EP1208370A4

EP1208370A4 - Dilutions in high throughput systems with a single vacuum source

Info

Publication number: EP1208370A4
Application number: EP00957753A
Authority: EP
Inventors: Anne R Kopf-Sill; Steven A Sundberg; Andrea W Chow; Claudia L Poglitsch
Original assignee: Caliper Life Sciences Inc
Current assignee: Caliper Life Sciences Inc
Priority date: 1999-08-25
Filing date: 2000-08-23
Publication date: 2006-05-31
Also published as: JP4119649B2; AU6932300A; EP1208370A1; CA2380897A1; JP2003507737A; WO2001014865A1

Abstract

Flow rates in a microfluidic device (100) with channels (104) are modulated after performing serial dilutions by flow reduction channels (106, 110).

Description

Dilutions in High Throughput Systems with a Single Vacuum Source

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C § 119(e) and any other applicable statute or rule, the present application claims benefit of and pπoπty to USSN 60/150,670, entitled "Dilutions in High Throughput Systems with a Single Vacuum Source," filed August 25, 1999 by Kopf-Sill et al ; USSN 60/159,014, entitled "Dilutions m High Throughput Systems with a Single Vacuum Source," filed October 12, 1999 by Kopf-Sill et al , and USSN 60/200.139, entitled "Dilutions in High Throughput Systems with a Single Vacuum Source," filed Apπl 27. 2000 by Kopf-Sill et al

BACKGROUND OF THE INVENTION When carrying out chemical or biochemical analyses, assays, syntheses, or preparations one performs a large number of separate manipulations on the mateπal or component to be assayed, including measuπng, a quottmg, transferring, diluting, mixing, separating, detecting, etc. Microfluidic technology miniatuπzes these manipulations and integrates them so that they can be performed within one or a few microfluidic devices. For example, methods of performing dilutions in microfluidic devices were descπbed in U.S. Patent No: 5,869,004, by Parce and Kopf-Sill. "Methods and Apparatus for in Situ Concentration and or Dilution of Mateπals in Microfluidic Systems." These methods successively draw off and add mateπals to microfluidic channels to seπally dilute mateπals. The methods allow large accurate dilutions to be performed within the microscale environment.

For some bioassays, a constant flow of mateπal is useful to maintain a fixed assay reaction time. Therefore, the ability to modulate a flow rate and obtain constant incubation and reaction times in a microfluidic system when performing dilutions would be useful to the integration of fluidic sample and reagent manipulations in a microfluidic assay format. In addition, a constant flow rate, e.g., in a microfluidic device with a single pressure source, would help to reduce the reagent usage for reagents added after the dilutions have been made. Another technique that would be useful to integrate into a microfluidic format would be the ability to measure a sample at different concentrations simultaneously, to concurrently perform reactions at varying concentrations, and/or to test one sample concurrently versus a panel of different reagents Improved methods for controlling flow rates dunng dilutions and multiple concentration assays are, accordingly desirable, particularly those which take advantage of high-throughput, low cost microfluidic systems The present invention provides these and other features by providing high throughput microscale systems for dilutions, reduced reagent consumption, multiple concentration measurements, and many other features that will be apparent upon complete review of the following disclosure

SUMMARY OF THE INVENTION

The present invention provides devices and methods for modulating flow rates and reducing reagent consumption in microfluidic devices In one aspect, the invention provides a device which includes flow reduction channels that are structurally configured to draw fluid from a mam channel, thereby reducing the flow rate in the mam channel

In addition to providing flow rate modulation, these channels also provide for reduced reagent consumption by reducing the flow rate of the mateπals at the point of sample mateπal introduction Additional reduction in reagent consumption is optionally obtained by providing flow reduction channels of a smaller cross-sectional dimension than the main channel. Secondary flow reduction channels are also optionally added for additional reduction of reagent consumption

In another aspect, the flow reduction channels provide a method of performing seπal dilutions on a sample and obtaining measurements corresponding to each dilution level. The flow reduction channels provide a way to perform seπal dilutions of a sample without increasing the flow rate with each dilution Alternatively, one can change the cross-sectional area of the channel downstream of a mixing point. With a detection region provided m each flow reduction channel, measurements are optionally obtained at each dilution level.

In one aspect, the microfluidic devices of the invention compπse a body structure and a mam pressure source. The body structure includes a ma channel disposed therein, wherein the main channel is fluidly coupled to the main pressure source. The body structure also includes one or more flow reduction channels that intersect the main channel at one or more intersection points The flow reduction channels are structurally configured to reduce fluid pressure or velocity in the ma channel as the fluid flows past the one or more intersection poιnt(s) In addition, the body structure can include a first pressure source fluidly coupled to the one or more flow reduction channels. The first pressure source is positioned on the one or more flow reduction channels downstream of the one or more intersection points in a direction of flow toward the first pressure source. In a preferred embodiment, the first pressure source and the main pressure source are the same, typically a single vacuum source fluidly coupled to the main channel at a position downstream from the flow reduction channel(s)

In another aspect, the microfluidic device compπses a body structure and a • mam pressure source, which is typically a single vacuum source. The body structure compπses a main channel disposed therein and fluidly coupled to the mam pressure source In addition, the body structure compπses one or more flow reduction channels that intersect the main channel at a first intersection point and a second intersection point, thus forming a bypass loop. The one or more flow reduction channels are structurally configured to reduce fluid pressure or velocity in the main channel as the fluid flows past the first intersection point in a direction toward the second intersection point. For example, typically the main pressure source is fluidly coupled to the mam channel at a position downstream from the one or more flow reduction channels in a direction of flow toward the main pressure source

The devices of the invention typically compπse 2 or more flow reduction channels in fluid communication with the main channel, but also optionally compπse about 3 to about 4 flow reduction channels or from about 5 to about 10 flow reduction channels or about 10 or more flow reduction channels. The cross-sectional dimension of the flow reduction channels is optionally the same as, larger than, or smaller than the cross-sectional dimension of the main channel.

In addition, the one or more flow reduction channels are optionally unmtersected channels or intersected channels. For example, the flow reduction channels are optionally intersected by sources or reservoirs for additional mateπals, such as reagents In one embodiment, the flow reduction channels are intersected by secondary flow reduction channels, which are typically of smaller cross-sectional dimension than the flow reduction channels. In other embodiments, the devices of the invention further compπse one or more detection regions within or proximal to the one or more flow reduction channels. The devices also optionally include a detection system compπsing one or more detectors located proximal to the detection regions or to both the main channel and at least one of the one or more flow reduction channels. Preferabl) , a single detector is positioned to simultaneous!) detect a signal in each of the one or more flow reduction channels. Alternatively, the single detector scans across the vaπous channels. A computer and software are optionally included in the devices for analyzing signals detected by the detection system. For example, the software can include instruction sets for detecting components of interest, their concentrations and the like.

In another embodiment, the device further compπses a source of a first fluidic mateπal in fluid communication with the main channel at a first position along the main channel; and, a source of a second fluidic mateπal in fluid communication with the main channel at a second position along the main channel. The sources of fluidic mateπals are typically upstream from at least one of the one or more flow reduction channels and are used to introduce fluidic materials into the device. The first fluidic material and the second fluidic material are optionally the same or different materials, and typically comprise a sample material and a diluent or buffer material.

In another embodiment, the fluidic mateπals of the device are directed through the channels by a fluid direction system. The fluid direction system directs the movement of the first fluidic material and the second fluidic material from their sources to the main channel, thus combining the first fluidic material with the second fluidic material to form a third fluidic material. The fluid direction system also directs movement of a first portion of the third fluidic material from the main channel to a flow reduction channel, with a second portion of the third fluidic material remaining in the main channel. The second portion of the third fluidic material is then optionally directed through the main channel.

During operation of the device, the first fluidic material, e.g., a sample, has a first flow rate as it flows through the main channel. Upon addition of the second fluidic material, e.g., a buffer, to form the third fluidic material, e.g., a diluted sample, the flow rate in the main channel increases. Therefore, the third fluidic material or diluted sample has a second flow rate that is higher than the flow rate of the first fluidic material or sample. The second flow rate decreases after movement of the first portion of the third fluidic material from the main channel into a flow reduction channel. The second flow rate in one embodiment decreases to substantially the same level of the first flow rate, so that the device maintains a substantially constant flow rate

In another embodiment, a device of the invention further compπses a source of a fourth fluidic mateπal in fluid communication with the main channel at a third position, which third position is downstream of at least one of the one or more flow reduction channels The fluid direction system directs movement of the fourth fluidic mateπal from its source into the main channel The fourth fluidic mateπal is typically a reagent mateπal that reacts with the third fluidic mateπal, e.g., a diluted sample, to produce a product One or more reagent mateπals, such as a substrate material and an enzyme, are optionally added to the device in this manner The device provides reduced consumption of the reagent mateπal by reducing the flow rate of the third fluidic mateπal pπor to the movement of the reagent mateπal into the mam channel

Alternatively, reagent mateπals are added to the flow reduction channels When the flow reduction channels are configured to have smaller cross-sectional dimensions than the main channel, the reagent consumption is reduced even farther. In addition, secondary flow reduction channels are optionally added to draw fluid from the flow reduction channels. Smaller dimensions in the secondary pressure channels reduce reagent consumption as well.

Methods for modulating a volumetπc flow rate of a fluid in microfluidic devices are also provided The method compπses providing a body structure, such as one descπbed above, and flowing a first fluidic mateπal through the main channel A second fluidic mateπal is flowed into the main channel, combining with the first fluidic mateπal and resulting in a third fluidic mateπal. A first portion of the third fluidic mateπal is flowed through a flow reduction channel and a second portion of the third fluidic mateπal is flowed through the ma channel, thereby modulating the flow rate of the third fluidic mateπal m the mam channel and in the one or more flow reduction channels. These steps are optionally iteratively repeated to perform seπal dilutions without substantially increasing the flow rate of mateπals withm the channels.

In one embodiment, the method further compπses flowing the third fluidic mateπal through a detection region which is optionally downstream of the position at which a fourth fluidic mateπal is added to the mam channel to react with the third fluidic mateπal. By adding a fourth mateπal after the flow rate has been reduced by the flow reduction channels, reagent consumption is decreased. Additionally, the method provides for obtaining multiple measurements of a single sample, at multiple or repeat concentrations, or of a single sample, after undergoing multiple assays, by providing detectors proximal to each of the flow reduction channels Alternatively, the channels are configured so that one detector detects signal from each of the channels, thereby concurrently measuπng multiple concentrations or assay products

In another embodiment, the present invention provides methods and devices to suppress pressure perturbations from spontaneous injection into a microfluidic device The devices descπbed above are optionally used to suppress pressure perturbations due to spontaneous injection Spontaneous injection aπses when samples are sipped into a microfluidic device through a capillary from an external sample source, e g , one or more microwell plate

Methods to reduce or eliminate pressure perturbations due to spontaneous injection compπse dipping an open end of a capillary into a sample source, thereby drawing a sample from the sample source into the capillary The capillary, which is typically maintained at a first pressure, is fluidly coupled to a microfluidic device into which samples are flowed from the capillary The method further compπses withdrawing the open end of the capillary from the sample source. A first portion of the sample remains on the open end and is spontaneously injected into the capillary due to the surface tension of the sample exerting a pressure on the capillary. From the capillary, the sample is flowed into the microfluidic device. A second portion of the sample is flowed from the capillary into a main channel, which intersects the capillary at a first intersection point A third portion of the sample is flowed through a flow reduction or shunt channel, which intersects the mam channel at the first intersection point or downstream of the first intersection point. Flowing a poπion of the sample through a flow reduction channel creates a higher flow rate in the capillary than that needed the main channel, thereby giving πse to a higher pressure gradient through the capillary. This higher pressure gradient in the capillary reduces the influence of the spontaneous injection pressure perturbation at the open end of the capillary, thereby suppressing pressure perturbations in the main channel.

In an example device to suppress spontaneous injection, the capillary compπses an inlet region and an outlet region. The mlet region, which is typically maintained at a first pressure, e.g., atmospheπc pressure, is fluidly coupled to at least a first sample source dunng operation of the device and to a microfluidic body structure. The body structure of the device typically compπses a plurality of microscale channels disposed therein The microscale channels compπse a main channel having an upstream region and a downstream region The upstream region of the mam channel is fluidly coupled to the outlet region of the capillary at a first intersection point. In addition to the main channel, the devices include a shunt channel or flow reduction channel as descπbed above The shunt channel is fluidly coupled to at least the upstream region of the mam channel and optionally to the downstream region of the main channel as well A fluid direction system typically directs fluid from the outlet region of the capillary into the main channel and shunts a portion of the fluid into a shunt or by-pass channel, thereby reducing pressure perturbations in the main channel due to spontaneous injection into the main channel.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Panels A, B, and C are schematic drawings of an integrated system of the invention, including a body structure, microfabπcated elements, and a pipettor channel.

Figure 2: Schematic drawing of an integrated system of the invention further depicting incorporation of a microwell plate, a computer, detector, and a fluid direction system. The integrated system is optionally used with either the device or body structure of Figure 3, 4, 5, 6, 7, 8, 9, 10, or 12, or any other suitable microfluidic device. Figure 3: Schematic drawing of a microfluidic device compπsing a single flow reduction channel.

Figure 4: Schematic drawing of a microfluidic device compnsing a single flow reduction channel in a bypass loop configuration.

Figure 5: Schematic drawing of a microfluidic device compnsing multiple flow reduction channels, e.g., for use m making seπal dilutions of a single sample or for performing multiple assays on a single sample.

Figure 6: Schematic drawing of a microfluidic device compπsing multiple flow reduction channels, multiple detectors, and multiple reagent wells, e.g., for obtaining multiple concentration measurements on a single sample or for assaying multiple test compounds or enzymes against a single sample. Figure 7. Schematic drawing of a microfluidic device compπsing multiple flow reduction channels and secondary flow reduction channels, e.g., for obtaining multiple concentration measurements on a single sample and reducing reagent consumption.

Figure 8: Panel A provides a schematic drawing of a microfluidic device compπsing multiple flow reduction channels configured in parallel and fluidly coupled to a single detection window, e.g., for simultaneously obtaining multiple measurements on a single sample, e.g., at vaπous concentrations or for different assay conditions Panel B depicts one possible embodiment of a channel configuration corresponding to the schematic of Panel A Figure 9. Panel A provides a schematic of a microfluidic device without a shunt channel and Panel B provides a schematic of a microfluidic device compπsing a shunt channel Both devices compπse a sipper capillary

Figure 10. A channel configuration for a microfluidic sipper device without a shunt channel. Figure 11. Data showing the spontaneous injection perturbations observed using the microfluidic device of Figure 10.

Figure 12' A channel configuration for a microfluidic sipper device compnsing a shunt channel.

Figure 13: Enzyme inhibition data obtained using the device of Figure 10, the device without a shunt channel.

Figure 14: Enzyme inhibition data obtained using the device of Figure 12, the device compπsing a shunt channel.

Figure 15: Inhibitor titration measured using a device as shown m Figure 8B using two substrate concentrations. Figure 16: K, determined by a Dixon plot.

Figure 17: Enzyme inhibition assay data showing inhibitor channel concentration vs. percent inhibition.

DETAILED DISCUSSION OF THE INVENTION

The present invention provides microfluidic methods and devices for modulating flow rates in microfluidic channel systems, particularly systems in which the flow of mateπals through the channels is pressure induced flow dπven by a single pressure source. The invention provides devices containing flow reduction channels structurally configured to draw material from a main channel of the device during flow of the material in the main channel, thus reducing pressure, volumetric flow rate, and/or velocity in the main channel. In brief, the invention controls the flow rate by dividing the flow of material into multiple portions, which are all controlled by a single vacuum source.

The methods are also useful in providing suppression of spontaneous injection perturbations, e.g., when performed in a microfluidic device comprising a sipper capillary. For example, a sample is typically flowed from a microwell plate into a sipper capillary and then into a microfluidic channel, e.g., a main channel. Flowing a poπion of the sample from the main channel into a flow reduction channel creates a pressure differential between the sipper capillary and the intersection point of the flow reduction channel and the main channel. This pressure differential suppresses pressure perturbations due to spontaneous injection of fluid from the tip of a sipper capillary into the microfluidic device. In addition, the invention provides methods for simultaneously obtaining multiple concentration measurements on a single sample by simultaneously detecting the signal in the various flow reduction channels, e.g., by providing channels that converge in a single detection region. Therefore, the present invention allows serial dilutions and multiple concentration measurements to be obtained in a microfluidic device with a single vacuum source without substantially increasing the flow rate or increasing reagent consumption. In other embodiments, the devices of the present invention are used to concurrently perform multiple assays on a single test compound. For example, a single sample is optionally divided into multiple portions and flowed into multiple parallel channels. Reagent wells fluidly coupled to each of the multiple channels are used to contact the various portions of sample with various reagents. A different reaction or assay, e.g., binding assay, is optionally carried out in each of the different channels. Alternatively, the same reaction is performed in each channel, the only difference being, e.g., a different enzyme isoform. By providing multiple channels that converge in a single detection region, the results of the various assays are optionally concurrently detected.

I. Microfluidic devices containing flow reduction channels

In microfluidic devices, the volumes of sample materials of interest are extremely small, but in order to dilute the concentration of the materials, larger volumes of secondary mateπals, e.g , diluents, buffers, and the like, are added to the sample mateπals The addition of larger volumes of fluids to microfluidic systems increases reagent consumption for reagents added to the samples after dilution, e.g., substrate and enzyme reagents are added to diluted inhibitor samples. For example, increased volume in the channels of a device in which the flow is dπven by a single vacuum source causes an increased flow rate which increases the reagent consumption The devices of the present invention solve this problem by drawing fluid from the channel in which the dilution takes place into a flow reduction channel. For example, as fluid is added to a mam channel, e.g , to dilute a sample, the flow rate of fluid in the ma channel increases When a channel, e.g., a flow reduction channel or bypass loop, is positioned to intake a portion of the fluid from the main channel, the reduced amount of fluid in the main channel causes the flow rate to decrease, e.g., to its pre-dilution level Thus, placement of channels in a microfluidic device provides reduced flow rates by removing fluid from a mam channel Additional flow rate reduction is optionally obtained, e.g., by drawing fluid from a flow reduction channel into a second flow reduction channel.

When the flow reduction channels are used m microfluidic sipper devices, e.g., devices compnsing an external sipper capillary, to shunt fluid from the mam channel, a pressure gradient is created with respect to the sipper capillary This pressure gradient reduces pressure perturbations due to the spontaneous injection of fluid into the sipper capillary, e.g., as the capillary is moved from well to well of a microwell plate.

The configuration of flow reduction channels used m the present invention to control flow rate also provides devices for multiple concentration assays Multiple concentration assays are accomplished by performing seπal dilutions in a device compπsing multiple flow reduction channels. After each dilution, a portion of the fluid is drawn from the mam channel into a flow reduction channel. When multiple flow reduction channels are used after multiple dilutions, each flow reduction channel contains a different concentration of the sample mateπal. The sample is then detected at multiple concentrations by one or more detectors.

Furthermore, reactions are optionally performed in the flow reduction channels by adding reagents to the flow reduction channels This allows reactions to be earned out concurrently at the vaπous sample concentrations, e.g., for determining enzyme kinetics in a high throughput system. Alternatively, multiple assays are earned out in the flow reduction channels or parallel channel regions, e.g , at the same or different concentrations Different reagents are optionally added to each channel to carry out different assays, e.g , on the same compound, e.g , hich has been divided mto multiple samples in the vaπous flow reduction channels Example applications include, but are not limited to, human serum albumin binding assays, genotyping assays, high throughput target screens, e.g . drug screening, selectivity screens, e g , of a panel of different enzymes or isozymes, measurement of intπnsic drug fluorescence, and the like The same assay is optionally repeated in each of the parallel channel regions, e g , at a different concentration or the same concentration, for repeat measurements

Additionally, the flow reduction channels provide for reduced reagent consumption in the assays due to the reduced flow rates The flow reduction channels descnbed above are typically incorporated into microfluidic devices and used as descnbed below The devices generally compπse a body structure with microscale channels disposed therein For example, the present system typically compnses, e.g., a main channel, one or more flow reduction channels, and one or more secondary flow reduction channels or reaction channels The channels are fluidly coupled to each other and to vaπous reservoirs or other sources of fluidic mateπals Matenals used in the present invention include, but are not limited to, buffers, diluents, substrate solutions, enzyme solutions, and sample solutions In addition, the channels optionally compnse detection regions

For example, vanous channels and channel regions are disposed throughout the microfluidic device The devices typically include a main channel into which a sample is introduced For example, a sample containing a potential modulator or activator of an enzyme of interest is introduced into a channel An assay to determine the effect of the modulator, e.g., an activator or an inhibitor, on the enzyme's reaction rate is then optionally performed by allowing the enzyme to react with the substrate in the presence of the modulator Reaction rates are often studied at multiple concentrations to determine the effect of concentration upon kinetic parameters. The devices of the present invention allow seπal dilutions to be made in the microfluidic device without increasing the flow rate and thus minimize reagent consumption while maintaining constant reaction times for fixed channel lengths In the present invention, devices are also provided for performing multiple concentration measurements simultaneously The flow and/or pressure reduction channels are channels that are structurally configured to reduce the pressure and/or flow rate in a main channel or in another flow reduction channel A channel that is "structurally configured to reduce pressure/flow rate" is one that is configured to provide a desired flow rate A device containing channels structurally configured to reduce pressure, flow rate, or velocity of the fluid in the channels typically relies on the structural configuration of the channels carrying the fluid to regulate the pressure and/or velocity in the channel, as opposed to relying on the modulation of forces such as a vacuum sources or electrokinetic forces By configunng the channels to modulate the flow rate, a single constant dnving force is optionally applied over the whole system, e g., a single vacuum source.

For example, where a plurality of channels and a source of samples are fluidly coupled, a single vacuum source can draw reagents from the source mto the channels and move the sample through the channels When channels are configured as in the present invention to serve as flow reduction channels, the vacuum will pull the fluid through the flow reduction channel as well as a main channel, thus dividing the flow mto two portions and decreasing the velocity Typically, the flow reduction channels in the present invention are spaced far enough apart that mixing of assay components is complete before part of the flow is diverted mto the flow reduction channel.

Typically, the channel is configured by varying the channel length or cross section or by the addition of a flow-retarding matπx These changes m the channel configuration alter the resistance to fluid flow in the channel, thus changing the flow rate For example, by narrowing channel width, the flow rate is decreased by providing greater resistance to flow. A preferable way to structurally configure the channels to reduce fluid flow is to design or place a plurality of channels such that one channel, e.g., a pressure and/or flow reduction channel, pulls fluid from another channel, e.g., a mam channel, thus decreasing resistance in the main channel. The flow rate of the matenals in the channels is thus precisely modulated and reagent consumption is reduced by the appropnate configuration of flow reduction channels. By using one or more of the above methods, the channels are optionally structurally configured to reduce pressure or flow rate by a specific desired amount. For more detail on structurally configunng channels for desired flow rates using channel length and dimensions, see, e.g., USSN 09/238,467, filed 1/28/99.

In the present invention, the reduction in flow rate and/or pressure is typically accomplished by drawing fluid from the main channel into a flow reduction channel or a shunt channel, e.g.. by tapping off pressure Secondary flow reduction channels are those flow reduction channels that draw fluid from another flow reduction channel These secondary flow reduction channels are preferably of smaller cross-sectional dimension than the channels from which they draw fluid In this case, the channels optionally function as reaction channels when they are fluidly coupled to sources of reagent matenals This type of reaction channel provides reduced reagent consumption because the flow rate has been reduced and the dimension of the channel is reduced so that smaller amounts of reagents are required to perform the assays of interest

Alternatively, the channels in the present invention are configured to provide assay channels, e.g . for performing multiple assays on a single compound For example, a microfluidic device is optionally configured to provide multiple parallel channels A single sample is optionally sipped into the device through a capillary fluidly connected to the parallel channels The sample is optionally divided into portions, each of which is flowed through a separate parallel channel region. A different assay is then optionally performed on each of the sample portions, e.g., by using different channel chemistnes or by adding different reagents into each channel. For example, different channels are optionally loaded with vaπous bead arrays compnsing different chemistnes or with different separation matπces In other embodiments, individual channels compπse different surface modifications, e.g., to provide different chemistnes in each channel. The term "downstream" refers to a location in a channel or microfluidic device that is farther along the channel or plurality of channels in a selected direction of fluid or mateπal flow, relative to a selected site or region. For example, the pressure source is optionally farther along in the direction of flow m the channel system than the buffer well or flow reduction channels; therefore, the fluid flows down the main channel past the buffer well and past the flow reduction channels towards the pressure source. In this embodiment, the pressure source is typically a vacuum source.

In another embodiment of the present device, the pressure source is optionally positioned at the upstream end of the main channel "Upstream" refers to a location in a channel or system of channels that is farther along the channel or plurality of channels in a direction that is opposite the flow of fluid or mateπal flow, relative to a selected site or region. For example, a pressure source is optionally upstream from the detection region. The pressure source is optionally positioned at the sample well, where sample mateπals are introduced mto the system. In this instance, the pressure source would push the fluid through the channels in a direction away from the pressure source and toward the opposite end of the channel, e g., the detection region or waste well

Reservoirs or wells are locations at which samples, components, reagents and the like are added into the device for assays to take place. Introduction of these elements into the system is earned out as descnbed below The reservoirs are typically placed so that the sample or reagent is added into the system upstream from the location at which it is used For example, a dilution buffer will be added upstream from the source of a reagent if the sample is to be diluted before reaction with the reagent

In the present case, a dilution buffer is typically added into the ma channel upstream of a flow reduction channel, so that the increase in flow rate due to the addition of buffer matenal may be counteracted by the reduction in pressure due to the flow reduction channel Reagent matenals, on the other hand, are typically added downstream of a flow reduction channel so that they are added after the flow rate has been reduced so that smaller quantities of reagent are added. In some embodiments, a different reagent well or multiple reagent wells are fluidly coupled to each of the flow reduction channels or to each channel or channel region, e.g., in a parallel channel configuration The reagent wells in this case, are optionally used to add a different reagent or reagents to each channel, e.g., to perform a different assay in each channel, e.g., to concurrently assay multiple binding sites on a single target or to screen a vaπety of enzyme isoforms.

Detection regions are also included in the present devices The detection region is optionally a subunit of a channel or of multiple channels that are close in space, or it optionally compnses a distinct channel that is fluidly coupled to the plurality of channels in the microfluidic device. The detection region is optionally located proximal to the main channel. For example, in Figure 5, detection region 532 is proximal to mam channel 504 Alternatively, detection regions are positioned proximal to one or more of the flow reduction channels, such as in Figure 6 where detection regions 634, 636, and 638 are proximal to flow reduction channels 612, 610, and 608 respectively. In another embodiment, detection regions are placed proximal to one or more of the secondary flow reduction channels. For example, detection regions 734, 736, and 738 are proximal to secondary flow reduction channels 742, 744, and 746.

Alternatively, the detection region may compπse a region that is proximal to all of the flow reduction channels and the ma channel in the device. For example, the detection region is optionally located at a point downstream of the main channel and all the flow reduction channels so that it is proximal to both the main channel and the flow reduction channels Such a device is depicted in Figure 8 When the flow reduction channels and the main channel are configured such that they all converge, then one detection region is sufficient for detection of signals from all the proximal channels For example, multiple assays are optionally performed in multiple parallel channels, e.g.. on a single sample under different assay conditions or at vaπous concentrations, and the results are optionally concurrently detected in a single detection region in which all of the multiple channel regions converge The detection window or region at which a signal is monitored typically includes a transparent cover allowing visual or optical observation and detection of the assay results, e.g , observation of a colonmetπc or fluorometπc signal or label Such regions optionally include one or more detectors Examples of suitable detectors for use in the detection regions are well known to those of skill in the art and are discussed in more detail below

The elements descπbed above, including but not limited to, flow reduction channels, bypass loops, detection regions, and reservoirs are optionally combined into microfluidic devices that are useful in controlling flow rates, reducing reagent consumption, and performing multiple concentration measurements or multiple assays, e.g., on a single sample Specific examples of channel configurations are provided in the figures, which are descnbed below Other possible configurations using substantially the same elements will be apparent upon review of the entire disclosure.

One embodiment of a device of the present invention is illustrated in Figure 3 As shown, the device compnses sample well 302, which is used to introduce a sample or fluidic mateπal into the device From sample well 302, the fluidic matenal is flowed through mam channel 304. Additional matenals are optionally added to the fluidic matenal as it flows through mam channel 304 For example, a buffer is optionally added to dilute the fluidic matenal. In addition, a reagent is optionally added to the fluidic matenal, which reacts with the reagent to form a product. The fluidic matenal is flowed through the device using, e.g., pressure source 306, which is optionally located at the downstream end of the mam channel. Alternatively the pressure source is located at sample well 302. In the configuration shown in Figure 3, pressure source 306 is fluidly coupled to mam channel 304 at the downstream end. When a second fluidic matenal, e.g., a dilution buffer, is added to the fluidic matenal in main channel 304, the flow rate of the fluidic mateπal in mam channel 304 increases To decrease the flow rate in the mam channel, a first portion of the fluidic mateπal is flowed into flow reduction channel 308 Flow reduction channel 308 is configured to draw fluid from main channel 304 and reduce the pressure and/or velocity in main channel 304. In this embodiment, flow reduction channel 308 is fluidly coupled at its downstream end to pressure source 306

The reduction in pressure or velocity in main channel 304 serves multiple purposes In one embodiment, the flow reduction reduces the volumetπc flow rate and thus reduces the amount of reagent that must be added to main channel 304 in any reactions earned out downstream of flow reduction channel 308 For example, a reagent that is used in an assay with the fluidic matenal is optionally added through reagent well 318 Smaller amounts of reagent from reagent well 318 are required after the flow rate has been decreased by drawing fluid into flow reduction channel 308

Alternatively, the flow reduction allows multiple concentrations of the same sample matenal to be measured in one device. For example, a dilution buffer is optionally added from buffer well 316 to the sample introduced from sample well 302. A first portion of the resulting fluid is then drawn from mam channel 304 mto flow reduction channel 308 and detected in detection region 332. Simultaneously, a second portion of the resulting fluid is flowed through main channel 304, where it is again diluted with a dilution buffer from reagent well 318, resulting in a successive dilution of the sample matenal This diluted matenal is then flowed through main channel 304 and detection region 332, where it is detected concurrently with the first dilution.

In another embodiment, shown in Figure 4, flow reduction channel 408 is structurally configured to draw fluid from mam channel 404 by forming a bypass loop off main channel 404. This embodiment works in substantially the same way as that in Figure 3. For example, fluid is flowed from sample well 402 mto main channel 404. Additional fluid is then added from reservoir 416 and then a portion of the fluid is drawn from ma channel 404 mto flow reduction channel 408, thereby reducing the pressure in main channel 404 and reducing the flow rate of fluid. The fluid flow is typically controlled by vacuum source 406, but is optionally any other type of pressure control system that draws fluid through or mto main channel 404.

Figure 5 illustrates an alternate embodiment of the device m Figure 4, in which multiple flow reduction channels are included m the device. For example, samples are introduced into the device through sample well 502, and transported through main channel 504 At this point, multiple dilutions are optionally performed in the device of Figure 5. For example, a dilution buffer is added to the sample through reservoir 516, resulting in a diluted sample, at which point the flow rate and/or pressure in main channel 504 increases. The pressure is reduced by drawing a portion of the diluted fluid into flow reduction channel 508. An additional dilution is optionally made by adding dilution buffer into the ma channel from reservoir 518, resulting in a second diluted sample. At this point, the pressure in main channel 504 is again increased, thus increasing the flow rate Therefore, flow reduction channel 510 (the second flow reduction channel or bypass loop) is used to reduce the pressure, thus controlling the flow rate in main channel 504. More dilutions are optionally made at this point, with pressure controlled by additional flow reduction channels. After the desired dilution concentration is obtained, the sample is optionally reacted with a vaπety of reagents that may be added to the sample through reservoirs 520 and/or 522 The sample is also optionally detected by a detector placed proximal to detection region 532.

Figure 6, showing an additional embodiment of Figure 4, provides a device in which assays are optionally run on the same sample at vaπous concentrations and simultaneously detected. The device in Figure 6 works like the one in Figure 5 descnbed above with an additional dilution step possible due to the addition of flow reduction channel 612 (in addition to flow reduction channels 608 and 610). Furthermore, the device of

Figure 6 contains detection regions 632, 634, 636. and 638. which are positioned proximal to main channel 604, and flow reduction channels 612, 610, and 608 respectively Thus, detectors placed proximal to detection regions 632, 634, 636, and 638 are optionally used to detect fluid as it flows through mam channel 604, and flow reduction channels 608, 610 and 612, which, when configured and operated as descnbed above, contain vanous concentrations of the sample matenal that was injected or sipped at sample well 602. Alternatively, flow reduction channels 608, 610 and 612 are optionally used to perform a different assay on the same sample, a portion of which is flowed through all three channels Reagents for each assay are added to the flow reduction channels, e.g., from reservoirs 622, 624, 626, and/or 628.

In addition, Figure 6 compnses additional reservoirs 622, 624, 626, and 628 for adding matenals, e.g., reagents, such as substrates and enzymes, mto mam channel 604 and flow reduction channels 608, 610 and 612. Thus, an assay of interest is optionally performed at all of the various concentrations of sample material in the flow reduction channels. Furthermore, the reagent usage is decreased by the decrease in flow rate produced by the flow reduction channels.

An additional embodiment of the invention provides an even greater reduction in reagent usage by varying the channel dimensions in which the assays of interest are performed. For instance, Figure 7 illustrates a device in which flow reduction channels of smaller dimension are added as bypass loops. For example, secondary flow reduction channels 742, 744, and 746 are fluidly coupled to flow reduction channels 708, 710, and 712. The secondary flow reduction channels function to reduce pressure and/or velocity in flow reduction channels 708, 710, and 712 by drawing fluid out of the flow reduction channels. Secondary flow reduction channels 742, 744, and 746 therefore reduce reagent consumption by decreasing flow rate in the channels.

In addition, secondary flow reduction channels 742, 744, and 746 reduce reagent consumption even more when used as reaction channels. As reaction channels, they are typically configured to have a smaller cross-sectional dimension than flow reduction channels, e.g., flow reduction channels 708, 710, and 712. Reservoirs 722, 724, 726, and 728 are fluidly coupled in this embodiment to secondary flow reduction channels 742, 744, and 746, so that the amount of reagents added from the reservoirs into the smaller dimension channels is smaller. Additionally, detection regions 732, 734, 736 and 738 are located proximal to secondary flow reduction channels 742, 744, and 746 for detection of materials in those channels. Thus assays of interest are optionally performed on small volumes, to reduce reagent consumption, and at various dilution levels concurrently and then concurrently detected at those levels. Furthermore the detection regions in the various flow reduction channels are optionally configured in such as way that one detector may be used to detect signals from all flow reduction channels and the main channels simultaneously.

Figure 8, Panels A and B, provides a schematic illustration and an actual embodiment of one possible channel configuration in which fluid in the various flow reduction channels, secondary flow reduction channels, and the main channels are detectable by the same detector. Other configurations are also possible. The devices in

Figure 8 provide capillary attachment point 802, 802B positioned on the main channel.

Samples are introduced into a device at capillary attachment point 802, 802B. The sample is flowed into parallel channel regions 808, 808B, 810, 810B, 812, 812B, and main channel region 804, 804B These channels act as flow reduction channels in that they are structurally configured to reduce volumetπc flow rate in main channel region 804, 804B, by drawing fluid from main channel region 804, 804B into parallel channel regions 808, 808B. 810, 810B, and 812, 812B. The fluid in channel regions 808, 808B, 810, 810B, and 812, 812B is optionally diluted with mateπal from reservoirs 816, 816B and 818, 818B, which are fluidly coupled to channel regions 808, 808B. 810, 810B, 812, 812B, 844. 846, 848, 850 and 852 After the dilutions, the pressure and/or flow rate is optionally further reduced by secondary flow reduction channels (or reaction channels) 842, 844, 846. 848. 850, and 852 By utilizing all of the secondary flow reduction channels available, multiple seπal dilutions are optionally made without significantly increasing the flow rate of the fluid through main channel region 804 and parallel channel regions 808, 810, and 812. After the desired dilution level is achieved, assays are optionally performed at multiple dilution levels, such as those obtained in channel regions 804, 804B, 808, 808B, 810, 810B, 812, 812B, 842, 844, and 846. The result of the vaπous assays or concentration levels is detected concurrently because channel regions 804, 804B, 808, 808B, 810, 810B, 812, 812B, 842, 844, and 846 all converge in detection region 832, 832B, before being discarded in waste well 806, 806B. In an additional embodiment, a single pressure source is optionally applied at waste well 806, 806B for inducing flow through the channel system.

In one embodiment, the parallel channel configuration of Figures 8A and 8B is used to perform multiple assays, e.g.. on the same test compound or on different test compounds. For example, a test compound, e.g., a single test compound, is optionally sipped from a capillary, e.g., from a microwell plate, mto a microfluidic device as shown in Figure 8A. The compound is then optionally divided into four portions. One portion is flowed through parallel channel region 804. A second portion is flowed through parallel channel region 808, a third portion through channel region 810, and a fourth portion through channel region 812. Each sample portion is then optionally subjected to a different assay. For example, reservoir 820 is optionally used to add reagents necessary for an assay to probe binding site I in a human serum albumin (HSA) assay in channel 804 and reservorr 822 is used to add reagents necessary to probe binding site II in an HSA assay m channel 808. Reservoirs 824 and 826 are also optionally used to add reagents for other assays mto channels 810 and 812. Alternatively, the reservoirs add the same reagents to each channel, so that the same assay is performed to obtain repeat measurements, e.g., four times, or the same assay at different concentrations. In other embodiments, the same reaction is performed in each channel, the only difference being, e g . a different enzyme isoform The results of the different assays are then optionally concurrently detected in detection window 832

A vaπety of microfluidic devices are optionally adapted for use in the present invention by the addition of flow reduction components as descnbed above These devices are descnbed in vanous PCT applications and issued U S Patents by the inventors and their coworkers, including U S Patent Nos 5.699,157 (J Wallace Parce) issued 12/16/97, 5,779,868 (J Wallace Parce et al ) issued 07/14/98, 5.800,690 (Calvin Y H Chow et al ) issued 09/01/98, 5,842,787 (Anne R Kopf-Sill et al ) issued 12/01/98, 5,852,495 (J Wallace Parce) issued 12/22/98, 5,869.004 (J Wallace Parce et al ) issued 02/09/99,

5,876,675 (Colin B Kennedy) issued 03/02/99, 5,880,071 (J Wallace Parce et al ) issued 03/09/99, 5,882,465 (Richard J McReynolds) issued 03/16/99, 5.885,470 ( J Wallace Parce et al ) issued 03/23/99, 5,942,443 (J Wallace Parce et al ) issued 08/24/99, 5,948,227 (Robert S Dubrow) issued 09/07/99, 5,955,028 (Calvin Y H Chow) issued 09/21/99, 5.957,579 (Anne R Kopf-Sill et al ) issued 09/28/99, 5,958,203 (J Wallace Parce et al ) issued 09/28/99, 5,958,694 (Theo T. Nikiforov) issued 09/28/99, and 5,959,291 ( Morten J Jensen) issued 09/28/199; and published PCT applications, such as, WO 98/00231, WO 98/00705, WO 98/00707, WO 98/02728, WO 98/05424, WO 98/22811, WO 98/45481, WO 98/45929, WO 98/46438, and WO 98/49548, WO 98/55852, WO 98/56505, WO 98/56956, WO 99/00649, WO 99/10735, WO 99/12016, WO 99/16162, WO 99/19056, WO 99/19516, WO 99/29497, WO 99/31495, WO 99/34205, WO 99/43432, and WO 99/44217

For example, pioneeπng technology providing cell based microscale assays are set forth in U.S Patent 5,942,443, by Parce et al "High Throughput Screening Assay Systems m Microscale Fluidic Devices" and, e g , m 60/128,643 filed Apnl 4, 1999, entitled "Manipulation of Microparticles In Microfluidic Systems," by Burd Mehta et al Complete integrated systems with fluid handling, signal detection, sample storage and sample accessing are available For example, U S Patent 5,942.443 provides pioneenng technology for the integration of microfluidics and sample selection and manipulation In addition, vaπous other elements are optionally included m the device, such as particle sets, separation gels, antibodies, enzymes, substrates, and the like These optional elements are used m performing vanous assays For example, in a kmase reaction a product and substrate are typically separated electrophoretically, e g., on a separation gel

Cell based microscale assays are also optionally performed in the devices of the invention With cell assays, for example, a constant flow rate is important for ascertaining and modulating cell incubation times Cell-based microscale systems are set forth in Parce et al "High Throughput Screening Assay Systems in Microscale Fluidic Devices" WO 98/00231 and, e g., in 60/128,643 filed Apπl 4, 1999, entitled "Manipulation of Microparticles In Microfluidic Systems," by Mehta et al

Complete integrated systems with fluid handling, signal detection, sample storage and sample accessing are also available For example WO 98/00231 (supra) provides pioneeπng technology for the integration of microfluidics and sample selection and manipulation Also included in the integrated systems of the invention are sources of sample mateπals, enzymes, and substrates. These fluidic matenals are introduced mto the devices by the methods descπbed below

Sources of assay components and integration with microfluidic formats

Reservoirs or wells are provided in the present invention as sources of buffers, diluents, substrates, enzymes, reagents, and the like. For example, Figure 3 illustrates vanous reservoirs, such as sample wells 302, buffer well 316, and reagent well 318 These reservoirs are fluidly coupled to main channel 304 Figure 6 illustrates alternate placement of reagent wells, such as reservoirs 622, 624, 626, and, 628. These reservoirs are positioned so that they are fluidly coupled to flow reduction channels 608. 610, and 612 In Figure 8A, for example, reservoirs 820, 822, 824, and 826 each optionally compnse a different reagent, e.g., for performing different assays in channels 804, 808, 810, and 812 In other embodiments, the different assay channels are fabπcated or pre-filled with different reagents to conduct a different assay in each channel. For example, channels 804, 808, 810, and 812 are each exposed to or filled with a different reagent either before to dunng the assay. Different chemistnes are achieved in each channel using, e.g., beads compnsing different reagents, e.g., having different properties, separation matnces, e.g., gels, and/or reagents that modify or react with the channel surface.

Sources of samples, buffers, and reagents, e.g., substrates, enzymes, and the like, are fluidly coupled to the mrcrochannels noted herein in any of a vanety of ways. In particular, those systems compnsing sources of matenals set forth in Knapp et al. "Closed Loop Biochemical Analyzers" (WO 98/45481; PCT/US98/06723) and Parce et al. "High Throughput Screening Assay Systems in Microscale Fluidic Devices" WO 98/00231 and, e g , in 60/128.643 filed Apnl 4, 1999, entitled "Manipulation of Microparticles In Microfluidic Systems," by Mehta et al are applicable

In these systems, a "pipettor channel" (a channel m which components can be moved from a source to a microscale element such as a second channel or reservoir) is temporanly or permanently coupled to a source of matenal The source can be internal or external to a microfluidic device compπsing the pipettor channel Example sources include microwell plates, membranes or other solid substrates compπsing lyophilized components, wells or reservoirs the body of the microscale device itself and others

For example, the source of a cell type, component, or buffer can be a microwell plate external to the body structure, having, e g , at least one well with the selected cell type or component Alternatively, the source is a well disposed on the surface of the body structure compπsing the selected cell type, component, or reagent, a reservoir disposed within the body structure compnsing the selected cell type, component, mixture of components, or reagent, a container external to the body structure compnsing at least one compartment compnsing the selected particle type, component, or reagent, or a solid phase structure compπsing the selected cell type or reagent in lyophilized or otherwise dned form

A loadmg channel region is optionally fluidly coupled to a pipettor channel with a port external to the body structure The loading channel can be coupled to an electropipettor channel with a port external to the body structure, a pressure-based prpettor channel with a port external to the body structure, a pipettor channel with a port internal to the body structure, an internal channel within the body structure fluidly coupled to a well on the surface of the body structure, an internal channel within the body structure fluidly coupled to a well withm the body structure, or the like

The integrated microfluidic system of the invention optionally includes a very wide vanety of storage elements for stoπng reagents to be assessed These include well plates, matπces, membranes and the like The reagents are stored in liquids (e.g , in a well on a microtiter plate), or in lyophilized form (e g , dned on a membrane or in a porous matnx), and can be transported to an array component, region, or channel of the microfluidic device using conventional robotics, or using an electropipettor or pressure pipettor channel fluidly coupled to a region or channel of the microfluidic system

The above devices, systems, features, and components are used in the methods descπbed below to modulate flow rate, make multiple concentration

79 measurements, perform multiple assays, and reduce reagent consumption, e.g., when performing serial dilutions in a single pressure source microfluidic system.

II. Movement of fluid through a microfluidic device

In the present invention, a sample material is flowed through a main channel and various materials are added to the main channel, e.g., to dilute the sample material or to react the sample material with a reagent material. For example, in high-throughput screening applications it is sometimes useful to dilute the samples coming into the device by a factor of 10, 100, 1000, or even 10,000-fold. In vacuum driven flow systems, this can be achieved by introducing a diluting buffer into the device and designing buffer and compound fluid paths to have hydrodynamic resistances proportional to the desired dilution. However, the volumetric flow rate goes up 10-10,000-fold. Subsequent additions of reagents must then be at 10 to 10,000 times higher volumes than would be required without the dilution.

The present invention provides a flow reduction channel or bypass arm that reduces the pressure, flow rate, and/or velocity in the main channel by pulling fluid through the flow reduction channel or bypass arm. In this way the flow rate or velocity of the fluid through the device is decreased. The channels are optionally configured to decrease the flow rate so that it is substantially equal to the initial flow rate before the diluent was added or so that it is less than the initial flow rate. By reducing the flow rate, subsequent additions of reagents do not need to 10 to 10,000 times higher to meet the demands of an increased flow rate.

Typically, movement of fluidic materials through the microfluidic devices of the invention is driven by a pressure source. The pressure source is typically a vacuum source applied at the downstream terminus of the main channel. For example, in Figure 4, vacuum source 406 is applied at one end of main channel 404. Vacuum source 406 or another type of pressure source such as those described below, applies a pressure to draw or pump fluid through the channels of the device, such as main channel 404 and flow reduction channel 408. These applied pressures, or vacuums, generate pressure differentials across the lengths of channels to drive fluid flow through them. In the interconnected channel networks described herein, such as the one shown in Figure 4, differential flow rates on volumes are optionally accomplished by applying different pressures or vacuums at multiple ports, or preferably, by applying a single vacuum at a common waste port and configuring the vanous channels with appropnate resistance to yield desired flow rates. For example, the channels in Figure 4 are configured to control flow rate through the device when vacuum source 406 is applied at the end of main channel 404 When a single vacuum source is used to draw fluid through main channel 404 and additional fluid is added through reservoir 416. the flow rate is controlled by the flow reduction channel configuration. A portion of the fluid is drawn into flow reduction channel 408, thus reducing the pressure in main channel 404.

A vanety of techniques are available to apply pressure forces to microscale elements to achieve the fluid movement descnbed above. Fluid flow (and flow of mateπals suspended or solubilized within the fluid, including cells or other particles) is optionally regulated by pressure based mechanisms such as those based upon fluid displacement, e.g , using a piston, pressure diaphragm, vacuum pump, probe or the like, to displace liquid and raise or lower the pressure at a site in the microfluidic system. The pressure is optionally pneumatic, e.g., a pressunzed gas, or uses hydraulic forces, e.g., pressuπzed liquid, or alternatively, uses a positive displacement mechanism, i.e., a plunger fitted into a mateπal reservoir, for forcing mateπal through a channel or other conduit, or is a combination of such forces.

In other embodiments, a vacuum source is applied to a reservoir or well, such as a waste well as shown in Figure 8. Waste well 806, 806B includes a vacuum source at one end of the channel system to draw the suspension through the channel Pressure or vacuum sources are optionally supplied external to the device or system, e.g., external vacuum or pressure pumps sealably fitted to the inlet or outlet of the channel, or they are internal to the device, e.g., microfabncated pumps integrated into the device and operably linked to the channel. Examples of microfabncated pumps have been widely descnbed m the art. See, e.g., published International Application No. WO 97/02357.

The systems of the present invention, while descπbed in terms of a single vacuum source, may compnse other sources of fluid movement, such as electrokinetic flow and other types of pressure dnven flow, including but not limited to pressure sources at multiple reservoirs or channels of the device. For example, electrokinetic techniques are optionally used to inject fluids into the device or to transfer fluids from one channel of the device to another channel in a cross-injection. The following techniques are optionally used in conjunction with those of the present invention to provide further alternatives to fluid control. Additional methods of controlling flow in a channel or portion of the devices include the use of hydrostatic, wickmg, and capillary forces to provide pressure for fluid flow of mateπals such as cells or sample mateπals. See, e.g., "METHOD AND APPARATUS FOR CONTINUOUS LIQUID FLOW IN MICROSCALE CHANNELS USING PRESSURE INJECTION, WICKTNG AND ELECTROKINETIC INJECTION," by Alajoki et al., USSN 09/245,627, filed February 5, 1999 In these methods, an adsorbent matenal or branched capillary structure is placed in fluidic contact with a region where pressure is applied, thereby causing fluid to move towards the adsorbent matenal or branched capillary structure. Mechanisms for focusing cells and other matenals into the center of microscale flow paths, which is useful in increasing assay throughput by regulanz g flow velocity, e.g., in pressure based flow, is descπbed in "FOCUSING OF MICROPARTICLES IN MICROFLUIDIC SYSTEMS" by H. Garrett Wada et al. USSN 60/134,472, filed May 17, 1999. In bπef, matenals are focused into the center of a channel by forcing fluid flow from opposing side channels into the main channel, or by other fluid manipulations.

In an alternate embodiment, microfluidic systems can be incorporated into centπfuge rotor devices, which are spun in a centnfuge. Fluids and particles travel through the device due to gravitational and centπpetal/centnfugal pressure forces.

Another method of achieving transport through microfluidic channels is by electrokinetic mateπal transport "Electrokinetic mateπal transport systems," as used herein, include systems that transport and direct mateπals within a microchannel and/or chamber containing structure, through the application of electπcal fields to the matenals, thereby causing mateπal movement through and among the channel and/or chambers, i.e., cations will move toward a negative electrode, while anions will move toward a positive electrode. A vaπety of electrokinetic controllers and systems are described, e.g., in Ramsey WO 96/04547, Parce et al. WO 98/46438 and Dubrow et al., WO 98/49548, as well as a vaπety of other references noted herein. In the present invention electrokinetic transport or electropumping is optionally used to introduce pressure dnven flow.

Pressure dnven flow, as descπbed above, is used m the present system to transport fluidic mateπals through the channel system to perform vanous assays. The flow rate m the vanous assays is controlled, e.g., to reduce reagent consumption and or to modulate reaction times, by the channel configuration as descπbed below. III. Modulating flow rate using bypass loops

The flow rate of a fluidic mateπal in a microfluidic device is optionally modulated by configunng the channels such that the pressure and/or velocity or flow rate of fluid in the channels is reduced Examples of vanous types of channel configurations that will reduce flow rate in microfluidic devices, e.g , in the main channels, are shown, e g., in Figures 3-10, and 12

In one embodiment, the flow reduction channel is a bypass loop, e g , flow reduction channel 508, as shown in Figure 5, which intersects mam channel 504 in two positions In Figure 4, for example, flow reduction channel 408 intersects mam channel 408 in a first position and a second position The flow rate in the device in Figure 4 is optionally modulated in the following way A sample is sipped from sample well 402 A buffer, diluent, or other fluidic matenal is added to the sample from reservoir 416 As this additional fluidic mateπal is added to the sample, the pressure increases in the main channel and the flow rate is elevated By drawing fluid from the mam channel into flow reduction channel 408, the pressure is decreased and the flow rate returns to its initial rate The flow reduction channel is configured such that it intersects the main channel downstream from the point of introduction of the additional fluidic matenal. The second position of intersection for the bypass loop is typically downstream from the first position. If additional matenals are to be added to the sample for reactions or assays, the second position is typically downstream of the reaction point or of the point of addition of the reagent mateπals For example, in Figure 4, flow reduction channel 408 intersects main channel 404 at a first position that is downstream of reservoir 416 and at a second position that is downstream of reagent wells 418 and 420 and upstream of pressure source 406.

Alternatively, a flow reduction channel is configured to intersect the main channel, e.g., main channel 408, upstream from the point of intersection of the additional fluidic matenal In this embodiment, fluid is drawn from, e.g , main channel 408, mto the flow reduction channel, thus reducing the flow rate of the fluid in the mam channel pnor to the addition of, e.g., a buffer, diluent, or the like The subsequent addition, e.g., of buffer, diluent or other fluidic mateπal, increases the flow rate, e.g., to its oπginal or normal value, i.e., the flow rate before the fluid was drawn mto the flow reduction channel. In this embodiment, the channels are configured such that the flow reduction channel is upstream from the reagent or diluent introduction channel. This way, the flow rate decreases before, e.g., the dilution mixing point, and returns to a normal value (as opposed to an elevated value) after the introduction of the diluent, buffer, or the like. This allows the mixing of the reagent or diluent into the sample to occur in a shorter distance.

In another embodiment, the flow reduction channel intersects the main channel in only one position, but is connected at its downstream end to a pressure source, typically the same pressure source that is fluidly coupled to the downstream end of the main channel. Examples of this flow reduction channel configuration include, but are not limited to. the devices of Figures 3 and 8 In Figure 3, flow reduction channel 308 intersects main channel 304 and is fluidly coupled to pressure source 306. In Figure 8. channel regions 808, 808B, 810, 810B, and 812, 812B intersect main channel region 804, 804B and are fluidly coupled to waste well 806, 806B at which a pressure source is applied.

By directing fluid through the flow reduction channels descπbed above, the flow rate in a microfluidic device is modulated in response to the addition of mateπal to the mam channel. An added advantage of this flow rate modulation is that, in addition to maintaining a continuous or constant flow rate, subsequent reagent addition need not be in larger amounts to meet an increased flow rate demand. This saves on the amount of reagents necessary to run assays in microfluidic devices.

To further reduce reagent consumption, the reagents for assays are optionally added to secondary flow reduction channels or reaction channels. A device containing secondary flow reduction channels is illustrated in Figure 7. For example, secondary flow reduction channels 742, 744, and 746 are fluidly coupled to flow reduction channels 712, 710, and 708 respectively. Therefore, fluid is drawn, e.g., from flow reduction channel 708 into secondary flow reduction channel 746, thus reducing the flow rate of matenal in channel 746. The substrates, reactants, or enzymes necessary for an assay are optionally added mto the flow reduction channels or into the secondary flow reduction channels. For example, reagents are optionally added from reservoir 726 or 728 mto reaction channel 742 If reaction channel 742 has a smaller cross-sectional dimension than flow reduction channel 712, then reagent consumption is additionally reduced due to the smaller dimension of the reaction channel. Alternatively, they are added into the mam channel after a portion of the fluid has been drawn off mto a flow reduction channel. In either case, the amount of reagent mateπal that must be added to conduct an assay is diminished, due to decreased flow rate and/or decreased channel dimensions. IV. Obtaining multiple concentration measurements and performing multiple assays in a microfluidic device

Another added benefit to using flow reduction channels in a microfluidic device is that the channels are optionally used to obtain multiple measurements on the same sample material in one assay. For example, a diluent is optionally added to create a 10:1 dilution of a sample material in the main channel. When a portion of the 10: 1 diluted material is drawn from the main channel into a flow reduction channel, a measurement is optionally obtained in a detection region positioned within the flow reduction channel. For every additional dilution level obtained in this manner, e.g., 100:1, 1000:1, or 10,000:1, a separate detection region is optionally placed within the flow reduction channel and a signal detected.

For example, see Figure 6. A detector is placed proximal to each flow reduction channel, e.g., 608, 610, and 612. Additionally, a detector is placed proximal to the main channel. Typically, when an assay is performed in the device, the detection region where the detector is placed is positioned downstream of the reagent reservoirs, e.g., reservoirs 626, 628, 622, and 624. Then the assay is run and the detector is used to detect a signal that is correlated with the assay results.

In another embodiment, a movable detector is used and is moved between detection regions such that a signal is detected from each of the detection regions of interest. Alternatively, a single detector is positioned in such a way that it detects a signal from all the flow reduction channels concurrently. Such a configuration is shown in Figure 8. The main channel and flow reduction channels comprise parallel channel regions 804, 804B, 808, 808B, 810, 810B, and 812, 812B, which converge in a single detection window 832, 832B. Using these channel configurations, it is possible to take measurements on a single sample at multiple concentrations using one assay. In addition, because the reagent reservoirs are optionally positioned such that they are fluidly coupled to the flow reduction channels, reaction or assays are optionally performed at the various dilution levels obtained from the serial dilution. For example, as shown in Figure 6, reservoirs 622 and 624 are fluidly coupled to flow reduction channels 608 and 610. Therefore reactions or assays are optionally performed in flow reduction channels 608 and 610 which contain different concentrations of sample when operated as described above for serial dilutions. The channel configurations descπbed herein are optionally used, e.g., to perform multiple assays or measure multiple samples, e.g., concurrently A single sample or test compound is optionally introduced into a microfluidic device, e.g., through a capillary fluidly coupled to the device. The test compound is optionally a drug, a potential drug, a chemical compound, an enzyme, a protein, a nucleic acid, or the like. The sample is optionally divided mto n portions, n ranging from about 2 to about 100. The portions are optionally used at their initial concentration or diluted as descπbed above

Each portion is flowed through one of x assay channels, e.g., parallel assay channels, x ranging from about 1 to about 100 As used herein, "assay channel" refers to a microscale channel, e.g.. in a parallel configuration, that is used for performing assays, tests, screens, or the like, e.g., biochemical assays, on a vanety of compounds, e.g.. chemical or biochemical compounds The flow reduction channels of the present invention are optionally used as assay channels. Portions of the sample or test compound are optionally flowed into the assay channels simultaneously to perform multiple simultaneous assays Once the sample portions are flowed into an assay channel, one or more reagents are added to each channel, thereby combining the reagents and the sample portions and performing an assay. A different reagent is optionally added to each assay channel, thereby performing y different assays in the x assay channels, y being between about 1 and about 100. The assays are optionally performed simultaneously, e.g., the vanous sample portions are concurrently flowed through the channels and concurrently reacted with one or more reagents, e.g., different reagents. The reagents are optionally used to perform HSA binding assays, target screens, drug screens, enzyme assays, fluorescence assays, dose- response assays, selectivity screens, protease assays, binding assays, and the like. An alternate method of performing different assays or subjecting a single compound to multiple chemistnes compπses using different channel chemistnes in each of the assay channels.

Each assay results in z products, z ranging from about 1 to about 1000. For example, each reaction produces one or more products, the products in each of the x channels from y assays are optionally simultaneously detected as they flow through a detection region, e.g., a detection region that is proximal to all x channels. In this manner, multiple assays are performed simultaneously on a single test compound using the devices and the methods of the present invention. V. Assays that are optionally performed using the devices of the invention.

A device, such as the one Figure 8, is optionally used to test compounds for inhibition of a target enzyme in a continuous-flow enzyme inhibition assay A device, such as that in Figure 8, incorporates a capillary attached to the microfluidic body structure at capillary attachment point 802, 802B to serve as a sample injection port, and four parallel reaction/processing channels, e.g., channel regions 804, 804B, 808, 808B, 810, 810B,and 812, 812B The channel regions incorporate one or more fixed-dilution stages that vary the final concentration of the test compound over a three order of magnitude range In addition, the four parallel channel regions compπse an assay region, in which the compounds are mixed with enzyme and substrate, and detection window 832, 832B, in which the four channel regions 804, 804B, 808, 808B, 810, 810B, and 812, 812B are brought into close proximity to facilitate simultaneous monitoπng of the resultant fluorescence using imaging or scanning techniques

Test compounds are injected into the microfluidic device via a sample capillary by applying a vacuum at waste well 806, 806B. In channel region 804, 804B, the main channel region, the test compound is brought into the device and mixed with enzyme and substrate without further dilution Channel region 808, 808B incorporates one dilution stage to accomplish a 10-fold reduction in concentration by adding additional assay buffer from reservoir 816, 816B and allowing the components to mix and drawing off additional fluid mto a waste channel to keep the overall flow rate constant. Similarly, channel region 810, 810B and 812, 812B incorporate two and three dilution stages respectively to accomplish 100-fold and 1000-fold reductions in concentration of the test compound By keeping the waste streams from the dilution stages separate instead of connected as shown in the figure, the test compounds are optionally monitored for autofluorescence (because the waste channels are also imaged or scanned) and this data is optionally used to correct raw fluorescence data for the assay before calculation of % inhibition.

To further modulate the flow rate, the channel drmensions are optionally adjusted, e.g., by incorporating loops and serpentine features, varyrng wrdths, depths, to vary relative hydrodynamic resrstances n such a way that the desired dilution factors, flow rates, and mixing times are achieved. The sample injection circuit is optionally adjusted in such a way that the test compound solution reaches the parallel channel regions simultaneously. Similarly, the overall length of the parallel channel regions (and waste streams if desired) are optionally adjusted so that the samples reach the detection window in synchronized fashion Similar designs are optionally used in kmase assays, binding assays, cell-based assays, etc.

Alternatively, the devices of the invention are used to concurrently perform multiple assays. For example, an HSA assay and a high throughput target screen are optionally performed m combination, e.g.. on a single sample plug divided into portions that are flowed into the vanous assay channels of the invention, each undergoing a different reaction chemistry. Alternatively, mtnnsic drug fluorescence is measured in combination with a high throughput target screen or a panel of similar enzymes is concurrently evaluated in a selectivity screen. In other embodiments, a dose-response expeπment is performed using in-line dilutions as descnbed above, to study a whole range of dissociation constants with a single sample.

In another embodiment, the same reaction is performed in each channel, the only difference being, e.g., a different enzyme isoform. For example, single nucleotide polymorphisms (SNP)are currently known and quickly being determined for all enzymes, i.e., gene products. The enzymes are optionally drug targets, enzymes important in metabolism, or the like, e.g., P450 enzymes. The enzymes are typically screened against potential drugs or drug compounds, e.g., in a high throughput format. Performing a screen in parallel versus multiple forms of an enzyme is typically probative with respect to reagent consumption and time Differences in individual enzymes, e.g., p450 enzymes, can be an important factor impacting differential drug-drug interactions and side-effects of drugs. The devices of the present invention are optionally used to simultaneously screen all desired forms of an enzyme or protein in parallel using one sample. This allows, e.g., consideration of key SNP differences between individual compounds early in the drug discovery process, e.g., before costly ensuing phases including clinical tnals with human subjects. In addition such methods are useful in high-throughput target screening and m non-target dependent high throughput screening.

Different reactions are performed concurrently in the same device, e.g., by the incorporation of multiple reagent wells to add different reagents to each channel as descπbed above. For example, reservoir 822 optionally adds reagents to channel region 804, reservoir 820 to channel 808, reservoir 826 to channel 810 and reservoir 824 to channel 812. Channels 842, 844, and 846 are optional since multiple reactions are optionally performed in a device without the flow reduction channels. Alternatively, the flow reduction channels are used as additional assay channels or to dilute the sample portions before subjecting them to the various assays.

In other embodiments, multiple assays are concurrently carried out in a single device with a single sample or test compound by altering channel chemistries of the various assay channels of the device, e.g., by using different channel coatings, materials, and the like, in the different assay channels. For example, one channel optionally contains a separation matrix or optionally comprises functionalized glass, e.g., silanized glass. In some embodiments, different reagents, beads comprising different reagents, or the like are pre-loaded into the channels to provide different chemistries. Surface modification of polymeric substrates may take on a variety of different forms, including coating with an appropriately charged material, derivatizing molecules present on the surface to yield charged groups on that surface, and/or coupling charged compounds to the surface. For descriptions regarding application and use of channel coatings, see, e.g., U.S. Patent No. 5,885,470, by Parce et al., entitled "Controlled Fluid Transport in Microfabricated Polymeric Substrates" and published PCT application WO 98/46438 of the same name.

VI. Suppression of pressure perturbations due to spontaneous injection into a microfluidic device.

Spontaneous injection typically occurs in microfluidic systems utilizing an external capillary, e.g., to transport samples from a sample plate into the microfluidic device. As used herein, the phrase "spontaneous injection" refers to the action of fluid or other material to move into a given passage or conduit under no externally applied forces, e.g., applied pressure differentials, applied electric fields, etc. Typically, and as used herein, spontaneous injection refers to the action of fluids at the tip of a fluid-filled capillary channel in moving into the channel as a result of capillary action within the channel, surface tension on the fluid outside the channel, or the like. Thus, a fluid or other material that is "spontaneously injected" into a channel, chamber or other conduit, moves into that channel, chamber or other conduit without the assistance of an externally applied motive force.

The phenomenon of spontaneous injection is generally viewed as a problem in capillary electrophoresis applications as it presents a constant volume error in sampling (independent of sampled volume) that can vary depending upon the geometry of the capillary channel and channel tip. Methods for reducing or eliminating this effect are provided in, e.g., USSN 09/416,288, "External Material Accession Systems" by Chow et al . which also provides methods for exploiting this phenomenon to provide improved sample accession, e g , sampling extremely small volumes of fluid

Spontaneous injection into a microfluidic device compnsing an extemal sipper capillary is also known to induce a perturbation in flow rate under a substantially constant dπving force, such as pressure, e.g . a single vacuum source, or electrok etically dnven flow As a sipper capillary is lifted out of a fluid reservoir, e g , a sample well, the curvature of the drop of liquid at the end of the capillary exerts an additional pressure inward into the capillary, resulting in a higher flow rate The perturbation in flow rate results in a perturbation in assay signal, which can interfere with quantitative analysis of the assay results See, e.g , Figure 11 For example, the perturbation in signal can obscure an inhibition in an enzymatic inhibition reaction Therefore, it is desirable to minimize the pressure perturbations due to increased flow rate when a drop of fluid is spontaneously injected into a microfluidic device

The present invention provides methods for suppressing the pressure perturbations due to spontaneous injection into a microfluidic device. The methods typically compnse dipping an open end of a capillary mto a sample source, e.g., a microwell plate, thereby drawing a sample from the sample source into the capillary The capillary is typically an external sipper capillary fluidly coupled to a microfluidic device. The method compnses withdrawing the open end of the capillary from the sample source A first portion of the sample remains on the open end and is spontaneously injected into the capillary due to surface tension exerting pressure on the capillary A second portion of the sample is flowed from the capillary into a main channel, which intersects the capillary at a first intersection point or pressure node. A third portion of the sample is flowed through a shunt channel to create a pressure differential between the first intersection point or pressure node and the open end of the capillary. The shunt channel typically intersects the main channel at the first intersection point or downstream of the first intersection point. The flow of fluid through the shunt or by-pass channel changes the pressure at the first intersection point, thereby suppressing pressure perturbations in the mam channel. The pressure at the first intersection point is optionally greater than or less than the pressure at the open end of the capillary, which is typically atmospheπc pressure.

The above method reduces the effect of spontaneous injection by flowing fluid from the sipper capillary into a by-pass channel to change the pressure points in the system For example, the pressure node at which an-on-chip reagent is mixed with or joins the reagent or sample introduced into the microfluidic device from a sipper capillary determines the extent of pressure perturbation in the microfluidic device Typically, the further away from atmosphenc pressure this pressure node is the smaller the spontaneous injection pressure perturbation effect The flow reduction channels or shunt channels of the present mvention are used to shape the pressure at this pressure node or intersection point and minimize the pressure perturbation In addition, the shunt channel is optionally a controllable channel in which the pressure is optionally adjusted to increase or decrease the pressure differential For example, a pressure source, e.g , coupled to a controller, is optionally fluidly coupled to a shunt channel to control the pressure in the shunt channel, e g , by applying a positive or negative pressure to the shunt channel Alternatively, the shunt channel is configured to provide a particular pressure differential, e.g., using width, depth, channel coatings or the like

For example, in a microfluidic device without a shunt channel, such as that shown in Figure 9A, the internal pressure node Pi is determined by the flow rate Qi, the hydrodynamic resistance Ri and the applied pressure P₀ as follows: Figure 9B shows a device with shunt channel 902 added. As fluid is flowed into shunt channel 902, e g., from capillary 904, Qi is increased to Qi', e.g., when all external pressure nodes, P₀, P₂, and P₃ are unchanged The internal pressure node Pi, at the intersection of capillary 904, shunt 902, and side channel 906 is as follows.

PΓ=P₀ +Q R,

Since QT IS greater then Qi, Pi' is also greater then Pi This pressure difference makes the device of Figure 9B more resistant to spontaneous injection pressure perturbations at P₀, e.g., intersection point 908, than the device of Figure 9A. Other benefits of the shunt design include, but are not limited to, a reduced level of relative hydrodynamic dispersion of the sample plugs through the capillary and a reduced tailing of the sample plugs through the sipper joint, connecting the capillary to the microchannel on the device

VII. Instrumentation Although the devices and systems specifically illustrated herein are generally descπbed 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 mtegration of additional operations into these devices For example, the devices and systems descnbed optionally include structures, reagents and systems for performing virtually any number of operations both upstream and downstream from the operations specifically descnbed herein Such upstream operations include sample handling and preparation operations, e.g., cell separation, extraction, punfication, amplification, cellular activation, labeling reactions, dilution, aliquotting, and the like Similarly, downstream operations may include similar operations, including, e g , separation of sample components, labeling of components, assays and detection operations, electrokinetic or pressure-based injection of components into contact with particle sets, or mateπals released from particle sets, or the like

In the present invention, mateπals such as cells, proteins, antibodies, enzymes, substrates, buffers, or the like are optionally monitored and/or detected, e.g., so that presence of a component of interest can be detected, an activity of a compound can be determined, or an effect of a modulator on. e.g , an enzyme^'s activity, can be measured Depending on the label signal measurements, decisions are optionally made regarding subsequent fluidic operations, e.g., whether to assay a particular component in detail to determine, e.g., kinetic information.

The systems descπbed herein generally include microfluidic devices, as descπbed above, in conjunction with additional instrumentation for controlling fluid transport, flow rate and direction withm 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, stoπng and interpreting the data, and providing the data and interpretations in a readily accessible reporting format.

Fluid Direction System A vaπety of controlling instrumentation is optionally utilized in conjunction with the microfluidic devices descπbed above, for controlling the transport and direction of fluidic mateπals and/or mateπals withm the devices of the present invention, e.g., by pressure-based or electrokinetic control

In the present system, the fluid direction system controls the transport, flow and/or movement of a sample through the microfluidic device. For example, the fluid direction system optionally directs the movement of a sample mto and through the main channel, where the sample is optionally diluted with a buffer or other diluent. It optionally directs movement of the buffer or other diluent from the source of the mateπal into the ma channel, resulting in a first diluted sample It also directs movement of a portion of the first diluted sample into a flow reduction channel, while a second portion of the first diluted sample remains flowing through the main channel The fluid direction system also optionally directs the movement of a second aliquot of buffer into the main channel to perform a seπal dilution, thus creating a second diluted sample Thereafter, the fluid direction system would direct a first portion of the second diluted mateπal into a second flow reduction channel, while the second portion of the second diluted mateπal remains in the main channel The fluid direction system also may iteratively repeat these movements to create more seπal dilutions of the sample matenal, reducing the pressure in the main channel after each dilution by directing a portion of the fluidic matenals mto a flow reduction channel.

After the desired dilution levels are obtained in the vaπous channels of the system, the fluid direction system optionally directs the movement of one or more reagent matenals, e.g., substrates, enzymes, and the like, from reagent reservoirs into the main channel and/or flow reduction channels to react with the sample matenals and/or diluted matenals. To decrease reagent usage the fluid direction system optionally directs movement of the diluted samples from the flow reduction channels mto secondary flow reduction channels or reaction channels. In addition, movement of the sample matenal and diluted matenals through the channels and into the detection region, where they are detected, is also controlled by the fluid direction system.

To perform multiple assays, e.g., on a single sample, the fluid direction system divides a sample into n portions and directs the n sample portions mto x assay channels, e.g., parallel assays channels such as those in Figures 8 A and 8B. The fluid direction system also optionally directs the addition of vanous reagents to each of the x channels. Different reagents are optionally added to each of the x channels, thereby exposing each portion of the sample to a different chemistry, e.g., to a different reaction or assay, thereby performing y different assays and producing z different products. The number of sample portions used, n, is typically between about 2 and about 100. Typically the number of sample portions is substantially equal to the number of channels, x. The fluid direction system typically directs one sample portion mto each different channel.

Alternatively, a device with 10 channels is used and the sample is only divided in 2 portions such that only 2 channels are used. In other embodiments, a different sample is directed into each of the 10 channels. The number of channels is typically between about 2 and about 100 Preferably, the number of channels ranges from about 2 to about 20 More preferably, the number of channels is from about 4 to about 10 channels Once the samples are in the channels, the fluid direction system directs vaπous reagents from the reservoirs into the assay channels, e g , to perform y different assays The number of different assays, \ , is typically between about 2 and about 100 However, the number of assays does not have to equal the number of channels or the number of sample portions A different assay is optionally performed in each one of the x channels For example, a number of different enzyme substrates are optionally screened simultaneously in the different channels of the invention or a number of different protein binding sites are optionally probed simultaneously by testing each binding site in a different channel Alternatively, one assay e g , site I HSA binding, is performed in some channels, e g , half of the channels, and another assay, e g , site II HSA binding, is performed in the remaining channels The y assays typically produce z different products, e g , each assay produces one or more products Therefore, the number of products is typically at least equal to the number of assays, but is often greater than the number of assays, y The number of products typically ranges from about 1 to about 10,000, more typically from about 2 to about 1000 and most typically from about 2 to about 100 Each assay typically results in one or more products, which the fluid direction system directs through the assay channels and into a detection region for detection, e.g , concurrent detection, e g , fluorescent detection, from assay channels that converge into a single detection region

To suppress pressure perturbations created by spontaneous injection of a fluid from an external capillary mto a microfluidic device, the fluid direction system, which compπses at least one fluid control element fluidly coupled to a main channel, to a shunt channel, and to a capillary, directs movement of a sample from a first sample source into an inlet region of the capillary and movement of the sample from the mlet region of the capillary to an outlet region of the capillary The sample is then directed from the outlet region of the capillary into the upstream region of a ma channel A first portion of the sample from the upstream region of the main channel is flowed mto a shunt channel, as descπbed above, and a second portion of the sample remains in the main channel This fluid movement maintains the intersection of the mam channel and the capillary at a pressure that is different from the pressure at the inlet region of the capillary The difference in pressure reduces the effect of the spontaneous injection pressure perturbations Fluid transport and direction in microfluidic devices, e.g., as descπbed above, are typically controlled in whole or in part, using pressure based flow systems that incorporate external or internal pressure sources to dnve fluid flow Internal sources include microfabncated pumps, e.g., diaphragm pumps, thermal pumps, lamb wave pumps and the like that have been descnbed in the art See, e.g., U.S. Patent Nos. 5,271,724, 5,277,556. and 5,375,979 and Published PCT Application Nos. WO 94/05414 and WO 97/02357 As noted above, the systems descπbed herein can also utilize electrokinetic mateπal direction and transport systems Preferably, external pressure sources are used, and applied to ports at channel termini More preferably, a single pressure source is used at a ma channel terminus Typically, the pressure source is a vacuum source applied at the downstream terminus of the main channel These applied pressures, or vacuums, generate pressure differentials across the lengths of channels to dnve fluid flow through them In the interconnected channel networks descπbed herein, differential flow rates on volumes are optionally accomplished by applying different pressures or vacuums at multiple ports, or preferably, by applymg a smgie vacuum at a common waste port and confrgunng the vaπous channels with appropnate resistance to yield desired flow rates. Example systems are descπbed in USSN 09/238,467, filed 1/28/99.

Typically, the controller systems are appropπately configured to receive or interface with a microfluidic device or system element as descπbed herein For example, the controller and/or detector, optionally includes a stage upon which the device of the invention is mounted to facilitate appropnate interfacing between the controller and/or detector and the device Typically, the stage includes an appropnate mounting/alignment structural element, such as a nesting well, alignment pins and/or holes, asymmetric edge structures (to facilitate proper device alignment), and the like. Many such configurations are descπbed m the references cited herein.

The controlling instrumentation discussed above is also optionally used to provide for electrokinetic injection or withdrawal of mateπal downstream of the region of interest to control an upstream flow rate. The same instrumentation and techniques descπbed above are also utilized to inject a fluid into a downstream port to function as a flow control element.

Detector

The devices herein optionally include signal detectors, e.g., which detect fluorescence, phosphorescence, radioactivity, pH, charge, absorbance, luminescence, temperature, magnetism, color, or the like Fluorescent and chemiluminescent detection are especially preferred

The detector(s) optionally monitors one or a plurality of signals from the one or more detection regions of the device, e.g., detection regions 332 or 832, 832B in Figures 3 and 8 The one or more detection regions may correspond to the vaπous sample concentrations achieved by the seπal dilutions or to the vaπous samples being assayed For example, the detector optionally monitors an optical signal that corresponds to a labeled component, such as a labeled antibody or protein located, e.g., in detection region 832, 832B. In one embodiment, the detection region spans multiple main channels and/or flow reduction channels and one detector is used to detect signal from all channels concurrently In Figure 8, for example, detection region 832, 832B monitors signals from ma channel region 804, 804B, which contains, e.g.. an undiluted sample, and parallel regions 808, 808B. 810, 810B, and 812, 812B, which contain e.g., a 1 : 10 dilution of the sample mateπal, a 1: 100 dilution of the sample mateπal, and a 1:1000 dilution of the sample matenal Alternatively, a single detector proximal to each of 2 or more assay channels, e.g., parallel assay channel regions, detects the results of the two or more different assays, e.g., performed on the same sample which has been divided into the 2 or more channels to undergo multiple assays The results of a plurality of enzyme assays, e.g., producing different products, are optionally detected simultaneously by a detector placed proximal to all relevant assays channels For example, the results of HSA binding to site I and site II are optionally detected concurrently when srmultaneously probed in two different channels.

Alternatively, if the flow reduction channels are not proximal to one another, a separate detector is optionally used to detect the signal from each channel. For example, m Figure 6 the channels are optionally configured so that the detectors are not proximal to one another. If the channels loop around to make the detectors proximal to each other, then a single detector would suffice. When a single detector does not detect all the signals or when different types of detection are required the channel configuration of Figure 6 is optionally used. Detectors are placed proximal to detection region 632 to detect a signal from mam channel 604, detection region 634 to detect a signal from flow reduction channel

612, detection region 636 to detect a signal from flow reduction channel 610, and detection region 638 to detect a signal from flow reduction channel 608. Alternatively, a single detector is moved between detection regions 632, 634, 636, and 638. In the above case detection optionally works as follows: an undiluted, unreacted sample is detected in detection region 632, an undiluted reacted sample in detection region 634, a twice diluted reacted sample in detection region 636, and a sample that has been diluted once and reacted with the reagents in detection region 638. Once detected, the flow rate and velocity of cells in the channels is also optionally measured and controlled as described above.

Examples of detection systems useful in the present invention 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 "proximal," to a particular element or region, 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 to 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. Example detectors include photo multiplier tubes, a CCD array, a scanning detector, a galvo-scanner or the like. For example, in preferred aspects, a fluorescence, chemiluminescence or other optical detector is used in the assay. Proteins, antibodies, or other components which emit a detectable signal can be flowed past the detector, or, alternatively, the detector can move relative to an array to determine protein position (or, the detector can simultaneously monitor a number of spatial positions corresponding to channel regions, e g , as in a CCD array)

In preferred aspects, the detector measures an amount of light emitted from the matenal, such as a fluorescent or chemiluminescent matenal As such, the detection system will typically include collection optics for gathenng a light based signal transmitted through the detection window, and transmitting that signal to an appropnate light detector Microscope objectives of varying power, field diameter, and focal length are readily utilized as at least a portion of this optical train The light detectors are optionally 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 a computer (descnbed in greater detail below), via an analog to digital or digital to analog converter, for transmitting detected light data to the computer for analysis, storage and data manipulation. In the case of fluorescent mateπals such as labeled cells, the detector typically includes a light source which produces light at an appropnate wavelength for activating the fluorescent mateπal, as well as optics for directing the light source through the detection window to the mateπal contained in the channel or chamber. The light source can be any number of light sources that provides an appropnate wavelength, including lasers, laser diodes and LEDs Other light sources are required for other detection systems For example, broad band light sources are typically used in light scattenng/transmissivity detection schemes, and the like Typically, light selection parameters are well known to those of skill in the art.

The detector can 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 a computer (descπbed below), by permitting the use of few or a single communication port(s) for transmittrng information between the controller, the detector and the computer Integration of the detection system with a computer system typically includes software for converting detector signal information into assay result information, e.g., concentration of a substrate, concentration of a product, presence of a compound of interest, or the like Computer

As noted above, either or both of the fluid direction 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 analog to digital or digital to analog 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, 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. For example, the software optionally directs the fluid direction system to transport the sample to the main channel, the buffer or other diluent to the main channel, a portion of the sample or diluted sample to a flow reduction channel, a portion of the sample or diluted material through the main channel, a portion of a sample or diluted material into a secondary flow reduction channel, a reagent material into the main channel, a flow reduction channel or a secondary flow reduction channel, and any other movement necessary to perform the assay of interest and/or detect a component of interest.

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.

In the present invention, the computer typically includes software for the monitoring of materials in the channels. Additionally the software is optionally used to control electrokinetic or pressure-modulated injection or withdrawal of material. The injection or withdrawal is used to modulate the flow rate as described above. In addition, the computer optionally includes software for deconvolution of the signal or signals from the detection system. For example, the deconvolution distinguishes between two detectably different spectral characteristics that were both detected, e.g., when a substrate and product comprise detectably different labels. Example Integrated System

Figure 1. Panels A, B, and C and Figure 2 provide additional details regarding example integrated systems that are optionally used to practice the methods herein. As shown, body structure 102 has main channel 104 disposed therein. A sample or mixture of components is optionally flowed from pipettor channel 120 towards reservoir 114. e.g., by applying a vacuum at reservoir 114 (or another point in the system) or by applying appropnate voltage gradients. Alternatively, a vacuum is applied at reservoirs 108, 112 or through pipettor channel 120 Additional matenals, such as buffer solutions, substrate solutions, enzyme solutions, and the like, as descnbed above are optionally flowed from wells 108 or 112 and into mam channel 104. Flow from these wells is optionally performed by modulating fluid pressure, or by electrokinetic approaches as descnbed (or both) As fluid is added to main channel 104, e.g., from reservoir 108, the flow rate increases The flow rate is optionally reduced by flowing a portion of the fluid from ma channel 104 into flow reduction channel 106 or 110. The arrangement of channels depicted in Figure 1 is only one possible arrangement out of many which are appropnate and available for use in the present invention. Alternatives are provided in Figures 3, 4, 5, 6, 7, 8, 9, 10 and 12 Additional alternatives can be devised, e.g., by combining the microfluidic elements descπbed herein, e.g., flow reduction channels, with other microfluidic devices descnbed in the patents and applications referenced herein. Furthermore the elements of Figures 3, 4, 5, 6, 7, 8, 9, 10 and/or 12 are optionally recombmed to provide alternative configurations.

Samples and mateπals are optionally flowed from the enumerated wells or from a source external to the body structure. As depicted, the integrated system optionally includes pipettor channel 120, e.g., protruding from body 102, for accessing a source of mateπals external to the microfluidic system. Typically, the external source is a microtiter dish or other convenient storage medium. For example, as depicted in Figure 2, pipettor channel 120 can access microwell plate 108. which includes sample matenals, buffers, substrate solutions, enzyme solutions, and the like, in the wells of the plate. Detector 206 is in sensory communication with channel 104, detecting signals resulting, e.g., from labeled matenals flowing through the detection region. Detector 206 is optionally coupled to any of the channels or regions of the device where detection is desired. Detector 206 is operably linked to computer 204, which digitizes, stores, and manipulates signal information detected by detector 206, e.g., using any of the instructions descnbed above, e.g., or any other instruction set, e.g , for determining concentration, molecular weight or identity, or the like.

Fluid direction system 202 controls voltage, pressure, or both, e.g., at the wells of the systems or through the channels of the system, or at vacuum couplings fluidly coupled to channel 104 or other channel descnbed above Optionally, as depicted, computer 204 controls fluid direction system 202. In one set of embodiments, computer 204 uses signal information to select further parameters for the microfluidic system. For example, upon detecting the presence of a component of interest in a sample from microwell plate 208, the computer optionally directs addition of a potential modulator of component of interest into the system.

Generally, the microfluidic devices descπbed herein are optionally packaged to include reagents for performing the device's preferred function. For example, the kits can include any of microfluidic devices descπbed along with assay components, reagents, sample mateπals, proteins, antibodies, enzymes, substrates, control matenals, or the like. Such kits also typically include appropnate instructions for using the devices and reagents, and in cases where reagents are not predisposed in the devices themselves, with appropnate instructions for introducing the reagents into the channels and/or chambers of the device. In this latter case, these kits optionally include special ancillary devices for introducing mateπals mto the microfluidic systems, e.g., appropπately configured syπnges/pumps, or the like (in one embodiment, the device itself compnses a pipettor element, such as an electropipettor for introducing matenal mto channels and chambers within the device). In the former case, such kits typically include a microfluidic device with necessary reagents predisposed in the channels/chambers of the device.

Generally, such reagents are provided in a stabilized form, so as to prevent degradation or other loss dunng prolonged storage, e.g., from leakage. A number of stabilizing processes are widely used for reagents that are to be stored, such as the inclusion of chemical stabilizers (i.e., enzymatic inhibitors, microcides/bactenostats, anticoagulants), the physical stabilization of the mateπal, e.g., through immobilization on a solid support, entrapment in a matπx (i.e., a gel), lyophilization, or the like. Kits also optionally include packaging mateπals or containers for holding microfluidic device, system or reagent elements

The discussion above is generally applicable to the aspects and embodiments of the invention descπbed in the claims. Moreover, modifications can be made to the method and apparatus descnbed herein without departing from the spint and scope of the invention as claimed, and the invention can be put to a number of different uses including the following:

The use of a microfluidic system for performing the assays set forth herein

The use of a microfluidic system for obtaining measurements at multiple concentrations as descπbed herein

The use of a microfluidic system for performing an assay at multiple concentrations as descπbed herein

The use of a microfluidic device for performing multiple assays as descπbed herein. The use of a microfluidic device for performing concurrent multrple assays on a single test compound as descπbed herein.

The use of a microfluidic system for reducing reagent consumption as descπbed herein.

The use of a microfluidic system as descπbed herein for performing seπal dilutions of a sample.

The use of a microfluidic system or device for controlling or modulating flow rate as descnbed herein.

The use of a microfluidic system or device for suppressing pressure perturbations due to spontaneous injection as descnbed herein. An assay utilizing a use of any one of the microfluidic systems or substrates descnbed herein.

VIII. Examples

Example 1 : Phosphatase Assays A microfluidic device compnsing the channel layout shown in Figure 8B was used to obtain data in a phosphatase assay. In a typical assay, approximately 60 microliters of enzyme solution are loaded into wells 820b and 824b. Substrate is added into wells 822b and 826b and wells 816b and 818b are loaded with buffer. The final concentration of reagents delivered to the main reaction channel is determined by the relative flow rates in the side channels and the reaction channels Reaction times on the chip are vaned either by moving the detection point to different locations along the reaction channels or by varying the negative pressure applied at waste well 806b. Reagents

Inhibitors, e.g , commercially available phosphatase inhibitors, at vaπous concentrations are loaded into a microtiter plate for loading into the device through a sipper capillary attached at capillary attachment point 802b For example, typical inhibitor concentrations include 1 mM. 0.625 mM, 1.25 mM, 2 5 mM, 5 mM, and 10 mM A typical buffer used for phosphatase reactions compnses 25 mM Tπs-HCl. pH 7.0, 50 mM NaCl, 2 mM EDTA, 0.01% Bπj 35, 5 mM DTT, and 500 NDSB.

For enzymes, a 1 100 dilution of phosphatase is prepared (approximately 100 nm) is typically placed in the well of the microfluidic device

A 100 μM phosphatase substrate solution is placed in substrate wells. A 1 nM solution of marker dye Cy5 from Beckman is placed in the microtiter plate along with the inhibitor samples Enzyme Inhibition and K, determination

Inhibitor titration expenments were earned out using a competitive peptide phosphatase inhibitor with high affinity for the phosphatase being assayed The peptide concentrations typically range from 0.625 to 10 mM in the microplate. Percent inhibition values were calculated from the decrease in fluorescence corresponding to the inhibitor injection, the enzyme + substrate baseline, and the measured substrate-only background. Analysis of this data (with the 10 highest inhibitor concentrations from 100% and 10% channel excluded from the analysis because they gave near-saturating responses) yielded a K, value of 169.55 μM when analyzed using a Dixon plot. From the plot of inhibitor concentration versus percent inhibition, the IC₅₀ value of 160 and 200 μM were estimated when using substrate concentration of 100 μM and 50 μM in the substrate well of the device, and the K, value of 113 μM and 109 μM were calculated using the following equation: IC₅₀ = K₁* (1 + [S] K_m)

Inhibitor titration measured m 4 different assay channels using 2 different substrate concentrations is shown m Figure 15. The Dixon plot used to determine K_! is shown Figure 16 and Figure 17, which provide inhibitor channel concentration data plotted against percent inhibition.

Example 2 Suppression of Pressure Perturbations A microfluidic device compnsing the channel layout shown in Figure 10, is typically used for high throughput screening assays, e.g., fluorogenic assays In a typical expenment, an enzyme is placed in well 1004 and a fluorogenic substrate is placed in well 1005 Potential inhibitors are placed in a microwell plate and brought onto the device through a capillary attached to the device at capillary connection point 1008 As descπbed above, spontaneous injection occurs as the capillary is lifted from one well in the microwell plate and moved to another well The effect of spontaneous injection on the dilution factor for the reagents added from internal wells, e.g , wells 1004 and 1005, is characteπzed by placing a dye, e.g., a fluorescent dye, in a buffer solution in wells 1004 and 1005 on the device and moving the capillary back and forth between 2 microplate wells containing the same buffer solution.

Figure 11 provides the corresponding fluorescent signal measured when a vacuum positioned at waste well 1010 is set at -1 psi. The double dip feature in the data results from the perturbation to the steady state value due to spontaneous injection of buffer from the capillary mto the microfluidic device. The magnitude of the dips is about 9% of the steady state value.

The microfluidic device channel layout shown in Figure 12 contains a modification that is optionally used to suppress the pressure perturbations descnbed above and illustrated by the data in Figure 11. The channel configuration contains a by-pass or shunt channel 1202 used to draw fluid from main channel 1210. A fluorogenic assay is optionally performed in the device of Figure 12 in the same manner as that descπbed above for the device in Figure 10. Enzyme and substrate are introduced into ma channel 1210 from wells 1204 and 1205. In main channel 1210, the enzyme and substrate contact a sample brought in from an external capillary via capillary connection point 1208. Pressure at capillary connection point 1208 is controlled by the shunting of fluid from main channel 1210 to shunt channel 1202.

Flowing fluid through shunt channel 1204 alters the pressure at the intersection point to reduce the effect of spontaneous injection. For example, when characteπzed using the dyes and buffers as descnbed above for the device of Figure 10, the signal produced using the device of Figure 12 results in dips having a magnitude that is onh 3% of the steady state value

Figures 13 and 14 provide data for a fluorogenic enzyme assay performed using a device without a shunt channel (Figure 10) and a device with a shunt channel (Figure 12), respectively. An approximately 10 nM solution of a phosphatase enzyme was placed in well 1204 and 100 μM phosphatase substrate was placed in well 1205 The buffer used was 50 MM Bis-Tπs at pH 6.3, 50 mM NaCl, 0.075% BSA, 0.1 % Bπj-35, and 2% DMSO Inhibitors of phosphatases were provided at 10 different concentrations 0 156, 0 313, 0.625, 1.25, 5, 10, 20, 40, and 80 μM The inhibitor samples were placed in a microwell plate and flowed into the device from the microwell plate via a sipper capillary attached at capillary connection point 1208 Applied pressure was set at -1.5 psi at waste reservoir 1212 The penodic wiggles or dips in the steady state signal due to spontaneous injection were much reduced in amplitude in the device compπsing a shunt channel

While the foregoing invention has been descnbed in some detail for purposes of claπty and understanding, it will be clear to one skilled m the art from a reading of this disclosure that vanous changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus descnbed above may be used in vaπous combinations. All publications, patent applications, patents, and other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document were individually so denoted

Claims

WHAT IS CLAIMED IS.

1. A microfluidic device compπsing:

(1) a body structure compπsing a main channel disposed therein; the body structure further compnsing- (a) one or more flow reduction channels that intersect the main channel at one or more intersection points, which one or more flow reduction channels are structurally configured to reduce flow rate of a fluid in the mam channel as the fluid flows past the one or more intersection points, (b) a first pressure source fluidly coupled to the one or more flow reduction channels, which first pressure source is positioned on the one or more flow reduction channels downstream of the one or more intersection points in a direction of flow toward the first pressure source; and, (n) a ma pressure source fluidly coupled to the main channel

2. The device of claim 1, wherein the first pressure source and the main pressure source are the same.

3. The device of claim 1, wherein the first pressure source and the main pressure source compnse a single vacuum source.

4. A mrcroflurdic device compπsing:

(I) a body structure compnsing a mam channel disposed therein; the body structure further compnsing: one or more flow reduction channels that intersect the main channel at a first intersection point and a second intersection point; which one or more flow reduction channels are structurally configured to reduce flow rate of a fluid in the mam channel as the fluid flows past the first intersection point in a direction toward the second intersection point; and,

(n) a main pressure source fluidly coupled to the mam channel.

5. The device of claim 1 or claim 4, wherein the one or more flow reduction channels are structurally configured to reduce flow rate of the fluid in the main channel as the fluid flows past the one or more intersection points.

6. The device of claim 1 or claim 4. compπsing 2 or more flow reduction channels, which flow reduction channels are in fluid communication with the main channel

7. The device of claim 1 or claim 4, compnsing about 3 to about 4 flow reduction channels, which flow reduction channels are in fluid communication with the main channel.

8. The device of claim 1 or claim 4, compnsing between about 5 and about 10 flow reduction channels, which flow reduction channels are m fluid communication with the mam channel.

9. The device of claim 1 or claim 4, compπsing about 10 or more flow reduction channels, which flow reduction channels are in fluid communication with the ma channel

10. The device of claim 1 or claim 4, wherein the main channel has a first cross-sectional dimension and the one or more flow reduction channels have a second cross- sectional dimension, and wherein the first cross-sectional dimension and the second cross- sectional dimension are the same.

11. The device of claim 1 or claim 4, wherein the mam channel has a first dimension and the one or more flow reduction channels have a second dimension, and wherein the first dimension and the second dimension are different.

12. The device of claim 11, wherein the second dimension is smaller than the first dimension.

13. The device of claim 1 or claim 4, wherein the one or more flow reduction channels are unintersected channels.

14. The device of claim 1 or claim 4, wherein the one or more flow reduction channels are intersected by one or more secondary flow reduction channels, which secondary flow reduction channels have a cross-sectional dimension smaller than the cross- sectional dimension of the one or more flow reduction channels.

15. The device of claim 1 or claim 4. wherein the main pressure source is fluidly coupled to the ma channel at a position downstream from the one or more flow reduction channels in a direction of flow toward the mam pressure source

16. The device of claim 1 or claim 4. wherein the main pressure source is a vacuum source

17. The device of claim 1 or claim 4. wherein the main pressure source is a single vacuum source

18. The device of claim 1 or claim 4, further compπsing one or more detection regions within or proximal to the one or more flow reduction channels

19. The device of claim 1 or claim 4, further compπsing a detection system compnsing one or more detectors located proximal to both the main channel and at least one of the one or more flow reduction channels

20. The device of claim 19, wherein the one or more detectors compnses a single detector, which detector detects a signal simultaneously in each of the one or more flow reduction channels.

21. The device of claim 19, further compnsing a computer and software operably coupled to the device for analyzing signals detected by the detection system

22. The device of claim 1 or claim 4, the device further compnsing a source of a first fluidic matenal in fluid communication with the main channel at a first position along the main channel; and, a source of a second fluidic mateπal in fluid communication with the main channel at a second position along the main channel.

23. The device of claim 22, wherein the first position and the second position are upstream from at least one of the one or more flow reduction channels

24. The device of claim 22, wherein the first position is upstream from at least one of the one or more flow reduction channels and the second position is downstream from the at least one of the one or more flow reduction channels.

25. The device of claim 22, wherein the first position and the second position are downstream from at least one of the one or more flow reduction channels.

26. The device of claim 22, wherein the first fluidic mateπal and the second fluidic mateπal are the same matenal.

27. The device of claim 22, wherein the first fluidic mateπal and the second fluidic matenal are different

28. The device of claim 22, wherein the first fluidic matenal is a sample mateπal and the second fluidic mateπal is a diluent matenal

29. The device of claim 28, wherein the diluent is a buffer

30. The device of claim 22, wherein the device further compnses a fluid direction system, which fluid direction system directs one or more of

(a) movement of the first fluidic mateπal from the source of the first fluidic matenal and the second fluidic mateπal from the source of the second fluidic mateπal to the main channel, combining the first fluidic mateπal with the second fluidic mateπal to form a third fluidic mateπal;

(b) movement of a first portion of the third fluidic matenal from the main channel to at least one of the one or more flow reduction channels, a second portion of the third fluidic mateπal remaining in the mam channel; and,

(c) movement of the second portion of the third fluidic matenal through the main channel.

31. The device of claim 30, wherein, dunng operation of the device, the first fluidic matenal has a first flow rate and the third fluidic matenal has a second flow rate; and wherein the second flow rate increases after combining the first fluidic matenal with the second fluidic matenal, resulting in a higher flow rate than the first flow rate; and wherein the second flow rate decreases after the movement of the first portion of the third fluidic mateπal from the mam channel to the at least one of the one or more flow reduction channels

32. The device of claim 31 wherein, dunng operation of the device, the second flow rate decreases to substantially the same as the first flow rate or less than the first flow rate after movement of the first portion of the third fluidic matenal from the main channel to the at least one of the one or more flow reduction channels

33. The device of claim 30, wherein the flow rate of the third fluidic mateπal is modulated by a change in pressure due to the movement of the first portion of the third fluidic mateπal from the mam channel into the at least one of the one or more flow reduction channels

34. The device of claim 30, wherein the device further compπses a source of a fourth fluidic matenal in fluid communication with the main channel at a third position, which third position is downstream of the one or more flow reduction channels, and wherein the fluid direction system directs movement of the fourth fluidic matenal from the source of the fourth fluidic mateπal into the ma channel

35. The device of claim 34, wherein the fourth fluidic matenal is a reagent matenal

36. The device of claim 35, wherein the system provides reduced consumption of the reagent mateπal by reducing the flow rate of the third fluidic matenal pπor to the movement of the reagent matenal into the mam channel

37. The device of claim 34, wherein the device further compnses a detection region within or fluidly coupled to the main channel at a fourth position, which fourth position is downstream of the third position

38. The device of claim 22, the device further compnsing a source of a third fluidic matenal in fluid communication with the one or more flow reduction channels at a first position along the one or more flow reduction channels, and, a source of a fourth fluidic matenal m fluid communication with the one or more flow reduction channels at a second position along the one or more flow reduction channels

39. The device of claim 38, wherein the third fluidic matenal and the fourth fluidic mateπal are reagent mateπals

40. The device of claim 38. wherein the first fluidic matenal is a sample mateπal. the second fluidic mateπal is a buffer matenal, the third fluidic mateπal is a substrate matenal, and the fourth fluidic matenal is an enzyme mateπal.

41. The device of claim 38, wherein the device further compπses a fluid direction system, which fluid direction system directs one or more of: movement of the first fluidic mateπal dunng operation of the device from the source of the first fluidic matenal and the second fluidic matenal from the source of the second fluidic mateπal to the main channel, combining the first fluidic mateπal with the second fluidic mateπal to form a diluted mateπal; movement of a first portion of the diluted mateπal dunng operation of the device from the main channel to at least one of the one or more flow reduction channels, a second portion of the diluted matenal remaining in the main channel; movement of the second portion of the diluted matenal dunng operation of the device through the main channel; movement of the fourth fluidic matenal dunng operation of the device into at least one of the one or more flow reduction channels; movement of the fourth fluidic matenal dunng operation of the device into the main channel; movement of the fifth fluidic mateπal dunng operation of the device mto at least one of the one or more flow reduction channels; movement of the fifth fluidic matenal dunng operation of the device into the main channel.

42. The device of claim 41, wherein the fluid direction system directs the movement of the fourth fluidic mateπal and the fifth fluidic mateπal into at least one of the one or more flow reduction channels, combining the fourth flurdic matenal, the fifth fluidic matenal and the diluted mateπal to form a reacted matenal.

43. The devrce of claim 22. wherein the device further compπses a fluid direction system, which fluid direction system directs one or more of:

(a) movement of the first fluidic mateπal from the source of the first fluidic matenal mto the mam channel; (b) movement of a first portion of the first fluidic mateπal from the main channel into at least one of the one or more flow reduction channels, a second portion of the first fluidic mateπal remaining m the main channel; (c) movement of the second fluidic mateπal from the source of the second fluidic mateπal into the ma channel, combining the second fluidic matenal and the second portion of the first fluidic matenal to form a third fluidic mateπal; and, (d) movement of the third fluidic matenal through the main channel.

44. The device of claim 43, wherein dunng operation of the device, the first fluidic matenal has a first flow rate, the second portion of the first fluidic matenal has a second flow rate, and the third fluidic mateπal has a third flow rate, wherein the second flow rate decreases after movement of the first portion of the first fluidic mateπal into the at least one of the one or more flow reduction channels, resulting in a lower flow rate than the first flow rate, and the third flow rate increases after combining the second fluidic matenal and the second portion of the first fluidic matenal, resulting in a higher flow rate than the second flow rate.

45. The device of claim 44, wherein the third flow rate increases to a flow rate substantially equal to the first flow rate.

46. The device of claim 1, wherein the mam channel compnses a first parallel region, a second parallel region, a third parallel region and a fourth parallel region fluidly connected to the main pressure source and to a source of a sample matenal; the one or more flow reduction channels compnse: a first flow reduction channel fluidly coupled to the first parallel region; a second flow reduction channel fluidly coupled to the second parallel region; a third flow reduction channel fluidly coupled to the third parallel region; the first pressure source and the mam pressure source are the same; and, wherein the device further compπses: a source of a diluent mateπal in fluid communication with the first parallel region, the second parallel, region, and the third parallel region; one or more sources of one or more reagent materials in fluid communication with the first parallel region, the second parallel, region, the third parallel region, and the fourth parallel region; and, a detection region proximal to the main channel, the first flow reduction channel, the second flow reduction channel, the third flow reduction channel, the first parallel region, the second parallel region, and the third parallel region.

47. The device of claim 4, wherein the main channel comprises an upstream end and a downstream end; and, wherein the one or more flow reduction channels comprise: a first flow reduction channel that intersects the main channel at a first position and a second position; a second flow reduction channel that intersects the main channel at a third position and a fourth position, which third position and fourth position are positioned on the main channel between the first position and the second position; a third flow reduction channel that intersects the main channel at a fifth position and at a sixth position, which fifth position and sixth position are positioned on the main channel between the third position and the fourth position; and, wherein the main pressure source is a vacuum source fluidly coupled to the main channel at the downstream end; and, wherein the device further comprises: a source of a sample material fluidly coupled to the main channel at the upstream end; a first source of a diluent material in fluid communication with the main channel at a seventh position, which seventh position is between the source of the sample material and the first position; a second source of the diluent material in fluid communication with the main channel at an eighth position, which eighth position is between the first position and the second position; a third source of a the diluent material in fluid communication with the main channel at a ninth position, which ninth position is between the third position and the fifth position; one or more sources of one or more reagent materials in fluid communication with the main channel and the one or more flow reduction channels; and, a detection region proximal to the first flow reduction channel, the second flow reduction channel, the third flow reduction channel, and the main channel

48. The device of claim 47, wherein the device further compnses one or more reaction channels, which reaction channels intersect the one or more flow reduction channels, and wherein the one or more sources of the one or more reagents are in fluid communication with the one or more reaction channels

49. The device of claim 48, wherein the one or more reaction channels compπse a first cross-sectional dimension and the one or more flow reduction channels compnse a second cross-sectional dimension, wherein the first cross-sectional dimension is smaller than the second cross-sectional dimension

50. A method for modulating a volumetnc flow rate of a fluid in a channel of a microfluidic system, the method compnsing

(0 providing a body structure compnsing a plurality of microscale channels disposed therein, the plurality of microscale channels compnsing: (a) a main channel; and,

(b) one or more flow reduction channels that intersect the mam channel at one or more intersection points, which one or more flow reduction channels are structurally configured to reduce flow rate of a fluid in the main channel as the fluid flows past the one or more intersection points;

(n) flowing a first fluidic mateπal through the mam channel;

(iii) flowing a second fluidic matenal into the ma channel; resulting m a third fluidic matenal; (IV) flowing a first portion of the third fluidic mateπal through the one or more flow reduction channels; and,

(v) flowing a second portron of the third fluidic matenal through the ma channel, thereby modulating the flow rate of the third fluidic matenal in the main channel and in the one or more flow reduction channels.

51. The method of claim 50, further compπsing structurally configunng the one or more flow reduction channels to reduce flow rate of the fluid in the main channel as the fluid flows past the one or more intersection points

52. The method of claim 50, wherein the body structure compnses 2 or more flow reduction channels in fluid communication with the mam channel, and wherein the method compπses iteratively repeating step (n) through step (v)

53. The method of claim 52, further compπsing structurally configunng the body structure to compnse about 3 to about 4 flow reduction channels in fluid communication with the main channel

54. The method of claim 52, further compnsing structurally configunng the body structure to compπse between about 5 and about 10 flow reduction channels in fluid communication with the mam channel

55. The method of claim 52, further compπsing structurally configunng the body structure to compπse about 10 or more flow reduction channels in fluid communication with the main channel.

56. The method of claim 50, further compπsing structurally configunng the mam channel to have a first dimension and the one or more flow reduction channels to have a second dimension, wherein the first dimension and the second dimension are different.

57. The method of claim 56, wherein the second dimension is smaller than the first dimension.

58. The method of claim 50, further compπsing providing a main pressure source operably coupled to the main channel

59. The method ol claim 58, wherein the mam pressure source compπses a vacuum source.

60. The method of claim 58, wherein the main pressure source compnses a single vacuum source.

61. The method of claim 50, further compπsing providing a first pressure source fluidly coupled to the one or more flow reduction channels, which first pressure source is positioned on the one or more flow reduction channels downstream of the one or more intersection points in a direction of flow toward the first pressure source, and a ma pressure source fluidh coupled to the main channel

62. The method of claim 61, wherein the first pressure source and the main pressure source are the same.

63. The method of claim 61, wherein the first pressure source and the main pressure source compnse a single vacuum source

64. The method of claim 50. wherein the first fluidic matenal and the second fluidic mateπal are the same

65. The method of claim 50, wherein the first fluidic mateπal and the second fluidic mateπal are different.

66. The method of claim 50, wherein the first fluidic mateπal is a sample matenal and the second fluidic mateπal is a diluent mateπal.

67. The method of claim 50, wherein the first fluidic mateπal has a first flow rate after step (n), the third fluidic matenal has a second flow rate after step (in), and the third fluidic mateπal has a third flow rate after step (iv).

68. The method of claim 67, wherein the second flow rate is greater than the third flow rate.

69. The method of claim 67, wherein the third flow rate is substantially the same as the first flow rate.

70. The method of claim 67. wherein the third flow rate is less than the first flow rate.

71. The method of claim 50, further compπsing flowing the third fluidic mateπal through a detection region, which detection region is downstream of at least one of the one or more intersection points.

72. The method of claim 50, further comprising adding a fourth fluidic material to the main channel at a first position that is downstream from at least one of the one or more intersection points, thereby combining it with the third fluidic material, resulting in a fifth fluidic material.

73. The method of claim 72, wherein the fourth fluidic material is a reagent material.

74. The method of claim 72, further providing reduced consumption of the reagent material by reducing the flow rate of the third fluidic material in the main channel prior to adding the reagent material to the main channel.

75. The method of claim 72, further comprising flowing the fifth fluidic material through a detection region, which detection region is positioned along the main channel downstream of the first position.

76. The method of claim 50, further comprising structurally configuring the one or more flow reduction channels to intersect the main channel at a first intersection point and a second intersection point.

77. The method of claim 76, further comprising flowing the third fluidic material through a detection region, which detection region is positioned between the first intersection point and the second intersection point.

78. The method of claim 76, further comprising adding a fourth fluidic material to the main channel at a first position, which first position is between the first intersection point and the second intersection point, thereby combining the fourth fluidic material with the third fluidic material, resulting in a fifth fluidic material.

79. The method of claim 78, further comprising flowing the fifth fluidic material through a detection region, which detection region is positioned between the first position and the first intersection or the second intersection point.

80. A method for modulating a volumetric flow rate of a fluid in a channel of a microfluidic system, the method comprising: (1) providing a body structure compπsing a plurality of microscale channels disposed therein, the plurality of microscale channels compnsing:

(a) a main channel; and,

(b) one or more flow reduction channels that intersect the main channel at one or more intersection points, which one or more flow reduction channels are structurally configured to reduce flow rate of a fluid in the main channel as the fluid flows past the one or more intersection points, (n) flowing a first fluidic mateπal through the main channel, (in) flowing a first portion of the first fluidic mateπal through at least one of the one or more flow reduction channels; flowing a second portion of the first fluidic mateπal through the main channel, thereby modulating the volumetπc flow rate of the first fluidic matenal in the main channel and in the at least one of the one or more flow reduction channels.

81. The method of claim 80, the method further compnsmg flowing a second fluidic matenal through the main channel, resulting in a third fluidic matenal.

82. The method of claim 81, compπsing flowing the second fluidic matenal through the mam channel after step (iv).

83. The method of claim 81, wherein the first fluidic matenal has a first flow rate and the third fluidic mateπal has a second flow rate, wherein the first flow rate is substantially equal to the second flow rate.

84. A method of performing multiple assays in a device of claim 1 or claim 4, the method compπsing: (i) providing a test compound;

(n) flowing a first portion of the test compound into a first flow reduction channel; (iii) flowing a second portion of the test compound into a second flow reduction channel; ) flow ing at least a first reagent into the first flow reduction channel, thereby performing a first assay on the test compound, resulting in at least a first product, and,

(v) flowing at least a second reagent into the second flow reduction channel, thereby performing a second assay on the test compound, resulting in at least a second product

85. The method of claim 84, wherein the test compound compπses a drug sample, a chemical compound, an enzyme, a protein, a nucleic acid, or a combination thereof

86. The method of claim 84, wherein the first reagent and the second reagent are different

87. The method of claim 84, wherein the first assay and the second assay are different.

88. The method of claim 84, wherein the first assay and the second assay are selected from one or more of: an enzyme assay, an HAS binding assay, a target screen, a fluorescence assay, a binding assay, a dose-response assay, a genotyping assay, and a selectivity screen

89. The method of claim 84, compnsmg simultaneously performing step (l) and step (n).

90. The method of claim 84. compnsing simultaneously performing step

91. The method of claim 84, wherein the first product is a first reacted test compound and the second product is a second reacted test compound.

92. The method of claim 84, the method further compnsing simultaneously detecting the first product and the second product.

93. The method of claim 84, compnsing dividing the test compound mto n portions, simultaneously flowing each portion into one of x flow reduction channels, performing y different assays on the test compound, resulting in z products, and simultaneously detecting the z products

94. The method of claim 93, wherein n is between about 2 and about 100, wherein x is between about 2 and about 100, y is between about 1 and about 100, and z is between about 1 and about 1000

95. A method of suppressing pressure perturbations from spontaneous injection into a microfluidic device, the method compnsing

(I) dipping an open end of a capillary into a sample source, thereby drawing a sample from the sample source into the capillary, which capillary is fluidly coupled to a microfluidic device,

(n) withdrawing the open end of the capillary from the sample source, wherein a first portion of the sample remains on the open end, which first portion has a surface tension, which surface tension exerts a first pressure on the capillary, thereby spontaneously injecting the first portion into the capillary; (in) flowing a second portion of the sample from the capillary mto a main channel, which main channel intersects the capillary at a first intersection point, and, (iv) flowing a third portion of the sample through a shunt channel, which shunt channel intersects the main channel, thereby maintaining the first intersection point at a second pressure, which second pressure is different from the first pressure, thereby suppressing pressure perturbations in the main channel

96. The method of claim 95, wherein the sample source compnses a microwell plate

97. The method of claim 95, wherein the shunt channel intersects the main channel at the first intersection point or downstream of the first intersection point.

98. The method of claim 95, wherein the second pressure is greater than the first pressure.

99. The method of claim 95, wherein the second pressure is less than the first pressure.

100. The method of claim 95, wherein the first pressure is atmospheric pressure.

101. The method of claim 95, wherein step (iv) creates a pressure gradient between the open end of the capillary and the first intersection point.

102. The method of claim 95, further comprising applying a third pressure to the shunt channel, thereby altering the second pressure at the first intersection point.

103. The method of claim 102, wherein the third pressure is a negative or a positive pressure.

104. A microfluidic apparatus for suppressing pressure perturbations due to spontaneous injection into the microfluidic apparatus, the apparatus comprising:

(i) a capillary, which capillary comprises an inlet region and an outlet region, which inlet region is fluidly coupled to at least a first sample source during operation of the device and which inlet region is maintained at a first pressure;

(ii) a body structure having a plurality of microscale channels disposed therein, the microscale channels comprising:

(a) a main channel having an upstream region and a downstream region, wherein the upstream region is fluidly coupled to the outlet region of the capillary at a first intersection point;

(b) a shunt channel fluidly coupled to at least the upstream region of the main channel; and,

(iii) a fluid direction system fluidly coupled to the microfluidic device, which fluid direction system comprises at least one fluid control element fluidly coupled to the main channel, to the shunt channel, and to the capillary, which fluid direction system directs:

(a) movement of a sample from the first sample source into the inlet region of the capillary; (b) movement of the sample from the inlet region of the capillary to the outlet region of the capillary,

(c) movement of the sample from the from the outlet region of the capillary into the upstream region of the main channel, and, (d) movement of a first portion of the sample from the upstream region of the main channel into the shunt channel, a second portion of the sample remaining in the mam channel, thereby maintaining the first intersection point at a second pressure, which second pressure is different from the first pressure

105. The apparatus of claim 104, wherein the at least first sample source compnses one or more multiwell plate

106. The apparatus of claim 104, wherein the first pressure is maintained at atmospheπc pressure.

107. The apparatus of claim 104, wherein the shunt channel is fluidly coupled to the upstream region of the main channel and to the downstream region of the mam channel.

108. The apparatus of claim 104, wherein the at least one fluid control element compπses a pressure source or an electrokinetic controller.

109. The apparatus of claim 104, wherein the first pressure is greater than or less than the second pressure.