JP4596776B2 - Microfluidic processing method and system - Google Patents

Description

  The present invention relates to a method and system for processing a sample using a microfluidic processing system.

A microfluidic device is typically formed of a substrate (made of silicon, glass, ceramic, plastic and / or quartz) containing a microchannel network, and fluid passes through the microchannel network under the control of a propulsion mechanism. And flow. At least one dimension of the microchannel is typically on the order of nanometers to 100 microns.
Microfluidic devices offer various advantages over conventional large scale instruments. For example, they typically require much less fluid sample and use much less reagents than large scale equipment, and process these fluids at a much higher rate. If the microfluidic device uses only a very small amount of sample, the chemical and physical characteristics of the sample can be determined by performing fluid processing on the sample.

  In many cases, the accuracy of these fluid treatments depends on the relative amount of sample and reagents used. For example, when analyzing a sample for DNA “fingerprints”, the results may vary depending on the concentration of reagents used to amplify the DNA present in the sample. Therefore, if an inappropriate sample to reagent ratio is used, the results may be inaccurate. Because microfluidic devices process very small amounts of samples and reagents, even small absolute uncertainties in the amount of reagents or samples used can introduce uncertainty into the results of microfluidic analysis.

  The variance in the amount of sample and reagent that the microfluidic device processes can come from a variety of sources. For example, some microfluidic devices handle a continuously flowing liquid stream. Changes in the viscosity of the liquid can change the flow rate of the stream, and correspondingly, the time required to introduce a given amount of material at a given location in the microflow device can also change. is there. When using a fluid flow stream to move sample components from one location of the microfluidic device to another, sample dilution can occur.

Microfluidic analysis of cells in body fluids is particularly troublesome because the number of cells available for analysis is relatively small and it is inherently difficult to handle such substances.
Sample manipulation may involve moving the sample between different locations within the microfluidic device. For example, in some microfluidic devices, an electric field is used as a propulsion mechanism. In such a device, an electric field is generated in the fine channel by applying a high voltage in kilovolts between the electrodes inside the device. The electric field applies a force to ions in the fluid, thereby propelling the ions and passing them through the fine flow path. The fluid itself may also be propelled by the movement of ions moving through the fluid.

  Gas pressure is also used to propel the fluid through the microchannel. In some devices, a pressurized gas source external to the microfluidic device is connected to the microfluidic device to provide gas pressure and propel the fluid. The gas pressure can also be generated by the heating chamber inside the microfluidic device itself, driving the fluid into the microchannel.

In general, a first aspect of the invention relates to a system and method for moving a sample, such as a fluid, within a microfluidic system. In one aspect, the invention relates to the use of a plurality of gas actuators that provide a force to move a sample by applying pressure at different locations within the microfluidic system. For example, in one embodiment, the first gas actuator provides sufficient gas pressure to move the first sample from the first position to the second position of the microfluidic device. The second gas actuator supplies a gas pressure that moves another sample from the third position to the fourth position of the microfluidic device.
In another example, multiple gas actuators move together to move the same fluid sample. The first gas actuator provides sufficient gas pressure to move the microdroplet between the first and second processing zones of the microfluidic device, and the second gas actuator moves the microdroplet to the third processing zone The gas pressure to be supplied is supplied.

In a preferred embodiment, a plurality of actuators are integrated with the microfluidic network through which the microfluidic sample passes. For example, multiple gas actuators can be fabricated in the same substrate that forms a microfluidic network. One such gas actuator is coupled to the net in a first position and provides a gas pressure that moves the microfluidic sample within the net. Another gas actuator is coupled to the mesh at the second position and provides a gas pressure that further moves at least a portion of the microfluidic sample within the mesh.
In another aspect, the invention relates to the use of a valve with a plurality of actuators. For example, in one embodiment, coupling the valve to a microfluidic network and closing the valve substantially isolates the second gas actuator from the first gas actuator. Such a valve can control the direction of the driving force of the actuator by expanding it in a desired direction while preventing the expansion gas from moving in a certain direction. They can also widen the range over which the actuator can propel the fine droplets by preventing the gas from dissipating in the area upstream from the fine droplets.

Another aspect of the invention relates to a microfluidic system and method for treating a particle-containing fluid, such as, for example, a liquid containing bacterial cells or human cells.
In one aspect, the invention relates to preparing a concentrated particle sample from a particle-containing fluid. For example, a microfluidic system for preparing a concentrated sample includes a concentration zone and a flow member disposed in fluid communication with the concentration zone. The flow member comprises the concentrated particle sample in the concentration zone by allowing the particle-containing fluid to pass through and out of the concentration zone while accumulating particles of the particle-containing fluid in the zone. The flow member can include a plurality of passages, such as pores, that are large enough to allow fluid to pass through, but have a size that is too small for particles to pass through. Suitable flow members are composed of, for example, filter elements such as filter paper, porous glass, and porous gel.

The microfluidic system can also include an actuator to move the concentrated particle sample out of the concentration zone without essentially diluting the concentrated particle sample. In one embodiment, the actuator is a gas actuator and moves the sample by increasing the gas pressure associated with the upstream portion of the enrichment zone relative to the gas pressure associated with the downstream channel. For example, a gas actuator can include a heat source that is in thermal contact with a volume of gas. Enough gas to move the concentrated particle sample from the concentration zone to another location within the microfluidic system, such as a location containing another fluid processing module, by activating the heat source to expand the gas Generate pressure.
In another aspect, the invention relates to a microfluidic system and method for processing a concentrated particle sample comprising cells entrained in a liquid. For example, the system includes a collapse zone that receives the concentrated cell-containing sample and a positioning element that places the concentrated cell-containing sample at a collapse location in the vicinity of the collapse mechanism. The decay mechanism releases intracellular substances such as DNA or RNA from the cell. Alternatively, the disintegration mechanism can disrupt cells using chemical, thermal and / or ultrasonic techniques or any combination of these techniques.

In one embodiment, the disruption zone releases intracellular contents from the cells of the cell-containing fluid, and then prepares fine droplets containing the intracellular contents released from the cells from the fluid. The fine droplets are preferably prepared from only a part of the cell-containing fluid. For example, suitable microdroplets comprise less than about 90 percent of the cell containing fluid. In another embodiment, the collapse zone receives a fine droplet of cell-containing fluid and releases the intracellular contents of the cells within the droplet.
In another aspect, the invention relates to a microfluidic substrate that processes the intracellular contents of cells suspended in a fluid. The substrate includes a concentration zone, a collapse module, a fine droplet formation module, a mixing module, and an amplification module. The concentration zone prepares a concentrated particle sample from the cell-containing fluid. The disruption module is coupled to the enrichment zone to receive the enriched particle sample and form a disintegrated sample by releasing intracellular material from cells within the sample. The microdroplet forming module then forms a first microdroplet of fluid from the collapsed sample and transfers it to the mixing module for mixing with the reagent microdroplets. The amplification module amplifies intracellular material in the fine droplets formed from the mixture.

Another aspect of the invention relates to microfluidic systems and methods for treating cell-containing fluids, such as liquids containing, for example, bacterial cells or human cells. For example, the system includes a collapse zone that receives a cell-containing sample and a positioning element that positions the cell-containing sample at a collapse location in the vicinity of the collapse mechanism. The decay mechanism releases intracellular substances such as DNA or RNA from the cell. In one embodiment, the collapse mechanism includes an electrode that generates an electric field sufficient to release intracellular contents from the cell. Alternatively, the disintegration mechanism can disrupt cells using chemical, thermal and / or ultrasonic techniques or any combination of these techniques.
In one embodiment, the disruption zone releases intracellular contents from the cells of the cell-containing fluid, and then prepares fine droplets containing the intracellular contents released from the cells from the fluid. The fine droplets are preferably prepared from only a part of the cell-containing fluid. For example, suitable microdroplets comprise less than about 90 percent of the cell containing fluid. In another embodiment, the collapse zone receives a fine droplet of cell-containing fluid and releases the intracellular contents of the cells within the droplet.
The positioning element assists in placing the cell-containing fluid sample in the vicinity of the disintegration mechanism and allowing the disintegration mechanism to release intracellular material from the cell. These elements preferably operate differently than the valves. The valve completely blocks the passage of material between upstream and downstream positions adjacent to the valve. In contrast, positioning elements typically control fluid placement by providing resistance to fluid flow at a desired position (collapse position).

In one embodiment, the positioning element is disposed downstream of the disruption mechanism and positions the upstream portion of the cell-containing sample (such as a microdroplet) in the collapse position. The positioning element preferably includes surface hearing of the downstream surface of the cell-containing sample, thereby preventing the sample from moving downstream. For example, the positioning element includes a quantity of a moisture reducing material, such as a hydrophobic material, and is positioned to contact a portion of the downstream surface of the cell-containing microdroplet.
In another embodiment, the positioning element is disposed upstream of the collapse zone and positions the downstream portion of the cell-containing microdroplet at the collapse position. The positioning element includes a vent, which causes the gas pressure upstream of the cell-containing droplet to be substantially equal to the gas pressure downstream of the cell-containing droplet, thereby downstream of the cell-containing droplet. Stop moving. When a fine droplet is in the collapsed position. Preferably, the valve is then arranged so as to block the passage between the collapse zone and the vent, and the upstream gas pressure again moves the droplets further downstream for further processing. For example, the microfluidic system includes mixing zones downstream of the concentration zone and / or the collapse zone and mixes the fine droplets emerging from these zones with a predetermined amount of reagent material.

  In another aspect, the invention relates to a microfluidic substrate that processes the intracellular contents of cells suspended in a fluid. The substrate includes a collapse module, a fine droplet formation module, a mixing module, and an amplification module. The disintegration module forms a disintegrating sample by releasing intracellular material from cells in the sample. The microdroplet formation module then forms a first microdroplet of fluid from the collapsed sample and transfers it to the mixing module for mixing with the reagent microdroplets. The amplification module amplifies intracellular substances in the fine droplets formed by mixing.

The present invention will be described below with reference to the drawings.
The present invention relates to microfluidic systems and methods for processing materials such as samples and reagents. More particularly, one aspect of the invention relates to a microfluidic system and a method for moving fluid in a microfluidic system. In one embodiment described below, the fluid includes particles that are easy to move with the fluid. The fluid component of the particle-containing fluid is a gas or, preferably, a liquid. The particles of the particle-containing fluid are preferably whole cells, such as bacterial cells or animal cells such as humans. However, they can also contain intracellular material from such cells. For example, the system of the present invention can be used to process a sample of bacterial cells to determine whether the bacteria are pathogenic.

A. System Overview FIG. 1 shows a microfluidic system 100 that includes a microfluidic device 110 and a corresponding cartridge 120, which are under control of a computer 127 and a data acquisition and control board (DAQ) 126. Receive a fluid sample and process the sample.
Computer 127 preferably provides a user interface that allows the user to select a desired action, notifies DAQ 126 about the selected action, and displays the results of such actions for the user. Execute the upper function. These operations include, for example, subjecting the sample to processing steps in various processing zones of the microfluidic device. If the computer 127 is a portable computer, the microfluidic system can be easily carried.

The computer 127 is connected to the DAQ 126 via the connection unit 128. The connection 128 provides data I / O, power, ground, reset, and other functional connections. Alternatively, a wireless link 132 may be provided between the computer 127 and the DAQ 126 to exchange data and control signals via the wireless elements 132 (a) and 132 (b). If the data link is a wireless link, for example, the DAQ 126 may have a separate power source such as a battery.
In general, the DAQ 126 controls the operation of the microfluidic device 110 in response to a higher order command received from the computer 127. More specifically, in order to realize the desired operation required by the computer 127, the DAQ 126 supplies an electric control signal to the cartridge 120 via the contact 125 accordingly.
The cartridge 120 provides an electrical and optical connection 121 for electrical and optical signals between the DAQ 126 and the microfluidic substrate 110 so that the DAQ 126 can control the operation of the substrate.

  The chip carrier cartridge 120 is shown inserted (or removed) from the interface hardware receptacle of the DAQ 126 and standardized to match the corresponding contact 121 of the chip carrier cartridge 120. It has electrical and optical contacts 125. Most contacts are for electrical signals, but some contacts are for optical signals (IR, visible, UV, etc.) in the case of optical monitoring microfluidic processors or photoexcited microfluidic processors. Alternatively (not shown), the entire DAQ 126 may be a single ASIC chip, which may be incorporated into the chip carrier cartridge 120, in which case the contacts 121, 125 provide a conductive path on the printed circuit board.

B. Microfluidic Device FIG. 2 shows the schematic structure of a preferred type of microfluidic device. The device includes an upper substrate 130 that is bonded to a lower substrate 132 to form a fluid network.
The upper substrate 130 shown in FIG. 2 is preferably made of glass and has a microfluidic network 134 on its bottom surface 136. Those skilled in the art will appreciate that substrates comprised of silicon, glass, ceramic, plastic, and / or quartz are also acceptable with respect to the present invention.
The microfluidic network includes a plurality of zones. The number of zones and the overall topology of the microfluidic network will depend on the particular application that the microfluidic device is designed to perform. The cross-sectional shape of the zone of the microfluidic device may be any of an overall arc shape or an overall polygonal shape. For example, a zone can include a channel, chamber, or other substantially enclosed space. By “substantially sealed” is meant that the material enters and exits the zone only through a given passage. Examples of such passages include channels, fine channels, etc., interconnecting various zones. The zone preferably has at least one microscale dimension, such as less than about 250 μm, or more preferably less than about 75 μm.

  The channels and chambers of the microfluidic network are etched into the bottom surface 136 of the upper substrate 130 using known lithographic techniques. More specifically, an object is optically defined on the surface of the substrate using a transparent template or mask containing opaque portions. The pattern on the template is generated by a computer aided design program and can draw structures with a line width of less than 1 micron. Once the template has been generated, it can be used almost infinitely to produce the same replica structure. As a result, even very complex microfluidic networks can be reproduced in large quantities with a low incremental cost per unit. Alternatively, when plastic material is used, the upper substrate can be formed using injection molding techniques and fine channels are formed during the molding process.

The lower substrate 132 can include a glass substrate 138 and an oxide layer 140. A resistive heater 142 and electrical leads 144 are formed in the oxide layer 40 using photolithography techniques. The lead 144 is connected to the terminal 146, and the terminal 146 is exposed at the edge of the substrate, so that the heater can be controlled by the DAQ 126. More specifically, to activate the heater 142, the DAQ 126 applies a voltage to the pair of terminals 146 (via the cartridge 120) and provides a current through the lead 146 and the heater 142 to provide resistance. Heat the heater element 142.
When a metal heater element 142 is placed and the upper and lower substrates are joined together, the heater is located directly below certain areas of the upper substrate fluid network so that the contents of these areas can be heated. It has become. The silicon oxide layer 140 prevents the heating element 142 from coming into direct contact with the material in the microfluidic network.

Oxide layer 140, heating element 142, and resistive lead 144 are fabricated using well-known photolithographic techniques, such as those used in etching microfluidic networks.
FIG. 3 is a comprehensive view of the microfluidic device 110. As shown in the figure, the substrate has a sample input module 150 and a reagent input module 152, and a sample and a reagent substance can be input to the device 110, respectively. Preferably, the input modules 150, 152 are arranged to allow automatic substance input using a computer controlled laboratory robot 154.
The substrate also includes process modules 156, 158, 160, 166 and 162 for processing sample and reagent materials. Within these process modules, the sample undergoes various physical and chemical processing steps. For example, the concentration module 156 prepares a fluid sample having a relatively high concentration of cell particles, the disruption module 160 releases intracellular material from the cell particles, and the mixing module 166 uses the resulting sample as a reagent. Mix. As another example, the amplification process module 162 can be used to amplify and detect very small amounts of DNA in a sample.

  The various modules of the microfluidic device are connected by channels 164 or the like so that the substance can move from one location inside the device 110 to another. Actuators 168, 170, 172 in conjunction with the microfluidic device generate an activation force, such as gas pressure, to move the sample and reagent material along the channels and zones. For example, the first actuator 168 moves material downstream from the process module 156 to the process module 158. When processing in process module 158 is complete, second actuator 170 moves the material downstream toward mixing process module 160. Subsequently, the actuator 170 or additional actuator moves the substance to the mixing module 166 where the substance mixes with the reagent moved by the actuator 172. Eventually, actuator 172, or another actuator, moves the mixed material to module 162.

  Since each actuator is preferably responsible for the movement of material only in a subset of the modules of the device 110, it can be controlled more accurately than if a single actuator is responsible for movement of material across the device. Various functional elements of the microfluidic device 110, including actuators, are preferably under computer control to allow automatic sample processing and analysis.

C. Multiple Actuators The various actuators of the microfluidic device 110 work together to move material between different locations of the microfluidic device 110. For example, the actuator 168 moves a substance, such as a concentrated sample, between the concentration zone 931 and the fine droplet preparation module 158. Actuator 170 prepares microdroplets from the concentrated sample, while moving the microdroplets to collapse zone 950. Actuator 170 is used to move material from collapse zone 950 to mixing module 166. However, it is noted that another actuator may be placed intermediate the collapse zone 950 and the fine droplet preparation zone to move the collapsed sample downstream toward the mixing module 166.
In addition, the actuator of the device 110 can co-operate when moving two kinds of substance amounts simultaneously. For example, as described above, the actuator 172 and the actuator 170 cooperate to mix the reagent and the broken fine droplets. Such co-actuating actuators can be controlled independently of each other to ensure proper mixing. For example, if one of the materials is known to have a higher viscosity, the starting force that moves the material can be increased independently of the moving force that moves the other material.

  The multiple actuators and modules of the microfluidic device 110 are preferably operably connectable and separable by the microfluidic device valves. For example, the microdroplet preparation module 170 is isolated from the concentration module 156 when either of the valves 915, 216 is closed. In this way, one or more actuators can be used to move material between predetermined locations within the microfluidic device 110, disrupting or otherwise disturbing material in an operatively isolated module. There is no contact. The ability to operably connect and disconnect the desired modules is advantageous in microfluidic devices having many process functions. Furthermore, these valves also control the direction of the thrust of the actuator by expanding it in the desired direction while preventing the expanding gas from moving in a certain direction. This prevents the gas from dissipating in a certain direction in the area upstream from the fine droplets, thereby expanding the range in which the actuator can propel the fine droplets.

  In the following, a number of such multiple actuators in an example embodiment having a plurality of processing modules, namely a concentration zone 915, a microdroplet preparation module 158, a cell disruption module 160, a mixing module 166, and a DNA manipulation module 167. The co-dynamic operation will be described with an example.

1. Concentration module a. Concentration Module Structure Referring to FIGS. 4 and 5, the microfluidic device 901 includes a concentration module 156 that concentrates a received sample. These samples include a particle containing fluid, such as a bacterial cell containing fluid. In general, the concentration module 156 receives a flow of a particle-containing fluid from the input port 180 of the input module 150 and accumulates particles in the zone while allowing the fluid to pass through the zone. Thus, the more fluid that passes through the zone, the higher the concentration of particles in the module. The obtained concentrated fluid sample is referred to herein as a concentrated particle sample.
The concentration module includes a concentration zone 931 (FIG. 5), a flow through member 900, valves 915, 919, and a sample introduction channel 929. Valve 919 is connected between flow member 900 and actuator 168 as shown, and valve 915 is connected between the flow member and downstream channel 937 leading to process module 158. These valves are suitable for use in microfluidic devices, such as the thermally actuated valves discussed in copending US patent application Ser. No. 09 / 953,921, filed Sep. 9, 2001. Any one can be used. If the valve can be reversed between open and closed states, the concentration module 931 can be reused.
A flow member is also connected to the sample input module 150 through the sample introduction channel 929, allowing fluid to pass through the concentration zone. A valve 913 is connected to the sample introduction channel and controls inflow and outflow of fluid from the input port.

  FIG. 5 is a cross-sectional view of the concentration zone, showing the flow member in more detail. As shown, the flow member 900 has first and second surfaces 941, 943. The first surface 941 is preferably adjacent to the concentration chamber 931. The second surface 941 is preferably separated from the concentration chamber 931 by the flow member 900. The flow member 900 is preferably formed of a material having passages that are smaller than the diameter of the particles to be concentrated, such as, for example, pores having a diameter of less than about 2 microns, for example, about 0.45 microns. Suitable materials for constructing the flow member 900 include, for example, filter media such as paper or fiber, polymers having a channel network, and glassy materials such as glass frit.

6 and 7 show cross-sectional views of the upper substrate 130 and illustrate the enrichment zone 931. As shown, the fluid exits the concentration zone 931 through the surface 941, passes through the flow member 900, and enters the space 400. The space 400 includes an absorbent material 402 and can absorb the flowing fluid. Thus, the space 400 preferably provides a substantially self-contained region in which fluid exiting the concentration zone can be collected without contacting the exterior of the microfluidic system 100. .
The space 400 is formed during the manufacture of the upper substrate 130. As discussed above, microfluidic structures such as zones and channels are created on the surface 136 of the substrate 130. However, the space 400 is created in the surface 137 and is preferably disposed on the other side of the substrate 130, ie, the opposite surface 136. In this way, even when the surface 136 is combined with the lower substrate 132, the fluid can flow out of the concentration zone 931 through the flow member 900.

The flow member 900 and the absorbent material 402 do not require adhesives or other fasteners to be positioned within the substrate 130. Rather, the flow member 900 and the absorbent material 402 can be formed in a shape and size substantially corresponding to the space 400. Thereby, once the flow member 900 and the absorbent material 402 are disposed in the space 400, the friction holds them in place. Any residual void at location 404 between flow member 900 and substrate 130 should be very small to prevent particles from exiting concentration zone 931 through void 404. Of course, the flow member 900 or the absorbent material 402 may be fixed using an adhesive or other fastening means.
In an alternative embodiment, the flow member is integrally formed with the substrate by using microfabrication techniques such as chemical etching that introduces pores or other passages into the substrate. The pores provide a fluid path between the concentration zone 931 and the outside of the substrate.

b. Operation of the Concentration Module To concentrate the sample, the device 901 operates as follows. Referring to FIG. 4, valves 915 and 919 are closed in the initial state, and valve 913 is open. Particle containing fluid is introduced into input port 180. Since valve 913 is open, the sample passes along channel 929 and reaches enrichment zone 931. Alternatively, the enrichment zone 931 can be configured to receive the sample directly, such as by injection. Since valves 915 and 919 are closed, fluid is substantially prevented from escaping to actuator 977 and downstream channel 937.
Thus, the flow member 900 is provided only with a path through which the fluid flows out of the concentration channel. The fluid passes through the surface 941 and out of the concentration zone 931 through the second surface 943, while the particles accumulate in the zone. The concentration zone 931 can therefore receive a volume of fluid that is larger than the volume of the concentration chamber 931. That is, as the fluid passes through the chamber, the concentration of the particles in the chamber increases as compared to the concentration in the particle-containing fluid supplied at the sample input. When the particle is a cell, the concentration or number of cells in the zone 931 should be such that a polymerase chain reaction (PCR) analysis of the polynucleotide released from the cell in the downstream processing module can be performed. Is preferred.

Thus, the concentration zone 931 prepares a concentrated particle sample from the particles of the particle-containing fluid received therein. The ratio of particles to fluid volume (PPVF) of the concentrated particle sample is much higher than the corresponding ratio of particle-containing fluid received by the concentration zone. The PPVF of the concentrated particle sample is preferably at least 25 times, preferably about 250 times, more preferably about 1,000 times that of the particle-containing fluid.
After receiving a sufficient volume of the particle-containing fluid by the concentration zone 931, the valve 913 is closed to prevent the fluid from entering the further concentration zone and the material in the zone 931 returns to the sample introduction port 180. To prevent. The valves 915, 919 are then preferably opened by actuating the associated heat source. When valve 919 is opened, actuator 168 can push the concentrated sample, and valve 915 can move the concentrated sample downstream.

The activation force generated by the actuator 168 moves the concentrated particle sample from the concentration zone 931. Actuator 168 is preferably a gas actuator and provides gas pressure upon activation of heat source 975, which is in thermal communication with a volume of gas 977. The operation of the heat source 975 increases the temperature, and thus the pressure of the gas 977 also increases. The flow member and the fluid therein substantially prevent gas from escaping from the concentration zone. Thus, the resulting gas pressure moves the concentrated particle sample downstream from the concentration zone 931.
The gas actuator can also include elements useful for alternative pressure generation techniques, such as chemical pressure generation. In another embodiment, the actuator moves the sample out of the concentration zone, creating a differential pressure between the samples by reducing the volume of gas involved in the upstream portion of the concentration zone. An example of such an element is a mechanical actuator, such as a plunger or a diaphragm.

  Instead of generating a positive pressure upstream from the enrichment zone, the gas actuator can also reduce the pressure downstream from this zone relative to the upstream pressure. For example, a gas actuator can include a cooling element that is in thermal contact with a volume of gas involved in the downstream portion of the zone. Actuating the cooling element to contract the gas creates a gas pressure differential between the upstream and downstream portions of the concentration zone, causing the concentrated particle sample to move out of the concentration zone. Alternatively, a mechanical actuator can be used to move the concentrated particle sample out of the concentration zone by decreasing the gas pressure by increasing the volume of gas involved in the downstream portion of the concentration zone.

Preferably, the concentrated particle sample moves downstream without substantial dilution thereof, i.e., the concentration of the concentrated particles does not substantially decrease when moving from the concentration zone 931. That is, the removal of the particles from the concentration channel of the present invention does not require diluting the particles with a fluid different from the fluid of the particle-containing fluid introduced into the concentration channel or otherwise contacting them. In contrast, systems that concentrate materials by surface adsorption require an elution fluid to remove the adsorbed material, which is diluted by contacting it.
In the present invention, the concentrated particle sample is preferably received by the downstream channel 937 when removed from the concentration zone. The downstream channel 937 leads to another processing module, which performs another processing of the concentrated particle sample. In the embodiment of FIG. 3, the concentrated particle sample is received by a fine droplet preparation module 158 to prepare a fine droplet sample consisting of a portion of the concentrated particle sample.

2. Fine droplet preparation module a. Microdroplet Characteristics The microdroplet 802 is a discrete sample having a predetermined volume, for example, between about 1.0 picoliter and about 0.5 microliter. That is, the fine droplets prepared by the fine droplet preparation module supply a known amount of sample for further processing. The volume of the microdroplet prepared by the microdroplet preparation module is preferably essentially independent of the viscosity, conductivity, and osmotic force of the microdroplet fluid.
The fine droplets 802 are preferably defined by upstream and downstream boundaries formed by respective gas-liquid interfaces 804, 806, respectively. The liquid at the interface is formed by the surface of the liquid that forms fine droplets. The interface gas is a gas present in the channel microfluid of the microfluidic device 901.

b. Structure and Operation of the Microdroplet Preparation Module Referring to FIGS. 8a, 8b and 9a, 9b, the microdroplet preparation module 158 prepares microdroplets 802 from the microfluidic sample received therein. This module includes a microdroplet preparation zone 800, a positioning element 979, a gas actuator 170, and a valve 216 that cooperate to prepare microdroplets 800 from a microfluidic sample received from the enrichment zone.
As previously described, the concentration zone actuator 168 pushes the concentrated sample into the microdroplet preparation zone 800. The concentrated sample moves until it reaches the positioning element 979. In general, the positioning element positions the sample in the desired location by preventing the microfluidic sample from traveling downstream. However, as described in more detail below, the positioning element does not permanently impede sample progression. Conversely, after a predetermined time, the microfluidic sample is allowed to flow downstream.
The tip of the microfluidic sample 808 that has reached the positioning element 979 is located downstream from the opening 920 of the gas actuator 170. Accordingly, the first portion 821 of the microfluidic sample 808 is disposed upstream of the opening 820 and the second portion of the microfluidic sample 808 is disposed downstream from the opening 820.

Referring to FIGS. 8 a and 8 b, the gas actuator 170 is actuated by DAQ 126 or the like, thereby generating sufficient gas pressure to separate the microdroplet 802 from the second portion 822 of the microfluidic sample 808. This gas pressure is preferably supplied by activation of a heat source 958. The heat source 958 heats the volume of gas involved in the gas actuator 957. As the pressure increases, the gas expands, thereby separating the fine droplets 802 from the rest of the sample 808. The microdroplet 802 need only consist of a portion, such as less than 75% or less than 50% of the microfluidic sample 808 received by the microdroplet preparation zone 800. The size of the microdroplet 802 is determined by the volume of the channel between the fluid barrier 979 and the opening 820. For example, in a channel having a uniform cross-sectional area, the length l ₁ of the microdroplet 802 corresponds to the distance d ₄ between the positioning element 979 and the opening 820. In this way, the microfluidic device can be configured to prepare any volume of microdroplets by varying the length between the fluid barrier and the corresponding actuator opening.
By continuously actuating the gas actuator 170, the restraining effect of the positioning element 979 is overcome, thereby driving the microdroplet 802 to a position downstream of the microdroplet preparation zone 800, while the microfluidic sample first count is reached. The two portions 822 move upstream from the microdroplet 802 to the cell disruption module 160.

3. Cell Disintegration Module Referring back to FIG. 3, the disintegration module 160 receives the microdroplet 802 prepared by the microdroplet preparation zone 800. In general, the collapse module 160 releases material from the inside of the particle, such as by releasing intracellular material from the cell.
As shown in FIGS. 4 and 12, the collapse module 160 includes a collapse zone 950, a collapse mechanism (such as an electrode 954) within the collapse zone, and a vent positioning element 200 located upstream of the collapse zone. The collapse mechanism preferably includes a set of electrodes or other structures for generating an electric field in the collapse zone. The vent positioning element preferably includes a vent 202, a valve 204, and a second positioning element 206 that prevents fluid from flowing into the vent.

As explained above, the actuator 170 of the microdroplet preparation module 158 feeds the microdroplet into the cell disruption module 160. As the microdroplet moves into the module 160, the vent positioning element 200 positions the microdroplet 802 in a collapsed position with respect to the electrode 954. More specifically, when the fine droplet reaches the collapse module 160, it passes through the opening of the positioning element 200. This is because the second positioning element 206 prevents fine droplets from flowing into the vent hole 202. When the trailing edge of the fine droplet passes through the opening of the barrier 200, the propelling gas from the actuator 170 is dissipated through the vent hole 202, thereby changing the gas pressure upstream of the droplet 802 to the pressure downstream of the fine droplet 802. And substantially equal. For this reason, the fine droplet stops moving at the collapse position just downstream of the barrier 200. Preferably, substantially all of the fine droplets 802 are disposed between the upstream edge 212 and the downstream edge of the electrode 954 at the collapsed position.
After the microdroplet 802 is placed at the cell disruption position, the pulse circuit of the DAQ 126 supplies a pulsed voltage signal between the electrodes 954. In response, the electrode 954 generates a pulsed electric field in the vicinity of the electrode. Since the fine droplet is located in the vicinity, the cells in the fine droplet receive a pulsed electric field. Preferably, substantially all of the microdroplet cells, such as greater than about 75%, are subjected to an electric field sufficient to release intracellular material therefrom. In this way, the collapse module prepares collapsed microdroplets containing a predetermined amount of sample.

A preferred pulse circuit is shown in FIG. In general, this circuit generates a series of voltage pulses and generates a series of electric field pulses corresponding to the vicinity of electrode 954, sufficient to release the desired amount of intracellular material from the cells in the microdroplet. Have a large amplitude and duration.
The intracellular substance present in the collapsing fine droplet can be used to proceed to the next processing step. For example, DNA and / or RNA released from cells can be used for increase by a polymerase chain reaction (polymerase chain reaction). As used herein, the term decay does not mean that the cell must be completely destroyed. Conversely, decay means the release of intracellular material. For example, instead of destroying cells, the electric field may increase the perforation of the cell membrane by an amount that allows the release of intracellular material without permanent destruction of the membrane.

Other decay mechanisms can also be used to release intracellular material from the cell. For example, to release the substance, other forces may be applied to the cell, including, for example, osmotic shock or pressure. A chemical selected from the group consisting of surfactants, solvents, and antibiotics may be contacted with the cells. Mechanical shearing methods can also be used to release intracellular material.
The collapsed fine droplets can move downstream to the mixing module 160 and undergo further processing. In order to move the collapsed fine droplets downstream, the valve 216 located upstream of the collapse zone 950 is closed. Valve 204 is also closed to prevent gas from exiting collapse zone 950 through the vent. Actuator 170 is then actuated as described above to provide sufficient gas pressure to move the collapsed fine droplets downstream of collapse zone 950.

In an alternative embodiment, a collapse module 300 as shown in FIGS. 13a and 13b includes a collapse zone 302 configured to prepare a predetermined volume of collapsed microdroplets 304 from a microfluidic sample 306 whose volume cannot be determined. . The collapse zone 302 preferably includes a collapse mechanism such as an electrode 308. Electrical leads 310 provide connections to the pulse circuit of DAQ 126 through contacts 112, chip carrier 120, and contacts 125. A positioning element 312 is arranged downstream of the collapse zone 302. An actuator 314 is disposed upstream of the collapse zone. Actuator 314 preferably includes a second positioning element 316 to prevent fluid from the microfluidic sample from entering it.
The collapse zone 302 operates as follows. The microfluidic sample 306 flows into the collapse zone 302 and moves downstream until the downstream interface 316 of the microfluidic sample 306 reaches the positioning element 312. The positioning element 312 preferably prevents further downstream movement by increasing the surface tension of the downstream interface of the microfluidic sample 306, and causes a portion of the microfluidic sample to collapse into the electrode 308. Position. The collapse position is defined as the location of a portion of the microfluidic sample placed downstream of the actuator 314 and upstream of the positioning element 312. Preferably, the actuator 314 and the positioning element 312 are disposed adjacent to the electrode 308 so that substantially all of the material in the collapsed position receives an electric field when the electrode 308 is activated.

  Driving the electrode 308 in the previous embodiment generates an electric field sufficient to release intracellular material from cells in the aforementioned portion of the microfluidic sample in the collapsed position. Once a sufficient amount of intracellular material has been released, the actuator 314 is actuated to prepare the collapsed microdroplet 304 from the microfluidic sample 306. Actuator 314 preferably provides sufficient gas pressure to move collapsing microdroplets 304 to a downstream portion of the microfluidic device, such as mixing module 166.

4). Mixing Module and Reagent Input Module Referring again to FIG. 4, the collapsed sample prepared by the collapse module 160 is received by the mixing module 166. The mixing module 166 includes a mixing zone 958. In this zone, for example, an amount of reagent received from the reagent source module 152 is mixed to contact the disrupted cell sample. The reagent source module 152 includes a reagent microdroplet preparation zone (RMPZ) 434, which preferably operates to prepare microdroplets having a predetermined volume of reagent.

a. Reagent Input Module Reagent input module 152 is essentially the same as microdroplet formation module 158, but provides a desired ratio of reagent to sample when mixed with microdroplets from cell disruption module 160. , Specially designed for the formation of fine droplets of reagents having a predetermined volume. Module 152 includes an input port 420, a valve 422, and an actuator 172, each of which joins a reagent source channel 428. Similarly, an overflow channel 424 that joins the reagent source channel 428 may also be provided. Actuator 172 can include a second positioning element 432 to prevent liquid from flowing into it.
The reagent material is preferably composed of at least one liquid, and is introduced through the input port 420 by a pipette or a syringe. Examples of suitable reagent materials include substances that facilitate the further processing of disrupted cell samples, such as enzymes, and other materials that amplify the DNA therein by polymerase chain reaction (PCR). The reagent material moves downstream in the reagent source channel 428 until the downstream portion of the reagent material contacts the positioning element 426. Any additional reagent material that continues to be received in the reagent source module preferably flows into the overflow channel 424. When reagent introduction is complete, valve 422 is closed to prevent the reagent from flowing out of the reagent source channel through reagent port 420.

b. Mixing Module The mixing module mixing zone 958 includes adjacent first and second channels 410, 412. The substances moving downstream toward the mixing zone 958 are preferably in contact with each other and mixed therein. Because the dimensions of the mixing zone 958 are fine, the sample and reagent material are preferably mixed by diffusion without other mass transport sources, such as mechanical agitation. However, it goes without saying that mixing in the mixing zone 958 increases when a stirring force such as an acoustic wave is applied.

c. Operation of Mixing Module and Reagent Input Module Reagent source module 152 and mixing module 166 preferably operate as follows. When the disintegrated sample from disintegration zone 950 is ready to be mixed with the reagent material, actuator 172 is actuated to prepare a fine droplet of reagent. Fine reagent droplets are prepared from portions of reagent material downstream of the opening 430 of the actuator 172 and upstream of the positioning element 427. Thus, assuming that the dimensions of the reagent source channel 428 are constant, the volume of the reagent microdroplet is determined by the distance between the positioning element 426 and the actuator opening 430.
Fine reagent droplets move downstream toward the channel 412 in the reagent mixing zone. Meanwhile, a sample of disintegrating material, such as a disintegrating fine liquid, moves downstream from disintegrating zone 950 toward channel 410 in mixing zone 958. The actuator 170 can supply an activation force that moves the collapsing fine droplets downstream. Alternatively, as discussed above, another actuator can be provided upstream of the collapse zone 950 and downstream of the actuator 170 to provide the necessary actuation force.

  Sample and reagent material flow into the downstream channel 438 of the mixing zone 958 where they contact and mix. Since both the collapsed sample and reagent material mix in the form of fine droplets, the mixing zone 958 prepares an amount of mixed material having a predetermined sample to reagent ratio. The volume of the microdroplet prepared in the microfluidic device 110 is preferably independent of physical properties such as the viscosity, conductivity, and osmotic force of the microdroplet. Thus, the mixing zone 958 prepares an amount of mixed material, and its sample to reagent material ratio is also independent of the physical and chemical properties of the mixed material. The vent 440 is downstream of the various zones of the microfluidic device 110 and provides confirmation that the pressure accumulated downstream does not interfere with the downstream movement of the sample within the microfluidic device 110.

5). DNA manipulation module The mixed disrupted cell sample and reagents are received in the DNA manipulation zone 971 of the DNA manipulation module 162. Module 162 can perform, for example, suppression, digestion, ligation, hybridization, and amplification of DNA material. In one embodiment, the DNA manipulation zone 971 is configured to perform PCR amplification of nucleic acids present in the disrupted cell sample. Vent 440 prevents pressure from increasing in zone 971 as disintegrated cell samples and reagents are introduced. By closing the valves 972 and 973 of the DNA manipulation module 162, it is possible to prevent the substances in the zones therein from coming out by evaporation or the like during PCR amplification. The DNA manipulation zone is provided with a heat source under the control of the computer 127 to allow thermal cycling of the DNA manipulation zone during amplification. This will be understood by those skilled in the art.
System 901 also includes a detector 981 that detects the presence of amplified polynucleotide generated by PCR. The detector 981 is preferably a photodetector that is in optical communication with the zone 971 by an optical fiber 981 or the like. A light source, such as a laser diode, introduces light into the DNA manipulation zone 971 and generates fluorescence indicative of the amount of amplified polynucleotide present therein. This fluorescence is generated from a fluorescent tag involved in the polynucleotide during amplification and contained in the reagent.

D. Preferred positioning elements Preferred positioning elements are discussed below.
1. Non-wetting positioning element The positioning element 979 can be formed of a non-wetting material that is placed in contact with the microfluidic sample. The physicochemical properties of the non-wetting material are selected when considering the type of liquid that forms the microfluidic sample. For example, if the microfluidic sample is a water soluble sample, the positioning element is preferably made of a hydrophobic material. Examples of hydrophobic materials include non-polar organic compounds such as aliphatic silanes and can be formed by modifying the inner surface of the microfluidic device 901. For microfluidic samples formed with organic solvents, the non-wetting material may be composed of a hydrophilic material.
When the microfluidic sample 808 reaches the positioning element 979, the liquid of the microfluidic sample undergoes an increase in surface tension at the downstream interface 810, which prevents the microfluidic sample 808 from moving downstream continuously. . The resistance is overcome by increasing the gas pressure difference between the upstream and downstream portions of the microfluidic sample and the microfluidic sample is moved downstream.

2. Capillary Assisted Positioning Element Referring to FIGS. 10a-10c, another type of positioning element is formed by changing the dimensions of the microfluidic channel to form a capillary assisted positioning element (CAFB) 700. be able to. The CAFB includes an upstream supply zone 702, a loading zone 704, and a stop zone 704. The microfluidic sample 720 arriving at the CAFB moves downstream until the downstream interface 710 of the microfluidic sample contacts the upstream surface 714 of the loading zone 706. At this point, capillary action moves the microfluidic sample downstream until the downstream sample interface 710 reaches the opening 712 between the loading zone 704 and the stop zone 706. The surface tension resists the microfluidic sample from going through the opening 714 and going downstream. For this reason, the microfluidic sample 720 is positioned at a predetermined location along the channel axis with respect to the positioning element 700.
The volume of the microfluidic sample reaching CAFB is preferably larger than the volume of the loading zone 704 to ensure that all the microfluidic sample proceeds to the opening. For fluids having surface tension and interfacial properties similar to water, the depth d ₁ of the loading zone 704 is preferably about 50% or less of the respective depths d ₂ and d ₃ of the stop and feed zones.
The tendency of the microfluidic sample to move in a given direction is adjusted by the ratio between the mean radius of curvature (MRC) of the front of the microfluidic sample and the back of the microfluidic sample. These curvatures depend on the contact angle between the sample fluid and the dimensions of the zone in which the microdroplet is moving. The MRCr _{1 at the} microdroplet interface in the loading zone is preferably smaller than the MRCr _{2 at} the drop interface in the feed zone or MRCr _{3 at the} drop interface in the stop zone. MRCr ₂ is preferably larger than MRCr ₃ . In this way, the radius of the downstream microdroplet interface increases upon reaching the stop zone, thereby preventing further downstream movement. Preferably, the contact angle with the fluid wall is substantially constant throughout the capillary auxiliary loading zone.

3. Aeration Positioning Element Referring to FIGS. 11 a-11 c, the positioning element 500 reduces the gas pressure acting on the upstream portion 504 of the microfluidic sample relative to the gas pressure acting on the downstream portion 506 of the microfluidic sample. And operate to position the microfluidic sample 502. The positioning element 500 includes a vent 508 provided in gas communication with a zone 510 through which the microfluidic sample 502 moves. Vent 508 is preferably in communication with zone 510 through passage 526. This zone is, for example, a channel or a conduit. The positioning element 500 can also include a second positioning element, such as a non-wetting material, to substantially prevent fluid from the microfluidic sample from contacting the vent.
When valve 512 is open, gas can pass between zone 510 and vent 508. When the valve 512 is closed, such gas passage is prevented. The valve 514 is preferably thermally activated and includes a TRS mass 514.
The actuator 518 is disposed upstream of the positioning element 500. The actuator is preferably a gas actuator and may include a heat source 520 that heats the gas involved in the actuator 518. Actuator 518 includes a positioning element 522, such as a non-wetting material, that can substantially prevent fluid from the microfluidic sample from flowing into it.

The positioning element 500 preferably operates as follows. Referring to FIG. 11 a, the microfluidic sample 502 moves downstream in the direction of arrow 524. The microfluidic sample is preferably moved by gas pressure supplied from an upstream actuator, not shown in FIGS. 9a-9c. The gas pressure acts on the upstream portion 504.
Referring to FIG. 11b, when the upstream portion 504 passes through the opening of the vent 508, the upstream gas is dissipated through the vent 508, thereby reducing the upstream pressure. The pressure drop preferably equalizes the downstream and upstream pressures, reducing or eliminating the starting force that tends to bias the microfluidic sample downstream.
Referring to FIG. 11 c, valve 512 is closed to prevent the passage of gas between zone 510 and vent 508. Preferably, TRS 514 moves into passage 526. When valve 512 is closed, actuation of actuator 518 provides an activation force that moves microfluidic sample 502 downstream in the direction of arrow 528 for subsequent processing.

4). Active Fluid Positioning Element Referring to FIGS. 15 a-15 c, the microdroplet preparation module 652 includes a microdroplet preparation zone 650, an active fluid positioning element 654, an actuator 656, and a valve 658. A second actuator is operatively associated with the active positioning element 654 and introduces the microfluidic sample 666 into the microdroplet preparation zone 650. The second actuator 660 is preferably disposed upstream from the valve 658. The microdroplet preparation module 652 prepares microdroplets 668 having a predetermined volume from the microfluidic sample 666 received therein.
In operation, the microfluidic preparation module 652 receives a microfluidic sample 666 that moves downstream due to the activation force provided by the second actuator 660. This activation force is preferably an upstream gas pressure that is higher than the downstream gas pressure acting on the microfluidic sample 666. The microfluidic sample moves downstream until its downstream portion 670 reaches the active positioning element 654. Active positioning element 654 preferably includes a sensor 672 having an electrical lead 674. Lead 674 is in electrical communication with the I / O pin of the microfluidic device and allows signals from sensor 672 to be received by DAQ.

Sensing element 672 is preferably a pair of electrical contacts. To detect the presence of liquid, DAQ 126 applies a small voltage across leads 674 and measures the resulting current. When the liquid of the microfluidic sample contacts the first and second contacts, the current passing between them changes to indicate to the DAQ 126 that the liquid has reached the sensor 672.
Upon detecting that the liquid has reached the sensor 672, the DAQ commands the second actuator 660 to reduce the downstream activation force acting on the microfluidic sample 666. For example, the DAQ reduces the temperature of the gas therein by reducing the current passing through the heat source 676 associated with the second actuator 660. This temperature drop reduces the gas pressure acting on the upstream portion 678 of the microfluidic sample, thereby preventing the microfluidic sample 666 from moving downstream. The microfluidic sample is positioned such that the first portion 680 is located downstream of the actuator 656 and the second portion is located upstream of the actuator 656.

To prepare the microdroplet 668, the DAQ 126 actuates an actuator to provide an actuation force and prepares the microdroplet 668 from the first portion of the microfluidic sample 666. The microdroplet 668 moves downstream while the second portion 682 of the microfluidic sample 666 moves upstream from the actuator 656. Closing valve 658 during microdroplet preparation can substantially isolate actuator 656 from the second actuator and other upstream portions of the microfluidic device.
The active positioning element preferably operates as a closed loop element and provides feedback from the sensor 672 to the DAQ. Feedback is indicated when the microfluidic sample reaches a predetermined position in the microfluidic device. Upon receiving feedback, the DAQ changes the state of the actuator that provides the starting force to move the fine droplets.

  Although the foregoing invention has been described with reference to certain preferred embodiments, it should be kept in mind that the scope of the invention is not limited thereto. Accordingly, those skilled in the art will be able to find variations of these preferred embodiments, which fall within the spirit of the invention, the scope of which is defined by the claims.

1 shows a microfluidic system according to the present invention. It is a figure which shows the enlarged view of a microfluidic device. It is a figure which shows the structure of the microfluidic device of the microfluidic system of FIG. FIG. 4 is a top view of the microfluidic device of FIG. 3. It is a fragmentary sectional view of the microfluidic device of FIG. FIG. 3 is a partial cross-sectional view of an upper substrate from the microfluidic device of FIG. 2. FIG. 3 is a second partial cross-sectional view of the upper substrate from the microfluidic device of FIG. 2. FIG. 5 is a top view of the microdroplet preparation zone of the blunt fluid device of FIG. 4 prior to microdroplet preparation. FIG. 8b is a cross-sectional view of the microdroplet preparation zone of FIG. 8a. FIG. 5 is a top view of a fine droplet preparation zone of the microfluidic device shown in FIG. 4 after preparation of the fine droplets. 9b is a cross-sectional view of the fine droplet preparation zone of FIG. 9a. a to c are cross-sectional views of the capillary auxiliary fluid barrier of the present invention. a to c are top views of a fluid barrier including an outlet. FIGS. 5a and 5b are top views of the collapse module of the microfluidic device shown in FIG. 4 before and after the preparation of the collapsed sample. a and b are figures which show 2nd Embodiment of the collapse module of this invention. It is a figure which shows the pulsation circuit interlock | cooperated with the collapse module of FIG. FIGS. 4A to 4C are diagrams showing a second microdroplet preparation module according to the present invention. FIGS.

Claims (13)

A microfluidic processing device for processing microfluids, comprising a microfluidic network comprising a plurality of modules, the network comprising:
A concentration module configured to receive a fluid sample containing particles and generate a concentrated sample from the fluid sample;
A microdroplet preparation module configured to receive the concentrated sample and generate microdroplets from the concentrated sample;
A disintegration module configured to release material from internal particles in the fine droplets of the concentrated sample;
A mixing module configured to mix one or more reagents with fine droplets of the concentrated sample;
Receiving a fine droplet of the concentrated sample mixed with the reagent, and performing a DNA manipulation module,
The modules provided in the microfluidic network are operatively connected via one or more channels integrated with the microfluidic network, and at least one of these modules is connected to the microfluidic network. can be separated by a valve that integrates, at least one actuator said became microfluidic network integrally with the fine droplets of the liquid material from one module to another module in the modules to move A microfluidic processing device configured to generate a difference between an upstream pressure and a downstream pressure acting on the microdroplets of fluid.   2. The microfluidic processing device according to claim 1, wherein the concentration module condenses particles in a fluid sample containing particles such that a ratio of particles per unit volume of the concentrated sample is higher than a ratio of particles in the fluid sample. A microfluidic processing device configured to be high. A microfluidic processing device for processing microfluids,
Comprising a microfluidic network, the network comprising:
A collapse module integrated with the microfluidic network configured to receive microdroplets containing cells;
An actuator integrated with the microfluidic network disposed upstream of the collapsing module, generating a difference between the upstream pressure and the downstream pressure acting on the microdroplet; An actuator configured to move the to the collapse module;
A vent positioning element having a vent hole disposed on the upstream side of the collapse module and the downstream side of the actuator, the vent positioning element having the vent hole having a micro droplet containing the cells. The vent positioning element is provided with a vent arranged to exchange gas with the moving area, and the vent positioning element in which the vent is opened has an upstream portion of the fine droplet containing the cells passing through the vent opening. Then, by dissipating the gas upstream of the fine droplets containing the cells through the vent hole , the fine droplets containing the cells are stopped at the collapse position of the collapse module, and the cells contain the cells. A vent positioning element configured to position a portion of a fine droplet downstream of the vent positioning element in the collapsed position;
A disintegration mechanism provided in the disintegration module, the disintegration mechanism configured to release intracellular substances from the cells in the fine droplets containing the cells at the disintegration position. A microfluidic processing device characterized by.   4. A microfluidic processing device according to claim 3, wherein the vent positioning element comprises a wetting reducing material for preventing the microdroplets from entering the holes. 5. The microfluidic processing device of claim 4, wherein the vent positioning element further comprises a valve in the microfluidic network to selectively block and pass gas between the wetting reducing material and the vent. A microfluidic processing device characterized by comprising: A microfluidic processing device for processing microfluids,
A disintegration module configured to receive a microdrop of a sample containing cells;
A disintegration mechanism provided in the disintegration module, the disintegration mechanism configured to release intracellular material from cells in the microdroplets of the sample in the disintegration module;
A first gas actuator integrated with a microfluidic network disposed upstream of the collapse module, wherein the microdroplet of the sample is moved downstream so as to partially overlap the collapse module A first gas actuator configured as follows:
A position-decided Me elements disposed downstream of the decay modules, by increasing the interfacial surface tension of the downstream side of the sample when the sample comes into contact with the positioning element, downstream of the sample and prevents movement of the, and position-decided Me element configured to position at least a portion of the sample to the collapse location of the decay modules,
A second gas actuator integrated with a microfluidic network disposed upstream of the collapse module and downstream of the first gas actuator, comprising: (a) from the cells of the sample in the collapse module a microdroplet is collapsed consisting released intracellular material, to prepare the fine droplets of a length equal to the distance between the second gas actuator before Symbol position-decided Me element, and, (b) a second gas actuator configured to provide the collapsed gaseous pressure which can be moved past the front Symbol position-decided Me element is moved downstream of the microdroplet the collapse module A microfluidic processing device comprising: A method for releasing intracellular material from cells in a fine droplet of liquid containing cells, comprising:
Introducing the microdroplet of liquid containing cells into a collapse module integrated with a microfluidic network of a microfluidic processing device;
Preventing the fine droplets of the liquid from moving downstream from the collapse module;
Activating a release mechanism for releasing intracellular material from the cells in the liquid in the collapse module;
Providing a gas pressure for separating a first portion of the liquid droplets of the liquid within the collapse module from a second portion of the liquid droplets of the liquid located upstream of the collapse module; Providing a disintegrated microdroplet composed of intracellular material released from the cells of the liquid in the disintegration module. A microfluidic processing device for processing microfluids,
Comprising a microfluidic network, the network comprising:
A concentration module comprising a flow member and a concentration chamber configured to receive fine droplets of fluid containing particles, wherein the flow member has a passage smaller than the diameter of the particles of fluid containing the particles. is formed as a concentrate module the fluid passes through the flow-through element, the concentrated particle sample consisting of particles of the fluid is configured to accumulate in the concentration chamber,
A downstream channel integral with the microfluidic network leading downstream from the concentration chamber to at least one downstream processing module;
An actuator integral with the microfluidic network configured to move a concentrated particle sample from the concentration module in a downstream direction along the downstream channel;
A microfluidic treatment device , comprising a first valve integrated with the microfluidic network, disposed between the actuator and the flow member . 9. The microfluidic processing device according to claim 8, further comprising a second valve disposed between the flow member and the downstream channel. 9. The microfluidic processing device according to claim 8, wherein the device includes opposing upper and lower substrates, the flow member includes first and second surfaces, and the first surface is the concentration chamber. The second surface is remote from the concentration chamber and adjacent to a space, the space containing a hygroscopic agent and opposite the lower substrate. A microfluidic treatment device provided on the surface of   The microfluidic processing device according to any one of claims 1, 2, 5, 8, 9, and 10, wherein the valve is made of a thermally actuated material.   9. The microfluidic processing device of claim 8, wherein the concentrated particle sample has a ratio of particles per unit volume of fluid that is greater than the fluid containing the particles received at the concentration module. Processing device. A microfluidic device for processing microfluids,
And a distribution member and enrichment chamber, a concentrate module configured to generate a particle sample enriched from a sample of fluid containing a particle element, the flow member of the particles of the fluid containing the particles diameter A concentration module formed to have a smaller passageway ;
A processing module disposed downstream of the concentration module;
An actuator disposed downstream of the concentration module and integrated with the microfluidic network of the microfluidic processing device to generate a difference between the upstream pressure and the downstream pressure to produce a concentrated particle sample; An actuator for moving the sample downstream from the concentration module in an undiluted state;
A microfluidic treatment device comprising a valve integrated with the microfluidic network disposed between the actuator and the flow member .

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