Methods and systems for monitoring and controlling fluid flow rates in microfluidic systems

The present invention generally provides methods and systems for monitoring and controlling electroosmotic flow rates in microfluidic systems. Generally, such methods and systems monitor flow rates in electroosmotically driven microfluidic systems by flowing signaling elements within these channels and measuring the flow rate of these signals. The methods of monitoring flow rates are also applied to methods and systems for continuously monitoring and controlling these flow rates in electroosmotically driven microfluidic systems.

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.

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 communication with the channel via an optical detection 
window that is disposed across the channel or chamber of the device. 
Optical detection systems include systems that are capable of measuring 
the light emitted from material within the channel, the transmissivity or 
absorbance of the material, as well as the materials spectral 
characteristics. In preferred aspects, the detector measures an amount of 
light emitted from the material, such as a fluorescent or chemiluminescent 
material. As such, the detection system will typically include collection 
optics for gathering a light based signal transmitted through the 
detection window, and transmitting that signal to an appropriate light 
detector. Microscope objectives of varying power, filed diameter, and 
focal length may be readily utilized as at least a portion of this optical 
train. The light detectors may be photodiodes, avalanche photodiodes, 
photomultiplier tubes, diode arrays, or in some cases, imaging systems, 
such as charged coupled devices (CCDs) and the like. In preferred aspects, 
photodiodes are utilized, at least in part, as the light detectors. The 
detection system is typically coupled to the computer (described in 
greater detail below), via an AD/DA converter, for transmitting detected 
light data to the computer for analysis, storage and data manipulation. 
In the case of fluorescent materials, the detector will typically include a 
light source which produces light at an appropriate wavelength for 
activating the fluorescent material, as well as optics for directing the 
light source through the detection window to the material contained in the 
channel or chamber. The light source may be any number of light sources 
that provides the appropriate wavelength, including lasers, laser diodes 
and LEDs. Other light sources may be required for other detection systems. 
For example, broad band light sources are typically used in light 
scattering/transmissivity detection schemes, and the like. Typically, 
light selection parameters are well known to those of skill in the art. 
The detector may exist as a separate unit, but is preferably integrated 
with the controller system, into a single instrument. Integration of these 
functions into a single unit facilitates connection of these instruments 
with the computer (described below), by permitting the use of few or a 
single communication port(s) for transmitting information between the 
controller, the detector and the computer. As noted above, either or both 
of the controller system and/or the detection system are coupled to an 
appropriately programmed processor or computer which functions to instruct 
the operation of these instruments in accordance with preprogrammed or 
user input instructions, receive data and information from these 
instruments, and interpret, manipulate and report this information to the 
user. As such, the computer is typically appropriately coupled to one or 
both of these instruments (e.g., including an AD/DA converter as needed). 
The computer typically includes appropriate software for receiving user 
instructions, either in the form of user input into a set parameter 
fields, e.g., in a GUI ("graphical user interface"), or in the form of 
preprogrammed instructions, e.g., preprogrammed for a variety of different 
specific operations. The software then converts these instructions to 
appropriate language for instructing the operation of the fluid direction 
and transport controller to carry out the desired operation. The computer 
then receives the data from the one or more sensors/detectors included 
within the system, and interprets the data, either provides it in a user 
understood format, or uses that data to initiate further controller 
instructions, in accordance with the programming, e.g., such as in 
monitoring and control of flow rates, temperatures, applied voltages, and 
the like. 
II. Flow Monitoring and Control 
As noted above, a number of factors can affect the flow rate of materials 
under the above-described electrokinetic material transport systems. For 
example, protein adsorption on channel surfaces can block charged groups 
on the surfaces of channels, which charged groups effect electrokinetic 
fluid movement. As such, in microfluidic systems which involve protein 
transport, such protein adsorption can result in reduced flow rates over 
the time of use of the device. Furthermore, the need for charged surface 
groups in electrokinetic systems generally prevents the use of surface 
treatments to prevent protein adsorption to these surfaces. The problems 
of protein adsorption are compounded in systems wherein protein containing 
fluids are only transported in one or a subset of the interconnected 
channels of the device. Specifically, protein adsorption in a subset of 
all channels of a system can result in variations in the flow rates of 
these channels whereas flow rates in other channels may be unaffected. As 
noted previously, variations in the relative flow rates of interconnected 
channels can result in variations of combination rates of materials from 
different channels, e.g., in performing dilutions, reactant addition and 
the like. 
In addition, factors which affect the level of voltage drop across the 
length of the microscale channels of these microfluidic devices, will also 
affect the efficacy with which electrokinetic systems are able to 
manipulate fluids. Such factors include, e.g., the pH and/or ionic 
concentration of the fluid within the channels. In particular, lower ionic 
strength fluids will have a greater voltage drop per unit channel length 
than higher ionic strength fluids, resulting in a greater flow rate in 
electrokinetic systems. As a result, variations in buffer conditions, 
either from one assay system to another, or within the context of a single 
assay system, can result in a wide variation in electrokinetic flow rates 
in microfluidic channels from device to device, as well as among the 
channels of a single device. 
Furthermore, in many applications or uses of microfluidic systems, the 
ultimate user, rather than the system manufacturer, will supply many of 
the fluids used in the device, e.g., in the form of samples or test 
compounds that are sought to be analyzed within the system. In such cases, 
these fluids may have widely varying compositions, ionic strengths, pH, 
and the like, which will affect flow rates in microfluidic systems. 
The present invention addresses these problems by providing an in situ 
method for monitoring and controlling flow rates within a microfluidic 
system, in order to maintain the flow rates of the system at desired, 
e.g., constant, levels. In particular, the methods described herein employ 
an internal flow standard which permits the real time monitoring of flow 
rates within the channels of a microfluidic device, and in preferred 
aspects, within each separate channel of such devices. Further, the 
systems provided herein provide for the automated monitoring and control 
of those flow rates. 
Generally, the methods and systems described herein, employ a source of a 
detectable signal, e.g., a signaling compound or composition, in fluid 
communication with a microfluidic channel in which the flow rate is to be 
monitored. A small, but detectable amount of the signal is injected into 
the channel in which the flow rate is to be determined, typically 
utilizing one of the electrokinetic injection schemes described above, 
e.g., pinched or gated injection. The amount of time for that signal to 
travel a predefined length of the channel is then determined and the flow 
rate is calculated. 
In some embodiments, the signals used in the methods and systems described 
herein are already provided as an integral part of the operation being 
performed by the device. For example, where a microfluidic device is used 
to monitor the level of binding between a receptor and a ligand, e.g., in 
screening for inhibitors or enhancers of that interaction, one element, 
either the receptor or the ligand, typically has a detectable label 
incorporated into its structure or otherwise associated therewith, i.e., 
fluorescent, chromophoric, chromogenic, chemiluminescent, radioactive, 
etc. As such, a small amount of the labeled component of the particular 
operation or reaction may be separately injected as the signal into the 
microscale channel in which it is desired to determine the flow rate. 
Typically, the amount of such material injected will be only sufficiently 
large to permit detection. Limiting the amount of the labeled component 
that is injected serves to prevent or minimize any changes in flow rate 
which could potentially result from that labeled component. Typically, the 
injection of such material solely for the purpose of determining the flow 
rate within a given channel are maintained at volumes that are less than 
10 nl, preferably, less than 1 nl, optimally less than 100 pl, and often, 
between about 1 pl and about 10 pl. 
In alternative embodiments, a separate source of a signal is provided from 
which a small but detectable amount of signal is injected into the channel 
of interest. Typically, such sources are provided as separate fluid 
reservoirs that are in fluid communication with the channels of interest, 
typically via an appropriate channel. 
As used herein, the term "signal" generally refers to a detectable property 
which can be transported along a microfluidic channel at the same, or at 
some readily calculable proportion of the flow rate of fluid within that 
channel. Typically, such signals comprise a detectable compound or 
composition, but may also comprise properties that are not, per se, a 
property of a particular compound or composition being transported, e.g., 
variations in temperature, optical or electrical properties, not 
necessarily associated with a single composition, e.g., such properties 
may result from the interface or interaction of one compound or 
composition with another. Generally, it is preferred to use signals that 
have little or no net electrophoretic mobility in the relevant pH, e.g., 
are substantially uncharged at the pH of the particular operation being 
performed by the device/system. Such materials are then capable of flowing 
at the flow rate of the bulk electroosmotic fluid flow within the channel. 
This obviates any need for providing a correction factor in the flow rate 
calculation to account for any electrophoretic biasing of the signal 
element during transport. 
Typically, the signals used in accordance with the present invention are 
compounds or compositions which bear a detectable labeling group or 
moiety. In particularly preferred aspects, such detectable labeling groups 
are capable of producing an optically detectable signal. Such compounds 
include, e.g., fluorescent compounds, chemiluminescent compounds, 
chromophoric or chromogenic compounds, and colloidal compounds, e.g., 
compounds having visually detectable particles, e.g., gold, platinum, 
iron, and the like, associated therewith. These signals are readily 
detected using an appropriate optical detector positioned to take optical 
measurements from the channel of interest. 
A number of non-optically detectable signals are optionally employed. Such 
non-optical signals include compositions which have variations in pH, 
ionic strength, or which incorporate radioabels, and the like. For 
example, the signal, e.g., the detectable compound or composition, may 
include a discrete volume of fluid having a varied pH or ionic strength 
over that of the remainder of fluid within the channels. Such a signal is 
readily detected by incorporating a pH or conductivity sensor within the 
channel. In the case of pH or ionic strength signals, as described herein, 
it will be appreciated that amounts of such signals injected into the 
microscale channels under electrokinetic systems must be sufficiently 
small so as to not substantially affect the flow rate that is sought to be 
determined. Typically, this is accomplished by injecting the detectable 
signal using a pinched injection scheme, such that only the amount of 
signal disposed in the channel intersection is injected into the channel, 
and which typically maintains the injected volume below 100 pl. 
Once the signal is injected into the channel of interest, the amount of 
time required for that signal to travel a given distance in the channel is 
determined. In the simplest embodiments, the distance is the distance from 
the injection intersection to the detection window of the device, and the 
time required is the time from the injection to the moment that the 
detector registers the signal. However, in some embodiments, multiple 
detectors are provided at different points in the channel of interest or 
at different points in the microfluidic system, and the flow rate is 
determined from the time required for the signal to travel from one 
detector to the other. In intersecting channel structures, this allows one 
to use a single injected signal plug, routed through multiple channels, to 
determine flow rate in each of those channels, provided that each channel 
includes its own detector or pair of detectors. 
In some cases, the signaling element may be provided that is readily 
distinguishable from the signal produced by the overall operation. For 
example, where the overall operation employs an optical signaling 
mechanism, e.g., fluorescence, a non-optical signaling mechanism is used 
for determination of flow rates, permitting distinction between the 
signals. Alternatively, optically detectable signals may be used in both 
the overall operation and the determination of flow rate, where those 
optical signals are distinguishable, e.g., fluorescent compounds which 
emit light at two different wavelengths, e.g., fluorescein and rhodamine. 
A wide variety of fluorescent dyes having varied fluorescence emission 
spectra are readily available from e.g., Molecular Probes, Inc. This 
latter scheme permits the use of a single detection system for monitoring 
the overall reaction as well as monitoring flow rates. Specifically, an 
optical detector such as a laser activated fluorescence microscope 
employing dual wavelength detection optics, can separately detect 
fluorescent signals at each of the two different wavelengths, e.g., 
through the incorporation of dichroic optics and dual detectors. 
Utilization of a flow rate signal that is distinguishable from the signal 
of the overall operation also permits the monitoring and determination of 
flow rates in a microfluidic system without significantly interrupting the 
performance of the overall operation the device, e.g., assay. This 
provides more accurate and useful "real-time" data regarding flow rates 
within the channels of the device. Further, such systems are readily used 
to inject periodic flow rate signals into the running system. This 
provides constant feedback of the flow rate in the system. In particular, 
regularly injected signaling compounds provide a regular detectable signal 
profile. Deviations in the flow rate of the system will result in 
deviations from this regular signal output, allowing correction by 
adjusting the voltage gradients applied to the system. 
Flow rate monitoring systems are also provided for monitoring flow rates in 
multiple channels within a single device. In such cases, each of the 
separate channels will typically be in fluid communication with a source 
of labeled compound or detectable signal. Each channel will also typically 
include a detector disposed in communication with the channel, e.g., 
optical or sensory communication, at a given distance from the point at 
which the source of signal is in communication with that channel. 
In many cases, the distance traveled is the distance from the point of 
injection of the signal, e.g., the point at which the source of signal 
compound is in fluid communication with the channel of interest, e.g., the 
intersection of the signal channel and the channel of interest, to a 
detection window disposed across the channel of interest. This method 
allows one to practice the present invention in the context of an 
unmodified microfluidic device, e.g., which does not include separate 
detection elements for determining flow rates. Alternatively, two discrete 
detection elements may be provided within a single channel a known 
distance apart. The flow rate is then calculated by determining the amount 
of time required for the signal to traverse that known distance. For more 
complex operations, more complex monitoring systems could be readily 
provided. For example, in complex channel networks, employing large 
numbers of intersecting channels, it is often desirable to determine the 
flow rates in each of several different channels. As such, separate flow 
rate monitoring elements or detectors may be placed in communication, 
e.g., optical or physical communication, with each channel in which flow 
rate information is desired. 
II. Devices and Systems 
As noted above, the methods of monitoring flow rates according to the 
present invention can often be practiced in the context of the operation 
for which a particular device was intended, without the need for modifying 
the device. For example, FIG. 2 shows a typical microfluidic device that 
is utilized in performing a standard competitive diagnostic immunoassay, 
e.g., where the amount of a given antigen in a serum sample is determined 
based upon the ability of the serum to compete with a labeled antigen for 
binding to a supplied amount of antibody. As shown the microfluidic device 
200 includes a body structure 202 having a main channel 204 disposed 
within the body structure. Serum reservoir 206 and a first buffer 
reservoir 208 are shown in fluid communication with one terminus of main 
channel 204 at channel intersection 210, via connecting channels 212 and 
214, respectively. The opposite terminus of main channel 204 is in fluid 
communication with second waste reservoir 216. Labeled antigen and 
antibody are provided in reservoirs 218 and 220, respectively, which are 
in fluid communication with main channel 204 via connecting channels 222 
and 224, respectively. A second buffer reservoir 226 and second waste 
reservoir 228 are provided at opposite termini of gating channel 230, 
which intersects and crosses main channel 204. 
In performing the immunoassay, serum is transported from reservoir 206, to 
main channel 204. This serum is optionally diluted by a concurrent flow of 
buffer from reservoir 208 to main channel 204. At the same time, labeled 
antigen is transported from reservoir 218 to main channel 204 via 
connecting channel 222 where it mixes with the serum. Antibody is then 
transported from reservoir 220 to main channel 204, via channel 224 where 
it mixes with the serum/labeled antigen mixture. The transport of these 
materials from their respective reservoirs to the main channel, is carried 
out by applying an appropriate voltage gradient between reservoirs 206, 
208, 218, 220 and first waste reservoir 228, causing the materials in each 
of these reservoirs to flow toward waste reservoir 228. A gating or 
constraining flow of fluid is provided from buffer reservoir 226 to second 
waste reservoir 216, in order to control flow at the intersection of 
channel 230, and main channel 204. Periodic injections of the 
serum/antibody/antigen mixtures are then made into the remaining portion 
of main channel 204, whereupon the labeled antigen:antibody complex is 
separated from unbound labeled antigen, and detection by a detector 
disposed adjacent to a detection window 232 disposed across main channel 
204 just prior to its turn toward second waste reservoir 216. 
In a first option, the flow rate within the main channel may be determined 
by pumping the labeled antigen into first waste reservoir 228 while a 
constraining or gating flow of buffer is transported from buffer reservoir 
226, to second waste reservoir 216. At time zero, the flow is switched 
such that the labeled antigen is transported toward second waste reservoir 
216. The time required for the label front to reach the detection point is 
then determined and used to calculate the flow rate based upon the channel 
distance between the injection intersection and the detection window. A 
second and preferred option for determining flow rates in the main channel 
is to inject an extremely small but detectable amount of the labeled 
antigen into the main channel, and determine the time required for that 
amount of label to reach the detection window. By injecting only a small 
amount of labeled antigen, any effects of the antigen on the flow rate are 
minimized. Typically, very small quantities of the labeled antigen can be 
readily injected using the microfluidic systems described above. In 
particular, injection schemes are readily achieved for injecting volumes 
that are less than 1 nl, preferably less than 100 pl and often in the 
range of from about 10 to about 50 pl. 
More complex comparative analyses of flow rates are also performed using 
the device and system shown in FIG. 2. For example, a dilution series of 
the labeled marker, e.g., the labeled antigen, is transported along main 
channel past the detection window. The dilution series is carried out by 
transporting successively larger proportions of buffer into the main 
channel from either of buffer reservoirs 208 or 226. In the electrokinetic 
systems, this is accomplished by applying a constant voltage gradient 
between both reservoirs 208 and 218, and second waste reservoir 216, e.g., 
the sum of the voltage gradient between reservoir 218 and waste reservoir 
216 and reservoir 228 and waste reservoir 216 remains constant. During the 
dilution however, the voltage gradient contributed by the buffer or 
diluent and labeled marker reservoirs 208 and 218, respectively, is varied 
proportionally with the dilution steps desired. 
If, based upon the applied voltages, the flow rates in the labeled antigen 
channel and the diluent or buffer channel are equal, then the dilution 
series will provide a linear output of detectable label vs. voltage or 
current applied to the detectable antigen reservoir. However, where these 
flow rates are different based upon the applied voltage gradients, the 
dilution series will provide a curved output of fluorescence as a function 
of voltage gradient applied to transport the labeled antigen. Where the 
detectable signal (antigen) stream has a higher relative flow rate than 
the nondetectable stream (buffer), the output curve for the dilution 
series will have the general shape shown in FIG. 5A. Where the detectable 
stream has a slower relative flow rate than the nondetectable stream, the 
output curve for the dilution series will have the general shape shown in 
FIG. 5B. The curvature of the plot can also be used to readily calculate 
the relative flow rates of fluids coming from each channel which can, in 
turn, be used to control the relative flow rates. 
In alternative, more complex aspects, as described above, additional 
reservoirs can be provided in fluid communication with any of the channels 
of the device, which reservoirs include a detectable signaling compound or 
composition. Small amounts of detectable signal are then injected into 
each channel and detected at a point in the channel that is removed from 
the injection point such that a flow rate can be calculated. 
FIG. 3 illustrates an overall microfluidic system capable of utilizing the 
monitoring and control methods and systems of the present invention. As 
shown, the system 300 includes a microfluidic device, such as microfluidic 
device 200, shown in FIG. 2, an electrical controller 304, a detector 306 
and a computer or other processor unit 308. 
As shown, the microfluidic device includes one or more channels, such as 
channels 204, 212, 214, 222, 224 and 230 (as shown in FIG. 2) having a 
plurality of reservoirs or ports disposed at and in electrical contact 
with the termini of these channels, such as ports 206, 208, 216, 218, 220, 
226 and 228 (as shown in FIG. 2). 
An electrode 310 is placed in electrical contact with each of the ports, 
and is electrical coupled to controller 304 via electrical line 312. 
Appropriate voltages are delivered through lines 312 to the electrodes 
310, in accordance with a flow profile desired for the given operation 
being performed by the system. Detector 306 is disposed adjacent the 
microfluidic device, and particularly disposed adjacent to the detection 
window 232, whereby the detector is capable of sensing a signal within 
that portion of the main channel 204 across which the detection window is 
disposed. 
The computer portion of the system 308 is capable of performing a number of 
functions in the context of the overall microfluidic system, generally, 
and specifically with respect to the monitoring and control methods 
described herein. Specifically, the computer typically includes 
appropriate programming for instructing the application of voltages to the 
channel termini by the voltage controller 304, in order to carry out a 
desired fluid transport profile, which is either input by the user, or is 
contained in a separate program. Additionally, the computer receives data 
transmitted from the detector, and is typically appropriately programmed 
to store this data, as well as manipulate the data to provide an output 
that is readily comprehended by the user. In accordance with the present 
invention, the computer typically includes appropriate programming for 
monitoring and controlling flow rates within the microfluidic device. Such 
programming is optionally embodied in software stored in an appropriate 
memory device, such as a compact disk read only memory ("CDROMs"), hard 
disks, floppy disks, high capacity disks (e.g., ZipDrive.TM. available 
from Iomega), field programmable gate arrays ("FPGAs") electrically 
erasable programmable read only memories ("EEPROMs"), read only memories 
("ROMs"), random access memories ("RAMs"), and the like. 
In particular, the computer instructs the voltage controller to apply an 
appropriate voltage gradient across the length of the main channel, as 
discussed above, with reference to FIG. 2, to provide fluid flow in that 
channel. The computer then instructs the voltage controller to apply 
appropriate voltages to the ports to inject a small volume of the 
signaling compound into the main channel at time 0. The computer then 
receives the data from the detector indicating that the signal has 
traversed the detection window. From these data, the computer then 
calculates the actual flow rate of fluid within main channel 204, and 
compares this flow rate to an expected or desired flow rate, e.g., a set 
point as input by the user, or as set in an initial flow rate 
determination step. If the computer determines that the actual flow rate 
is less than the expected or desired flow rate, the computer increases the 
voltage gradient applied across the main channel and retests the flow 
rate. If, however, the actual flow rate is greater than the desired flow 
rate, the computer decreases the voltage gradient applied across the main 
channel. The size of the increase or decrease may be a small incremental 
increase or decrease, or may be an increase or decrease calculated from 
the size of the original voltage gradient and the actual flow rate. 
An example of process steps dictated by appropriate software programming 
are shown in FIG. 4. Briefly, the process is initiated at step 400. The 
computer instructs the voltage controller at step 402, to commence fluid 
flow in the channel in which a flow rate determination is desired, by 
applying a first voltage gradient across the length of the channel. At 
step 404, the computer instructs the system to inject a small amount of 
signal into the main channel by appropriate modulation of the applied 
voltages at the various ports of the device, as described previously. The 
signal is then detected as it passes the detection point at step 405, 
e.g., typically by a sensor device, such as an optical or potentiometric 
detector, which is operaably linked to the computer. The time required 
(t.sub.actual) for the signal to travel from the injection point to the 
detection point or window (the distance "d") is then determined at step 
406. At step 408, this time is then compared with the expected time for 
the signal to travel distance "d" (t.sub.expected). In alternative 
aspects, the computer optionally compares any of a variety of parameters, 
e.g., calculated flow rates, signal strengths, signal periodicity, etc., 
which can be used as measures of flow rate. 
Where t.sub.expected is less than t.sub.actual the computer proceeds to 
step 410, whereupon the computer instructs the voltage controller to 
decrease the voltage gradient applied across the main channel, to decrease 
the actual flow rate. If however, t.sub.expected is greater than 
t.sub.actual, then the computer proceeds to step 412, whereupon it 
instructs the computer to increase the voltage gradient applied across the 
length of the main channel. After performing one of steps 410 or 412, or 
in the case where the actual and expected times or flow rates are equal, 
the computer then repeats steps 404 through 414, to continuously monitor 
the flow rate, or verify the changes made to the voltage gradient/flow 
rates. 
Although the present invention has been described in some detail by way of 
illustration and example for purposes of clarity and understanding, it 
will be apparent that certain changes and modifications may be practiced 
within the scope of the appended claims. All publications, patents and 
patent applications referenced herein are hereby incorporated by reference 
in their entirety for all purposes as if each such publication, patent or 
patent application had been individually indicated to be incorporated by 
reference.