U.S. patent application number 13/212685 was filed with the patent office on 2012-02-23 for chemical coating of microwell for electrochemical detection device.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to James BUSTILLO, Wolfgang HINZ, Shifeng LI, John MAURO.
Application Number | 20120045368 13/212685 |
Document ID | / |
Family ID | 45594240 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120045368 |
Kind Code |
A1 |
HINZ; Wolfgang ; et
al. |
February 23, 2012 |
Chemical Coating of Microwell for Electrochemical Detection
Device
Abstract
The described embodiments may provide a method of fabricating a
chemical detection device. The method may comprise forming a
microwell above a CMOS device. The microwell may comprise a bottom
surface and sidewalls. The method may further comprise applying a
first chemical to be selectively attached to the bottom surface of
the microwell, forming a metal oxide layer on the sidewalls of the
microwell, and applying a second chemical to be selectively
attached to the sidewalls of the microwell. The second chemical may
lack an affinity to the first chemical.
Inventors: |
HINZ; Wolfgang;
(Killingworth, CT) ; MAURO; John; (Burlingame,
CA) ; LI; Shifeng; (Fremont, CA) ; BUSTILLO;
James; (Castro Valley, CA) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
CARLSBAD
CA
|
Family ID: |
45594240 |
Appl. No.: |
13/212685 |
Filed: |
August 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61374676 |
Aug 18, 2010 |
|
|
|
Current U.S.
Class: |
422/69 ;
257/E21.002; 438/1; 438/49 |
Current CPC
Class: |
G01N 27/4145 20130101;
G01N 27/26 20130101; G01N 27/414 20130101; G01N 27/4148 20130101;
H01L 29/78 20130101; C12Q 1/6874 20130101; Y10T 436/143333
20150115 |
Class at
Publication: |
422/69 ; 438/49;
438/1; 257/E21.002 |
International
Class: |
G01N 30/00 20060101
G01N030/00; H01L 21/02 20060101 H01L021/02 |
Claims
1. A method of fabricating a chemical detection device, comprising:
forming a microwell above a CMOS device, wherein the microwell
comprises a bottom surface and sidewalls; applying a first chemical
to be selectively attached to the bottom surface of the microwell;
forming a metal oxide layer on the sidewalls of the microwell; and
applying a second chemical to be selectively attached to the
sidewalls of the microwell, wherein the second chemical lacks an
affinity to the first chemical.
2. The method of claim 1, wherein the microwell comprises one or
more top edges also covered by the metal oxide layer, wherein the
metal oxide layer is a mono-molecular layer.
3. The method of claim 2, wherein the metal oxide layer is coated
by a neutral PEG phosphate or PEG phosphonate.
4. The method of claim 2, wherein the metal oxide layer is a
solvent based deposition of zirconium oxide (ZrO.sub.2).
5. The method of claim 1, wherein the second chemical comprises a
silane group.
6. The method of claim 1, wherein the first chemical comprises a
phosphate, phosphonate, catechol, nitrocatechol, boronate,
phenylboronate, imidazole, silanol.
7. The method of claim 1, wherein the first chemical comprises a
ph-sensing group.
8. The method of claim 1, wherein the CMOS device comprises a
charge-sensitive transistor having a floating gate terminal and the
bottom surface of the microwell comprises a passivation layer on
top of the floating gate terminal.
9. A chemical detection device, comprising: a microwell having a
bottom surface and sidewalls, the bottom surface being covered by a
first chemical, the sidewalls being covered by a metal oxide layer
and a second chemical on top of the metal oxide layer, wherein the
second chemical lacks an affinity to the first chemical; and a CMOS
device placed underneath the bottom of the microwell.
10. The chemical detection device of claim 9, wherein the CMOS
device comprises a charge-sensitive transistor having a floating
gate terminal.
11. The chemical detection device of claim 10, wherein the bottom
of the microwell comprises a passivation layer on top of the
floating gate terminal.
12. The chemical detection device of claim 11, wherein the
passivation layer is a layer of metal oxide on top of one or more
other layers of metal oxide.
13. The chemical detection device of claim 9, wherein the first
chemical comprises a phosphate, phosphonate, catechol,
nitrocatechol, boronate, phenylboronate, imidazole, silanol,
14. The chemical detection device of claim 9, wherein the first
chemical comprises a pH-sensing group.
15. The chemical detection device of claim 9, wherein the microwell
has SiO.sub.2 sidewalls.
16. The chemical detection device of claim 9, wherein metal oxide
layer covering the sidewalls is a mono-molecular layer.
17. The chemical detection device of claim 16, wherein top edges of
the microwell are also covered by the metal oxide layer.
18. The chemical detection device of claim 17, wherein the metal
oxide layer is a solvent based deposition of one of zirconium oxide
(ZrO.sub.2).
19. The chemical detection device of claim 9, wherein the second
chemical is a neutral PEG phosphate or PEG phosphonate.
20. A method comprising: forming a microwell above a CMOS device,
wherein the microwell comprises a bottom surface and sidewalls, and
the microwell is configured to receive a solid phase support that
has a plurality of analytes attached thereto; applying a first
chemical to be selectively attached to the bottom surface of the
microwell; forming a metal oxide layer on the sidewalls of the
microwell; and applying a second chemical to be selectively
attached to the sidewalls of the microwell, wherein the second
chemical lacks an affinity to the first chemical, wherein the CMOS
device is configured to sense a charge at the bottom surface of the
microwell due to one or more byproducts generated by at least one
chemical reaction with the plurality of the analytes, wherein the
one or more byproducts lack an affinity to the sidewalls of the
microwell due to the second chemical.
21. The method of claim 20, wherein the first chemical is
positively charged prior to receiving the solid phase support to
facilitate depositing of the solid phase support.
22. The method of claim 20, wherein the solid phase support is one
of: a microparticle, a nanoparticle, a bead, solid and porous
comprising gels.
23. The method of claim 20, wherein the plurality of analytes are
multiple copies of a DNA sample.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to
previously filed U.S. provisional patent application Ser. No.
61/374,676 filed Aug. 18, 2010, and incorporates its disclosure by
reference in its entirety.
[0002] This application also incorporates by reference in its
entirety the U.S. patent application Ser. No. 12/785,716 filed May
24, 2010.
BACKGROUND
[0003] Electrochemical detection is attractive because it provides
high sensitivity, small dimensions, low cost, fast response, and
compatibility with microfabrication technologies. (See, e.g.,
Hughes et al., Science, 254: 74-80 (1991); Mir et al.,
Electrophoresis, 30: 3386-3397 (2009); Trojanowicz, Anal. Chim.
Acta, 653: 36-58 (2009); and, Xu et al., Talanta, 80: 8-18 (2009).)
These characteristics have led to the development of a variety of
sensors based on amperometric, potentiometric or impedimetric
signals and their assembly into arrays for chemical, biochemical
and cellular applications. (See, e.g., Yeow et al., Sensors and
Actuators B 44: 434-440 (1997); Martinoia et al., Biosensors &
Bioelectronics, 16: 1043-1050 (2001); Hammond et al., IEEE Sensors
J., 4: 706-712 (2004); Milgrew et al., Sensors and Actuators B 103:
37-42 (2004); Milgrew et al., Sensors and Actuators B, 111-112:
347-353 (2005); Hizawa et al., Sensors and Actuators B, 117:
509-515 (2006); Heer et al., Biosensors and Bioelectronics, 22:
2546-2553 (2007); Barbaro et al., Sensors and Actuators B, 118:
41-46 (2006); Anderson et al., Sensors and Actuators B, 129: 79-86
(2008); Rothberg et al., U.S. patent publication 2009/0127589; and,
Rothberg et al., U.K. patent application GB24611127.) Typically in
such systems, analytes are randomly distributed among an array of
confinement regions, such as microwells (also referred to herein as
"wells") or reaction chambers, and reagents are delivered to such
regions by a fluidics system that directs flows of reagents through
a flow cell containing the sensor array. Microwells in which
reactions take place, as well as empty wells where no reactions
take place, may be monitored by one or more electronic sensors
associated with each of the microwells.
[0004] In one type of electrochemical detection, the fundamental
reaction product, or "signal", is a pH change. The pH change is
detected by measuring a change in the surface charge at the bottom
of the well. The surface at the bottom of the well buffers the pH
change produced as a result of the biological reaction. The
resulting change in surface charge due to the biological reaction
is sensed by capacitive coupling of the bottom of the well to a
floating gate of a chemically-sensitive field effect transistor
(chemFET) below the surface. However, the sidewalls of the well are
too far removed from the chemFET to contribute to the chemFET
signal. Unfortunately, in current implementations, the sidewalls of
the well buffer as well as surface at the bottom of the well. For
example, FIG. 2A shows a prior art microwell structure with native
metal oxide, nitride, or oxinitride surface on both the bottom and
sidewall. Hence the sidewall's buffering reduces the signal
detected at the bottom of the well.
[0005] In view of the above, it would be advantageous to have
available a microwell structure and a method of conformal coating
and selective etching of microwell sidewalls that reduce sidewall
buffering, which overcome the deficiencies of current
approaches.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
[0006] FIG. 1 illustrates a cross-sectional view of a flow cell
according to an embodiment of the present teachings.
[0007] FIG. 2A illustrates a well structure of the prior art with a
native metal oxide surface.
[0008] FIG. 2B illustrates a well structure with a conformal
silanized surface according to an embodiment of the present
teachings.
[0009] FIG. 2C illustrates a well structure with a conformal
silanized surface, and all horizontal surfaces etched to the
original metal oxide according to an embodiment of the present
teachings.
[0010] FIG. 3A illustrates the molecular structure of a PEG-Silane
of one type of silane that can be used for the conformal coating
process of the microwells according to an embodiment of the present
teachings.
[0011] FIG. 3B illustrates the molecular structure of another type
of silane that can be used for the conformal coating process of the
microwell according to an embodiment of the present teachings.
[0012] FIG. 3C illustrates the molecular structure of a zwitter
ionic species silane that can be used for the conformal coating
process of the microwells according to an embodiment of the present
teachings.
[0013] FIGS. 4A-4C illustrate different stages of fabricating a
well structure according to an embodiment of the present
teachings.
[0014] FIG. 5 illustrates a process of fabricating a well structure
of FIG. 4C according to an embodiment of the present teachings.
[0015] FIGS. 6A-6F illustrate different stages of fabricating
another well structure according to an embodiment of the present
teachings.
[0016] FIG. 7 illustrates a process of fabricating a well structure
of FIG. 6F according to an embodiment of the present teachings.
[0017] FIG. 8 illustrates a well structure according to another
embodiment of the present teachings.
[0018] FIGS. 9A-9E illustrate different stages of fabricating a
well structure according to another embodiment of the present
teachings.
[0019] FIG. 10 illustrates a process of fabricating a well
structure of FIG. 9E according to an embodiment of the present
teachings.
[0020] FIG. 11 illustrates a chemical sensing process according to
an embodiment of the present teachings.
DETAILED DESCRIPTION
[0021] The described embodiments may provide a chemical detection
device with an improved signal-to-noise ratio (SNR). The chemical
detection device may include a microwell coated with a chemical
that binds to the bottom of the microwell and facilitates binding
of an ion and a CMOS device placed underneath the bottom of the
microwell. In different embodiments, the sidewalls of the microwell
may be made of silicon dioxide (SiO.sub.2) or plastic. In one
embodiment, the sidewalls of the microwell may be coated with a
silane group that reduces buffering of protons in solutions. The
SNR may be improved by a reduction of buffering of protons by the
sidewalls.
[0022] One embodiment may provide a method of fabricating a
chemical detection device with an improved signal-to-noise ratio.
The method may comprise forming a plastic layer on top of a CMOS
device, forming a layer of metal oxide on top of the plastic layer,
forming a microwell on the plastic layer on top of the CMOS device
by anisotropic plastic etching, and applying a chemical that binds
to the bottom of the microwell but does not bind to the plastic
sidewall.
[0023] Another embodiment may provide a method of fabricating a
chemical detection device with an improved signal-to-noise ratio.
The method may comprise forming an opening of a microwell on a
metal oxide layer on top of a silicon dioxide (SiO.sub.2) layer.
The SiO.sub.2 layer may be on top of a CMOS device. The method may
further comprise forming a circular undercut on the SiO.sub.7 layer
underneath an edge of the opening of the microwell, forming the
microwell on the SiO.sub.2 layer on top of the CMOS device by
anisotropic SiO.sub.2 etching, applying one chemical that binds to
the sidewalk and at the bottom of the microwell, deactivating the
chemical at the bottom of the microwell, and applying another
chemical that locates at the bottom of the microwell and
facilitates binding of ions.
[0024] A further embodiment may provide a method of fabricating a
chemical detection device with an improved signal-to-noise ratio.
The method may comprise forming a metal oxide layer on top of a
silicon dioxide (SiO.sub.2) layer. The SiO.sub.2 layer may be on
top of a CMOS device. The method may further comprise forming a
microwell on the SiO.sub.2 layer on top of the CMOS device,
applying a chemical that binds to a sidewall and a bottom of the
microwell, and etching away the chemical at the bottom of the
microwell.
[0025] Another embodiment may provide a method of fabricating a
chemical detection device with an improved signal-to-noise ratio.
The method may comprise forming a microwell in a silicon dioxide
(SiO.sub.2) layer on top of a CMOS device, applying a chemical to
be selectively attached to the bottom of the microwell, forming a
metal oxide layer on the top edges and sidewalk of the microwell,
and applying another chemical to be selectively attached to the top
edges and sidewalk of the microwell.
[0026] FIG. 1 is an expanded and cross-sectional view of flow cell
100 showing a portion of a flow chamber 106 with reagent flow 108
moving across the surface of microwell array 102 over the open ends
of the microwells. Microwell array 102 and sensor array 105
together may form an integrated unit forming a bottom wall or floor
of flow cell 100. In one embodiment, reference electrode 104 may be
fluidly connected to flow chamber 106. A microwell 101 and sensor
114 are shown in an expanded view. Microwell 101 may be formed in
the bulk material 110 by any conventional microfabrication
technique. The volume, shape, aspect ratio (such as base
width-to-well depth ratio), and other dimensional characteristics
of the microwells may be design choices that depend on a particular
application, including the nature of the reaction taking place, as
well as the reagents, byproducts, and labeling techniques (if any)
that are employed. The sensor 114 may be a chemFET with a floating
gate 118 having a sensor plate 120 separated from the microwell
interior by a passivation layer 116. The sensor 114 may be
predominantly responsive to (and generates an output signal related
to) the amount of charge 124 present on the passivation layer 116
opposite of the sensor plate 120. Changes in charge 124 may cause
changes in the current between source 121 and drain 122 of the FET,
which may be used directly to provide a current-based output signal
or indirectly with additional circuitry to provide a voltage-based
output signal. Reactants, wash solutions, and other reagents may
move into microwells from flow chamber 106 primarily by diffusion
140.
[0027] In one embodiment, reactions carried out in microwell 101
may be analytical reactions to identify or determine
characteristics or properties of an analyte of interest. Such
reactions may generate directly or indirectly byproducts that
affect the amount of charge adjacent the sensor plate 120.
(Indirect detection may occur, for example, if byproduct chelators
or other binding compounds are used that affect the sensor after
binding an analyte of interest or if labeling moieties are
employed, such as enzymes that may generate a secondary byproduct
as the result of a binding event, or the like). If such byproducts
are produced in small amounts or rapidly decay or react with other
constituents, then multiple copies of the same analyte may be
analyzed in microwell 101 at the same time in order to increase the
output signal ultimately generated. In one embodiment, multiple
copies of an analyte may be attached to a solid phase support 112,
either before or after deposition into a microwell. The solid phase
support 112 may be microparticles, nanoparticles, beads, solid and
porous comprising gels, and the like. For nucleic acid analyte,
multiple, connected copies may be made by rolling circle
amplification (RCA), exponential RCA, and like techniques, to
produce an amplicon without the need of a solid support.
[0028] In one embodiment, the byproduct produced as a result of
genomic DNA fragment sequencing is a pH change due to the
incorporation of a nucleotide. In an embodiment, this may occur at
an appropriate operating pH of between 7.5 and 7.8. Approximately
one proton is released per nucleotide that is incorporated under
the given conditions. The pH change is detected by measuring the
change in the charge of the surface on the bottom of the well. The
surface on the bottom and the sides of a well are typically
composed of a metal oxide or nitride. The surface may comprise a
number of surface groups that undergo charging reactions. These
charging reactions can be described using the equations below:
M-OHM-O.sup.-+H.sup.+ (1)
M-OH+H.sup.+M-OH.sub.2.sup.+ (2)
[0029] The number of surface groups, along with the characteristic
equilibrium constants associated with the charging reactions above,
impart a buffering capacity to the surface. The surface buffering
capacity in a limited sample volume, such as a microwell, will
buffer the pH change associated with the biological incorporation
reaction. The change in surface charge due to the charging
reactions (equations (1) and (2) above) may be sensed by
capacitively coupling the bottom of the well to a chemFET located
below the bottom surface of the well. As discussed in the
Background section, however, the sidewalls of the well are too far
removed from the chemFET to contribute to the chemFET signal.
Therefore, embodiments of the present invention use conformal
coating to differentiate the sidewalls and the bottom of the well,
and thus reduce the sidewall buffering.
[0030] FIGS. 26 and 2C show one embodiment of the present invention
which solves the problem of sidewall buffering by conformal coating
of the native metal oxide, nitride, or oxinitride surface layer,
followed by selective etching of all horizontal surfaces. FIG. 2B
shows the conformal coating of all surfaces of the microwell with
silane, in accordance with one embodiment of the present invention.
As shown in FIG. 26, microwells 202 may be covered (both on the
bottom and sidewalls) by a layer 204. The layer 204, for example,
may be a layer of chemical that lacks an affinity for protons. The
conformal coating reduces or eliminates surface groups that can
undergo reactions with protons in solution and hence reduces or
eliminates the buffering capacity of said surfaces. For example,
when the layer 204 is removed from the bottom of the microwells
202, the layer 204 on the sidewalls may help reduce or eliminate
buffering capacity of the sidewalls of the microwells. FIG. 2C
shows the results of the selective etching of horizontal surfaces
only (including the bottom of the wells and top edges between
wells), restoring the native metal oxide, nitride, or oxinitride
surface, and hence allows the pH sensing to occur selectively at
the bottom of the well. Different techniques for etching the
horizontal surfaces can be employed. In an embodiment, the etching
is accomplished using a directional reactive ion etch of the
horizontal surfaces (e.g., Bosch process).
[0031] FIGS. 3A-3C shows different types of silane that can be used
for the conformal coating process. FIG. 3A shows the structure of
PEG-Silane that can be used in the conformal coating process. It
may be available from NANOCS.com (Product code PEG6-0 I 02) and has
an average MW of PEG=2000. It may impart a hydrophilic surface that
does not change in charge with small changes in pH around the
operating pH (e.g., pH range of 7.5 to 7.8). FIG. 3B shows the
structure of N,N,N-trimethyI-3-(trimethoxysilyI)-1-propanaminium
chloride silane that can be used in the conformal coating process.
It may be available from Sigma Aldrich (Product #595888). It may
impart a hydrophilic positive charge to all vertical surfaces. The
charge on the surface does not respond to small changes in pH
around the operating pH. FIG. 3C shows the structure of a zwitter
ionic species silane that can be used in the conformal coating
process. It is available in a single step reaction using propane
sultone and APTMS. It may impart a highly hydrophilic but overall
neutral coating to the sidewalls.
[0032] FIGS. 4A-4C illustrate cross-sectional views of different
stages of fabricating a well structure according to an embodiment
of the present teachings. FIG. 4A shows a cross-sectional view of a
part of a chemical sensing device 400 before microwells are formed.
The chemical sensing device 400 may comprise a metal oxide layer
402, a microwell layer 404, a CMOS device layer 406 and charge
sensitive devices 408.1 and 4082 between the microwell layer 404
and the CMOS device layer 406. The metal oxide layer 402 may be on
top of the microwell layer 404, which in turn may be on top of the
CMOS device layer 406. The charge sensitive devices 408.1 and 408.2
may each represent a top portion of a chemFET. For example, the
charge sensitive devices 408.1 and 408.2 may be the passivation
layers of the chemFETs (e.g., passivation layer 116 of FIG. 1),
while the floating gate structures of the chemFETs may be
underneath the charge sensitive devices 408.1 and 408.2 (not
shown). In one embodiment, the microwell layer 404 may be a layer
of plastic (e.g., Cytop, TEFLON, Parylene, etc.). Further, in one
embodiment, the charge sensitive devices 408.1 and 408.2 may each
comprise two or more layers of metal oxides. For example, each of
the charge sensitive devices 408.1 and 408.2 may comprise a top
layer of tantalum pentoxide (Ta.sub.2O.sub.5) and a bottom layer of
aluminum oxide (Al.sub.2O.sub.3). The metal oxide layer 402 may
comprise one or more layers of Ta.sub.2O.sub.5, Hafnium dioxide
(HfO.sub.2), zirconium oxide (ZrO.sub.2), or Al.sub.2O.sub.3. The
CMOS device layer may be a layer of semiconductor material (e.g.,
Si).
[0033] FIG. 4B shows a cross-sectional view of the chemical sensing
device 400 with a pair of microwells 410. The microwells 410 may be
etched on the metal oxide layer 402 and the microwell layer 404. In
one embodiment, the microwells 410 may be etched to expose the top
of the charge sensitive devices 408.1 and 408.2. The microwells 410
may be etched, for example, by anisotropic etching. FIG. 4C shows a
cross-sectional view of the chemical sensing device 400 after a
chemical may be applied to it. The applied chemical, shown as
wiggly lines 412, may be coated only at the bottom of the
microwells because of inert property of the plastic sidewall. In
one embodiment, the plastic sidewalls may have zero buffering
because of the inert property. In one embodiment, the chemical may
be phosphate, phosphonate, or silane.
[0034] FIG. 5 illustrates a process 500 of fabricating a well
structure of FIG. 4C according to an embodiment of the present
teachings. At block 502, the process 500 may form a layer of
plastic on top of a CMOS device layer. As shown in FIG. 4A, the
plastic layer 404 may be formed on top of the CMOS device layer
406. Then at block 504, the process 500 may form a layer of metal
oxide on top of the plastic layer. For example, the metal oxide
layer 402 may be formed on top of the plastic layer 404 in FIG. 4A.
The process 500 may then proceed to block 506. At block 506, a
microwell may be formed on the plastic layer on top of the CMOS
device layer. As described above with respect to FIG. 4B, the
etching on the plastic layer may be performed by anisotropic
plastic etching. At block 508, a chemical may be applied. The
chemical (e.g., the wiggly lines 412) may bind only to the bottom
of the microwell, for example, because of the inert property of the
plastic sidewall.
[0035] FIGS. 6A-6F illustrate cross-sectional views of different
stages of fabricating a well structure according to another
embodiment of the present teachings. FIG. 6A shows a
cross-sectional view of a part of a chemical sensing device 600
before microwells are formed. The chemical sensing device 600 may
comprise a metal oxide layer 602, a microwell layer 604, a CMOS
device layer 606 and charge sensitive devices 608.1 and 608.2
between the microwell layer 604 and the CMOS device layer 606. The
metal oxide layer 602 may be on top of the microwell layer 604,
which in turn may be on top of the CMOS device layer 606. The
charge sensitive devices 608.1 and 608.2 may each represent a top
portion of a chemFET. For example, the charge sensitive devices
608.1 and 608.2 may be the passivation layers of the chemFETs
(e.g., passivation layer 116 of FIG. 1), while the floating gate
structures of the chemFETs may be underneath the charge sensitive
devices 608.1 and 608.2 (not shown). In one embodiment, the
microwell layer 604 may be a layer of silicon oxide (SiO.sub.2).
Further, in one embodiment, the charge sensitive devices 608.1 and
608.2 may each comprise two or more layers of metal oxides. For
example, each of the charge sensitive devices 608.1 and 608.2 may
comprise a top layer of Ta.sub.2O.sub.5 and a bottom layer of
Al.sub.2O.sub.3. The metal oxide layer 602 may comprise one or more
layers of Ta.sub.2O.sub.5, HfO.sub.2, ZrO.sub.2, or
Al.sub.2O.sub.3. The CMOS device layer 606 may be a layer of
semiconductor material (e.g., Si).
[0036] FIG. 6B shows a cross-sectional view of the chemical sensing
device 600 with a pair of microwells 610 being formed. The openings
of the microwells 610 may be etched on the metal oxide layer 602
and the upper portion of the microwell layer 604. As shown in FIG.
6B, underneath the opening edges of the metal oxide layer 602,
upper portion of the microwell layer 604 may be etched to create
undercuts. In one embodiment, the undercuts may be created by
isotropic SiO.sub.2 etching. FIG. 6C shows a cross-sectional view
of the chemical sensing device 600 with the pair of microwells 610
having been formed. In one embodiment, the microwells 610 may be
etched to expose the top of the charge sensitive devices 608.1 and
608.2. The microwells 610 may be formed by anisotropic SiO.sub.2
etching. FIG. 6D shows a cross-sectional view of the chemical
sensing device 600 after application of a chemical. The applied
chemical, shown as wiggly lines 612, may be coated to cover
sidewalls and bottom of the microwells 610. In one embodiment, the
chemical may be a silane group.
[0037] FIG. 6E shows a cross-sectional view of the chemical sensing
device 600 with the bottom of the pair of microwells 610 having
been cleaned of the applied chemical. In one embodiment, the
microwells 610 may be etched to clean the applied chemical and
expose the top of the charge sensitive devices 608.1 and 6082. The
bottom of the microwells 610 may be cleaned by directional etching,
reactive ion etching, sacrificial layer etching, or combination of
these etching techniques. If the sacrificial layer etching is used,
a sacrificial layer may be created on top of the metal oxide
sensing layers of the charge sensitive devices 608.1 and 608.2
before the chemical is applied. After the chemical is applied, the
sacrificial layer may be etched to strip the chemical at the bottom
of the microwells 610 to expose the metal oxide sensing layers of
the charge sensitive devices 608.1 and 608.2. In another
embodiment, the chemical may be deactivated by other means, such
as, for example, covered or chemically rendered inactive.
[0038] FIG. 6F shows a cross-sectional view of the chemical sensing
device 600 after application of another chemical. The other applied
chemical, which may be referred to as the first chemical and shown
as bolded wiggly lines 614, may be coated to cover the bottom of
the microwells 610. In one embodiment, the other chemical may be
one or more chemicals selected from a group including: phosphate,
phosphonate, catechol, nitrocatechol, boronate, phenylboronate,
imidazole, silanol or other pH-sensing group. The other chemical
may be positively charged to help bead loading and help
accumulation of electrical charges caused by chemical
reactions.
[0039] In one embodiment, the other chemical may be absent. That
is, the chemical sensing device 600 may have the sidewalls of
microwells 610 coated with one group of chemicals and may leave the
bottom of the microwells 610 exposed. By coating only the sidewalls
with one group of chemicals, the buffering of electrical charges by
the sidewalls may be reduced or eliminated, and the SNR may be
improved.
[0040] FIG. 7 illustrates a process of fabricating a well structure
of FIG. 6F according to an embodiment of the present teachings. At
block 702, an opening of a microwell may be formed on a metal oxide
layer on top of a silicon dioxide (SiO.sub.2) layer and the
SiO.sub.2 layer may be on top of a CMOS device layer. Then at block
704, a circular undercut may be formed on the SiO.sub.2 layer
underneath an edge of the opening of the microwell. As shown in
FIG. 6B, opening and undercuts around the opening for the
microwells 610 may be formed on the metal oxide layer 602 and
microwell layer 606. As described above with respect to FIG. 6B,
the undercuts may be created by isotropic SiO.sub.2 etching. At
block 706, a microwell may be formed on the SiO.sub.2 layer on top
of the CMOS device layer. For example, anisotropic SiO.sub.2
etching may be used to etch the microwells. At block 708, a
chemical may be applied to the microwell. The chemical may bind to
the sidewall and bottom of the microwell, and cover the sidewall
and bottom of the microwell. At block 710, the chemical at the
bottom of the microwell may be deactivated. For example, the
chemical may be removed (e.g., by directional etching, reactive ion
etching, sacrificial layer etching, or combination thereof),
inactivated by chemicals, or covered. At block 712, another
chemical may be applied to the microwell. The other chemical may
bind to the bottom of the microwell and facilitate binding of
ions.
[0041] FIG. 8 illustrates a well structure according to another
embodiment of the present teachings. The well structure of FIG. 8
may comprise a metal oxide layer 802, a microwell layer 804, a CMOS
device layer 806 and a chemical sensing device 808.1 or 808.2. The
metal oxide layer 802 may be on top of the microwell layer 804,
which in turn may be on top of the CMOS device layer 806. The
charge sensitive devices 808.1 and 808.2 may be exposed in the
microwells 810. The sidewalls and peripheral edges 816 of the
bottom of the microwells 810 may be covered by a second chemical
812 (non-bolded wiggly lines). The peripheral edges 816 may be
portions of the bottom of the microwell 810 around the charge
sensitive devices 818.1 and 808.2. The top surfaces of the chemical
sensing devices 808.1 and 808.2 may be covered by a first chemical
814 (bolded wiggly lines). In one embodiment, as shown in FIG. 8,
the microwells 810 may each have an inversed tapered shape. That
is, the bottom of the microwells 810 may have a larger diameter
than the top of the microwells 810 and thus, the sidewalls may be
inversely inclined. In one embodiment, the metal oxide layer 802
may comprise one or more layers of Ta.sub.2O.sub.5, HfO.sub.2,
ZrO.sub.2, or Al.sub.2O.sub.3; the microwell layer 806 may be a
layer of SiO.sub.2; and the CMOS device layer 806 may be a layer of
semiconductor material (e.g., Si). The microwells 810 may be etched
using directional etching, reactive ion etching, sacrificial layer
etching, or combination thereof.
[0042] FIGS. 9A-9E illustrate cross-sectional views of different
stages of fabricating a well structure according to another
embodiment of the present teachings. FIG. 9A shows a
cross-sectional view of a part of a chemical sensing device 900
before any microwells are formed. The chemical sensing device 900
may comprise a microwell layer 902 on top of a CMOS device layer
904, and a charge sensitive device 906 embedded between the
microwell layer 902 and CMOS device layer 904. The embedded device
906 may represent a top portion of a chemFET. For example, the
embedded device 906 may be the passivation layer of a chemFET
(e.g., passivation layer 116 of FIG. 1), the floating gate
structure of the chemFET may be underneath the device 906 (not
shown). In one embodiment, the microwell layer 902 may be a layer
of SiO.sub.2. Further, in one embodiment, the device 906 may
comprise two or more layers of metal oxides. For example, the
device 906 may comprise a top layer of Ta.sub.2O.sub.5 and a bottom
layer of Al.sub.2O.sub.3. The CMOS device layer may be a layer of
semiconductor material (e.g., Si).
[0043] FIG. 9B shows a cross-sectional view of the chemical sensing
device 900 with a microwell 908. The microwell 908 may be etched on
the microwell layer 902. In one embodiment, the microwell 908 may
be etched to expose the top of the device 906. The microwells 908
may be etched by anisotropic SiO.sub.2 etching. FIG. 9C shows a
cross-sectional view of the chemical sensing device 900 after
application of a chemical. The applied chemical, shown as the
bolded wiggly lines 910, may be coated only at the bottom of the
microwell 908 because of chemical properties of the surface of the
charge sensitive device 906. For example, the surface of the charge
sensitive device 906 may have selective affinity of phosphate or
phosphonate, or a silane group. In one embodiment, the chemical may
be a phosphate, phosphonate, catechol, nitrocatechol, boronate,
phenylboronate, imidazole, silanol or other pH-sensing group.
[0044] FIG. 9D shows a cross-sectional view of the chemical sensing
device 900 after the sidewalls and top horizontal portions 916
(also referred to herein as "top edge" or "horizontal surface
proximate to top edge of sidewall") of the microwell 908 is covered
by a layer 912 of metal oxide. In one embodiment, the metal oxide
layer 912 may be a mono-molecular layer. For example, the
mono-molecular layer may be a metal oxide formed by a chemo
selective reaction of well wall oxide (such as silicon dioxide)
with Zirconium alkoxides followed by hydrolysis post reaction. That
is, the metal oxide layer 912 may be a layer of a solvent based
deposition of ZrO.sub.2. The metal oxide layer 912 is not disposed
along the bottom portion of the well, as shown in FIG. 9D, due to
the lack of affinity between the metal oxide layer 912 and the
chemical 910, according to an embodiment of the present teachings.
In effect, the chemical 910 may serve as a "mask" or a
protective/barrier layer to prevent the metal oxide layer 912 from
being disposed along the bottom of the microwell. FIG. 9E shows a
cross-sectional view of the chemical sensing device 900 after
application of another chemical. The other chemical, shown as the
non-bolded wiggly lines 914, may be coated at the sidewalls and top
horizontal portions of the microwell 908 because of chemical
properties of the surface of the metal oxide layer 912. In one
embodiment, the other chemical may be a PEG phosphate or
phosphonate. To be consistent with previous embodiments, the
chemical coated at the bottom of the microwell 908 may be referred
to as the first chemical and the chemical coated at the sidewalls
may be referred to as the second chemical.
[0045] The neutral PEG surfaces at the sidewalls of the microwell
908 may reduce, or eliminate, the proton buffering at the
sidewalls. Further, the phosphate or phosphonate coated at the
bottom of the microwell may give positive charge to improve bead
loading or DNA primers to further boost sequencing signal-to-noise
ration (SNR).
[0046] FIG. 10 illustrates a process 1000 of fabricating a well
structure of FIG. 9E according to an embodiment of the present
teachings. At block 1002, a microwell may be formed in a silicon
dioxide (SiO.sub.2) layer on top of a CMOS device. As shown in FIG.
9B, the microwell 908 may be formed in the SiO.sub.2 layer 902.
Then at block 1004, a chemical may be applied to be selectively
attached to the bottom of the microwell. For example, a first
chemical may be applied and selectively attached to the top surface
of the charge sensitive device 906 in the microwell 908. At block
1006, a metal oxide layer on the sidewalls and top horizontal
portions of the microwell may be formed. As described above with
respect to FIG. 9D, a solvent based deposition of ZrO.sub.2
mono-molecular layer may be created at the sidewalls and top
horizontal portions of the microwell 908. At block 1008, a second
chemical may be applied that selectively attaches to the sidewalls
and top horizontal portions of the microwell. The second chemical
(e.g., the non-bolded wiggly lines 914 of FIG. 9E) may bind only to
the metal oxide layer 912 of the microwell at the sidewalls and top
horizontal portions, for example, because of the chemical
properties of the other chemical and metal oxide layer 912.
[0047] In one embodiment, microwells may be made on a layer of
metal that is placed on top of a CMOS device layer. That is, for
example, the microwell layer 902 may be a layer of metal, such as,
Al, Cu or Ti. In this embodiment, the bottom of the microwells may
be coated by the first chemical, or the sidewalls of the microwells
may be coated by the second chemical, or both. Further, in this
embodiment, the top edges of the microwells may be covered by a
metal oxide layer, such as one or more layers of tantalum pentoxide
(Ta.sub.2O.sub.5), aluminum oxide (Al.sub.2O.sub.3), Hafnium
dioxide (HfO.sub.2), or zirconium oxide (ZrO.sub.2). The
fabrication for metal microwells may use Damascene or dual
Damascene process. For example, use photolithography to make
negative pillar patterns, deposit a seed layer, perform metal
electrical plating, polish the metal and then etch the negative
pillar patterns to form metal microwells. See, e.g., Ohmori, et
al., Japanese Journal of Applied Physics 49 05FD01: 1-4 (2010), the
content of which is incorporated herein by reference.
[0048] In one embodiment, the silane group for the second chemical
may be R--[(CH.sub.2).sub.n]--Si--[X1X2X3] where R is an
organofunctional group, [(CH2).sub.n] is a hydrocarbon linker (n=1
to 20) Si is a silicon atom, and [X1X2X3] comprises one or more
independent hydrolysable groups, including alkoxy or halogen
groups. In another embodiment, the silane group for the second
chemical may be R--[(C2H4O)n]--Si--[X1X2X3] where R is an
organofunctional group, [(C2H4O)n] (n=1 to 100) is a polyether
linker, Si is a silicon atom, and [X1X2X3] comprises one or more
hydrolysable groups, including alkoxy or halogen groups. In either
of the embodiments, organofunctional groups R include, but are not
limited to methyl, methylene, phenyl, benzyl, anilino, amino,
amide, hydroxyl, aldehyde, alkoxy, halo, mercapto, carboxy, acyl,
vinyl, allyl, styryl, epoxy, isocyanato, glycidoxy, and
acryloxy.
[0049] Examples of the silane group for the second chemical may
include: [0050] N-(6-AMINOHEXYL)AMINOMETHYLTRIETHOXYSILANE, [0051]
(MERCAPTOMETHYL)METHYLDIETHOXYSILANE, [0052]
CHLOROMETHYLTRIETHOXYSILANE, [0053]
(ISOCYANATOMETHYL)METHYLDIMETHOXYSILANE, [0054]
N-PHENYLAMINOMETHYLTRIETHOXYSILANE, [0055] TRIETHOXYSILYLUNDECANAL,
[0056] 11-MERCAPTOUNDECYLTRIMETHOXYSILANE, [0057]
10-UNDECENYLTRIMETHOXYSILANE, [0058]
N-(2-AMINOETHYL)-11-AMINOUNDECYLTREETHOXYSILANE, [0059]
11-BROMOUNDECYLTRIMETHOXYSILANE, [0060] n-OCTYLTRIETHOXYSILANE,
[0061] 2-[METHOXY(POLYETHYLENEOXY)PROPYL]TRIMETHOXYSILANE, [0062]
3-METHOXYPROPYLTRIMETHOXYSILANE, [0063]
METHOXYTRIETHYLENOXYPROPYLTRISILANE, [0064] METHOXYSILANE, [0065]
METHOXYETHOXYUNDECYLTRICHLOROSILANE, [0066]
2-[METHOXY(POLYETHYLENEOXY)PROPYL]-TRICHLOROSILANE,
[0067] FIG. 11 illustrates a chemical sensing process 1100
according to an embodiment of the present teachings. At block 1102,
a solid phase support with a plurality of analytes attached thereto
may be deposited into a microwell. For example, a plurality of
analytes may be attached to the solid phase support 112 and the
solid phase support 112 may be deposited into the microwell 101.
The microwell may have a bottom surface and sidewalls. In one
embodiment, the bottom surface may be covered in a first chemical
that facilitates pH sensing and the sidewalls may be covered in a
second chemical that reduces buffering of protons in the solution.
At block 1104, a charge at the bottom surface of the microwell may
be sensed. As described with respect to equations (1) and (2), the
charge may be due to one or more byproducts generated by at least
one chemical reactions with the plurality of analytes. In one
embodiment, the chemical sensing process 1100 may be a DNA sequence
sensing process. That is, each of the plurality of analytes may be
a sample DNA fragment. The DNA sequence may be determined by
subsequently supplying different reagents to the microwell 101 and
detecting byproducts of chemical reactions.
[0068] Several embodiments of the present invention are
specifically illustrated and described herein. However, it will be
appreciated that modifications and variations of the present
invention are covered by the above teachings. In other instances,
well-known operations, components and circuits have not been
described in detail so as not to obscure the embodiments. It can be
appreciated that the specific structural and functional details
disclosed herein may be representative and do not necessarily limit
the scope of the embodiments. For example, some embodiments are
described using CMOS technology. A skilled artisan would appreciate
that a CMOS device may be used to refer to a pure PMOS device or a
pure NMOS device, or a combination of PMOS and NMOS devices.
[0069] Those skilled in the art may appreciate from the foregoing
description that the present invention may be implemented in a
variety of forms, and that the various embodiments may be
implemented alone or in combination. Therefore, while the
embodiments of the present invention have been described in
connection with particular examples thereof, the true scope of the
embodiments and/or methods of the present invention should not be
so limited since other modifications will become apparent to the
skilled practitioner upon a study of the drawings, specification,
and following claims.
[0070] Various embodiments may be implemented using hardware
elements, software elements, or a combination of both. Examples of
hardware elements may include processors, microprocessors,
circuits, circuit elements (e.g., transistors, resistors,
capacitors, inductors, and so forth), integrated circuits,
application specific integrated circuits (ASIC), programmable logic
devices (PLD), digital signal processors (DSP), field programmable
gate array (FPGA), logic gates, registers, semiconductor device,
chips, microchips, chip sets, and so forth. Examples of software
may include software components, programs, applications, computer
programs, application programs, system programs, machine programs,
operating system software, middleware, firmware, software modules,
routines, subroutines, functions, methods, procedures, software
interfaces, application program interfaces (API), instruction sets,
computing code, computer code, code segments, computer code
segments, words, values, symbols, or any combination thereof.
Determining whether an embodiment is implemented using hardware
elements and/or software elements may vary in accordance with any
number of factors, such as desired computational rate, power
levels, heat tolerances, processing cycle budget, input data rates,
output data rates, memory resources, data bus speeds and other
design or performance constraints.
[0071] Some embodiments may be implemented, for example, using a
computer-readable medium or article which may store an instruction
or a set of instructions that, if executed by a machine, may cause
the machine to perform a method and/or operations in accordance
with the embodiments. Such a machine may include, for example, any
suitable processing platform, computing platform, computing device,
processing device, computing system, processing system, computer,
processor, or the like, and may be implemented using any suitable
combination of hardware and/or software. The computer-readable
medium or article may include, for example, any suitable type of
memory unit, memory device, memory article, memory medium, storage
device, storage article, storage medium and/or storage unit, for
example, memory, removable or non-removable media, erasable or
non-erasable media, writeable or re-writeable media, digital or
analog media, hard disk, floppy disk, Compact Disc Read Only Memory
(CD-ROM), Compact Disc Recordable (CD-R), Compact Disc Rewriteable
(CD-RW), optical disk, magnetic media, magneto-optical media,
removable memory cards or disks, various types of Digital Versatile
Disc (DVD), a tape, a cassette, or the like. The instructions may
include any suitable type of code, such as source code, compiled
code, interpreted code, executable code, static code, dynamic code,
encrypted code, and the like, implemented using any suitable
high-level, low-level, object-oriented, visual, compiled and/or
interpreted programming language.
[0072] "Amplicon" means the product of a polynucleotide
amplification reaction. That is, a clonal population of
polynucleotides, which may be single stranded or double stranded,
which are replicated from one or more starting sequences. The one
or more starting sequences may be one or more copies of the same
sequence, or they may be a mixture of different sequences that
contain a common region that is amplified, for example, a specific
exon sequence present in a mixture of DNA fragments extracted from
a sample. Amplicons are formed by the amplification of a single
starting sequence. Amplicons may be produced by a variety of
amplification reactions whose products comprise replicates of the
one or more starting, or target, nucleic acids. In one aspect,
amplification reactions producing amplicons are "template-driven"
in that base pairing of reactants, either nucleotides or
oligonucleotides, have complements in a template polynucleotide
that are required for the creation of reaction products. In one
aspect, template-driven reactions are primer extensions with a
nucleic acid polymerase or oligonucleotide ligations with a nucleic
acid ligase. Such reactions include, but are not limited to,
polymerase chain reactions (PCRs), linear polymerase reactions,
nucleic acid sequence-based amplification (NASBAs), rolling circle
amplifications, and the like, disclosed in the following references
that are incorporated herein by reference: Mullis et al., U.S. Pat.
Nos. 4,683,195, 4,965,188, 4,683,202, and 4,800,159 (PCR); Gelfand
et al., U.S. Pat. No. 5,210,015 (real-time PCR with "taqman"
probes); Wittwer et al., U.S. Pat. No. 6,174,670; Kacian et al.,
U.S. Pat. No. 5,399,491 ("NASBA"); Lizardi, U.S. Pat. No.
5,854,033; Aono et al., Japanese patent publ. JP 4-262799 (rolling
circle amplification); and the like. In one aspect, amplicons are
produced by PCRs. As used herein, the term "amplifying" means
performing an amplification reaction. A "reaction mixture" means a
solution containing all the necessary reactants for performing a
reaction, which may include, but not be limited to, buffering
agents to maintain pH at a selected level during a reaction, salts,
co-factors, scavengers, and the like, A "solid phase amplicon"
means a solid phase support, such as a particle or bead, having
attached a clonal population of nucleic acid sequences, which may
have been produced by a process such as emulsion PCR, or like
technique.
[0073] "Analyte" means a molecule or biological cell of interest
that directly affects an electronic sensor at a sample retaining
region, such as a microwell, or that indirectly affects such an
electronic sensor by a byproduct from a reaction involving such
molecule or biological cell located in such a sample retaining
region, or reaction confinement region, such as a microwell. In one
aspect, analyte is a nucleic acid template that is subjected to a
sequencing reaction which, in turn, generates a reaction byproduct,
such as hydrogen ions, that affects an electronic sensor. The term
"analyte" also comprehends multiple copies of analytes, such as
proteins, peptide, nucleic acids, or the like, attached to solid
supports, such as beads or particles. In a one embodiment, the term
"analyte" means a nucleic acid amplicon or a solid phase
amplicon.
[0074] "Microfluidics device" means an integrated system of one or
more chambers, ports, and channels that are interconnected and in
fluid communication and designed for carrying out an analytical
reaction or process, either alone or in cooperation with an
appliance or instrument that provides support functions, such as
sample introduction, fluid and/or reagent driving means,
temperature control, detection systems, data collection and/or
integration systems, and the like. Microfluidics devices may
further include valves, pumps, and specialized functional coatings
on interior walls to, for example. prevent adsorption of sample
components or reactants, facilitate reagent movement by
electroosmosis, or the like. Such devices are usually fabricated in
or as a solid substrate, which may be glass, plastic, or other
solid polymeric materials, and typically have a planar format for
case of detecting and monitoring sample and reagent movement,
especially via optical or electrochemical methods. Features of a
microfluidic device usually have cross-sectional dimensions of less
than a few hundred square micrometers and passages typically have
capillary dimensions, e.g. having maximal cross-sectional
dimensions of from about 0.1 .mu.m to about 500 .mu.m.
Microfluidics devices typically have volume capacities in the range
of from a few nL, e.g. 10-100 nL to 1 .mu.L. The fabrication and
operation of microfluidics devices are well-known in the art as
exemplified by the following references that are incorporated by
reference: Ramsey, U.S. Pat. Nos. 6,001,229, 5,858,195, 6,010,607,
and 6,033,546; Soane et al., U.S. Pat. Nos. 5,126,022 and
6,054,034; Nelson et al., U.S. Pat. No. 6,613,525; Maher et al.,
U.S. Pat. No. 6,399,952; Ricco et al., International patent
publication WO 02/24322; Bjornson et al., International patent
publication WO 99/19717; Wilding et al., U.S. Pat. Nos. 5,587,128;
5,498,392; Sia et al., Electrophoresis, 24: 3563-3576 (2003); Unger
et al., Science, 288: 113-116 (2000); Enzelberger et al., U.S. Pat.
No. 6,960,437.
[0075] "Microwell," which is used interchangeably with "reaction
chamber," means a special case of a "reaction confinement region,"
that is, a physical or chemical attribute of a solid substrate that
permit the localization of a reaction of interest. Reaction
confinement regions may be a discrete region of a surface of a
substrate that specifically binds an analyte of interest, such as a
discrete region with oligonucleotides or antibodies covalently
linked to such surface. Usually reaction confinement regions are
hollows or wells having well-defined shapes and volumes which are
manufactured into a substrate. These latter types of reaction
confinement regions are referred to herein as microwells or
reaction chambers, and may be fabricated using conventional
microfabrication techniques, for example, as disclosed in the
following references: Doering and Nishi, Editors, Handbook of
Semiconductor Manufacturing Technology, Second Edition (CRC Press,
2007); Saliterman, Fundamentals of BioMEMS and Medical Microdevices
(SPIE Publications, 2006); Elwenspoek et al., Silicon
Micromachining (Cambridge University Press, 2004); and the like.
Configurations (e.g., spacing, shape and volumes) of microwells or
reaction chambers are disclosed in Rothberg et al., U.S. patent
publication 2009/0127589; Rothberg et al., U.K. patent application
GB24611127, which are incorporated by reference. Microwells may
have square, rectangular, or octagonal cross sections and be
arranged as a rectilinear array on a surface. Microwells may also
have hexagonal cross sections and be arranged as a hexagonal array,
which permit a higher density of microwells per unit area in
comparison to rectilinear arrays. Exemplary configurations of
microwells have 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6 or
10.sup.7 reaction chambers.
[0076] As used herein, an "array" is a planar arrangement of
elements such as sensors or wells. The array may be one or two
dimensional. A one dimensional array is an array having one column
(or row) of elements in the first dimension and a plurality of
columns (or rows) in the second dimension. The number of columns
(or rows) in the first and second dimensions may or may not be the
same. The array may include, for example, at least 100,000
chambers. Further, each reaction chamber has a horizontal width and
a vertical depth with, for example, an aspect ratio of about 1:1 or
less. The pitch between the reaction chambers is no more than about
10 microns, for example. Briefly, in one embodiment, microwell
arrays may be fabricated after the semiconductor structures of a
sensor array are formed, in which the microwell structure is
applied to such structure on the semiconductor die. That is, the
microwell structure can be formed on the die or it may be formed
separately and then mounted onto the die.
[0077] To form the microwell structure on the die, various
fabrication processes may be used. For example, the entire die may
be spin-coated with, for example, a negative photoresist such as
Microchem's SU-8 2015 or a positive resist/polyimide such as HD
Microsystems HD8820, to the desired height of the microwells. The
desired height of the wells (e.g., about 3-12 .mu.m in the example
of one pixel per well, though not so limited as a general matter)
in the photoresist layer(s) can be achieved by spinning the
appropriate resist at predetermined rates (which can be found by
reference to the literature and manufacturer specifications, or
empirically), in one or more layers. (Well height typically may be
selected in correspondence with the lateral dimension of the sensor
pixel for a nominal 1:1-1.5:1 aspect ratio, height:width or
diameter.) Alternatively, multiple layers of different photoresists
may be applied or another form of dielectric material may be
deposited. Various types of chemical vapor deposition may also be
used to build up a layer of materials suitable for microwell
formation therein. In one embodiment, microwells are formed in a
layer of tetra-methyl-ortho-silicate (TEOS). The invention
encompasses an apparatus comprising at least one two-dimensional
array of reaction chambers, wherein each reaction chamber is
coupled to a chemically-sensitive field effect transistor
("chemFET") and each reaction chamber is no greater than 10.sup.3
.mu.m.sup.3 (i.e., 1 pL) in volume. Each reaction chamber is no
greater than 0.34 pL, and no greater than 0.096 pL or even 0.012
pL. in volume. A reaction chamber can optionally be 0.5.sup.2, 1,
2.sup.2, 3.sup.2, 4.sup.2, 5.sup.2, 6.sup.2, 7.sup.2, 8.sup.2.
9.sup.2, or 10.sup.2 square microns in cross-sectional area at the
top. The array can have at least 10.sup.2, 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, or more reaction
chambers. The reaction chambers may be capacitively coupled to the
chemFETS.
[0078] "Primer" means an oligonucleotide, either natural or
synthetic that is capable, upon forming a duplex with a
polynucleotide template, of acting as a point of initiation of
nucleic acid synthesis and being extended from its 3' end along the
template so that an extended duplex is formed. Extension of a
primer is usually carried out with a nucleic acid polymerase, such
as a DNA or RNA polymerase. The sequence of nucleotides added in
the extension process is determined by the sequence of the template
polynucleotide. Usually primers are extended by a DNA polymerase.
Primers usually have a length in the range of from 14 to 40
nucleotides, or in the range of from 18 to 36 nucleotides. Primers
are employed in a variety of nucleic amplification reactions, for
example, linear amplification reactions using a single primer, or
polymerase chain reactions, employing two or more primers. Guidance
for selecting the lengths and sequences of primers for particular
applications is well known to those of ordinary skill in the art,
as evidenced by the following references that are incorporated by
reference: Dieffenbach, editor, PCR Primer: A Laboratory Manual,
2nd Edition (Cold Spring Harbor Press, New York, 2003).
* * * * *