube within which an electrophoretic
separation is conducted. Exemplary separation media include
crosslinked gels, un-crosslinked polymer solutions, or polymer-free
solvents, e.g., buffered water. Optionally, separation media may
include denaturants such as detergents, e.g., SDS, or organics,
e.g., urea, formamide, or pyrrolidinone.
[0034] II. Capillary Array Assembly
[0035] The capillary array assembly of the present invention
provides a means for arranging an array of capillary
electrophoresis tubes in an automated multi-channel capillary
electrophoresis system. More particularly, the capillary array
assembly (1) allows the capillary array to be easily removed from
and introduced into the capillary electrophoresis system, e.g., to
facilitate replacement of the capillary tubes, (2) provides an
interface between the capillary tubes and a sheath-flow detection
system, (3) facilitates alignment of the capillary outlets with an
optical detection system, and (4) effects alignment of the
capillary inlets with sample reservoirs.
[0036] Generally, the capillary array assembly of the present
invention comprises (1) a plurality of capillary tubes, (2) an
inlet support for supporting inlet ends of the capillaries in
registration wiih a pluraty of sample reservoirs, and (3) an outlet
support for supporting oulet ends of the capillaries and for
locating such outlet ends with respect to a sheath-flow detection
system. FIGS. 1 and 2 show perspective views of a preferred
embodiment of a capillary assay assembly according to the present
invention having 104 capillary tubes 5, an inlet support 10, and an
outlet support 15.
[0037] 1. Capillaries. The capillaries of the present invention are
tubes or channels or other structure capable of supporting a volume
of separation medium suitable for carrying out an electrophoretic
separation. The geometry of a capillary may vary widely and
includes tubes with circular, rectangular or square cross-sections,
channels, groves, plates, and the like, and may be fabricated by a
wide range of well known technologies. An important feature of a
capillary for use with the invention is the surface-to-volume ratio
of the capillary lumen. High values of this ratio permit efficient
dissipation of the Joule heat produced in the separation medium
during electrophoresis. Preferably, ratios in the range of about
0.4 to 0.04 .mu.m.sup.-1 are employed. These ratio values
correspond to the surface-to-volume ratios of tubular capillaries
with circular cross-secuons having inside diameters in the ranoge
of about 10 .mu.m to about 100 .mu.m.
[0038] Preferably, capillaries for use with the invention are made
of silica, fused silica, quartz, silicate-based glass, such as
borosilicare glass, phosphate glass, alumina-containing glass, or
other silica-like materials, or from plastics, e.g., polycarbonate
or acrylic.
[0039] Where the capillaries are in the form of discrete capillary
tubes, e.g., fused silica capillary tubes, preferably the outside
surfaces of the capillaries are coated with a material to protect
the capillaries from breakage, e.g., a polyimide, Teflon, acrylic
or other polymeric coating (e.g., Polymicro Technologies, AZ).
However, as will be discussed in more detail below, if the
capillaries have a coating or cladding on their outside walls, and
the properties of the coating are such that the coating interferes
with the detection process, e.g., fluorescent detection is used and
the coating material is fluorescent, the coating should be removed
adjacent to the capillary outlets, e.g., by laser ablation.
[0040] Referring to FIG. 3, the capillary tubes of a preferred
embodiment of the invention are characterized by a lumen 20 having
an inner radius r.sub.1, a wall 25 having a thickness
r.sub.2-r.sub.1, an outer coaling 30 having a thickness
r.sub.3-r.sub.2, and a length L. Preferably the inner radius is
between about 5 .mu.m and 100 .mu.m, the wall has a thickness of
between about 20 .mu.m and about 150 .mu.m, and the outer coating
has a thickness of between about 2 .mu.m and 10 .mu.m. Preferred
outer coatings include polyimide, Teflon, acrylic, and the like.
The preferred length of the capillary tubes used in the present
invention will depend upon the speed and resolution of the
separation required in a particular application. Generally, as the
capillary length is increased, the resolution of the separation is
increased while the speed of the separation is decreased. However,
typically the capillaries will be between about 10 cm and 100 cm in
length.
[0041] To increase the throughput of the capillary electrophoresis
device of the present invention, a plurality of capillaries are
used. Preferably between about 10 to 1000 capillaries, and more
preferably between about 20 and 200 capillaries are used.
[0042] The capillaries may be multiple individual capillary tubes,
as shown in FIGS. 1 and 2, or they may be formed in a monolithic
substrate, e.g., in a micromachined device (e.g., Soane and Soane,
U.S. Pat. No. 5,750,015; and Mathies et al., Analytical Chemistry,
69: 2181-2186 (1997)). Preferably, in the present invention, the
capillary tubes are individual capillary tubes formed from fused
silica having an outside surface coated with a polyimide
coating.
[0043] 2. Inlet support. The inlet support of the capillary array
assembly of the present invention serves to position the capillary
inlets in registration with sample reservoirs containing samples to
be analyzed. Such registration is required to effect efficient and
reproducible injection of samples into each capillary of the
capillary array.
[0044] FIG. 4 shows a perspective view of a preferred embodiment of
an inlet support 10 of the present invention including capillary
tubes 5 mounted therein. The inlet support of this preferred
embodiment comprises a body 35, registration features 40 and 45,
and upper 55 and lower 50 capillary alignment grooves.
[0045] Registration features 40 and 45 serve to align the inlet
support with respect to sample reservoirs containing samples to be
analyzed. In particular, registration features 40 serve to align
the inlet support with respect to a contact surface 41 associated
with the sample reservoirs 70, and registration features 45 serve
to guide the inlet support into a proper position with respect to
the sample wells. Registration features 40 determine the length of
the capillary tubes that enter into the sample wells. FIG. 5 shows
the inlet support 10 fitted into frame members 65 with which the
registration features 45 are engaged.
[0046] Alignment grooves 50 and 55 serve to hold inlets 60 of
capillaries 5 in a defined and fixed position relative to the body
35 of the inlet support. Preferably, the alignment grooves have a
V-shape to more accurately locate the capillary tubes therein. The
pitch of the grooves may be any pitch that conforms to a pitch of
the sample reservoirs to be addressed. However, it is preferred
that the pitch of the grooves be an integral fraction of 9 mm in
order to effect registration of a multi-channel pipette with both
sample reservoirs arranged in a traditional 96-well microtiter
plate configuration and the capillary inlets. Thus, exemplary
preferred pitches are 9 mm, 9/2 mm, 9/3 mm, etc. The alignment
grooves of the inlet support shown in FIG. 4 have a two-tier
configuration comprising an upper tier of V-grooves 55 and a lower
tier of V-grooves 50. This multi-tier configuration is advantageous
because it allows for more wells to be located in a given linear
dimension. This is important because the maximum spacing of the
capillary inlets is constrained by the spacing of the capillary
outlets, and the spacing of the capillary outlets is typically made
as small as possible to facilitate sample detection. In addition,
the multi-tier arrangement facilitates access to the sample wells
by a sample delivery device, e.g., a robotically-controlled
micro-pipette.
[0047] Preferably, to secure the capillary inlets into the
alignment grooves 50 and 55, portions of the capillaries adjacent
to the inlets are potted into the alignment grooves with a potting
agent (not shown). Particularly preferred potting agents include
epoxy and silicone adhesives.
[0048] As most clearly illustrated in FIG. 6, the capillary inlets
are positioned in the inlet support such that the capillary inlets
60 are suspended away from the body of the inlet support in order
to facilitate insertion of the inlets into sample reservoirs
70.
[0049] 3. Outlet Support. The outlet support of the present
invention performs a number of important functions including (1)
aligning the capillary outlets with respect to an optical detection
system, (2) aligning the capillary outlets with respect to a
sheath-flow fluid delivery system such that a sheath fluid carries
sample material from the capillary outlets into a sheath-flow
stream, (3) creating a pressure-tight seal between the capillary
tubes of the capillary array and the sheath-flow flow delivery
system, and (4) providing a mechanism whereby the capillaries may
be easily replaced by a user.
[0050] A preferred outlet support 15 of the present invention is
shown in FIGS. 7-10. The major components of this preferred outlet
support include a base 75 and a platform 80. The base 75 includes
guide-holes 85 into which guide-pins (not show) may be inserted in
order to locate the outlet support with respect to a cuvette
assembly. The base further includes fasteners 90 to securely attach
the outlet support to the cuvette assembly and create a
pressure-tight seal between the cuvette assembly and the outlet
support. Preferably, these fasteners are stainless steel thumb
screws. A front face 95 of the base further includes a sealing
member 100 to form a pressure-tight seal between the outlet support
and the cuvette assembly to which it mates such that the platform
80 is circumscribed by the sealing member. The sealing member is
preferably an o-ring formed from an elastomeric polymer, e.g.,
ethylene-propylene rubber, or a fluoroelastomer, e.g., Viton. The
o-ring is located in an o-ring registration groove 106 to precisely
locate the o-ring with respect to the platform.
[0051] Details of the platform 80 of the preferred outlet support
are shown in FIGS. 8-10. The platform 80 includes a support surface
105 includes grooves 110 located thereon to precisely position
capillary outlets 61 with respect to the platform and to each
other. As was the case for the inlet support, the capillaries are
preferably potted into the grooves of the outlet support with a
potting material (not shown). Preferably, the platform holds the
capillary outlets 61 in 2 linear array such that the capillary
outlets are located on a line perpendicular to a longitudinal axis
of the capillary outlets. This arrangement provides for
simultaneous "side" illumination of a detection zone proximate to
the capillary outlets using a single stationary light beam.
[0052] The platform 80 further includes guide rails 125 and chamfer
features 130 and 135 that serve to effect smooth insertion of the
platform into a receiving slot of a cuvette assembly. As most
clearly shown in FIG. 19, the guide rails further serve to define
an upper flow path 140 and a lower flow path 145 above and below
the support surface 105 of the platform when the platform is
inserted into a receiving slot 190 of the cuvette 150. Guide rails
125 include a bottom surface 155 for contact with an interior
surface 160 of the cuvette. To ensure rigid positioning of the
platform within the cuvette the contours of the bottom surface 155
should be such that the bottom surface is in intimate contact with
the interior surface 160 of the cuvette over a substantial portion
of the length of the guide rail.
[0053] While the figures show only two guide rails, one on each
edge of the platform, more guide rails may be used, e.g., one or
more central rails running along the top and/or bottom center
portion of the platform parallel to the peripheral guide rails 125.
In a preferred embodiment, the top or bottom central rails are made
from a resilient material such that the rails cause the platform to
be securely seated in the receiving slot. These additional rails
may serve to inhibit bowing of the platform when in the receiving
slot. Such bowing could be disadvantageous because it could cause a
lack of uniformity of the flow of sheath fluid across the platform
and loss of optical alignment of the capillary outlets,
particularly as the width of the platform is increased.
[0054] As most clearly illustrated in FIGS. 9 and 10, a top surface
165 of the guide rail preferably includes one or more flexure
features 170 for producing a compressive force between the interior
surface 160 of the receiving slot of the cuvette and the platform
thereby fixedly positioning the platform within the receiving slot.
The flexure feature 170 of the preferred embodiment comprises a
compressible protrusion located on the top surface 165 of the guide
rail. This flexure feature may be formed by molding or machining
the top surface of the guide rail.
[0055] To facilitate insertion of the platform 80 into the
receiving slot 190 of the cuvette 150, the guide rails 125 may
further include chamfer features 130 and 135. These chamfer
features serve to guide the platform into the receiving slot such
that there is a reduced likelihood that the capillary outlets will
be mispositioned or broken during the insertion of the outlet
support into the receiving slot. In a preferred embodiment depicted
in FIGS. 9a and 9b, the chamfer feature consists of an upper
chamfer surface 135 and a lower chamfer surface 130. An angle
.theta..sub.1 between the bottom surface 155 and the lower chamfer
surface 130, and an angle .theta..sub.2 between the top surface 165
and the upper chamfer surface 135 preferably ranges between about
10 and 80 degrees. More preferably, .theta..sub.1 ranges between
about 20 and 70 degrees, and .theta..sub.2 ranges between about 20
and 70 degrees.
[0056] In an alternative embodiment of the platform portion of the
outlet support shown in FIG. 10, the platform is divided into a
first portion 80a and a second portion 80b, wherein the first and
second portions are connected by the capillary tubes themselves. In
this configuration, the capillary tubes act as a flexible hinge
allowing second portion 80b to align itself in the receiving slot
with reduced interference from the base 75 or the first portion
80a, thereby facilitating the positioning of the platform in the
receiving slot.
[0057] II. Cuvette Assembly
[0058] The cuvette assembly of the present invention provides a
sheath-flow detection cell that operates in concert with the outlet
support of the capillary array assembly to provide for easy removal
and insertion of the capillary array. Specifically, the cuvette
assembly provides (1) a detection zone within which to perform
simultaneous optical measurements of material eluting from outlets
of a plurality of capillary electrophoresis tubes with a minimum of
light scattering or other optical nonidealities, (2) a sheath-flow
cell including means for providing a sheath fluid, (3) means for
introducing fluids into the capillary outlets under high pressure,
e.g., to wash the interior of the capillary tubes and/or to
introduce fresh electrophoretic separation media into the capillary
tubes, and (4) means for removably mounting the capillary array
assembly into the cuvette assembly.
[0059] Generally, a cuvette assembly in accordance with preferred
embodiment of the invention comprises (1) a cuvette to receive the
outlet support of the capillary array and within which to perform
optical measurements, (2) a clamping block for providing support
for the cuvette, and (3) a plumbing block with associated fluidics
for conducting fluids into and out of the cuvette.
[0060] 1. The Cuvette. A cuvette 180 of the preferred embodiment of
the invention is shown in FIGS. 11-13. The cuvette 180 comprises a
body 185, a receiving slot 190 for removably receiving the platform
80 of the outlet support 15 and for forming flow channel 140 and
145 in cooperation therewith, and a gap region 195 within which to
perform an optical measurement.
[0061] In an important feature of the present invention, an
interior surface 160 of the receiving slot 190 is adapted to
confrom with the guide rails 125 of the platform 80 such that when
the platform is removably inserted into the receiving slot the
platform is securely seated therein, where, as used herein, the
term "removably inserted" or "removably received" means that the
platform is received by the receiving slot of the cuvette in such a
way that the platform may be removed by a user without disassembly
of the cuvette or capillary array. In particular, the interior
surface 160 of the receiving slot 190 includes a surface for
contacting a bottom surface 155 of the guide rails, and a
flexure-contact surface for contacting a top surface 165 of the
guide rails, and for contacting the flexure-features 170 located on
the of the top surface 165 of the guide rails. When the platform is
securely seated in the receiving slot, upper 140 and lower 145 flow
paths are formed.
[0062] The gap region 195 comprises a channel that is in fluid
communication with the receiving, slot 190. Thus, an inlet-end 210
of the gap region connects with the receiving slot and an
outlet-end 215 of the gap region is distal to the receiving slot.
Preferably, the gap region 195 has a vertical dimension that is
smaller than that of the receiving slot, where, the vertical
dimension is orthogonal to the plane of the array of capillary
tubes. Preferably, the vertical dimension of the gap region is
approximately equal to the outside diameter of the capillary tubes.
Thus, typically the vertical dimension of the gap region 195 is
between about 100 .mu.m to about 1000 .mu.m.
[0063] The gap region 195 further includes detection zone 220 in
which samples emerging from the outlets of the capillary tubes are
detected by a detector 227, e.g., a photomultiplier tube (PMT),
charged coupled device (CCD), and the like, and, if fluorescent
detection is used, where the samples are excited with a light beam.
The detection zone is located adjacent to the capillary outlets.
The location of the detection zone with respect to the capillary
outlets should be far enough away from the capillary outlets so as
to reduce light scattering caused by the capillary tubes, but, not
so far away from the capillary outlets that samples emerging from
the capillary tubes are excessively diluted and/or deformed by the
sheath flow such that a band profile is substantially distorted
thereby leading to a loss of resolution. Preferably, the detection
zone 220 is located between about 20 .mu.m and 2000 .mu.m from the
capillary outlets, and more preferably, between about 100 and 500
.mu.m from the capillary outlets.
[0064] In an important feature of the gap region 195 of the
cuvette, at least one of the walls of the cuvette 180 bounding the
detection zone 220 of the gap region includes a window that is
substantially transparent to light so as to provide optical
communication between the detection zone and a detector located
outside of the cuvette thereby facilitating detection of samples
emerging from the capillary tubes into the gap region. For example,
as shown in FIG. 17, a top wall 225 of the cuvette bounding the
detection zone contains a window 226 that is transparent to light.
Where fluorescence detection is used, it is also preferred that one
or both side walls of the cuvette, 230 and/or 235, also include a
window that is transparent to light to allow for an excitation
radiation to enter the detection zone in the plane of the capillary
outlets, e.g., a laser beam or other light beam, to effect
fluorescence excitation of samples in the detection zone.
[0065] In a particularly preferred embodiment, the entire cuvette
is formed from a rigid, chemically inert and optically transparent
material, e.g., glass, quartz or fused silica.
[0066] 2. Clamping Block. In another significant aspect of the
preferred embodiment of the present invention, exterior walls of
the cuvette are supported by a clamping block. The purpose of the
clamping block is to restrain exterior surfaces of the cuvette in
order to prevent bowing and/or deflection of such surfaces due to
high pressures within the cuvette. Such bowing and/or deflection is
disadvantageous because it can lead to tensile stresses within the
cuvette e.g., at corners, that may cause mechanical failure of the
cuvette.
[0067] In a preferred embodiment of the clamping block of the
present invention, the clamping block comprises a first support for
contacting a first external surface of the cuvette, a second
support for contacting a second external surface of the cuvette,
and a clamp for urging the first support and the second support
against the first and second external surfaces of the cuvette.
[0068] A front view of a cuvette 180 located in a clamping block
308 is shown in FIG. 14. The clamping block depicted in FIG. 14
includes bottom support 310 for supporting a bottom surface 311 of
the cuvette 180, a top support 325 for supporting a top surface 312
of the cuvette, and biasing means 320 for providing a compressive
force for urging the top support, cuvette and bottom support
together. Preferably, the top support 325 includes a window for
providing optical communication between a surface of the cuvette
and a detector and/or height source located proximate to the top
support. For example, the top support may be made from optical
quality glass, e.g., BK7 glass.
[0069] The compressive force supplied by the biasing means 320
should be greater than the lifting force generated by the internal
operating pressure of the cuvette. In the preferred embodiment
described here, typical compressive forces range from about 50 lbs.
to about 400 lbs..
[0070] 3. Plumbing Block and Associated Fluidics
[0071] In another important aspect of the cuvette assembly of the
preferred embodiment, the cuvette assembly includes a plumbing
block (1) for controlling the flow of sheath fluids through the
cuvette, and (2) for directing fluids into the outlets of the
capillary tubes making up the capillary array, e.g., solutions for
washing the capillary tubes and/or fresh separation media. The
plumbing block is located such that it is in fluid communication
with the receiving slot 190 and the gap region 195 of the cuvette
180.
[0072] A preferred plumbing block according to the present
invention is composed of a front plumbing block and a rear plumbing
block wherein the front plumbing block abuts and is in fluid
communication with an inlet 313 of the receiving slot of the
cuvette, and the rear plumbing block abuts and is in fluid
communication with an outlet 215 of the gap region of the
cuvette.
[0073] Various views of a front plumbing block 239 of a preferred
embodiment of the invention are shown in FIGS. 15 and 16. Referring
to FIG. 15, the front plumbing block 239 comprises an inlet port
240, an inlet channel 245, a front plenum 250, an outlet channel
255, and an outlet port 260. The front plenum includes an entrance
slot 251 and an exit slot 252. The inlet port 240 and inlet channel
245 serve to conduct fluid into the front plenum 250 of the front
plumbing block. An inlet valve (not shown) may be included to
regulate the flow of fluid into and through the inlet channel 245.
The outlet channel 255 and outlet port 260 serve to conduct fluid
from the front plenum 250 and out of the front plumbing block to
facilitate flushing fluid out of the cuvette and associated fluid
passages. An outlet valve (not shown) may be included to regulate
the flow of fluid into and through the outlet channel.
[0074] FIG. 16 shows the spatial relationship between the cuvette
180 and the front plumbing block 239. As indicated in the figure,
the entrance 313 to the receiving slot 190 of the cuvette 180 abuts
and is fluid communication with the exit slot 252 of the front
plenum. Thus, fluid leaving the front plenum 250 through the exit
slot 252 will enter the entrance 313 to the receiving slot 190 of
the cuvette 180.
[0075] FIGS. 17-19 show various views of the spatial relationship
among the platform 80 of the outlet support 15, the front plumbing
block 239, and the cuvette 180 of a preferred embodiment of the
invention when the platform is inserted through the front plumbing
block and into the receiving slot of the cuvette.
[0076] Thus, as can be seen in the figures, fluid enters the front
plumbing block 239 through the inlet channel 249 and into the front
plenum 250 of the front plumbing block. Fluid is prevented from
leaving the front plenum through the entrance slot 251 of the front
plenum by the seal formed between the front face 95 of the base 75
of the outlet support and the front plumbing block 239. As most
clearly shown in FIGS. 18 and 19, fluid then leaves the front
plenum 250 through exit slot 252 of the front plenum and enters the
receiving slot 190 through upper 140 and lower 145 flow channels
formed between the guide rails 125, the interior surface of the
receiving slot 190 and the support surface 105 of the platform 80.
Fluid then flows out of the receiving slot into the gap region 195
of the cuvette, around the outlets 61 of the capillary tubes 5 and
out of the gap region through the outlet end 215 of the gap
region.
[0077] In a preferred embodiment, the plumbing block further
includes a rear plumbing block abutting and in fluid communication
with the outlet 215 of the gap region 195 of the cuvette 180. A
see-through view of the rear plumbing block of a preferred
embodiment is shown in FIG. 20. The rear plumbing block 265
includes rear plenum 270, rear plenum exit channel 275, waste valve
port 280 with an associated waste valve (not shown), weir 285, weir
head space 286, waste channel 290, fill channel 295, and fill port
300 with associated fill valve (not shown). The rear plenum
includes an entrance slot 271 that is in fluid communication with
the outlet 215 of the gap region 195 of the cuvette 180. The waste
valve port generally includes a waste valve (not shown) located
therein such that flow between the plenum exit channel 275 and the
weir 285 may be controlled. The weir head space 286 is open to
atmosphere to ensure atmospheric pressure in the weir. The rear
plenum serves to conduct fluid leaving the gap region of the
cuvette into the rear plumbing block such that the pressure at the
capillary outlets is uniform across the capillary array, i.e., the
pressure at each of the capillary tube outlets is substantially the
same. The rear plenum exit channel 275 serves to conduct fluid out
of the rear plenum 270 and into the waste valve port 280.
[0078] In an important feature of the present invention, the height
of the weir 285 is adjusted so as to substantially eliminate any
pressure drop between the inlet and outlet ends of the capillary
tubes. This is important because any pressure drop across the
capillaries may cause a pressure-driven flow having a parabolic
flow profile that can lead to a substantial loss of electrophoretic
resolution. Thus, the height of the weir is preferably set such
that an hydraulic elevation at the top of the weir is approximately
equal to an hydraulic elevation of the fluid in which the capillary
inlets are submerged. In certain circumstances, it may be
preferable to set the hydraulic elevation of the weir slightly less
than the hydraulic elevation at the capillary inlet in order to
take into account any pressure caused by the flow of the sheath
fluid. As used herein, the term "hydraulic elevation" refers to a
distance normal to the earth's surface above a reference
elevation.
[0079] Fill channel 295 and fill port 300 serve to provide means
for flowing a fluid into the rear plenum 270 of the rear plumbing
block 265, and into the gap region 195 of the cuvette, and into the
outlets or the capillary tubes. A fill valve (not shown) is
positioned adjacent to the fill port to control flow therethrough.
Typically, the fill port and fill channel are used to conduct fresh
separation media and/or wash solutions into the outlets of the
capillary tubes.
[0080] The fluid is driven into the fill port by a pumping system.
Preferably, the pumping system is capable of high-pressure
operation, i.e., above 200 psi, and is constructed such that
materials used in parts of the pump that contact the fluid are
formed from materials that are chemically inert to common fluids
used in the system, e.g., water, acid, and organic solvents.
Preferred materials include glass, and certain plastics, e.g.
Teflon and Kel-F. In addition, preferably the pumping system
includes a pressure sensor for monitoring the output pressure of
the pump, and a multi-port distribution valve. Exemplary components
of a preferred pumping system include a Cavro Model XL 3000 syringe
pump, an Entran Model EPX-VO pressure sensor, and a Rheodyne RV
Series motorized multi-position valve.
[0081] Preferably, the rear plumbing block 263 serves to house an
electrode (not shown) that is positioned in an electrode reservoir
284 located between the waste valve port 280 and the weir 285. The
electrode is in electrical communication with the capillary outlets
61. The electrode reservoir is preferably vented to atmosphere to
eliminate any back-pressure caused by the build up of gases formed
by electrolysis at the electrode during electrophoresis.
[0082] During electrophoresis, sheath fluid is conducted through
the plumbing block and the cuvette assembly as follows. The inlet
valve associated with the inlet port 240 is open, the outlet valve
associated with outlet port 260 is closed, the fill valve
associated with the fill port 300 is closed, and the waste valve
associated with the waste valve port 280 is open. Thus, during
electrophoresis, the sheath fluid is pumped into the front plumbing
block through the inlet port 240, flows through the inlet channel
245, into the front plenum 250, out the front plenum exit slot 252,
through the receiving slot 190 of the cuvette, across the support
surface 105 of the platform 80, into the gap region 195 of the
cuvette 180, past the capillary outlets 61, into the rear plenum
270, through the rear plenum exit channel 275, over the weir 285,
and out the waste channel 290. Note that in a particularly
preferred embodiment of the invention, in order to eliminate flow
and/or electrical discontinuities in the system, the sheath fluid
and the separation medium are the same material, e.g., a flowable
non-crosslinked polymer solution.
[0083] When a fluid is being introduced into the capillary tubes
through the capillary outlets, e.g., when the capillary tubes are
being filled with fresh separation media or washed/regenerated with
a wash solution, e.g., nitric acid or sodium hydroxide, the valves
are positioned as follows. The inlet valve associated with the
inlet port 240 is closed, the outlet valve associated with outlet
port 260 is closed, the fill valve associated with the fill port
300 is open, and the waste valve associated with the waste valve
port 280 is closed. Thus, fresh separation medium is directed into
the fluid port 300, through the fill channel 295, into the rear
plenum 270, through the gap region 195 and into the capillary
outlets 61, through the capillary tubes 5, and out the capillary
inlets 60. Alternatively, when the cuvette is being flushed to
exchange fluids, remove bubbles or simply wash the cuvette, each of
the valves is positioned as above, with the exception that the
outlet valve associated with outlet port 260 is open.
[0084] IV. Additional Features
[0085] The electrophoresis device of the invention course also
includes other elements required to conduct a capillary
electrophoresis process, e.g., electrodes in electrical
communication with the capillary inlets, a power supply connected
to the electrodes for creating an electrical field within the lumen
of the capillaries, optionally a computer to control the functions
of the device and for data collection and analysis, a detector for
detecting samples in the cuvette, and a temperature control device
for controlling the temperature of the capillary tubes and cuvette.
Details of these and other common features of an operable capillary
electrophoresis device may be found in any number of available
publications, e.g., Capillary Electrophoresis Theory and Practice,
Grossman and Colbur, eds., Academic Press (1992).
[0086] All publications and patent applications referred to are
herein incorporated by reference to the same extent as if each
individual publication or patent application was specifically and
individually indicated to be incorporated by reference.
[0087] Although only a few embodiments have been described in
detail above, those having ordinary skill in the analytical
chemistry art will clearly understand that many modifications are
possible in the preferred embodiment without departing from the
teachings thereof. All such modifications are intended to be
encompassed within the following claims.
* * * * *