JP2003507737A - Dilution in high throughput systems with a single vacuum source - Google Patents

Abstract

(57) Summary The flow rate in a microfluidic device (100) with a channel (104) is adjusted after performing a continuous dilution with a flow reduction channel (106, 110).

Description

Detailed Description of the Invention

Cross Reference of Related Applications 35U. S. C. By § 119 (e) and other applicable statutes or regulations, this application is hereby filed by Kopf-Sill et al. In USSN 60/150, filed August 25, 1999,
670 "Dilutions in High Througput Systems with a Single Vacuum Source
USPF 60/15 filed Oct. 12, 1999 by Kopf-Sill et al.
9,014 "Dilutions in High Througput Systems with a Single Vacuum Sou
rce ", and USSN 60 filed April 27, 2000 by Kopf-Sill et al.
/ 200,139 "Dilutions in High Througput Systems with a Single Vacu
um Source's interests and priorities.

BACKGROUND OF THE INVENTION In conducting chemical or biochemical analyses, assays, syntheses or manufactures, the substances or components to be assayed are very sensitive, including measuring, aliquoting, transferring, diluting, mixing, separating, detecting, etc. Do many separate operations. Microfluidic technology scales down and integrates these operations so that they can be performed in one or a few microfluidic devices. For example, a method of diluting in a microfluidic device is described by Parce and Kopf-Sill in US Pat. No. 5,869,004, "Methods and Apparatus for in Situ Concen".
tration and / or Dilution of Materials in Microfluidic Systems ”. These methods continuously withdraw material and add it to the microfluidic channel, continuously diluting the material. By these methods, highly accurate dilution can be performed in a microscale environment.

In some bioassays, a constant flow of material is effective in maintaining a fixed assay reaction time. Thus, the ability to control flow rates and obtain constant incubation and reaction times during dilutions in microfluidic systems is useful for integrating fluid sample and reagent operations in the form of microfluidic assays. Also, a constant flow rate, eg, in a microfluidic device with a single pressure source, helps to reduce reagent usage as the reagent is added after dilution. Another technique useful for integration into microfluidic formats is the ability to simultaneously measure different concentrations of samples and perform different concentrations of reactions at the same time, and / or test one sample against a panel of different reagents at the same time. Have the ability. Thus, improved methods of controlling flow rates during dilution and multiple concentration assays are desirable, especially with the advantages of high throughput, low cost microfluidic systems. The present invention provides these and other features and presents a high throughput microscale system that contributes to dilution, reduced reagent consumption, multiple concentration measurements and many other features that will be apparent from the entire description below. .

SUMMARY OF THE INVENTION The present invention provides devices and methods for regulating flow rates and reducing reagent consumption in microfluidic devices. In one aspect, the invention provides an apparatus for reducing flow in a main channel by structurally including a flow reduction channel configured to draw fluid from the main channel. In addition to regulating the flow rate, these channels also reduce reagent consumption by reducing the flow rate of material at the point of introduction of sample material. An additional control of reagent consumption is
It can be obtained by using a flow reduction channel having a cross-sectional size smaller than that of the main channel. A second flow reduction channel is also added as appropriate for additional suppression of reagent consumption.

In another aspect, a flow reduction channel provides a method for performing serial dilutions of a sample and obtaining measurements corresponding to each dilution level. The flow reduction channel provides a way to perform continuous dilution of the sample without increasing the flow rate at each dilution. Alternatively, the cross-sectional area of the channel downstream of the mixing point can be varied. A detection region is provided in each flow reduction channel, and a measurement value is appropriately obtained at each dilution level.

In one aspect, the microfluidic device of the present invention includes a body structure and a main pressure source.
The body structure includes a main channel disposed therein, the main channel being fluidly connected to a main pressure source. The body structure also includes one or more flow reduction channels that intersect the main channel at one or more intersections. Structurally, the flow reduction channel is configured to reduce the pressure or velocity of the fluid in the main channel as the fluid flow passes through one or more intersections. Also, the body structure can include a first pressure source fluidly coupled to one or more flow reduction channels. The first pressure source is arranged in the one or more flow reduction channels downstream of the one or more intersections in the direction of flow towards the first pressure source. In a preferred embodiment, the first pressure source and the main pressure source are the same, generally a single vacuum source fluidly connected to the main channel at a location downstream from the flow reduction channel.

In another aspect, a microfluidic device includes a body structure and a main pressure source, which is generally a single pressure source. The body structure includes a main channel disposed therein and fluidly connected to a main pressure source. In addition, the main body structure portion includes one or more flow reduction channels that intersect the main channel at the first intersection and the second intersection, and forms a bypass loop. Structurally, the one or more flow reduction channels are configured to reduce the pressure or velocity of the fluid in the main channel as the fluid flow passes through the first intersection in a direction toward the second intersection. For example, typically the main pressure source is fluidly coupled to the main channel at a location downstream from one or more flow reduction channels in the direction of flow towards the main pressure source. The device of the present invention generally comprises two or more flow reduction channels fluidly connected to the main channel, but, where appropriate, about 3 to about 4 flow reduction channels, about 5 to about 10 flow reduction channels or about. 10 or 11
It includes the above flow reduction channels. The cross-sectional dimension of the flow reduction channel is larger or smaller than the cross-sectional dimension of the main channel, as appropriate. The one or more flow reduction channels are channels that do not intersect or channels that intersect as appropriate. For example, the flow reduction channel optionally intersects a source or reservoir for additional substances such as reagents. In one embodiment, the flow reduction channel intersects a second flow reduction channel that is generally smaller in cross-sectional dimension than the flow reduction channel.

In another embodiment, the device of the present invention further comprises one or more detection regions in or near said one or more flow reduction channels. This device
Optionally, it comprises a detection system consisting of one or more detectors, which detectors are arranged in the vicinity of the detection area or both of the main channel and at least one of said one or more flow reduction channels. It Preferably, a single detector is arranged to simultaneously detect the signals in each of the one or more flow reduction channels. Alternatively, a single detector scans over the various channels. Computer and software are optionally included in the device for analyzing the signals detected by the detection system. For example, the software may include a set of instructions that detect components of interest, their concentrations, and the like. In another embodiment, the device comprises a source of a first fluid substance in fluid communication with the main channel at a first position along the main channel and a flow with the main channel at a second position along the main channel. Further included is a freely connected source of second fluid material. Generally, these fluid substance sources are upstream of at least one of the one or more flow reduction channels and are used to introduce the fluid substance into the device. The first fluid material and the second fluid material are, where appropriate, the same or different materials and generally include the sample material and the diluent or buffer material.

In another embodiment, the fluidic substance of the device is directed through the channel by a fluidic pointing system. The fluid-indicating system directs the movement of the first fluid substance and the second fluid substance from their sources into the main channel, thereby combining the first fluid substance and the second fluid substance to form a third fluid substance. . Also, the fluid indicating system is a third
The movement of the first portion of the fluid substance is directed from the main channel to the flow reduction channel, while the second portion of the third fluid substance remains in the main channel. The second portion of the third fluid material is then optionally routed through the main channel. During operation of the device, a first fluid material (eg, sample) has a first flow rate as it flows through the main channel. Add a second fluid material (eg, a buffer) and add a third
In forming the fluid material (eg, diluted sample), the flow rate in the main channel is increased. Thus, the third fluid material or diluted sample has a second flow rate that is greater than the flow rate of the first fluid material or sample. The second flow rate decreases after the first portion of the third fluid material has moved from the main channel into the flow reduction channel. The second flow rate in one embodiment is reduced to substantially the same level as the first flow rate,
As a result, the device maintains a substantially constant flow rate.

In another embodiment, the apparatus of the invention comprises a fourth fluid in fluid communication with the main channel at a third position downstream of at least one of the one or more flow reduction channels. Further includes a source of material. The fluid indicating system is the fourth
Direct the movement of fluid material from its source to the main channel. Generally, the fourth fluid material is a reagent material that reacts with a third fluid material (eg, a diluted sample) to produce a product. One or more reagent substances (eg substrate substance and enzyme) are optionally added to the device in this manner. The device is designed to allow a third
Reducing the flow rate of the fluid substance reduces the consumption of the reagent substance.

Alternatively, the reagent substance is added to the flow reduction channel. For configurations where the flow reduction channel has a smaller cross-sectional dimension than the main channel,
The consumption of reagents is further reduced. Also, a second flow reduction channel is added as appropriate to draw fluid from the flow reduction channel. The small size of the second pressure channel likewise reduces reagent consumption. A method for adjusting the volumetric flow rate of a fluid in a microfluidic device is also provided. The method includes providing a body structure as described above and flowing a first fluid substance through the main channel. A second fluid material is flowed into the main channel to combine with the first fluid material to obtain a third fluid material. Flowing a first portion of a third fluid material into a flow reduction channel and a second portion of a third fluid material into a main channel, thereby causing a third fluid material in the main channel and in one or more flow reduction channels. Adjust the flow rate of. Where appropriate, these steps are repeated in series to effect continuous dilution without substantially increasing the flow rate of material in the channel.

[0012] In one embodiment, the method further comprises flowing a third fluidic substance through the detection region. This detection region is optionally downstream of the position at which the fourth fluid material is added to the main channel to react with the third fluid material. Reagent consumption is reduced by adding a fourth substance after reducing the flow rate by the flow reduction channel. The method also provides multiple measurements of a single sample at multiple or repeated concentrations, or multiple single samples after multiple assays, by providing a detector near each flow reduction channel. Get the measured value of. Alternatively, the channels are configured so that one detector can measure multiple concentrations or assay products at the same time by detecting the signal from each channel. In another embodiment, the present invention provides a spontaneous infusion of a microfluidic device.
section and method for suppressing pressure perturbations. The devices described above are optionally used to suppress pressure perturbations due to spontaneous injection. External sample source (
Spontaneous injection occurs when sucking a sample from one or more microwell plates, for example, through a capillary into a microfluidic device.

A method of reducing or eliminating pressure perturbations due to natural injection involves extracting a sample from the sample source into the capillary by dipping the open end of the capillary into the sample source. A capillary, which is generally maintained at a first pressure, is fluidly coupled to a microfluidic device and a sample is flowed from the capillary to the device. The method further includes withdrawing the open end of the capillary tube from the sample source. The first portion of the sample remains at this open end and is naturally injected into the capillary by the surface tension of the sample exerting pressure on the capillary. The sample is flowed from the capillary into a microfluidic device. The second portion of the sample is flowed from the capillary into the main channel that intersects the capillary at the first intersection. A third portion of the sample is flowed through a flow reduction channel or a shunt channel that intersects the main channel at or downstream of the first intersection. By flowing a portion of the sample through the flow reduction channel, a larger flow rate is created in the capillary than is needed in the main channel, which results in a larger pressure gradient in the capillary. This higher pressure gradient in the capillaries reduces the effects of pressure perturbations of spontaneous injection at the open ends of the capillaries, thereby suppressing pressure perturbations in the main channel.

In an example device for suppressing spontaneous injection, the capillary tube includes an inlet region and an outlet region. The inlet region, which is generally maintained at a first pressure (e.g., atmospheric pressure), is fluidly connected to at least a first sample source and to the microfluidic body structure during operation of the device. Generally, the body structure of the device includes a plurality of microscale channels disposed therein. Microscale channels include a main channel having an upstream region and a downstream region. The upstream region of the main channel is fluidly connected to the outlet region of the capillary at the first intersection. In addition to the main channel, the device comprises a shunt channel or a flow reduction channel as described above. The shunt channel is fluidly connected to at least an upstream region of the main channel and, where appropriate, also to a downstream region of the main channel. Generally, the fluid-indicating system directs fluid from the exit region of the capillary into the main channel and diverts some of the fluid to a shunt or bypass channel, thereby reducing pressure perturbations in the main channel due to spontaneous injection into the main channel. .

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a microfluidic method and apparatus for regulating flow rate in a microfluidic channel system, in particular, the flow of material through the channel is driven by a single pressure source. It provides a system that is pressure induced flow. The present invention provides a device that includes a flow reduction channel, the flow reduction channel being structurally configured to withdraw material from the main channel of the device as the material flows into the main channel, whereby the main channel Reduce the pressure, volumetric flow rate and / or velocity at. In essence, the present invention controls the flow rate by dividing the material flow into multiple parts, all controlled by a single vacuum source. These methods are also effective in suppressing spontaneous injection perturbations, for example, when performed in microfluidic devices that include suction capillaries. For example, a sample is typically run from a microwell plate into a suction capillary, and then the microfluidic channel (
For example, the main channel). Flowing a portion of the sample from the main channel into the flow reduction channel creates a pressure differential between the suction capillary and the intersection of the flow reduction channel and the main channel. This differential pressure suppresses pressure perturbations due to spontaneous injection of fluid from the tip of the suction capillary into the microfluidic device.

The present invention also provides for multiple concentration measurements on a single sample by, for example, providing channels gathered within a single detection region to simultaneously detect signals in various flow reduction channels. It provides a way to get the values at the same time. Thus, the present invention allows for serial dilutions and acquisition of multiple concentration measurements in a microfluidic device with a single vacuum source, without substantially increasing flow rate or reagent consumption. In another embodiment, the device of the invention is used to perform multiple assays simultaneously on a single test compound. For example, a single sample can be divided into multiple portions and run in multiple parallel channels, as appropriate. Reagent wells fluidly coupled to each of the plurality of channels are used to contact different portions of the sample with different reagents. Different reactions or assays (eg binding assay)
Are performed in each of the different channels, as appropriate. Alternatively, the same reaction is carried out in each channel, with the only difference being, for example, the different enzyme isoforms. By providing multiple channels centered on a single detection region, the results of various assays are detected simultaneously, as appropriate.

I. Microfluidic Devices Containing Flow Reduction Channels In microfluidic devices, the volume of sample material of interest is extremely small, but in order to dilute the concentration of these materials, a larger volume of a second material, such as diluent or A buffer or the like is added to the sample material. Adding a larger volume of fluid to the microfluidic system increases reagent consumption as reagents added to the diluted sample, such as substrate and enzyme reagents, are added to the diluted inhibitor sample. For example, increasing the volume for the channels of a device whose flow is driven by a single vacuum source increases the flow rate, which increases reagent consumption. The device of the present invention solves this problem by withdrawing fluid from the channel in which it is diluted and entering the flow reduction channel. For example, as fluid is added to the main channel to dilute the sample, for example, the flow rate of the fluid in the main channel increases. If a channel (eg, a flow reduction channel or bypass loop) is arranged to draw a portion of the fluid from the main channel, the flow rate will decrease, for example, to its pre-dilution level as the amount of fluid in the main channel decreases. ). Thus, the arrangement of channels in a microfluidic device reduces the flow rate by removing fluid from the main channel. Additional flow reductions are conveniently achieved, for example, by withdrawing fluid from the flow reduction channel and entering it into the second flow reduction channel.

When a flow reduction channel is used in a microfluidic suction device (eg, a device that includes an external suction capillary) to shunt fluid from the main channel, a pressure gradient is created across the suction capillary. This pressure gradient reduces pressure perturbations due to spontaneous injection of fluid into the suction capillary as the capillary moves from well to well of a microwell plate. The flow reduction channel configuration used in the present invention to control the flow rate also provides an apparatus for multiple concentration assays. Multiple concentration assays are achieved by performing serial dilutions in a device containing multiple flow reduction channels. After each dilution, a portion of the fluid is withdrawn from the main channel and placed in the flow reduction channel. When multiple flow reduction channels are used after multiple dilutions, each flow reduction channel contains a different concentration of sample material. The sample is then detected at multiple concentrations by one or more detectors. Further, by adding a reagent to the flow reduction channel, a reaction is appropriately caused in the flow reduction channel. This allows reactions to be run simultaneously at various sample concentrations, for example to determine enzyme kinetics in a high throughput system.

Alternatively, multiple assays are performed in flow reduction or parallel channel regions, eg at the same or different concentrations. For example, for the same compound divided into multiple samples in different flow reduction channels, different reagents are added to each channel as appropriate to perform different assays. Applications include, but are not limited to, human serum albumin binding assays, genotype assays, high throughput target screens such as drug screening, eg selectivity screens for panels of different enzymes or isozymes, measurement of intrinsic drug fluorescence, etc. Is included. For repeated measurements, the same assay is optionally repeated in each of the parallel channel regions, eg at different or the same concentration. The flow reduction channel also reduces reagent consumption in the assay due to the reduced flow rate. The flow reduction channel described above is typically coupled to a microfluidic device and used as described below. In general, the device includes a body structure in which microscale channels are located. For example, the system generally includes, for example, a main channel, one or more flow reduction channels, and one or more second flow reduction or reaction channels. These channels are fluidly connected to each other and to various reservoirs or other sources of fluid material. Materials used in the present invention include, but are not limited to, buffers, diluents, substrate solutions, enzyme solutions, and sample solutions. In addition, the channel appropriately includes a detection region.

For example, various channels and channel regions are located throughout the microfluidic device. Generally, the device includes a main channel in which the sample is introduced. For example, a sample containing a potential modulator or activator of the enzyme of interest is introduced into the channel. An assay to determine the effect of the modulator (eg, activator or inhibitor) on the reaction rate of the enzyme is then optionally performed by allowing the enzyme to react with the substrate in the presence of the modulator. To determine the effect of concentration on kinetic parameters, the kinetics are often examined at multiple concentrations. The device of the present invention allows for continuous dilution in a microfluidic device while maintaining constant reaction time for a fixed channel length while minimizing reagent consumption without increasing flow rate. The present invention also provides an apparatus for making multiple concentration measurements simultaneously.

A flow and / or pressure reduction channel is a channel that is structurally configured to reduce pressure and / or flow in the main channel or another flow reduction channel. Channels that are "structurally configured to reduce pressure / flow rate" are those that are configured to provide the desired flow rate. Devices that include structurally configured channels that reduce the pressure, flow rate, or velocity of a fluid in the channel are generally based on regulation of forces, such as vacuum sources or electrokinetic forces, as opposed to In particular, it is based on the structural configuration of the channel that carries fluid to regulate pressure and / or velocity in the channel.
By configuring the channels to regulate the flow rate, a single, constant driving force is conveniently applied across the entire system (eg, a single vacuum source). For example, if multiple channels and sample sources are fluidly connected,
A single vacuum source can draw reagents from the source into the channel and move the sample through the channel. When the channel is configured as a flow reduction channel in the present invention, the vacuum pulls fluid through the flow reduction channel as well as the main channel, splitting the flow into two parts and reducing velocity. In general, the flow reduction channels of the present invention are spaced apart so that mixing of assay components is complete before a portion of the flow is diverted into the flow reduction channel.

In general, channels are constructed by varying the channel length or cross section, or by adding a flow delay matrix. These changes in the channel configuration change the resistance to fluid flow in the channel, which in turn changes the flow rate.
For example, narrowing the channel width increases the resistance to flow and thus reduces the flow rate. Structurally, the preferred method of configuring the channels to reduce fluid flow is to create a channel in the main channel by pulling fluid from one channel (eg, pressure and / or flow reduction channel) from another channel (eg, main channel). Designing or arranging multiple channels to reduce resistance. Thus, the flow rate of the substance in the channel is precisely adjusted, and the reagent consumption is suppressed by the proper configuration of the flow reduction channel. By using one or more of the methods described above, the channels are structurally configured to reduce the pressure or flow rate by the desired specific amount. Details of constructing a channel structure with a desired flow rate using channel lengths and dimensions can be found, for example, in USS Jan. 28, 1999.
See N09 / 238,467.

In the present invention, reducing the flow rate and / or pressure generally involves, for example, depressurizing (tappi
ng off) to extract the fluid from the main channel and put it in the flow reduction channel or the shunt channel. The second flow reduction channel is a flow reduction channel that draws fluid from another flow reduction channel. Preferably, these second flow reduction channels have a smaller cross-sectional dimension than the fluid withdrawal channels. In this case, the channel is
When fluidly connected to the source of the reagent substance, it functions appropriately as a reaction channel. This type of reaction channel reduces the consumption of reagents. Because the flow rate is reduced and the channel dimensions are reduced, resulting in less reagent needed to perform the assay of interest.

Alternatively, the channels in the present invention are configured to provide assay channels, eg, to perform multiple assays on a single compound. For example, the microfluidic device is optionally configured to provide multiple parallel channels. A single sample is optionally aspirated into the device through a capillary fluidly connected to parallel channels. The sample is conveniently divided into portions, each of which is flowed through a separate parallel channel region. Different assays are then performed on each portion of the sample, as appropriate, for example, by using different channel chemistries or by adding different reagents to each channel. For example, different channels are filled with different bead arrays with different chemistries, or different separation matrices, as appropriate. In another embodiment, the individual channels include different surface modifications, for example to impart different chemistries to each channel. The term "downstream" is a position in a channel or microfluidic device that is farther along a channel or channels in a selected direction of fluid or substance flow with respect to a selected site or region. Means position. For example, the pressure source is optionally farther along the direction of flow in the channel system than the buffer well or flow reduction channel. Thus, the fluid travels down the main channel past the flow buffer well, past the flow reduction channel and towards the pressure source. In this embodiment, the pressure source is typically a vacuum source.

In another embodiment of the device, the pressure source is optionally located at the upstream end of the main channel. "Upstream" means a position in a channel or channel system that is farther along a channel or channels in a direction opposite a fluid or substance flow to a selected site or region. For example, the pressure source is
It is located upstream of the detection area. A pressure source is optionally placed in the sample well where sample material is introduced into the system. In this case, the pressure source pushes fluid through the channel in a direction away from the pressure source and toward the opposite end of the channel (eg, the detection region or waste well). A reservoir or well is a place where samples, components, reagents, etc. are added to the device for assaying. The introduction of these elements into the system is done as follows. Generally, the reservoir is arranged so that the sample or reagent is added to the system upstream of where it is used. For example, if the sample must be diluted prior to reaction with the reagent, a dilution buffer is added upstream of the reagent source. In this case, the dilution buffer is generally added to the main channel upstream of the flow reduction channel so that the increase in flow rate due to the addition of the buffer substance is offset by the reduction in pressure due to the flow reduction channel. On the other hand, the reagent substance is generally added downstream of the flow reduction channel so that it is added after the flow rate has been reduced to add a smaller amount of reagent.

In certain embodiments, different reagent wells or multiple reagent wells are fluidly coupled to each of the flow reduction channels, eg, in a parallel channel configuration, or to each channel or channel region. In this case, for example, to perform different assays in each channel, for example to simultaneously assay multiple binding sites for a single target, or to screen for different enzymatic isoforms.
Where appropriate, reagent wells are used to add different reagent or reagents to each channel. The detection area is also included in the device. The detection region is optionally a channel or a sub-unit of channels in close proximity in space, which optionally comprises a separate channel in fluid communication with the channels in the microfluidic device. The detection region is appropriately arranged near the main channel. For example, in FIG. 5, the detection region 532 is near the main channel 504. Alternatively, the detection region is located near one or more flow reduction channels as in FIG. 6, and in FIG.
The detection regions 634, 636 and 638 are respectively flow reduction channels 61.
It is near 2, 610 and 608. In another embodiment, the detection area is 1 or 2
It is arranged in the vicinity of the above second flow reduction channel. For example, the detection areas 734, 736 and 738 are in the vicinity of the second flow reduction channels 742, 744 and 746.

Alternatively, the detection region may include all of the flow reduction channels in the device and the regions in the vicinity of the main channel. For example, the detection region is appropriately located at all downstream points of the main channel and the flow reduction channel so as to be located near both the main channel and the flow reduction channel. Such a device is shown in FIG. If the flow reduction channel and the main channel are all arranged to be concentrated, one detection area is sufficient to detect the signals from all adjacent channels. For example, multiple assays may be performed on multiple samples in parallel channels, eg, under different assay conditions or at different concentrations, as appropriate, with the result being that a single channel in which all of the multiple channel regions are concentrated. It is detected in the detection area at the same time. The detection window or area where the signal is typically monitored includes a transparent cover to allow visual or optical observation or detection of assay results, such as colorimetric or fluorometric signal or label observation. Such regions include one or more detectors, as appropriate. Examples of detectors suitable for use in the detection area are well known to those skilled in the art and are described in detail below.

The above-mentioned elements, including, but not limited to, flow reduction channels, bypass loops, detection areas and reservoirs, control flow rates, reduce reagent consumption and provide multiple concentration measurements or multiple measurements, eg, on a single sample. It is optionally coupled to a microfluidic device that is effective in performing the assay. Specific examples of channel configurations are given in the figures described below. Other possible configurations with substantially the same elements will be apparent with reference to the entire disclosure.

One embodiment of the device of the present invention is shown in FIG. As shown, the device includes a sample well 302 and is used to introduce a sample or fluidic substance into the device. Fluid material is flowed from the sample well 302 through the main channel 304. Additional material is optionally added to the fluid material as it flows through the main channel 304. For example, an appropriate buffer is added to dilute the fluid material. Also, where appropriate, a reagent is added to the fluid material and reacts with the reagent to form a product. For example, a fluid source is flowed through the device using a pressure source 306 suitably located at the downstream end of the main channel. Alternatively, a pressure source is placed in sample well 302. In the configuration shown in FIG. 3, the pressure source 306 is at the downstream end of the main channel 304.
Fluidly connected to. The second fluid material (eg, dilution buffer) is the main channel 30.
When added to the fluid material at 4, the flow rate of the fluid material in the main channel increases. A first portion of fluid material is flowed into the flow reduction channel 308 to reduce the flow rate in the main channel. The flow reduction channel 308 is configured to draw fluid from the main channel 304 to reduce pressure and / or velocity in the main channel 304. In this embodiment, the flow reduction channel 3
08 is fluidly connected to the pressure source 306 at its downstream end.

Reducing the pressure or velocity in the main channel 304 serves multiple purposes. In one embodiment, this flow rate reduction reduces the volumetric flow rate and, thus, the amount, of reagents that must be added to the main channel 304 in any reaction performed downstream of the flow reduction channel 308. For example, the sample used in the assay with the fluid substance is added through reagent well 318, as appropriate. After the flow rate is reduced by withdrawing fluid into the flow reduction channel 308, less reagent is needed from the reagent well 318. Alternatively, this flow reduction allows multiple concentrations of the same sample material to be measured in a single device. For example, dilution buffer is optionally added from buffer well 316 to the sample introduced from sample well 302. The first portion of the resulting fluid is then withdrawn from the main channel 304 and placed in the flow reduction channel 308 and detected at the detection region 332. At the same time, a second portion of the resulting fluid is flowed through the main channel 304 where it is again diluted with the dilution buffer from the reagent well 318 to provide a continuous dilution of the sample material. This diluted material is then flowed through main channel 304 and detection region 332 where it is simultaneously detected by the first dilution.

In another embodiment, shown in FIG. 4, the flow reduction channel 408 is structurally configured to draw fluid from the main channel 404 by forming a bypass loop away from the main channel 404. This embodiment works in substantially the same way as in FIG. For example, fluid may flow from sample well 402 to main channel 40.
Pour into 4. Next, additional fluid is added from reservoir 416 and a portion of the fluid is withdrawn from main channel 404 into flow reduction channel 408, thereby reducing pressure in main channel 404 and fluid flow rate. To reduce. Generally, fluid flow is controlled by vacuum source 406, but is controlled by any other type of pressure control system that draws or admits fluid from main channel 404, as appropriate.

FIG. 5 shows an alternative embodiment of the device of FIG. 4 in which multiple flow reduction channels are included in the device. For example, a sample is introduced into the device through sample well 502 and transferred through main channel 504. At this point, multiple dilutions are optionally performed in the apparatus of FIG. For example, the dilution buffer is reservoir 51
6 is added to the sample to produce a diluted sample, at which point the flow rate and / or pressure in the main channel 504 increases. The pressure is reduced by withdrawing a portion of the diluted fluid into the flow reduction channel 508. Optionally, an additional dilution is performed by adding dilution buffer from reservoir 518 into the main channel to obtain a second diluted sample. At this point, the pressure in the main channel 504 increases again and the flow rate increases. Therefore, a flow reduction channel 510 (second flow reduction channel or bypass loop) is used to reduce the pressure, which controls the flow rate in the main channel 504. If appropriate, further dilution is performed at this point by controlling the pressure with an additional flow reduction channel. After the desired dilution concentration is obtained, the sample is optionally reacted with various reagents that may be added to the sample through reservoirs 520 and / or 522. In addition, the sample is appropriately detected by a detector arranged near the detection region 532.

FIG. 6, which illustrates an alternative embodiment of FIG. 4, shows a device in which the assay is suitably performed on the same sample at various concentrations and is detected simultaneously. The device of FIG. 6 operates similarly to the device of FIG. 5 above, with the additional dilution step possible by the addition of flow reduction channels 612 (in addition to flow reduction channels 608 and 610). The apparatus of FIG. 6 also includes a detection region 632 located near the main channel 604 and flow reduction channels 612, 610 and 608, respectively.
634, 636 and 638. Thus, the detectors located proximate the detection regions 632, 634, 636 and 638 are suitable for detecting the fluid as it flows through the main channel 604 and flow reduction channels 608, 610 and 612. used. These flow reduction channels 608, 610, and 612, when constructed and operative as described above, contain various concentrations of sample material injected or aspirated in the sample well 602. Alternatively, flow reduction channel 6 can be used to perform different assays on the same sample, as appropriate.
08, 610 and 612 are used and a portion of the sample is flowed through all three channels. The reagents for each assay may be, for example, reservoirs 622, 624, 6
26 and / or 628 to the flow reduction channel. FIG. 6 also includes additional reservoirs 622, 624, 626 and 628 for adding substances (eg reagents such as substrates and enzymes) into the main channel 604 and flow reduction channels 608, 610, 612. Therefore, the assay of interest is optionally run at all different concentrations of sample material in the flow reduction channel. Also, reducing the flow rate by the flow reduction channel reduces the use of reagents.

In another embodiment of the invention, the use of reagents is further reduced by varying the channel size in which the assay of interest is performed. For example, in the device shown in FIG. 7, a smaller size flow reduction channel has been added as a bypass loop. For example, second flow reduction channels 742, 744 and 746 are fluidly coupled to flow reduction channels 708, 710 and 712. The second flow reduction channels are flow reduction channels 708, 710 by withdrawing fluid from the flow reduction channels.
And 712 to reduce pressure and / or velocity. Thus, the second flow reduction channels 742, 744 and 746 reduce reagent consumption by reducing the flow rate in the channels. Also, the second flow reduction channels 742, 744 and 746, when used as reaction channels, further reduce reagent consumption. In the case of reaction channels, they are generally configured to have smaller cross-sectional dimensions than the flow reduction channels (eg, flow reduction channels 708, 710 and 712). In this embodiment, reservoirs 722, 724, 726 and 72
8 is fluidly connected to the second flow reduction channels 742, 744 and 746 so that less reagent is added from the reservoir into the smaller sized channels. Further, the detection areas 732, 734, 736 and 738 are
For detection of substances in the second flow reduction channels 742, 744 and 746, they are placed in the vicinity of these channels. Thus, the assays of interest are optionally run concurrently at various dilution levels in small volumes to reduce reagent consumption, and then simultaneously detected at those levels. Also, the detection regions in the various flow reduction channels are appropriately configured such that one detector can be used to detect signals from all flow reduction channels and the main channel simultaneously.

Panels A and B of FIG. 8 are schematic illustrations, which are a practical implementation of one possible channel configuration, in which the fluids in different flow reduction channels, the second flow reduction channel and the main channel have the same detection. It can be detected by a container. Other configurations are possible. The device of FIG. 8 has a capillary attachment point 80 located on the main channel.
Give 2,802B. The sample is introduced into the device at capillary attachment points 802, 802B. The samples are parallel channel regions 808, 808B, 810, 8
10B, 812, 812B and main channel regions 804, 804B. These channels are configured to reduce volumetric flow in the main channel regions 804, 804B by drawing fluid from the main channel regions 804, 804B into the parallel channel regions 808, 808B, 810, 810B and 812, 812B. In terms of the above configuration, it operates as a flow reduction channel. Channel regions 808, 808B, 810, 810B and 812, 812B
The fluid therein is channel regions 808, 808B, 810, 810B, 812, 81.
2B, 844, 846, 848, 850 and 852, diluted appropriately with material from reservoirs 816, 816B and 818, 818B. After dilution, the pressure and / or flow rate is optionally further reduced by the second flow reduction channels (or reaction channels) 842, 844, 846, 848, 850 and 852. By utilizing all available second flow reduction channels, multiple serial dilutions are optionally performed without significantly increasing the fluid flow rate through main channel region 804 and parallel channel regions 808, 810, 812. Once the desired dilution level is reached, the assay is run through the channel regions 804, 804B.
, 808, 808B, 810, 810B, 812, 812B, 842, 844 and 846, as appropriate. Channel regions 804, 804B, 808, 808B, 810, 810B, 812, 812B,
Since all 842, 844 and 846 are concentrated within the detection area 832, 832B before reaching the waste well 806, 806B, different assay or concentration level results are detected simultaneously. In another embodiment, a single pressure source is optionally applied at waste wells 806, 806B to generate flow through the channel system.

In one embodiment, the parallel channel configurations of FIGS. 8A and 8B are used to perform multiple assays, eg, on the same test compound or different test compounds. For example, a test compound (eg, a single test compound) is optionally aspirated from a capillary tube (eg, a microwell plate) into the microfluidic device shown in FIG. 8A. The compound is then divided into four parts as appropriate. One portion is flowed through the parallel channel region 804. The second portion is flowed through parallel channel region 808, the third portion is flowed through channel region 810, and the fourth portion is flowed through channel region 812. Then, each sample portion is appropriately assayed differently. For example, reservoir 820 is optionally used to add the reagents necessary for the assay for binding site I in the human serum albumin (HSA) assay in channel 804. Reservoir 822 is used to add the reagents necessary to probe binding site II in the HSA assay in channel 808. Reservoirs 824 and 826 are also used to add reagents for other assays to channels 810 and 812, as appropriate. Alternatively, the reservoir adds the same reagent to each channel, thereby performing the same assay to obtain repeated measurements (eg, 4) or the same assay at different concentrations. In other embodiments, the same reaction is performed in each channel, the only different being, for example, different enzyme isoforms. The results of the different assays are then simultaneously detected in the detection window 832, as appropriate.

Various microfluidic devices are optionally adapted for use in the present invention by the addition of flow reduction components as described below. These devices are described in US Pat.
699,157 (J. Wallace Parce) (issued December 16, 1997),
US Pat. No. 5,779,868 (J. Wallace Parce et al.) (Issued July 14, 1998), US Pat. No. 5,800,690 (Calvin YH Chow et a.
l.) (issued September 1, 1998), US Pat. No. 5,842,787 (Anne
R. Kopf-Sill et al.) (Issued December 1, 1998), US Pat.
52,495 (J. Wallace Parce) (issued December 22, 1998), US Pat. No. 5,869,004 (J. Wallace Parce et al.) (February 9, 1999).
Issued date), US Pat. No. 5,876,675 (Colin B. Kennedy) (199
Issued March 2, 1997), US Pat. No. 5,880,071 (J. Wallace Parce
et al.) (issued March 9, 1999), US Pat. No. 5,882,465 (
Richard J. McReynolds) (issued March 16, 1999), US Pat.
No. 85,470 (J. Wallace Parce et al.) (Issued March 23, 1999)
U.S. Pat. No. 5,942,443 (J. Wallace Parce et al.) (1999 Aug.
Issued March 24), US Pat. No. 5,948,227 (Robert S. Dubrow) (
Issued September 7, 1999), US Pat. No. 5,955,028 (Calvin Y.
H. Chow) (issued September 21, 1999), US Pat. No. 5,957,579 (Anne R. Kopf-Sill et al.) (Issued September 29, 1999), US Pat. , 958,203 (J. Wallace Parce et al.) (Issued September 28, 1999), U.S. Pat. No. 5,958,694 (Theo T. Nikiforov) (September 1999).
Issued March 28), and US Pat. No. 5,959,291 (Morten J. Jensen)
) (Issued September 28, 1999) and published PCT applications, eg W
O98 / 00231, WO98 / 00705, WO98 / 00707, WO98
/ 02728, WO98 / 05424, WO98 / 22811, WO98 / 45.
481, WO98 / 45929, WO98 / 46438, and WO98 / 495.
48, WO98 / 55852, WO98 / 56506, WO98 / 56956,
WO99 / 00649, WO99 / 10735, WO99 / 12016, WO9
9/16162, WO99 / 19056, WO99 / 19516, WO99 / 2
9497, WO99 / 31495, WO99 / 34205, WO99 / 4343
2 and WO 99/44217, including various PCT applications and issued US patents by the inventor and their co-workers.

[0038] For example, pioneering techniques for performing cell-based microscale assays are described in Parce
U.S. Pat. No. 5,942,443 "High Throughput Screening Ass by et al.
1 by ay Systems in Microscale Fluidic Devices "and Burd Mehta et al.
No. 60 / 128,643 "Manipulation of Mic, submitted April 4, 1999.
roparticles In Microfluidic Systems ". A fully integrated system with fluid handling, signal detection, sample storage and sample access is available. For example, US Pat. No. 5,942,443 A pioneering technique for integrating microfluidics and sample selection and manipulation is presented, and various other elements such as particle sets, separation gels, antibodies, enzymes, substrates, etc., where appropriate. These optional elements are used in performing various assays, eg, in a kinase reaction, product and substrate are typically electrophoretically separated, eg, on a separation gel. Microscale assays based on
Appropriately implemented in the apparatus of the present invention. For example, in cell assays, a constant flow rate is important to ascertain and regulate cell culture time. A cell-based microscale system is described in WO98 / 00231 (Parce et al., "High Throughput S.
creening Assay Systems in Microscale Fluidic Devices ") and Mehta et al.
No. 60 / 128,643 "Manipulatio filed April 4, 1999 by
n of Microparticles In Microfluidic Systems ". A fully integrated system with fluid handling, signal detection, sample storage and sample access is also available. For example, WO98 / 00231 (supra)
A groundbreaking technique for integrating microfluidics and sample selection and manipulation is presented. Sources of sample material, enzymes and substrates are also included in the integrated system of the present invention.
These fluid substances are introduced into the device by the method described below.

Source of Assay Components and Integration of Microfluidic Format In the present invention, reservoirs or wells are provided as sources of buffers, diluents, substrates, enzymes, reagents and the like. For example, FIG. 3 illustrates various reservoirs such as sample well 302, buffer well 316 and reagent well 318. These reservoirs are fluidly connected to the main channel 304. FIG. 6 shows reservoirs 622, 6
Alternative arrangements of reagent wells such as 24, 626 and 628 are shown. These reservoirs are arranged to fluidly connect to flow reduction channels 608, 610 and 612. For example, in FIG. 8A, each of reservoirs 820, 822, 824 and 826 optionally contains different reagents, eg, to perform different assays in channels 804, 808, 810 and 812. In another embodiment, different assay channels are manufactured or pre-loaded with different reagents to perform different assays in each channel. For example, channels 804, 808, 810
And 812 are each exposed or filled with different reagents before or during the assay. Different chemistries are achieved in each channel, for example using beads, eg separation matrices such as gels, of different reagents with different properties and / or reagents which modify or react with the channel surface.

Sources of samples, buffers, reagents (eg, substrates, enzymes, etc.) are flowably coupled to the microchannels described herein in any of a variety of ways. In particular, Knapp et
al. "Closed Loop Biochemical Analyzers" (WO98 / 45481; PCT
/ US98 / 06723) and Parce et al. “High Throughput Screening As”
say Systems in Microscale Fluidic Device ”(WO98 / 00231) and for example 60 / 128,64 filed April 4, 1999 by Mehta et al.
A system including a substance source described in No. 3, “Manipulation of Microparticles In Microfluidic Systems” can be applied. In these systems, a "pipette channel", in which components can move from a source to a second channel or microscale element such as a reservoir, is temporarily or permanently connected to a source of material. . This source may be located inside or outside the microfluidic device containing the pipette channel. Examples of sources include microwell plates, other solid substrates containing membranes or lyophilized components, wells or reservoirs in the microscale device itself and other bodies.

For example, the source of cell type, component or buffer may be a microwell plate external to the body structure, eg having at least one well with the selected cell type or component. Alternatively, the source is a well located on the surface of the body structure and contains the selected cell type, component or reagent, and the reservoir is located within the body structure and the selected cell type, component. , A mixture of components or reagents, the container being external to the body structure, containing at least one compartment, containing the selected particle type, components or reagents, or the solid phase structure containing the selected cell type. Or a reagent in lyophilized or otherwise dried form. The loading channel region is optionally fluidly connected to a pipette channel having a port external to the body structure. The loading channel is an electric pipette channel with a port outside the body structure, a pressure-based pipette channel with a port outside the body structure, a pipette channel with a port inside the body structure, on the surface of the body structure , The internal channel in the body structure that is fluidly connected to the well, the internal channel in the body structure that is fluidly connected to the well in the body structure, and the like.

The integrated microfluidic system of the present invention optionally comprises a wide variety of storage elements for storing the reagents to be evaluated. These include well plates, matrices, membranes and the like. Reagents can be stored in liquids (eg in wells on microtiter plates) or in lyophilized form (eg dried on membranes or in porous matrices) and also using conventional robotics or Electropipettes or pressure pipette channels fluidly coupled to a region or channel of the microfluidic system may be used to transfer to array components, regions or channels in the microfluidic device. In the methods described below, the above-described devices, systems, features and components adjust flow rates, perform multiple concentration measurements, perform multiple assay measurements, for example, during serial dilutions in a single pressure source microfluidic system. Used to reduce reagent consumption.

II. Movement of Fluid Through a Microfluidic Device In the present invention, sample material is flowed through the main channel and various materials are added to the main channel, eg, to dilute the sample material or to react the sample material with the reagent material. For example, in high throughput screening applications, it is often useful to dilute the sample entering the device by a factor of 10, 100, 1000 or 10,000. In a vacuum driven flow system, this can be accomplished by introducing a diluent buffer into the device and designing the buffer and compound fluid passages to have a hydrodynamic resistance proportional to the desired dilution. However, the volume flow rate increases 10 to 10,000 times. Therefore, subsequent reagent additions should be 10 to 10,000 times larger in volume than would be required without 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 drawing fluid through the flow reduction channel or bypass arm. In this way, the flow rate or velocity of fluid through the device is reduced. The channel is optionally configured to reduce the flow rate such that the flow rate is equal to or less than the initial flow rate before the diluent is added. By reducing the flow rate, subsequent reagent additions are not required to be 10 to 10,000 times greater to meet the increased flow rate requirements.

Generally, the movement of fluidic material through the microfluidic device of the present invention is driven by a pressure source. Generally, the pressure source is a vacuum source applied to an end point downstream of the main channel.
For example, in FIG. 4, vacuum source 406 is applied to one end of main channel 404.
A vacuum source 406, or another type of pressure source as described below, applies pressure to draw or pump fluid through the channels of the device, such as the main channel 404 and flow reduction channel 408. These applied pressures or vacuums
A pressure differential is created across the channel length, driving the flow of fluid therethrough. Figure 4
In the interconnected channel networks described herein, such as those shown in Figure 1, the differential flow rate over volume is determined by applying different pressures or vacuums to multiple ports, or preferably a single vacuum in common. In addition to the waste ports of the above, and by configuring various channels with appropriate resistance to produce the desired flow rate. For example, the channel of FIG. 4 is configured to control the flow rate through the device when vacuum source 406 is applied to the end of main channel 404. A single vacuum source is used to draw fluid through the main channel 404, and when additional fluid is added through the reservoir 416, the flow rate is controlled by the flow reduction channel configuration. A portion of the fluid is withdrawn and entered into the flow reduction channel 408, reducing the pressure in the main channel 404.

A variety of techniques are available for achieving the fluid movements described above by applying pressure forces to the microscale element. The flow of fluid (and the flow of substances suspended or solubilized in the fluid, including cells or other particles) will displace the liquid and increase or decrease the pressure at the sites in the microfluidic system, as appropriate. Regulated by a pressure-based mechanism such as a mechanism based on displacement of fluid using a piston, a pressure diaphragm, a vacuum pump, a probe, or the like. The pressure may be pneumatic, for example using a pressure gas, or water pressure, for example a pressure liquid, as appropriate, or alternatively to a positive displacement mechanism or substance reservoir for exerting a force on the substance through a channel or other conduit. With the provided plunger, or a combination of these forces. In other embodiments, a vacuum source is applied to the reservoir or a well, such as a waste well as shown in FIG. Waste wells 806, 806B contain a vacuum source at one end of the channel system to draw suspension from the channel. The pressure or vacuum source is externally supplied to a device or system (eg, an external vacuum or pressure pump that is hermetically attached to the inlet or outlet of the channel), or where appropriate, or they are integrated into the device (eg, channel and channel). Micro-manufacturing pump operatively linked to) is internally supplied. Examples of micromanufactured pumps are widely described in the art. See, for example, published international application WO 97/02357.

Although the system of the present invention has been described with respect to a single vacuum source, it includes electrokinetic flow including, but not limited to, pressure sources at multiple reservoirs or channels of the device. And other sources of fluid movement such as other types of pressure driven flows. For example, electrokinetic techniques may be used to inject fluid into the device, or one of the devices, as appropriate.
Used to move from one channel to another in cross injection. The following techniques are optionally used in connection with the techniques of the present invention to provide further fluid control alternatives. Another method for controlling flow in channels or sections of the device is the use of hydrostatics, wicking and capillary forces to provide pressure for fluid flow of substances such as cells and sample substances. Accompanied by. For example, Alajoki et al.
METHOD AND APPARATUS FOR CONTINUOUS LIQUID FLOW IN MICROSCALE CHANNELS U
SING PRESSURE INJECTION, WICKING AND ELECTROKINETIC INJECTION ", USSN
09 / 245,627, filed February 5, 1999. In these methods, an adsorbent material or branched capillary structure is placed in fluid contact with an area to which pressure is applied, thereby displacing fluid toward the adsorbent material or branched capillary structure. Mechanisms for concentrating cells and other substances in the center of microscale flow pathways are effective in increasing assay throughput, for example by regulating flow rates in pressure-based flow, and H. Garrett Wada et al. By “FOCUSING OF MICROPAR
TICLES IN MICROFLUIDIC SYSTEMS ", USSN 60/134, 472, 1999
Filed on May 17, 2015. In essence, the substance is collected in the center of the channel by forcing the fluid flow from the opposite channel to the main channel or by other fluid manipulation.

In an alternative embodiment, the microfluidic system may be incorporated into a centrifuge rotor device that rotates in a centrifuge. Fluid and particles travel through the device due to gravity and centripetal / centrifugal pressure. Another way to achieve transmission by microfluidic channels is by transmission of electrokinetic materials. As used herein, "electrokinetic substance delivery system" refers to the transmission and orientation of a substance within a chamber containing microchannels and / or structures by the application of an electric field to the substance, through the channel and / or chamber. And a system for moving the substance therebetween, ie, the cations move towards the negative electrode,
The negative ions include a system that moves towards the positive electrode. A variety of electrokinetic controllers and systems are available, not only for the various references described herein, but also for example Ramsey W.
O96 / 04547, WO98 / 46438 of Parce et al. And Dubrow et al.
WO 98/49548. In the present invention, electrokinetic transmission or electric pumping is used to introduce pressure driven flow, as appropriate. The pressure driven flow described above is used in the system to transport fluidic substances through the channel system to perform various assays. Flow rates in various assays are controlled by the channel configurations described below, eg, to reduce reagent consumption and / or to regulate reaction times.

III. Adjusting Flow Rate Using Bypass Loops The flow rate of fluidic material in a microfluidic device is adjusted accordingly by configuring the channel to reduce the pressure and / or velocity or flow rate of the fluid in the channel. Various example channel configurations that reduce the flow rate in a microfluidic device, such as in the main channel, are shown, for example, in Figures 3-10 and 12. In one embodiment, the flow reduction channel is a bypass loop, such as the flow reduction channel 508 as shown in FIG. 5, which intersects the main channel 504 at two points. For example, in FIG. 4, the flow reduction channel 408 intersects the main channel 408 at the first and second positions. Figure 4
The flow rate in the device is appropriately adjusted by the following method. The sample is aspirated from the sample well 402. Buffers, diluents or other fluid substances may
Added to the sample from. When this additional fluid substance is added to the sample, the pressure increases in the main channel and the flow rate increases. By withdrawing fluid from the main channel and entering it into the flow reduction channel 408, the pressure is reduced and the flow rate returns to the initial flow rate. The flow reduction channel is configured such that it intersects the main channel downstream of the point of introduction of the additional fluid material. The second location of the intersection for bypass is generally downstream of the first location. If additional material is added to the sample for the reaction or assay, the second location is generally downstream of the reaction point or the point of addition of the reagent material. For example, in FIG. 4, the flow reduction channel 408 intersects the main channel 404 at a first position downstream of the reservoir 416 and at a second position downstream of the reagent wells 418 and 420 and upstream of the pressure source 406. .

Alternatively, the flow reduction channel is configured to intersect a main channel (eg, main channel 408) upstream of the intersection of the additional fluid material. In this embodiment, fluid is for example withdrawn from the main channel 408 and placed in a flow reduction channel, which reduces the flow rate of the fluid in the main channel prior to addition of, for example, buffers, diluents and the like. Subsequent addition of, for example, buffers, diluents or other fluid substances will increase the flow rate to, for example, its original or standard value, i.e. the flow rate before the fluid is withdrawn and placed in the flow reduction channel. . In this embodiment, the channel is configured such that the flow reduction channel is upstream of the reagent or diluent introduction channel. Thus, the flow rate decreases, for example, before the dilution mixing point and returns to the standard value (as opposed to the increase value) after the introduction of diluents, buffers, etc. This allows mixing of reagents or diluents into the sample to occur at shorter distances.

In another embodiment, the flow reduction channel intersects the main channel at only one location, but at its downstream end is a pressure source, generally the same pressure fluidly connected to the downstream end of the main channel. Connected to the source. Examples of configurations for this flow reduction channel include, but are not limited to, the devices of FIGS. 3 and 8. In FIG. 3, a flow reduction channel 308 intersects the main channel 304 and is fluidly connected to the pressure source 306. In FIG. 8, channel regions 808, 808B, 810, 81
0B and 812, 812B intersect the main channel regions 804, 804B and are fluidly connected to waste wells 806, 806B to which a pressure source is applied. By directing the fluid through the flow reduction channel described above, the flow rate in the microfluidic device is adjusted in response to the addition of material to the main channel. Another advantage of this flow regulation is that, in addition to maintaining a continuous or constant flow rate, subsequent reagent additions need not be higher to meet the increased flow rate requirements.
This saves the amount of reagents needed to perform the assay in a microfluidic device.

To further reduce reagent consumption, assay reagents are optionally added to the second flow reduction or reaction channels. An apparatus including a second flow reduction channel is shown in FIG. For example, the second flow reduction channel 7
42, 744 and 746 are flow reduction channels 712, 71, respectively.
0 and 708 are fluidly connected. Thus, fluid is withdrawn, for example, from flow reduction channel 708 and enters second flow reduction channel 746, thereby reducing the flow rate of material in channel 746. Substrates, reactants or enzymes required for the assay are added to the flow reduction channel or the second flow reduction channel as appropriate. For example, if the reagent is reservoir 726 or 72
8 to the reaction channel 742 as appropriate. If the reaction channel 742 has a smaller cross-sectional dimension than the flow reduction channel 712, reagent consumption is further suppressed due to the smaller size of the reaction channel. Alternatively, they are added into the main channel after a portion of the fluid has been withdrawn and placed in the flow reduction channel. In both cases, the smaller flow rates and / or channel dimensions reduce the amount of reagent material that must be added to perform the assay.

IV. Acquiring Multiple Concentration Measurements and Performing Multiple Assays in a Microfluidic Device Another advantage of using flow reduction channels in a microfluidic device is the use of these channels, where appropriate, for multiple samples of the same sample material in one assay. Is to obtain the measured value of. For example, if appropriate, a diluent is added to make a 10: 1 dilution of sample material in the main channel. When a part of the 10: 1 diluted substance is extracted from the main channel and put into the flow reduction channel, appropriate measurement values are obtained in the detection region located in the flow reduction channel. For all additional dilution levels thus obtained, eg 100: 1, 1000: 1 or 10,000: 1, separate detection regions
Optionally, the signal is detected by being placed in the flow reduction channel. See, for example, FIG. A detector is located near each flow reduction channel, eg, 608, 610 and 612. Also, the detector is located near the main channel. Generally, when the assay is performed in the device, the detection area in which the detector is located is a reagent reservoir, such as reservoirs 626, 628, 622 and 62.
4 is arranged downstream. The assay is then run and the detector is used to detect the signal that correlates to the result of the assay.

In another embodiment, moveable detectors are used to move between detection regions to detect a signal from each of the detection regions of interest. Alternatively, a single detector is arranged to detect signals from all flow reduction channels simultaneously. Such a configuration is shown in FIG. The main channel and flow reduction channel include parallel channel regions 804, 804B, 808, 808B, 810, 810B and 812, 812B centered at a single detection window 832, 832B. Using these channel configurations, one assay can be used to obtain measurements for a single sample at multiple concentrations. Also, because the reagent reservoirs are appropriately positioned to be fluidly connected to the flow reduction channels, the reaction or assay is optionally performed at various dilution levels resulting from serial dilutions. For example, as shown in FIG. 6, reservoirs 622 and 624 are fluidly coupled to flow reduction channels 608 and 610. Accordingly, reactions or assays are optionally performed in flow reduction channels 608 and 610 containing different concentrations of sample when operating as described above to perform serial dilutions.

The channel configurations described herein are optionally used, for example, to simultaneously perform, eg, multiple assays or measure multiple samples. A single sample or test compound is optionally introduced into the device, for example through a capillary fluidly connected to the microfluidic device. The test compound may be any drug, potential drug, compound, enzyme,
Examples include proteins and nucleic acids. The sample is conveniently divided into n parts, where n ranges from about 2 to about 100. These portions are optionally used at their initial concentration or diluted as described below. Each portion is flowed through one of x assay channels (eg, parallel assay channels), where x ranges from about 1 to about 100. As used herein, "assay channel" refers to, for example, a parallel configuration used to perform assays, tests, screens, etc. (eg, biochemical assays) on various compounds (eg, chemical or biochemical compounds). Refers to microscale channels. The flow reduction channels of the present invention are optionally used as assay channels. A portion of the sample or test compound is optionally co-flowed into the assay channel to perform multiple simultaneous assays.

Once the sample portion is cast into the assay channel, one or more reagents are added to each channel and the assay is performed by combining the reagent and sample portion. Different reagents are added to each assay channel as appropriate, thereby performing y different assays in x assay channels (y ranging from about 1 to about 100).
Optionally, these assays are performed simultaneously, eg, different sample portions are simultaneously flowed through the channel and react simultaneously with one or more reagents (eg, different reagents). These reagents are suitable for HSA binding assay, target screening, drug screening, enzyme assay, fluorescence assay, dose-resp
onse) assay, selectivity screen, protease assay, binding assay, etc. An alternative method of performing different assays or imposing multiple chemistries on a single compound involves using different channel chemistries in each of the assay channels. Each assay yields z products, where z ranges from about 1 to about 1000. For example, each reaction produces one or more products, and the products in each of the x channels from the y assays are appropriately labeled such that they are in the detection region (eg, a detection region in the vicinity of all x channels). ) Is detected as it flows through. In this way
Using the apparatus and methods of the invention, multiple assays are run simultaneously on a single test compound.

V. Assays Optionally Performed Using Devices of the Invention Devices, such as those shown in Figure 8, are optionally used to test compounds for inhibition of target enzymes in a continuous flow enzyme inhibition assay. For example, a device such as that shown in FIG. 8 has a capillary attached to the microfluidic body structure at capillary attachment points 802, 802B to act as a sample injection port, and four parallel reaction / treatment channels, eg, channel regions. 804, 804B, 808,
808B, 810, 810B and 812, 812B are incorporated. The channel region incorporates one or more fixed dilution stages that alter the final concentration of test compound over a range of three orders of magnitude. Also, the four parallel channel regions are
It includes an assay region in which the compound is mixed with enzyme and substrate and detection windows 832, 832B. In the detection windows 832 and 832B, four channel regions 804 and 804B are provided.
, 808, 808B, 810, 810B and 812, 812B are placed in close proximity to facilitate simultaneous monitoring of the resulting fluorescence using imaging or scanning techniques.

The test compound was applied to the waste wells 806, 806B by applying a vacuum.
It is injected into the microfluidic device through the sample capillary. In the main channel regions, channel regions 804, 804B, the test compound is delivered into the device and mixed with the enzyme and substrate without further dilution. The channel regions 808, 808B incorporate one dilution stage to reduce the concentration by a factor of 10. This is the reservoir 81
6,816B with additional assay buffer to allow mixing of the components and withdrawal of additional fluid into the waste channel to maintain a constant total flow rate. Similarly, channel regions 810, 810B and 812, 812B incorporate two and three dilution stages, respectively, to increase test compound concentrations by a factor of 100 and 10.
It is reduced by 00 times. The test compounds are appropriately autofluorinated by keeping the waste streams from the dilution stage separated instead of ligating as shown.
luminescence) (since the waste channel is also imaged or scanned) and this data is optionally used to correct the raw fluorescence data for the assay prior to calculation of% inhibition.

To further adjust the flow rate, the channel dimensions are adjusted accordingly, for example by incorporating loops or serpentine features. This changes the width and depth,
It modifies the relative hydrodynamic drag to achieve the desired dilution factor, flow rate and mixing time. The sample injection circuit is optionally adjusted so that the test compound solution reaches the parallel channel regions simultaneously. Similarly, the total length of the parallel channel region (and waste stream if desired) is adjusted accordingly so that the samples arrive at the detection window synchronously. Similar designs are used in kinase assays, binding assays, cell-based assays, etc., as appropriate. Alternatively, the device of the invention is used to perform multiple assays simultaneously.
For example, for a single sample plug divided into parts, HS
The A assay and high throughput target screening are performed in an appropriate combination. This divided portion is cast into the various assay channels of the invention, each of which undergoes a different reaction chemistry. Alternatively, intrinsic drug fluorescence is measured in combination with a high throughput target screen, or a panel of similar enzymes is evaluated simultaneously in a selectivity screen. In another embodiment, dose response experiments are performed using in-line dilution as described above to study the entire range of dissociation constants for a single sample.

In another embodiment, the same reaction is carried out in each channel, the only difference being eg the different enzyme isoforms. For example, a single nucleotide polymorphism (SN
P) is now known and is being rapidly determined for all enzymes or gene products. These enzymes are drug targets, enzymes important in metabolism, etc., such as P450 enzymes, as appropriate. Generally, enzymes are screened against potential drugs or drug compounds, eg, in a high throughput format. Performing screening in parallel for multiple types of enzymes is generally tentative in terms of reagent consumption and time. Differences in individual enzymes, such as p450 enzymes, can be important factors that have a large impact on different drug-drug interactions and drug side effects. The device of the present invention is optionally used to simultaneously screen all desired forms of an enzyme or protein in parallel with one sample. This allows significant SNP differences between individual compounds to be taken into account, eg, early in the drug discovery process, before the next expensive steps, including clinical trials on human subjects, for example. Further, such a method is effective in high-throughput target screening and non-target-dependent high-throughput screening.

For example, different reactions can be performed simultaneously in the same device by incorporating multiple reagent wells and adding different reagents to each channel as described above. For example, reservoir 822 may optionally add a reagent to channel region 804 to cause reservoir 820 to
In addition to channel 808, reservoir 826 in addition to channel 810, reservoir 8
24 is added to channel 812. Channels 842, 844 and 846 are optional, as multiple reactions are optionally performed in the apparatus without flow reduction channels. Alternatively, flow reduction channels are used as additional assay channels or to dilute sample portions before subjecting to various assays.

In another embodiment, a single sample or test compound is given a single sample by varying the channel dimensions of the various assay channels of the device, eg, by using different channel coatings, substances, etc. in different assay channels. Multiple assays are run simultaneously on the device. For example, one channel is
A glass optionally containing or functionalized with a separation matrix, such as silanized
ized) glass is included as appropriate. In some embodiments, different reagents, beads composed of different reagents, etc. are preloaded into the channels to provide different chemistries. Surface modification of polymeric substrates can take a variety of different forms, including coatings with suitably charged materials, derivatizing the molecules present on the surface to give charged groups on the surface, and / or Alternatively, the charged compound is coupled to the surface. For a description of the application and use of channel coatings, see, eg, Parce et al., US Pat. No. 5,885,470, “Controlled Fluid Transport in Microf.
abricated Polymeric Substrates ”and published PCT application WO 9 by the same person
See 8/46438.

VI. Suppression of Pressure Perturbations by Natural Injection into Microfluidic Devices In general, natural injection occurs in microfluidic systems that use external capillaries, eg, to transfer a sample from a sample plate to the microfluidic device. The term "spontaneous injection" as used herein refers to the movement of a fluid or other substance into a given passage or conduit in the absence of external forces, such as differential pressure or electric fields. In general, as also used here, spontaneous injection results in fluid migration into the channel at the tip of the fluid-filled capillary channel as a result of capillary action within the channel, surface tension of the fluid outside the channel, etc. Refers to operation. Thus, fluids or other substances that are "naturally injected" into channels, chambers or other conduits move into the channels, chambers or other conduits without the assistance of externally applied motive forces. In general, the reduction of spontaneous injection is considered a problem in capillary electrophoresis applications because it provides a constant volumetric error in sampling (independent of sampled volume) that may vary depending on the shape of the capillary channel and channel tip. Be done. Methods for reducing or eliminating this effect are described, for example, by Chow et al. By USS
N09 / 416,288 "External Material Accession Systems", which is a method for utilizing this phenomenon to obtain improved sample acquisition,
It also provides a method for sampling, for example, extremely small volumes of fluid.

Spontaneous injection into a microfluidic device that includes an external suction capillary can also be performed under a substantially constant driving force, such as pressure (eg, a single vacuum source), or electrokinetically driven flow. Is known to cause flow rate perturbations in. The suction capillary is a fluid reservoir (
When fried from a sample well, for example, the curvature of the drop of liquid at the end of the capillary
It exerts additional pressure inwardly of the capillaries, resulting in a higher flow rate. Perturbations in flow rate cause perturbations in the assay signal, which can interfere with the quantitative analysis of assay results. See, eg, FIG. For example, perturbation of the signal can obscure inhibition in the enzyme's inhibition reaction. Therefore, it is desirable to minimize pressure perturbations due to increased flow rate as drops of fluid spontaneously infuse into the microfluidic device.

The present invention presents a method for suppressing pressure perturbations due to spontaneous injection into a microfluidic device. The method generally involves dipping the open end of the capillary into a sample source (eg, a microwell plate), thereby withdrawing the sample from the sample source into the capillary. Capillaries are generally external suction capillaries fluidly coupled to a microfluidic device. The method includes withdrawing the open end of the capillary from the sample source. The first portion of the sample remains at the open end and is naturally injected into the capillary by surface tension exerting pressure on the capillary. A second portion of the sample is flowed from the capillary into the main channel that intersects the capillary at the first crossing point or pressure node. A third portion of the sample is flowed through the shunt channel, creating a differential pressure between the first crossing point or pressure node and the open end of the capillary. Generally, the shunt channel intersects the main channel at or downstream of the first intersection. Fluid flow through the shunt or bypass channel changes the pressure at the first crossing point, thereby suppressing pressure perturbations in the main channel. The pressure at the first crossing point is suitably greater or less than the pressure at the open end of the capillary (typically atmospheric pressure).

The method described above diminishes the effect of spontaneous injection by flowing fluid from the suction capillary into the bypass channel to change the pressure point in the system. For example, the pressure nodes that mix the reagents on the chip or merge the reagents or samples introduced into the microfluidic device from the suction capillary determine the degree of pressure perturbation in the microfluidic device. In general, the further away this pressure node is from atmospheric pressure, the less effective the pressure perturbation of spontaneous injection becomes. The flow reduction or shunt channel of the present invention is used to shape the pressure at this pressure node or intersection and to minimize pressure perturbations. The shunt channel is also a controllable channel whose pressure is adjusted accordingly to increase or decrease the differential pressure. For example, a pressure source coupled to the controller is fluidly coupled to the shunt channel to control the pressure in the shunt channel, for example by applying positive or negative pressure to the shunt channel. Alternatively, the shunt channel is configured to provide a particular differential pressure, for example using width, depth, channel coating, etc.

[0066]   For example, in a microfluidic device without shunt channels as shown in FIG. 9A,
Part pressure node P₁Is the flow rate Q₁, Hydrodynamic resistance R₁And the applied pressure P₀ Is determined as follows.     P₁= P₀+ Q₁R₁ FIG. 9B shows a device with the addition of shunt channel 902. Fluid shunt channel 90
2 when, for example, flowing from the capillary 904, all external pressure nodes P₀, P ₂ And P₃If does not change, Q₁Is Q₁’ Capillary tube 904, shunt 9
02 and side channel 906 at the internal pressure node P₁Is as follows
is there.     P₁’= P₀+ Q₁’R₁ Q₁’Is Q₁Because it is larger, P₁’P₁Greater than Due to this pressure difference, FIG.
B device is P₀FIG. 9A for spontaneous injection pressure perturbation (eg, at intersection 908).
It has a greater resistance than the other devices. Other benefits of shunt design include, but are not limited to,
The level of relative hydrodynamic dispersion of the sample plug through the capillary is reduced.
And through a suction joint that connects the capillary to the microchannel on the device.
Including that the tailing of the pull plug is reduced.

VII. Equipment Although the devices specifically described herein are generally described in terms of performing a few or one particular operation, it is herein noted that the flexibility of these systems allows other operations to be readily integrated into these devices. It will be easily understood from the calligraphy. For example, the described devices and systems optionally include structures, reagents and systems to perform virtually any number of operations both upstream and downstream of the operations specifically described herein. Such upstream operations include sample handling and preparation operations such as cell activation, labeling reactions, dilutions, aliquoting and the like. Similarly, downstream operations include, for example, separation of sample components, labeling of components, assay and detection operations, electrokinetic or pressure injection of components in contact with or released from particle sets. , And similar operations may be included.

In the present invention, substances such as cells, proteins, antibodies, enzymes, substrates, buffers, etc.
It is optionally monitored and / or detected so that, for example, the presence of the component of interest can be detected, the activity of the compound can be determined, or the effect of the modulator on, for example, the activity of the enzyme can be measured. Depending on the measured value of the label signal, subsequent fluid manipulations, such as whether or not to specifically assay a particular component to determine kinetic information, are appropriately determined. Generally, the system described herein includes another device for controlling the transfer, flow rate and direction of fluid within the device, a detection device for detecting or sensing the result of the operation performed by the system, a pre-programmed device. Command to the control device according to the command given,
It includes a microfluidic device as described above, with a processor (e.g., a computer) for receiving data from the detection instrument, analyzing, storing, interpreting the data, and providing the data and interpretation in an easily accessible reporting format.

Fluid Directing System Various control devices, such as the pressure-based or electrokinetic controls, for controlling fluidic substances and / or the transmission and direction of substances within the device of the present invention, are microfluidic devices as described above. Is used together with. In the system, a fluid pointing system controls the transfer, flow and / or movement of the sample through the microfluidic device. For example, the fluid-indicating system optionally directs sample transfer into and through the main channel, where the sample is optionally diluted with a buffer or other diluent. The fluid-indicating system optionally directs the transfer of buffer or other diluent from the source of material into the main channel, thereby obtaining a first diluted sample. The system also directs the transfer of a portion of the first diluted sample into the flow reduction channel, while the second portion of the first diluted sample remains flowing in the main channel. The fluid-indicating system also optionally directs the transfer of a second aliquot of buffer into the main channel to effect a serial dilution to produce a second diluted sample. The fluid indicating system then sends a first portion of the second diluted substance into the second flow reduction channel while leaving a second portion of the second diluted substance in the main channel. The fluid-indicating system also repeats these movements to provide further successive dilutions of the sample material, sending a portion of the fluid material into the flow reduction channel to reduce the pressure in the main channel after each dilution.

After the desired dilution levels have been obtained in the various channels of the system, the fluid-indicating system may optionally utilize one or more reagent substances (eg, substrate, (Eg, enzyme) to react with sample material and / or diluted material. To reduce reagent usage, the fluid-indicating system optionally directs the movement of the diluted sample from the flow reduction channel to the second flow reduction channel or reduction channel. The movement of sample material and diluted material through the channel into the detection area (where they are detected) is also controlled by the fluid indicating system.

For example, to perform multiple assays on a single sample, the fluid-indicating system divides the sample into n portions and divides the n sample portions into x assay channels (eg, in FIGS. 8A and 8B). Parallel assay channels as shown). The fluid indicating system also directs the addition of various reagents to each of the x channels, as appropriate. Different reagents are suitably added to each of the x channels, thereby exposing each portion of the sample to a different chemistry (eg, different reaction or assay) to perform y different assays, and Make z different products. Generally, the number n of sample portions used ranges from about 2 to about 100. In general, the number of sample portions is substantially equal to the number of channels x. Generally, the fluid pointing system sends one sample portion to each of the different channels. Alternatively, 1
A device with 0 channels is used, the sample is split into only two parts and only two channels are used. In another embodiment, 10 different samples
Sent to each of these channels. Generally, the number of channels is in the range of about 2 to about 100. Preferably, the number of channels is in the range of about 2 to about 20. More preferably, the number of channels is from about 4 to about 10. Once the sample is in the channel, the fluid indicating system sends various reagents from the reservoir to the assay channel to perform, eg, y different assays. Generally, the number y of different assays will range from about 2 to about 100. However, the number of assays need not equal the number of channels or sample portions. Different assays are run in each of the x channels, as appropriate. For example, several different enzyme substrates
Optionally, they are screened simultaneously in different channels of the invention, or several different protein binding sites are probed simultaneously by examining each binding site in different channels, as appropriate. Alternatively, one assay (eg, site IHSA binding) is performed on some channels (eg, half of the channels) and another assay (eg site IIHSA binding) is performed on the remaining channels. Generally, y assays produce z different products, eg, each assay produces one or more products. Therefore, the number of products is generally at least equal to the number of assays, but is often greater than the number of assays y. Generally, the number of products will range from about 1 to about 10,000, more typically from about 2 to about 1000, and most commonly from about 2 to about 100. In general, each assay produces one or more products that the fluid-indicating system directs through the assay channel to the detection region and is detected from the assay channels that are concentrated in a single detection region (eg, simultaneous detection, Fluorescence detection).

In order to suppress pressure perturbations caused by spontaneous injection of fluid from an external capillary into the microfluidic device (including at least one fluid control element fluidly connected to the main channel, the shunt channel and the capillary). ) A fluid-indicating system directs the movement of the sample from the first sample source to the inlet region of the capillary and the movement of the sample from the inlet region of the capillary to the outlet region of the capillary. The sample is then delivered from the exit region of the capillary to the upstream region of the main channel. As mentioned above, the first portion of the sample from the upstream region of the main channel is flowed into the shunt channel and the second portion of the sample remains in the main channel. This fluid movement is maintained at the intersection of the main channel and the capillary at a pressure different from the pressure in the inlet region of the capillary. Due to this pressure difference, the effect of pressure perturbation of natural injection is suppressed.

In general, fluid transfer and direction in microfluidic devices such as those described above may be performed in whole or in part using a pressure-based flow system that incorporates an external or internal pressure source to drive the fluid flow. Controlled by. Internal sources include microfabricated pumps, such as diaphragm pumps, heat pumps, Lamb wave pumps, etc. as described in the art. For example, US Pat. No. 5,271,7
24, 5,277,556, and 5,375,979, and published PCT applications WO 94/05414 and WO 97/02357. As mentioned above, the systems described herein may also utilize electrokinetic material directing and transfer systems. Preferably using an external pressure source,
Add to port at end of channel. More preferably, a single pressure source is used at the end of the main channel. Generally, the pressure source is a vacuum source applied to an end point downstream of the main channel. These applied pressures or vacuums create a differential pressure over the length of the channel, driving fluid flow through the channel. In the interconnected channel network described herein, different pressures or vacuums are applied to multiple ports, preferably with a single vacuum applied to a common waste port and with appropriate resistance to produce the desired flow rate. By configuring the various channels, flow rates with different volumes are realized appropriately. Example system is USSN0 dated January 28, 1999
9 / 238,467.

In general, the controller system is suitably configured to receive or interface with microfluidic devices or system elements as described herein. For example, the controller and / or detector may optionally include a stage upon which the apparatus of the present invention may be mounted to facilitate a suitable interface between the controller and / or detector and the apparatus. Generally, the stage will include suitable mounting / alignment structural elements such as, for example, mating wells, alignment pins and / or holes, asymmetric edge structures (to facilitate proper device alignment), and the like. Including. Many of these configurations are described in the references cited herein. The control devices described above are also used as appropriate to provide electrokinetic injection or withdraw material downstream of the region of interest to control the upstream flow rate. The same equipment and techniques described above are also used to inject fluid into downstream ports to act as flow control elements.

Detector The device herein suitably includes, for example, a signal detector for detecting fluorescence, phosphorescence, radioactivity, pH, charge, absorbance, luminescence, temperature, magnetism, color and the like. Detection of fluorescence and chemiluminescence is particularly preferred. The detector optionally monitors one or more signals from one or more detection areas of the device, for example detection areas 332 or 832, 832B in FIGS. 3 and 8. The one or more detection regions may correspond to different sample concentrations achieved by serial dilution, or different samples being assayed. For example, the detector optionally monitors the optical signal corresponding to labeled components such as labeled antibodies or proteins located in the detection regions 832, 832B, for example.

In one embodiment, the detection region spans multiple main channels and / or flow reduction channels, and one detector is used to detect signals from all channels simultaneously. For example, in FIG. 8, the detection regions 832, 832B include, for example, the main channel regions 804, 804B containing undiluted sample as well as, for example, 1
: 10 dilution sample material, 1: 100 dilution sample material and 1: 100
Parallel regions 808, 808B, 810, 810B containing zero dilution of sample material
And monitor the signals from 812 and 812B. Alternatively, a single detector in proximity to each of two or more assay channels, eg, parallel assay channel regions, may be performed on the same sample, eg, divided into two or more channels to perform multiple assays. The results of two or more different assays performed are detected.
The results of multiple enzyme assays, eg, producing different products, are optionally simultaneously detected by detectors located in close proximity to all relevant assay channels. For example,
The consequences of HSA binding to Site I and Site II are optionally detected simultaneously when simultaneously probed in two different channels.

Alternatively, if the flow reduction channels are not in close proximity to each other, then separate detectors are optionally used to detect the signal from each channel. For example, in FIG. 6, the channels are appropriately configured so that the detectors are not in close proximity to each other. A single detector is sufficient if the channels are looped and the detectors are in close proximity to each other. When a single detector does not detect all signals, or when different types of detection are required, the channel configuration of Figure 6 is used accordingly. The detector is the main channel 6
04 to detect the signal from the detector region 632, to detect the signal from the flow reduction channel 612 to detect the region 634, to detect the signal from the flow reduction channel 610 to detect the signal. Located near 636 and near the detection region 638 to detect the signal from the flow reduction channel 608. Alternatively, a single detector may be used in the detection area 632,
It is moved between 634, 636 and 638. In the above case, detection is appropriately performed as follows. That is, an undiluted unreacted sample is detected in the detection area 632, an undiluted reacted sample is detected in the detection area 634, and a double diluted and reacted sample is detected in the detection area 636, The sample once diluted and reacted with the reagent is detected in the detection region 638. Once detected, the flow rate and velocity of cells in the channel are also appropriately 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 easily incorporated into the microfluidic system described herein. In these systems, these sensors include a microfluidic device in the device or one or more channels, chambers, or the like, such that the detector is in sensory communication with the device, channel or chamber. Located inside or near the conduits. In general, the term “vicinity” as used herein with respect to a particular element or region is a property of the contents of the microfluidic device, the part of the microfluidic device or the part of the microfluidic device in which the detector is intended (detector is intended). The arrangement of the detector at a position where it can detect For example, a pH sensor arranged to be in sensor communication with a microscale channel may have a pH of a fluid arranged in that channel.
Can be determined. Similarly, a temperature sensor arranged in sensor communication with the body of the microfluidic device can determine the temperature of the device itself.

Particularly preferred detection systems include photodetection systems for detecting the optical properties of substances within the channels and / or chambers of microfluidic devices incorporated into the microfluidic systems described herein. Generally, such photodetection systems are placed adjacent to a microscale channel of a microfluidic device and are in sensor communication with the channel via a photodetection window located across the channel or chamber of the device. Photodetection systems include systems that can measure the properties of the material as well as the light emitted from the material in the channel, the transmittance or absorbance of the material. Examples of detectors include photomultiplier tubes, CCD arrays, scanning detectors, galvo scanners and the like. For example, in a preferred embodiment, fluorescence, chemiluminescence or other optical detectors are used during the assay. A protein, antibody or other component that emits a detectable signal is flowed past the detector, or alternatively the detector can be moved relative to the array to locate the protein (or Several spatial positions corresponding to the channel area in the CCD array can be monitored simultaneously).

In a preferred embodiment, the detector measures the amount of light emitted from a substance, eg a fluorescent or chemiluminescent substance. Generally as such, detection systems include collection optics for collecting a signal based on the light transmitted through the detection window and transmitting the signal to a suitable photodetector. Refractive power, field diameter (field d
The objective of the microscope, which alters the iameter) and the focal length, is easily utilized at least as part of this optics train. The photodetector may be an avalanche photodiode, a photomultiplier tube, a diode array or in some cases a charge coupled device (CC), as appropriate.
An imaging device such as D). In a preferred embodiment, the photodiode is at least partially used as a photodetector. Generally, the detection system is coupled to a computer (described in detail below) via an analog-to-digital or digital-to-analog converter for transmitting the detected optical signal to the computer for analysis, storage and data manipulation. .

In the case of fluorescent substances such as labeled cells, the detector generally activates the fluorescent substance as well as the optics that direct the light source through the detection window to the substance contained in the channel or chamber. Includes a light source that produces light of the appropriate wavelength. The light source can be any of several light sources that provide suitable wavelengths, including lasers, laser diodes and LEDs. Other light sources are required for other detection systems. For example, a broadband light source is generally used for measuring light scattering / transmittance. Light selection parameters are generally well known to those skilled in the art. The detector can be provided as a separate device, but is preferably integrated in a single instrument with the controller system. The integration of these functions into a single device allows these devices and computers (described below) to communicate with the controller, detector and computer by enabling a few or a single communication port. Facilitate connection. In general, the integration of a detection system with a computer system includes software for converting detector signal information into assay result information (eg, substrate concentration, product concentration, presence of the compound of interest, etc.).

Computer As noted above, either or both of the fluid pointing system and / or the detection system are coupled to a suitably programmed processor or computer.
The processor or computer directs the operation of these devices according to pre-programmed or user-entered instructions, receives data and information from these devices, interprets this information, manipulates and reports to the user. . As such, a computer is typically suitably coupled to one or both of these devices (including, for example, analog-to-digital converters or digital-to-analog converters if necessary). Generally, the computer will, for example, in the form of user input to a GUI set parameter field, or in the form of pre-programmed instructions (eg, pre-programmed for various different specific actions). Includes suitable software for accepting. The software then translates these instructions into the appropriate language to direct the operations of the fluid directing and transfer controller to perform the desired operations. For example, the software may instruct the fluid indicating system to transfer the sample to the main channel, transfer buffer or other diluent to the main channel, transfer sample or a portion of the diluted sample to the flow reduction channel. And transferring a portion of the sample or diluted sample through the main channel, transferring a portion of the sample or diluted sample into the second flow reduction channel, and transferring the reagent substance to the main channel, the flow reduction channel or the second channel. It can be transferred into the flow reduction channel and can also carry out other transfers necessary to perform the assay of interest and / or detect the component of interest.

The computer then receives data from one or more sensors / detectors contained within the system and interprets this data and provides it in a user-understandable format or uses that data. Program to initiate another controller command to monitor and control, for example, flow rate, temperature, applied pressure, etc. In the present invention, the computer typically includes software for monitoring the substance in the channel. Also, software is optionally used to control electrokinetic or pressure regulated injection or material withdrawal. This injection or withdrawal is used to regulate the flow rate as described above. The computer also optionally includes software for deconvolution of the signal (s) from the detection system. For example, this deconvolution distinguishes two detectable different spectral properties that were both detected, eg, when the substrate and product contained different detectable labels.

Integrated System Example Panels A, B and C of FIG. 1 and FIG. 2 provide further details on an example of an integrated system that may be suitably used to carry out the methods described herein. As shown, the body structure 102 has a main channel 104 disposed therein.
The sample or mixture of components is suitably flushed from the pipette channel 120 towards the reservoir 114, for example by applying a vacuum to the reservoir 114 (or another point in the system) or by applying an appropriate voltage gradient. Alternatively, a vacuum is applied at the reservoirs 108, 112 or through the pipette channel 120. Additional substances, such as buffer solutions, substrate solutions, enzyme solutions, etc., may be flushed from wells 108 or 112, as described above, and the main channel 104 may be used.
Shed inside. The flow from these wells is controlled by adjusting the fluid pressure.
Alternatively, it is appropriately realized by the electrokinetic approach as described above (or both purposes). The flow rate increases as fluid is added to the main channel 104, eg, from the reservoir 108. This flow rate is suitably reduced by flowing a portion of the fluid from the main channel 104 to the flow reduction channel 106 or 110. The channel configuration shown in FIG. 1 is one possible configuration among many suitable and available for use in the present invention. Alternatives are Figures 3, 4, 5, 6, 7, 8, 9, 1
Given to 0 and 12. Other alternatives are possible, for example, by combining the microfluidic elements described herein (eg, flow reduction channels) with other microfluidic devices described in the patents and applications referenced herein. Also, as appropriate, the elements of FIGS. 3, 4, 5, 6, 7, 8, 9, 10, and / or 12 may be rearranged to provide alternative configurations.

Samples and materials are optionally flushed from the wells listed or from sources external to the body structure. As shown, the integrated system optionally includes a pipette channel 120, eg, protruding from the body 102, for accessing a substance source external to the microfluidic system. Generally, the external source is a microtiter dish (mic
a rotiter dish) or other convenient storage medium. For example, as shown in FIG. 2, pipette channel 120 provides access to microwell plate 108, which contains sample material, buffer, substrate solution, enzyme solution, etc. within the wells of the plate. The detector 206 is in sensor communication with the channel 104 and detects, for example, the signal resulting from the leveled material flowing through the detection region. The detector 206 is suitably coupled to either the channel or the region in the device where detection is desired. Detector 2
06 is operatively coupled to a computer 204, which may use, for example, any of the above-described instructions, or any other instruction set, for example, to determine concentration, molecular weight, or identity, etc. , Digitize, store and manipulate the signal information detected by the detector 206. The fluid-indicating system 202 controls voltage, pressure, or both, for example, in the wells of the system, or through channels in the system, or in vacuum fittings fluidly coupled to channels 104 or other channels described above. Optionally, computer 204 controls fluid indicating system 202 as shown. In one set of embodiments, the computer 204 uses the signal information to select another parameter for the microfluidic system. For example, microwell plate 2
Upon detecting the presence of the component of interest in the sample from 08, the computer will optionally direct the addition of a potential modulator of the component of interest into the system.

Kits In general, the microfluidic devices described herein are packaged and contain reagents to perform the desired functions of the device. For example, a kit includes assay components, reagents, sample substances, proteins, antibodies, enzymes, substrates, regulatory substances, etc., along with any of the previously described microfluidic devices. In general, such kits will also include appropriate instructions for using the device and reagents, and, if the reagents have not been pre-positioned on the device itself, will place the reagents in the channels and / or chambers of the device. Have the appropriate instructions to install. In this latter case, these kits use special aids for introducing the substance into the microfluidic system, such as a suitably configured dropper (syr).
inges) / pumps, etc. (in one embodiment, the device itself includes pipette elements such as electronic pipettes for introducing substances into channels and chambers within the device). In the former case, such kits generally include a microfluidic device with the necessary reagents pre-placed in the channels / chambers of the device. Generally, these reagents are provided in a stabilized form to prevent disintegration and other losses during extended storage, such as by leakage. Incorporation of eg chemical stabilizers (ie enzyme inhibitors, microcides / bacteriostats, anticoagulants), eg physical stabilization of substances by immobilisation on solid supports, matrices (ie gels) Several stabilization processes are widely used for storage of reagents such as containment, lyophilization and the like. The kit also optionally includes packaging materials or containers for holding the microfluidic device, system or reagent elements.

In general, the above description is applicable to the features and embodiments of the invention as claimed. Also, the methods and apparatus described herein may be modified without departing from the spirit and scope of the claimed invention, and the invention may be subject to several different uses, including the following. Use of a microfluidic system to perform the assay described herein. Use of a microfluidic system to obtain measurements at multiple concentrations as described herein. Use of a microfluidic system to perform an assay at multiple concentrations as described herein. Use of a microfluidic device to perform multiple assays as described herein. Use of a microfluidic device to perform multiple assays simultaneously on a single test compound as described herein. Use of a microfluidic system to suppress reagent consumption as described herein. Use of the microfluidic system described herein for making serial dilutions of a sample. Use of a microfluidic system or device to control or regulate a flow rate as described herein. Use of a microfluidic system or device to suppress pressure perturbations due to spontaneous injection as described herein. An assay using any one of the microfluidic systems or substrates described herein.

VIII. Examples Example 1: In order to obtain data in phosphatase assay Phosphatase assay, using microfluidic device having a channel layout shown in Figure 8B. In a typical assay, wells 820b and 824b are filled with approximately 60 microliters of enzyme solution.
Substrate is added in wells 822b and 826b to form wells 816b and 818b.
Is filled with buffer. The final concentration of reagent delivered to the main reaction channel is determined by the relative flow rates in the side and reaction channels. By moving the detection point to a different location along the reaction channel, or in the waste well 806b.
By varying the negative pressure applied to the, the reaction time on the chip can be altered.

Reagents Various concentrations of reagents (eg, commercially available phosphatase inhibitors) are loaded into the microtiter plate for loading into the device through the suction capillary attached at capillary attachment point 802b. For example, the concentration of a general inhibitor is 1
Examples include mM, 0.625 mM, 1.25 mM, 2.5 mM, 5 mM and 10 mM. Common buffers used for phosphatase reactions are 25 mM Tris
-HCL (pH 7.0), 50 mM NaCl, 2 mM EDTA, 0.01%
Brij35, 5 mM DTT, and 500 NDSB. In the case of enzymes, a 1: 100 dilution of phosphatase was made (near 100 nm).
Generally located within the well of a microfluidic device. 100 μM phosphatase substrate solution is placed in the substrate well. A 1 nM solution of the marker dye Cy5 from Beckman is placed in a microtiter plate with an inhibitor sample.

Enzyme Inhibition and K _i Determination Inhibitor titration experiments were performed with competitive peptide phosphatase inhibitors having a high affinity for phosphatase in the assay. Generally, peptide concentrations range from 0.625 to 10 mM in microplates. Percent inhibition values were calculated from the decrease in fluorescence corresponding to the injection of inhibitor, the enzyme + substrate baseline, and the measured substrate-only background. Analysis of this data (1
The 10 highest inhibitor concentrations from the 00% and 10% channels showed an almost saturated response and were excluded from the analysis), resulting in a K _i of 169.55 μM when analyzed using the Dixon diagram. Got the value. From the graph of inhibitor concentration versus percent inhibition, IC ₅₀ values of 160 and 200 μM were estimated using substrate concentrations of 100 μM and 50 μM in the substrate wells of the device, and also the following equation: IC ₅₀ = K _i ^* ( 1+ [S] / K _m ) was used to calculate Ki values of 113 μM and 109 μM. The inhibitor titration measured in 4 different assay channels with 2 different substrate concentrations is shown in FIG.
Shown in. The Dickson diagrams used to determine K _i are shown in FIGS.
And gives the inhibitor channel concentration data plotted against percent inhibition.

Example 2: Suppression of Pressure Perturbation Generally, a microfluidic device having the channel layout shown in Figure 10 is used for high throughput screening assays, such as fluorogenic assays. In a typical experiment, the enzyme is placed in well 1004 and the fluorogenic substrate is placed in well 1005. Potential inhibitors are delivered to the device through a capillary placed in the microwell plate and attached to the device at a capillary junction 1008. As mentioned above, spontaneous injection occurs when a capillary tube is lifted from one well in a microwell plate and transferred to another well. The effect of spontaneous injection in dilution factor on reagents added from internal wells (eg, wells 1004 and 1005) is the same as placing a dye (eg, fluorescent dye) in the buffer solution in wells 1004 and 1005 on the device, and Characterized by moving the capillaries back and forth between two microplate wells containing buffer solution. FIG. 11 gives the corresponding fluorescence signal measured when the vacuum located in waste well 1010 was set to −1 psi. The double sinking feature in the data results from perturbations to the steady-state value due to the spontaneous injection of buffer from the capillary into the microfluidic device. The size of this sink is about 9% of the steady state value.

The channel layout of the microfluidic device shown in FIG. 12 includes the variations described above and optionally used to suppress the pressure perturbations shown by the data in FIG. The channel configuration includes a bypass or shunt channel 1202 used to draw fluid from the main channel 1210. The fluorogenic assay is suitably performed in the device of Figure 12 in the same manner as described above for the device of Figure 10. Enzymes and substrates are introduced into the main channel 1210 from wells 1204 and 1205. In the main channel 1210, the enzyme and substrate come into contact with the sample sent from the external capillary via the capillary junction 1208. The pressure at capillary connection point 1208 is controlled by shunting fluid from main channel 1210 to shunt channel 1202. Flowing the fluid through the shunt channel 1204 alters the pressure at the intersection, reducing the effect of spontaneous injection. For example, if characterized using dyes and buffers as described above for the device of FIG. 10, the signal produced using the device of FIG. 12 will be only 3% of the steady state value. It produces a subduction having a size.

13 and 14 provide fluorogenic enzyme assay data performed with a device without a shunt channel (FIG. 10) and a device with a shunt channel (FIG. 12), respectively. Approximately 10 nM solution of phosphatase enzyme was placed in well 1204 and
0 μM phosphatase substrate was placed in well 1205. The buffer used was 5
0MM Bis-Tris (pH 6.3), 50 mM NaCl, 0.075%
BSA, 0.1% Brij-35, and 2% DMSO. Inhibitors of phosphatase have ten different concentrations: 0.156, 0.313, 0.625, 1,
． Giving at 25, 5, 10, 20, 40 and 80 μM. The inhibitor sample was placed in the microwell plate and flowed from the microwell plate into the device via an aspiration capillary attached at capillary connection point 1208. The applied pressure is
The waste reservoir 1212 was set to -1.5 psi. Periodic wobbling or subduction in the steady-state signal due to spontaneous injection was well suppressed in the device containing the shunt channel.

While the above invention has been described in some detail for purposes of clarity and understanding, it should be understood that various changes in form and detail are possible without departing from the true scope of the invention. It will be apparent to those skilled in the art. For example, all of the techniques and apparatus described above can be used in various combinations. All publications, patent applications, patents and other references cited in this application are used for all purposes to the same extent as if each individual publication or patent document so indicated. Incorporated as a reference in its entirety.

[Brief description of drawings]

1 Panels A, B and C are schematic representations of the integrated system of the present invention including a body structure, microfabrication elements and pipette channels.

FIG. 2 is a schematic diagram of the integrated system of the present invention showing the combination of a microwell plate, a computer, a detector and a fluid detection system. The integrated system is shown in FIGS.
5, 6, 7, 8, 9, 10, or 12 devices or body structures or any other suitable microfluidic device.

[Figure 3]   1 is a schematic representation of a microfluidic device including a single flow reduction channel.

FIG. 4 is a schematic representation of a microfluidic device including a single flow reduction channel in a bypass loop configuration.

FIG. 5 is a schematic representation of a microfluidic device including multiple flow reduction channels for use in performing, for example, serial dilutions of a single sample or multiple assays of a single sample.

FIG. 6: Multiple flow reduction channels, multiple detectors and multiple detectors, eg, to obtain multiple concentration measurements for a single sample or to assay multiple test compounds or enzymes on a single sample. 1 is a schematic diagram of a microfluidic device including reagent wells.

FIG. 7 is a schematic diagram of a microfluidic device including multiple flow reduction channels and a second flow reduction channel, eg, to obtain multiple concentration measurements for a single sample and to reduce reagent consumption.

FIG. 8A shows a plurality of panels A configured in parallel and fluidly linked to a single detection window, eg, to obtain multiple measurements for a single sample at various concentrations or different assay conditions. 2 is a schematic diagram of a microfluidic device including the flow reduction channel of FIG.

8B shows one possible embodiment of the channel configuration corresponding to the schematic diagram of panel A. FIG.

FIG. 9: Panel A is a schematic of a microfluidic device without shunt channels, Panel B:
1 is a schematic diagram of a microfluidic device including shunt channels. Both devices include suction capillaries.

[Figure 10]   3 is a channel configuration of a microfluidic suction device without shunt channels.

FIG. 11 is data showing the perturbation of spontaneous injection observed using the microfluidic device of FIG.

[Fig. 12]   3 is a channel configuration of a microfluidic suction device including a shunt channel.

[Fig. 13]   11 is enzyme inhibition data obtained using the device of FIG. 10 without the shunt channel.

FIG. 14   13 is enzyme inhibition data obtained using the device of FIG. 12 including a shunt channel.

FIG. 15 is an inhibitor titration measured using the device shown in FIG. 8B with two substrate concentrations.

FIG. 16 is K _i determined by a Dickson diagram.

FIG. 17 is enzyme inhibition assay data showing inhibitor channel concentration for percent inhibition.

[Explanation of sign]

  100 Microfluidic device   102 body structure   104 Main channel   106 Flow reduction channel   108 reservoir   110 flow reduction channel   112 reservoir   120 pipette channels   202 fluid indicating system   204 computer   206 detector   208 Microwell plate   302 Sample well   304 Main channel   306 Pressure source   308 Flow reduction channel   316 buffer well   318 Reagent well   332 Detection area   402 sample well   404 Main channel   406 Vacuum source   408 flow reduction channel

─────────────────────────────────────────────────── ─── Continued front page    (31) Priority claim number 60 / 200,139 (32) Priority date April 27, 2000 (April 27, 2000) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Andrea W. Chow             United States California             94024 Los Altos Custa             Live 670 (72) Inventor Claudia El Poglish             United States California             94086 Sunnyvale S-Burner             Door Avenue Number 32 151 F-term (reference) 2G058 DA07 GE02

Claims (109)

[Claims] 1. A body structure including a main channel disposed therein, and further comprising: (a) one or more or more intersecting main channels at one or more intersections. A flow reduction channel, wherein the one or more flow reduction channels are structurally configured to reduce the flow rate of the fluid in the main channel as the fluid flows past the one or more intersections. (B) a first pressure source fluidly connected to the one or more flow reduction channels, the first pressure source being downstream of the one or more intersections in the direction of flow towards the first pressure source. A first pressure source disposed in the one or more flow reduction channels; a body structure including the first pressure source; and (ii) a main pressure source fluidly connected to the main channel. Fluidic device. 2. The apparatus of claim 1, wherein the first pressure source and the main pressure source are the same. 3. The apparatus of claim 1, wherein the first pressure source and the main pressure source comprise a single vacuum source. 4. (i) A main body structure part, which includes a main channel disposed therein, and further comprises one or more flows intersecting the main channel at a first intersection and a second intersection. A reduction channel, the one or more flow reduction channels being structurally configured to reduce the flow rate of fluid in the main channel as the fluid flows past the first intersection in a direction toward the second intersection. A microfluidic device comprising: the body structure; and (ii) a main pressure source fluidly connected to the main channel. 5. The one or more flow reduction channels are structurally configured to reduce the flow rate of fluid in the main channel as the fluid flows past the one or more intersections. The device according to claim 1 or claim 4. 6. The apparatus of claim 1 or claim 4 including two or more flow reduction channels in fluid communication with the main channel. 7. The apparatus of claim 1 or claim 4 including about 3 to about 4 flow reduction channels in fluid communication with the main channel. 8. The apparatus of claim 1 or claim 4 including from about 5 to about 10 flow reduction channels in fluid communication with the main channel. 9. The apparatus of claim 1 or claim 4 including about 10 or 11 or more flow reduction channels in fluid communication with the main channel. 10. The main channel has a first cross-sectional dimension, the one or more flow reduction channels have a second cross-sectional dimension, and the first cross-sectional dimension and the second cross-sectional dimension are the same. The device according to claim 1 or claim 4, wherein 11. The main channel has a first dimension, the one or more flow reduction channels have a second dimension, and the first dimension and the second dimension are different. Or the apparatus according to claim 4. 12. The apparatus of claim 11, wherein the second dimension is smaller than the first dimension. 13. The apparatus of claim 1 or claim 4, wherein the one or more flow reduction channels are non-intersecting channels. 14. The one or more flow reduction channels intersect with one or more second flow reduction channels, the second flow reduction channels having cross-sectional dimensions of the one or more flow reduction channels. An apparatus according to claim 1 or claim 4 having a smaller cross-sectional dimension. 15. The main pressure source comprises the one in the direction of flow towards the main pressure source.
Alternatively, the apparatus of claim 1 or claim 4 fluidly coupled to the main channel at a location downstream of the two or more flow reduction channels. 16. The apparatus according to claim 1 or 4, wherein the main pressure source is a vacuum source. 17. The apparatus according to claim 1 or 4, wherein the main pressure source is a single vacuum source. 18. The apparatus of claim 1 or claim 4, further comprising one or more detection regions in or near the one or more flow reduction channels. 19. A detection system further comprising at least one of a main channel and said one or more flow reduction channels.
An apparatus according to claim 1 or claim 4 comprising one or more detectors located in proximity to both of the two. 20. The apparatus of claim 19, wherein the one or more detectors comprises a single detector, which detector simultaneously detects signals in each of the one or more flow reduction channels. . 21. The apparatus of claim 19, further comprising a computer and software operably coupled to the apparatus for analyzing the signal detected by the detection system. 22. A source of first fluid material in fluid communication with the main channel at a first position along the main channel and a fluid source in fluid communication with the main channel at a second position along the main channel. The device of claim 1 or claim 4, further comprising a source of two-fluid material. 23. The apparatus of claim 22, wherein the first and second positions are upstream of at least one of the one or more flow reduction channels. 24. The first position is upstream of at least one of the one or more flow reduction channels and the second position is at least one of the one or more flow reduction channels. Downstream from one,
23. The device of claim 22. 25. The apparatus of claim 22, wherein the first and second positions are downstream of at least one of the one or more flow reduction channels. 26. The device of claim 22, wherein the first fluid material and the second fluid material are the same material. 27. The device of claim 22, wherein the first fluid material and the second fluid material are different. 28. The device of claim 22, wherein the first fluid material is a sample material and the second fluid material is a diluent material. 29. The device of claim 28, wherein the diluent is a buffer. 30. A fluid indicating system further comprising: (a) moving a first fluid substance from a source of the first fluid substance to a main channel and a second fluid substance of the source of the second fluid substance. From the main channel to the first fluid substance and the second
Combining fluid materials to form a third fluid material; (b) moving a first portion of the third fluid material from a main channel to at least one of the one or more flow reduction channels; 23. Indicating one or more of: leaving a second portion of the three-fluid material in the main channel; and (c) moving the second portion of the third-fluid material through the main channel. Equipment. 31. During operation of the device, the first fluid material has a first flow rate, the third fluid material has a second flow rate, and the second flow rate is such that the first fluid material is the second fluid material. And then increases to a flow rate greater than the first flow rate, and again, the second flow rate is
31. The apparatus of claim 30, wherein a first portion of a third fluid material decreases from a main channel after moving to at least one of the one or more flow reduction channels. 32. During operation of the device, the second flow rate is substantially second after the first portion of the third fluid material has moved from the main channel to at least one of the one or more flow reduction channels. 32. The device of claim 31, wherein the device is reduced to the same as one flow rate or to a flow rate less than the first flow rate. 33. A pressure change due to a first portion of a third fluid material moving from a main channel into at least one of the one or more flow reduction channels adjusts a flow rate of the third fluid material. 31. The apparatus of claim 30, wherein 34. A source of a fourth fluid material in fluid communication with the main channel at a third location downstream of the one or more flow reduction channels, the fluid indicating system further comprising: 31. The device of claim 30, which directs transfer of a fourth fluid material from a source of the four fluid material into the main channel. 35. The device of claim 34, wherein the fourth fluid substance is a reagent substance. 36. The system reduces consumption of reagent material by reducing a flow rate of a third fluid material prior to transfer of the reagent material into the main channel.
The described device. 37. The apparatus of claim 34, further comprising a detection region within the main channel or a detection region fluidly coupled to the main channel at a fourth position downstream of the third position. 38. A source of a third fluid material in fluid communication with said one or more flow reduction channels at a first location along said one or more flow reduction channels, and said one or more. 23. The apparatus of claim 22, further comprising a source of a fourth fluid material in fluid communication with the first or more flow reduction channels at a second location along the flow reduction channel of. 39. The device of claim 38, wherein the third fluid substance and the fourth fluid substance are reagent substances. 40. The first fluid material is a sample material, the second fluid material is a buffer material, the third fluid material is a substrate material, and the fourth fluid material is an enzyme material. 39. The device of claim 38. 41. Moving the first fluid material from a source of a first fluid material to a main channel and the second fluid material from a source of a second fluid material to a main channel during operation of the device. And combining a first fluid material and a second fluid material to form a diluted material, wherein during operation of the device, a first portion of the diluted material is removed from the main channel by the first or more flow reductions. Moving to at least one of the channels, leaving the second portion of the diluted substance in the main channel, moving the second portion of the diluted substance through the main channel during operation of the device Moving a fourth fluidic substance into at least one of the one or more flow reduction channels during operation of the device; Moving a fifth fluid material into at least one of the one or more flow reduction channels during operation of the apparatus, and moving a fifth fluid material into the main channel during operation of the apparatus 39. The apparatus of claim 38, further comprising a fluid indicating system indicating at least one or more of: 42. The fluid indicating system directs movement of the fourth and fifth fluid substances into at least one of the one or more flow reduction channels, the fourth fluid substance. 42. The apparatus of claim 41, wherein a substance, a fifth fluid substance and the diluted substance are combined to form a reactant. 43. The following items are provided: (a) moving a first fluid material from a source of the first fluid material into a main channel; (b) a first portion of the first fluid material from the main channel. Or moving into at least one of the two or more flow reduction channels, leaving a second portion of the first fluid material in the main channel, (c) a source of the second fluid material. To the main channel from the second
Combining a fluid material and a second portion of the first fluid material to form a third fluid material;
23. The apparatus of claim 22, further comprising: (d) moving a third fluid material through the main channel, the fluid indicating system indicating one or more of the following. 44. During operation of the device, the first fluid material has a first flow rate, the second portion of the first fluid material has a second flow rate, and the third fluid material has a third flow rate. Also, the first
The second flow rate decreases to a flow rate less than the first flow rate after the first portion of the fluid material has moved into at least one of the one or more flow reduction channels, and the second fluid material and 44. The apparatus of claim 43, which combines with the second portion of the first fluid material to produce a flow rate greater than the second flow rate. 45. The apparatus of claim 44, wherein the third flow rate increases to a flow rate substantially equal to the first flow rate. 46. The apparatus according to claim 1, wherein the main channel is fluidly connected to the main pressure source and the sample substance source, a first parallel region, a second parallel region, a third parallel region, and A first flow reduction channel including a fourth parallel region, wherein the one or more flow reduction channels are fluidly connected to the first parallel region, a second flow reduction channel fluidly connected to the second parallel region, A third flow reduction channel fluidly connected to the third parallel region, wherein the first pressure source and the main pressure source are the same, and the device comprises a first parallel region, a second parallel region and a third parallel region. A source of diluent material in fluid communication with a region, one or more reagent substances in fluid communication with a first parallel region, a second parallel region, a third parallel region and a fourth parallel region 2 or more sources, and the main channel, the first flow reduction channel, second flow reduction channel, the third flow reduction channels, first parallel region, the second parallel region and the third
The above device comprising a detection region proximate to the parallel region. 47. The apparatus according to claim 4, wherein the main channel includes an upstream end and a downstream end, and wherein the one or more flow reduction channels are at the first position and the second position. A first flow reduction channel intersecting the channel, a second flow reduction channel intersecting the main channel at a third position and a fourth position located on the main channel between the first position and the second position, the third A third flow reduction channel intersecting the main channel at a fifth position and a sixth position located on the main channel between the position and the fourth position, the main pressure source being at the downstream end at the main channel. A fluently connected vacuum source, the apparatus comprising a source of sample material fluently connected to a main channel at the upstream end, between the source of sample material and a first position. A first source of diluent material in fluid communication with the main channel at position 7 and a second source of diluent material in fluid communication with the main channel at an eighth position between the first and second positions. A source, a third source of diluent material in fluid communication with the main channel at a ninth position between the third and fifth positions, a flow channel to the main channel and the one or more flow reduction channels. The apparatus further comprising one or more sources of one or more reagent substances in communication, and a first flow reduction channel, a second flow reduction channel, a third flow reduction channel and a detection region proximate the main channel. . 48. Further comprising one or more reaction channels intersecting said one or more flow reduction channels, wherein one or more sources of said one or more reagents are said 1 or 2 48. The apparatus of claim 47, which is in fluid communication with the reaction channel. 49. The one or more reaction channels have a first cross-sectional dimension,
49. The apparatus of claim 48, wherein the one or more flow reduction channels have a second cross sectional dimension, the first cross sectional dimension being less than the second cross sectional dimension. 50. A method for adjusting the volumetric flow rate of a fluid in a channel of a microfluidic system, comprising: (i) providing a body structure, wherein the body structure is disposed therein. A plurality of microscale channels, the plurality of microscale channels being (a) a main channel, and (b) one or more flows intersecting the main channel at one or more intersection points. A reduction channel, the flow reduction channel being one or more structurally configured to reduce the flow rate of fluid in the main channel as the fluid flows past the one or more intersections. (Ii) flowing a first fluid substance through the main channel, (iii) flowing a second fluid substance in the main channel to obtain a third fluid substance, (iv) the one or more flow Flowing a first portion of a third fluid material through the reduction channel, and (v) flowing a second portion of the third fluid material through the main channel, whereby a first channel in the main channel and the one or more flow reduction channels is Adjusting the flow rate of the three-fluid material. 51. The one or more flow reduction channels are structurally configured to reduce the flow rate of the fluid in the main channel as the fluid flows past the one or more intersections. 51. The method of claim 50, including. 52. The method of claim 50, wherein the body structure comprises two or more flow reduction channels in fluid communication with the main channel, the method comprising steps (ii)-(v). ) Iteratively repeating, 53. The method of claim 52, further comprising structurally configuring the body structure to include about 3 to about 4 flow reduction channels in fluid communication with the main channel. 54. The method of claim 52, further comprising structurally configuring the body structure to include about 5 to about 10 flow reduction channels in fluid communication with the main channel. 55. The method of claim 52, further comprising structurally configuring the body structure to include about 10 or 11 or more flow reduction channels in fluid communication with the main channel. 56. A main channel is structurally configured to have a first dimension, and
51. The method of claim 50, further comprising structurally configuring the one or more flow reduction channels to have a second dimension that is different than the dimension. 57. The method of claim 56, wherein the second dimension is less than the first dimension. 58. The method of claim 50, further comprising providing a main pressure source operably coupled to the main channel. 59. The method of claim 58, wherein the main pressure source comprises a vacuum source. 60. The method of claim 58, wherein the main pressure source comprises a single vacuum source. 61. The method further comprises providing a first pressure source fluidly connected to the one or more flow reduction channels and a main pressure source fluidly connected to the main channel, wherein the first pressure source is provided. 51. The method of claim 50, wherein a pressure source is disposed on the one or more flow reduction channels downstream of the one or more intersections in a flow direction towards a first pressure source. 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 comprise a single vacuum source. 64. The first fluid material and the second fluid material are the same.
The method described in 0. 65. The method of claim 50, wherein the first fluid material and the second fluid material are different. 66. The method of claim 50, wherein the first fluid material is a sample material and the second fluid material is a diluent material. 67. The first fluid material has a first flow rate after step (ii), the third fluid material has a second flow rate after step (iii), and the third fluid material has step (iv). 51. The method of claim 50, having a third flow rate afterwards. 68. The method of claim 67, wherein the second flow rate is greater than the third flow rate. 69. The third flow rate is substantially equal to the first flow rate.
7. The method according to 7. 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 comprising flowing the third fluid material through a detection region downstream of at least one of the one or more intersections. 72. At a first location downstream of at least one of the one or more intersections, a fourth fluid material is added to the main channel to combine with a third fluid material and a fifth fluid material is provided. 51. The method of claim 50, further comprising obtaining. 73. The method of claim 72, wherein the fourth fluid substance is a reagent substance. 74. The method of claim 72, further comprising reducing the consumption of the reagent substance by reducing the flow rate of the third fluid substance in the main channel prior to adding the reagent substance to the main channel. 75. The method of claim 72, further comprising flowing a fifth fluid substance through a detection region located downstream of the first position along the main channel. 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 and a second intersection. 77. The method of claim 76, further comprising flowing a third fluid material through a sensing region located between the first intersection and the second intersection. 78. A fourth fluid material is added to the main channel at a first position between the first intersection and the second intersection to combine the fourth fluid material and the third fluid material,
77. The method of claim 76, further comprising obtaining a fifth fluid substance. 79. The method of claim 78, further comprising flowing a fifth fluid substance through a detection region located between the first location and the first or second intersection. 80. A method for adjusting the volumetric flow rate of a fluid in a channel of a microfluidic system, comprising: (i) providing a body structure, wherein the body structure is disposed therein. A plurality of microscale channels, the plurality of microscale channels being (a) a main channel, and (b) one or more flows intersecting the main channel at one or more intersection points. A reduction channel, the flow reduction channel being one or more structurally configured to reduce the flow rate of fluid in the main channel as the fluid flows past the one or more intersections. (Ii) flowing a first fluid substance through a main channel, (iii) a first portion of a first fluid substance through at least one of the one or more flow reduction channels. And (iv) flowing a second portion of the first fluid material through the main channel, thereby increasing the volumetric flow rate of the first fluid material in the main channel and at least one of the one or more flow reduction channels. Adjusting. 81. The method of claim 80, further comprising flowing a second fluid material through the main channel to obtain a third fluid material. 82. The method of claim 81, comprising flowing a second fluid substance through the main channel after step (iv). 83. The first fluid material has a first flow rate, the third fluid material has a second flow rate, and the first flow rate is substantially equal to the second flow rate. the method of. 84. A method of performing a plurality of assays in the apparatus of claim 1 or 4, comprising: (i) providing a test compound, (ii) a first portion of the test compound being a first flow reduction channel. Flowing to
(Iii) flowing a second portion of the test compound into the second flow reduction channel, (iv) flowing at least a first reagent into the first flow reduction channel to perform a first assay on the test compound and removing at least a first product. Obtaining, and (v) flowing at least a second reagent through a second flow reduction channel to perform a second assay on the test compound to obtain at least a second product. 85. The method of claim 84, wherein the test compound comprises a drug sample, compound, enzyme, protein, nucleic acid or combination thereof. 86. The method of claim 84, wherein the first and second reagents are different. 87. The method of claim 84, wherein the first assay and the second assay are different. 88. The first and second assays are enzymatic assays, HA
85. The method of claim 84, selected from one or more of an S-binding assay, target screen, fluorescence assay, binding assay, dose-response assay, genotype assay, and selectivity screen. 89. The method of claim 84, comprising performing step (i) and step (ii) simultaneously. 90. The method of claim 84, comprising performing step (iii) and step (iv) simultaneously. 91. The method of claim 84, wherein the first product is a first reaction test compound and the second product is a second reaction test compound. 92. The method of claim 84, further comprising simultaneously detecting the first product and the second product. 93. Dividing the test compound into n parts, flowing each part simultaneously into one of the x flow reduction channels, performing y different assays on the test compound to produce z products. 85. The method of claim 84, comprising: obtaining z and detecting z products simultaneously. 94. n is in the range of about 2 to about 100, x is in the range of about 2 to about 100, y is in the range of about 1 to about 100, and z is in the range of about 1 to about 1000. 94. The method according to claim 93. 95. A method of suppressing pressure perturbations due to natural injection into a microfluidic device, comprising: (i) immersing an open end of a capillary tube in a sample source fluidly connected to the microfluidic device. (Ii) pulling the open end of the capillary from the sample source, leaving the first portion of the sample at the open end, the first portion of which has surface tension Tension exerts a first pressure on the capillary, thereby spontaneously injecting the first portion into the capillary, (iii) a second portion of the sample from the capillary into the main channel that intersects the capillary at the first intersection point. And (iv) flowing a third portion of the sample through a shunt channel that intersects the main channel, maintaining a first crossing point at a second pressure different from the first pressure, Suppressing pressure perturbations in the channel. 96. The sample source comprises a microwell plate
The method of claim 95. 97. The method of claim 95, wherein the shunt channel intersects the main channel at or downstream of the first intersection. 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 ends of the capillaries and the first intersection. 102. The method of claim 95, further comprising applying a third pressure to the shunt channel and varying a second pressure at the first intersection. 103. The method of claim 102, wherein the third pressure is a negative or positive pressure. 104. A microfluidic device for suppressing pressure perturbations due to spontaneous injection into a microfluidic device, comprising: (i) a capillary including an inlet region and an outlet region, the inlet region of the device A capillary, (ii) a plurality of body structures disposed therein, the capillaries being fluidly coupled to at least a first sample source during operation, and wherein the inlet region is maintained at a first pressure; It has a microscale channel, which is (a) a main channel having an upstream region and a downstream region, said upstream region being fluidly connected to an outlet region of a capillary at a first intersection. A main channel comprising: (b) a shunt channel fluidly connected to at least an upstream region of the main channel; and (iii) fluidly connected to a microfluidic device. A fluid indicating system,
A main channel, a shunt channel, and at least one fluid control element fluidly coupled to the capillary, the fluid indicating system comprising: (a) movement of the sample from a first sample source into the inlet region of the capillary; (B) migration of the sample from the inlet region of the capillary to the outlet region of the capillary, (c) migration of the sample from the outlet region of the capillary into the upstream region of the main channel, and (d) shunting from the upstream region of the main channel. Fluid of the first portion of the sample into the channel, leaving the second portion of the sample in the main channel and maintaining the first crossing point at a second pressure different from the first pressure. An indicator system, the microfluidic device. 105. The apparatus of claim 104, wherein at least the first sample source comprises one or more multiwell plates. 106. The apparatus of claim 104, wherein the first pressure is maintained at atmospheric pressure. 107. The apparatus of claim 104, the shunt channel fluidly connected to an upstream region of the main channel and a downstream region of the main channel. 108. The device of claim 104, wherein the at least one fluid control element comprises a pressure source or an electrokinetic controller. 109. The first pressure is greater than or less than the second pressure,
105. The device of claim 104.