U.S. patent application number 11/702368 was filed with the patent office on 2007-07-12 for multi-reservoir pressure control system.
This patent application is currently assigned to Caliper Life Sciences, Inc.. Invention is credited to Ring-Ling Chien, Andrea W. Chow, Anne Kopf-Sill, J. Wallace Parce.
Application Number | 20070157973 11/702368 |
Document ID | / |
Family ID | 26880102 |
Filed Date | 2007-07-12 |
United States Patent
Application |
20070157973 |
Kind Code |
A1 |
Chien; Ring-Ling ; et
al. |
July 12, 2007 |
Multi-reservoir pressure control system
Abstract
Improved microfluidic devices, systems, and methods allow
selective transportation of fluids within microfluidic channels of
a microfluidic network by applying, controlling, and varying
pressures at a plurality of reservoirs. Modeling the microfluidic
network as a series of nodes connected together by channel segments
and determining the flow resistance characteristics of the channel
segments may allow calculation of fluid flows through the channel
segments resulting from a given pressure configuration at the
reservoirs. To effect a desired flow within a particular channel or
series of channels, reservoir pressures may be identified using the
network model. Viscometers or other flow sensors may measure flow
characteristics within the channels, and the measured flow
characteristics can be used to calculate pressures to generate a
desired flow. Multi-reservoir pressure modulator and pressure
controller systems can optionally be used in conjunction with
electrokinetic or other fluid transport mechanisms.
Inventors: |
Chien; Ring-Ling; (San Jose,
CA) ; Parce; J. Wallace; (Palo Alto, CA) ;
Chow; Andrea W.; (Los Altos, CA) ; Kopf-Sill;
Anne; (Portola Valley, CA) |
Correspondence
Address: |
CALIPER LIFE SCIENCES, INC.
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043-2234
US
|
Assignee: |
Caliper Life Sciences, Inc.
Mountain View
CA
|
Family ID: |
26880102 |
Appl. No.: |
11/702368 |
Filed: |
February 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10916270 |
Aug 11, 2004 |
7171983 |
|
|
11702368 |
Feb 3, 2007 |
|
|
|
09792435 |
Feb 23, 2001 |
6915679 |
|
|
10916270 |
Aug 11, 2004 |
|
|
|
60216793 |
Jul 7, 2000 |
|
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60184390 |
Feb 23, 2000 |
|
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Current U.S.
Class: |
137/565.29 ;
137/806; 137/825 |
Current CPC
Class: |
B01L 3/502715 20130101;
B01L 3/502746 20130101; Y10T 137/86131 20150401; B01L 2400/082
20130101; Y10T 137/218 20150401; Y10T 137/2076 20150401; B01L
3/502738 20130101; G05D 7/0694 20130101; B01L 3/50273 20130101;
G01N 27/44791 20130101; B01L 2400/0415 20130101; B01L 2200/027
20130101; G01N 11/08 20130101; B01L 2400/0487 20130101; B01L 9/527
20130101; B01L 2200/146 20130101; B01L 2300/0816 20130101; G01N
2035/1044 20130101; B01L 2300/0867 20130101; B01L 2200/025
20130101 |
Class at
Publication: |
137/565.29 ;
137/825; 137/806 |
International
Class: |
F15C 1/04 20060101
F15C001/04; F04B 41/06 20060101 F04B041/06 |
Claims
1. A method of controlling the movement of fluid through an
intersection between a first channel and a second channel in a
microfluidic device, the method comprising: applying a first
pressure differential across the first channel with a first
pressure source to, and applying a second pressure differential
across the second channel with a second pressure source.
2. The method of claim 1, wherein the first channel interfaces with
two reservoirs on opposite sides of the intersection, wherein the
second channel interfaces with two reservoirs on opposite sides of
the intersection, wherein the first pressure source applies the
first differential pressure across the two reservoirs interfacing
with the first channel, and wherein the second pressure source
applies the second pressure differential across the two reservoirs
interfacing with the second channel.
3. The method of claim 2, wherein the two reservoirs interfacing
with the first channel are at the termini of the first channel.
4. The method of claim 1, wherein the first and second channels
have different flow resistances.
5. The method of claim 1, wherein the first and second pressure
differentials are transmitted from the first and second pressure
sources respectively through a pressure transmission system
comprising lumens.
6. The method of claim 5, wherein the flow resistance of the lumens
is less than the flow resistance of the first and less than the
flow resistance of the second channel.
7. The method of claim 6, wherein the flow resistance of each of
the first and second channels is at least ten times higher than the
flow resistance of the pressure transmission system.
8. The method of claim 6, wherein the flow resistance of each of
the first and second channels is at least one hundred times higher
than the flow resistance of the pressure transmission system.
9. The method of claim 1, wherein the steps of applying the first
and second pressure differentials further comprise the steps of
measuring the first and second pressure differentials with first
and second pressure sensors respectively.
10. The method of claim 9, wherein the steps of applying the first
and second pressures further comprise providing feedback control to
the first and second pressure sources.
11. The method of claim 1, wherein the first and second pressure
sources are positive displacement pumps.
12. The method of claim 11, wherein the first and second pressure
sources are syringes.
13. The method of claim 1, wherein each of the first and second
channels comprise at least one fabricated dimension of less than
500.mu..
14. The method of claim 1, wherein the intersection is a cross
intersection.
15. The method of claim 1, wherein the intersection is a "T"
intersection.
16. The method of claim 1, wherein the microfluidic device
comprises a silica-based substrate.
17. The method of claim 1, wherein the microfluidic device
comprises a polymeric substrate.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/916,270, filed Aug. 11, 2004, which is a continuation of U.S.
Ser. No. 09/792,435, filed Feb. 23, 2001, now U.S. Pat. No.
6,915,679, which is a nonprovisional of U.S. Ser. Nos. 60/184,390,
filed Feb. 23, 2000 and 60/216,793, filed Jul. 7, 2000, which are
hereby incorporated herein by reference in their entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0002] The present invention is generally related to analytical
tools for the biological and chemical sciences, and in particular,
provides microfluidic devices, systems, and methods for selectively
transporting fluids within microfluidic channels of a microfluidic
network, often using a plurality of selectively variable
pressures.
[0003] Microfluidic systems are now in use for the acquisition of
chemical and biological information. These microfluidic systems are
often fabricated using techniques commonly associated with the
semiconductor electronics industry, such as photolithography, wet
chemical etching, and the like. As used herein, "microfluidic"
means a system or device having channels and chambers which are at
the micron or submicron scale, e.g., having at least one
cross-sectional dimension in a range from about 0.1 .mu.m to about
500 .mu.m.
[0004] Applications for microfluidic systems are myriad.
Microfluidic systems have been proposed for capillary
electrophoresis, liquid chromatography, flow injection analysis,
and chemical reaction and synthesis. Microfluidic systems also have
wide ranging applications in rapidly assaying compounds for their
effects on various chemical, and preferably, biochemical systems.
These interactions include the full range of catabolic and anabolic
reactions which occur in living systems, including enzymatic,
binding, signaling, and other reactions.
[0005] A variety of methods have been described to effect the
transport of fluids between a pair of reservoirs within a
microfluidic system or device. Incorporation of mechanical micro
pumps and valves within a microfluidic device has been described to
move the fluids within a microfluidic channel. The use of acoustic
energy to move fluid samples within a device by the effects of
acoustic streaming has been proposed, along with the use of
external pumps to directly force liquids through microfluidic
channels.
[0006] The capabilities and use of microfluidic systems advanced
significantly with the advent of electrokinetics: the use of
electrical fields (and the resulting electrokinetic forces) to move
fluid materials through the channels of a microfluidic system.
Electrokinetic forces have the advantages of direct control, fast
response, and simplicity, and allow fluid materials to be
selectively moved through a complex network of channels so as to
provide a wide variety of chemical and biochemical analyses. An
exemplary electrokinetic system providing variable control of
electro-osmotic and/or electrophoretic forces within a
fluid-containing structure is described in U.S. Pat. No. 5,965,001,
the full disclosure of which is incorporated herein by
reference.
[0007] Despite the above-described advancements in the field of
microfluidics, as with all successes, still further improvements
are desirable. For example, while electrokinetic material transport
systems provide many benefits in the micro-scale movement, mixing,
and aliquoting of fluids, the application of electrical fields can
have detrimental effects in some instances. In the case of charged
reagents, electrical fields can cause electrophoretic biasing of
material volumes, e.g., highly charged materials moving to the
front or back of a fluid volume. Where transporting cellular
material is desired, elevated electrical fields can, in some cases,
result in a perforation or electroporation of the cells, which may
effect their ultimate use in the system.
[0008] To mitigate the difficulties of electrokinetic systems,
simplified transport systems for time domain multiplexing of
reagents has been described in WO 00/45172 (assigned to the
assignee of the present invention), the full disclosure of which is
incorporated herein by reference. In this exemplary time domain
multiplexing system, structural characteristics of channels
carrying reagents can, at least in part, regulate the timing and
amount of reagent additions to reactions (rather than relying
solely on the specific times at which pumps are turned on and/or
valves are actuated to regulate when and how much of a particular
reagent is added to a reaction). While other solutions to the
disadvantageous aspects of electrokinetic material transport within
a microfluidic system have been described, still further
alternative fluid transport mechanisms and control methodologies
would be advantageous to enhance the flexibility and capabilities
of known microfluidic systems.
[0009] Regardless of the mechanism used to effect movement of fluid
and other materials within a microfluidic channel network, accuracy
and repeatability of specific flows can be problematic. There may
be variations in, for example, electroosmotic flow between two
chips having similar designs, and even between different operations
run on a single chip at different times. Quality control can be
more challenging in light of this variability, as accurate control
over microfluidic flows in applications such as high throughput
screening would benefit significantly from stable and reliable
assays.
[0010] In light of the above, it would be advantageous to provide
improved microfluidic devices, systems, and methods for selectively
transporting fluids within one or more microfluidic channels of a
microfluidic network. It would be desirable if these improved
transport techniques provided selective fluid movement capabilities
similar to those of electrokinetic microfluidic systems, while
mitigating the disadvantageous aspects of the application of
electrical fields to chemical and biochemical fluids in at least
some of the microfluidic channels of the network.
[0011] It would also be beneficial to provide improved devices,
systems, methods and kits for enhancing the accuracy, reliability,
and stability of microfluidic flows within a microfluidic network.
It would be beneficial if these enhanced flow control techniques
provided real-time and/or quality control feedback on the actual
flows, ideally without relying on significantly increased system
complexity or cost.
SUMMARY OF THE INVENTION
[0012] The present invention generally provides improved
microfluidic devices, systems, and methods. The devices and systems
of the invention generally allow flexible and selective
transportation of fluids within microfluidic channels of a
microfluidic network by applying, controlling, and varying
pressures at a plurality of reservoirs or ports. By modeling the
microfluidic network as a series of nodes (including the
reservoirs, channel intersections, and the like) connected together
by channel segments, and by determining the flow resistance
characteristics of the channel segments, the fluid flows through
the channel segments resulting from a given pressure configuration
at the reservoirs can be determined. Reservoir pressures to effect
a desired flow profile may also be calculated using the network
model. A simple multi-reservoir pressure modulator and pressure
controller system can optionally be used in conjunction with
electrokinetic or other fluid transport mechanisms. The invention
also provides techniques to avoid fluid mixture degradation within
a microfluidic channel by maintaining sufficient oscillation to
avoid separation of the fluid mixture when no gross movement of the
fluid is desired. Microfluidic systems and methods having
viscometers or other flow sensors are particularly useful for
determining pressures so as to hydrodynamically induce a desire to
flow in response to a measured flow within a microfluidic channel.
Regardless of the mechanism used to effect movement of fluids
within a microfluidic network, the techniques of the present
invention may be used to provide feedback on the actual flow and/or
network system characteristics, allowing (for example) more
accurate, stable and reliable assays.
[0013] In a first aspect, the invention provides a microfluidic
system comprising a body defining a microfluidic channel network
and a plurality of reservoirs in fluid communication with the
network. The network includes a channel. A plurality of pressure
modulators are also included, each pressure modulator providing a
selectably variable pressure. A plurality of pressure transmission
lumens transmit the pressures from the pressure modulators to the
reservoirs so as to induce a desired flow within the channel.
[0014] Generally, the lumens will transmit the pressures to the
ports with significantly less resistance to the lumen flow than the
resistance of the channel to the associated microfluidic flow. Each
pressure modulator will typically be in fluid communication with an
associated port via an associated lumen. In many embodiments, a
network flow controller will be coupled to the pressure modulators
and will send signals to the pressure modulators so that the
modulators vary the pressures. The network controller will
generally include channel network data which correlates the channel
flows with the pressures from the pressure modulators.
[0015] In some embodiments, the network will comprise a plurality
of microfluidic channels in fluid communication at channel
intersections. The intersections and reservoirs will define nodes
coupled by channel segments. The network data can indicate
correlations between the flows in the channel segments and the
plurality of pressures.
[0016] In other embodiments, a network data generator may be
coupled to the network controller. The network data generator may
comprise a network flow model, a viscometer coupled to the channel,
and/or a network tester adapted to measure at least one parameter
indicating the pressure-flow correlation. The pressure controller
or controllers will often make use of signals from pressure sensors
so as to provide a pressure feedback path. Optionally, the pressure
controllers may include calibration data correlating drive signals
with the resulting reservoir pressures. Preferably, the pressure
modulators will comprise pneumatic displacement pumps.
[0017] Typically, at least one sample test liquid will be disposed
in the channel network. A pressure-transmission fluid can be
disposed in the lumens, with a fluid/fluid-pressure-transmission
interface disposed therebetween. Typically, the
pressure-transmission fluid will comprise a compressible gas, which
can compliantly couple the pressure modulators with the channel
flow.
[0018] Typically, the system will include at least four
independently variable pressure modulators. Preferably, the system
will make use of at least eight independently variable pressure
modulators. A pressure interface manifold can be used to releasably
engage the microfluidic body, the manifold providing sealed fluid
communication between the lumens and the associated reservoirs.
Ideally, a plurality of electrodes will also be coupled to the
microfluidic network with an electrokinetic controller coupled to
the electrodes so as to induce electrokinetic movement of fluids
within the network. In general, when a hydrodynamic pressure
differential is used to move fluid within the microfluidic network,
the pressure differential will be significantly greater than a
capillary pressure of fluids within the reservoirs.
[0019] In another aspect, the invention provides a body defining a
microfluidic channel network with a plurality of reservoirs in
fluid communication with the network. The network includes a first
channel. A plurality of pressure modulators is also provided, with
each pressure modulator in fluid communication with a reservoir for
varying a pressure applied thereto. A network flow controller is
coupled to the pressure modulators. The network controller
comprises channel network data correlating a flow within the first
channel and the pressures from the pressure modulators. The network
controller independently varies the pressures from the pressure
modulators in response to a desired flow within the first channel
in the network data.
[0020] Optionally, the system may further include means for
generating the network data coupled to the network controller. The
network data generating means may comprise a model of the network,
a viscometer, an electrical resistance sensor for sensing
electrical resistance within the network, or the like.
[0021] In another aspect, the invention provides a microfluidic
system comprising a body defining a microfluidic channel network
and a plurality of ports in fluidic communication with the network.
The network includes a first channel. A network flow controller
generates independent desired pressure signals in response to a
desired flow within the first channel. A plurality of pressure
modulators coupled to the network flow controller are each in fluid
communication with an associated reservoir. A pressure controller
with calibration data couples the pressure modulators with the
network controllers. The pressure controllers transmit drive
signals to the pressure modulators in response to desired pressure
signals from the network flow controller and the calibration
data.
[0022] In a first method aspect, the invention provides a
microfluidic method comprising transmitting a first plurality of
pressures to an associated plurality of reservoirs using a
plurality of pressure transmission systems. A first flow is induced
within a first microfluidic channel of a microfluidic network in
response to the first pressures. A second plurality of pressures in
determined so as to effect a desired second flow within the first
microfluidic channel. The determined second plurality of pressures
are applied with the pressure transmission systems and the second
flow is induced within the first microfluidic channel with the
second pressures.
[0023] The methods of the present invention are particularly well
suited for precisely combining selected fluids within a
microfluidic network, such as for multiport dilution in which
concentrations of first and second fluids from first and second
reservoirs can be combined at different concentrations.
[0024] In another method aspect, the invention provides a
microfluidic method comprising determining pressure-induced flow
characteristics of a microfluidic channel within a microfluidic
network. A first plurality of pressures are derived from the
characteristics of the microfluidic network so as to provide a
first desired flow in a first microfluidic channel. The first
desired flow is induced by applying the first pressures to a
plurality of ports in communication with the microfluidic
network.
[0025] In yet another method aspect, the invention provides a
method for use with a fluid mixture which can degrade when held
stationary. The method comprises introducing the fluid mixture into
a microfluidic channel of a microfluidic network. The mixture is
maintained by oscillating the fluid mixture within the channel. The
maintained fluid mixture is then transported along the channel.
[0026] While analysis of the microfluidic network based on the
known channel geometry can significantly facilitate calculation of
pressures to be applied for generation of a desired hydrodynamic
flow, work in connection with the present invention has shown that
the complex nature of the flows within a microfluidic channel can
make calculation of effective fluid viscosity within a microfluidic
network highly problematic. Specifically, the flows within a single
channel of a microfluidic network may include differing dilutions
of test fluids separated by a plurality of different buffering
solutions, and the like. To over come this complication, the
invention often makes use of viscometers and other flow sensing
systems to determine actual flow characteristics from a known
microfluidic driving force. Based on these measurements, a desired
flow may then be generated hydrodynamically by adjusting the
appropriate reservoir pressures.
[0027] In a related method aspect, the invention provides a
microfluidic method comprising inducing flow within a microfluidic
channel of a microfluidic network. The flow is measured and a
pressure is calculated from the measured flow so as to generate a
desired flow. The desired flow is generated within the channel by
applying the calculated pressure to the microfluidic network.
[0028] The flow is optionally measured by generating a detectable
signal within the flow at a first location, and by measuring a time
for the signal to reach a second location. The signal may comprise
a change in a fluid of the flow, particularly where the first
location comprises an intersection between a plurality of
microfluidic channels. Such a change in the flow may be initiated
hydrodynamically by applying a pressure pulse to a reservoir in
communication with the intersection, and/or electrokinetically by
varying an electrical field across the first intersection.
Optionally, a plurality of detectable signals from a plurality of
channel intersections may be sensed as each of these signals
reaches the second location. In many embodiments, a signal will
comprise a change in an optical quality of fluid in the flow. For
example, the signal may comprise a change in a concentration of a
dye from a channel intersection, as described above. Alternatively,
where the fluid comprises a photobleachable dye, the dye may be
photobleached by a laser at the first location with the
photobleaching sensed at the second location. Many of these methods
will allow a speed of the flow to be determined, particularly when
a distance between the first and second locations is known. In some
embodiments, the speed of the flow may be determined by, for
example, Dopler velocimetry, tracer particle videography, or the
like. Ideally, a viscosity of the flow can be calculated using a
first pressure (which induces the measured flow) and the speed of
the flow. This viscosity can then be used in determination of the
calculated pressure so as to generated the desired flow.
[0029] In a related system aspect, the invention provides a
microfluidic system comprising a body defining a microfluidic
channel network and a plurality of reservoirs in fluid
communication with the network. The network includes a microfluidic
channel. A viscometer is coupled to the channel for determining a
viscosity of a flow therein.
[0030] In yet another system aspect, the invention provides a
microfluidic system comprising a body defining a microfluidic
channel network and a plurality of reservoirs in fluid
communication with the network. The network includes a microfluidic
channel. A plurality of pressure modulators are in fluid
communication with the reservoirs. A sensor is coupled to the
channel for transmission of flow signals in response to flow within
the channel. The controller couples the sensor to the pressure
modulators. The controller transmits pressure commands in response
to the flow signals to provide a desired flow.
[0031] In yet another aspect, the invention provides a microfluidic
system comprising a body defining a microfluidic channel network
and a plurality of reservoirs in fluid communication with the
network. The system also includes means for selectively and
independently varying pressures within the reservoirs. The pressure
varying means is in fluid communication with the reservoirs.
[0032] In yet another aspect, the invention provides a microfluidic
method comprising inducing a perturbation in a flow through a
microfluidic channel of a microfluidic network by applying a
pressure transient to the microfluidic network. A characteristic of
the flow or microfluidic network is determined by monitoring
progress of the perturbation.
[0033] The pressure transient may conveniently be applied by
spontaneous injection of an introduced fluid into an injection
channel of the microfluidic network. Such spontaneous injection may
draw the introduced fluid into the injection channel using
capillary forces between the injection channel and the introduced
fluid.
[0034] Typically, the perturbation will comprise a change in a
material of the flow downstream of an intersection. This change
will often comprise a change in quantity of a fluid from a first
channel, with the pressure transient being applied at the first
channel.
[0035] The use of pressure induced flow perturbations may be used
to determine flow or network characteristics in systems having flow
that is pressure induced, electrically induced, or any mixture of
flow inducing mechanisms. Typically, flow characteristics such as
effective flow viscosity, flow speed, and the like may be
determined. In some embodiments, network characteristics such as
flow resistance of one or more channels may be determined.
[0036] The progress of the perturbation may be monitored at least
in part with a sensor disposed downstream of a perturbation source
location (such an intersection of channels). A speed of the flow
may be determined from, for example, a time interval extending from
the pressure transient to detection of the perturbation at the
sensor location, and from a distance along the channel or channels
extending from the source location to the sensor location. More
complex analyses are also possible, such as determining a second
speed of a second flow. This second speed may be generated in
response to a time interval defined in part by detection of a
second flow perturbation, and a second distance defined in part by
a second perturbation source location (such as a second channel
intersection). As the different speeds along intersecting channels
may be determined, the amount of materials combined from different
channels at an intersection may be calculated.
[0037] In a related system aspect, the invention provides a
microfluidic system comprising a body having channel walls defining
a microfluidic network. A pressure transient generator is in
communication with a channel intersection of the microfluidic
network for initiation of a flow perturbation. A sensor is coupled
to the flow within the network at a sensor location. A processor
coupled to the pressure generator and the sensor determines a
characteristic of the flow or the network in response to detection
of the perturbation at the sensor location.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 schematically illustrates a microfluidic system
having a multi-reservoir pressure modulation system according to
the principles of the present invention.
[0039] FIG. 2 is a plan view of a representative microfluidic
device having microfluidic channels with enhanced fluid flow
resistance for use in the microfluidic system of FIG. 1.
[0040] FIGS. 3A and 3B are perspective views of a pressure manifold
for releasably sealing reservoirs of the microfluidic device of
channel 2 in fluid communication with the pressure modulators of
the system of FIG. 1.
[0041] FIG. 4 schematically illustrates a control system for
independently varying reservoir pressures in the microfluidic
system of FIG. 1.
[0042] FIGS. 5A-C schematically illustrate a method and computer
program for determining pressures to provide a desired flow within
a channel of the microfluidic network in the microfluidic device of
FIG. 2.
[0043] FIG. 6 schematically illustrates a microfluidic system
having both a multi-reservoir pressure modulation system and an
electrokinetic fluid transportation and control system according to
the principles of the present invention.
[0044] FIGS. 7A and 7B illustrate well-pair dilution in which
concentration variations are produced by selectively varying the
relative flow rates from two reservoirs connected at an
intersection.
[0045] FIGS. 7C-E graphically illustrate measured dilution verses
set or intended dilution for a multi-reservoir pressure controlled
well-pair dilution.
[0046] FIGS. 8 and 8A-8D graphically illustrate an enzyme assay
using a multi-reservoir pressure controlled microfluidic system,
and more specifically: FIG. 8 illustrates the reaction, FIG. 8A is
a titration curve for different substrate concentrations, FIG. 8B
is a plot of the corrected signal verses substrate concentration,
FIG. 8C is a plot for determination of the Michaelis constant, and
FIG. 8D is a substrate titration plot.
[0047] FIGS. 9A-C illustrate a microfluidic Protein Kinase A (PKA)
reaction assay with variations in concentration achieved using
hydrodynamic pressure modulation.
[0048] FIGS. 10A and 10B illustrate a mobility shift assay
microfluidic network and assay test results at different
concentrations.
[0049] FIGS. 11A and 11B are a perspective and plane view,
respectively, of an exemplary hydrodynamic and electrokinetic
interface structure for coupling to a microfluidic body.
[0050] FIG. 12 schematically illustrates an exemplary microfluidic
viscometer.
[0051] FIGS. 13A and 13B schematically illustrate a microfluidic
network and method for imposing detectable signals on a
microfluidic flow for measurement of flow characteristics which can
be used to calculate pressures to affect a desired flow.
[0052] FIGS. 14A and 14B graphically illustrate flow characteristic
signals which may be used to determine effective viscosity.
[0053] FIG. 15 is a perspective view of a microfluidic chip having
a plurality of capillaries for spontaneous injection of fluids into
the microfluidic network.
[0054] FIG. 16 is a top view of a simple microfluidic chip having a
single capillary for spontaneous injection.
[0055] FIGS. 16A-16C graphically illustrate methods for monitoring
progress of perturbations induced by spontaneous injection of
fluids, for use in determining characteristics of a flow and/or
microfluidic network.
[0056] FIGS. 17A and 17B are perspective and plan view of
fluorogenic multi-capillary chips.
[0057] FIGS. 18A and 18B are perspective and plan view of a
mobility-shift capillary chip.
[0058] FIG. 19 graphically illustrates the detection of a
perturbation generated at an intersection of microfluidic channels
by spontaneous injection.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0059] The present invention generally makes use of a
multi-reservoir pressure controller coupled to a plurality of
independently variable pressure modulators to effect movement of
fluids within microfluidic networks. By selectively controlling and
changing the pressure applied to the reservoirs of a microfluidic
device, hydrodynamic flow at very low flow rates may be accurately
controlled within intersecting microfluidic channels. Such
pressure-induced flows can help to decrease (or entirely avoid) any
detrimental effects of the electrical fields associated with
electrokinetic transportation methods, such as sample bias, cell
perforation, electroporation, and the like. Additionally, such
pressure-induced microfluidic flows may, through proper chip
design, reduce flow variabilities as compared to electrokinetic
techniques through the use of pressure differentials (and/or
channel resistances that are significantly greater than flow
variations induced by secondary effects, such as inflow/outflow
capillary force differentials within the reservoirs).
Advantageously, the pressure-induced flows of the present invention
may also be combined with electrokinetic and/or other fluid
transportation mechanisms thereby providing composite
pressure/electrokinetic microfluidic systems.
[0060] The techniques of the present invention will often make use
of data regarding the network of channels within a microfluidic
device. This network data may be calculated using a model of the
microfluidic network, measured by testing a microfluidic device,
sensed using a sensor, and/or the like. The network data will often
be in the form of hydrostatic resistances along microfluidic
channel segments connecting nodes, with the nodes often being
intersections between channels, ports or reservoirs, connections
between channel segments having differing cross-sectional
dimensions and/or flow characteristics, and the like. As used
herein, the term "reservoir" encompasses ports for interfacing with
a microfluidic network within a microfluidic body, including ports
which do not have cross-sections that are much larger than the
microfluidic channel to enhance fluid capacity.
[0061] By selectively controlling the pressure at most or all of
the reservoirs of a microfluidic system, very small flow rates may
be induced through selected channel segments. Such small
pressure-induced flows can be accurately controlled at flow rates
which might be difficult and/or impossible to control using
alternative fluid transportation mechanisms. Advantageously, the
present invention may provide flow rates of less than 0.1
nanoliters per second, the flow rates often being less than 1
nanoliters per second, and the pressure induced flow rates
typically being less than 10 nanoliters per second within the
microfluidic channel.
[0062] To accurately apply the pressures within the microfluidic
network, the invention generally makes use of a pressure
transmission system having relatively large lumens coupling the
pressure modulators to the reservoirs of the microfluidic device,
with the pressure transmission lumens ideally containing a
compressible gas. Pressure is often transmitted through this
relatively low resistance pressure transmission system to fluids
disposed within the reservoirs of the microfluidic system via a
gas/fluid interface within the reservoir. The resistance of the
microfluidic channels to the fluid flows therein is typically much
greater than the resistance of the pressure transmission lumens to
the associated flow of compressible gas. Generally, the channel
resistance is at least 10 times the transmission system resistance,
preferably being at least 100 times, and ideally being at least
1000 times the transmission system resistance of the compressible
gas used to induce the channel flows. In other words, a response
time constant of the pressure transmission system will generally be
lower than the time constant of the channel network, preferably
being much lower, and ideally being at least one, two, or three
orders of magnitude lower. The head space of a fluid (for example,
in the pressure modulator pump and/or in the port or reservoir)
times the resistance of the fluid flow (for example, in the
channels or lumens) may generally define the response time
constant.
[0063] Surprisingly, it is often advantageous to enhance the
resistance of the microfluidic channels to provide the desired
relative resistance factors. The channels may have reduced
cross-sectional dimensions, pressure drop members (such as a small
cross-section pressure orifice, a flow restricting substance or
coating, or the like), and/or lengths of some, most, or even all of
the microfluidic channel segments may be increased by including
serpentine segment paths. As the resistance of the pressure
transmission system can be several orders of magnitude less than
the resistance of the channels, pressure differentials can be
accurately transmitted from the pressure modulators to the
reservoirs of the microfluidic device. Additionally, reduced
transmission system resistances can help to enhance the response of
the pressure system, providing a faster response time constant.
[0064] Referring now to FIG. 1, a microfluidic system 10 includes a
microfluidic device 12 coupled to a bank of pressure modulators 14
by a pressure transmission system 16. Pressure modulator bank 14
includes a plurality of pressure modulators 14a, 14b, . . .
Modulator bank 14 will generally include at least three
independently, selectively variable pressure modulators, typically
having at least four modulators, and ideally having eight or more
modulators. Each modulator is in fluid communication with a
reservoir 18 of microfluidic device 12 via an associated tube 20,
the tube having a pressure transmission lumen with a compressible
gas therein.
[0065] Modulator bank 14 generally provides independently
selectable pressures to the lumens of tubing 20 under the direction
of a controller(s) 22. Feedback may be provided to controller 22
from pressure sensors 24, as will be described hereinbelow.
Processor 22 will often comprise a machine-readable code embodied
by a tangible media 26, with the machine-readable code comprising
program instructions and/or data for effecting the methods of the
present invention. Processor 22 may comprise a personal computer
having at least an Intel Pentium.RTM. or Pentium II.RTM. processor
having a speed of at least 200 MHz, 300 MHz, or more. Tangible
media 26 may comprise one or more floppy disks, compact disks, or
"CDs," magnetic recording tape, a read-only memory, a random access
memory, or the like. In some embodiments, the programming
instructions may be input into controller 22 via a disk drive or
other input/output system such as an internet, intranet, modem
reservoir, or the like. Suitable programs may be written in a
variety of programming languages, including the LabView.TM.
language, as available from National Instruments of Austin, Tex.
Controller 22 transmits drive signals to modulator bank 14, ideally
via an RS232/RS485 serial connection.
[0066] In addition to tubing 20, pressure transmission system 16
includes a manifold 28. Manifold 28 releasably seals the lumen of
each tube 20 with an associated reservoir 18 of microfluidic device
12. Tubing 20 may comprise a relatively high-strength polymer such
as polyetheretherketone (PEEK), or a polytetrafluoroethylene (such
as a Teflon.TM. material), or the like. The tubing typically has an
inner diameter in a range from about 0.01'' to about 0.05'', with a
length from about 1 m to about 3 m. A "T" connector couples the
pressure output from each pressure modulator to an associated
pressure sensor 24.
[0067] Each modulator 14a, 14b . . . generally comprises a pump or
other pressure source which pressurizes the compressible gas within
the lumen of associated tubing 20. The modulators preferably
comprise positive displacement pumps, with the exemplary modulators
comprising a piston which is selectively positioned within a
surrounding cylinder by an actuator. Preferably, the actuators are
adapted to allow accurate positioning of the piston in response to
drive signals from controller 22, the exemplary actuators
comprising stepper motors. The exemplary piston/cylinder
arrangement is similar to a syringe. Exemplary modulator banks may
be provided by (or modified from components available through) a
variety of commercial sources, including Kloehn of Las Vegas, Nev.,
Cavaro of Sunnyvale, Calif., and the like.
[0068] Microfluidic device 12 is seen more clearly in FIG. 2.
Microfluidic device 12 includes an array of reservoirs 18a, 18b, .
. . coupled together by microscale channels defining a microfluidic
network 30. As used herein, the term "microscale" or
"microfabricated" generally refers to structural elements or
features of a device which have at least one fabricated dimension
in the range of from about 0.1 .mu.m to about 500 .mu.m. Thus, a
device referred to as being microfabricated or microscale will
include at least one structural element or feature having such a
dimension. When used to describe a fluidic element, such as a
passage, chamber or conduit, the terms "microscale",
"microfabricated" or "microfluidic" generally refer to one or more
fluid passages, chambers or conduits which have at least one
internal cross-sectional dimension, e.g., depth, width, length,
diameter, etc., that is less than 500 .mu.m, and typically between
about 0.1 .mu.m and about 500 .mu.m. In the devices of the present
invention, the microscale channels or chambers preferably have at
least one cross-sectional dimension between about 0.1 .mu.m and 200
.mu.m, more preferably between about 0.1 .mu.m and 100 .mu.m, and
often between about 0.1 .mu.m and 50 .mu.m.
[0069] The microfluidic devices or systems of the present invention
typically include at least one microscale channel, usually at least
two intersecting microscale channel segments, and often, three or
more intersecting channel segments disposed within a single body
structure. Channel intersections may exist in a number of formats,
including cross intersections, "T" intersections, or any number of
other structures whereby two channels are in fluid
communication.
[0070] The body structures of the devices which integrate various
microfluidic channels, chambers or other elements may be fabricated
from a number of individual parts, which when connected form the
integrated microfluidic devices described herein. For example, the
body structure can be fabricated from a number of separate
capillary elements, microscale chambers, and the like, all of which
are connected together to define an integrated body structure.
Alternatively and in preferred aspects, the integrated body
structure is fabricated from two or more substrate layers which are
mated together to define a body structure having the channel and
chamber networks of the devices within. In particular, a desired
channel network is laid out upon a typically planar surface of at
least one of the two substrate layers as a series of grooves or
indentations in that surface. A second substrate layer is overlaid
and bonded to the first substrate layer, covering and sealing the
grooves, to define the channels within the interior of the device.
In order to provide fluid and/or control access to the channels of
the device, a series of reservoirs or reservoirs is typically
provided in at least one of the substrate layers, which reservoirs
or reservoirs are in fluid communication with the various channels
of the device.
[0071] A variety of different substrate materials may be used to
fabricate the devices of the invention, including silica-based
substrates, i.e., glass, quartz, fused silica, silicon and the
like, polymeric substrates, i.e., acrylics (e.g.,
polymethylmethacrylate) polycarbonate, polypropylene, polystyrene,
and the like. Examples of preferred polymeric substrates are
described in commonly owned published international patent
application no. WO 98/46438 which is incorporated herein by
reference for all purposes. Silica-based substrates are generally
amenable to microfabrication techniques that are well-known in the
art including, e.g., photolithographic techniques, wet chemical
etching, reactive ion etching (RJE) and the like. Fabrication of
polymeric substrates is generally carried out using known polymer
fabrication methods, e.g., injection molding, embossing, or the
like. In particular, master molds or stamps are optionally created
from solid substrates, such as glass, silicon, nickel electro
forms, and the like, using well-known micro fabrication techniques.
These techniques include photolithography followed by wet chemical
etching, LIGA methods, laser ablation, thin film deposition
technologies, chemical vapor deposition, and the like. These
masters are then used to injection mold, cast or emboss the channel
structures in the planar surface of the first substrate surface. In
particularly preferred aspects, the channel or chamber structures
are embossed in the planar surface of the first substrate. Methods
of fabricating and bonding polymeric substrates are described in
commonly owned U.S. patent application Ser. No. 09/073,710, filed
May 6, 1998, and incorporated herein by reference in its entirety
for all purposes.
[0072] Further preferred aspects of the microfluidic devices of the
present invention are more fully described in co-pending U.S.
patent application Ser. No. 09/238,467, as filed on Jan. 28, 1999
(commonly assigned with the present application), the full
disclosure of which is incorporated herein by reference. These
preferred aspects include, for example, a reaction zone disposed
within the overall body structure of the device, a reagent or other
component of an "biochemical system" (generally referring to a
chemical interaction that involves molecules of the type generally
found within living organisms), sensing systems for detecting
and/or quantifying the results of a particular reaction (often by
sensing an optical or other detectable signal of the reaction), and
the like.
[0073] Referring once again to FIG. 2, reservoirs 18 will often be
defined by openings in an overlaying substrate layer. Reservoirs 18
are coupled together by channels 32 of microfluidic network 30,
with the channels generally being defined by indentations in an
underlying layer of the substrate, as was also described above.
[0074] Microfluidic channels 32 are in fluid communication with
each other at channel intersections 34a, 34b, . . . (generally
referred to as intersections 34). To simplify analysis of
microfluidic network 30, channels 32 may be analyzed as channel
segments extending between nodes defined at reservoirs 18 and/or
channel intersections 34.
[0075] To provide enhanced control over movement of fluids within
microfluidic network 30 by reducing the effects of secondary
hydrostatic forces (such as capillary forces within reservoirs 18),
the resistance of channels 32 to flow through the microfluidic
network may be enhanced. These enhanced channel resistances may be
provided by having a channel length greater than the normal
separation between the nodes defining the channel segment, such as
by having serpentine areas 36 along the channel segments.
Alternatively, a cross-sectional dimension of the channel may be
decreased along at least a portion of the channel, or flow may be
blocked by a flow restrictor such as a local orifice, a coating or
material disposed in the channel, or the like. In general, to take
advantage of the full range of flow control provided by the
pressure modulators, microfluidic device 12 should be optimized for
hydrodynamic flow. Flow control is generally enhanced by providing
sufficient flow resistance between each reservoir 18 and the
adjacent nodes so as to allow a sufficient variation in flow rate
to be achieved within the various channel segments given the
dynamic operating pressure range of the pressure modulators.
[0076] Pressure manifold 28 can be seen more clearly in FIGS. 3A
and 3B. Manifold 28 has at least one device engaging surface 40 for
engaging microfluidic device 12, with the engagement surface having
an array of pressure lumens 42 corresponding to reservoirs 18 of
the device. Each of pressure lumens 42 is in fluid communication
with a fitting 44 for coupling each reservoir with an associated
pressure modulator via an associated tube. Sealing body 46 helps
maintain a seal between the associated pressure modulator and
reservoir, and manifold 28 is releasably secured to device 12 by a
securing mechanism 48, which here includes openings for threaded
fasteners, or the like.
[0077] Manifold 28 may comprise a polymer, a metal such as 6061-T6
aluminum, or a wide variety of alternative materials. Lumens 42 may
have a dimension in a range from about 2 mm to about 3 mm. Fittings
44 optionally comprise standard 1/4-28 fittings. Sealing body 46
will often comprise an elastomer such as a natural or synthetic
rubber.
[0078] The pressure transmission system (including manifold 28)
will preferably maintain a seal when transmitting pressures greater
than atmospheric pressure (positive gauge pressures) and less than
atmospheric pressure (negative gauge pressures or vacuum). The
pressure transmission system and modulator bank 14 will generally
be capable of applying pressure differentials which are
significantly higher than hydrostatic and capillary pressures
exerted by, for example, a buffer or other fluid in reservoirs 18,
so as to avoid variability or noise in the pressure differential
and resulting flow rates. As capillary pressures within reservoirs
18 are typically less than 1/10 of a psi, often being less than
1/100th of a psi, the system will preferably be capable of varying
pressure at reservoirs 18 throughout a range of at least 1/2 psi,
more often having a pressure range of at least 1 psi, and most
often having a pressure range of at least +/-1 psig (so as to
provide a 2 psi pressure differential.) Many systems will be
capable of applying at least about a 5 psi pressure differential,
optionally having pressure transmission capabilities so as to apply
pressure anywhere throughout a range of at least about +/-5
psig.
[0079] A control system for selecting the pressures applied to
reservoirs 18 is schematically illustrated in FIG. 4. Controller 22
generally includes circuitry and/or programming which allows the
controller to determine reservoir pressures which will provide a
desired flow within a channel of microfluidic network 30 (here
schematically illustrated as microfluidic network controller 52)
and also includes circuitry and/or programming to direct the
modulators of modulator bank 14 to provide the desired individual
reservoir pressures (here schematically illustrated as a plurality
of pressure controllers 54.) It should be understood that network
controller 52 and pressure controller 54 may be integrated within a
single hardware and/or software system, for example, running on a
single processor board, or that a wide variety of distributing
process techniques might be employed. Similarly, while pressure
controllers 54 are schematically illustrated here as separate
pressure controllers for each modulator, a single pressure
controller might be used with data sampling and/or multiplexing
techniques.
[0080] In general, pressure controller 54 transmits drive signals
to an actuator 56, and the actuator moves a piston of displacement
pump or syringe 58 in response to the drive signals. Movement of
the piston within pump 58 changes a pressure in pressure
transmission system 20, and the change in pressure is sensed by
pressure sensor 24. Pressure sensor 24 provides a feedback signal
to the pressure controller 54, and the pressure controller will
optionally make use of the feedback signal so as to tailor the
drive signals and accurately position the piston.
[0081] To enhance the time response of the pressure control system,
pressure controller 54 may include pressure calibration data 60.
The calibration data will generally indicate a correlation between
drive signals transmitted to actuator 56 and the pressure provided
from the pressure modulator. Pressure calibration data 60 will
preferably be determined by initially calibrating the pressure
change system, ideally before initiation of testing using the
microfluidic network.
[0082] Generation of calibration data 60 may be effected by
transmitting a calibration drive signal to actuator 56 and sensing
the pressure response using pressure sensor 24. The change of
pressure from this calibration test may be stored in the program as
calibration data 60. The calibration signal will typically cause a
known displacement of the piston within pump 58. Using this known
displacement and the measured change in pressure, the overall
pressure system response may be calculated for future drive signals
using the ideal gas law, PV=nRT (in which P is pressure, V is the
total compressible air volume, n is the number of moles of gas in
the volume, R is the gas constant, and T is the temperature).
Calibration may be preformed for each modulator/pressure
transmission systems/reservoir (so as to accommodate varying
reagent quantities within the reservoirs, and the like), or may be
preformed on a single reservoir pressurization system as an
estimate for calibration for all of the modulators of the
system.
[0083] Once calibration data 60 has been generated, pressure
controller 54 can generate drive signals for actuator 56 quite
quickly in response to a desired pressure signal transmitted from
network controller 52. It should be noted that these estimate will
preferably accommodate the changing overall volume of the
compressible gas within the system, so that the calculated change
in pressure for a given displacement of the piston within pump 58
at low pressures may be different than the same displacement of the
piston at high pressures (i.e., the displacement/pressure
correlation plot is not linear, but curves.)
[0084] In the exemplary embodiment, actuator 56 comprises a stepper
motor coupled to a linear output mechanism. Pump 58 comprises a
syringe having a length of about 100 mm, and a diameter of about 20
mm. Overall response time for the system may depend on a variety of
parameters, including dead volume, syringe size, and the like.
Preferably, the response time will be less than about 1 sec/psi of
pressure change, ideally being less than about 500 msecs/psi for a
pressure change from zero to 1 psi.
[0085] Network controller 52 generally calculates the desired
pressure from each pressure modulator in response to a desired flow
in one or more of the channels of microfluidic network 30. Given a
desired channel flow, network controller 52 derives these pressures
using network data 62, with the network data typically being
supplied by either a mathematical model of the microfluidic network
64 and/or a tester 66. Network data 62 will generally indicate a
correlation between pressure differentials applied to reservoirs 18
and flows within the microfluidic channels.
[0086] Network model 64 preferably comprises programming to help
translate desired hydrodynamic flow rates into pressures to be
applied at reservoirs 18. An exemplary network model 64 generates a
hydrodynamic multi-level resistance network correlating to each
microfluidic network 30, as can be understood with reference to
FIGS. 5A-5C.
[0087] Referring now to FIGS. 5A and 2, nodes can be defined at
each well 18 and at each intersection 34. Hydrodynamic resistances
of channel segments coupling the nodes can be calculated from the
chip design. More specifically, calculation of hydrodynamic
resistances may be preformed using hydrostatic pressure loss
calculations based on the cross sectional dimensions of channels
32, the length of channel segments connecting the nodes, the
channel surface properties, the fluid properties of the fluids
included in the flows, and the like.
[0088] Analysis of the multi-level flow resistance network may be
performed using techniques often used for analysis of current in
electrical circuits, as can be understood with reference to FIGS.
5B-5C. Hydrodynamic resistances of the channel segments connecting
reservoirs 18 to adjacent nodes may be analyzed as the lowest level
of a multi-level network. The channel segments adjoining these
lowest level segments form the second level of hydrodynamic
resistances of the network. This level-by-level analysis continues
until all channels of microfluidic network 30 are included in the
network model. The relative flow rate of any channel in the
microfluidic network can then be obtained once the flow rates from
each of the reservoirs 18 in the lowest level have been
calculated.
[0089] As described above, flow resistances maybe calculated based
upon hydrodynamic chip design alone. It is also possible to measure
these resistances using, for example, electrical sensors, pressure
drop sensors, or the like. In other words, resistances to
hydrodynamic flow of the channels and channel segments may be
measured by, for example, measuring electrical resistance between
reservoirs 18 while a conductive fluid is disposed within the
network. Regardless, once the channel resistances are known, the
pressure drop in each channel segment in the network can be
obtained by simply multiplying the flow rate of that channel with
its associated channel resistance. The pressure of each reservoir
18 can then be calculated by summing up all the pressure drops
along the network 30 starting at the top level of the network.
[0090] Referring now to the exemplary program for calculating
pressures illustrated in FIGS. 5B and 5C, hydrodynamic flow rate Q
is related to flow resistance R.sub.e and pressure differential
.DELTA.P by the equation: .DELTA.P=QR.sub.e This relationship is
quite similar to that used in electrokinetic calculations, in which
current I and electrical resistance R are related to voltage V by
the equation: V=IR This simplifies the application of circuit
analysis techniques to the hydrodynamic analysis.
[0091] Determination of reservoir pressures so as to provide a
desired flow rate will preferably be performed using a pressure
calculation program 70, as illustrated in FIG. 5C. Desired flow
rates are input in step 72 from each reservoir 18. These flow rates
may be input by the user, by an automated test matrix generation
program, or the like. Flow resistances are obtained 74 as described
above, and the input flow rate propagates through the network to
obtain flow rates for each branch 76. The pressure drop of each
branch is then determined using the network resistance circuit 78.
These pressure branches are then allowed to propagate through the
network to obtain reservoir pressures 80 so as to effect the
desired flow.
[0092] Referring to FIG. 6, an alternative embodiment of a
microfluidic system makes use of both electrokinetic transport and
hydrodynamic transport mechanisms to move fluids within
microfluidic channels of the system. Electrokinetic transfer of
fluids has significant advantages when electro osmosis and/or
electrophoresis are desired. Electrokinetic fluid transport is also
both fast and convenient, and modifications of the channel surfaces
are possible to avoid and/or eleviate electrokinetic transport
disadvantages. The plug profiles of fluid plugs moved within a
electrokinetic transport system can also be well-controlled and
defined.
[0093] Electrokinetic/hydrodynamic system 90 also provides the
advantages of hydrodynamic transport described above. This
hydrodynamic transport is quite reliable, and is independent of
charges and electrical surface properties of the channels.
Hydrodynamic transport is particularly well-suited for biocompounds
which are sensitive to electrical fields.
[0094] Electrokinetic/hydrodynamic microfluidic system 90 includes
many of the pressurization, microfluidic network and control
components described above. In this embodiment, manifold 92
includes fittings 44 opening laterally from the manifold to provide
sealed fluid communication from each pressure transmission tube 20
to an associated reservoir 18 of the microfluidic device 12.
Additionally, electrodes 94 are coupled to each reservoir 18 via
manifold 92. In the exemplary embodiment, the electrodes comprise
platinum surfaces which extend down from manifold 92 into
electrical contact with fluids disposed within reservoirs 18 when
the manifold provides a sealing engagement between fittings 44 and
the reservoirs. Coupling of the electrodes with the fittings 44 may
be provided by using "T" connectors within the manifold for each
well, and inserting a platinum electrode across and through the
"T". The appropriate (upper, in this example) connector branch of
the T-connector can be sealed and the electrode affixed in place
with a sealing material such as epoxy.
[0095] By coupling electrodes 94 to computer 22, and by including
within computer 22 an electrokinetic fluid transport controller
capable of inducing electro-osmosis and electrophoresis, the system
of FIG. 6 is capable of emulating pumps, valves, dispensers,
reactors, separation systems, and other laboratory fluid handling
mechanisms, often without having to resort to moving parts on
microfluidic device 12. Electrokinetic transportation and control
are described in, for example, U.S. Pat. No. 5,965,001, previously
incorporated herein by reference.
[0096] One particular advantageous use of the pressure modulated
flow control can be understood with reference to FIGS. 7A and 7B.
In many chemical analysis, it is desirable to vary the relative
flow rates from two reservoirs connected to a common node so as to
vary a concentration of a test solution, reagent, or the like,
particularly for defining standard curves of chemical reactions. As
illustrated in FIG. 7A, it is possible to vary the flows from two
reservoirs electrokinetically, with the relative fluid
concentrations being indicated by the changes in fluorescence
intensity over time. Unfortunately, control over the relative flow
rates (and hence, the concentration) may be less than ideal due to
variation in capillary forces within the reservoirs and the
like.
[0097] An alternative well-pair dilution plot in FIG. 7B can be
generated by varying concentrations using multi-pressure control.
This plot illustrates the reduced noise and enhanced flow control
provided by the pressure control systems of the present invention.
As generally described above, hydrodynamic control can be enhanced
by increasing resistance of the channel segments. In the exemplary
microfluidic device 12 illustrated in FIG. 2, channels 32 coupling
wells 18b, 18c, 18d, 18e, 18f, and 18g to the adjacent nodes have a
resistance of 1.3.times.10.sup.11 g/cm.sup.4 s. Channel 32 coupling
reservoir 18a to the adjacent intersection 34 has a resistance of
4.8.times.10.sup.10 g/cm.sup.4 s. Such a chip is well-suited for
use with flows having a pressure drop between reservoirs of about 2
psi, so as to provide a mixing time of about 6 seconds, and a
reaction time of about 20 seconds.
[0098] FIG. 7C is a plot of measured dilution vs. set dilution for
a dilution well-pair with a hydrodynamic flow system, showing the
accuracy and controllability of these dilution methods. FIGS. 7D
and 7E are plots of the measured dilution near the upper and lower
extremes, respectively, showing that a small amount of mixing at a
channel intersection may occur when flow from a channel is at least
substantially halted. As can be understood with reference to these
figures, some modification of the overall flow from one or more
channels at an intersection may be used to effect a desired
dilution percentage adjacent a maximum and/or a minimum of the
dilution range. For example, relative flow adjustments within 5% of
a maximum or minimum desired dilution, and often within 2.5% of a
desired maximum and/or minimum may be employed. More specifically,
to achieve a near 0% actual dilution from a given channel at an
intersection, fluid may flow into the channel at the intersection.
Similarly, to achieve 100% measured dilution from the channel, more
than 100% of the desired flow may be provided from the supply
channel into the intersection.
[0099] Characterization of an enzyme often involves determination
of maximum reaction velocity and a Michaelis constant for each
substrate. The enzymatic reaction of Alkaline Phosphatase on dFMUP
(as illustrated in FIG. 8) was studied on a microfluidic device 12
optimized for pressure driven flow. FIG. 8A is a titration curve
for different concentrations with and without substrate. A plot of
background corrected signal vs. substrate concentration is shown in
FIG. 8B, while a Lineweaver-Burk plot for the Michaelis constant
(Km) is provided in FIG. 8C. Results of a substrate titration assay
for the reaction are shown in FIG. 8D.
[0100] Additional exemplary assay reactions, assay results, and
microfluidic networks to provide those results are illustrated in
FIGS. 9A through 10B. More specifically, FIGS. 9A-C illustrate the
reaction and assay results for a Protein Kinase A (PKA) assay
performed at different ATP concentrations. FIG. 10A illustrates a
chip design having a microfluidic network 130 of microfluidic
channels 32 connecting reservoirs 18, in which the network is
adapted for a mobility shift assay. FIG. 10B are exemplary results
of a mobility shift assay at different concentrations of ATP as may
be measured using the chip design of FIG. 10A.
[0101] Referring now to FIGS. 11A and 11B, an exemplary manifold or
chip interface structure 92' is illustrated in more detail.
Exemplary manifold 92' is adapted to provide both hydrodynamic
coupling and electrokinetic coupling between a microfluidic body
and an associated controller, as described above. Electrical
conduit passages 140 for coupling electrodes 94 to a system
controller 22 (see FIG. 6) are illustrated in FIG. 11A. FIG. 11B
illustrates manifold pressure transmission lumens 142 which provide
fluid communication between fittings 44 and a microfluidic body
interface surface 144 within manifold 92'. Manifold lumens 142 are
illustrated in phantom.
[0102] Accurate control of the flow of fluids within a network of
microfluidic channels can be quite challenging within even a
relatively simple network of channels. More specifically, in many
microfluidic applications, a variety of different fluids (with
different characteristics) may be present in a single channel
segment. As described above, where the hydro-resistance of each
channel segment can be obtained, it may be possible to simulate and
calculate the flow of fluids throughout the network for a given
pressure configuration. Unfortunately, it can be quite difficult to
accurately calculate viscosities (and, hence, resistances and flow
rates) when several different buffers are used within a channel,
often together with one or more different test fluid samples.
[0103] Fortunately, a relatively simple flow sensor can be provided
to measure an actual flow within a channel of a microfluidic
network. Where the measured flow results from a known driving force
(such as a known pressure differential) can be determined,
pressures to be applied at the fluid reservoirs so as to affect a
desired flow condition may then be calculated.
[0104] Referring now to FIG. 12, a relatively simple viscometer 150
makes use of a channel intersection 152 at a first location and a
detector 154 at a second location to measure fluid flow
characteristics. In general, a steady-state flow within a
microfluidic channel 32 between intersection 152 and sensor 154 may
be produced using a pressure differential between reservoirs 18, as
described above. Intersection 152 may impose a signal on the
steady-state flow by applying a pressure pulse to one or more of
the reservoirs 18, by applying an electrokinetic pulse across
intersection 152, or the like. The signal imposed at intersection
152 will often be in the form of a small flow perturbation,
typically for a short duration. For example, where reservoir 18d
includes a detectable dye, the flow perturbation or signal may
comprise an increase or decrease in the dye concentration in the
flow of microfluidic channel 32 from intersection 152 toward
detector 154.
[0105] Detector 154 is downstream from intersection 152, and can be
used to detect the arrival time of the signal, for example, as a
peak or dip in the intensity of a fluorescent signal from the dye.
Thus, the time difference between imposition of the signal at
intersection 152 and sensing of the signal flow at detector 154 may
be readily measured. Calling this time differential .DELTA.t, and
knowing the distance along channel 32 between intersection 152 and
detector 154, .DELTA.d, from the microfluidic network geometry, the
flow rate Q can be calculated from the equation:
Q=A(.DELTA.d/.DELTA.t) in which A is the cross-sectional area of
the channel. This measured flow rate of a steady-state flow for a
given initial driving force greatly facilitates calculation of an
appropriate pressure configuration to achieve a desired flow.
[0106] Where viscosity is to be determined by the system of FIG.
12, reservoirs 18d and 18e coupled to channel 32 by intersection
152 may individually or in combination introduce fluid of known or
unknown viscosity into the microfluidic channel at the intersection
to provide a flow within the channel having an unknown total flow
resistance. With channel 32 optionally containing only a trace
amount of fluorescent dye (to inhibit any effect of the dye on the
unknown overall viscosity), a substantially constant pressure
configuration at ports 18 may drive flow from intersection 152
toward detector 154. This steady-state flow condition may be
effected by a constant vacuum at reservoir 18a adjacent detector
154, positive pressures applied at reservoirs 18d, 18e adjacent
intersection 152, or a combination of both. Regardless, the
steady-state flow with a constant pressure differential will result
in a volumetric flow rate Q in channel 32 which is linearly
proportional to the pressure differential .DELTA.P and inversely
proportional to the fluid viscosity .eta. as follows:
Q=K.DELTA.P/.eta. K is a proportionality constant which depends on
the geometry of the channel network. K can be calculated from the
channel geometry, or can be determined through a calibration
standard test, or the like.
[0107] A variety of alternative structures may be used to sense
flow characteristics so as to apply a proper pressure configuration
to generate a desired flow. For example, a signal may be imposed on
a flow within a microfluidic channel by photobleaching of a
fluorescent dye, rather than imposing a flow perturbation at a
intersection. Alternative flow velocimetry approaches such as laser
Dopler velocimetry, tracer particle videography, and the like are
also possible. Using such techniques, a simple straight channel
connecting a fluid supply reservoir and a waste fluid reservoir may
suffice, with the fluid supply reservoir containing a fluid
comprising a photobleachable fluorescent tracer dye or appropriate
tracer particles.
[0108] As can be understood with reference to the calculations of
flow rate Q above, sensors may also be used to determine
alternative flow characteristics within a microfluidic channel,
including flow rate, viscosity, the proportionality constant for a
segment or network (by use of fluids having known and/or uniform
viscosities) and/or other flow characteristics. In fact, in
addition to providing a tool to study effective viscosity of two or
more mixed fluids (of optionally unequal viscosity) still further
measurements are possible. Mixing of DMSO and an acquiesce buffer
can yield a non-monotonic viscosity-composition relationship. By
applying different levels of pressure differential .DELTA.P and
measuring the flow rate Q, viscometer 150 could be used to
establish a relationship of the effective viscosity during mixing
as a function of mixing length. This information may be pertinent
to chip design for tests which involve geometric dilution.
[0109] Where temperature dependency of viscosity is of interest,
systems such as viscometer 150 can be coupled to a temperature
control system comprising an external heater block in contact with
the body defining the microfluidic channel network, by using joule
heating to selectively control the temperature of fluids within the
channel network, or the like. In a still further alternative, a
structure similar to viscometer 150 might be used to measure
non-Newtonian viscosity. Non-Newtonian fluids have viscosities
which are a function of the sheer rate experienced by the fluid.
One example of a non-Newtonian fluid is a polymer solution
containing high molecular weight molecules. A microfluidic
viscometer similar to viscometer 150 of FIG. 12 might have a
channel geometry and/or channel network intersection structure
and/or flow arranged so that the application of a pressure
differential creates a range of sheer stresses so as to accurately
measure such non-Newtonian viscosity.
[0110] Real-time flow and viscosity measurements for microfluidic
systems based on transient pressure pulse techniques can be further
understood with reference to FIGS. 13A and 13B. A microfluidic
network structure 30 with a single branch channel coupling each
node to a main channel 32' is used. Each branch can be connected to
a single reservoir 18 for a different buffer, sample, enzyme, or
like. In the simplest embodiment, reservoir 18e at the end of the
microfluidic channel network contains a dye solution to provide a
detectable signal.
[0111] A steady flow can be directed toward reservoir 18a by
applying initial pressures on wells 18. A short pressure pulse may
be applied to well 18e and/or some or all of the other reservoirs
of the microfluidic system. This pressure pulse will propagate
substantially instantly to alter flow at some or all of the
intersections 34 of network 30. This disturbance of the flow at the
node points can change the dilution ratio from one or more of the
side branches. After the pressure pulse, steady state flow is
resumed.
[0112] As can be understood with reference to FIG. 13B, a time
series of signals 160a, 160b, and 160c occur at times T.sub.1,
T.sub.2, and T.sub.3, respectively. The flow rate from some or all
of the side branches may then be obtained from the difference of
flow rates between successive node points. Once the flow rates of
the branches have been obtained, as the pressures at reservoirs 18
are known, the resistances of the branch channels may then be
calculated. From the known channel geometry, the viscosity of the
solution in the side branches may also be determined. This
information can then be fed back to the network model to derive the
pressures for a desired flow rate from each reservoir.
[0113] Referring now to FIGS. 14A and 14B, exemplary time signature
data indicates that pressure pulse signals can effectively be
imposed on the flow within a microfluidic system, and can
accurately and repeatedly be sensed by a detector (such as an
optical detector, or the like) for measurement of flow
characteristics.
[0114] Hydrodynamic, electrokinetic, and other fluid transport
mechanisms may be used in a variety of ways to provide specialized
functions within a microfluidic system. For example, fluid mixtures
such as biological fluid samples having particulates and/or cells
in suspension within a liquid are often introduced into
microfluidic systems. A particularly advantageous system and method
for introducing a large number of samples into a microfluidic
system is described in U.S. Pat. Nos. 5,779,868 and 5,942,443, the
full disclosure of which is incorporated herein by reference. In
that system, a vacuum may be used to draw a sequential series of
fluid samples from the wells of a multi-well plate into a capillary
tube in fluid communication with the microfluidic system.
[0115] In the above-described system, it may be desirable to
maintain fluids at a substantially stationary location within the
microfluidic channel, for example, during the time delay while a
sample in a last well of a first multi-well plate is moved away
from the capillary tube and before a sample in a first well of a
second multi-well plate is in fluid communication with the
capillary tube. Maintaining the fluids within the microfluidic
channel at a substantially fixed location can avoid introducing
significant amounts of air into the microfluidic system, which
might interfere with its operation. In general, it may be desirable
to maintain fluid mixtures at a given location within a
microfluidic network for a wide variety of reasons.
[0116] Unfortunately, work in connection with the present invention
has found that halting movement of some fluid mixtures within a
microfluidic network may have significant disadvantages.
Specifically, cell-based assays performed using a fluid mixture
including cells suspended in a liquid are susceptible to sticking
of the cells to the channel walls if flow is completely halted.
Similarly, other fluids may deteriorate if flow within the channel
is sufficiently low for a sufficient amount of time.
[0117] To avoid deterioration of fluid mixtures, the present
invention can provide a small amplitude oscillatory movement of a
fluid mixture so as to maintain the fluid mixture within a
microfluidic channel. Modulator bank 14 is capable of providing a
small amplitude oscillatory pressure such that there is no
significant inflow or outflow of materials from the channel. This
small amplitude oscillatory pressure will preferably be sufficient
to continuously move the fluid mixture (and, for example, the cells
within the liquid) continuously back and forth. The oscillation
frequency should be high enough such that the instantaneous fluid
mixture velocity is sufficiently high to avoid deterioration of the
mixture, while amplitude should be small enough such that there is
little or no unintended net transportation into or out of the
channel from adjacent reservoirs, reservoirs and intersecting
channels. Once the desired delay in fluid mixture movement has been
provided it will often be desirable to flow an intervening liquid
such as a buffer into the channel to help insure that unintended
flows and/or mixtures at the channel ends have been flushed.
[0118] It should be noted that this small amplitude oscillatory
motion may optionally be provided using electrokinetic forces, such
as providing an alternating current, particularly if the
alternating current is not harmful to cells or other components of
the fluid mixture. It may also be beneficial to insure that cells
in the channel do not lyse when subjected to the alternating
current if electrokinetic forces are to be used to induce the
oscillatory motion.
[0119] Referring now to FIG. 15, the systems and methods described
above may optionally take advantage of a wide variety of pressure
transient generators so as to initiate a flow perturbation. A
multiple capillary assembly 170 includes a microfluidic body or
chip 172 mounted a polymer interface housing 174. A plurality of
capillaries 176 contain fluid introduction channels. As explained
in detail in U.S. Pat. No. 6,149,787, the full disclosure of which
is incorporated herein by reference, the capillary channels can be
used to spontaneously inject fluids into the microfluidic network
of chip 172 using capillary forces between the injected fluid and
the capillary channels. Such spontaneous injection is sufficient to
induce a pressure transient for measurement of hydrodynamic and/or
electrokinetic flow. Such flow measurements allow the derivation of
information regarding the properties of the chip, microfluidic
network, and/or fluids.
[0120] The use of multiple capillary assembly 170 is beneficial for
parallel assays using a plurality of test samples, and the like.
Referring now to FIG. 16, a simple chip 178 having a relatively
straightforward microfluidic network may be used to understand the
derivation of flow and/or chip properties from spontaneous
injection. In many embodiments, the open end of capillary 176 will
be placed in a fluid, typically by introducing the end of the
capillary into a microtiter plate (or any other structure
supporting one or more fluid test samples). This may be effected by
moving the capillary 176 and chip 178 relative to the microtiter
plate, by moving the microtiter plate relative to the capillary or
by moving both structures relative to each other. Regardless,
placing capillary 176 into a fluid results in spontaneous
introduction of the fluid into the capillary channel. By applying a
constant vacuum on at least one well of the microfluidic system, a
steady flow may then be provided along a channel coupling the
capillary to the well.
[0121] If, for example, a steady-state flow is induced from
capillary 176, a substrate reservoir 180a, and/or an enzyme
reservoir 180b toward a vacuum reservoir or waste well 180c along a
channel 182, a flow perturbation can be initiated at intersection
186 between the capillary channel and the microfluidic network at
the time the capillary is withdrawn out from the well containing
the introduced fluid. This flow perturbation may, for example,
comprise a change in composition of the flow progressing along
channel 182 toward vacuum reservoir 180c. This change in
composition may be sensed at a detection location 184 as, for
example, a change in fluorescent intensity. Similar flow
perturbations might be induced by applying other pressure
transients at intersection 186, for example, when capillary 176 is
introduced into the spontaneously injected fluid, or by applying a
change in pressure using a pressure modulation pump as described
above, again changing the composition of the flow within channel
182. By monitoring the property of the composition at detection
point 184, progress of the perturbations may be detected. A time
delay between initiation of the perturbation and their respective
detections at the detection point, when combined with a known
length of channel 182, can be used to determine a speed of the flow
within the channel. From this actual, real-time speed, a variety of
information regarding the fluid and/or network system may be
determined.
[0122] Referring now to FIGS. 16A and 16B, each time capillary 176
is dipped into and are removed from a fluid well, a perturbation
will be generated at a capillary intersection 186 coupling the
capillary channel with the microfluidic network. Additionally, as
the pressure perturbation will propagate throughout the
microfluidic network, another flow perturbation may be
simultaneously initiated at a second intersection 186a downstream
of the sipper intersection 186. If we assume that fluid is flowing
from reservoirs affixed to the microfluidic network toward a vacuum
reservoir 180c, the pressure transient applied by spontaneous
injection at capillary 176 will alter the mixtures occurring at
each intersection.
[0123] Where the channel lengths may be designated, and
.DELTA.d.sub.1, .DELTA.d.sub.2 a time delay may be measured at
detector 184 between initiation of the pressure transient (at t=0)
and sensing of a first flow perturbation as a signal 188a at
detector 184. The first signal 188a may be said to have occurred
after a time delay of .DELTA.t.sub.1, with this time being the time
required for flow to propagate from the intersection immediately
upstream of detector 184. A similar time delay .DELTA.t.sub.2 will
then be required for the flow to propagate from the second upstream
intersection (186 in the simple network of FIG. 16A). Where the
channel lengths between intersection are known, the various time
delays can be used to determine the various fluid speeds between
intersections. Where the channel cross-sections are known, this
information can be used to determined contributions from branch
channels to the flow volume, and the like, regardless of whether
the flows throughout the microfluidic system are induced
hydrodynamically, electrokinetically, electroosmotically, or the
like.
[0124] Referring now to FIG. 16C, capillary 176 may be dipped into
and removed from a variety of fluids in a sequential series. P
indicates pressure, S.sub.1 is a signal indicating a flow
perturbation caused at a first intersection by spontaneous
injection into the capillary, and signals S.sub.2 indicates a flow
perturbation signal generated at a second intersection by the same
spontaneous injection at the capillary. A series of pressure
transients 190 will be generated by capillary 176 when the
capillary is, for example, dipped into and removed from a dye,
followed by dipping of the capillary into a buffer solution,
followed dipping of the capillary into a first test substance well,
and the like. This sequence of spontaneous injection events at
capillary 176 may result in generation of a series of S.sub.1
signals due to a series of flow perturbations at, for example,
intersection 186a. Simultaneously, a series of second flow
perturbation signals S.sub.2 will also be generated at intersection
186, with detection of the second series following the first series
by a time delay .DELTA.t.sub.2 which is dependent on the speed of
fluid within the network channels. The total signal S.sub.t
measured at detector 184 will be a combination of this offset
series of signals with the more immediate S.sub.1 signals.
Furthermore, the composition of the overall flow arriving at the
detector may vary significantly with the different materials
introduced by capillary 176. Regardless, by properly identifying
the time delays between signals, flows between the nodes of the
microfluidic system may be calculated.
[0125] Referring now to FIG. 16A, placing a detector 184a
downstream of an electrode v.sub.1 may facilitate measurements of
electrically induced flow, such as electroosmotic flows induced by
a differential voltage between V.sub.1 and V.sub.2. As described
above, pressure perturbations will be initiated at the channel
intersections, so that an initial signal may be generated at the
detector from the downstream electrode V.sub.1, followed by another
signal generated at the upstream electrode V.sub.2. Setting
.DELTA.t.sub.1, as the time delay between these electrode
intersections and .DELTA.t.sub.2, as the time delay for a
subsequent signal generated by a reaction channel at intersection
186, and knowing the lengths of the channels .DELTA.d.sub.1,
.DELTA.d.sub.2 we can calculate the electroosmotic EO flow as
follows:
[0126] With voltage between the electrodes off, using only pressure
to drive fluids within the network, we can determine velocities
along the channels between nodes caused by pressure v.sub.1P,
v.sub.2P from: .DELTA. .times. .times. t 2 .DELTA. .times. .times.
d 2 = v 2 .times. p .times. .times. and .times. .times. .DELTA.
.times. .times. t 1 .DELTA. .times. .times. d 1 = v 1 .times. p
##EQU1## While leaving the same pressure differential on, the
voltage differential may then be turned on, allowing us to
calculate the electroosmotic flow velocity as follows: .DELTA.
.times. .times. t 2 1 .DELTA. .times. .times. d 2 = v 2 .times. p
.times. .times. and .times. .times. .DELTA. .times. .times. t 1 1
.DELTA. .times. .times. d 1 = v 1 .times. p + v eo ; ##EQU2## which
give us v eo = .DELTA. .times. .times. t 1 1 - .DELTA. .times.
.times. t 1 .DELTA. .times. .times. d 2 ##EQU3##
[0127] This electroosmotic velocity may then be used to calculate
electroosmotic mobility using the equation: .mu. eo = v eo E 1 ,
##EQU4## in which E.sub.1 is the electric field strength between
the first and second voltages V.sub.1, V.sub.2. FIG. 19 graphically
illustrates data from a detector or sensor from which the time
delays discussed above may be taken.
[0128] The multiple capillary assembly and simplified capillary
networks of FIGS. 15, 16 and 16A are examples of microfluidic
devices which might benefit from monitoring of pressure induced
flow perturbations for analysis and/or control of flows, quality
control, and the like. Additional examples of microfluidic
structures which may benefit from these techniques are illustrated
in FIGS. 17A, 17B, 18A and 18B.
[0129] Referring now to FIGS. 17A and 17B, more complex
microfluidic networks may include a plurality of capillary joints
or intersections 192 and substrate wells or reservoirs 194, enzyme
wells 196, wastewells 198, and the like. One or more detection or
sensor windows or locations 200 may be provided for monitoring of
propagation of the flow perturbations. The microfluidic assembly
and network of FIGS. 17A and 17B may be useful for multi-capillary
fluorogenic assays. A multi-capillary basic mobility-shift
microfluidic assembly and network having similar structures is
illustrated in FIGS. 18A and 18B. This structure also includes a
plurality of electrode wells 202 for applying voltages to the
microfluidic network, as described above.
[0130] While the exemplary embodiments have been described in some
detail, by way of example and for clarity of example, a variety of
modifications, changes, and adaptation will be obvious to those of
skill in the art. Hence, the scope of the present invention is
limited solely by the appended claims.
* * * * *