U.S. patent application number 10/137525 was filed with the patent office on 2002-09-19 for methods and systems for performing superheated reactions in microscale fluidic systems.
This patent application is currently assigned to Caliper Technologies Corp.. Invention is credited to Kopf-Sill, Anne R..
Application Number | 20020132265 10/137525 |
Document ID | / |
Family ID | 21816668 |
Filed Date | 2002-09-19 |
United States Patent
Application |
20020132265 |
Kind Code |
A1 |
Kopf-Sill, Anne R. |
September 19, 2002 |
Methods and systems for performing superheated reactions in
microscale fluidic systems
Abstract
The present invention is generally directed to methods and
systems for performing chemical and biochemical reactions at
superheated temperatures by carrying out the reactions in
microscale fluidic channels. Also provided are applications of
these methods and systems, as well as ancillary systems for use
with these methods and systems in monitoring and controlling the
performance of the methods of the invention.
Inventors: |
Kopf-Sill, Anne R.; (Portola
Valley, CA) |
Correspondence
Address: |
CALIPER TECHNOLOGIES CORP
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043
US
|
Assignee: |
Caliper Technologies Corp.
605 Fairchild Drive
Mountain View
CA
94043
|
Family ID: |
21816668 |
Appl. No.: |
10/137525 |
Filed: |
May 2, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10137525 |
May 2, 2002 |
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09023693 |
Feb 13, 1998 |
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6420143 |
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Current U.S.
Class: |
435/6.19 ;
435/287.2; 435/91.2 |
Current CPC
Class: |
C12P 19/34 20130101 |
Class at
Publication: |
435/6 ; 435/91.2;
435/287.2 |
International
Class: |
C12Q 001/68; C12P
019/34; C12M 001/34 |
Claims
What is claimed is:
1. A system for performing at least one reaction at superheated
temperature comprising: a microfluidic device comprising: at least
a first substrate; and a microscale channel disposed in the first
substrate; a heating system operable to apply energy to the
microscale channel to heat a fluid in the channel to superheated
temperatures, without boiling the fluid in the microscale channel;
and a controller which is operable to control energy applied from
the heating system to the microscale channel.
2. The system of claim 1, wherein the first substrate comprises a
silica substrate.
3. The system of claim 2, wherein the silica substrate is a silica
capillary.
4. The system of claim 1, further comprising a source of a first
reactant.
5. The system of claim 4, wherein the source of the first reactant
comprises a nucleic acid.
6. A system for performing at least one reaction at superheated
temperature, comprising: a microfluidic device comprising: at least
a first substrate having at least a first planar surface, a first
microscale channel fabricated into the first planar surface; and a
second planar substrate having at least a first planar surface, the
first planar surface of the second planar substrate overlaying and
being bonded to the first planar surface of the first planar
substrate, thereby defining the first microscale channel
therebetween; means for applying energy to the microscale channel
to heat a fluid in the channel to superheated temperatures, without
boiling the fluid in the channel; and means for controlling energy
applied from the applying energy means to the microscale
channel.
7. The system of claim 6, wherein at least one of the first and
second planar substrates comprises a silica substrate, and the
microscale channel is etched into the first planar surface of the
first planar substrate.
8. The system of claim 6, wherein at least one of the first and
second planar substrates comprises a polymeric substrate.
9. The system of claim 1 or 6, further comprising a sensor for
determining a temperature of a fluid in the microscale channel.
10. The system of claim 9, wherein the sensor comprises a
conductivity sensor integrated into the controller.
11. The system of claim 1, wherein the heating system comprises a
heating element disposed in thermal contact with the microscale
channel for delivering thermal energy to the microscale channel,
the thermal energy heating a fluid in the channel to a superheated
temperature.
12. The system of claim 6, wherein the means for applying energy
comprises a heating element disposed in thermal contact with the
microscale channel for delivering thermal energy to the microscale
channel, the thermal energy heating a fluid in the channel to a
superheated temperature.
13. A system for performing at least one reaction at superheated
temperature, comprising: a microfluidic device comprising: at least
a first substrate; and a microscale channel disposed in the first
substrate; means for applying energy to the microscale channel to
heat a fluid in the channel to superheated temperatures, without
boiling the fluid in the microscale channel; and means for
controlling energy applied from the applying energy means to the
microscale channel.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. Ser. No.
09/023,693, filed Feb. 13, 1998, which is incorporated herein by
reference for all purposes.
BACKGROUND OF THE INVENTION
[0002] Microfluidic analytical systems have been gaining
substantial interest for use in performing myriad chemical and
biochemical analyses and syntheses. For example, such systems have
been described for use in performing nucleic acid amplification
reactions (See U.S. Pat. Nos. 5,498,392 and 5,587,128), for use in
performing high throughput screening assays, e.g., in drug
discovery operations (See commonly owned Published International
Application No. WO 98/00231), for use in nucleic acid separations
(See Published PCT Application No. WO 96/04547), and for a variety
of other uses. These microfluidic systems generally combine the
advantages of low volume/high throughput assay systems, with the
reproducibility and ease of use of highly automated systems.
[0003] Because of the above advantages, it would generally be
desirable to expand the applications for which these systems are
used, as well as expand the scope of the advantages which such
systems offer over conventional assay systems, e.g., faster
throughput, lower volumes, etc. One area of particular interest is
the performance of temperature responsive reactions, e.g.,
reactions that progress faster at higher temperatures, or require a
substantially elevated base temperature to occur. In many cases,
desirable chemical and biochemical reactions can be substantially
expedited by performing the reaction at substantially elevated
temperatures. However, in fluid systems, and especially aqueous
fluid systems, a practical limit on the temperature of the
operation generally is imposed by the boiling point of the fluid.
For example, in aqueous systems, the boiling temperature of the
fluid at or near 100.degree. C. is the effective maximum achievable
temperature at ambient pressures of approximately 1 atm.
[0004] In order to perform reactions that utilize or even require
temperatures that are above the boiling point for the fluid
reactants, the use of pressure sealed reaction vessels are
typically required to elevate the boiling temperature of the fluid
by increasing the ambient pressure for the reaction. Unfortunately,
in many reaction systems, the use of such sealed containers is
impracticable. For example, in microfluidic systems, the extremely
small scale of the fluid carrying elements of the system and thus
the fluid volumes used, as well as the nature of the fluid
transport systems employed, typically prohibit the use of pressure
sealed reaction containers.
[0005] Additional concerns are raised in microfluidic systems where
the presence of a bubble or bubbles, e.g., from inadvertent boiling
of fluids within the system, can have extremely detrimental effects
on the system by significantly fouling or plugging channels of the
system. Such fouling can inhibit or completely block the ability to
move fluids through the channels of the system, as well as the
ability to monitor the contents of the system, e.g., using
amperometric or potentiometric means. Further, in microfluidic
devices employing electrokinetic material transport systems to move
materials through the microscale channels of the device, such
fouling can result in a cascade effect where the blockage results
in higher current densities through the remaining portions of the
channel which leads to greater heating. This greater heating, in
turn, leads to more bubbles within the channels from boiling of the
fluids.
[0006] It would therefore be desirable to be able to perform
reactions at temperature levels that are at or substantially above
the boiling point of the fluids used in the reaction, while
benefiting from the advantages of microfluidic systems. The present
invention meets these and a variety of other needs.
SUMMARY OF THE INVENTION
[0007] The present invention is generally directed to methods and
systems for performing chemical and biochemical reactions at
superheated temperatures by carrying out the reactions in
microscale fluidic channels. Also provided are applications of
these methods and systems, as well as ancillary systems for use
with these methods and systems in monitoring and controlling the
performance of the methods of the invention.
[0008] In one aspect, the present invention provides methods for
performing reactions at superheated temperatures, which comprise
placing at least a first reactant in a microscale fluidic channel.
An effective level of energy then is applied to the fluid in the
microscale channel, whereby the fluid is heated to a superheated
temperature without boiling the fluid within the channel.
[0009] In a related aspect, the invention also provides a method
for performing a reaction at a superheated temperature, which
comprises providing a substrate having at least a first microscale
channel disposed therein. The substrate is in communication with an
energy source that delivers the sufficient level of energy to the
contents of the microscale channel to heat said contents to
superheated temperatures. The first reactant then is placed into
the microscale channel, and the sufficient level of energy from
said energy source is applied to the microscale channel to heat the
contents of the channel to superheated temperatures.
[0010] In a further aspect, the present invention also provides
systems for carrying out the methods described herein. In
particular, these systems comprise a microfluidic device that
includes at least a first substrate having a microscale channel
disposed therein, where the microscale channel has at least first
and second unintersected termini. A heating system is also included
to apply energy to the microscale channel to heat a fluid in the
channel to superheated temperatures, without boiling the fluid in
the channel. Further, a controller is also provided for maintaining
the energy applied from the heating system to the microscale
channel at a level sufficient to superheat contents of the
microscale channel without boiling the contents of the channel.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 illustrates an expanded view of a microfluidic device
and channel structure for performing superheated reactions
according to the present invention.
[0012] FIG. 2 is a temperature profile for fluids disposed within
microscale channels of a microfluidic device while the device was
globally heated in an oven.
[0013] FIG. 3 is a profile of the temperature of fluid, as
calculated from the fluid conductivity, in a microscale channel
versus time while increasing current was incrementally applied
through the channel.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention generally provides methods and systems
for performing chemical, biological or biochemical reactions in
fluid systems. More particularly, the methods and systems of the
invention permit the performance of such reactions at superheated
temperatures, i.e., above the boiling temperature for the fluid at
the ambient pressure of the system. Such methods and systems are
generally useful in speeding reactions that are temperature
dependant, as well as for carrying out reactions that require
temperatures in excess of the boiling temperatures for the fluids
used. Examples of such reactions include reactions that require a
thermal denaturation step such as enzyme inactivation reactions,
nucleic acid amplification reactions, and the like.
[0015] Typically, the methods and systems of the invention operate
by placing the fluid reactant or reactants into a microscale
fluidic channel, and heating the fluid within the channel to
superheated temperatures. Included among the benefits of the
present invention is the fact that the systems and methods
described herein allow fluid contained within one or more
microscale channels to be heated to superheated temperatures
without boiling the fluid that is contained within those channels.
In addition to the benefits normally available in performing
superheated reactions, e.g., higher reaction temperatures in
aqueous systems, the present invention is also particularly useful
in microscale systems where the generation of a bubble within a
channel can have fatal consequences for the system, e.g., blocking
material transport, current flow, etc. through that channel.
[0016] Without being bound to a particular theory of operation, it
is believed that the microscale channels fabricated by traditional
microfabrication methods, e.g., photolithography, chemical vapor
deposition, wet chemical etching, injection molding, etc., have
surfaces that are resistant to bubble nucleation during the boiling
process. As such, fluids within channels having such surfaces will
not boil at their expected boiling points.
[0017] As used herein and as noted above, the term "superheated
temperature" for a given fluid or mixture of fluids, refers to a
temperature that is greater than the temperature at which the
particular fluid or fluids will boil at the ambient pressure for
the system. In preferred aspects, the present invention provides
heating of fluids to temperatures more than 5.degree. C. above the
boiling point of the fluids, more preferably, greater than
10.degree. C., 20.degree. C., 30.degree., 40.degree. C. and even
50.degree. C. over the boiling point of the fluids. For example, in
the case of a pure water system, superheated temperatures are
generally greater than 100.degree. C. at 1 atm pressure. In many
instances therefore, the methods and systems of the present
invention provide heating of fluids, and particularly aqueous
fluids, within microscale channels to temperatures well in excess
of 100.degree. C., 105.degree. C., 110.degree. C., 120.degree. C.,
130.degree. C., often in excess of 140.degree. C., and in some
cases in excess of 150.degree. C., without boiling the aqueous
fluid that is contained within the heated channel. Of course,
variations in ambient pressure also bring corresponding changes in
the boiling temperature of fluids at that pressure. As used herein,
the term "aqueous system" generally refers to a fluid composition
that is made up substantially of water, e.g., greater than 30%
(v/v), typically greater than 50%, often greater than 80%,
preferably greater than 90% and more preferably greater than 95%
water (v/v). Although described in terms of aqueous systems, it
will be appreciated that the present invention is equally
applicable to non-aqueous systems, e.g., organic solutions,
etc.
[0018] As described above, the methods and systems of the present
invention operate through the placement of the fluid reactants into
a microscale channel that is typically incorporated into the body
of a microfluidic device. As used herein, the term microscale or
microfluidic refers to a structural element, and typically a
fluidic element, e.g., a channel or chamber, which has at least one
cross-sectional dimension, e.g., depth width or both, that is
between about 0.1 .mu.m and about 500 .mu.m, preferably between
about 1 .mu.m and about 200 .mu.m, and in many cases, between about
10 .mu.m and about 100 .mu.m.
[0019] The body structures including the microscale channel or
channels, as described herein, can be fabricated from a variety of
different substrate materials. For example, in many instances, the
body structure and channel or channel networks are microfabricated.
As such, substrate materials are often selected based upon their
compatibility with known microfabrication techniques, e.g.,
photolithography, wet chemical etching, laser ablation, air
abrasion techniques, injection molding, embossing, LIGA, and other
techniques. The substrate materials are also generally selected for
their compatibility with the full range of conditions to which the
microfluidic devices may be exposed, including extremes of pH,
temperature, salt concentration, and application of electric
fields. Accordingly, in some preferred aspects, the substrate
material may include materials normally associated with the
semiconductor industry in which such microfabrication techniques
are regularly employed, including, e.g., silica based substrates,
such as glass, quartz, silicon or polysilicon, as well as other
substrate materials, such as gallium arsenide and the like.
[0020] In the case of semiconductive materials, it will often be
desirable to provide an insulating coating or layer, e.g., silicon
oxide, over the substrate material, and particularly in those
applications where electric fields are to be applied to the device
or its contents.
[0021] In alternate preferred aspects, the substrate materials will
comprise polymeric materials, e.g., plastics, such as
polymethylmethacrylate (PMMA), polycarbonate,
polytetrafluoroethylene (TEFLON.TM.), polyvinylchloride (PVC),
polydimethylsiloxane (PDMS), polysulfone, and the like. Such
polymeric substrates are readily manufactured using available
microfabrication techniques, as described above, or from
microfabricated masters, using well known molding techniques, such
as injection molding, embossing or stamping using metal
electroforms, e.g., LIGA methods, or by polymerizing the polymeric
precursor material within the mold (See U.S. Pat. No. 5,512,131).
Such polymeric substrate materials are preferred for their ease of
manufacture, low cost and disposability, as well as their general
inertness to most extreme reaction conditions. Again, these
polymeric materials may include treated surfaces, e.g., derivatized
or coated surfaces, to enhance their utility in the microfluidic
system, e.g., provide enhanced fluid direction, e.g., as described
in U.S. Pat. No. 5,885,470, and which is incorporated herein by
reference in its entirety for all purposes.
[0022] Substrates can also come in a variety of shapes and forms,
including planar forms, e.g., in a chip format, or tubular forms,
e.g., a capillary format. The specific shape will typically vary
depending upon the particular application for which the system is
utilized. For example, systems employing complex networks of
intersecting channels for performance of multiple successive or
parallel integrated operations or reactions typically comprise
planar structures to permit the incorporation of the more complex
channel networks that are required. Simpler reactions, on the other
hand, may be carried out in less complex systems, e.g., a single
channel capillary.
[0023] In particularly preferred aspects, the substrates, and thus
the overall structure of the microfluidic devices used in
accordance with the present invention, are planar. Typically, such
devices are fabricated from at least two different planar substrate
layers. The channel or channels of the device are typically
fabricated as grooves into one surface of one of the substrate
layers. A second substrate layer is then overlaid and bonded onto
the surface of the first, thereby sealing and defining the
microscale channels of the device between the two layers.
Generally, at least one of the substrate layers has one or more
holes or ports disposed through the planar substrate, such that the
hole or port is in fluid communication with one or more of the
microscale channels when the substrate layers are mated. These
holes or ports are typically used both as fluid reservoirs for
introducing fluids into the channels of the device, as well as
providing electrical access, e.g., contact points for electrodes
that are placed in electrical contact with the fluids contained in
the device.
[0024] Examples of microfluidic devices employing these planar
structures are described in U.S. Pat. Nos. 5,965,410 and 5,976,336
and U.S. Patent Application No. 60/060,902, filed Oct. 3, 1997,
each of which is incorporated herein by reference in its entirety
for all purposes. Three layer substrate structures may also be
employed having an optional third interior layer placed between the
first and second planar layers, where the interior layer defines
the side walls of the channels of the device while the first and
second layers make up the top and bottom walls of the channels,
respectively.
[0025] The electrical access ports are useful in heating
applications, as is discussed in greater detail below, as well as
in the transport and direction of materials through the channels
that are contained in the device. In particular, in preferred
aspects, the microfluidic devices and systems that are used in
practicing the present invention employ electrokinetic material
transport systems. These electrokinetic transport systems utilize
controlled electrokinetic forces, e.g. electrophoretic and/or
electroosmotic, to controllably move materials and fluids through
the channels and their respective intersections. Examples of
controlled electrokinetic transport in microfluidic systems are
described in e.g., published PCT Application No. 96/04547, to
Ramsey, which is incorporated herein by reference.
[0026] Once the liquid reactants are placed into the microscale
channels, superheating is initiated by applying an effective level
of an appropriate energy source for heating the contents of the
channel or channels. A variety of energy sources may optionally be
used to heat the fluid within the channels of the microfluidic
device. For example, the contents of the microscale channels may be
heated using conductive methods, e.g., by applying thermal energy
to the external surfaces of the body structure of the microfluidic
element, e.g., substrate or capillary. A variety of thermal energy
sources may be readily utilized in this capacity. For example, in a
simple aspect, the body of the device may be placed into an oven or
adjacent to or in contact with a heating element, such that the
body structure and thus the contents of the channels disposed
within the body structure are heated to superheated levels.
Examples of suitable heating elements are well known to those of
skill in the art, and range from simple laboratory hot plates,
heating blocks or ovens, to resistive thin film heating elements
that may be integrated into an internal or external surface of a
microfluidic device or within an appliance adapted for use with the
device, e.g., into which the device is inserted.
[0027] Alternative energy sources can also be readily utilized in
heating the contents of microscale channels, including, e.g., light
sources such as lasers, lamps and the like, which can be directed
at the channels of the device, and preferably, precisely directed
at the channels within a microfluidic device where superheating is
desired.
[0028] As noted above, however, in preferred aspects, the
microfluidic devices described herein have electrodes associated
with the channels of the device. As such, it is generally preferred
to utilize electrical energy in superheating the contents of the
channels of the device by resistive methods. Not only does this
provide advantages of efficiency, e.g., in using a preexisting
energy interface in the electrodes, but it also provides a more
precise method of controlling and monitoring the temperature within
the system. Specifically, applying a current through the liquid
content of a reaction channel results in a resistive heating of
that liquid.
[0029] Electrical resistive heating of fluids in microscale
channels is described in substantial detail in U.S. Pat. No.
5,965,410, which is incorporated herein by reference. By applying
enough current, e.g., a sufficient current density, through a given
channel, the contents of that channel are superheated. Briefly,
electric current passing through the fluid in a channel produces
heat by dissipating energy through the electrical resistance of the
fluid. Power dissipates as the current passes through the fluid,
going into the fluid as energy over time to heat the fluid. The
following mathematical expression generally describes a
relationship between power, electrical current, and fluid
resistance:
POWER=I.sup.2R
[0030] where POWER=power dissipated in fluid; I=electric current
passing through fluid; and R=electric resistance of fluid. The
above equation provides a relationship between power dissipated
("POWER"), current ("I") and resistance ("R").
[0031] Thus, temperature within a given channel can be increased by
either increasing the resistance of the channel or increasing the
amount of current passing through the channel, or a combination of
the two. Increasing resistance of a channel can be readily
accomplished by narrowing the cross-sectional area of the channel
through which the current is applied. Further, by increasing the
resistance and/or current within a channel to sufficiently high
levels, one can achieve superheated temperatures within the
channels of the device.
[0032] In preferred aspects, sufficient current densities are
achieved by using one or both of (1) narrowed channel
cross-sectional areas, and (2) increased applied current through
the fluid. A simplified example of a microfluidic device having a
channel with a region of narrowed cross-sectional area is shown in
FIG. 1. In particular, as shown in FIG. 1, a microfluidic device
100 comprises a body structure 102, typically fabricated from two
overlaid and bonded planar substrates (not separately shown) where
one substrate has a series of channels 104, 106, 108 and 110,
etched into one planar surface. Overlaying the second substrate
provides the cover and sealing wall for the etched channels,
forming conduits between the substrate layers. Each of the channels
shown, e.g., channel 106, include a region of narrowed
cross-sectional area (112) relative to the remaining regions of the
channel 114. Reservoirs, e.g., reservoirs 116 and 118, are disposed
at the termini of the channels, typically as apertures disposed
through the overlaying planar substrate, for fluid introduction and
to provide electrical access to the channel.
[0033] As noted above, one or both of the channel cross-sectional
area or the applied current can be varied to elevate the
temperature of fluid within the channel. As such, microscale
channels for use in carrying out superheated reactions according to
the present invention may fall within a wide range of suitable
cross-sectional areas. Similarly, the currents applied to such
channels are similarly widely variable. However, in preferred
microfluidic systems, e.g., those having typical non-heating
channel dimensions in the microscale range, as set forth above,
where it is desired to heat fluids to superheated temperatures, the
cross-sectional area of the channels or channel regions in which
heating is desired will typically range from 10 .mu.m.sup.2 to
about 500 .mu.m.sup.2. This corresponds to channels having
dimensions of, e.g., from about 10 .mu.m wide by 1 .mu.m deep, to
about 50 .mu.m wide by 10 .mu.m deep. However, wider and deeper
channels my also be used.
[0034] Similarly, currents applied to the fluids within such
narrowed channels typically range from about 5 .mu.A to about 500
.mu.A, and preferably from about 10 .mu.A to about 100 .mu.A.
[0035] The systems of the invention typically include a controller
operably coupled to the energy source, for monitoring and
controlling the temperature within the reaction channels of the
device. This is particularly useful in those instances where
reaction temperatures are desired that far exceed the expected
boiling point of the fluid reactants. Specifically, careful
monitoring and control of applied energy better allows maintenance
of superheated temperatures without overshooting the desired
temperature and/or inadvertently boiling the fluid reactants, and
thereby fouling the channels of the device.
[0036] The controller aspect of the system typically includes a
processor, e.g., a computer, that is appropriately programmed to
receive temperature data from a sensor placed in thermal
communication with the device or its fluid contents. The processor
is also typically coupled to the energy source that delivers the
heating energy to the device, e.g., the oven, hot plate, resistive
heater, or electrical power supply. The processor is also
appropriately programmed to instruct the energy source to increase
or decrease the amount of applied energy depending upon whether the
sensed temperature of the fluid within the device is above or below
a set point temperature, e.g., chosen by the user. The processor
may further include appropriate programming that indicates whether
the fluid within the device is beginning to boil, e.g., as
indicated by a significant, sudden increase in the resistance of
the channel.
[0037] The sensor aspect of the controller is typically coupled to
the processor, and is in contact with the channels of the device,
and preferably, with the fluid content of those channels. Such
sensors may include traditional thermal sensors, such as
thermocouples, thermistors, IC temperature sensors. In preferred
aspects, however, the temperature within the channels is determined
from the conductivity of the fluid disposed therein, which is
dependent in part upon the fluid temperature (See U.S. Pat. No.
5,965,410 and previously incorporated herein). As such, the sensor
aspect of the controller typically comprises electrodes placed into
electrical contact with different points of the microscale channels
of the device. Preferably, the same electrodes used for heating
and/or for material transport/direction are utilized to determine
the conductivity of the fluid, and thus the temperature.
[0038] The methods and systems of the invention have broad
applicability. For example, as noted above, many reactions that
progress faster at higher temperatures can be carried out in
accordance with the present invention at still faster rates. For
example, performance of the polymerase chain reaction for
amplification of nucleic acids generally utilizes temperatures
approaching the boiling point of the aqueous reactants, e.g., in
the range of 95 to 100.degree. C., in order to expedite the process
of denaturing hybridized strands of template nucleic acids.
However, such reactions are generally further expedited at
superheated temperatures, without adverse effects on the overall
reaction.
[0039] Similarly, a number of reactions, e.g., enzyme assays,
require the denaturation of certain enzyme components of the
material to be tested, prior to performance of the overall
reaction, so that those components do not interfere with the
desired reaction. The ability to superheat the reaction components,
in situ, permits the performance of such denaturation more quickly
and efficiently. Similarly, such superheated temperatures are also
useful in the destruction and/or lysis of cells for performance of
cell-based operations, e.g., preparative or analytical.
[0040] The present invention is further illustrated with reference
to the following non-limiting examples.
EXAMPLES
[0041] A planar microfluidic device having the channel geometry
illustrated in FIG. 1 was used in each of the following
superheating examples. Reagents were introduced into the channels
of the device by placing the reagents into the reservoirs and
allowing capillary action to draw the reagents through the
channels.
[0042] The present invention is further illustrated with reference
to the following non-limiting examples.
Example 1
Conductive Superheating in Microfluidic Systems
[0043] PCR buffer was placed into the channel of the device that
included a narrowed region that was 20 .mu.m wide.times.5 .mu.m
deep, by 2 mm long (channel 106 in FIG. 1), and mineral oil was
placed over the buffer in the reservoirs (reservoirs 116 and 118)
to reduce evaporative losses within the reservoirs.
[0044] Temperature changes within the fluid filled channel were
monitored by measuring the conductivity of the fluid. At room
temperature, the conductivity measured at 41.8 nA when a 1V
potential was applied.
[0045] The substrate was placed in an oven at 100.degree. C. for
approximately one hour, at which point the temperature of the oven
was increased above 100.degree. C. FIG. 2 shows a plot of the
temperature of the fluid within the channels over the duration of
the experiment. The arrows above the plot indicate the point at
which the oven temperature was raised to the next incremental
setting. Boiling of the fluid within the reservoirs was visually
observed at just above 100.degree. C., however, no boiling was
observed within the channel, as shown by the labeled arrow below
the plot. This was confirmed by measuring the conductivity of the
fluid within the channels after removal from the oven.
Specifically, production of bubbles within the channel would have
resulted in a substantial decrease in the conductivity of that
channel, as even small bubbles will significantly constrict the
channels used, e.g., having narrow dimensions of 20 .mu.m.times.5
.mu.m. However, conductivity through the channels did not
decrease.
Example 2
Resistive Superheating in Microfluidic Systems
[0046] PCR buffer was again placed into a channel (channel 106) of
a microfluidic device having the channel geometry shown in FIG. 1
as described above, and the conductivity of the buffer at room
temperature was determined. Electrodes were placed into the
reservoirs at the termini of the channel network. The electrodes
were coupled to an electrical power supply having a 100 .mu.A,
1000V capability, for passing current through the channel network
and for concomitantly determining the conductivity of the fluid
through the channels. The temperature of the fluid within the
channels was estimated from the conductivity of the solution using
a calibration table.
[0047] The device was placed upon a hot plate at between 65 and
70.degree. C. to elevate the ambient temperature of the device and
minimize the amount of current required to superheat the fluid in
the channels. The current applied through the channel was stepped
up over time from a minimum of 2 .mu.A to a maximum of 80 .mu.A.
During the experiment, the applied current was stepped up over time
to: 2, 5, 10, 20, 30, 40, 50, 55, 60, 65, 70, 75 and 80 .mu.A. A
plot of fluid temperature (from calibrated conductivity) versus the
time period of the experiment is shown in FIG. 3. The temperature
of the fluid within the channel of the device increased over time
from a measured temperature of 70.degree. C., which was
substantially equal to the temperature of the hot plate as measured
by conventional means, to a temperature of approximately 140 to
150.degree. C. For an aqueous buffer at or near sea level, this
represents superheating of the fluid by 40 to 50.degree. C. The
ability to monitor temperature within the channel by the
conductivity through that channel indicates a lack of bubble
formation within the channel, as noted above.
[0048] All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference. Although the present
invention has been described in some detail by way of illustration
and example for purposes of clarity and understanding, it will be
apparent that certain changes and modifications may be practiced
within the scope of the appended claims.
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