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BACKGROUND OF THE INVENTION
Microfluidic systems have been gaining increasing interest for use in chemical and biochemical analysis and synthesis. Miniaturization of a variety of laboratory analyses provides myriad benefits, including providing substantial savings in time
of analysis, cost of analysis, and space requirements for the equipment which performs this analysis. Another touted advantage of microfluidic systems is their suggested adaptability as automated systems, thereby providing additional savings associated
with the costs of the human factor of performing analyses, e.g., labor costs, costs associated with operator error, and generalized costs associated with the imperfection of human operations, generally.
A number of different microfluidic technologies have been proposed for realizing the potential of these systems. For example, microfluidic systems have been proposed that are based upon microscale channels or conduits through which fluid is
transported by internal or external pressure sources, e.g., pressure pumps, and wherein fluid direction, e.g., as between two potential fluid paths, is carried out using microfabricated mechanical valve structures. Other unrealized technologies have
proposed utilizing acoustic energy, or electrohydrodynamic pumping of fluids to effect fluid movement. However, due to fundamental problems with these technologies, e.g., excessive costs or inoperability, they have largely floundered in the research
institutions where they were originally conceived.
Electrokinetic material transport systems have shown the ability to fulfill the promise of microfluidics by providing an accurate, automatable, easily manufacturable system for manipulating fluids within microscale systems. Despite the advances
of electrokinetic flow systems, it would generally be desirable to provide more and more complex systems for performing a wide variety of different fluidic operations, integrating multiple operations in a single microfluidic system, as well as provide
systems capable of performing massively parallel experimentation. In order to provide such systems, it would generally be desirable to provide such systems with advanced abilities to monitor and control the relevant parameters of any and all fluidic
elements within a given system, including variables such as temperature, time of reaction, length of separations, and the like. The present invention provides methods and systems that meet these and other needs by providing an operator with greater
ability to monitor and control microfluidic systems.
SUMMARY OF THE INVENTION
The present invention is generally directed to methods and systems utilized in monitoring and controlling flow rates within microfluidic channel systems. As such, in a first aspect, the present invention provides a method of monitoring an
electroosmotic flow rate of fluid in a microfluidic device having at least first and second intersecting microscale-channels disposed therein. The method comprises flowing a fluid along the first channel by applying a voltage gradient across a length of
the first channel. A detectable amount of a signaling compound is then injected into the first channel. The flow rate of fluid in the first channel is then determined from the rate at which the signaling compound flows from a first point in the first
channel to a second point in the first channel. This is repeated in a second channel. Specifically, a fluid is also flowed along the second channel by applying a voltage gradient across a length of the second channel, a detectable amount of a signaling
compound is injected into the second channel, and the flow rate of fluid in the second channel is determined from the rate at which the signaling compound flows from a first point in the second channel to a second point in the second channel.
In an alternate embodiment, the present invention provides a microfluidic system employing at least first and second intersecting microscale channels disposed in a body structure, wherein the system is used for analyzing a result of a chemical
reaction which produces a first detectable signal. In particular, the present invention provides a method of monitoring a flow rate of a fluid in the first channel, which comprises flowing a fluid in the first channel and injecting into the first
channel, a detectable amount of a signaling compound. In this aspect, the signaling compound produces a second detectable signal that is capable of being distinguished from the first detectable signal. The second detectable signal is then detected and
distinguished from the first detectable signal. The flow rate of fluid in the main channel is then calculated from the amount of time between the injecting step and the detecting step.
In still another aspect, the present invention provides methods of continuously monitoring electroosmotic flow rate of a fluid in a microscale channel of a microfluidic device having at least first and second intersecting microscale channels
disposed therein. The method comprises electroosmotically flowing the fluid along the first channel by applying a voltage gradient across the length of the first channel. A detectable amount of a signaling compound is periodically injected into the
first channel at a first point. The periodic signal from the signaling compound is then detected at a second point in the first channel, the second point being removed from the first point. Variation in flow rate is then identified from a variation in
the periodic signal detected in the detecting step.
In an additional aspect, the present invention provides a microfluidic device for use in accordance with the monitoring methods described herein. In particular, the device comprises a body structure having at least first, second and third
channels disposed therein. The first channel comprises first and second reservoirs in fluid communication with its first and second termini. The first reservoir has the fluid deposited therein. The second channel intersects the first channel at a
first terminus of the second channel, and has a third reservoir in fluid communication with a second terminus of the second channel. The third reservoir has a signaling compound disposed therein, which signaling compound is capable of producing a
detectable signal. The third channel intersects the first channel at a first terminus of the third channel and has a fourth reservoir in fluid communication with a second terminus of the third channel. The device also comprises a detection window
disposed across at least one of the first and second microscale channels, wherein the detection window is capable of transmitting the detectable signal therethrough.
The monitoring methods described herein are also useful in methods of controlling the electroosmotic flow rate of a fluid in a microfluidic device having at least a first microscale channel disposed therein. In particular, an electroosmotic flow
rate is controlled by a method which comprises flowing the fluid along the first channel by applying a voltage gradient across a length of the first channel. A detectable amount of a signaling compound is injected into the first channel at a first point
in the first channel. The actual flow rate of fluid is then determined from the rate at which the signaling compound flows along the first channel. The actual flow rate is then compared to a desired flow rate. The voltage gradient applied across the
length of the first channel is then increased or decreased until the actual flow rate is approximately equal to the desired flow rate.
In a related aspect, the present invention provides a system for controlling an electroosmotic flow rate of a fluid in a microfluidic system. The system comprises a microfluidic device comprising at least first, second and third channels
disposed therein, the first channel having first and second reservoirs in fluid communication with its first and second termini, the first reservoir having the fluid deposited therein, the second channel intersecting the first channel at a first terminus
of the second channel, and having a third reservoir in fluid communication with a second terminus of the second channel the third reservoir having a signaling compound disposed therein, the third channel intersecting the first channel at a first terminus
of the third channel, and having a fourth reservoir in fluid communication with a second terminus of the third channel. The system also comprises an electrical controller for concomitantly applying and modulating voltages at at least three of the first,
second, third and fourth reservoirs, to flow a fluid in the first channel from the first reservoir to the second reservoir, and periodically injecting a detectable amount of the signaling compound into the first channel from the third reservoir. The
system further includes a detector disposed adjacent to and in sensory communication with a point in the first channel, whereby the detector is capable of detecting the signaling compound at the first point in the first channel. In addition, the system
comprises an appropriately programmed computer for receiving signal data from the detector, calculating the actual flow rate of the fluid in the channel from the signal data, comparing the actual flow rate, and instructing the electrical controller to
increase or decrease the voltage gradient across the channel based upon a difference between the actual flow rate and the desired flow rate.
In still another aspect, the present invention provides a computer or processor for use in accordance with the monitoring and controlling methods and systems described herein. The computer or processor comprises appropriate programming for
determining an actual electroosmotic flow rate of a fluid in a first microscale channel. The computer then compares the actual electroosmotic flow rate to a desired electroosmotic flow rate in the first microscale channel, and increases or decreases the
voltage gradient applied across the first microscale channel depending upon the comparison of the actual electroosmotic flow rate to the desired electroosmotic flow rate, until the actual electroosmotic flow rate is approximately equal to the desired
electoosmotic flow rate.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the fabrication of a typical microfluidic device incorporating a multilayer fabrication strategy.
FIG. 2 illustrates a microfluidic device useful in practicing the methods of the present invention.
FIG. 3 illustrates an overall view of a control and monitoring system in accordance the present invention.
FIG. 4 is a flow chart illustrating process steps carried out by the control system and control software, in accordance with the present invention.
FIGS. 5A and 5B show plots of fluorescence vs. voltage or current applied to a fluorescent signal reservoir during a dilution series, for isentifying variations in flow rates.
DETAILED DESCRIPTION OF THE INVENTION
I. General
Microfluidic systems have been described for use in the performance of a large number of useful operations. Of increasing interest is the use of such systems in the performance of wide varieties of chemical and biochemical reactions, including
analytical and synthetic reactions.
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 5 .mu.m and 20 .mu.m. Accordingly, the microfluidic devices or systems prepared in accordance with the present invention typically include at least one microscale channel, usually at least two intersecting microscale channels,
and often, three or more intersecting channels 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.
The body structure of the microfluidic devices described herein typically comprises an aggregation of two or more separate layers which when appropriately mated or joined together, form the microfluidic device of the invention, e.g., containing
the channels and/or chambers described herein. Typically, the microfluidic devices described herein will comprise a top portion, a bottom portion, and an interior portion, wherein the interior portion substantially defines the channels and chambers of
the device.
FIG. 1 illustrates a two layer body structure 10, for a microfluidic device. In preferred aspects, the bottom portion of the device 12 comprises a solid substrate that is substantially planar in structure, and which has at least one
substantially flat upper surface 14. A variety of substrate materials may be employed as the bottom portion. Typically, because the devices are microfabricated, substrate materials will be selected based upon their compatibility with known
microfabrication techniques, e.g., photolithography, wet chemical etching, laser ablation, air abrasion techniques, injection molding, embossing, 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. 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.
In additional 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, 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. patent application Ser. No. 08/843,212, filed Apr. 14, 1997, and which is incorporated herein by reference in its entirety for all purposes.
The channels and/or chambers of the microfluidic devices are typically fabricated into the upper surface of the bottom substrate or portion 12, as microscale grooves or indentations 16, using the above described microfabrication techniques. The
top portion or substrate 18 also comprises a first planar surface 20, and a second surface 22 opposite the first planar surface 20. In the microfluidic devices prepared in accordance with the methods described herein, the top portion also includes a
plurality of apertures, holes or ports 24 disposed therethrough, e.g., from the first planar surface 20 to the second surface 22 opposite the first planar surface.
The first planar surface 20 of the top substrate 18 is then mated, e.g., placed into contact with, and bonded to the planar surface 14 of the bottom substrate 12, covering and sealing the grooves and/or indentations 16 in the surface of the
bottom substrate, to form the channels and/or chambers (i.e., the interior portion) of the device at the interface of these two components. The holes 24 in the top portion of the device are oriented such that they are in communication with at least one
of the channels and/or chambers formed in the interior portion of the device from the grooves or indentations in the bottom substrate. In the completed device, these holes function as reservoirs for facilitating fluid or material introduction into the
channels or chambers of the interior portion of the device, as well as providing ports at which electrodes may be placed into contact with fluids within the device, allowing application of electric fields along the channels of the device to control and
direct fluid transport within the device.
These devices may be used in a variety of applications, including, e.g., the performance of high throughput screening assays in drug discovery, immunoassays, diagnostics, genetic analysis, and the like. As such, the devices described herein,
will often include multiple sample introduction ports or reservoirs, for the parallel or serial introduction and analysis of multiple samples. Alternatively, these devices may be coupled to a sample introduction port, e.g., a pipettor, which serially
introduces multiple samples into the device for analysis. Examples f such sample introduction systems are described in e.g., U.S. patent application Ser. Nos. 08/761,575, 08/760,446 (Attorney Docket Nos. 100/00310 and 100/00210) each of which was
filed on Jun. 28, 1996, and is hereby incorporated by reference in its entirety for all purposes.
Microfluidic systems have been employed in the separation of biological macromolecules, in the performance of assays, e.g., enzyme assays, immunoassays, receptor binding assays, and other assays in screening for affectors of biochemical systems.
Generally, such systems employ microscale channels and/or chambers through which various reactants are transported, where they may be mixed with additional reactants, subjected to changes in temperature, pH, ionic concentration, etc., separated into
constituent elements and/or detected.
The result of the performance of these functions is often greatly affected by the rate at which the reactants are transported within the microscale channels of these microfluidic devices. In particular, the rate at which materials flow within
these systems directly affects a number of parameters upon which the outcome of the reaction depends, at least in part. For example, where two reactants are being transported from separate channels into a common channel or chamber for reaction and
subsequent detection, the flow rate of two reactants into the common channel affects the concentration of each reagent. Further, the rate at which the mixed reactants are transported to the detection region of the device affects the amount of time the
mixed reagents are allowed to react, thereby directly affecting the amount of reaction product.
In microfluidic systems that employ pressure driven systems, e.g., external pressure sources, integrated micropumps and the like, the flow rate of fluids within a given channel is directly related to the viscosity of the fluid, the amount of
pressure applied to the system, and the dimensions of the channel. While these parameters remains constant, the flow rate will also remain constant. As such, in these pressure driven systems, flow rates can be easily and accurately determined, either
experimentally, or based upon well known physical principles.
In electrokinetically driven microfluidic systems, e.g., systems employing electrokinetic material transport systems, however, a number of additional factors can affect the flow rate of fluids within the channels of the device. As with pressure
driven systems, where all of these factors can be maintained as a constant, flow rate will also remain constant. Unfortunately, however, in a large number of applications for which it is desired to use these microfluidic systems, maintaining all of
these factors constant is not reasonably practicable. As such, it is highly desirable to be able to monitor and control flow rates in microfluidic systems employing these electrokinetic material transport systems.
As used herein, "electrokinetic material transport systems" include systems which transport and direct materials within an interconnected channel and/or chamber containing structure, through the application of electrical fields to the materials,
thereby causing material movement through and among the channel and/or chambers, i.e., cations will move toward the negative electrode, while anions will move toward the positive electrode.
Such electrokinetic material transport and direction systems include those systems that rely upon the electrophoretic mobility of charged species within the electric field applied to the structure. Such systems are more particularly referred to
as electrophoretic material transport systems. Other electrokinetic material direction and transport systems rely upon the electroosmotic flow of fluid and material within a channel or chamber structure which results from the application of an electric
field across such structures. In brief, when a fluid is placed into a channel which has a surface bearing charged functional groups, e.g., hydroxyl groups in etched glass channels or glass microcapillaries, those groups can ionize. In the case of
hydroxyl functional groups, this ionization, e.g., at neutral pH, results in the release of protons from the surface and into the fluid, creating a concentration of protons at near the fluid/surface interface, or a positively charged sheath surrounding
the bulk fluid in the channel. Application of a voltage gradient across the length of the channel, will cause the proton sheath to move in the direction of the voltage drop, i.e., toward the negative electrode.
"Controlled electrokinetic material transport and direction," as used herein, refers to electrokinetic systems as described above, which employ active control of the voltages applied at multiple, i.e., more than two, electrodes. Rephrased, such
controlled electrokinetic systems concomitantly regulate voltage gradients applied across at least two intersecting channels. Controlled electrokinetic material transport is described in Published PCT Application No. WO 96/04547, to Ramsey, which is
incorporated herein by reference in its entirety for all purposes. In particular, the preferred microfluidic devices and systems described herein, include a body structure which includes at least two intersecting channels or fluid conduits, e.g.,
interconnected, enclosed chambers, which channels include at least three unintersected termini. The intersection of two channels refers to a point at which two or more channels are in fluid communication with each other, and encompasses "T"
intersections, cross intersections, "wagon wheel" intersections of multiple channels, or any other channel geometry where two or more channels are in such fluid communication. An unintersected terminus of a channel is a point at which a channel
terminates not as a result of that channel's intersection with another channel, e.g., a "T" intersection. In preferred aspects, the devices will include at least three intersecting channels having at least four unintersected termini. In a basic cross
channel structure, where a single horizontal channel is intersected and crossed by a single vertical channel, controlled electrokinetic material transport operates to controllably direct material flow through the intersection, by providing constraining
flows from the other channels at the intersection. For example, assuming one was desirous of transporting a first material through the horizontal channel, e.g., from left to right, across the intersection with the vertical channel. Simple
electrokinetic material flow of this material across the intersection could be accomplished by applying a voltage gradient across the length of the horizontal channel, i.e., applying a first voltage to the left terminus of this channel, and a second,
lower voltage to the right terminus of this channel, or by allowing the right terminus to float (applying no voltage). However, this type of material flow through the intersection would result in a substantial amount of diffusion at the intersection,
resulting from both the natural diffusive properties of the material being transported in the medium used, as well as convective effects at the intersection.
In controlled electrokinetic material transport, the material being transported across the intersection is constrained by low level flow from the side channels, e.g., the top and bottom channels. This is accomplished by applying a slight voltage
gradient along the path of material flow, e.g., from the top or bottom termini of the vertical channel, toward the right terminus. The result is a "pinching" of the material flow at the intersection, which prevents the diffusion of the material into the
vertical channel. The pinched volume of material at the intersection may then be injected into the vertical channel by applying a voltage gradient across the length of the vertical channel, i.e., from the top terminus to the bottom terminus. In order
to avoid any bleeding over of material from the horizontal channel during this injection, a low level of flow is directed back into the side channels, resulting in a "pull back" of the material from the intersection.
In addition to pinched injection schemes, controlled electrokinetic material transport is readily utilized to create virtual valves which include no mechanical or moving parts. Specifically, with reference to the cross intersection described
above, flow of material from one channel segment to another, e.g., the left arm to the right arm of the horizontal channel, can be efficiently regulated, stopped and reinitiated, by a controlled flow from the vertical channel, e.g., from the bottom arm
to the top arm of the vertical channel. Specifically, in the `off` mode, the material is transported from the left arm, through the intersection and into the top arm by applying a voltage gradient across the left and top termini. A constraining flow is
directed from the bottom arm to the top arm by applying a similar voltage gradient along this path (from the bottom terminus to the top terminus). Metered amounts of material are then dispensed from the left arm into the right arm of the horizontal
channel by switching the applied voltage gradient from left to top, to left to right. The amount of time and the voltage gradient applied dictates the amount of material that will be dispensed in this manner.
Although described for the purposes of illustration with respect to a four way, cross intersection, these controlled electrokinetic material transport systems can be readily adapted for more complex interconnected channel networks, e.g., arrays
of interconnected parallel channels.
Although the devices and systems specifically illustrated herein are generally described in terms of the performance of a few or one particular operation, it will be readily appreciated from this disclosure that the flexibility of these systems
permits easy integration of additional operations into these devices. For example, the devices and systems described will optionally include structures, reagents and systems for performing virtually any number of operations both upstream and downstream
from the operations specifically described herein. Such upstream operations include sample handling and preparation operations, e.g., cell separation, extraction, purification, amplification, cellular activation, labeling reactions, dilution,
aliquoting, and the like. Similarly, downstream operations may include similar operations, including, e.g., separation of sample components, labeling of components, assays and detection operations. Assay and detection operations include without
limitation, probe interrogation assays, e.g., nucleic acid hybridization assays utilizing individual probes, free or tethered within the channels or chambers of the device and/or probe arrays having large numbers of different, discretely positioned
probes, receptor/ligand assays, immunoassays, and the like.
The systems described herein generally include microfluidic devices, as described above, in conjunction with additional instrumentation for controlling fluid transport and direction within the devices, detection instrumentation for detecting or
sensing results of the operations performed by the system, processors, e.g., computers, for instructing the controlling instrumentation in accordance with preprogrammed instructions, receiving data from the detection instrumentation, and for analyzing,
storing and interpreting the data, and providing the data and interpretations in a readily accessible reporting format. A variety of controlling instrumentation may be utilized in conjunction with the microfluidic devices described above, for
controlling the transport and direction of fluids and/or materials within the devices of the present invention. As noted above, the systems described herein preferably utilize electrokinetic material direction and transport systems. As such, the
controller systems for use in conjunction with the microfluidic devices typically include an electrical power supply and circuitry for concurrently delivering appropriate voltages to a plurality of electrodes that are placed in electrical contact with
the fluids contained within the microfluidic devices. Examples of particularly preferred electrical controllers include those described in, e.g., International Patent Application No. PCT/US 97/12930, the disclosures of which are hereby incorporated
herein by reference in their entirety for all purposes. In brief, the controller uses electric current control in the microfluidic system. The electrical current flow at a given electrode is directly related to the ionic flow along the channel(s)
connecting the reservoir in which the electrode is placed. This is in contrast to the requirement of determining voltages at various nodes along the channel in a voltage control system. Thus the voltages at the electrodes of the microfluidic system are
set responsive to the electric currents flowing through the various electrodes of the system. This current control is less susceptible to dimensional variations in the process of creating the microfluidic system in the device itself. Current control
permits far easier operations for pumping, valving, dispensing, mixing and concentrating subject materials and buffer fluids in a complex microfluidic system. Current control is also preferred for moderating undesired temperature effects within the
channels.
In the microfluidic systems described herein, a variety of detection methods and systems may be employed, depending upon the specific operation that is being performed by the system. Often, a microfluidic system will employ multiple different
detection systems for monitoring the output of the system. Examples of detection systems include optical sensors, temperature sensors, pressure sensors, pH sensors, conductivity sensors, and the like. Each of these types of sensors is readily
incorporated into the microfluidic systems described herein. In these systems, such detectors are placed either within or adjacent to the microfluidic device or one or more channels, chambers or conduits of the device, such that the detector is within
sensory communication with the device, channel, or chamber. The phrase "within sensory communication" of a particular region or element, as used herein, generally refers to the placement of the detector in a position such that the detector is capable of
detecting the property of the microfluidic device, a portion of the microfluidic device, or the contents of a portion of the microfluidic device, for which that detector was intended. For example, a pH sensor placed in sensory communication with a
microscale channel is capable of determining the pH of a fluid disposed in that channel. Similarly, a temperature sensor placed in sensory communication with the body of a microfluidic device is capable of determining the temperature of the device
itself.
Particularly preferred detection systems include optical detection systems for detecting an optical property of a material within the channels and/or chambers of the microfluidic devices that are incorporated into the microfluidic systems
described herein. Such optical detection systems are typically placed adjacent a microscale channel of a microfluidic device, and are in sensory com | | |
