U.S. patent application number 17/091620 was filed with the patent office on 2021-03-04 for fluidic devices with freeze-thaw valves with ice-nucleating agents and related methods of operation and analysis.
The applicant listed for this patent is The University of North Carolina at Chapel Hill. Invention is credited to Joseph Carl Gaiteri, William Hampton Henley, John Michael Ramsey.
Application Number | 20210060564 17/091620 |
Document ID | / |
Family ID | 1000005210118 |
Filed Date | 2021-03-04 |
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United States Patent
Application |
20210060564 |
Kind Code |
A1 |
Ramsey; John Michael ; et
al. |
March 4, 2021 |
FLUIDIC DEVICES WITH FREEZE-THAW VALVES WITH ICE-NUCLEATING AGENTS
AND RELATED METHODS OF OPERATION AND ANALYSIS
Abstract
Embodiments of the invention provide fluidic devices such as,
but not limited to, microfluidic chips, with one or more freeze
thaw valves (FTVs) employing one or more ice-nucleating agents
(INAs), that can reliably operate to freeze at relatively higher
temperatures and/or at faster rates than conventional microfluidic
devices with FTV systems.
Inventors: |
Ramsey; John Michael;
(Chapel Hill, NC) ; Henley; William Hampton;
(Chapel Hill, NC) ; Gaiteri; Joseph Carl; (Chapel
Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill |
Chapel Hill |
NC |
US |
|
|
Family ID: |
1000005210118 |
Appl. No.: |
17/091620 |
Filed: |
November 6, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15742662 |
Jan 8, 2018 |
10864520 |
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PCT/US2016/043463 |
Jul 22, 2016 |
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17091620 |
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62195652 |
Jul 22, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/0816 20130101;
C07K 14/21 20130101; F16K 2099/0084 20130101; F16K 99/0032
20130101; F16K 99/0044 20130101; B01L 3/502738 20130101; C12N
2310/16 20130101; C07K 14/27 20130101; B01L 2300/1894 20130101;
C07K 14/195 20130101; F16K 99/0036 20130101; G01N 1/42 20130101;
C12N 15/115 20130101; B01L 2400/0677 20130101; B01L 7/50
20130101 |
International
Class: |
B01L 7/00 20060101
B01L007/00; F16K 99/00 20060101 F16K099/00; C07K 14/21 20060101
C07K014/21; C07K 14/195 20060101 C07K014/195; C07K 14/27 20060101
C07K014/27; B01L 3/00 20060101 B01L003/00; C12N 15/115 20060101
C12N015/115; G01N 1/42 20060101 G01N001/42 |
Goverment Interests
STATEMENT OF FEDERAL SUPPORT
[0002] This invention was made with government support under Grant
No. HR0011-12-2-0001 awarded by DOD Defense Advanced Research
Projects Agency (DARPA). The government has certain rights in the
invention.
Claims
1. A liquid buffer comprising at least one ice nucleating agent
(INA), wherein the at least one INA contains a carbon atom.
2. The liquid buffer of claim 1, wherein the at least one INA is
present in the liquid buffer in an amount in a range of 1
molecule/4 to 10 billion molecules/4.
3. The liquid buffer of claim 1, wherein the at least one INA is
present in the liquid buffer in a concentration range of about 1 nM
and about 1 mM.
4. The liquid buffer of claim 1, wherein the at least one INA is
present in the liquid buffer in a concentration range of 10 nM to
50 nM.
5. The liquid buffer of claim 1, wherein the at least one INA is
extracted or derived from an organism.
6. The liquid buffer of claim 1, wherein the at least one INA
comprises an ice-nucleating protein (INP) and/or a functional
fragment thereof.
7. The liquid buffer of claim 6, wherein the INP and/or the
functional fragment thereof is encoded by a gene selected from the
group consisting of: iceE, iceH, inaA, inaE, inaF, inaK, inaPb,
inaQ, inaU, inaV, inaW, inaX, and inaZ, and wherein the gene is
found in or obtained from an organism selected from the group
consisting of: Pseudomonas syringae, Ps. fluorescens, Erwinia
herbicola, E. uredovora, Pantoea ananatis, and Xanthomonas
campestris.
8. The liquid buffer of claim 1, wherein the at least one INA
comprises one or more aptamers.
9. The liquid buffer of claim 1, wherein the at least one INA
comprises an ice-nucleating protein and/or a functional fragments
thereof, an ice-nucleating nucleic acid, ice-nucleating lipid,
and/or an ice-nucleating carbohydrate.
10. The liquid buffer of claim 1, wherein the at least one INA
comprises a structure similar to ice.
11. The liquid buffer of claim 1, wherein the at least one INA
comprises a structure that aligns water molecules into an ice-like
lattice.
12. The liquid buffer of claim 1, wherein the liquid buffer is
provided with a fluidic analysis device comprising at least one
fluid channel comprising at least one freeze thaw valve.
13. The liquid buffer of claim 1, wherein the liquid buffer is
provided with a fluidic analysis device and a liquid sealing agent
for the fluidic analysis device.
14. The liquid buffer of claim 1, wherein the liquid buffer
comprises a Tris buffer.
15. The liquid buffer of claim 1, wherein the liquid buffer
comprises a phosphate buffer.
16. The liquid buffer of claim 2, wherein the liquid buffer is a
liquid buffer solution comprising the liquid buffer and the at
least one INA, wherein the at least one INA is diluted by 1:10 to
1:10,000, relative to the liquid buffer in the liquid buffer
solution.
17. The liquid buffer of claim 1, wherein the liquid buffer freezes
at -20.degree. C.
18. The liquid buffer of claim 1, wherein the liquid buffer freezes
at a temperature in a range of about -45.degree. C. and about
-20.degree. C.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 15/742,662, filed Jan. 8, 2018, which is a 35
U.S.C. .sctn. 371 national stage application of PCT/US2016/043463,
filed Jul. 22, 2016, which claims the benefit of and priority to
U.S. Provisional Application Ser. No. 62/195,652, filed Jul. 22,
2015, the contents of which are hereby incorporated by reference as
if recited in full herein.
RESERVATION OF COPYRIGHT
[0003] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner, The University of North Carolina at Chapel Hill, N.C., has
no objection to the reproduction by anyone of the patent document
or the patent disclosure, as it appears in the United States Patent
and Trademark Office patent file or records, but otherwise reserves
all copyright rights whatsoever.
FIELD OF THE INVENTION
[0004] This invention relates to fluidic devices for processing
analytes for analysis.
BACKGROUND OF THE INVENTION
[0005] Microfluidic devices or chips have received a great deal of
attention for their ability to manipulate and study minute
quantities of liquid. See, e.g., Whitesides, G. M., The Origins and
the Future of Microfluidics. Nature 2006, 442, 368-373. A variety
of applications have been demonstrated with these devices,
including chromatographic and electrophoretic separations, cell
culture, and point-of-care medical diagnostics. In microfluidic
systems that utilize multiple liquids or reagents, valves are often
useful or necessary to modulate fluid flow. Freeze-thaw valves
(FTVs) utilize an external cooling source such as a thermoelectric
cooler (TEC) to freeze a plug of fluid in a channel, thus stopping
fluid flow in the desired regions of the fluidic chip. The channel
can then be opened by warming the plug until it thaws. However,
implementation of FTVs has been hindered by the low temperatures
needed to nucleate ice crystals on a microfluidic scale.
[0006] Liquid water does not necessarily nor immediately freeze
below its melting point. When the temperature of liquid water drops
below 0.degree. C., it enters a metastable thermodynamic state
(supercooling) in which small aggregates of a solid-like phase
("embryos") exist through the liquid phase. These embryos are
decaying and growing in equilibrium with the liquid phase, and
their average size can be proportional to the degree of
supercooling. In order for the metastable liquid phase to
transition to the stable solid phase (ice/frozen phase), embryo
growth should be energetically more favorable and likely than
decay. This occurs during a process known as "nucleation," of which
there are two types. In pure liquids, nucleation occurs
homogenously when the embryos reach a critical size and trigger
continued growth of the solid phase. The probability of homogenous
nucleation increases as the temperature of the supercooled phase
drops. At approximately -35.degree. C., the probability begins to
increase by a factor of 50 with each successive 1.degree. C. drop,
and at approximately -40.degree. C., the likelihood of homogenous
nucleation approaches or reaches 100%. Above this temperature
range, water can be nucleated heterogeneously with a foreign
substance such as a mineral, dust, or pollen. Deep supercooling is
not often observed on the macro scale because these nucleating
impurities can be abundant.
[0007] Microfluidic devices typically have smooth channel walls and
very small volumes, making nucleation sites rare. A high degree of
supercooling, often to temperatures below -20.degree. C., is
typically needed to freeze water in most microfluidic devices. This
increases the system's requirements for heat pumping and, in turn,
power. Strong thermal contact between the microfluidic device and
the cooling elements can also be a nontrivial design effort. Thin
materials such as D263 glass are often used to increase or maximize
heat pumping out of the channels. These thin glass materials can be
fragile. Additionally, in the absence of nucleation sites, the
reproducibility of successful valving events can have a high
percent relative standard deviation (% RSD). These problems may be
circumvented with the use of agents capable of nucleating ice at
warm temperatures, but most have limited applications in
microfluidic devices due to their water insolubility and/or
constituents that can inhibit many biological processes and cause
precipitation of many salts used in biological buffers. Also, many
nucleators function best when dry and can lose much of their
effectiveness when suspended in water. These considerations have
limited the implementation of FTVs in microfluidic devices.
SUMMARY OF EMBODIMENTS OF THE INVENTION
[0008] Embodiments of the invention provide microfluidic chips with
one or more FTVs employing one or more ice-nucleating agents (INAs)
(e.g., one or more ice-nucleating proteins "INP" and/or functional
fragment thereof and/or a microorganism comprising the same), that
can reliably operate to freeze at relatively high temperatures
and/or at faster rates than conventional microfluidic devices with
FTV systems.
[0009] Embodiments of the invention provide INAs that can reliably
function in aqueous solution and do not need to be dry, thereby
allowing multiple valve actuations on a respective microfluidic
device.
[0010] Embodiments of the invention can employ a substrate, such
as, for example, polymer, glass, silicon, metal, or other
substrates suitable for a microfluidic chip with FTVs.
[0011] Embodiments of the invention are directed to fluidic
analysis devices that include at least one fluid channel with at
least one freeze thaw valve and at least one ice nucleating agent
(INA).
[0012] The at least one INA can include one or more of an
ice-nucleating protein, an ice-nucleating nucleic acid,
ice-nucleating lipid, and/or an ice-nucleating carbohydrate.
[0013] The at least one INA can be extracted and/or derived from an
organism.
[0014] The at least one INA can include an ice-nucleating protein
(INP) and/or a functional fragment thereof. Optionally, the INP
and/or functional fragment thereof can be extracted from a membrane
of Pseudomonas syringae.
[0015] The INP can be encoded by one or more of the following
genes: iceE, iceH, inaA, inaE, inaF, inaK, inaPb, inaQ, inaU, inaV,
inaW, inaX, and inaZ found in and/or obtained from one or more of
the following organisms: Pseudomonas syringae, Ps. fluorescens
KUIN-1, Erwinia herbicola, E. uredovora, Pantoea ananatis, and
Xanthomonas campestris. Other genes and/or organisms may also be
used.
[0016] The fluidic analysis device can have first and second
substrates attached together to define a microfluidic chip with at
least one fluid transport channel and the fluid transport channel
with a plurality of spaced apart freeze thaw valves.
[0017] The fluidic analysis device can be in fluid communication
with a liquid buffer comprising the at least one INA. The at least
one INA can be flowably introduced into a fluid port on the fluidic
analysis device.
[0018] The at least one freeze thaw valve can be a plurality of
spaced apart freeze thaw valves, each including a thermoelectric
cooler in thermal communication with a defined region of the at
least one fluid channel.
[0019] The at least one fluid channel can have at least a segment
that is sized to be a microfluidic or nanofluidic channel.
[0020] The at least one fluid channel can have a primary transport
channel and at least one reagent channel.
[0021] The fluidic analysis device can have a sample input, a
buffer input and a bead well array, all in fluid communication with
the primary transport channel. The at least one freeze thaw valve
can be a plurality of spaced apart freeze thaw valves in fluid
communication with the primary transport channel and/or at least
one reagent channel.
[0022] The at least one fluid channel can include at least one
fluid channel with the at least one INA bonded and/or coated to a
surface thereof.
[0023] The fluid analysis device can have a bead storage/sample
incubation chamber, a waste reservoir and a sample metering loop.
The sample metering loop can reside between the bead
storage/incubation chamber and the sample input.
[0024] The sample metering loop can have a volumetric capacity of
between about 1 .mu.L to about 1 mL and is in fluid communication
with first and second freeze thaw valves on respective end portions
of the sample metering loop. INAs can be used to improve freezing
of the sample in the sample metering loop as a part of sample
preparation including, but not limited to, cell lysis.
[0025] The at least one INA may comprise a structure similar to ice
and/or a structure that aligns water molecules into an ice-like
lattice.
[0026] Other embodiments are directed to methods of analyzing a
target analyte. The methods include: providing a fluidic analysis
device with at least one fluidic channel in fluid communication
with at least one freeze thaw valve; introducing at least one ice
nucleating agent (INA) into the at least one fluidic channel; and
electronically selectively cooling the at least one freeze thaw
valve to freeze the at least one freeze thaw valve using the at
least one INA.
[0027] The at least one freeze thaw valve can be a plurality of
spaced apart freeze thaw valves. The method can include
(electronically) setting coolers thermally communicating with the
at least one fluidic channel to a temperature, typically in a range
of -100 degrees C. to -1 degree C., more typically between -50
degrees C. and -2 degrees C., to freeze the freeze thaw valves.
[0028] The fluidic device can be a fluidic microchip with a
thickness between 0.01 mm and 10 mm.
[0029] The at least some of the freeze thaw valves have
thermoelectric coolers set to a desired temperature, such as, for
example, between about -50 degrees C. and about -2 degrees C., for
a freeze operation that occurs in three minutes or less and the
electronically selectively cooling can be carried out a plurality
of times for a respective fluidic device, analyte and/or
analysis.
[0030] The device can be a fluidic microchip.
[0031] The analyte can be or include a molecule of DNA, RNA,
peptide, protein, glycan, pharmaceutical, or other biological or
synthetic macromolecule or an elemental or inorganic compound.
[0032] The at least one INA can include an ice-nucleating protein,
an ice-nucleating nucleic acid, ice-nucleating lipid, and/or an
ice-nucleating carbohydrate.
[0033] The at least one INA can be extracted or derived from an
organism.
[0034] The at least one INA can include an ice-nucleating protein
(INP) and/or functional fragments thereof.
[0035] Optionally, the INP and/or functional fragment thereof is
extracted from a membrane of Pseudomonas syringae.
[0036] The INP can be encoded by one or more of the following
genes: iceE, iceH, inaA, inaE, inaF, inaK, inaPb, inaQ, inaU, inaV,
inaW, inaX, and inaZ found in and/or obtained from one or more of
the following organisms: Pseudomonas syringae, Ps. fluorescens
KUIN-1, Erwinia herbicola, E. uredovora, Pantoea ananatis, and
Xanthomonas campestris. Other genes and/or organisms may also be
used.
[0037] The at least one INA is flowably introduced into the at
least one fluidic channel in a liquid.
[0038] The liquid can be a buffer. The at least one INA can be
diluted in the buffer.
[0039] The at least one INA can be introduced from a surface
comprising a coating of the at least one INA and/or the at least
one INA is adsorbed and/or chemically bonded to a surface of the at
least one fluidic flow channel.
[0040] The at least one INA can have structure similar to ice
and/or a structure that aligns water molecules into an ice-like
lattice.
[0041] Yet other embodiments are directed to liquid buffers for DNA
processing.
[0042] The liquid buffers can include at least one ice nucleating
agent (INA), optionally diluted to between 1 molecule/4 and 10
billion molecules/4. Concentrations of the INA can also exceed 10
billion molecules/.mu.L and may span the nanomolar, micromolar,
and/or millimolar concentration ranges.
[0043] The at least one INA can be extracted or derived from an
organism.
[0044] The at least one INA can include an ice-nucleating protein
(INPs) and/or a functional fragment thereof. Optionally, the INP
and/or functional fragment thereof can be extracted from a membrane
of Pseudomonas syringae.
[0045] The INP can be encoded by one or more of the following
genes: iceE, iceH, inaA, inaE, inaF, inaK, inaPb, inaQ, inaU, inaV,
inaW, inaX, and inaZ found in and/or obtained from one or more of
the following organisms: Pseudomonas syringae, Ps. fluorescens
KUIN-1, Erwinia herbicola, E. uredovora, Pantoea ananatis, and
Xanthomonas campestris. Other genes and/or organisms may also be
used.
[0046] The at least one INA can include one or more aptamers.
[0047] The at least one INA can include one or more of an
ice-nucleating protein, an ice-nucleating nucleic acid,
ice-nucleating lipid, and/or an ice-nucleating carbohydrate.
[0048] The at least one INA can have a structure similar to ice
and/or a structure that aligns water molecules into an ice-like
lattice.
[0049] It is noted that aspects of the invention described with
respect to one embodiment, may be incorporated in a different
embodiment although not specifically described relative thereto.
That is, all embodiments and/or features of any embodiment can be
combined in any way and/or combination. Applicant reserves the
right to change any originally filed claim and/or file any new
claim accordingly, including the right to be able to amend any
originally filed claim to depend from and/or incorporate any
feature of any other claim or claims although not originally
claimed in that manner. These and other objects and/or aspects of
the present invention are explained in detail in the specification
set forth below. Further features, advantages and details of the
present invention will be appreciated by those of ordinary skill in
the art from a reading of the figures and the detailed description
of the preferred embodiments that follow, such description being
merely illustrative of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1A is a schematic end cutaway view of a microfluidic
chip with at least one thermoelectric cooler positioned proximate a
transport channel according to embodiments of the present
invention.
[0051] FIG. 1B is a schematic top view of the device shown in FIG.
1A, illustrating the thermoelectric cooler below the substrate
holding at least part of the transport channel according to
embodiments of the present invention.
[0052] FIG. 2A is an enlarged top view of an exemplary microfluidic
chip according to embodiments of the present invention.
[0053] FIG. 2B is an enlarged top view of the exemplary
microfluidic chip shown in FIG. 2A and further illustrating
additional components/features including exemplary freeze-thaw
valve foot-prints according to embodiments of the present
invention.
[0054] FIG. 2C is an enlarged top view of the microfluidic chip
shown in FIG. 2B with annotations indicating valve numbers
according to some embodiments of the present invention.
[0055] FIG. 3A is an enlarged top view of an exemplary microfluidic
chip according to embodiments of the present invention.
[0056] FIG. 3B is an enlarged top view of the exemplary
microfluidic chip shown in FIG. 3A and further illustrating
additional components/features including exemplary freeze-thaw
valve foot-prints according to embodiments of the present
invention.
[0057] FIG. 3C is an enlarged top view of the microfluidic chip
shown in FIG. 3B with annotations indicating valve numbers
according to some embodiments of the present invention.
[0058] FIG. 4A is a digital photograph of a dual substrate
microfluidic chip according to embodiments of the present
invention.
[0059] FIG. 4B is a digital photograph of a dual substrate
microfluidic chip according to other embodiments of the present
invention.
[0060] FIG. 5A is a digital photograph of a dual substrate
microfluidic chip illustrating placement of a PTFE air-only
membrane over a defined region of the chip according to embodiments
of the present invention.
[0061] FIG. 5B is a digital photograph of an enlarged segment of
the microfluidic chip shown in FIG. 5A but with the PTFE air-only
membrane not in position over the bead storage/sample incubation
chamber according to embodiments of the present invention.
[0062] FIG. 6 is a flow chart of exemplary actions of methods for
processing and/or analyzing analytes that can be carried out
according to embodiments of the present invention.
[0063] FIG. 7 is a side, front perspective view of a portion of an
automated test system that can releasably engage one or more
microfluidic chips for analysis according to embodiments of the
present invention.
[0064] FIG. 8 is a schematic illustration of an exemplary system
with freeze-thaw valves for fluidic control according to
embodiments of the present invention.
[0065] FIG. 9 is an exemplary graphic user interface for
controlling freeze thaw valve ON/OFF and/or temperature operation
according to embodiments of the present invention.
[0066] FIG. 10 is an exploded view of an exemplary mounting
assembly with TEC mounts for holding a microfluidic chip according
to embodiments of the present invention.
[0067] FIG. 11 is a graph of freeze time (seconds) versus
temperature for a buffer with no INP ("INP-free") shown as the left
most bar at each temperature) and for different INP extract
dilutions in Tris buffer according to embodiments of the present
invention. The INP-free buffer did not freeze at -20 degrees C. in
the time allotted. The error bars illustrate one standard deviation
in each direction.
[0068] FIG. 12 is a graph of thaw time (seconds) versus temperature
used to freeze the sample for five different INP extract dilutions
in Tris buffer according to embodiments of the present invention.
The left most bar for each set of bars is for a buffer with no INP
(INP-free). Thaw times are not applicable at the -20 degree C.
freeze temperature for the INP-free buffer because it did not
freeze at that temperature. The error bars illustrate one standard
deviation in each direction.
[0069] FIG. 13 is a graph of freeze time (seconds) versus
temperature used to freeze a sample for different chip thicknesses
and different INP extract dilutions according to embodiments of the
present invention. Thaw times are not applicable at the -20 degree
C. freeze temperature for the INP-free buffer for the standard and
thin thickness microfluidic chips because the sample did not freeze
at that temperature. The error bars illustrate one standard
deviation in each direction.
DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0070] The present invention will now be described more fully
hereinafter with reference to the accompanying figures, in which
embodiments of the invention are shown. This invention may,
however, be embodied in many different forms and should not be
construed as limited to the embodiments set forth herein. Like
numbers refer to like elements throughout. In the figures, certain
layers, components or features may be exaggerated for clarity, and
broken lines illustrate optional features or operations unless
specified otherwise. In addition, the sequence of operations (or
steps) is not limited to the order presented in the figures and/or
claims unless specifically indicated otherwise. In the drawings,
the thickness of lines, layers, features, components and/or regions
may be exaggerated for clarity and broken lines illustrate optional
features or operations, unless specified otherwise. The
abbreviations "FIG. and "Fig." for the word "Figure" can be used
interchangeably in the text and figures.
[0071] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. As used herein, the singular forms, "a", "an" and
"the" are intended to include the plural forms as well, unless the
context clearly indicates otherwise. It will be further understood
that the terms "comprises," "comprising," "includes," and/or
"including" when used in this specification, specify the presence
of stated features, regions, steps, operations, elements, and/or
components, but do not preclude the presence or addition of one or
more other features, regions, steps, operations, elements,
components, and/or groups thereof. As used herein, the term
"and/or" includes any and all combinations of one or more of the
associated listed items. As used herein, phrases such as "between X
and Y" and "between about X and Y" should be interpreted to include
X and Y. As used herein, phrases such as "between about X and Y"
mean "between about X and about Y." As used herein, phrases such as
"from about X to Y" mean "from about X to about Y."
[0072] It will be understood that when a feature, such as a layer,
region or substrate, is referred to as being "on" another feature
or element, it can be directly on the other feature or element or
intervening features and/or elements may also be present. In
contrast, when an element is referred to as being "directly on"
another feature or element, there are no intervening elements
present. It will also be understood that, when a feature or element
is referred to as being "connected", "attached" or "coupled" to
another feature or element, it can be directly connected, attached
or coupled to the other element or intervening elements may be
present. In contrast, when a feature or element is referred to as
being "directly connected", "directly attached" or "directly
coupled" to another element, there are no intervening elements
present. Although described or shown with respect to one
embodiment, the features so described or shown can apply to other
embodiments.
[0073] Unless otherwise defined, all terms (including technical and
scientific terms) used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. It will be further understood that terms, such
as those defined in commonly used dictionaries, should be
interpreted as having a meaning that is consistent with their
meaning in the context of the present application and relevant art
and should not be interpreted in an idealized or overly formal
sense unless expressly so defined herein. Well-known functions or
constructions may not be described in detail for brevity and/or
clarity.
[0074] Spatially relative terms, such as "under", "below", "lower",
"over", "upper" and the like, may be used herein for ease of
description to describe one element or feature's relationship to
another element(s) or feature(s) as illustrated in the figures. It
will be understood that the spatially relative terms are intended
to encompass different orientations of the device in use or
operation in addition to the orientation depicted in the figures.
For example, if the device in the figures is inverted, elements
described as "under" or "beneath" other elements or features would
then be oriented "over" the other elements or features. Thus, the
exemplary term "under" can encompass both an orientation of over
and under. The device may be otherwise oriented (rotated 90 degrees
or at other orientations) and the spatially relative descriptors
used herein interpreted accordingly. Similarly, the terms
"upwardly", "downwardly", "vertical", "horizontal" and the like are
used herein for the purpose of explanation only unless specifically
indicated otherwise.
[0075] It will be understood that, although the terms first,
second, etc. may be used herein to describe various elements,
components, regions, layers and/or sections, these elements,
components, regions, layers and/or sections should not be limited
by these terms. These terms are only used to distinguish one
element, component, region, layer or section from another region,
layer or section. Thus, a first element, component, region, layer
or section discussed below could be termed a second element,
component, region, layer or section without departing from the
teachings of the present invention.
[0076] All of the document references (patents, patent applications
and articles) are hereby incorporated by reference as if recited in
full herein.
[0077] The terms "microchip" and "microfluidic chip" are used
interchangeably and refer to a substantially planar, thin device.
The microfluidic chip can be rigid, semi-rigid or flexible. The
term "thin" refers to a thickness dimension that is 10 mm or less
such as between 10 mm and 0.01 mm, and can be about 3 mm, about 2.5
mm, about 2 mm, about 1.5 mm, about 1 mm, about 0.5 mm, about 0.1
mm, and about 0.01 mm. The microchip typically has a width and
length that is less than about 6 inches, more typically between
about 1 inch and 6 inches. The microchip can have a width dimension
that is less than a length dimension. The microfluidic chip can
have a width dimension that is about 2.13 inches (54 mm) and a
length dimension that is about 3.4 inches (85.5 mm), in some
embodiments. The microchip can include micro-sized and/or
nano-sized fluidic channels.
[0078] The term "primary dimension" refers to a width and/or depth
dimension of a fluidic channel.
[0079] The terms "micro-sized" and "microfluidic" with respect to a
fluidic channel refer to a fluid flow channel that has millimeter,
sub-millimeter, or smaller size width and/or depth (e.g., the term
includes millimeter, micrometer, and nanometer size channels) and
includes channels with at least a segment having a width and/or
depth in a size range of hundreds of microns or less, typically
less than 900 microns and greater than 1 nm.
[0080] In some embodiments, the primary transport channel can be a
microfluidic channel having a major length with at least one
primary dimension that is between 1 nm to about 500 .mu.m. The
primary transport channel can, for example, have primary dimensions
of about 250 .mu.m/250 .mu.m (width/depth, i.e., width/height)
dimensions. The term "primary fluid transport channel" refers to a
fluidic channel, typically comprising at least a sub-length that is
micro-sized channel or nanochannel through which an analyte flows
for analysis.
[0081] The term "nanochannel" refers to a channel or trench having
a critical dimension that is at a nanometer scale. The primary
(also known as "critical") dimensions of a nanochannel are both
typically below about 100 nm, including between about 1-70 nm. In
some embodiments, at least one primary dimension can be about 5 nm
or less (on average or at a maxima).
[0082] A fluidic channel has sidewalls and a floor. One or more
fluidic transport channels can be formed into a solid substrate to
have an open top surface and a closed bottom surface with the
sidewalls extending therebetween. One or more top substrates,
membranes or covers may be used to seal, cover or otherwise close
the upper surface of the fluidic channel(s) and/or associated
ports.
[0083] The term "about" refers to parameters that can vary between
+/-20% or less, such as +/-10%.
[0084] The term "transverse" channel refers to a fluidic channel
that crosses a respective fluid transport channel. A fluidic device
can include a plurality of transverse channels that each merge into
a primary transport channel.
[0085] The analyte in a sample can be any analyte of interest from
a sample including, for example, various mixtures including
synthetic and biological macromolecules, nanoparticles, small
molecules, DNA, nucleic acids/polynucleic acids, peptides,
proteins, glycans, pharmaceuticals, elemental compounds, inorganic
compounds, organic compounds, and/or the like. The analyte can be
one or more single analyte molecules. The sample or analyte of a
sample can include one or more polar metabolites such as amino
acids or charged molecules, molecules, peptides, and proteins. The
sample and/or analyte may also or alternatively include molecules
extracted from biofluids, blood, serum, urine, dried blood, cell
growth media, lysed cells, beverages or food. The sample may also
or alternatively include environmental samples such as water, air
or soil.
[0086] The transport through the transport channel can be carried
out using one or more of electrokinetics, concentration
polarization and/or hydraulic or pneumatic pressure (forced
pressure or pressure gradients), capillary forces or gravity.
[0087] An "ice-nucleating agent", "INA" or grammatical variations
thereof, as used herein, refer to an agent that can and/or has the
ability to catalyze and/or initiate ice crystal formation. Thus, an
INA can and/or has the ability to nucleate ice. An INA may reduce
the FTV actuation time at a given temperature. In some embodiments,
the INA is a carbon-containing INA (i.e., contains at least one
carbon atom). In some embodiments, an INA includes and/or is an
ice-nucleating protein ("INP" in the singular, "INPs" in the
plural) and/or a functional fragment thereof, an ice-nucleating
nucleic acid, ice-nucleating lipid, and/or an ice-nucleating
carbohydrate. In some embodiments, the INA is not a
silver-containing agent, such as, for example, silver iodide and/or
a nanoparticle comprising silver. In some embodiments, the INA does
not cause and/or provide for the precipitation of a salt in a fluid
in which it is present. In some embodiments, the INA does not
interfere with a biological process and/or a process in an assay
being studied and/or carried out using a microfluidic device. In
some embodiments, the INA does not interfere with a component in
the fluid in which is it present.
[0088] Accordingly, in some embodiments, an INA may be an organism,
such as, for example, an invertebrate (e.g., a multicellular
invertebrate), bacterium, fungi, and/or lichen, that includes
and/or expresses an INP and/or a functional fragment thereof, an
ice-nucleating nucleic acid, ice-nucleating lipid, and/or an
ice-nucleating carbohydrate. In some embodiments, an INA may be
obtained from an organism such as a bacterium and/or yeast that
includes and/or expresses (e.g., by protein engineering) an INP
and/or a functional fragment thereof, an ice-nucleating nucleic
acid, ice-nucleating lipid, and/or an ice-nucleating carbohydrate.
In some embodiments, an INA may be a component and/or derivative of
an organism (e.g., a vertebrate and/or microorganism), such as, for
example, a cell and/or cell component (e.g., a cell membrane) of
the organism in which an INP and/or a fragment thereof, an
ice-nucleating nucleic acid, ice-nucleating lipid, and/or an
ice-nucleating carbohydrate is present. In some embodiments,
nucleation effectiveness may be maximized when an INP and/or a
functional fragment thereof remains in a cell membrane and/or is
capable of interacting with other membrane components (e.g., other
cell membrane proteins or lipids). In some embodiments, an
ice-nucleating lipid may comprise all or a portion of a cell
membrane, or may comprise all or a portion of a synthetic lipid
membrane. An ice-nucleating lipid may enhance the effects of other
ice-nucleating agents. In some embodiments, an INA may be an INP
and/or a functional fragment thereof. In some embodiments, an INA
may be an ice-nucleating nucleic acid, ice-nucleating lipid, and/or
an ice-nucleating carbohydrate, such as, for example, a synthetic
nucleic acid, a synthetic lipid, and/or synthetic carbohydrate that
can nucleate ice. In some embodiments, an INA may be a naturally
occurring, synthetic, or selectively evolved nucleic acid
sequence.
[0089] Two or more (e.g., 3, 4, 5, 6, or more) different INAs may
be present in a device, fluid channel, and/or FTV of the present
invention. For example, a device of the present invention may
comprise a plurality of FTVs and one FTV of the plurality of FTVs
may include the same INA, the same combination of INAs, a different
INA, and/or a different combination of INAs as another FTV of the
plurality of FTVs. An INA may be present in a device, fluid
channel, and/or FTV of the present invention in any suitable
concentration. In some embodiments, an INA may be present in a
device, fluid channel, and/or FTV at a concentration in a range of
about 1 molecule or organism per 4 to about 10 billion molecules or
organisms per 4 or any range and/or individual value therein, such
as, for example, about 1,000 molecules or organisms per 4 to about
3 million molecules or organisms per 4. In some embodiments, the
concentration of an INA can range from nanomolar to millimolar
concentrations, such as, for example, from about 1 nM to about 1
mM. In some embodiments, an INA may be present in a device, fluid
channel, and/or FTV at a concentration greater than 10 billion
molecules/4. In some embodiments, an INA may be present in a
device, fluid channel, and/or FTV at a concentration in a range
that spans the nanomolar, micromolar, and/or millimolar
concentration range(s).
[0090] In some embodiments, an INA (e.g., an INP, an ice-nucleating
nucleic acid, ice-nucleating lipid, and/or an ice-nucleating
carbohydrate) comprises a structure (e.g., a secondary, tertiary,
or quaternary structure or portion thereof) that can nucleate ice
formation. In some embodiments, an INA (e.g., an aptamer, such as a
DNA, RNA, and/or peptide aptamer) may comprise a structure similar
to ice and/or may align water molecules into an ice-like lattice.
In some embodiments, an INA may efficiently nucleate ice (i.e., the
INA may not randomly nucleate ice like pollen and/or dust
particles). In some embodiments, an INA may efficiently nucleate
ice by freezing water and/or a solution at a given temperature in
less time, on average, than the average time at which water and/or
the solution freezes in the absence of the INA. In some
embodiments, an INA may efficiently nucleate ice by freezing water
and/or a solution in a respective freeze-thaw valve of a
microfluidic device at a given temperature in less time with a
standard deviation of less than 50 seconds (e.g., less than 45, 40,
35, 30, 25, 20, 15, 10, 5, 4, 3, 2 or 1 seconds) over successive
freeze actuations, which can be assessed using ten trials, compared
to the time at which water and/or the solution freeze in the
freeze-thaw valve in the absence of the INA.
[0091] Example INAs include, but are not limited to, those
described in U.S. Pat. Nos. 4,200,228; 4,706,463; 4,978,540;
5,233,412; 5,489,521; 5,532,160; 5,554,368; 5,620,729; 5,843,506;
5,972,686; 6,361,934; and 7,624,698 and European Patent Application
No. 88114120.4, the portions of each of which are incorporated
herein by reference in their entirety for the teachings relevant to
this paragraph.
[0092] As used herein, the terms "polypeptide," "peptide" and
"protein" refer to a polymer of amino acid residues. The terms
encompass amino acid polymers in which one or more amino acid
residue is an artificial chemical analogue of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers.
[0093] The terms "nucleic acid," "nucleic acid molecule,"
"nucleotide sequence" and "polynucleotide" can be used
interchangeably and encompass both RNA and DNA, including cDNA,
genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or
RNA and chimeras of RNA and DNA. The term polynucleotide,
nucleotide sequence, or nucleic acid refers to a chain of
nucleotides without regard to length of the chain. A nucleic acid
may comprise one or more non-naturally occurring nucleotides (e.g.,
an artificial chemical analogue of a naturally occurring
nucleotide), which may or may not base pair in a manner similar to
a naturally occurring nucleotide. In some embodiments, a nucleic
acid may comprise one or more bases that do not normally occur in
nature, and that may not necessarily base pair in a manner similar
to naturally occurring nucleotides. The nucleic acid can be
double-stranded or single-stranded. The term "nucleic acid," unless
otherwise limited, encompasses analogues having the essential
nature of natural nucleotide sequences in that they hybridize to
single-stranded nucleic acids in a manner similar to naturally
occurring nucleotides (e.g., peptide nucleic acids).
[0094] In some embodiments, an INP and/or a functional fragment
thereof, an ice-nucleating nucleic acid, ice-nucleating lipid,
and/or an ice-nucleating carbohydrate can confer the ice
nucleation-active (Ice.sup.+) phenotype on an organism and/or cell
in which it is present and/or expressed. The Ice.sup.+ phenotype as
used herein refers to the ability of an organism and/or cell to
catalyze and/or initiate ice crystal formation at relatively high
temperatures, such as at temperatures below 0.degree. C. and above
-20.degree. C. and/or any range and/or individual value therein.
Thus, the Ice.sup.+ phenotype provides the organism and/or cell
with the ability to produce ice nuclei at temperatures in a range
of -20.degree. C. to 0.degree. C. As those of skill in the art will
recognize, an INP and/or a functional fragment thereof (e.g., an
INP and/or functional fragment thereof harvested and/or extracted
from an organism in which it is expressed), ice-nucleating nucleic
acid, ice-nucleating lipid, and/or ice-nucleating carbohydrate
itself may have the ability to catalyze and/or initiate ice crystal
formation.
[0095] In some embodiments, an INP may be a cell membrane protein,
such as, for example, an outer cell membrane protein. An INP may be
naturally expressed by and/or present in an organism, such as, for
example, a vertebrate; invertebrate (e.g., a multicellular
invertebrate); bacterium; fungi; and/or lichen. In some
embodiments, an INP may be recombinantly expressed in an
organism.
[0096] Example bacteria that naturally express an INP include, but
are not limited to, Pseudomonas syringae, P. antarctica, P.
viridiflava, P. fluorescens, Erwinia herbicola, E. ananas, and
Xanthomonas campestris. In some embodiments, an INP may be present
in an outer membrane of an organism (e.g., bacteria) that expresses
the INP. In some embodiments, an INP may be harvested and/or
extracted from a membrane of a bacterium that expresses the INP,
such as, for example, Pseudomonas syringae.
[0097] As used herein with respect to polypeptides, the term
"fragment" refers to a polypeptide that is reduced in length
relative to a reference polypeptide (e.g., a full-length INP, such
as naturally occurring INP) and that comprises, consists
essentially of and/or consists of an amino acid sequence of
contiguous amino acids identical or almost identical (e.g., 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a
corresponding portion of the reference polypeptide. Such a
polypeptide fragment may be, where appropriate, included in a
larger polypeptide of which it is a constituent. In some
embodiments, the polypeptide fragment comprises, consists
essentially of or consists of at least about 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 300,
350, 400, 450, 500, or more consecutive amino acids. In some
embodiments, the polypeptide fragment comprises, consists
essentially of or consists of less than about 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,
175, 200, 225, 250, 300, 350, 400, 450 or 500 consecutive amino
acids.
[0098] As used herein with respect to polypeptides, the term
"functional fragment" refers to a polypeptide fragment that retains
at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,
99.5% or more of at least one biological activity of the
full-length polypeptide (e.g., the ability to catalyze ice
formation and/or confer the ice nucleation-active (Ice.sup.+)
phenotype). In some embodiments, the functional fragment may have a
higher level of at least one biological activity of the full-length
polypeptide. In some embodiments, an organism containing a
functional fragment of an INP (e.g., a bacterium containing an INP
functional fragment) may have a higher level of at least one
biological activity of the full-length polypeptide.
[0099] In some embodiments, an INP and/or a functional fragment
thereof may comprise at least 1 repeat of two or more consecutive
amino acids, such as, for example, a repeat of an 8, 16, and/or 48
amino acid sequence. In some embodiments, the two or more
consecutive amino acids (e.g., the 8, 16, and/or 48 amino acid
sequence) may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times
in the INP and/or functional fragment. The two or more consecutive
amino acids (e.g., the 8, 16, and/or 48 amino acid sequence) may be
consecutively repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times in
the INP and/or functional fragment. In some embodiments, the
repeating sequence can be imperfect and/or exhibit a natural
variation. Example amino acid sequences include, but are not
limited to, those described in Green & Warren, Nature 1985,
vol. 317, pp 645-648, the contents of which are incorporated herein
by reference for the portions relevant to this paragraph. In some
embodiments, an INP and/or a functional fragment thereof may
comprise a tandemly repeated 8, 16, and/or 48 amino acid sequence,
which may be present in the internal region of an INP.
[0100] In some embodiments, an INP can be encoded by one or more
genes, such as, for example, but not limited to, iceE, iceH, inaA,
inaE, inaF, inaK, inaPb, inaQ, inaU, inaV, inaW, inaX, and/or inaZ.
An INP may be present or found in and/or obtained from an organism,
such as, for example, but not limited to, Pseudomonas syringae, Ps.
fluorescens KUIN-1, Erwinia herbicola, E. uredovora, Pantoea
ananatis, Xanthomonas campestris, E. carotovora, Ps. antarctica,
Ps. aeruginosa, Ps. putida, Ps. viridiflava, Pa. agglomerans, E.
ananas, and/or Ps. borealis. In some embodiments, an INP may be
encoded by a gene present or found in and/or obtained from an
organism as shown in Table 1. For example, the INP may be a
polypeptide encoded by the iceE gene found or present in and/or
obtained from Erwinia herbicola.
TABLE-US-00001 TABLE 1 Example ice-nucleating genes/proteins and
the species from which they may be found or present in and/or
obtained from. Ice-nucleating gene/protein Species iceE Erwinia
herbicola iceH E. herbicola inaA Pantoea ananatis, E. ananas inaE
E. herbicola inaF Pseudomonas fluorescens KUIN-1 inaK Ps. syringae
inaPb Ps. borealis inaQ Ps. syringae inaU Pa. ananatis, E.
uredovora inaV Ps. syringae inaW Ps. fluorescens inaX Xanthomonas
campestris inaZ Ps. syringae
[0101] In some embodiments, an INP and/or a functional fragment
thereof may be a polypeptide encoded by the inaZ gene, such as, for
example, a polypeptide as set forth in Accession No. P06620
(ICEN_PSESY). The central domain of an INP (e.g., inaZ) may
comprise 122 imperfect repeats of an octapeptide, and it may have a
structure similar to that of ice. In some embodiments, an INP
and/or a functional fragment thereof may align water molecules into
an ice-like lattice. In some embodiments, an INP and/or a
functional fragment thereof may have a structure comprising at
least one beta hairpin (e.g., 1, 2, 3, 4, 5, or more), and
optionally at least one beta hairpin may comprise 1, 2, or more
octapeptide repeat(s). In some embodiments, an ice-nucleating
nucleic acid, ice-nucleating lipid, and/or an ice-nucleating
carbohydrate may have a structure similar to at least part of a
structure of an INP.
[0102] In some embodiments, an INP and/or functional fragment
thereof may be naturally, synthetically, and/or recombinantly
obtained and/or produced. In some embodiments, an INP and/or
functional fragment thereof may be isolated, obtained, harvested,
and/or extracted from an organism in which it may naturally and/or
recombinantly be expressed. In some embodiments, an INP and/or
functional fragment thereof may be isolated, obtained, harvested,
and/or extracted from an organism or microorganism (e.g., bacteria,
fungi, etc.) and used as an INA.
[0103] In some embodiments, an INP and/or functional fragment
thereof may be obtained through recombinant techniques and may be
based on plasmids from natural and/or synthetic DNA. A recombinant
INP and/or functional fragment thereof may be expressed in an
organism such as a bacterium (e.g., Escherichia coli) and/or a
yeast (e.g., Saccharomyces cerevisiae). Similarly, the INP agent
can be expressed in other suitable organisms such as plants or
animals. A recombinant INP and/or functional fragment thereof may
be modified to aid in the expression, purification, modification,
and/or stability of the INP and/or functional fragment. For
example, a recombinant INP and/or functional fragment thereof may
be modified to include a polyhistidine tag to facilitate
purification of the polypeptide from an organism in which it is
expressed and/or may be modified to have more preferable
characteristics, such as, for example, improved resistance to
denaturation under different conditions such as a range of
temperatures, pH levels, ionic strengths, and solvent strengths. A
recombinant INP and/or functional fragment thereof may be modified
to include a functional group for attachment to a surface or
another molecule. In some embodiments, a recombinant INP and/or
functional fragment thereof may be modified to improve ice
nucleation. For example, recombinant INP and/or functional fragment
thereof may be modified to include a repeating the sequence to link
two or more INPs/functional domains together, to link phospholipids
during PTMs, or to include spacers to optimize the distance between
INP domains. In some embodiments, a recombinant INP and/or
functional fragment thereof may be modified to group two or more
INPs and/or functional fragments thereof together.
[0104] In some embodiments, an INP and/or functional fragment
thereof, ice-nucleating nucleic acid, ice-nucleating lipid, and/or
ice-nucleating carbohydrate may be isolated. As used herein with
respect to polypeptides, nucleic acids, lipids, and/or
carbohydrates, the term "isolated" refers to a polypeptide, nucleic
acid, lipid, and/or carbohydrate that, by the hand of man, exists
apart from its native environment and is therefore not a product of
nature. In some embodiments, the ice-nucleating polypeptide,
ice-nucleating nucleic acid, ice-nucleating lipid, and/or
ice-nucleating carbohydrate exist in a purified form that is
substantially free of cellular material, viral material, culture
medium (when produced by recombinant DNA techniques), or chemical
precursors or other chemicals (when chemically synthesized). An
"isolated fragment" is a fragment of a polypeptide, nucleic acid,
lipid, and/or carbohydrate that is not naturally occurring in a
substantial concentration as a fragment and would not be found in
the natural state above trace levels. "Isolated" does not mean that
the preparation is technically pure (homogeneous), but rather that
it is sufficiently pure to provide the ice-nucleating polypeptide,
ice-nucleating nucleic acid, ice-nucleating lipid, and/or
ice-nucleating carbohydrate in a form in which it can be used for
the intended purpose. In certain embodiments, a composition
comprising an ice-nucleating polypeptide, ice-nucleating nucleic
acid, ice-nucleating lipid, and/or ice-nucleating carbohydrate is
at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% or more pure.
[0105] In some embodiments, an INA may nucleate ice crystals on a
microfluidic scale. Thus, an INA as described herein may catalyze
and/or initiate ice crystal formation in water and/or a solution
(e.g., an aqueous solution) on a microfluidic scale, such as, for
example, in a microfluidic chip, to thereby freeze the water and/or
solution. The volume of fluid (e.g., water) that may freeze in a
microfluidic chip (e.g., in a fluidic channel and/or FTV) may be in
a range of about 1 femtoliter to about 100 microliters and/or any
range and/or individual value therein. In some embodiments, a
larger fluid volume that can freeze in one or more freeze-thaw
valves may be used, such as about 1 milliliter or a plurality of
milliliters. "Freeze" as used herein refers to the formation of a
solid phase in an amount sufficient to block and/or stop the flow
of a fluid present in a FTV on a microfluidic device associated
with an external cooling source, such as a thermoelectric cooler.
Thus, after freezing, the solid phase in the microfluidic chip
(e.g., in a fluidic channel and/or FTV) may have a volume greater
than that of the fluid prior to freezing.
[0106] In some embodiments, the use of an INA in a microfluidic
chip may freeze water and/or a solution at a higher temperature
and/or in less time, on average, than the temperature and/or time
at which water and/or the solution freeze in the absence of the
INA. In some embodiments, an INA may freeze water and/or a solution
at a temperature that is at least 5.degree. C. higher (e.g., 6, 7,
8, 9, 10.degree. C., or more) than the temperature at which water
and/or the solution freeze in a given amount of time in the absence
of the INA. In some embodiments, an INA may reduce the average time
water and/or a solution is held below its melting point (e.g.,
0.degree. C. for water) before crystallization occurs. In some
embodiments, an INA may reduce the average time water and/or a
solution is held below its melting point (e.g., 0.degree. C. for
water) before crystallization occurs by at least about 5% or more,
such as, for example, by at least 10%, 15%, 20%, 25%, 50%, or
more.
[0107] An INA may catalyze and/or initiate ice crystal formation in
water and/or a solution (e.g., an aqueous solution) at a
temperature below 0.degree. C. and above -50.degree. C. and/or any
range and/or individual value therein, such as, for example, about
-1.degree. C. to about -20.degree. C., about -6.degree. C. to about
-14.degree. C., about -2.degree. C. to about -12.degree. C., about
-1.degree. C. to about -30.degree. C., about -2.degree. C. to about
-8.degree. C., about -2.degree. C. to about -50.degree. C., or
about -5.degree. C. to about -25.degree. C. In certain embodiments,
an INA may catalyze and/or initiate ice crystal formation in water
and/or a solution at a temperature of about 0, -1, -2, -3, -4, -5,
-6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19,
-20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32,
-33, -34, -35, -36, -37, -38, -39, -40, -41, -42, -43, -44, -45,
-46, -47, -48, -49 or -50.degree. C. and/or any range and/or
individual value therein. The freeze/thaw temperatures can be
measured in a microfluidic transport channel holding a solution
with the INA at a desired freeze-thaw region. The actual
temperature (setting) of a thermo-electric source providing the
freeze and/or thaw input will typically be different than the
temperature in the transport channel at the freeze/thaw region,
e.g., a freeze temperature setting such as -45.degree. C. to
-20.degree. C., will result in a freeze temperature in the material
containing the INA in the transport channel that is above the
setting, typically by about 5 to 25.degree. C. above the
setting.
[0108] One, and typically all, respective FTVs 22 can operate at a
freeze time of less than three minutes, typically under about 45
seconds, more typically between 2 seconds and 33 seconds. Thaw
times for one or more FTVs 22 can be less than corresponding freeze
times, typically between 1-30 seconds, such as between 1-15
seconds.
[0109] An INA may be designed to function in water and/or a
solution, such as an aqueous solution, and may not need to be dry.
An INA may be provided in a number of ways. In some embodiments, an
INA may be diluted into a buffer for input into and/or onto a
microfluidic chip. In some embodiments, an INA may be provided in a
buffer or other liquid suitable for use with immunoassays (e.g., a
Tris-based or phosphate-based buffer). Other formulations/delivery
mechanisms of the INPs are described below.
[0110] The term "circuit" refers to an entirely hardware embodiment
or an embodiment combining software and hardware.
[0111] The term "homogenous nucleation" refers to when small
aggregates of a solid-like phase (embryos) exist throughout the
liquid phase, the embryos reach a critical size, and the embryos
trigger continued growth of the solid phase. A homogeneous
nucleation generally occurs at about -37.degree. C. or below for
water.
[0112] The term "heterogeneous nucleation" refers to a phase
transition from a metastable supercooled state to a stable solid
state due to the presence of foreign substance (e.g., an INA) in
the liquid phase. Heterogeneous nucleation typically occurs at
nucleation sites on a surface of a liquid and, for water, generally
occurs at a temperature in a range of about 0.degree. C. and above
-40.degree. C. In some embodiments, an INA may heterogeneously
nucleate a solution and/or liquid in which it is present.
[0113] The term "oligonucleotide" refers to a nucleic acid sequence
of about five nucleotides to about 500 nucleotides (e.g. 5, 6, 7,
8, 9, 10, 12, 15, 18, 20, 21, 22, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350,
400, 450 or 500 nucleotides). In some embodiments, for example, an
oligonucleotide can be from about 15 nucleotides to about 40
nucleotides, or about 20 nucleotides to about 25 or to about 30
nucleotides, which can be used, for example, as a primer in a
polymerase chain reaction (PCR) amplification assay and/or as a
probe in a hybridization assay or in a microarray. Oligonucleotides
for processing can be natural or synthetic, e.g., DNA, RNA, PNA,
LNA, modified backbones, etc., or any combination thereof as are
well known in the art.
[0114] Probes and primers, including those for either amplification
and/or detection, are oligonucleotides (including naturally
occurring oligonucleotides such as DNA and synthetic and/or
modified oligonucleotides) of any suitable length, but are
typically from 5, 6, or 8 nucleotides in length up to 40, 50 or 60
nucleotides in length, or more. Such probes and or primers may be
immobilized on or coupled to a solid support such as a bead, chip,
pin, or microtiter plate well, and/or coupled to or labeled with a
detectable group such as a fluorescent compound, a chemiluminescent
compound, a radioactive element, or an enzyme.
[0115] In some embodiments, an INA may be immobilized on, attached
to, or coupled to a solid support, such as, e.g., a particle or a
bead. An INA may be immobilized on, attached to, or coupled to a
solid support prior to being introduced into a fluidic analysis
device. The INA immobilized on, attached to, or coupled to the
solid support may be introduced into at least one fluidic channel
of a fluidic analysis device and/or may be used to freeze and thaw
at least one freeze thaw valve of the fluidic analysis device.
[0116] In some particular embodiments, fluidic devices with
freeze-thaw valves and comprising INAs (e.g., an INP and/or
functional fragment thereof and/or a microorganism comprising the
same) can carry out PCR in accordance with known techniques. See,
e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and
4,965,188. In general, PCR involves, first, treating a nucleic acid
sample (e.g., in the presence of a heat stable DNA polymerase) with
one oligonucleotide primer for each strand of the specific sequence
to be detected under hybridizing conditions so that an extension
product of each primer is synthesized which is complementary to
each nucleic acid strand, with the primers sufficiently
complementary to each strand of the specific sequence to hybridize
therewith so that the extension product synthesized from each
primer, when it is separated from its complement, can serve as a
template for synthesis of the extension product of the other
primer, and then treating the sample under denaturing conditions to
separate the primer extension products from their templates if the
sequence or sequences to be detected are present. These steps can
be cyclically repeated until the desired degree of amplification is
obtained. Detection of the amplified sequence may be carried out by
adding to the reaction product an oligonucleotide probe capable of
hybridizing to the reaction product (e.g., an oligonucleotide probe
of the present invention), the probe carrying a detectable label,
and then detecting the label in accordance with known techniques,
or by direct visualization on a gel. The amplified sequence can
also be detected by an intercalating dye in the reaction mixture
and monitoring the fluorescence signal strength, which will be
proportional to the total mass of double stranded DNA. Although
embodiments according to the present invention are described with
respect to PCR reactions, it should be understood that other
nucleic acid amplification methods can be used, such as reverse
transcription PCR (RT-PCR) including isothermal amplification
techniques such as rolling circle amplification or loop-mediated
isothermal amplification (LAMP).
[0117] DNA amplification techniques such as the foregoing can
involve the use of a probe, a pair of probes, or two pairs of
probes which specifically bind to DNA containing a polymorphism or
mutation of interest, but do not bind to DNA that does not contain
the polymorphism of interest under the same hybridization
conditions, and which serve as the primer or primers for the
amplification of the DNA or a portion thereof in the amplification
reaction. Such probes are sometimes referred to as amplification
probes or primers herein.
[0118] The term "reagent" refers to any substance or compound,
including primers, a nucleic acid template and/or an amplification
enzyme(s) that is added to a system in order to bring about a
chemical reaction, or added to see if a reaction occurs.
Amplification reagents or reagent refer to those reagents
(deoxyribonucleotide triphosphates, buffer, etc.) generally used
for amplification except for primers, nucleic acid template and the
amplification enzyme. Typically, amplification reagents along with
other reaction components are placed and contained in a reaction
vessel (test tube, microwell, etc.).
[0119] The term "magnetic" as used herein includes ferromagnetic,
paramagnetic and super paramagnetic properties.
[0120] In some embodiments, the fluidic devices contemplated by
embodiments of the invention may employ an oligonucleotide probe
which is used to detect DNA containing a polymorphism or mutation
of interest and is an oligonucleotide probe which binds to DNA
encoding that mutation or polymorphism, but does not bind to DNA
that does not contain the mutation or polymorphism under the same
hybridization conditions. The oligonucleotide probe can be labeled
with a suitable detectable group, such as those set forth below.
Such probes are sometimes referred to as detection probes or
primers herein.
[0121] Although embodiments according to the present invention are
described herein with respect to PCR reactions, it should be
understood that the microfluidic devices and methods described
herein may be used in various other processes, e.g., reactions. For
example, any nucleic acid transcription and/or
amplification-related reaction is within the scope of the current
invention, including but not limited to PCR reactions, real-time
PCR (rt-PCR), digital PCR (dPCR), reverse transcription of RNA into
cDNA (RT), PCR of cDNA from previous RT step (RT-PCR), RT-PCR using
real-time or digital quantification, immuno-PCR (iPCR) and its
variants, loop-mediated isothermal amplification (LAMP), rolling
circle replication, and/or non-enzymatic nucleic acid amplification
methods (e.g., "DNA circuits"). Methods of sequencing nucleic acids
including next generation sequencing methods including sequencing
by synthesis or hybridization-based methods are also included
within the scope of the invention. Other reactions that are
included within the scope of the present invention include but are
not limited to enzyme-linked immunosorbent assays (ELISA), single
molecule array (SiMoA) or digital ELISAs, reactions in which
multiple beads are used to deliver different reagents for
combinatorial chemistry, reactions where the beads deliver a
catalyst reagent, and/or reactions where "click" chemistry reagents
are delivered in stoichiometries determined by stochastic bead
loading. See, e.g., U.S. Ser. No. 14/402,565, for examples of
processes using cleaved reagents, the contents of which are hereby
incorporated by reference as if recited in full herein.
[0122] Turning now to the figures, by way of simple illustration,
one example of a fluidic device contemplated by embodiments of the
invention can include a microfluidic chip 10 as shown in FIGS. 1A
and 1B. The microfluidic chip 10 has at least one fluid transport
channel 20. Some embodiments can comprise a plurality of transport
channels. A respective transport channel 20 has at least one
defined flow region 20r with a freeze thaw "valve" 22. The freeze
thaw valve 22 is in thermal communication with at least one cooler
25; typically, the fluidic device comprises a plurality of freeze
thaw valves 22, each with a respective cooler 25, optionally one or
more thermoelectric cooler (TEC) 25t, that can be used to both
freeze and thaw liquid in respective defined regions of a transport
channel 20. The at least one thermoelectric cooler 25t can be, for
example, a Peltier cooler.
[0123] In some embodiments, one or more of the FTVs 22 can include
a plurality of coolers 25, such as one over and one under a
corresponding channel segment 20r associated with a respective FTV
22, which may increase a freeze action, i.e., shorten a time to
freeze.
[0124] In some particular embodiments, the thermoelectric cooler
25t can be configured as a solid-state device composed of n- and
p-type semiconductors sandwiched between thin sheets of
electrically insulating yet thermally conductive material. When
direct current is applied to the array of n- and p-type elements,
one side of the device cools due to the Peltier effect and absorbs
heat from the surrounding environment. The heat is transported
through the device to the opposite side and released, creating a
temperature gradient between the two sides of the TEC. See, e.g.,
Sgro et al., Thermoelectric Manipulation of Aqueous Droplets in
Microfluidic Devices, Anal Chem. 2007, 79(13): 4845-4851, the
contents of which are hereby incorporated by reference as if
recited in full herein. Microscale and larger TECs can be embedded
in, integrally attached to and/or releasably attached directly or
indirectly, to a microfluidic chip 10 for localized cooling of
nanoliter and/or microliter sized volumes. For other examples of
thermoelectric coolers, see, e.g., Maltezos et al., Thermal
management in microfluidics using micro-Peltier junctions, Applied
Phys. Lett. 87, 154105 (2005), the contents of which are hereby
incorporated by reference as if recited in full herein.
[0125] The cooler 25 can have a thermal footprint "F" in thermal
communication with the microfluidic chip 10 that can have a length
L measured in a flow direction toward and/or along the transport
channel 20, some footprints F of FTV regions 22 can be larger
(width and/or length) than others (FIGS. 2B, 2C, 3B, 3C, for
example). In some particular embodiments, the footprint F can be
defined by the size of a respective TEC 25t. For example, the TEC
25t can have a surface area of about 16 mm.sup.2, e.g., have a
width and length dimension of about 4 mm by 4 mm, for at least some
of the FTV regions 22.
[0126] FIGS. 2A-2C and 3A-3C illustrate examples of microfluidic
chips 10, each with a plurality of freeze thaw valves 22 in thermal
contact with respective coolers 25, bead well array 40, a sample
input 45, a bead storage/sample incubation chamber 50 and a waste
reservoir 55. A sample metering loop 47 can reside between the bead
storage/incubation chamber 50 and the sample input 45. The sample
metering loop 47 can optionally have a serpentine shape as shown.
The sample metering loop 47 may have a volumetric capacity of
between about 1 .mu.L to about 1 mL, more typically between 1 and
500 .mu.L, such as between about 10.75 .mu.Land 29 .mu.L, in some
embodiments. The loop 47 can have opposing open ends merging into
the primary fluid transport channel 20.
[0127] As shown, for example, in FIGS. 2A and 3A, some of the
fluidic channel segments associated with freeze-thaw valves 22 can
have curvilinear elbows 22b that can be sized and configured to
increase volume cooled by a cooler to facilitate a freezing
action.
[0128] The bead well array 40 can have a temperature control member
125 residing under or above the well array 40 over its entire
length or substantially over its entire length, potentially
terminating adjacent, before or after the inlet/outlet necks
thereof.
[0129] The microfluidic chip 10 and/or one layer of the chip 10 can
have through-holes (i.e., access apertures and/or ports) and/or
fluid paths extending through one or both sides of the chip 10 at
defined locations for fluid intake/exit regions.
[0130] As shown in FIGS. 2A and 3A, the sample input 45 can include
a sample input through an access aperture or via 45a. The bead
storage/sample incubation chamber 50 can include an access via or
aperture 50a. The bead washing and secondary reagent incubation
chamber 52 can include an access aperture or via 52a. The buffer
input 60 can include an access via or aperture 60a (FIG. 3A). The
sealing agent input 66 can include an access aperture or via 66a.
The detection substrate (and/or PCR master mix) 70 can include an
access aperture or via 70a. The waste reservoir 55 can include a
through-hole through one side or via 55a. The secondary reagent
inputs 63 can include access apertures or vias 63a.
[0131] FIG. 3A illustrates the microfluidic chip 10 can include an
array pre-treat reagent chamber 68 with a through hole or via 68a
and an array wash and oil purge waste chamber 69 with a through
hole or via 69a.
[0132] FIGS. 2A-2C illustrate fifteen freeze thaw valves 22 with
coolers 25 and FIGS. 3A-3C illustrate fourteen freeze thaw valves
22 with coolers 25 (e.g., TEC members/elements 25t), some on a top
surface 10t (FIG. 4) of the microfluidic chip 10 and a plurality
under the bottom 10b of the microfluidic chip 10.
[0133] As shown in FIG. 2C, coolers 25 (e.g., TEC members 25t) for
freeze thaw valves 22 can be positioned on each side of a channel
segment 20s leading to the bead well array 40, e.g., valves 7 and
8, valve 7 being upstream of and adjacent the bead
storage/incubation chamber 50, can reside on the top side of the
fluidic chip 10 and the remainder can reside under the microfluidic
chip 10. As shown in FIG. 3C, coolers 25 for freeze-thaw valves 8
and 9 can reside on top of the microfluidic chip 10, one upstream
of and adjacent the bead storage/incubation chamber 50 and one
inside the channel segment 20s leading to the bead well array
40.
[0134] The top side coolers 25 for the freeze-thaw valves 22 (i.e.,
valves 7/8, FIG. 2C, valves 8/9, FIG. 3C) can reside in a thin
region, e.g., a region that is about 30-95% the thickness of the
top substrate 10t and/or primary body of the chip itself, e.g.,
about a 900 .mu.m thick region on a top of the substrate that has a
nominal or average thickness of between 1-2 mm outside this
area/volume. That is, the microfluidic chip 10 can have a top
substrate 10t (FIGS. 4A, 4B) that is molded and holds the fluidic
channels 20, 63i, 20s, for example. The thinner regions 22t can be
sized and configured to hold and/or be in thermal communication
with the top-mounted coolers 25t.
[0135] The microfluidic chip 10 can include a buffer input 60 and a
plurality of reagent inputs 63 (e.g., fluid reservoirs 63f, FIG.
7). FIGS. 2A-2C illustrate three secondary reagent inputs 63 while
FIGS. 3A-3C illustrate two secondary reagent inputs 63. The reagent
inputs 63 can each be associated with an input channel 63i having a
corresponding freeze thaw valve 22 with a cooler 25.
[0136] The microfluidic chip 10 can include a bead
washing/secondary incubation chamber 52 in fluid communication with
one or all of the secondary reagent inputs 63.
[0137] The microfluidic chip 10 can also include a detection
substrate region and/or a PCR master mix region 70. Where used, the
PCR master mix can comprise a premixed, ready-to-use solution
containing a thermostable DNA polymerase, dNTPs, MgCl.sub.2 and
reaction buffers at optimal concentrations for efficient
amplification of DNA templates by PCR, for example.
[0138] The microfluidic chip 10 can include a sealing agent input
66. The sealing agent can comprise mineral, silicone, hydrocarbon,
fluorocarbon-based, a mixture of such oils, or a
multiply-functionalized synthetic oil and/or waxes.
[0139] FIGS. 2B, 2C, 3B and 3C illustrate exemplary footprints F of
freeze-thaw valves 22 associated with a cooler 25, illustrated by a
closed perimeter drawn about respective defined localized regions
of fluidic channels. These figures also illustrate air permeable
membranes 35, 135. FIGS. 2B and 2C illustrate one membrane 35 over
the reagent inputs 63, bead washing and secondary reagent
incubation chamber 52 and another membrane 135 over the bead
storage and sample incubation chamber 50. FIGS. 3B and 3C
illustrate two discrete spaced membranes 35 covering the waste
reservoir 55 and the reagent inputs 63, respectively. Another
air-permeable membrane 135 resides over the bead storage/incubation
chamber 50 and adjacent detection substrate and/or PCR master mix
70 and array pre-treat reagent 68.
[0140] Referring to FIGS. 4A and 4B, the microfluidic chip 10 can
comprise a plurality of stacked layers, typically first and second
substrate layers attached together. FIG. 4A illustrates that both
the top and bottom substrates 10t, 10b can be visually
transmissive, typically transparent or translucent. FIG. 4B
illustrates that the bottom substrate 10b can be opaque (e.g.,
opaque black) while the top 10t can be visually transmissive.
[0141] The top substrate layer 10t can hold at least a portion of
the at least one fluid transport channel 20. The transport channel
20 and other fluidic channels may be an internal channel (encased
by the top substrate 10t) or may be formed as an open upper surface
in the substrate 10t. If the latter, an overlay or cover may attach
to an upper surface of the top layer 10t to seal the transport
channel 20 or the transport channel 20 may be open to atmosphere
over its length or a portion thereof. The bottom substrate layer
10b can hold the bead well array 40. As discussed above, vias
and/or fluid intake/exit channels may be used to connect reservoirs
or chambers with one or more of external fluid supplies and/or
drains.
[0142] As shown in FIGS. 4A and 4B, some of the chambers or
reservoirs may be open to atmosphere/environmental conditions,
including sample intake 45, bead storage 50, secondary reagent
incubation chamber 52, waste reservoir 55, secondary reagent inputs
63 and detection substrate and/or PCR master mix 70. An air
permeable membrane or membranes 35, 135 can reside over these
features/components.
[0143] The top and/or bottom substrates 10t, 10b can comprise any
suitable material and can be rigid, semi-flexible or flexible. The
substrate of the bottom and top layers 10t, 10b can be the same or
different materials. The top layer and/or the bottom layer 10t, 10b
can comprise silicon, glass, hard plastic, hard polymeric material,
a flexible polymeric material, or any hybrid or composite of such
materials. Freezing can be used to modulate fluid flow on
microfluidic chips through freeze-thaw valves 22. The microfluidic
chips 10 can hold one or more primary transport and secondary
fluidic buffer and/or reagent channels in a substrate that is
elastic/flexible (e.g. polydimethylsiloxane) and/or in a substrate
that is inelastic/inflexible (e.g. glass and hard plastic).
[0144] FIG. 5 illustrates a microfluidic chip 10 with an air
permeable membrane 35, 135. The membrane 35 can, in some
embodiments reside over the waste reservoir 55, secondary reagent
inputs 63, and detection substrate and/or PCR master mix 70. The
membranes 35, 135 can comprise PTFE or other suitable materials and
may be "air-only" membranes. The term "air-only" means allowing gas
to escape or transverse the membrane while allowing no liquid or
particulate matter to pass through under applied pressure
differentials of zero to at least 1 psi (or greater, such as to 2,
3, 4 or 5 psi).
[0145] FIG. 5B illustrates beads dried down in the bead
storage/sample incubation chamber 50 without the air-permeable
membrane 135 in position over the chamber 50.
[0146] In some particular embodiments, the top substrate 10t can be
injection molded with the fluidic channel 20 and other fluid
channels. The transport channel 20 can, for example, comprise a 250
.mu.m wide, 250 .mu.m deep channel. The sample metering channel 47
can be wider, typically by between 20-60%, such as about 50% wider
than the flow channel 20 on either end of the sample metering
channel 47. In some embodiments, the metering channel 47 can be
about 500 .mu.m wide and translate up and down a plurality of times
before merging back into a straight segment of the channel 20. The
array chamber 40 can be relatively shallow, e.g., more shallow by
10-50% than the transport channel 20 between the metering channel
47 and the array 40, such as about 200 .mu.m deep. The array 40 can
include physical structures such as laterally and longitudinally
spaced apart dividers 40d in the array chamber 40c to keep the
chamber from collapsing during bonding of the top substrate 10t to
the bottom substrate 10b. The dividers 40d can facilitate control
of fluid flow through the wide array chamber 40.
[0147] The bottom substrate 10b can be either visually
transmissive, e.g., transparent or clear, or opaque, typically
opaque black and is also typically plastic. The bottom substrate
10b can contain an array 40 of between about 1-2 million "slit" or
"comet" shaped bead and reaction wells. The array 40 can hold about
2 million cylindrical wells. Holes in the top substrate 10t can be
used as reservoirs. They can contain dried beads and/or dried
reagents.
[0148] Once covered with one or more air permeable (filter)
membranes 35, 135, the holes (e.g., access apertures or vias) allow
air to be vented when a reader forces buffer into the chip 10.
Vacuum on a waste reservoir 55 can be used with the freeze-thaw
valves 22 to draw rehydrated reagents into the incubation
chamber(s) and/or reaction chamber(s) 50, 52. Oil or other suitable
sealing agent via input 66 (FIGS. 2A-2C, 3A-3C) can be used to seal
individual reactions from one another before imaging the bead array
40 to readout a fluorescence signal. Beads can be loaded and moved
through the chip 10 using actuator arms with magnets attached to
them as is well known to those of skill in the art.
[0149] A buffer 60f (FIGS. 2A, 3A) comprising INAs (e.g., an INP
and/or functional fragment thereof and/or a microorganism
comprising the same) can be flowably introduced into the transport
channel 20 from buffer input 60 (FIG. 2A, 3A), typically after the
microfluidic chip 10 is placed in thermal contact with the at least
one cooler 25. More typically, the microfluidic chip 10 is placed
in position in a test fixture/mounting assembly to be in thermal
contact with a plurality of spaced apart thermoelectric coolers
25t, positioned at defined positions, then the buffer with solvated
INAs 60f is flowably introduced into the microfluidic chip 10 to
reside/fill the fluidic channels, including channel 20 and reagent
inputs 63i.
[0150] The freeze-thaw valves 25 can be operated by setting a
cooler 25, such as the TECs 25t, to defined temperatures for a
"freeze" operation/mode, typically between -100 degrees C. and -1
degrees C. In some embodiments, the freeze temperature can be
between about -45.degree. C. (estimated steady state internal
temperature of -32.degree. C.) and about -20.degree. C. (estimated
steady state internal temperature of -11.degree. C.). Temperatures
below -45 degrees C. may also be used, in some embodiments the
temperatures can be about -50 degrees C. or above to -1 degree C.
Temperatures above -20 degrees C. may be used. Different freeze
actuations of one or more FTVs 22 can have different temperature
and/or time settings. At -45.degree. C., INA-containing buffers are
found to freeze more than twice as quickly than an INA-free
control, as well as more reproducibly. At -20.degree. C. applied to
external surface of the chip, the INA-containing buffers exhibited
concentration-dependent effects in terms of freeze time, while
INA-free buffers did not freeze at all in the observed timeframe.
It is contemplated that embodiments of the invention may be used to
facilitate microfluidic freeze-thaw valving that can reduce the
freeze time and temperature requirements of the system over
conventional FTV systems. The freeze-thaw systems can be compatible
with different microfluidic substrates including
inflexible/inelastic materials such as glass and silicon and may
expand the number of device designs available to users.
[0151] While certain embodiments described herein propose INAs
solvated in a primary (working) buffer such as via buffer input 60
(FIGS. 2A, 3A, for example), other ways of providing and/or
introducing INAs to a fluidic microchip 10 are also contemplated.
For example, one or more fluidic channels of a microfluidic chip 10
may be filled (partially or totally) with INA-containing buffer and
then dried, thus passively coating channels or channel segments
with the INAs. These proteins may then be used to nucleate freezing
after rehydrating the device. While an indefinite number of valving
steps may not be possible with this method if the proteins are
washed away over time, it may work well if a few actuations are
used for the device's operation. Examples of this include devices
in which freezing is only required during early phases of the
device's use, or there are otherwise only a few freezing steps with
minimal exchange of buffer that would wash away the INAs.
[0152] It may also be possible to covalently attach INAs to channel
walls, e.g. with PEG-Sulfo-NHS linking chemistry, and achieve a
similar effect with the advantage of fixed nucleating locations
with resistance to removal by washing alone.
[0153] Targeted deployment of the INAs may also be possible through
noncovalent attachments. One instance of this would be coupling
streptavidin to the INAs (e.g., either directly during their
recombinant expression, or covalently afterwards) and biotin to the
regions of the device in which the INAs are desired. The
streptavidin-modified INAs would then strongly couple to the areas
in which they are desired. Another instance of non-covalent
targeted deployment might involve modifying regions of the device
with a target molecule, then coupling INAs to antibodies or
aptamers that bind to sites on the target molecule. Large particles
such as polystyrene microspheres might also be used as carriers for
INAs, following their attachment (either covalent or passive) to
the particles. Additionally, the INAs may be adsorbed to a
microfluidic channel wall by hydrophobic or hydrophilic surface
interactions or other non-covalent or electrostatic forces.
Finally, if a system does not require the protein to be extracted
and solvated, the extraction process may be forgone entirely in
place of using whole organisms (e.g., intact P. syringae) or their
INA-containing membranes. Use of the intact organisms or membranes
may be necessary for systems in which the warmest possible ice
nucleation temperatures (approximately -2.degree. C. to -6.degree.
C.) are needed, as >99.9% of these nucleation sites may be lost
after removing the INPs from cell membranes.
[0154] Freezing of solutions (e.g., aqueous solutions) using
freeze-thaw valves with INAs can be initiated at higher
temperatures relative to known conventional microfluidic chips
without such INAs. The INAs may optionally be harvested from
bacteria as a fluid additive. The freezing process can occur at
relatively warm temperatures compared to those required in the
absence of the INAs. INAs can initiate the ice crystallization
process at temperatures of about -2.degree. C., whereas water can
be supercooled to approximately -40.degree. C. and not crystallize
in the absence of nucleation sites (e.g., in pure water or in
microfluidic devices with smooth channels). In addition and/or
alternatively to triggering freezing at warmer temperatures, INAs
can significantly reduce the average time water must be held below
the freezing point before crystallization occurs. Implementation of
the INA freeze-thaw processing technique can be used to allow
precise fluid control with simple and easy to manufacture chip
architectures without requiring moving valve parts, greatly
reducing fabrication and operation costs.
[0155] In some embodiments, an INA may be used in a method of
preparing a sample. For example, an INA may be used to improve
freezing of a sample in a sample metering loop in a fluidic
analysis device as part of sample preparation including, but not
limited to, cell lysis. In some embodiments, a sample may comprise
a plurality of cells, and at least a portion of the plurality of
cells may be lysed by electronically selectively cooling the sample
metering loop using at least one freeze thaw valve to freeze the at
least one freeze thaw valve and/or at least a portion of the sample
in the sample metering loop. In some embodiments, the sample
metering loop and/or at least one freeze thaw valve may be heated
to thaw the sample metering loop, the at least one freeze thaw
valve, and/or at least a portion of the sample. Heating the sample
metering loop and/or at least one freeze thaw valve may be active
heating or passive heating. Active heating can be electronically
selectively heating the sample metering loop and/or at least one
freeze thaw valve. Active heating can use a heat source (e.g., a
thermo-electric source) to increase the temperature of the freeze
thaw valve and/or sample metering loop. Passive heating is when a
freeze thaw valve and/or sample metering loop is allowed to warm up
(e.g., thaw) to room temperature based on the surrounding
environment without the active application of heat from a heat
source.
[0156] FIG. 6 illustrates exemplary actions that can be carried out
for methods of processing and/or analyzing analytes. As shown, a
fluidic device with a plurality of spaced apart freeze-thaw valves
and fluidic channels comprising INAs is provided (block 160). The
freeze thaw valves are cooled to a freeze temperature to stop
liquid flow through the freeze thaw valves (block 170). The freeze
thaw valves are heated to a thaw temperature to allow liquid to
flow through the freeze thaw valves (block 180). Heating of a
freeze thaw valve may be active or may be passive.
[0157] The INAs can be harvested and/or derived from organisms such
as bacteria (block 162).
[0158] The INAs can be flowably introduced in a liquid into at
least one fluidic channel associated with the freeze-thaw valves
(block 165). The flowable introduction can be in a single or
successive defined amount or bolus (i.e., batch), or carried out in
a continuous manner. The amounts of the INAs may be constant,
increase or decrease over successive processing operations in a
single microfluidic device.
[0159] The device can be a microfluidic chip that has a bottom
substrate and a top substrate (attached directly or indirectly
together). The bottom substrate can have a thickness between 0.01
mm and 10 mm (block 164).
[0160] The cooling and heating can be carried out using
thermoelectric coolers (TECs) in thermal communication with the
fluidic microchip (block 175). Some of the TECs can reside adjacent
the bottom substrate and some may reside adjacent to the top
substrate. The TECs can be set to a temperature of between -100
degrees C. and -1 degree C. for the cooling to the freeze
temperature (block 177).
[0161] The extracted INAs solution can be added to a buffer
solution diluted in any suitable amount, by way of example only,
e.g., 1:10 to 1:10,000, relative to the buffer (block 166). In some
embodiments, an INA may be present in a buffer solution in an
amount of about 1 molecule or organism per .mu.L to 10 billion
molecules or organisms per .mu.L and/or any range and/or individual
value therein. In some embodiments, an INA may be present in a
buffer solution in an amount of about 1 nanomolar to 100
millimolar, including 10-50 nM, in some particular embodiments.
[0162] The INAs can be provided as a coating and/or bonded to at
least one surface associated with a fluidic flow channel associated
with the freeze thaw valves (block 172). The bonded/coated
surface(s) can be on-device or in a conduit or other device
associated with a travel path in fluid communication with the
fluidic device.
[0163] The cooling can be controllably carried out to serially or
concurrently (i.e., selectively) freeze some or all of the
freeze-thaw valves (block 173). Similarly, the heating can be
controllably carried out to serially or concurrently (i.e.,
selectively) heat some or all of the freeze-thaw valves. Some or
all of the freeze-thaw valves may be actively heated or passively
heated.
[0164] The INAs can comprise or be one or more INP(s),
ice-nucleating (IN) nucleic acid(s), IN lipid(s), and/or IN
carbohydrate(s) (block 174).
[0165] The INAs can comprise or be one or more synthetic aptamer(s)
(block 176).
[0166] FIG. 7 illustrates an exemplary automated test system 200
with an integrated reader 300 and holding assembly 250 for a
microfluidic chip 10. The system 200 can be have a support module
200h with control inputs for coolers 25, such as voltage and fluid
inputs, for example. Wiring and tubing W, T can connect the holding
assembly 250 to the control/fluid inputs of module 200h. The
voltage and/or fluid inputs can be integrated into the same module
that holds the microfluidic chip 10 or may be a separate
cooperating sub-assembly or module 200h, for example. The system
200 can include a base 202 that supports the mounting assembly 250
and reader 300. The base 202 can also support secondary reagent
fluids 63f and/or a buffer, typically comprising INAs for fluidic
introduction to a fluidic flow channel for operation with a freeze
thaw valves 22.
[0167] The system 200 can include or be in communication with a
controller 200c (FIG. 8) with a display 210 and a graphic user
interface (GUI) 260 (FIG. 9) for allowing a user to select
timing/temperature inputs for controlling and/or scripting the
freeze thaw valves 22 operation. Default operations and/or
electronically selectable defined menus of operations for sets of
freeze thaw valves for particular samples or types or chip size
and/or configurations can be preset for ease of use. The display
210 may be onboard the system 200, module 200h, or be remote from
the system 200 and/or module 200h.
[0168] Referring to FIG. 8, the system controller 200c can be
onboard the system 200 or remote from the system and be configured
to provide the GUI 260 for allowing a user to adjust settings such
as a temperature parameter or script for one or more and/or sets of
different FTVs 22. The system controller 200c may be held totally
in a local computer, partially in a local computer and/or
distributed over a plurality of databases/servers (e.g., be CLOUD
based). The term "computer" is used broadly to include any
electronic device, typically comprising at least one digital signal
processor, allowing for control and communication with the FTVs 22
to control operation. The computer can be local or remote from a
site with the module 200h.
[0169] The display 210 can be onboard or remote from the system
200. The display 210 can comprise a display associated with a
pervasive computing device such as a smartphone, electronic
notebook and the like. The GUI 260 may be provided by an APP (the
APP typically has defined functionality accessible via one or more
icons). The system controller 200c can control one or more
temperature controllers 205 for one or more of the coolers 25,
e.g., the TEC elements 25t. The temperature controller 205 can be
any suitable temperature controller such as a Wavelength
Electronics (Bozeman, Mont.) PTCSK-CH 5 A temperature controller,
which can be controlled by a National Instruments data
acquisition/voltage output card that, in turn, can be controlled by
a computer running a LabVIEW or other suitable operating program.
Each TEC element 25t can have its own PTCSK-CH controller or sets
or all can share a temperature controller. In some embodiments, a
custom temperature control circuit can be used to control the
operation of the TEC elements 25t. Electrical paths 128 can connect
the temperature controller(s) 205 to one or more TEC element(s)
25t.
[0170] FIG. 9 illustrates the intuitive operating screen of a GUI
260 with steps and timing adjustments as well as user-selectable
temperature adjustments (Ti) for the freeze thaw valves 22 (valves
10-15, as shown). A graphic representation of the layout of the
chip 10 can be presented on the display 210. A user can adjust
settings and durations of respective coolers 25 associated with
freeze-thaw valves 22 using the GUI 260.
[0171] FIG. 10 illustrates a mounting assembly 250 for holding the
microfluidic chip 10 so that TEC members/elements 25t have
appropriate thermal contact with a thermally conductive block or
direct contact with a TEC member 25t. The mounting assembly 250 can
include a plurality of thermally conductive members 26, such as
metal (e.g., copper or other suitable thermally conductive
material) shims, that respectively abut the top or bottom substrate
10t, 10b of the chip 10 on one side and a TEC 25t on the other. In
some embodiments, a thermally conductive material and/or block 26
can be attached to the substrate 10t and/or 10b, then placed into
the mounting assembly 250 in thermal communication with the TEC
members 25t. As shown in FIG. 10, the TEC members 25t can be placed
on a holding substrate (e.g., plate) 251b aligned with valve
locations on the microfluidic chip 10.
[0172] Foil or thermally conductive coatings can be applied to the
substrate 10b, 10t, at least in regions of the freeze thaw valves
22 (not shown).
[0173] The TEC members 25t and mounting devices can be configured
to allow for easy replacement and the modular design can facilitate
future changes in designs.
[0174] As shown in FIG. 10, the mounting assembly 250 can include a
rigid top plate 251t that can have a perimeter that extends about a
window 251w. FIG. 8 also illustrates that the system 200 can
include a computer 200c with a circuit and/or at least one
processor 200p that can obtain the analysis data for the analyte in
the transport channel 20. The term "computer" is used broadly to
include any electronic device, typically comprising at least one
digital signal processor, to control operation. The computer can be
local or remote from a site with the device 10.
[0175] The reader 300 can include a detector and excitation source
that can take a series of images of an analyte molecule in the
transport channel 20 and/or array 40. The reader 300 for the system
200 can include an excitation light source (typically for
generating light that excites fluorescently labeled molecules)
(which can optionally include a mirror and lens or other objective)
and image generating device or detector such as one or more of a
camera, photomultiplier tube or photodiode. The objective/lens,
where used, can reside under or over a primary surface of the
device 10. The electric inputs/outputs and flow operation can
reside on an opposing side of the device 10. The device 10 may also
be flipped to operate on its side (with the flat primary surfaces
being upright or angled) rather than substantially horizontal as
shown.
[0176] The present invention is explained in greater detail in the
following non-limiting Examples.
Use of Ice-Nucleating Proteins to Reduce Valve Actuation Time and
Variability at Low Temperatures
[0177] A blocking buffer suitable for immunoassays (50 mM Tris+10%
newborn calf serum+0.1% Tween-20+0.05% sodium azide, pH=7.4) was
used to supply a 2 mm thick plastic microfluidic chip containing
channels with a 250 .mu.m width and 250 .mu.m depth comprised of
two substrates, each with a thickness of approximately 1 mm. The
chip was placed atop TECs so that its channels were positioned over
the TEC elements (FIGS. 1A/1B) in places where valving was desired.
Freezing and thawing of the channel contents were effected on-chip
by setting the TECs to -45.degree. C. and +25.degree. C.,
respectively. It should be noted that the actual temperature seen
at the FTV region of the channel was significantly higher than
-45.degree. C., as heat loss through the plastic can be modeled to
show a resulting steady state temperature of approximately
-32.degree. C. Each freeze and thaw event was timed according to
bright field microscopic monitoring of the channels, with the time
starting upon initiation of the cooling of the TEC from 25.degree.
C. or warming of the chip from -45.degree. C. (i.e. the time
includes the time required for the TEC to cool or heat itself and
the chip). The rapid appearance of ice was used as the endpoint of
each freeze event, while its disappearance was used as the endpoint
of each thaw event. Decade dilutions (1:10, 1:100, 1:1,000,
1:10,000) of the INP extract were then prepared in the Tris buffer,
and the same timing experiment was carried out with each of these
samples. INP-free Tris buffer required an average time of 37 s with
a standard deviation of 2.0 s (estimated channel
temperature.apprxeq.-32.degree. C.). Addition of the INP extracts
significantly impacted the freeze time and reproducibility, and no
concentration-dependent effects were observed across the four INP
concentrations tested, as freezing occurred in approximately 18-20
s in all cases with a standard deviation of approximately 1 s.
[0178] Using temperature measurements from an IR camera with a
single 1 mm thick plastic substrate atop the TEC, the internal
temperature is estimated to be at .apprxeq.-22.degree. C. at the
time of freezing. Ten freeze/thaw trials were run for each sample,
and these data are shown in FIG. 11.
[0179] Thaw times were completely unaffected by INP concentrations,
being approximately 20-22 s in all cases, including for the
INP-free buffer (as shown in FIG. 12).
[0180] FIG. 11 illustrates the effect of INP extract dilution on
freeze times in Tris blocking buffer. Compared to an INP-free
control, all four INP extract dilutions froze approximately twice
as quickly at -45.degree. C. with no concentration-dependent
effects. At -20.degree. C., INP-free buffer did not freeze during
five 30 min trials. Required freeze time and standard deviation
(both absolute and percent relative) decrease as a function of INP
concentration at this temperature. The 1:10,000 INP buffer at
-20.degree. C. (*) froze only ten times in fifteen 3 min trials.
N=10 for -45.degree. C. trials and N=15 for -20.degree. C. trials.
Error bars represent one standard deviation in each direction.
[0181] FIG. 12 illustrates thaw times for five different INP
concentrations in Tris buffer. Initial temperature refers to the
temperature used to freeze the channel, as channels were set to
thaw directly from this temperature. No concentration-dependent
effects are evident for thawing at either temperature. Thaw times
were not applicable for the INP-free buffer at -20.degree. C.
because it did not freeze at that temperature. N=10 for -45.degree.
C. trials and N=15 for -20.degree. C. trials. Error bars represent
one standard deviation in each direction.
Use of Ice-Nucleating Proteins to Reduce Temperature Requirements
for Warmer Valving Temperatures
[0182] The system and INP extract dilutions used for
low-temperature free-thaw valving were tested again in the same
manner described for the low-temperature tests, except the freezing
temperature used was -20.degree. C. (estimated steady state
temperature of -11.degree. C. inside the channel). Fifteen trials
were run, and the data are shown in FIG. 11. The INP-free blocking
buffer was not observed to freeze during five 30 min trials with
the TECs set at -20.degree. C. All INP-containing samples froze at
this temperature with concentration-dependent effects. The 1:10 and
1:100 INP extract dilutions took longer to freeze than at
-45.degree. C., with average freeze times of 33 s .+-.2.0 s and 36
s .+-.2.0 s, respectively (estimated channel temperatures of
-11.degree. C. in both cases). The 1:1,000 INP extract dilution
exhibited behavior similarly to that of Tris buffer at -45.degree.
C., with similar freeze times (44 s average) and a large standard
deviation (6.5 s). Over fifteen 3 min trials, the 1:10,000 INP
extract dilution froze ten times with an average of 91 s with an
extremely large standard deviation of 45 s; its shortest freeze
time was 41 s and the longest was 174 s. As with the trials at
-45.degree. C., the thaw times were not affected by the INP
concentration. All trials -20.degree. C. thawed in approximately 15
s with less than 1 s standard deviation. These data are shown in
FIG. 12.
Use of Ice-Nucleating Proteins to Reduce Actuation Time in Thin
Chips
[0183] A final set of experiments was conducted utilizing a thinner
chip that was identical to the ones used in the above trials
(standard chips) except its bottom substrate thickness was 0.5 mm
instead of 1 mm. Freeze times were again tested at -45.degree. C.
and -20.degree. C. TEC temperature using Tris buffer with and
without INPs. Only the 1:100 dilution of INP extract was tested in
these experiments. The effect of halving the substrate thickness
was pronounced. At -45.degree. C., freeze times were 13 s .+-.0.48
s and 22 s .+-.0.92 s with and without INPs, respectively, versus
19 s .+-.1.1 s and 37 s .+-.2.0 s for the standard chips under
those conditions. At -20.degree. C. with INPs, freezing required 18
s .+-.0.74 s, which is approximately the same as -45.degree. C.
with INPs in a standard chip. As with standard chips, no freezing
was observed at -20.degree. C. in the allotted time without INPs.
These data and a comparison to relevant standard chip data are
shown in FIG. 13.
[0184] FIG. 13 illustrates a comparison of freeze times between
standard (1 mm substrate) and thinner chips (0.5 mm substrate) with
and without INPs. For each freeze temperature and buffer condition,
freezing occurred substantially faster in a thin chip than a
standard chip, except at -20.degree. C. with no INPs, where neither
chip content froze in the allotted time. The fastest observed
freeze times occurred in the thinner chip with INPs at -45.degree.
C. (.about.13 s vs. .about.19 s for standard chips). The largest
performance difference occurred at -20.degree. C., where the thin
chip froze approximately twice as fast as the standard chip
(.about.18 s vs. .about.36 s). The temperature increase from
-45.degree. C. to -20.degree. C. added only .about.5 s to the
required freeze times for the thin chip with INPs, whereas it added
.about.18 s to the required freeze time for standard chips,
effectively doubling it. N=10 for all thin chip trials. The error
bars represent one standard deviation in each direction.
[0185] The foregoing is illustrative of the present invention and
is not to be construed as limiting thereof. Although a few
exemplary embodiments of this invention have been described, those
skilled in the art will readily appreciate that many modifications
are possible in the exemplary embodiments without materially
departing from the novel teachings and advantages of this
invention. Accordingly, all such modifications are intended to be
included within the scope of this invention as defined in the
claims. The invention is defined by the following claims, with
equivalents of the claims to be included therein.
* * * * *