U.S. patent application number 10/665389 was filed with the patent office on 2004-07-15 for method and apparatus for performing biological reactions on a substrate surface.
Invention is credited to Hawkins, George, Kahn, Peter, McGarry, Mark W., Tuggle, Todd.
Application Number | 20040137605 10/665389 |
Document ID | / |
Family ID | 29270858 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137605 |
Kind Code |
A1 |
McGarry, Mark W. ; et
al. |
July 15, 2004 |
Method and apparatus for performing biological reactions on a
substrate surface
Abstract
The present invention provides methods and apparatus for
performing biological reactions on a substrate surface that use a
low volume of sample fluid, accommodate substrates as large as or
larger than a conventional microscope slide, accommodate a
plurality of independent reactions, and accommodate a substrate
surface having one or more hydrogel-based microarrays attached
thereto. The invention further provides an apparatus that allows
introduction of fluids in addition to sample fluid into each
reaction chamber via standard pipet tips and associated pipettor
apparatus. The invention further an apparatus that increases
reaction reproducibility, increases reaction efficiency, and
reduces reaction duration. The preferred embodiment of the
invention is configured to accommodate a standard microscope slide
substrate having four hydrogel-based microarrays attached thereto
and comprises a base plate having a well structure corresponding to
each microarray and two fluid ports extending through the base
plate into each well structure, a means for temporarily clamping
the substrate against the base plate such that the microarrays face
into the well structures, a means for sealing the perimeter around
each microarray and well structure, and a means for sealing the
fluid ports from the environment.
Inventors: |
McGarry, Mark W.;
(Scottsdale, AZ) ; Kahn, Peter; (Phoenix, AZ)
; Tuggle, Todd; (Chandler, AZ) ; Hawkins,
George; (Gilbert, AZ) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
INTELLECTUAL PROPERTY DEPARTMENT
4 EMBARCADERO CENTER
SUITE 3400
SAN FRANCISCO
CA
94111
US
|
Family ID: |
29270858 |
Appl. No.: |
10/665389 |
Filed: |
September 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10665389 |
Sep 18, 2003 |
|
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|
09458534 |
Dec 9, 1999 |
|
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6642046 |
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Current U.S.
Class: |
435/287.2 ;
435/6.1; 435/6.11; 435/6.12 |
Current CPC
Class: |
C40B 60/14 20130101;
G01N 2035/00158 20130101; G01N 30/6069 20130101; B01L 7/52
20130101; G01N 30/6095 20130101; B01J 2219/00317 20130101; G01N
2035/00237 20130101; B01J 2219/00495 20130101; B01J 19/0093
20130101; F28F 2260/02 20130101; G01N 2030/025 20130101; B01J
2219/00351 20130101 |
Class at
Publication: |
435/287.2 ;
435/006 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
What is claimed is:
1. An apparatus for performing biological reactions on a substrate
surface, the apparatus comprising: a biochip comprising: a
substrate; and a plurality of arrays of biologically reactive sites
on said substrate; a base plate; and a gasket affixed between the
biochip and the base plate, the gasket defining a plurality of
reaction chambers, each reaction chamber corresponding to one of
the arrays.
2. An apparatus according to claim 1, wherein the base plate
defines at least one well structure, at least one reaction chamber
being positioned to include the well structure.
3. An apparatus according to claim 1, further comprising: a first
port extending from at least one of said reaction chambers through
the base plate.
4. An apparatus according to claim 3, further comprising a second
port extending from at least one said reaction chambers through the
base plate.
5. An apparatus according to claim 1, wherein at least one of the
plurality of arrays of biologically reactive sites comprises
oligonucleotide probes.
6. An apparatus according to claim 1, further comprising a heating
element positioned to heat at least one reaction chamber.
7. An apparatus for performing biological reactions on a substrate
layer comprising: a substrate having a first surface containing a
plurality of biologically reactive sites disposed thereon; a base
plate having a first surface and a second surface, wherein the
first surface further comprises a cavity comprising one or a
plurality of well structures; a sealing member disposed in each
well structure, wherein each sealing member defines a reaction
chamber between the surface of the substrate layer containing the
biologically reactive sites and the first surface of the base
plate; and a fluid port connected to at least one reaction
chamber.
8. An apparatus according to claim 7, further comprising a second
port extending from at least one said reaction chambers through the
base plate.
9. An apparatus according to claim 7, wherein at least one of the
biologically reactive sites comprise oligonucleotide probes.
10. An apparatus according to claim 7, further comprising a heating
element positioned to heat at least one reaction chamber.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuing application of U.S. patent
application Ser. No. 09/458,534 filed Dec. 9, 1999.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to methods and apparatus for
performing biological reactions on a substrate surface. More
specifically, the invention relates to methods and apparatus for
performing thermally controlled biological reactions on a substrate
surface having one or more arrays of biologically reactive sites
attached thereon. In particular, the invention provides a reusable
and thermally controllable reaction apparatus having one or more
biologically inert reaction chambers into which biologically
reactive sample fluid mixtures are introduced for reaction on a
substrate surface having one or more arrays of biologically
reactive sites attached thereon.
[0004] 2. Description of the Prior Art
[0005] As research into gene expression and nucleic acid sequencing
has progressed in recent years, the need has arisen for
high-capacity assaying methods and equipment. Much of the progress
in the fields of nucleic acid sequencing and gene expression has
resulted from the use of nucleic acid hybridization techniques and
antigen/antibody binding techniques, respectively. Assays utilizing
specific binding pairs such as complementary nucleic acids
including DNA/DNA, DNA/RNA, and RNA/RNA hybrids or antigen/antibody
are widely used in the art. The art also discloses various
techniques for nucleic acid sequencing based on complementary
binding and differential hybridization. Techniques for
manufacturing and utilizing microfluidic apparatus for conducting
such thermally controlled biological reactions are also well
known.
[0006] Recent technology utilizes the binding of molecules
contained within a biologically reactive sample fluid, hereinafter
referred to as target molecules, onto molecules contained within
biologically reactive sites, hereinafter referred to as probe
molecules. The primary enabler of this technology is a apparatus
commonly referred to as a biochip, which comprises one or more
2-dimensional microscopic arrays of biologically reactive sites
immobilized on the surface of a substrate. A biologically reactive
site is created by dispensing a small volume of biologically
reactive fluid onto a discrete location on the surface of a
substrate, also commonly referred to as spotting. To enhance
immobilization of probe molecules, many biochips include a
2-dimensional array of 3-dimensional polymeric anchoring structures
(for example, polyacrylamide gel pads) attached to the surface of
the substrate. Probe molecules such as oligonucleotides covalently
attach to polyacrylamide-anchoring structures by forming an amide,
ester or disulfide bond between the biomolecule and a derivatized
polymer comprising the cognate chemical group. Covalent attachment
of probe molecules to the polymeric anchoring structure is usually
performed after polymerization and chemical cross-linking of the
polymer to the substrate is completed.
[0007] Existing apparatus for performing thermally-controlled
biological reactions on a substrate surface are deficient in that
they either require unacceptably large volumes of sample fluid to
operate properly, cannot accommodate substrates as large as or
larger than a conventional microscope slide, cannot independently
accommodate a plurality of independent reactions, or cannot
accommodate a substrate containing hydrogel-based microarrays. Most
existing apparatus also do not allow introduction of fluids in
addition to the sample fluid such as wash buffers, fluorescent
dyes, etc., into the reaction chamber. Disposable apparatus require
disassembly and reapplication of a new apparatus to the substrate
surface every time a new fluid must be introduced. Other existing
apparatus are difficult to use in a laboratory environment because
they cannot be loaded with standard pipet tips and associated
pipettor apparatus.
[0008] Many existing apparatus also exhibit unacceptable reaction
reproducibility, efficiency, and duration. Reaction reproducibility
may be adversely affected by bubble formation in the reaction
chamber or by the use of biologically incompatible materials for
the reaction chamber. Reaction duration and efficiency may be
adversely affected by the presence of concentration gradients in
the reaction chamber.
[0009] Bubbles can form on introduction of sample fluid to the
reaction chamber, at elevated temperatures during the reaction due
to the potential high gas content of the fluid, or by outgassing of
the reaction chamber materials. When gas bubbles extend over the
substrate surface in an area containing biologically reactive
sites, the intended reaction may intermittently fail or yield
erroneous results because the intended concentration of the sample
fluid mixture has been compromised by the presence of gas bubbles.
To aggravate the problem, gas bubbles in the reaction chamber
attempt to expand at elevated temperatures during the reaction and
periodically cause the seal between the substrate surface and
reaction chamber apparatus to fail, allowing leakage and
evaporation of the sample fluid.
[0010] Biologically incompatible reaction chamber materials may
cause unacceptable reaction reproducibility, by interacting with
the sample fluid, thus causing the intended reaction to
intermittently fail or yield erroneous results.
[0011] Incomplete mixing of the sample fluid can introduce
concentration gradients within the sample fluid that adversely
impact reaction efficiency and duration. This effect is most
pronounced when there is depletion of target molecules in the local
volume surrounding a biologically reactive site. During a
biological reaction, the probability that a particular target
molecule will bind to a complementary (immobilized) probe molecule
is determined by the given concentration of target molecules
present within the sample fluid volume, the diffusion rate of the
target molecule through the reaction chamber, and the statistics of
interaction between the target molecule and the complementary probe
molecule. For diagnostic assays, target DNA molecules are often
obtained in minute (<picomol) quantities. In practice, it can
take tens of hours for a hybridization reaction to be substantially
complete at the low target nucleic acid molecule levels available
for biological samples. Concentration gradients further exacerbate
this problem.
[0012] U.S. Pat. No. 5,948,673 to Cottingham discloses a
self-contained multi-chamber reactor for performing both DNA
amplification and DNA probe assay in a sealed unit wherein some
reactants are provided by coating the walls of the chambers and
other reactants are introduced into the chambers prior to starting
the reaction in order to eliminate flow into and out of the
chamber. -Unfortunately, no provisions are made for pressurization
or mixing of the sample fluid introduced to the chambers, and the
apparatus cannot accommodate substrates including microscope
slides.
[0013] There remains a need in the art for methods and apparatus
for performing biological reactions on a substrate surface that use
a low volume of sample fluid, accommodate substrates as large as or
larger than a conventional microscope slide, accommodate a
plurality of independent reactions, and accommodate a substrate
surface having one or more hydrogel-based microarrays attached
thereto. There also remains a need in the art for an apparatus that
allows introduction of fluids in addition to sample fluid into each
reaction chamber via standard pipet tips and associated pipettor
apparatus. There also remains a need in the art for such an
apparatus that increases reaction reproducibility, increases
reaction efficiency, and reduces reaction duration. These needs are
particularly striking in view of the tremendous interest in biochip
technology, the investment and substantial financial rewards
generated by research into biochip technology, and the variety of
products generated by such research.
SUMMARY OF THE INVENTION
[0014] The present invention provides methods and apparatus for
performing biological reactions on a substrate surface that use a
low volume of sample fluid, accommodate substrates as large as or
larger than a conventional microscope slide, accommodate a
plurality of independent reactions, and accommodate a substrate
surface having one or more hydrogel-based microarrays attached
thereto. The invention further provides an apparatus that allows
introduction of fluids in addition to sample fluid into each
reaction chamber via standard pipet tips and associated pipettor
apparatus. The invention further provides an apparatus that
increases reaction reproducibility, increases reaction efficiency,
and reduces reaction duration.
[0015] The invention broadly comprises a base plate having a first
surface and a cavity disposed in the first surface, wherein the
cavity comprises one or more well structures and a biochip
comprising one or more microarrays of biologically reactive sites
disposed on a first surface can be inserted into the apparatus such
that the first surface of the biochip is in direct communication
with the well structures and is removably clamped to the base plate
using a compression plate. A sealing member is disposed between the
first surface of the substrate and the first surface of the base
plate in each well structure, thereby defining one or more reaction
chambers. Each well structure has at least two fluid ports for
introducing fluid samples into and removing fluid samples from the
reaction chambers. The invention further comprises a seal for the
fluid ports.
[0016] A preferred embodiment of the invention is configured to
accommodate a biochip comprising a standard microscope slide having
a plurality of hydrogel-based microarrays attached thereto. A
further preferred embodiment of the apparatus includes the
biochip.
[0017] In preferred embodiments of the present invention, the
sealing member around the perimeter of each well structure
comprises an O-ring or sheet of gasket material.
[0018] In further preferred embodiments, the fluid ports allow
introduction of fluid sample via a standard pipet tip or tubing. In
still further preferred embodiments, the fluid ports allow
interface to an external pumping system that provides mixing and
pressurization of the fluid in each reaction chamber to provide
uniform target molecule concentration and dissolve gas bubbles,
respectively.
[0019] In preferred embodiments, the fluid port seal comprises a
layer of flexible, thermally conductive material on which is
disposed a layer of pressure-sensitive adhesive.
[0020] In other preferred embodiments of the invention, the
biological compatibility of the base plate material is enhanced by
the addition of a biologically compatible surface coating to the
first surface of the base plate. The adhesion of the surface
coating to the first surface of the base plate may be further
enhanced by application of a layer of primer on the first surface
of the base plate prior to application of the surface coating.
[0021] In further preferred embodiments of the invention, the
compression plate is removably affixed to the base plate by a
plurality retaining pins disposed along the perimeter of the base
plate which fit into corresponding locking apertures disposed along
the perimeter of the retaining plate. In yet further preferred
embodiments, the compression plate comprises a cavity wherein a
compliance layer is seated.
[0022] In preferred embodiments of the microfluidic reaction
apparatus, the retaining plate, compression plate and compliance
layer further comprise one or more viewing ports corresponding in
position to the reaction chambers for observation of the biological
reactions taking place inside the reaction chambers.
[0023] The invention is advantageously used for performing
thermally controlled biological reactions, and in preferred
embodiments comprises a heating element and a thermal cycling
device.
[0024] Specific preferred embodiments of the present invention will
become evident from the following more detailed description of
certain preferred embodiments and the claims.
DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is an exploded perspective view from the upper side
of a specific embodiment the present invention, illustrating the
relationships between the various components and a biochip.
[0026] FIG. 2 is an exploded perspective view from the lower side
of the apparatus of FIG. 1, illustrating the proper orientation of
a biochip.
[0027] FIG. 3 is a perspective view from the upper side of the
apparatus of FIG. 1, illustrating the apparatus as assembled and
ports for viewing the contents of each reaction chamber.
[0028] FIG. 4 is a perspective view from the lower side of the
apparatus of FIG. 1, illustrating the relationship of the fluid
port-sealing member to the base plate.
[0029] FIG. 5 is an enlarged partial view of the apparatus of FIG.
1, illustrating details of the base plate and the relationship of
the retaining pins to the base plate.
[0030] FIG. 6 is an enlarged partial view of the biochip as shown
in FIG. 2, illustrating a hydrogel-based microarray attached to a
substrate surface.
[0031] FIG. 7 is a top view of the apparatus of FIG. 1,
illustrating ports for viewing the contents of each reaction
chamber.
[0032] FIG. 8 is a cross-sectional view of the apparatus of FIG. 1
taken along line 8-8 in FIG. 7, illustrating a reaction
chamber.
[0033] FIG. 9 is an enlarged partial view of the apparatus of FIG.
1, illustrating the spatial relationship between a reaction chamber
and a biochip.
[0034] FIG. 10 is an enlarged partial view of the apparatus of FIG.
1, illustrating a reaction chamber seal.
[0035] FIG. 11 is a cross-sectional view of the apparatus of FIG. 1
taken along line 8-8 in FIG. 7, illustrating a pipet tip inserted
into a fluid port.
[0036] FIG. 12 is a front-end plan view of the apparatus of FIG. 1,
illustrating the application of a heating element for temperature
cycling.
[0037] FIG. 13 is a top view of the apparatus of FIG. 1,
illustrating an O-ring groove in relation to a well structure and
microarray.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0038] The present invention provides methods and apparatus for
performing biological reactions on a substrate layer having a
multiplicity of biologically reactive sites disposed thereon. The
invention comprises a microfluidic reaction apparatus having one or
more individual reaction chambers in direct communication with a
biochip, preferably comprising one microarray of oligonucleotide
probes, corresponding to each reaction chamber, disposed on the
surface of the substrate, wherein each probe is anchored to the
substrate by a polyacrylarnide gel pad. The apparatus is
advantageously used for performing multiple, parallel, thermally
controlled biological reactions, most preferably hybridization
reactions. Use of the reaction apparatus of the present invention,
however, is not limited to DNA hybridization or
thermally-controlled biological reactions. Those skilled in the art
will recognize various additional uses for the apparatus. For
example, the amplification of nucleic acids or the addition of
labels to nucleic acids generally results in the presence of
various unwanted components in the sample fluid, e.g.,
unincorporated nucleotides, enzymes, or DNA molecules that are of
no interest. With this apparatus, probes can be used to capture
nucleic acids of interest and allow the reaction by-products to be
washed out of the reactor.
[0039] The preferred embodiments of the present invention and its
advantages over previously investigated apparatus are best
understood by reference to FIGS. 1-13 and Examples 1-2.
[0040] As used herein, the term "biochip" refers to one or more
microarrays of biologically reactive sites immobilized on the
surface of a substrate. Commonly used substrates include soda lime
glass sheets and microscope slides.
[0041] As used herein, the term "microarray" refers to an
addressable 2-dimensional microscopic spatial arrangement,
particularly an arrangement of biologically reactive sites.
[0042] As used herein, the terms "biomolecular probe" and
"oligonucleotide probe" refer to a nucleic acid sequence used to
detect the presence of a complementary target sequence by
hybridization with the target sequence.
[0043] As used herein, the term "standard pipet tip" refers to any
commonly used commercially available pipet tip, including but not
limited to the "Yellow Tip" 1-200 microliter tip and the "Blue Tip"
100-1000 microliter tip.
[0044] As used herein, the term "thermal cycling" refers to the
process of rapidly and repeatedly increasing and decreasing
temperature in a cyclic fashion, specifically the temperature of a
reaction chamber.
[0045] FIG. 1 is an exploded perspective view from the upper side
of a preferred embodiment of the present invention, illustrating
the relationships between the various components. In this
embodiment, the apparatus comprises a base plate 32 having a first
surface, a second surface, a first cavity 40 comprising four well
structures 34 disposed in the first surface, and a second cavity 41
disposed in the first surface. A biochip 20 having a first surface
containing a plurality of biologically reactive sites is inserted
in the apparatus such that the biochip is removably seated in the
second cavity 41 and the first surface of the biochip is in direct
communication with the first cavity 40. Each well structure 34
includes a groove 36 for seating an O-ring 48 between the biochip
20 and the base plate 32, wherein the O-ring 48 defines a reaction
chamber 30 between the biochip 20 and the base plate 32. A first
fluid port 38 and a second fluid port 39 extend through base plate
32 into each well structure 34. A port seal 46 can be removably
applied to the second surface of base plate 32 to temporarily close
fluid ports 38 and 39, thereby isolating the contents of reaction
chamber 30 from the environment.
[0046] Biochip 20 comprises one or more microarrays 24 of
biologically reactive sites 26 disposed on a first surface of the
substrate 22 facing a first surface of the base plate 32. A
compliance layer 50 is permanently affixed in a cavity 60 in
compression plate 54, and the compression plate 54 is then
removably seated on base plate 32, thereby removably locking
substrate 22 into base plate cavity 40.
[0047] The assembly is locked together with retaining plate 62 and
retaining pins 72, having a body 74, a neck 76, and a head 78. The
body 74 of each retaining pin 72 is press fit into a pin aperture
44 disposed along the perimeter of base plate 32. Retaining pin
body 74 extends through a corresponding pin aperture 56 in
compression plate 54. The neck 76 and head 78 of retaining pin 72
extend through a corresponding pin aperture 66 in retaining plate
62. The retaining pin aperture 66 in retaining plate 62 comprises a
substantially circular main section 68 configured to accept the
diameter of pin head 78, and a notch 70 extending from the main
section 68 configured to accept the diameter of pin neck 76, but
smaller than the diameter of pin head 78.
[0048] FIG. 2 is an exploded perspective view from the lower side
of reaction apparatus 28, illustrating the orientation of biochip
20 in relation to base plate 32. FIG. 3 is a perspective view, from
the upper side of apparatus 28, illustrating apparatus 28 as
assembled. FIG. 4 is a perspective view from the lower side of
apparatus 28, illustrating the relationship of sealing member 46 to
base plate 32.
[0049] FIG. 5 is an enlarged partial view of apparatus 28,
illustrating details of base plate 32 and the relationship of
retaining pins 72 to base plate 32. Base plate 32 is most
preferably 5 millimeters thick, 44 millimeters wide, and 82
millimeters long, and comprises two notches 42, six pin apertures
44, first cavity 40, second cavity 41, and well structures 34, each
having an O-ring ring groove 36, and first and second fluid ports
38 and 39. The base plate material is preferably thermally
conductive in order to conduct heat from heating element 82 to the
fluid inside each reaction chamber 30. The conductivity of the base
plate material is most preferably selected to provide for
alteration of the fluid temperature by at least 2 degrees
centigrade per second over a range from zero degrees centigrade to
100 degrees centigrade. The base plate material is preferably
titanium, copper, aluminum, ceramic, or any other material having
similar mechanical and thermal properties that will not introduce
gas bubbles into the reaction chamber by outgassing, and most
preferably is grade 2 commercially pure titanium.
[0050] Optional base plate notch 42 is located on either end of
base plate 32 as shown in FIGS. 1, 2, 4, and 5. Notch 42 is
configured to allow laboratory technicians to easily remove a
biochip 20 with their fingers, and is most preferably 20
millimeters wide and extends laterally most preferably 4
millimeters into base plate 32.
[0051] Base plate second cavity 41 is most preferably 25
millimeters wide, 75 millimeters long, and 1 millimeter deep. Each
dimension of cavity 41 is slightly larger than the corresponding
size of biochip 20 to ensure minimum play of biochip 20. In an
alternative configuration, biochip 20 is permanently affixed to the
base plate 32, thus forming a single integrated component.
[0052] Each pin aperture 44 is disposed along the perimeter of base
plate 32 as shown in FIG. 7 and extends entirely through base plate
32. The pin aperture 44 is preferably circular, having a diameter
of most preferably 5 millimeters, and allows heavy press-fit around
body 74 of retaining pin 72.
[0053] The depth of each well structure 34 is preferably between 25
micrometers and 150 micrometers, more preferably between 75
micrometers and 150 micrometers, and most preferably between 100
micrometers and 150 micrometers. The depth selected is critical for
developing the capillary action required to avoid gas bubble
formation upon introduction of fluid into each reaction chamber 30.
It is also critical to minimize the depth of well structure 34 in
order to correspondingly reduce the volume of fluid required to
fill reaction chamber 30. The volume of reaction chamber 30 is most
preferably 33 microliters when well structure 34 is 125 micrometers
deep and ports 38 and 39 are each 1.4 millimeters in diameter.
[0054] As shown in FIG. 13, each O-ring groove 36 is configured so
that a seated O-ring 48 completely surrounds one microarray 24 of
biologically reactive sites 26 on biochip 20. As shown in FIG. 10,
each O-ring groove 36 preferably comprises an oblong channel that
extends most preferably 1.6 millimeters into base plate 32 relative
to the first surface of base plate 32. Groove 36 has circular end
portions most preferably 11.5 millimeters in diameter, measured
from the center of groove 36 to the inner perimeter of the groove,
and most preferably 9.5 millimeters apart from center-to-center.
The width of the groove, as shown in FIG. 10, is chosen such that
it makes a slight interference fit with an O-ring 48, and is most
preferably 1.6 millimeters in the embodiment illustrated. This
condition reduces the opportunity for trapped gas bubbles to form
at the interface surface between each O-ring 48 and O-ring groove
36. Such trapped gas bubbles could expand during heating and cause
seal breach. The dimensions of groove 36 are limited only by the
size and shape of microarray 24. As shown in FIG. 13, the boundary
of each well structure 34 extends slightly outward from the
outermost perimeter of O-ring groove 36, allowing room for O-ring
48 to deform during compression of biochip 20 into the surface of
second cavity 41, thereby forming a tighter seal between biochip 20
and base plate 32.
[0055] A first fluid port 38 is located in the well structure 34
immediately adjacent to the circular end portion of the inner
perimeter of O-ring groove 36 as shown in FIG. 13. A second fluid
port 39 is located in the well structure 34 immediately adjacent to
the opposite circular end portion of the inner perimeter of O-ring
groove 36. The circular end portions of each O-ring groove 36
provide a gradual change in flow geometry which considerably
reduces the potential for bubble formation during introduction of a
fluid though fluid port 38 and removal through fluid port 39. End
portions that are parabolic or triangular in profile, or any shape
that provides a gradual change in flow geometry, could also be used
to create the same effect.
[0056] Each fluid port 38 and 39 is intended for interfacing to
pipet tip 80 as shown in FIG. 11 and has a diameter preferably
between 0.25 millimeters and 1.5 millimeters, more preferably
between 0.75 millimeters and 1.5 millimeters, and most preferably
between 1.25 millimeters and 1.5 millimeters. Pipet tip 82 is
preferably disposable and made of polypropylene, and can interface
with a standard pipettor for manual loading of the reaction
chambers. Many other similar types of pipet tips are commonly
available and would be useful in the present invention.
[0057] A biologically compatible outer surface coating is
optionally applied to base plate 32 and retaining pins 72 after all
retaining pins 72 are press-fitted into each pin aperture 44 of
base plate 32. To enhance adhesion performance of the outer surface
layer to base plate 32, a layer of biologically compatible primer
is optionally first applied to base plate 32. Preferably the
surface coating is selected from fluorinated ethylene propylene
(commonly known under the trademark Teflon.RTM.), gold, platinum,
polypropylene, an inert metal oxide, or any material having similar
biological compatibility and mechanical properties. Most
preferably, the surface coating is Teflon.RTM.. The primer material
is preferably Xylan.RTM., Teflon.RTM., polypropylene, an inert
metal oxide, or any material having similar biological
compatibility and mechanical properties.
[0058] Each O-ring 48 preferably has a circular cross-section of
most preferably 1.8 millimeters in diameter, and a circular profile
the inside diameter of which is most preferably 14 millimeters.
Preferably the O-ring material is selected from nitrile, silicone,
Kalrez.RTM., or any biologically inert material having similar size
and mechanical properties, that will not introduce gas bubbles into
the reaction chamber due to outgassing. Most preferably, the O-ring
is made of nitrile. As shown in FIG. 10, each O-ring 48 fits into a
corresponding O-ring groove 36 in base plate 32 such that no air
gaps form between O-ring 48 and O-ring groove 36. When reaction
chamber apparatus 28 is assembled correctly, each well structure 34
allows deformation of a corresponding O-ring 48 as shown in FIG.
10.
[0059] Biochip 20 broadly comprises substrate 22 and one or a
plurality of microarrays 24 disposed on a first surface thereof. In
a preferred embodiment, biochip 20 includes four microarrays 24.
The dimensions of substrate 22 are preferably between 25
millimeters wide by 75 millimeters long by 1 millimeter thick and
325 millimeters long by 325 millimeters wide by 2 millimeters
thick. Most preferably, substrate 22 is a standard soda lime glass
microscope slide 25 millimeters wide by 75 millimeters long by 1
millimeter thick. Alternative substrate materials include silicon,
fused silica, borosilicate, or any rigid and biologically inert
glass, plastic, or metal. As shown, biochip 20 must be oriented
with the microarray 24 bearing surface facing toward base plate 32.
When assembled as shown, four reaction chambers 30 are formed, each
defined by a volume bounded by biochip 20, each O-ring 48, and each
corresponding well structure 34.
[0060] As shown in FIG. 13, in a preferred embodiment, each
microarray 24 has twenty seven biologically reactive sites 26 in
one direction and twenty seven in a direction normal to the first
direction. As shown in FIG. 6, each site 26 contains a biologically
reactive three-dimensional polymerized polyacrylamide gel structure
27 affixed to substrate 22. Each gel structure 27 is preferably
cylindrical, most preferably having a 113 micron diameter and a 25
micron thickness. The distance between each site 26 within each
microarray 24 is most preferably 300 micrometers, and the distance
between each microarray 24 is most preferably 15 millimeters. Each
microarray 24 is also preferably isolated by a polyacrylamide gel
boundary 25. Each site 26 could alternatively comprise biologically
reactive reagents attached directly to substrate 22.
[0061] Optional compliance member 50 is intended to provide a
uniform distribution of clamping pressure over biochip 20 without
cracking substrate 22. The general size of compliance member is
intended to substantially match the overall size of substrate 22.
Compliance member 50 is most preferably 65 millimeters long, 26
millimeter wide, and 3 millimeter thick, and is formed of a layer
of pressure-sensitive adhesive disposed on a layer of
low-compression material, preferably selected from silicone sponge
rubber, natural sponge rubber, neoprene sponge rubber, or any
material having similar mechanical properties. Compliance member 50
further preferably includes four viewing ports 52, each of which
allows visual inspection of a corresponding reaction chamber 30 and
corresponds in size and shape to the inner perimeter of each O-ring
groove 36 in base plate 32 as shown in FIG. 8. The adhesive layer
permanently attaches compliance member 50 to cavity 60 of
compression plate 54 as shown in FIG. 8.
[0062] Compression plate 54 is most preferably 44 millimeters wide,
69 millimeters long, and 4 millimeters thick. Compression plate 54
is preferably formed of fluorinated ethylene propylene, acetal
resin, polyurethane, polypropylene, acrylonitrile-butadiene-styrene
(ABS), or any material having similar mechanical properties, and is
most preferably formed of Teflon. Compression plate 54 further
preferably includes six retaining pin apertures 56, four viewing
ports 58, and cavity 60. Retaining pin apertures 56 corresponding
to the six retaining pins 72 in base plate 32 are located around
the periphery of compression plate 54 and pass entirely through
compression plate 54 and as shown in FIG. 1. The pin apertures 56
are most preferably 5.5 millimeters in diameter. Each viewing port
58 allows visual inspection of a corresponding reaction chamber 30
and corresponds in size, shape, and location to each corresponding
viewing port 52 in compliance member 50 as shown in FIG. 8.
Compression plate cavity 60 is most preferably 2.2 millimeters
deep, 26 millimeters wide, and 65 millimeters long, and is
configured to contain compliance member 50 with minimum play.
[0063] Retaining plate 62 is most preferably 44 millimeters wide,
69 millimeters long, 1.5 millimeters thick. The retaining plate 62
is preferably stainless steel, copper, aluminum, titanium, or any
material having similar mechanical properties, more preferably is
stainless steel, and most preferably is 300 series stainless steel.
Retaining plate 62 further preferably comprises four viewing ports
64 located around the periphery of retaining plate 62 and six
retaining pin apertures 66, all of which pass entirely through the
thickness of retaining plate 62. Each viewing port 64 allows visual
inspection of a corresponding reaction chamber 30 and corresponds
in size, shape, and location to each corresponding viewing port 58
in compression plate 54. Each retaining aperture 66 further
includes a main section 68 that is substantially circular and a
notch 70 extending from the main section 68. Each main section 68
is most preferably 5.5 millimeters in diameter, allowing a pin head
78 to pass through. Each notch 70 is most preferably 2.2
millimeters in diameter, having a center 4 millimeters from the
center of the corresponding main section 68 as shown in FIG. 1.
[0064] As shown in FIG. 8, each retaining pin 72 is generally
cylindrical and is formed of stainless steel, aluminum, titanium,
ceramic, or any material having similar mechanical properties. More
preferably, retaining pin 72 is stainless steel, and most
preferably is 300 series stainless steel. Retaining pin 72
preferably comprises body 74, neck 76, and head 78. Body 74 has a
circular cross section most preferably 5 millimeters in diameter
and is most preferably 7.5 millimeters long. Body 74 is designed
specifically to be press-fitted into a pin aperture 44 such that
the end of body 74 is flush to the outer surface of base plate 32.
Alternatively, retaining pins 72 could be an integral molded
portion of base plate 32. Substrate 22 could also be clamped to
base plate 32 using standard fasteners including screws in place of
retaining pins 72. In large throughput embodiments, an automated
clamping mechanism could be used to simultaneously clamp one or
more substrates 22 to a base plate 32.
[0065] Pin neck 76 has a circular cross-section most preferably 2
millimeters in diameter and 3 millimeters long and is designed
specifically to engage notch 70 in the retaining pin aperture 66 of
retaining plate 62. Head 78 has a circular cross-section most
preferably 5 millimeters in diameter and is most preferably 2
millimeters long.
[0066] Port seal 46 is most preferably 52 millimeters long, 24
millimeters wide, 0.1 millimeters thick, and comprises a layer of
thermally conductive material having a biologically inert
pressure-sensitive adhesive backing attached thereto. The
conductivity of port seal 46 is preferably selected to allow
alteration of the fluid temperature by heating element 82 at a rate
of at least 2 degrees centigrade per second over a range from zero
degrees centigrade to 100 degrees centigrade. Most preferably, the
thermally conductive material is aluminum foil. After reaction
chamber apparatus 28 is assembled and loaded with fluid, port
sealing member 46 is temporarily affixed to base plate 32 such that
it completely seals off all ports 38 and 39 as shown in FIG. 4.
[0067] Heating element 82 heats reaction chamber apparatus 28 by
conduction directly through port sealing member 46 and base plate
32, and preferably is capable of altering the temperature of fluid
inside each reaction chamber 30 by at least 2 degrees centigrade
per second over a range from zero degrees centigrade to 100 degrees
centigrade. The embodiment describe herein is intended to interface
with a flat block style Alpha Module heating element and a
corresponding PTC-220 DNA Engine Tetrad available through MJ
Research, Inc. as shown in FIG. 12, although many other types of
thermal cycling systems that provide conductive or convective
heating could be used.
[0068] The preferred embodiment of the reaction apparatus is
assembled as follows. Retaining pins 72 are press-fit into base
plate pin apertures 44. A layer of primer is then applied to base
plate 32 containing retaining pins 72, followed by a layer of
biologically compatible surface coating. The substrate is then
positioned in base plate cavity 40, with the surface containing the
microarrays 24 of biologically reactive sites 26 facing the first
surface of the base plate 32. Compliance layer 50 is permanently
affixed in compression plate cavity 60 by application of the
adhesive layer to the compression plate 54. The pin apertures 56 in
compression plate 54 are aligned with the retaining pins 72, and
compression plate 54 is then seated on base plate 32. The main
sections 68 of retaining pin apertures 66 in retaining plate 62 are
aligned with retaining pin heads 78, retaining plate 62 is seated
on compression plate 54, and retaining plate 62 is then compressed
towards base plate 32 such that pin head 78 extends above retaining
plate 62. Retaining plate 62 is shifted laterally such that notch
70 engages each corresponding pin neck 76. Other methods of
temporarily locking the compression plate to the base plate,
including the use of an external clamp around the base plate and
the compression plate or a layer of adhesive between the base plate
and the compression plate, could also be used.
[0069] The reaction chambers 30 are loaded by inserting pipet tip
82 into first fluid port 38 as far as is necessary to create a seal
between tip 82 and port 38, and then slowly introducing fluid into
the corresponding reaction chamber 30 using a standard pipettor.
Second fluid port 39 allows air to escape as fluid enters reaction
chamber 30 through first port 38. Pipet tip 82 is removed from
first port 38 when reaction chamber 30 and second fluid port 39 are
completely loaded with fluid. If substrate 22 is visually
transparent, each reaction chamber 30 may be visually inspected
through each compression plate viewing port 58 and retaining plate
viewing port 64 immediately after loading for the presence of gas
bubbles. If gas bubbles are present over any microarray 24, the
fluid loading process must be performed again, or the reaction
chamber must be pressurized. Pressurization may be provided
manually by inserting additional fluid through a pipet tip inserted
into the first fluid port while the second fluid port is sealed, or
may be provided automatically by use of a pump and tubing attached
to the first fluid port. Preferably the chamber is pressurized to
between 27 and 207 kPa (4 and 30 psi), more preferably between 55
ad 69 kPa (8 and 10 psi), and most preferably to about 55 kPa (8
psi). Any other gas bubbles including those away from the edges of
any microarray 24, especially those near ports 38 and 39, are
harmless and can be ignored. After inspection, port seal 46 is
affixed to the lower surface of base plate 32 by applying the
pressure-sensitive adhesive side of the port seal port 46.
[0070] Once assembled, the reaction chamber apparatus 28 is placed
onto heating element b 84 as shown in FIG. 12, and thermal cycling
is commenced. Upon completion of the reaction, reaction chamber
apparatus 28 is removed from heating element 84, port seal 46 is
removed, retaining plate 62 and compression plate 54 are removed by
following the corresponding assembly steps in reverse, and finally
biochip 20 is removed.
[0071] Although the detailed description and operational
description previously recited contain many specific details, these
should not be construed as limitations on the scope of the
invention, but rather as an exemplification of one preferred
embodiment thereof. Those with skill in the art will recognize the
generality of the exemplified chamber, and the capacity for the
recited components as disclosed herein to be varied for any
particular purpose or reaction. For example, reaction chamber
apparatus 28 could be configured to accommodate a multitude of
different configurations of biochip 20, or apparatus 28 could be
configured to accommodate a biochip 20 comprising two microarrays
each having forty biologically reactive sites in one direction and
one hundred in a direction normal to the first direction. The size
of reaction chamber apparatus 28 could be scaled to accommodate a
substrate up to 310 millimeters wide, 310 millimeters long, and 3
millimeters thick. A high-throughput embodiment of reaction chamber
apparatus 28 that can accommodate a plurality of biochips 20 is
also possible.
[0072] The apparatus could be configured for automatic loading of
reaction chamber 30 by integrating an automated fluid pumping
system to interface to each fluid port 38 and 39. Such a pumping
system would allow introduction of a plurality of fluids into each
reaction chamber 30, and agitation and pressurization of fluids in
each reaction chamber 30.
[0073] Alternative means for creating a sealed reaction chamber
around each microarray 24 on substrate surface 22 of biochip 20
also exist. For example, well structures 34, O-rings 48, and O-ring
grooves 36 could be replaced with a single shaped gasket member
made from a biologically compatible sealing material such as
silicone rubber. The thickness of the gasket can easily be selected
such that when the substrate is clamped against the base plate the
resulting gap between the base plate and substrate is most
preferably the same as the depth of a well structure. The
disposable gasket reduces the complexity of the apparatus by
reducing the number of required elements and alleviates the
preventive maintenance required for O-rings.
[0074] The Examples that follow are illustrative of specific
embodiments of the invention and various uses thereof. They are set
forth for explanatory purposes only, and are not to be taken as
limiting the invention.
EXAMPLE 1
Preparation, Assembly and Loading of a Microfluidic Reaction
Chamber
[0075] Six retaining pins of 300 series stainless steel were
press-fitted into apertures on a grade 2 commercially pure titanium
base plate containing four well structures. A layer of Xylan 8840
black primer (Whitford Worldwide) was applied to the base plate,
followed by a layer of Dupont 856-200 Teflon-FEP clear. The base
plate and O-rings were soaked in a 1% Alconox Solution for at least
30 minutes, then thoroughly rinsed in distilled, de-ionized water,
and dried with compressed nitrogen or air to ensure proper
cleaning.
[0076] A clean O-ring (Parker Seal Group, O-Ring Division, Part No.
2-015) was pressed completely down into each O-ring groove on the
base plate. A soda glass microscope slide containing four
27.times.27 microarrays of polyacrylamide gel pads was then
inserted into the base plate cavity such that the microarrays faced
the base plate.
[0077] A low-compression silicone sponge rubber compliance layer
(McMaster-Carr Supply Co., Part No. 8623K82) was affixed in the
cavity of a Teflon.RTM. compression plate by application of the
adhesive side of the compliance member to the cavity. The retaining
pin apertures in the compression plate were then aligned with the
retaining pin heads, and the plate was seated on the base plate
with the compliance member seated on the microscope slide.
[0078] The pin apertures in a 300 series stainless steel retaining
plate were aligned with the retaining pin heads, and the retaining
plate was compressed towards the base plate such that the heads
extended through and above the retaining plate. The retaining plate
was then shifted laterally so that the pin necks engaged the notch
of the pin aperture, thereby locking the various components of the
apparatus together.
[0079] The reaction chambers were loaded by inserting a pipet tip
82 (VWR Scientific Products Corporation, Prod. No. 53510-084) into
a fluid port until a seal was created between the tip the port. The
reaction fluid was slowly introduced into the reaction chamber
using a pipettor (Rainin Instrument Company, P-200). When the
reaction chamber and the second fluid port were completely filled
with fluid, loading was halted. Each reaction chamber was visually
inspected for the presence of gas bubbles immediately after
loading. If gas bubbles were present over any microarray, the fluid
loading process was restarted. The fluid ports were then sealed by
applying the pressure-sensitive adhesive side of a piece of
aluminum foil tape (Beckman Instruments, Inc., Part No.
270-538620-A) to lower side of the base plate such that all fluid
ports were covered.
EXAMPLE 2
DNA Hybridization and Labeling
[0080] Nucleic acid probe molecules immobilized to each site 26 are
single-stranded; therefore, nucleic acid target molecules present
within the sample fluid introduced to each site must also be
single-stranded and contain a region complementary to the
oligonucleotide probe molecules for hybridization to occur. Nucleic
acids, however, naturally occur as double-stranded molecules.
Directly introducing single-stranded target molecules to the
single-stranded oligonucleotide probes immobilized to each site can
involve several time consuming steps that require costly reagents
and reduce the yield of the starting material. An additional
complication arises because single-stranded target molecules are
typically longer than the immobilized probe molecules, and often
have regions complementary to each other along the same target
molecule in addition to the region complementary to the immobilized
probe molecule, which may result in hybridization of the target
molecule to itself. This anomaly is commonly referred to as a
hairpin, and may preclude hybridization of the target molecule with
a complementary immobilized probe molecule.
[0081] Rapid thermal cycling in reaction chamber device alleviates
the problem of hairpins formation. During the thermal cycling
process, the heating element first increases the temperature of
reaction chamber contents to a level just below that required to
cause denaturing of any properly hybridized, double-stranded
target/probe molecules in the microarray. Improperly hybridized
target/probe molecules in the microarray, however, are denatured at
this temperature, as are any long double-stranded molecules.
[0082] The apparatus described in Example 1 is used to perform
nucleic acid amplification assays as follows. As an example,
oligonucleotide probe molecules are used having a sequence length
corresponding to a denaturing temperature of 60 degrees centigrade.
As shown in Table 1, after the apparatus is assembled, loaded and
sealed, the heating element first rapidly increases the temperature
of the sample fluid within each reaction chamber to 85 degrees
centigrade for 2 minutes and 30 seconds, creating conditions
sufficient to denature double-stranded target molecules into
single-stranded target molecules free from hairpin anomalies. The
heating element then rapidly decreases the temperature of the
sample fluid within each reaction chamber to 60 degrees
centigrade--the calculated melting temperature of the immobilized
probe molecules--for 10 minutes. The region of a single-stranded
target molecule complementary to an immobilized probe molecule may
then hybridize to that immobilized probe molecule before the target
molecule has a chance to form a hairpin or hybridize with another
complementary single-stranded target molecule.
[0083] In addition to target molecules, the sample fluid contains
DNA polymerase and a specific type of free nucleotide, for example
a fluorescently-labeled terminating nucleotide. After the target
molecules have hybridized to the immobilized probe molecules, the
DNA polymerase will covalently attach the free nucleotide to the
three prime terminal ends of the five prime linked immobilized
probe molecules. The polymerase can synthesize, depending on
sequence complementarity, a sister molecule to the target molecule
by using the immobilized probe molecule as a template. This allows
identification of specific nucleotide bases within the nucleic acid
sequence.
[0084] As shown in Table 1, the heating element again rapidly
increases the temperature of the sample fluid within each reaction
chamber again to 85 degrees centigrade for 30 seconds, again
creating the conditions required for denaturing of all
double-stranded target molecules in reaction chamber 30. Heating
and cooling steps are repeated many times to repeat the process of
covalently attaching free nucleotides to as many immobilized probe
molecules as possible. As shown in Table 1, this may take up to 4
hours to complete.
1 TABLE 1 Temperature Time Step (Degrees centigrade) (min:sec) 1 85
2:30 2 85 0:30 3 60 10:00 4 Go to step 2 and N/A repeat 20
times
[0085] This process can be used to query polymorphic nucleotides
within a given region by using two oligonucleotide probes that are
identical with the exception of a polymorphic base at the 3'
terminal ends. The free nucleotides present in the sample fluid are
fluorescently-labeled terminating nucleotides. When the target
molecules hybridize completely with the oligonucleotide probes, the
DNA polymerase is able to add exactly one fluorescent base to the
probe molecule. The result can be interpreted as a digital "on/off"
signal for each probe site.
[0086] An example is shown in FIG. 14. In this example, a blood
sample from patient A and a blood sample from patient B are
contained in the sample fluid. Two oligonucleotide probes having a
polymorphic base at the 3' terminal end are used to hybridize the
samples. FIG. 14A illustrates complete hybridization of a region of
patient A's sample with a probe having adenine as the 3' base. FIG.
14B illustrates complete hybridization of a region of patient B's
sample with a probe having guanine as the 3' terminal base. In each
of these cases, the complete hybridization of the target with the
probe, allows the DNA polymerase in the sample fluid to attach one
labeled base to the probe, and the site will be "on." FIG. 14C,
however, illustrates an incomplete hybridization due to a base
mismatch between the probe and target molecules at the 3' terminal
position on the probe, where the probe contains an adenine and the
target contains a guanine. In this case, the DNA polymerase will be
unable to attach a labeled base to the probe, and the site will be
"off."
[0087] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims.
* * * * *