U.S. patent application number 17/444487 was filed with the patent office on 2022-03-03 for molecular analysis system and use thereof.
The applicant listed for this patent is AKONNI BIOSYSTEMS, INC.. Invention is credited to Arial BUENO, Christopher G. COONEY, Peter Qiang QU.
Application Number | 20220062909 17/444487 |
Document ID | / |
Family ID | 1000005960398 |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220062909 |
Kind Code |
A1 |
COONEY; Christopher G. ; et
al. |
March 3, 2022 |
MOLECULAR ANALYSIS SYSTEM AND USE THEREOF
Abstract
A molecular testing device comprises a heating and cooling
module having a thin-film thermoelectric heating and cooling
device, and a removable test module having a combined amplification
and hybridization reaction chamber. The reaction chamber comprises
a thermo-conductive exterior surface and a microarray on an
interior surface. The thin-film thermoelectric heating and cooling
device has a heat transfer surface that is adapted to make contact
with the thermo-conductive exterior surface of the reaction
chamber. The molecular testing device may be used to perform a PCR
in the reaction chamber.
Inventors: |
COONEY; Christopher G.;
(Ellicott City, MD) ; QU; Peter Qiang; (New
Market, MD) ; BUENO; Arial; (Frederick, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AKONNI BIOSYSTEMS, INC. |
FREDERICK |
MD |
US |
|
|
Family ID: |
1000005960398 |
Appl. No.: |
17/444487 |
Filed: |
August 5, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16140101 |
Sep 24, 2018 |
11097278 |
|
|
17444487 |
|
|
|
|
16107468 |
Aug 21, 2018 |
10882046 |
|
|
16140101 |
|
|
|
|
14743389 |
Jun 18, 2015 |
10081016 |
|
|
16107468 |
|
|
|
|
62014329 |
Jun 19, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2200/12 20130101;
B01L 2300/1822 20130101; B01L 2300/0819 20130101; B01L 7/52
20130101; B01L 2200/147 20130101; B01L 2300/0636 20130101 |
International
Class: |
B01L 7/00 20060101
B01L007/00 |
Claims
1. A molecular testing device, comprising: a heating and cooling
module comprising a thin-film thermoelectric heating and cooling
device; and a removable test module comprising a combined
amplification and hybridization reaction chamber comprising a
thermo-conductive exterior surface and a microarray on an interior
surface; wherein said thermoelectric heating and cooling device
comprises a heat transfer surface that is adapted to make contact
with said thermo-conductive exterior surface of said reaction
chamber.
2. The molecular testing device of claim 1, wherein said thin-film
thermoelectric heating and cooling device is a Peltier device.
3. The molecular testing device of claim 2, wherein said Peltier
device is a ceramic Peltier device.
4. The molecular testing device of claim 1, wherein said thin-film
thermoelectric heating and cooling device comprises a thin-film
semiconductor comprising bismuth antimony, bismuth telluride, lead
telluride or silicon germanium.
5. The molecular testing device of claim 4, wherein said thin-film
semiconductor comprises bismuth telluride.
6. The molecular testing device of claim 1, wherein the thin-film
thermoelectric heating and cooling device is a thermoelectric
couple made of p and n type semiconductors.
7. The molecular testing device of claim 6, wherein the p and n
type semiconductors are selected from the group consisting of
bismuth antimony, bismuth telluride, lead telluride, and silicon
germanium.
8. The molecular testing device of claim 1, wherein the microarray
is a gel spot microarray.
9. The molecular testing device of claim 1, wherein said reaction
chamber further comprises an exterior surface that is insulated
with a thermal insulation material.
10. The molecular testing device of claim 1, wherein said removable
test module further comprises a waste chamber.
11. The molecular testing device of claim 1, wherein said removable
test module comprises a plurality of combined amplification and
hybridization reaction chambers, wherein each chamber comprises a
thermo-conductive exterior surface, and wherein said heating and
cooling module comprises a plurality of thermoelectric heating and
cooling device, wherein each of said plurality of thermoelectric
heating and cooling device comprises a heat transfer surface
adapted to make contact with a thermo-conductive exterior surface
of an amplification and hybridization reaction chamber.
12. The molecular testing device of claim 1, wherein said heating
and cooling module further comprises a temperature sensor.
13. The molecular testing device of claim 12, wherein said
temperature sensor comprises a thermistor or resistance thermal
device.
14. A device for performing a polymerase chain reaction (PCR),
comprising: a heating and cooling module comprising a thin-film
thermoelectric heating and cooling device comprising a heat
transfer surface; a holder for receiving a removable test module
comprising a reaction chamber having a thermo-conductive exterior
surface; a moving system that brings said heat transfer surface in
contact with said thermo-conductive exterior surface when said test
module is placed in said holder; and a programmable control module
that regulates temperature of said heat transfer surface.
15. The device of claim 14, wherein said thermoelectric device is a
Peltier device.
16. The device of claim 14, wherein said thermoelectric heating and
cooling device comprises a thin-film semiconductor and a heat
sink.
17. The device of claim 14, wherein said heating and cooling module
further comprises a temperature sensor.
18. The device of claim 17, wherein said temperature sensor
comprises a thermistor or resistance thermal device.
19. The device of claim 14, wherein said heating and cooling module
comprises a plurality of thin-film thermoelectric heating and
cooling devices each comprising a heat transfer surface, wherein
said removable test module comprises a plurality of reaction
chambers each having a thermo-conductive exterior surface, wherein
said programmable control module is capable of regulating
temperature of each of said heat transfer surface individually in
order to perform PCR under different conditions in each reaction
chamber.
20. A method for performing a polymerase chain reaction (PCR) on a
microarray in a reaction chamber, comprising: (a) placing a test
module comprising a reaction chamber into a PCR device, wherein
said reaction chamber comprises a thermo-conductive exterior
surface and a microarray mounted on an interior surface, and
wherein said PCR device comprises a heating and cooling module
comprising a thin-film thermoelectric heating and cooling device
with a heat transfer surface, and a programmable control module
that regulates temperature of said heat transfer surface; (b)
bringing said heat transfer surface of said thin-film
thermoelectric heating and cooling device into contact with said
thermo-conductive exterior surface of said reaction chamber; and
(c) completing a PCR by heating and cooling said reaction chamber
through said heat transfer surface based on a PCR program stored in
said control module.
Description
[0001] This application is a continuation application of U.S.
application Ser. No. 16/140,101, filed Sep. 24, 2018, now U.S. Pat.
No. 11,097,278, issued on Aug. 24, 2021 which is a continuation
application of U.S. application Ser. No. 16/107,468, filed on Aug.
21, 2018, now U.S. Pat. No. 10,882,046, issued on Jan. 5, 2021,
which is a continuation application of U.S. application Ser. No.
14/743,389, filed Jun. 18, 2015, now U.S. Pat. No. 10,081,016,
issued on Sep. 25, 2018, which claims priority of U.S. Provisional
Application No. 62/014,329, filed on Jun. 19, 2014, both of which
are incorporated herein in their entirety by reference.
FIELD
[0002] The present application relates generally to molecular
analysis systems and, in particular, to molecular analysis systems
with thermoelectric heating and cooling devices for detection of
biological materials in a sample using the polymerase chain
reaction (PCR).
BACKGROUND
[0003] Molecular testing is a test carried out at the molecular
level for detection of biological materials, such as DNA, RNA
and/or proteins, in a test sample. Molecular testing is beginning
to emerge as a gold standard due to its speed, sensitivity and
specificity. For example, molecular assays were found to be 75%
more sensitive than conventional cultures when identifying
enteroviruses in cerebrospinal fluid and are now considered the
gold standard for this diagnostic (Leland et al., Clin. Microbiol
Rev. 2007, 20:49-78).
[0004] Molecular assays for clinical use are typically limited to
identification of less than six genetic sequences in a single
reaction (i.e, real-time PCR assays). Microarrays, which are
patterns of molecular probes attached to a solid support, are one
way to increase the number of sequences that can be uniquely
identified. However, the workflow is typically complex and requires
molecular amplification prior to incubation, or hybridization, with
the microarray. Separate amplification and hybridization allows the
vessels for amplification to be designed for efficient thermal
transfer; however, the fluidic complexity is considerable.
Combining amplification and hybridization is one way to simplify
the fluidics and operational complexity; however, this approach can
suffer from thermal transfer inefficiencies because the reaction
vessel often consists of a thermally inefficient boundary or
support to which the microarrays can be attached.
SUMMARY
[0005] One aspect of the present application relates to a molecular
testing device. The device comprises a heating and cooling module
comprising a thermoelectric heating and cooling device, and a
removable test module comprising a combined amplification and
hybridization reaction chamber comprising a thermo-conductive
exterior surface and a microarray on an interior surface, wherein
the thermoelectric device comprises a heat transfer surface that is
adapted to make contact with the thermo-conductive exterior surface
of said reaction chamber.
[0006] Another aspect of the present application relates to a
device for performing PCR. The device comprises a heating and
cooling module comprising a thermoelectric heating and cooling
(TEHC) device comprising a heat transfer surface, a holder for
receiving a removable test module comprising a reaction chamber
having a thermo-conductive exterior surface, a moving system that
brings the heat transfer surface in contact with the
thermo-conductive exterior surface when the test module is placed
in the holder, and a programmable control module that regulates
temperature of the heat transfer surface.
[0007] Another aspect of the present application relates to a
method for performing PCR on a microarray in a reaction chamber.
The method comprises the steps of (a) placing a test module
comprising a reaction chamber into a PCR device, wherein the
reaction chamber comprises a thermo-conductive exterior surface and
a microarray mounted on an interior surface, and wherein the PCR
device comprises a heating and cooling module comprising a
thermoelectric heating and cooling device with a heat transfer
surface and a programmable control module that regulates
temperature of the heat transfer surface, (b) bringing the heat
transfer surface of the thermoelectric heating and cooling device
into contact with the thermo-conductive exterior surface of the
reaction chamber; and (c) completing a PCR by heating and cooling
the reaction chamber through the heat transfer surface based on a
PCR program stored in the control module.
BRIEF DESCRIPTION OF DRAWINGS
[0008] FIG. 1 is a diagram of an example of a heating and cooling
module.
[0009] FIG. 2 is a diagram of an example of an array of flow cell
reaction chambers and a waste chamber.
[0010] FIGS. 3A-3B are diagrams of an example of an array of flow
cell reaction chambers.
[0011] FIG. 4 is a diagram of an example of an array of flow cell
chambers on top of heating and cooling modules.
[0012] FIG. 5 is a diagram of an example of a heating and cooling
module that is lowered on top of a flow cell.
[0013] FIG. 6 is a diagram of a flow cell on top of a light
absorbing layer, an insulation layer and a supporting base.
[0014] FIG. 7 is a diagram showing a thermoelectric heating and
cooling (TEHC) device with two thin-film thermoelectric heating and
cooling chips within the heat and cooling unit.
[0015] FIGS. 8A-8F are diagrams showing different views of a
heating and cooling module with multiple TEHC devices.
[0016] FIG. 9 shows that insulating the exposed portions of the
reaction chamber reduces the temperature offset between the set
temperature and the actual temperature measured by a resistance
temperature detector (RTD) at the center of the reaction
chamber.
[0017] FIG. 10 shows exemplary fluorescent signal intensities from
microarray spots.
[0018] FIG. 11 shows results when performing PCR with the heating
and cooling module lowered on top of the reaction chamber.
[0019] FIG. 12 shows combined PCR and hybridization in the reaction
chamber when the heating and cooling module is lowered on top of
the reaction chamber.
DETAILED DESCRIPTION
[0020] The following detailed description is presented to enable
any person skilled in the art to make and use the invention. For
purposes of explanation, specific nomenclature is set forth to
provide a thorough understanding of the present application.
However, it will be apparent to one skilled in the art that these
specific details are not required to practice the invention.
Description of specific embodiments and applications is provided
only as representative examples. This description is an
exemplification of the principles of the invention and is not
intended to limit the invention to the particular embodiments
illustrated.
[0021] This description is intended to be read in connection with
the accompanying drawings, which are considered part of the entire
written description of this invention. The drawing figures are not
necessarily to scale and certain features of the invention may be
shown exaggerated in scale or in somewhat schematic form in the
interest of clarity and conciseness. In the description, relative
terms such as "front," "back" "up," "down," "top" and "bottom," as
well as derivatives thereof, should be construed to refer to the
orientation as then described or as shown in the drawing figure
under discussion. These relative terms are for convenience of
description and normally are not intended to require a particular
orientation. Terms concerning attachments, coupling and the like,
such as "connected" and "attached," refer to a relationship wherein
structures are secured or attached to one another either directly
or indirectly through intervening structures, as well as both
movable or rigid attachments or relationships, unless expressly
described otherwise.
[0022] As used herein, the term "sample" includes biological
samples such as cell samples, bacterial samples, virus samples,
samples of other microorganisms, samples obtained from a mammalian
subject, preferably a human subject, such as tissue samples, cell
culture samples, stool samples, and biological fluid samples (e.g.,
blood, plasma, serum, saliva, urine, cerebral or spinal fluid,
lymph liquid and nipple aspirate), environmental samples, such as
air samples, water samples, dust samples and soil samples.
[0023] The term "nucleic acid," as used in the embodiments
described hereinafter, refers to individual nucleic acids and
polymeric chains of nucleic acids, including DNA and RNA, whether
naturally occurring or artificially synthesized (including analogs
thereof), or modifications thereof, especially those modifications
known to occur in nature, having any length. Examples of nucleic
acid lengths that are in accord with the present invention include,
without limitation, lengths suitable for PCR products (e.g., about
50 to 700 base pairs (bp)) and human genomic DNA (e.g., on an order
from about kilobase pairs (Kb) to gigabase pairs (Gb)). Thus, it
will be appreciated that the term "nucleic acid" encompasses single
nucleic acids as well as stretches of nucleotides, nucleosides,
natural or artificial, and combinations thereof, in small
fragments, e.g., expressed sequence tags or genetic fragments, as
well as larger chains as exemplified by genomic material including
individual genes and even whole chromosomes. The term "nucleic
acid" also encompasses peptide nucleic acid (PNA) and locked
nucleic acid (LNA) oligomers.
[0024] The term "hydrophilic surface" as used herein, refers to a
surface that would form a contact angle of 45.degree. or smaller
with a drop of pure water resting on such a surface. The term
"hydrophobic surface" as used herein, refers to a surface that
would form a contact angle greater than 45.degree. with a drop of
pure water resting on such a surface. Contact angles can be
measured using a contact angle goniometer.
[0025] One aspect of the present application relates to a molecular
testing device. The device comprises a heating-and-cooling module
and a combined amplification and hybridization reaction chamber. In
some embodiments, the heating and cooling module comprises a heat
transfer surface that is adapted to make contact with an exterior
surface of the reaction chamber, and the reaction chamber comprises
a microarray.
[0026] In some embodiments, the heating-and-cooling module
comprises a plurality of TEHC devices and the same number of
combined amplification and hybridization reaction chambers. The
temperature in each reaction chamber is controlled by an individual
TEHC device such that different heating/cooling programs may be
applied to different reaction chambers. In some embodiments, the
heating-and-cooling module comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more TEHC devices and the same number of combined amplification and
hybridization reaction chambers.
Heating and Cooling Module
[0027] In some embodiments, the heating and cooling module includes
a thermoelectric heating and cooling (TEHC) device. One or more
TEHC devices can be integrated into the module. In other
embodiments, the heating and cooling module further comprises a
temperature sensor. Examples of temperature sensors are resistance
temperature detectors (RTDs), thermocouples, thermopiles, and
thermistors. In some embodiments, the temperature sensors are RTDs.
In other embodiments, the temperature sensors are thermistors,
which have higher resolution, a smaller temperature range and
larger drift over time. In some embodiments, a thermistor of the
heating and cooling unit couples to an electronic analog-to-digital
convertor (ADC).
[0028] In some embodiments, the TEHC device is a Peltier device. A
Peltier device is a thermoelectric heating and cooling device that
uses the Peltier effect to create a heat flux between the junction
of two different types of materials. A Peltier device functions as
a solid-state active heat pump that uses electrical energy to
transfer heat from one side of the device to the other, depending
on the direction of the current. Such an instrument can be used for
either heating or cooling and is also called a Peltier heat pump,
solid state refrigerator, or thermoelectric cooler (TEC). In some
embodiments, the Peltier device is made of ceramic materials (e.g.,
Ferrotec Peltier cooler model 72001/127/085B). Examples of ceramic
materials include: Alumina, Beryllium Oxide, and Aluminum
Nitride.
[0029] In other embodiments, the TEHC device is a thin-film
semiconductor (e.g., bismuth telluride). In other embodiments, the
TEHC device is a thermoelectric couple made of p and n type
semiconductors. Examples of p and n type semiconductors are bismuth
antimony, bismuth telluride, lead telluride, and silicon germanium.
This type of TEHC device has a response time that is shorter than
the 1 to 3 second response time of ceramic TEHC devices. This
characteristic allows rapid ramp rates and finer temperature
control. In some embodiments, the TEHC device is a thin-film
semiconductor having a response time less than 300 ms, 100 ms, 30
ms, 10 ms, 5 ms, 2 ms or 1 ms. In some embodiments, the TEHC
devices have footprints (e.g., 2.4 mm.times.3.5 mm) that offer an
ability to focus the heating and cooling towards a target area,
such as the exterior surface of the reaction chambers of a flow
cell. In some embodiments, the TEHC devices have footprints of 150
mm.sup.2 or less, 50 mm.sup.2 or less, 40 mm.sup.2 or less, 30
mm.sup.2 or less, 20 mm.sup.2 or less, or 10 mm.sup.2 or less. In
other embodiments, the TEHC devices have footprints of about 8.7
mm.times.15 mm, 5 mm.times.10 mm, 4 mm.times.8 mm, 3 mm.times.6 mm
or 2.4 mm.times.3.5 mm.
[0030] Furthermore, the high heat transfer power (e.g.,
Qmax/cm.sup.2.about.80 W/cm.sup.2 as compared to 3 W/cm.sup.2 for
ceramic Peltier devices) of these devices make them well suited for
heating and cooling small flow cell reaction chambers. In some
embodiments, the thin-film semiconductor thermoelectric devices are
coupled to heat spreaders of larger geometries to interface with
irregularly-shaped flow cell reaction chambers. These devices also
offer resistance to vibration and are less susceptible to failure,
caused by thermal cycling stress, than ceramic Peltiers.
[0031] FIG. 1 shows an embodiment of a heating and cooling module
200. In this embodiment, the heating and cooling module 200
includes a plurality of TEHC devices 204, each containing a heat
spreader 208 with a heat transfer surface 202 and a heating and
cooling unit 207; a platform (209, as shown, is a bezel to protect
TEHC devices 204) holding the TEHC devices 204; and a heat sink 201
coupled to the other side of the TEHC devices 204. Examples of heat
sinks 201 and heat spreader 208 are copper, aluminum, nickel, heat
pipes, and/or vapor chambers. During operation, the heat transfer
surface 202 makes intimate contact with an exterior surface of a
reaction chamber of a flow cell (shown in FIGS. 2 and 3) and thus
controls the temperature inside the reaction chamber of the flow
cell. In some embodiments, the heating and cooling module 200
further comprises an integrated printed circuit board 203 and a fan
205 under the heat sink 201.
[0032] In some embodiments, the heat sink 201 and/or heat spreader
208 are coupled to the heating and cooling unit 207 of the TEHC
device 204 with thermally conductive epoxy, thermally conductive
adhesives, liquid metal (e.g., Gallium) or solder (e.g., Indium).
In one embodiment the heat transfer surface 202 is flat. In some of
these embodiments the heat spreader 208 has a heat transfer surface
202 in a rectangular shape with dimensions that range from 3
mm.times.3 mm to 75 mm.times.80 mm, and preferably 8 mm.times.10 mm
to 10 mm.times.20 mm. In some embodiments, the heat transfer
surface 202 of the heat spreader 208 has an inlet section to heat a
fluidic channel of the flow cell where the inlet section is smaller
in size than the region that heats the reaction chamber. This inlet
section can be rectangular and has the size range of 0.1 to 5 mm
wide and 1 mm to 20 mm long. In another embodiment, the heat
transfer surface 202 of the heat spreader 208 has an outlet section
to heat a fluidic channel of the flow cell with a size range of 0.1
to 15 mm wide and 1 mm to 75 mm long. In some embodiments, the heat
transfer surface 202 of the heat spreader 208 has three sections,
an inlet heating section, a reaction chamber heating section, and
an outlet heating section. The thickness of the heat spreader 208
is preferably 0.05 to 5 mm, and more preferably 0.1 to 1 mm, and
even more preferably 0.15 to 0.6 mm.
Flow Cell
[0033] The term "flow cell," as used herein, refers to a
microarray-based detection device. In some embodiments, the flow
cell comprises a reaction chamber having a sample inlet, a sample
outlet and a microarray located therein. In some embodiments, the
reaction chamber is a combined amplification and hybridization
reaction chamber capable of performing both an amplification
reaction, such as a PCR, and a hybridization reaction in the same
chamber. In some embodiments, the flow cell further comprises a
waste chamber that is in fluid communication with the reaction
chamber. In some embodiments, the reaction chamber is coated with a
hydrophilic material and has a hydrophilic surface positioned to
facilitate complete filling of the reaction chamber and the fluid
flow from the reaction chamber to the waste chamber. The
hydrophilic surface contacts a liquid as it enters the reaction
chamber from the sample inlet and allows complete filling of the
microarray chamber. In certain embodiments, the reaction chamber is
in the shape of an elongated channel of variable width and is
directly connected to the waste chamber. In other embodiments, the
microarray chamber is connected to the waste chamber through a
waste channel.
[0034] In other embodiments, the flow cell comprises two or more
reaction chambers, or an array of reaction chambers. In other
embodiments, the flow cell comprises two or more reaction chambers
or an array of reaction chambers and two or more waste chambers or
an array of waste chambers, each reaction chamber is connected to a
waste chamber through a waste channel. In still other embodiments,
the flow cell comprises two or more reaction chambers or an array
of reaction chambers and a single waste chamber, wherein each
reaction chamber is connected to the waste chamber through a waste
channel.
[0035] In some embodiments, the microarray is located on the bottom
surface of the reaction chamber and the top surface, or at least a
portion of the top surface, of the reaction chamber is coated with
a hydrophilic material. Examples of the hydrophilic material
include, but are not limited to, hydrophilic polymers such as
polyethylene glycols, polyhydroxyethyl methacrylates, Bionite,
poly(N-vinyl lactams), poly(vinylpyrrolidone), poly(ethylene
oxide), poly(propylene oxide), polyacrylamides, cellulosics, methyl
cellulose, polyanhydrides, polyacrylic acids, polyvinyl alcohols,
polyvinyl ethers, alkylphenol ethoxylates, complex polyol
mono-esters, polyoxyethylene esters of oleic acid, polyoxyethylene
sorbitan esters of oleic acid, and sorbitan esters of fatty acids;
inorganic hydrophilic materials such as inorganic oxide, gold,
zeolite, and diamond-like carbon; and surfactants such as Triton
X-100, Tween, Sodium dodecyl sulfate (SDS), ammonium lauryl
sulfate, alkyl sulfate salts, sodium lauryl ether sulfate (SLES),
alkyl benzene sulfonate, soaps, fatty acid salts, cetyl
trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethyl
ammonium bromide, alkyltrimethylammonium salts, cetylpyridinium
chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium
chloride (BAC), benzethonium chloride (BZT), dodecyl betaine,
dodecyl dimethylamine oxide, cocamidopropyl betaine, coco ampho
glycinate alkyl poly(ethylene oxide), copolymers of poly(ethylene
oxide) and poly(propylene oxide) (commercially called Poloxamers or
Poloxamines), alkyl polyglucosides, fatty alcohols, cocamide MEA,
cocamide DEA, cocamide TEA.
[0036] In some embodiments, one or more surfactants are mixed with
reaction polymers such as polyurethanes and epoxies to serve as a
hydrophilic coating. In other embodiments, the top surface or the
bottom surface of the reaction chamber is made hydrophilic by
surface treatment such as atmospheric plasma treatment, corona
treatment or gas corona treatment.
[0037] The microarray in the reaction chamber can be any type of
microarray, including but not limited to oligonucleotide
microarrays and protein microarrays. In one embodiment, the
microarray is an antibody microarray and the microarray system is
used for capturing and labeling target antigens. In one embodiment,
the microarray is formed using the printing gel spots method
described in e.g., U.S. Pat. Nos. 5,741,700, 5,770,721, 5,981,734,
6,656,725 and U.S. patent application Ser. Nos. 10/068,474,
11/425,667 and 60/793,176, all of which are hereby incorporated by
reference in their entirety. In certain embodiments, the microarray
comprises a plurality of microarray spots printed on a microarray
substrate that forms the bottom of the microarray chamber.
[0038] FIG. 2 shows an exemplary array of flow cell reaction
chambers and a waste chamber. In this embodiment, the flow cell
comprises multiple reaction chambers 110, each having a channel 118
that connects the sample outlet of the reaction chamber 110 to the
inlet of the waste chamber 120. In one embodiment, the sidewall of
channel 118 is hydrophobic to trap bubbles. In some embodiments,
the cross-sectional area at the waste chamber end of the channel is
at least 2-times, 3-times, 4-times or 5-times larger than the
cross-sectional area at the reaction chamber end of the channel
118. In some embodiments, the channel 118 comprises a switchback
section that contains two turns to form an S-shaped or Z-shaped
channel section. In a further embodiment, the two turns are
90.degree. turns.
[0039] FIG. 3A shows another embodiment of a flow cell 100 with
multiple reaction chambers 110. In this embodiment, the reaction
chambers 110 are formed by a substrate 211, a spacer 212, and a
cover 213 (FIG. 3B). Materials used to create the substrate 211,
spacer 212, or the cover 213 include, but are not limited to,
ceramics, plastics, elastomers and metals. Examples of ceramics
include, but are not limited to, glass, silicon, silicon nitride,
and silicon dioxide. Examples of plastics include polycarbonate,
polyethylene (Low Density, High Density, UltraHigh Molecular
Weight), polyoxymethylene, polypropylene, polyvinylidene chloride,
polyester, polymethylmethacrylate, polyamide, polyvinylchloride,
polystyrene, acrylonitrile butadiene styrene, and polyurethane.
Examples of elastomers include, but are not limited to, natural
polyisoprene, synthethic polyisoprene, polybutadiene, chloroprene,
butyl rubber, styriene butadiene rubber, nitrile rubber, ethylene
propylene rubber, ethylene propylene diene rubber, epichlorohydrin
rubber, polyacrylic rubber, silicone rubber, fluorosilicone rubber,
fluoroelastomers, perfluoroelastomers, polyether block amides,
chlorosulfonated polyethylene, ethylene-vinyl acetate,
thermoelectric elastomers, protein resilin, elastin, polysulfide
rubber, and elastolefin. Examples of metals include, but are not
limited to, aluminum, platinum, gold, nickel, copper, and alloys of
these metals. These materials can be cast, extruded (e.g., films),
machined, and/or molded into the proper shape.
[0040] In some embodiments, the substrate material is plastic with
thermal conductivities of approximately 0.2 W/mK. In other cases
the substrate material is glass with a thermal conductivity of
about 1 W/mK. In some embodiments, the substrate material has a
thermal conductivity in the range of 0.2 to 3 W/mK. In some
embodiments, the substrate material has a thermal conductivity in
the range of 3 to 30 W/mK. In some embodiments, the substrate
material has a thermal conductivity in the range of 30 to 400 W/mK.
In other embodiments, the substrate material has a thermal
conductivity of at least 1, 3, 10, 30, 100 or 300 W/mK. In some
embodiments, the spacer 212 is bonded to the cover 213 and the
substrate 211. Bonding methods include adhesives, ultrasonic
welding, laser welding, heat staking, solvent bonding, thermal
bonding, and compression of an elastomeric spacer. Adhesives used
for bonding can be in a liquid or viscoelastic form. Examples of
adhesives include, but are not limited to, epoxies, acrylics,
silicones, polysaccharides, and rubbers. Adhesive curing can be
achieved with heat, pressure, ultraviolet irradiation, exposure to
air, and or catalysts.
[0041] In another embodiment the spacer 212 and the substrate 211
are a single monolithic part. In yet another embodiment the spacer
212 and the cover 213 are a single monolithic part. In still yet
another embodiment the substrate 211, spacer 212, and cover 213 are
a single monolithic part.
[0042] In some embodiments, the reaction chamber 110 comprises one
or more microarrays 130 formed on the substrate 211. In some
embodiments, the one or more microarrays 130 are DNA microarrays,
protein microarrays or mixtures thereof. As used herein, the term
"microarray` refers to an ordered array of spots presented for
binding to ligands of interest. A microarray consists of at least
two spots. In some embodiments, the microarray consists of a single
row of spots. In other embodiments, the microarray consists of a
plurality of rows of spots. The ligands of interest include, but
are not limited to, nucleic acids (e.g., molecular beacons,
aptamers, locked nucleic acids, peptide nucleic acids), proteins,
peptides, polysaccharides, antibodies, antigens, viruses, and
bacteria.
Interface Between Heating and Cooling Module and Reaction
Chamber
[0043] In some embodiments, the flow cell 100 is placed on top of
the heating and cooling module 200 so that the reaction chamber 110
is located on top of the heat transfer surface 202 of the heat and
cooling devices. See FIG. 4. In some embodiments the heating and
cooling module 200 is mounted to a moving system. In some
embodiments the heat transfer surface of the heat and cooling
devices absorbs light. Examples of how to achieve light absorption
include painting the heat transfer surface 202 black, black
anodizing, or coating it with black chrome by electroplating. Light
absorption reduces scatter that can interfere with imaging
microarrays. In some embodiments, thermal cycling occurs prior to
imaging. In some embodiments thermal cycling occurs simultaneously
with imaging.
[0044] In another embodiment the heating and cooling module 200 is
adapted to descend down on the flow cell 100 sitting on flow cell
holder 300, or flow cell holder 300 ascends up to the heating and
cooling module 200, such that the reaction chambers 110 of the flow
cell 100 make contact with the heat transfer surface 202 of the
TEHC devices (see FIG. 5). In some embodiments, compressible
devices are used to limit the force applied to the flow cell 100.
In some embodiments, the compressible devices are located above the
platform 209 on which the TEHC devices are mounted (See FIG. 5). In
other embodiments, the compressible devices 260 are located below
the flow cell 100 (see FIG. 6). In still other embodiments, the
compressible devices are located both above the platform 209 and
below the flow cell 100. Examples of compressible devices include,
but are not limited to, springs, foam, memory foam, leaf springs,
and deformable plastic or other materials such as silicon.
[0045] In some embodiments the external surfaces of the reaction
chamber 110 that do not interface with the heat transfer surface
202 are insulated. In some embodiments the insulation is a
component of the consumable. In other embodiments the insulation is
a component of the instrument. In still other embodiments the
insulation is a component on both the consumable and the
instrument. Examples of insulation include dead air, Styrofoam,
polyurethane foam, Aerogel, fiberglass, and plastic. In some
embodiments, the insulation layer 270 absorbs light. The effect of
insulation can be modeled as follows:
T offset = T TEC - T liquid .varies. 1 R insulation .times. ( T TEC
- T ambient ) ##EQU00001##
where T.sub.offset is the difference between the set temperature
and the actual temperature, T.sub.TEC is the temperature of the
heat spreader, T.sub.liquid is the temperature of the liquid, and
R.sub.insulation is the thermal resistance of the insulation
layer.
[0046] FIG. 6 shows an embodiment wherein the flow cell 100 is
insulated on one side with the insulation layer 270. In this
embodiment, a light absorbing material 271, such as black foil,
separates the insulation layer 270 from the flow cell 100. The
compressible devices are mounted below the insulation layer 270.
The base 250 comprises locating features 261 for the compressible
device. In some embodiments, the locating feature 261 is a stud,
pin or peg. In other embodiments, the locating feature 261 is a
cavity, hole or depression. In still other embodiments, the
locating feature 261 is a cavity, hole or depression with a stud,
pin or peg in its center.
[0047] In other embodiments, a single reaction chamber 110 may
interface with two or more TEHC devices 204. In one embodiment, one
TEHC device 204 interfaces with the top surface of the reaction
chamber 110, while another TEHC device 204 interfaces with the
bottom surface of the reaction chamber 110.
[0048] In another related embodiment, the heating-and-cooling
modules 200 comprises a plurality of TEHC devices 204 that
interface with an equal number of reaction chambers 110 in a flow
cell 100, wherein each TEHC device 204 comprises a heat transfer
surface 202 that is adapted to make contact with an exterior
surface of a corresponding reaction chamber 110. In some
embodiments, the TEHC devices 204 are attached to a common heat
sink 201. In some embodiments, all the TEHC devices 204 are
controlled by a single controller. In other embodiments, each TEHC
device 204 is separately controlled so that a different reaction
may be performed in each reaction chamber 110.
[0049] FIG. 7 shows an embodiment of a TEHC device 204 with two
thin-film thermoelectric chips 280 mounted within the heating and
cooling unit 207. The thin-film thermoelectric chips 280 are
manufactured with aluminum nitride semiconductors and are mounted
to a primary heat sink 221 with Indium solder and to a heat
spreader 208 with Gallium liquid metal. The heat spreader 208 is
0.6 mm thick copper with a nickel coating. A polyimide sheet spacer
serves as a standoff between the heat sink and the heat spreader
202. A thin-film RTD 281 is attached to the heat spreader 208 as
well.
[0050] FIGS. 8A-8F show different views of another exemplary
heating and cooling module 200 with multiple TEHC devices 204. Each
TEHC device 204 comprises a heat spreader 208 with a heat transfer
surface 202 and a heating/cooling unit with the primary heat sink
221. The multiple TEHC devices 204 are attached with a common,
secondary heat sink 201 with multiple fans 205.
Control Scheme for Thermal Cycling
[0051] In some embodiments the heating-and-cooling module is
controlled such that the set point temperature changes during the
ramping state as a means of accelerating the approach to the
desired temperature. In some embodiments the set point is
artificially set within a range of -5.degree. C. to 5.degree. C.
above the desired temperature
[0052] The heating-and-cooling module 250 performs thermal cycling
protocols that might include cycling between two temperatures,
cycling across three temperatures, a prolonged hold temperature for
storage or hybridization, and Touch Down PCR protocol. Temperature
transitions may follow a step change, a sawtooth waveform, or
sinusoidal waveform. These waveforms can also occur about a
specific set temperature to induce thermally-convective mixing.
[0053] An aspect of the present application relates to a molecular
testing device, comprising: a heating and cooling module comprising
a thin-film thermoelectric heating and cooling device; and a
removable test module comprising a combined amplification and
hybridization reaction chamber comprising a thermo-conductive
exterior surface and a microarray on an interior surface; wherein
said thermoelectric heating and cooling device comprises a heat
transfer surface that is adapted to make contact with said
thermo-conductive exterior surface of said reaction chamber.
[0054] In some embodiments, the thin-film thermoelectric heating
and cooling device is a Peltier device. In some further
embodiments, the Peltier device is a ceramic Peltier device.
[0055] In other embodiments, the thin-film thermoelectric heating
and cooling device comprises a thin-film semiconductor comprising
bismuth antimony, bismuth telluride, lead telluride or silicon
germanium. In some further embodiments, the thin-film semiconductor
comprises bismuth telluride.
[0056] In still other embodiments, the thin-film thermoelectric
heating and cooling device is a thermoelectric couple made of p and
n type semiconductors. In some further embodiments, the the p and n
type semiconductors are selected from the group consisting of
bismuth antimony, bismuth telluride, lead telluride, and silicon
germanium.
[0057] In yet other embodiments, the microarray is a gel spot
microarray.
[0058] In some embodiments, the reaction chamber further comprises
an exterior surface that is insulated with a thermal insulation
material.
[0059] In other embodiments, the removable test module further
comprises a waste chamber.
[0060] In still other embodiments, the removable test module
comprises a plurality of combined amplification and hybridization
reaction chambers, wherein each chamber comprises a
thermo-conductive exterior surface, and wherein said heating and
cooling module comprises a plurality of thermoelectric heating and
cooling device, wherein each of said plurality of thermoelectric
heating and cooling device comprises a heat transfer surface
adapted to make contact with a thermo-conductive exterior surface
of an amplification and hybridization reaction chamber.
[0061] In yet other embodiments, the heating and cooling module
further comprises a temperature sensor. In some further
embodiments, the temperature sensor comprises a thermistor or
resistance thermal device.
[0062] Another aspect of the present application relates to a
device for performing a polymerase chain reaction (PCR),
comprising: a heating and cooling module comprising a thin-film
thermoelectric heating and cooling device comprising a heat
transfer surface; a holder for receiving a removable test module
comprising a reaction chamber having a thermo-conductive exterior
surface; a moving system that brings said heat transfer surface in
contact with said thermo-conductive exterior surface when said test
module is placed in said holder; and a programmable control module
that regulates temperature of said heat transfer surface.
[0063] In some embodiments, the thermoelectric device is a Peltier
device. In some further embodiments, the thermoelectric heating and
cooling device comprises a thin-film semiconductor and a heat
sink.
[0064] In other embodiments, the heating and cooling module further
comprises a temperature sensor. In some further embodiments, the
temperature sensor comprises a thermistor or resistance thermal
device.
[0065] In still other embodiments, the heating and cooling module
comprises a plurality of thin-film thermoelectric heating and
cooling devices each comprising a heat transfer surface, wherein
said removable test module comprises a plurality of reaction
chambers each having a thermo-conductive exterior surface, wherein
said programmable control module is capable of regulating
temperature of each of said heat transfer surface individually in
order to perform PCR under different conditions in each reaction
chamber.
[0066] Yet another aspect of the present application relates to a
method for performing a polymerase chain reaction (PCR) on a
microarray in a reaction chamber. The method comprises several
steps, including placing a test module comprising a reaction
chamber into a PCR device, wherein said reaction chamber comprises
a thermo-conductive exterior surface and a microarray mounted on an
interior surface, and wherein said PCR device comprises a heating
and cooling module comprising a thin-film thermoelectric heating
and cooling device with a heat transfer surface, and a programmable
control module that regulates temperature of said heat transfer
surface. The method further comprises the step of bringing said
heat transfer surface of said thin-film thermoelectric heating and
cooling device into contact with said thermo-conductive exterior
surface of said reaction chamber. The method also comprises the
step of completing a PCR by heating and cooling said reaction
chamber through said heat transfer surface based on a PCR program
stored in said control module.
[0067] The present invention is further illustrated by the
following examples which should not be construed as limiting. The
contents of all references, patents and published patent
applications cited throughout this application, as well as the
Figures and Tables are incorporated herein by reference.
EXAMPLES
Example 1: Demonstration of the Effects of Insulation
[0068] A thin-film RTD (Minco RTD Model S39) is incorporated into a
reaction chamber (0.5 mm thick), filled with thermal paste, and
placed on a flat block Quanta thermocycler. One reaction chamber
includes a one inch thick Styrofoam insulation layer and the other
does not have insulation. The two reaction chambers are
sequentially introduced onto the thermocycler. The thermal cycling
protocol is 30 cycles of 88.degree. C. for 60 seconds followed by
55.degree. C. for 60 seconds. Only the denaturing temperatures are
plotted. Temperature measurements represent a moving average of 20
seconds. As can be seen from FIG. 9, there can be a temperature
offset of 1.degree. C. from the 88.degree. C. set point when the
reaction chamber is not insulated.
Example 2: Demonstration of PCR when Using Heating and Cooling
Module and Reaction Chamber
[0069] The reaction chamber is comprised of a Questar.TM.
substrate, an 0.5 mm double-sided pressure sensitive adhesive
spacer tape, and a cover film. The reaction chamber volume is
filled with approximately 50 .mu.L. The reaction chamber has an
inlet and an outlet hole.
[0070] The reaction chamber is filled 1.times.Qiagen QuantiFast
RT-PCR mix (Qiagen, Valencia, Calif., US) containing primer mix, 10
ng of human genomic DNA from NIST SRM 2372 kit, and 10.sup.4 copies
of purified Streptococcus pyogenes and influenza A nucleic
acid.
[0071] Primers are asymmetric in concentration, and the higher
concentration of primer is labeled with a fluorophore. Following
PCR, the fluorescently-labeled amplicon hybridizes to probes in the
gel spots on the microarray surface.
[0072] The thermal cycling protocol was 12.5 min at 47.degree. C.;
5 min at 88.degree. C.; and 35 cycles of 88.degree. C. for 30 s and
52.5.degree. C. for 35 s.
[0073] A control experiment was performed using amplification in a
PCR tube on a conventional MJ thermocycler using the same mastermix
as above and the following thermal cycling protocol was 12.5 min at
47.degree. C.; 5 min at 88.degree. C.; 35 cycles at 88.degree. C.
for 15 s and 52.5.degree. C. for 20 s.
[0074] Following PCR, the mastermix was removed from the chamber
and hybridized for 1 hr at 50.degree. C. to a microarray printed on
a glass substrate.
[0075] FIG. 10 shows fluorescent signal intensities from the
microarray spots for the S. pyogenes and influenza A probes. The
data show comparable results between the heating and cooling device
with reaction chamber and the conventional thermal cycler with PCR
tube.
Example 3: Demonstration of PCR when Heating and Cooling Module is
Lowered onto Reaction Chamber
[0076] A heating and cooling module 200 as described in Example 2
is mounted to a mechanical device that has a linear actuator that
is used to lower the assembly onto the reaction chamber (see FIG.
5). The assembly consists of 4 springs that compress when lowered
onto the reaction chamber.
[0077] Six reaction chambers similar to that of Example 2 are
constructed and attached to PVC Foam Insulation foam with double
sided tape.
[0078] The reaction chambers are filled with PCR mastermix and 33
pg of purified Mycobacterium tuberculosis (MTB) DNA from ATCC.
[0079] The following thermal cycling protocol is 88.degree. C. for
7.5 min, and 50 cycles of 88.degree. C. for 30 seconds and
55.degree. C. for 60 seconds.
[0080] The product from the PCR mastermix is mixed with a
hybridization buffer and incubated on a gel drop microarray, which
includes probes for katG (a gene with possible mutations that
confer drug resistance to isoniazid) and MTB. This is added to
25-.mu.L Frame seal chambers (Biorad) with a Parafilm cover and
incubated for 3 h at 55.degree. C. Following incubation, the slides
are agitated for 5 min in a bath consisting of 1.times.SSPE buffer
with 0.01% Triton X-100. The slides are then dried by
centrifugation at 2,300 rpm for 2 min.
[0081] Imaging is accomplished on an Akonni imaging system (see
U.S. Pat. No. 8,623,789; herein incorporated by reference in its
entirety) for 0.2 seconds and analyzed with Akonni software.
[0082] Signal intensities from the software are shown in FIG. 11.
The data in FIG. 11 shows positive amplification and detection from
the microarray spots that have probes for MTB and katG when
challenged with wild-type MTB DNA.
Example 4: Combined PCR and Hybridization in Reaction Chamber
[0083] N-acetyl cysteine, sodium hydroxide digested sputum was
amended with 10.sup.7 cfu/mL of H37Ra cells. Homogenization and
lysis was accomplished using the device described in U.S. Pat. No.
8,399,190 (herein incorporated by reference in its entirety).
Extraction of DNA was accomplished using the device and method
described in U.S. Pat. Nos. 8,236,553 and 8,574,923 (herein
incorporated by reference in their entirety).
[0084] Purified MTB DNA was mixed with PCR reagents described in
Example 3 and added to a reaction chamber, similar to that of
Example 2. The combined PCR and hybridization protocol was as
follows: 7.5 min at 90.5.degree. C., followed by 50 cycles of
90.5.degree. C. for 30 seconds and 56.degree. C. for 60 seconds,
and 3 hr of hybridization at 55.degree. C.
[0085] Following this protocol, the reaction chamber is washed with
300 .mu.L of 1.times.SSPE and imaged for 0.2 seconds using a
similar optical train as described in U.S. Pat. No. 8,623,789. The
image is analyzed and signal intensities from gel drops are
extracted and plotted in FIG. 12. FIG. 12 shows successful
amplification and detection of markers for MTB, katG, inhA (a gene
with possible mutations that confer drug resistance to isoniazid;
this isolate is wildtype), and rpoB (a gene with possible mutations
that confer drug resistance to rifampin; this isolate is
wildtype).
[0086] The above description is for the purpose of teaching the
person of ordinary skill in the art how to practice the present
invention, and it is not intended to detail all those obvious
modifications and variations of which will become apparent to the
skilled worker upon reading the description. It is intended,
however, that all such obvious modifications and variations be
included within the scope of the present invention.
* * * * *