U.S. patent application number 10/227164 was filed with the patent office on 2003-06-26 for flow cell for synthesis of arrays of dna probes and the like.
This patent application is currently assigned to Wisconsin Alumni Research Foundation. Invention is credited to Green, Roland D., Singh-Gasson, Sangeet, Yue, Yongjian.
Application Number | 20030118485 10/227164 |
Document ID | / |
Family ID | 23736133 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030118485 |
Kind Code |
A1 |
Singh-Gasson, Sangeet ; et
al. |
June 26, 2003 |
Flow cell for synthesis of arrays of DNA probes and the like
Abstract
A flow cell which can be used in the synthesis of DNA probes on
an active surface of the substrate includes a base having a central
window opening and a registration surface against which the
substrate may be mounted. A gasket having a central opening
defining an active area is mounted on the active surface of the
substrate and has inlet and output extension openings which extend
away from the central opening. A press block is engaged against the
gasket to fully enclose an active volume between the press block,
the peripheral walls of the central opening in the gasket, and the
active surface of the substrate. A press is mounted to the base to
selectively press the press block against the gasket and hold it in
position. Channels in the press block extend to the extension
openings in the gasket to allow flow of reagent into and out of the
active volume, which is confined to the thickness of the gasket and
can thus be minimized. The press may include a press screw which
can be turned to engage against the press block to hold it into
position, and which can be turned to release the press block from
the gasket, allowing rapid and easy replacement of substrates.
Inventors: |
Singh-Gasson, Sangeet;
(Mundelein, IL) ; Yue, Yongjian; (Chengdu, CN)
; Green, Roland D.; (Madison, WI) |
Correspondence
Address: |
FOLEY & LARDNER
150 EAST GILMAN STREET
P.O. BOX 1497
MADISON
WI
53701-1497
US
|
Assignee: |
Wisconsin Alumni Research
Foundation
|
Family ID: |
23736133 |
Appl. No.: |
10/227164 |
Filed: |
August 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10227164 |
Aug 23, 2002 |
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10053368 |
Nov 9, 2001 |
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6444175 |
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10053368 |
Nov 9, 2001 |
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09437369 |
Nov 10, 1999 |
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6315958 |
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Current U.S.
Class: |
422/400 |
Current CPC
Class: |
G01N 35/0098 20130101;
C40B 40/06 20130101; B01J 2219/00596 20130101; B01J 2219/00612
20130101; C40B 60/14 20130101; B01J 2219/00281 20130101; B01J
2219/00605 20130101; G01N 2035/00158 20130101; B01J 19/0046
20130101; B01J 2219/00722 20130101; B01J 2219/00527 20130101; B01J
2219/00351 20130101 |
Class at
Publication: |
422/102 |
International
Class: |
B01L 003/00 |
Claims
What is claimed is:
1. A flow cell of the type that may be used in the synthesis of
arrays of DNA probes and the like, comprising: (a) a transparent
substrate with two parallel flat surfaces, one of the surfaces
forming an active surface of the substrate; (b) a base having a
central window opening and a registration surface against which the
substrate is mounted; (c) a gasket mounted on the active surface of
the substrate and having a central opening defining an active area
surrounded by material of the gasket, an inlet extension opening
extending from the central opening of the gasket, and an outlet
extension opening extending from the central opening of the gasket;
and (d) press block having an engagement surface which is engaged
against the gasket to press the gasket against the substrate
mounted on the base to form a sealed volume defined by the
periphery of the openings in the gasket and the active surface of
the substrate and the engagement surface of the press block.
2. The flow cell of claim 1 further including an inlet channel in
the press block extending from an exterior surface thereof to
communication with the inlet extension opening in the gasket and an
outlet channel in the press block extending from communication with
the outlet extension opening in the gasket to an external surface
of the press block.
3. The flow cell of claim 1 wherein the registration surface of the
base is raised above adjacent areas of the base and surrounds the
central window opening in the base, the registration surface formed
flat to allow the precise location of the active surface of the
transparent substrate to be defined with respect to an optical
image projected onto the substrate through the window opening of
the base.
4. The flow cell of claim 3 further including a reference surface
on the base that is parallel to the registration surface whereby
the base can be mounted with the reference surface against a
surface of an image former to thereby locate the parallel
registration surface.
5. The flow cell of claim 1 wherein the gasket is formed of a thin,
non-reactive material having parallel opposite flat surfaces.
6. The flow cell of claim 5 wherein the gasket thickness is less
than one mm.
7. The flow cell of claim 5 wherein the total volume enclosed by
the central opening of the gasket between the engagement surface
and the substrate surface is less than 100 microliters.
8. The flow cell of claim 7 wherein the thickness of the gasket is
about 0.25 mm.
9. The flow cell of claim 1 including a standing frame secured to
the base having an upright section extending from the base and an
arm section extending from the upright section over the window
opening in the base, and a press screw threadingly engaged with the
arm section with a drive end thereof positioned to engage against
an external surface of the press block as the press screw is turned
to thread it toward the press block.
10. The flow cell of claim 9 wherein the drive end of the press
screw is rounded.
11. The flow cell of claim 10 wherein the external surface of the
press block opposite to the engagement surface has a concave
depression formed therein which is fitted to receive the rounded
drive end of the press screw to form a ball-and-socket
engagement.
12. A flow cell of the type that may be used in the synthesis of
arrays of DNA probes and the like, comprising: (a) a transparent
substrate with two parallel flat surfaces, one of the surfaces
forming an active surface of the substrate; (b) a base having a
central window opening and a registration surface against which the
substrate is mounted; (c) a press block having an engagement
surface such that the substrate is engaged between the press block
and the registration surface of the base, the press block having an
external surface opposite to the engagement surface with a concave
depression formed therein; and (d) a press comprising a standing
frame secured to the base and having an upright section extending
from the base and an arm section extending from the upright section
over the central window opening in the base, and a press screw
threadingly engaged with the arm section and positioned to engage
the concave depression in the external surface of the press block
with a rounded drive end thereof as the press screw is turned to
thread it toward the press block.
13. The flow cell of claim 12 further including a gasket having a
central opening defining an active area surrounded by the material
of the gasket, an input extension opening extending from the
central opening and an output extension opening extending from the
central opening, the gasket mounted on the substrate between the
substrate active surface and the engagement surface of the press
block.
14. The flow cell of claim 13 wherein the press block includes an
inlet channel extending from an external surface to communication
with the inlet extension opening in the gasket and an outlet
channel in the press block extending from communication with the
outlet extension opening in the gasket to an external surface of
the press block.
15. The flow cell of claim 13 wherein the gasket is formed of a
thin, non-reactive material having parallel opposite flat
surfaces.
16. The flow cell of claim 15 wherein the gasket thickness is less
than one mm.
17. The flow cell of claim 15 wherein the total volume enclosed by
the central opening of the gasket between the engagement surface
and the substrate surface is less than 100 microliters.
18. The flow cell of claim 17 wherein the thickness of the gasket
is about 0.25 mm
19. The flow cell of claim 12 wherein the registration surface of
the base is raised above adjacent areas of the base and surrounds
the central window opening in the base, the registration surface
formed flat to allow the precise location of the active surface of
a transparent substrate to be defined with respect to an optical
image projected onto the substrate through the window opening of
the base.
20. The flow cell of claim 19 further including a reference surface
on the base that is parallel to the registration surface whereby
the base can be mounted with the reference surface against a
surface of an image former to thereby locate the parallel
registration surface.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of application Ser. No.
10/053,368, filed Nov. 9, 2001, which is a continuation of
application Ser. No. 09/437,369, filed Nov. 10, 1999, now U.S. Pat.
No. 6,315,958, which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention pertains generally to the field of biology
and particularly to apparatus for use in the analysis and
sequencing of DNA and related polymers.
BACKGROUND OF THE INVENTION
[0003] The sequencing of deoxyribonucleic acid (DNA) is a
fundamental tool of modern biology and is conventionally carried
out in various ways, commonly by processes which separate DNA
segments by electrophoresis. See, e.g., Current Protocols In
Molecular Biology, Vol. 1, Chapter 7, "DNA Sequencing," 1995. The
sequencing of several important genomes has already been completed
(e.g., yeast, E. coli), and work is proceeding on the sequencing of
other genomes of medical and agricultural importance (e.g., human,
C. elegans, Arabidopsis). In the medical context, it will be
necessary to "re-sequence" the genome of large numbers of human
individuals to determine which genotypes are associated with which
diseases. Such sequencing techniques can be used to determine which
genes are active and which inactive either in specific tissues,
such as cancers, or more generally in individuals exhibiting
genetically influenced diseases. The results of such investigations
can allow identification of the proteins that are good targets for
new drugs or identification of appropriate genetic alterations that
may be effective in genetic therapy. Other applications lie in
fields such as soil ecology or pathology where it would be
desirable to be able to isolate DNA from any soil or tissue sample
and use probes from ribosomal DNA sequences from all known microbes
to identify the microbes present in the sample.
[0004] The conventional sequencing of DNA using electrophoresis is
typically laborious and time consuming. Various alternatives to
conventional DNA sequencing have been proposed. One such
alternative approach, utilizing an array of oligonucleotide probes
synthesized by photolithographic techniques is described in Pease,
et al., "Light-Generated Oligonucleotide Arrays for Rapid DNA
Sequence Analysis," Proc. Natl. Acad. Sci. USA, Vol. 91, pp.
5022-5026, May 1994. In this approach, the surface of a solid
support modified with photolabile protecting groups is illuminated
through a photolithographic mask, yielding reactive hydroxyl groups
in the illuminated regions. A 3' activated deoxynucleoside,
protected at the 5' hydroxyl with a photolabile group, is then
provided to the surface such that coupling occurs at sites that had
been exposed to light. Following capping, and oxidation, the
substrate is rinsed and the surface is illuminated through a second
mask to expose additional hydroxyl groups for coupling. A second 5'
protected activated deoxynucleoside base is presented to the
surface. The selective photodeprotection and coupling cycles are
repeated to build up levels of bases until the desired set of
probes is obtained. It may be possible to generate high density
miniaturized arrays of oligonucleotide probes using such
photolithographic techniques wherein the sequence of the
oligonucleotide probe at each site in the array is known. These
probes can then be used to search for complementary sequences on a
target strand of DNA, with detection of the target that has
hybridized to particular probes accomplished by the use of
fluorescent markers coupled to the targets and inspection by an
appropriate fluorescence scanning microscope. A variation of this
process using polymeric semiconductor photoresists, which are
selectively patterned by photolithographic techniques, rather than
using photolabile 5' protecting groups, is described in McGall, et
al., "Light-Directed Synthesis of High-Density Oligonucleotide
Arrays Using Semiconductor Photoresists," Proc. Natl. Acad. Sci.
USA, Vol. 93, pp. 13555-13560, November 1996, and G. H. McGall, et
al., "The Efficiency of Light-Directed Synthesis of DNA Arrays on
Glass Substrates," Journal of the American Chemical Society 119,
No. 22, 1997, pp. 5081-5090.
[0005] A disadvantage of both of these approaches is that four
different lithographic masks are needed for each monomeric base,
and the total number of different masks required are thus four
times the length of the DNA probe sequences to be synthesized. The
high cost of producing the many precision photolithographic masks
that are required, and the multiple processing steps required for
repositioning of the masks for every exposure, contribute to
relatively high costs and lengthy processing times.
[0006] An improved process for synthesizing arrays of DNA probe
sequences, polypeptides, and the like, rapidly and efficiently by a
patterning process utilizing a computer controlled image former, is
described in published PCT application International Publication
No. WO 99/42813, published Aug. 26, 1999, entitled Method and
Apparatus for Synthesis of Arrays of DNA Probes. This process
eliminates the need for a lithographic mask, significantly reducing
the costs and time delays that have been associated with processes
requiring such masks. In the patterning process described in the
foregoing published PCT application, a substrate with an active
surface to which, e.g., DNA synthesis linkers have been applied, is
used to support probes to be activated. To activate the surface a
high precision two-dimensional light image is projected onto the
substrate by an image former, illuminating those pixels on the
active surface which are to be activated to bind a first base. The
light incident on the pixels in the array to which the light is
applied deprotects OH groups and makes them available for binding
the bases. After this development step, a fluid containing the
appropriate base is provided to the active surface of the substrate
and the selected base binds to the exposed sites. The process is
repeated until all of the elements of the two-dimensional array on
the substrate surface have an appropriate base bound thereto. The
process is repeated for other pixel locations and desired levels of
bases until the entire selected two-dimensional array of probe
sequences has been completed. To provide the various chemicals in
an appropriate sequence to the substrate, the substrate may be
mounted within a flow cell having an enclosure which seals off the
active surface of the substrate, allowing the appropriate reagents
to flow through the flow cell and over the active surface.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to an improved flow cell
of the type that may be utilized in the synthesis of arrays of DNA
probe sequences, polypeptides and the like, and is particularly
adapted to be used with image formers for projecting an array of
patterned light onto a substrate held by the flow cell. The flow
cell of the invention is formed to precisely align a substrate with
respect to an image former while distributing the fluid containing
the appropriate chemicals through the active volume and over the
active exposed surface of the flow cell, while minimizing the total
volume of fluid contained within the flow cell to conserve the
reagents being utilized. The flow cell allows fast and simple
removal and replacement of substrates while insuring a tight seal
around the substrate to minimize the leakage of reagents in the
flow cell, and it locates the active surface of the substrate at
the focal plane of the image former with a high degree of accuracy
and repeatability.
[0008] A flow cell of a preferred construction in accordance with
the invention includes a base having a central window opening and a
registration surface against which a substrate may be mounted with
its active surface opposite to that which is engaged against the
registration surface. A gasket having a central opening defining an
active area surrounded by the material of the gasket is mounted on
the active surface of the substrate. The gasket has inlet and
outlet extension openings which optionally and preferably extend
away from the central opening in the gasket. A press block has an
engagement surface which is adapted to the engaged against the
gasket to fully enclose an active volume between the press block,
the peripheral walls of the central opening in the gasket, and the
active surface of the substrate. A press mounted to the base is
formed to selectively press the press block against the gasket and
hold it in position. The press block preferably includes a channel
therein which extends from an exterior surface of the press block
to a position in communication with the inlet opening extension in
the gasket and another channel extending from the exterior surface
of the press block to communication with the outlet extension
opening in the gasket, thereby allowing reagents to flow into the
active volume between the inlet opening extension and the outlet
opening extension across the active surface area defined by the
central opening in the gasket. The registration surface of the base
is preferably raised above adjacent areas of the base and surrounds
the central window opening to define a flat plane. The plane of the
registration surface is utilized to precisely locate the active
surface of the substrate with respect to an optical image former
which projects an image through the window opening of the base and
through a transparent substrate to a focal plane at the active
surface of the substrate. The gasket is preferably formed of a thin
non-reactive material having parallel flat surfaces. The thin
gasket allows the active volume within the flow cell through which
reagents flow to be minimized, with the extension openings in the
gasket allowing inlet and outlet of the reagent into and out of the
active volume in a manner which allows substantially the full
central opening area of the gasket to be utilized as the active
volume, with uniform flow of reagent across the active volume.
[0009] A press structure is preferably utilized to selectively
press the press block against the gasket and the substrate. The
press structure includes a standing frame secured to the base and
having an upright section and an arm section which extends
therefrom over the central opening in the base. A press screw is
threadingly engaged with the arm and has a drive end positioned to
engage an external surface of the press block as the press screw is
turned to thread it toward the press block. The drive end is
preferably rounded and fits into a rounded concave depression in
the press block to provide a ball-and-socket engagement between the
press screw and the press block that allows the press block to seat
against the gasket and provide even pressure by the press block
over the entire surface area of the gasket. When a substrate is to
be changed, the press screw can be easily unscrewed by the operator
until the press block is free of the press screw, allowing the
press block and substrate to be removed, a new substrate to be
inserted into position and the gasket and press block repositioned
onto the active substrate, after which the press screw can be
threaded down into contact with the press block to drive it into
engagement with the gasket to seal the active volume in the flow
cell.
[0010] Further objects, features and advantages of the invention
will be apparent from the following detailed description when taken
in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] In the drawings:
[0012] FIG. 1 is an exploded perspective view of the flow cell of
the present invention showing the parts separated and in position
for assembly.
[0013] FIG. 2 is a cross sectional view through the assembled flow
cell of the invention.
[0014] FIG. 3 is a top view of the base of the flow cell.
[0015] FIG. 4 is a top view of the press block of the flow
cell.
[0016] FIG. 5 is a bottom view of the press block.
[0017] FIG. 6 is a cross sectional view of the press block taken
generally along the line 6-6 of FIG. 4.
[0018] FIG. 7 is a plan view of a preferred gasket for use in the
flow cell of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0019] With reference to the drawings, an exploded view of a flow
cell in accordance with the invention in position for assembly is
shown generally at 10 in FIG. 1. The flow cell 10 includes a base
11, a standing frame 12 which is secured to the base (and may be
integrally formed therewith), and a press block 13. A substrate 15,
typically a transparent glass slide, has two parallel flat surfaces
16 and 17, with one of the surfaces 16 forming an active surface of
the substrate. A gasket 18 having a central opening 19 is mounted
between the substrate 15 and the press block 13 and is pressed
between the active surface 16 of the substrate and an engagement
surface 20 of the press block when the flow cell is in use.
[0020] The base 11 is preferably formed of a solid piece of
material, e.g., aluminum, having a central window opening 21 which
is surrounded by a registration surface 22 which is preferably
raised above the adjacent surfaces of the base. The registration
surface 22 is preferable machined to be flat to a high degree of
precision so that when the surface 17 of the substrate 15 is
mounted in engagement with the registration surface 22, the
parallel active surface 16 of the substrate will be precisely
located with respect to a light image projected by an image former
(not shown) through the window opening 21 of the base onto a focal
plane which should be located at and parallel to the active surface
16. The image former may be of the type described in the published
PCT application WO 99/42813, although it is understood that the use
of the flow cell 10 is not limited to such systems. The base 11 has
an outwardly extending rim 23 with a flat reference surface 24 on
the underside of the rim 23 that is parallel to the registration
surface 22. When the reference surface 24 is engaged against a
surface of a mounting ring (not shown) of the image former having a
known location in the image former, the registration surface 22
will thereby be located in proper position with respect to the
image former.
[0021] The standing frame 12 is secured to the base 11, as by a set
screw 25 which threads through a threaded bore in an upright
section 26 of the standing frame 12 and into a threaded blind hole
27 in the base, as best shown in the cross sectional view of FIG.
2. The standing frame 12 may also be formed integrally with the
base or may be secured to the base by any other means, e.g., by a
clamp, etc. An arm section 28 of the standing frame extends
outwardly from the upright section 26 and, when the standing frame
is mounted to the base, over the central opening 21 in the base. A
press screw 30 is threadingly engaged to the arm 28 through a
threaded bore in the arm and has an expanded head 31 which is
adapted to be grasped and turned by a user. The press screw 30
terminates in a drive end 32 which is preferably rounded as shown.
The standing frame 12 with the drive screw 30 engaged therewith
forms a press which, when the standing frame is secured to the base
11, is adapted to selectively apply pressure to the press block 13
to engage it tightly against the gasket 18 so that the gasket is
held tightly between the press block 13 and the substrate 15.
Although not preferred, other means may be used to press the press
block against the gasket, e.g., a C-clamp, screw(s) threaded
between the press block and base, spring-loaded clamps, etc. The
use of the press screw 30 is preferred because it provides a one
point type pressing system to exert a uniform pressure and thereby
ensure good sealing.
[0022] The press block 13 has a top external surface 34, opposite
the engagement surface 20, which has formed therein a concave
rounded depression 35 which is positioned to receive a rounded
drive end 32 of the press screw 30. When the press block 13 is in
its assembled position, as best shown in the cross sectional view
of FIG. 2, the press screw 30 may be turned by the user to draw it
downwardly such that the drive end 32 fits into the depression 35
to engage therewith with a ball-and-socket arrangement so that the
press block 13 can seat itself against the gasket 18, applying
uniform pressure throughout the area of the gasket. The engagement
of the press screw 32 with the depression 35 also helps to center
the press block in its proper position.
[0023] The press block 13 has an inlet fitting 38 and an outlet
fitting 39 engaged to the press block at the top surface 34. The
fittings 38 and 39 are connected to supply tubes 40 and 41 by which
reagent may be supplied to and removed from the flow cell. As best
shown in the top and bottom views of FIG. 4 and 5 and the cross
sectional view of FIG. 6, the press block 13 has threaded bores 43
and 44 formed in its top surface 34 into which the fittings 38 and
39 are threadingly engaged, with the bores 43 and 44 terminating at
a position above the bottom surface 19, with an inlet channel 48
and an outlet channel 49 extending through the press block from the
bores 43 and 44 to define and an inlet channel and an outlet
channel, respectively. As best shown in FIG. 7, the central opening
19 in the gasket 18 defines an active area (which may be square or
rectangular as shown), with an inlet extension opening 50 and an
outlet extension opening 51 formed in the gasket which extend
outwardly from the central opening 19 in the gasket. When the
gasket 18 is mounted in place on the substrate with the press block
13 engaged with it, the inlet channel 48 in the press block will be
in communication with the inlet opening extension in the gasket,
and the outlet channel 49 will be in communication with the outlet
extension opening 51 in the gasket. Reagent flowing in through the
inlet channel 48 will pass into the region defined by the extension
opening 50 and then through and across the central opening 19 in
the gasket to the outlet extension opening 51, and from thence to
the outlet channel 49.
[0024] The entire active volume of the flow cell is defined between
the periphery of the central opening in the gasket, acting as the
side walls of the active volume, and the engagement surface 20 of
the press block at the top and the substrate active surface 16 at
the bottom. This active volume can be made very small while still
insuring ample flow of reagent across the entire active area by
utilizing a thin gasket 18 with parallel bottom and top surfaces.
For example, the gasket, which may be formed of a non-reactive
plastic or synthetic material such as Kalrez.RTM.
perfluoroelastomer from DuPont Dow elastomers, may have a thickness
of, e.g., 0.25 mm, and still provide adequate flow volume for the
reagent. For a gasket 18 having a central opening 19 of one
cm.sup.2, the entire active volume may be less than 100
microliters. Typical satisfactory dimensions for the gasket are
0.25 mm thickness with a central square opening approximately 1.5
cm on a side, with a total reaction volume of about 65 microliters,
significantly less than that required for flow cells in comparable
systems.
[0025] The press block 13 is preferably formed of a strong and
non-reactive synthetic material, e.g., Kel-F.RTM.
chlorotrilfluoroethylen- e polymer from Minnesota Mining and
Manufacturing Company, in a block with an engagement surface 20
lapped flat. The standing frame 22 may be formed of metal, e.g.,
aluminum. Various other materials may be utilized for the parts of
the flow cell.
[0026] In preparation for use of the flow cell, the base 11 may be
fixed in position to a mounting ring (not shown) on an optical
breadboard. The base is preferably formed such that the
registration surface 22 is in a precise location with respect to
the optical system so that the active surface 16 of the substrate
is at a focal point within a few microns. The standing frame 12 is
secured to the base 11 utilizing the set screw 25, and the
substrate 15 is then mounted in position onto the base 11 so as to
cover the central opening 21 and be in full engagement (at its
surface 17) with the registration surface 22. The gasket 18 is then
positioned on the active surface 16 of the substrate so that the
central opening 19 in the gasket is fully within the window opening
21 of the base at a proper position, and the press block 13 is then
positioned over the gasket 18 so that the engagement surface 20
properly seats against the gasket 18. The standing frame 12
preferably includes a flat machined upright surface 58 against
which a flat side 59 of the press block 13 may be engaged to locate
the press block in proper position. An edge of the substrate 15 may
also be engaged against the flat surface 58 to conveniently locate
the substrate in a proper position. Accurate positioning of the
gasket 18 may be readily facilitated in this manner by placing the
gasket 18 onto the engagement surface 20 of the press block, after
which the press block and gasket are then engaged with the
substrate 15 with the press block surface 59 located against the
upright surface 58.
[0027] If desired, two or more of the flow cells 10 may be
connected together, such that reagent flowing out of one flow cell
flows into another flow cell. In this way, several substrates may
be treated simultaneously, while minimizing the use of the
reagent.
[0028] It is understood that the invention is not confined to the
particular embodiments set forth herein as illustrative, but
embraces all such modified forms thereof as come within the scope
of the following claims.
* * * * *