EP1438567B1 - Methods and systems for microfluidic processing - Google Patents
Methods and systems for microfluidic processing Download PDFInfo
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- EP1438567B1 EP1438567B1 EP02715213.1A EP02715213A EP1438567B1 EP 1438567 B1 EP1438567 B1 EP 1438567B1 EP 02715213 A EP02715213 A EP 02715213A EP 1438567 B1 EP1438567 B1 EP 1438567B1
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- Prior art keywords
- microfluidic
- sample
- lysing
- zone
- cell
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0636—Integrated biosensor, microarrays
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0442—Moving fluids with specific forces or mechanical means specific forces thermal energy, e.g. vaporisation, bubble jet
Definitions
- Another aspect of the present disclosure relates to a microfluidic system and method for processing a particle-containing fluid, such as, for example, a liquid containing bacterial cells or human cells.
- a particle-containing fluid such as, for example, a liquid containing bacterial cells or human cells.
- the system includes a lysing zone to receive the cell-containing sample and a positioning element to position the cell-containing sample in a lysing position in the vicinity of a lysing mechanism.
- the lysing mechanism releases intracellular material, such as DNA or RNA, from the cells.
- the lysing mechanism includes electrodes for generating an electric field sufficient to release intracellular contents from the cells.
- the lysing mechanism may lyse the cells using chemical, heat and/or ultrasonic techniques or any combination of these techniques.
- actuator 170 of the microdroplet preparation module 158 drives a microdroplet into cell lysis module 160.
- vented positioning element 200 positions microdroplet 802 in a lysing position with respect to electrodes 954. More specifically, as the microdroplet arrives in lysing module 160 it passes the opening of positioning element 200, because second positioning element 206 inhibits the microdroplet from flowing into vent 202.
- the propulsion gas from actuator 170 dissipates through vent 202, thereby substantially equalizing gas pressure upstream of microdroplet 802 with a pressure downstream of microdroplet 802.
- the microdroplet stops movement at a lysing position just downstream from barrier 200.
- substantially all of microdroplet 802 is disposed between an upstream edge 212 and a downstream edge 214 of electrodes 954.
- Reagent input module 152 is essentially the same as microdroplet formation module 158, however, it is specifically designed for formation of a microdroplet of reagent having a predetermined volume which will yield a desired ratio of reagent to sample when mixed with the microdroplet from cell lysing module 160.
- Module 152 includes an input port 420, a valve 422, and an actuator 172, each of which joins a reagent source channel 428.
- An overflow channel 424, which also joins reagents source channel 428, may also be provided.
- Actuator 172 may include a second positioning element 432 to prevent liquid from entering therein.
- the upstream gas dissipates through vent 508, thereby reducing the upstream pressure.
- the pressure reduction which preferably equalizes the downstream and upstream pressures, reduces or eliminates the motive force tending to urge the microfluidic sample downstream.
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Dispersion Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
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Description
- The present invention relates to methods and systems for processing samples using microfluidic systems.
- Microfluidic devices are typically formed of substrates (made of silicon, glass, ceramic, plastic and/or quartz) which include a network of micro-channels through which fluid flows under the control of a propulsion mechanism. The micro channels typically have at least one dimension which is on the order of nanometers to hundreds of microns.
- Microfluidic devices offer several advantages over a traditional macro-scale instrumentation. For example, in general, they require substantially smaller fluid samples, use far less reagent, and process these fluids at substantially greater speeds than macro-scale equipment. A microfluidic device may utilize only minute amounts of sample to determine chemical and physical properties of the sample, such as by subjecting the sample to fluid processing.
- In many cases, the accuracy of such fluid processing depends upon the relative amounts of sample and reagent used. For example, when a sample is analyzed for a DNA "fingerprint," the results may depend upon the concentration of reagents used to amplify DNA present in the sample. Thus, if an improper ratio of sample to reagent is used, the result may be inaccurate. Because microfluidic devices process samples and reagents in minute amounts, even a small absolute uncertainty in the amount of reagent or sample used can introduce uncertainty to the results of a microfluidic analysis.
- Variances in the amount of samples and reagents processed by a microfluidic device may originate from several sources. For example, some microfluidic devices manipulate continuous, flowing streams of liquid. Changes in the viscosity of the liquid can alter the flow rate of the streams and, correspondingly, the time required to introduce a predetermined amount of material to a given location of the microfluidic device. Sample dilution may occur where a liquid flow stream is used to move sample components from one location to another within a microfluidic device.
- Microfluidic analysis of cells within body fluids is especially challenging due to the relatively small number of cells available for analysis and the inherent difficulty in manipulating such materials.
- Manipulation of samples may involving moving a sample between different locations within a microfluidic device. For example, electric fields are used as a propulsion mechanism for some microfluidic devices. In such devices, a high voltage, on the order of kilovolts, is applied across electrodes within the device to thereby generate an electric field in the micro channels. The field imposes a force on ions within the fluid, thereby propelling the ions through the micro channel. The fluid itself may also be propelled by the motion of ions moving within the fluid.
- Gas pressure is also used to propel fluid through micro channels. In some devices, a source of pressurized gas, external to the microfluidic device, is connected to the microfluidic device to supply a gas pressure, which propels the fluid. Gas pressure may also be generated by a heated chamber within the microfluidic device itself to propagate fluid within a micro channel.
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WO98/22625 A1 US 6,130,098 discloses microscale devices which allow movement and mixing of microdroplets through microchannels. - The scope of protection is defined by the independent claims, to which reference should now be made. Advantageous features are set out in the dependent claims.
- According to one aspect of the claimed invention, there is provided a microfluidic device for processing a cell-containing microfluidic sample, comprising: a lysing module configured to receive a cell-containing microfluidic sample, wherein the lysing module comprises a lysing zone, wherein the microfluidic sample is a liquid; a positioning element configured to inhibit downstream movement of the microfluidic sample to position the microfluidic sample in a lysing position; a lysing mechanism within the lysing zone, to release intracellular contents from cells within the lysing zone; and a gas actuator disposed upstream from the positioning element and the lysing zone such that a first portion of the microfluidic sample is disposed upstream from the gas actuator and a second portion of the microfluidic sample is disposed downstream from the gas actuator and upstream of the positioning element, the gas actuator configured to provide a gas pressure sufficient to prepare a microdroplet having a predetermined volume comprising intracellular contents released from cells of the cell-containing microfluidic sample within the lysing zone by separating the second portion of the microfluidic sample from the first portion of the microfluidic sample and moving the second portion of the microfluidic sample to a location downstream of the lysing mechanism and the positioning element.
- According to another aspect of the claimed invention, there is provided a microfluidic method for processing a cell-containing microfluidic sample, comprising: positioning, using a positioning element, the cell-containing microfluidic sample in a lysing position with respect to a lysing mechanism of a lysing zone of a lysing module of a microfluidic device, the cell-containing microfluidic sample comprising a cell-containing liquid; actuating the lysing mechanism to release intracellular material from cells of the cell-containing microfluidic sample; actuating a gas actuator disposed upstream of the positioning element and the lysing zone to provide a gas pressure sufficient to separate a portion of the cell-containing microfluidic sample that is downstream of the gas actuator and upstream of the positioning element from a portion of the cell-containing microfluidic sample that is upstream of the gas actuator and move the downstream portion of the cell-containing microfluidic sample to a location downstream of the lysing mechanism and the positioning element, the downstream portion of the microfluidic sample being a microdroplet comprising intracellular material released from cells of the cell containing microfluidic sample and having a predetermined volume.
- In general, a first aspect of the disclosure relates to a system and method for moving samples, such as fluids, within a microfluidic system. In one aspect, the disclosure relates to the use of a plurality of gas actuators for applying pressure at different locations within the microfluidic system to thereby supply force for moving samples. For example, in one embodiment, a first gas actuator provides a gas pressure sufficient to move a first sample from a first location to a second location of the microfluidic device. A second gas actuator provides a gas pressure to move another sample from a third location to a fourth location of the microfluidic device.
- In another example, a plurality of gas actuators cooperate to move the same fluid sample. A first gas actuator provides a gas pressure sufficient to move the microdroplet between first and second processing zones of the microfluidic device, and a second gas actuator provides a gas pressure to move the microdroplet to a third processing zone.
- In preferred embodiments, the plurality of actuators are integral with a microfluidic network through which the microfluidic samples flow. For example, a plurality of gas actuators can be fabricated in the same substrate which forms the microfluidic network. One such gas actuator is coupled to the network at a first location for providing gas pressure to move a microfluidic sample within the network. Another gas actuator is coupled to the network at a second location for providing gas pressure to further move at least a portion of the microfluidic sample within the network.
- In another aspect, the disclosure relates to the use of valves with the plurality of actuators. For example, in one embodiment, a valve is coupled to a microfluidic network so that, when the valve is closed, it substantially isolates the second gas actuator from the first gas actuator. Such valves can control the direction of the propulsive force of the actuators by preventing the expanding gas from traveling in certain directions, while permitting it to expand in the desired direction. They also extend the range over which an actuator can propel a microdroplet, by preventing the gas from dissipating in certain in areas upstream from the microdroplet.
- Another aspect of the present disclosure relates to a microfluidic system and method for processing a particle-containing fluid, such as, for example, a liquid containing bacterial cells or human cells.
- In one aspect, the disclosure relates to preparing an enriched particle sample from the particle-containing fluid. For example, a microfluidic system for preparing an enriched sample includes an enrichment zone and a flow through member disposed in fluid communication with the enrichment zone. The flow through member allows fluid of the particle containing fluid to pass through and exit the enrichment zone, while causing particles of the particle-containing fluid to accumulate within the zone, thereby preparing an enriched particle sample in the enrichment zone. The flow through member may include a plurality of pathways, such as pores, having a sufficient size to allow passage of the fluid therethrough but of a size too small to allow passage of the particles therethrough. Suitable flow through members are composed of, for example, filter elements, such as filter paper, porous glasses, and porous gels.
- The microfluidic system may include an actuator that moves the enriched particle sample from the enrichment zone with essentially no dilution of the enriched particle sample. In one embodiment, the actuator is a gas actuator that moves the sample by increasing a gas pressure associated with an upstream portion of the enrichment zone relative to a gas pressure associated with the downstream channel. For example, the gas actuator may include a source of heat in thermal contact with a volume of gas. Expansion of the gas upon actuating the heat source creates a gas pressure sufficient to move the enriched particle sample from the enrichment zone to another location within the microfluidic system, such as a location containing modules for further fluid processing.
- In another aspect, the disclosure relates to a microfluidic system and method for processing an enriched particle sample which includes cells entrained in a liquid. For example, the system includes a lysing zone to receive the enriched cell-containing sample and a positioning element to position the enriched cell-containing sample in a lysing position in the vicinity of a lysing mechanism. The lysing mechanism releases intracellular material, such as DNA or RNA, from the cells. In one embodiment, the lysing mechanism includes electrodes for generating an electric field sufficient to release intracellular contents from the cells. Alternatively, the lysing mechanism may lyse the cells using chemical, heat and/or ultrasonic techniques or any combination of these techniques .
- In one embodiment, the lysing zone releases intracellular contents from cells of the cell-containing fluid and then prepares from this fluid a microdroplet which contains intracellular contents released from the cells. The microdroplet is preferably prepared from only a portion of the cell-containing fluid. For example, a preferred microdroplet includes less than about 90 percent of the cell-containing fluid. In another embodiment, the lysing zone receives a microdroplet of cell-containing fluid and releases the intracellular contents of the cells within the droplet.
- In another aspect, the disclosure relates to a microfluidic substrate for processing the intracellular contents of cells suspended in fluids. The substrate includes an enrichment zone, a lysing module, a microdroplet formation module, mixing module and an amplification module. The enrichment zone preparing an enriched particle sample from the cell-containing fluid. The lysing module, which is coupled to the enrichment zone for receiving the enriched particle sample, releases intracellular material from cells within the sample to thereby forming a lysed sample. The microdroplet formation module then forms a first microdroplet of fluid from the lysed sample and forwards it to a mixing module for mixing with a microdroplet ofreagent. The amplification module amplifies intercellular material within the microdroplet formed from the mixture.
- Another aspect of the disclosure relates to a microfluidic system and method for processing a cell-containing fluid, such as, for example, a liquid containing bacterial cells or human cells. For example, the system includes a lysing zone to receive the cell-containing sample and a positioning element to position the cell-containing sample in a lysing position in the vicinity of a lysing mechanism. The lysing mechanism releases intracellular material, such as DNA or RNA, from the cells. In one embodiment, the lysing mechanism includes electrodes for generating an electric field sufficient to release intracellular contents from the cells. Alternatively, the lysing mechanism may lyse the cells using chemical, heat and/or ultrasonic techniques or any combination of these techniques.
- In one embodiment, the lysing zone releases intracellular contents from cells of the cell-containing fluid and then prepares from this fluid a microdroplet which contains intracellular contents released from the cells. The microdroplet is preferably prepared from only a portion of the cell-containing fluid. For example, a preferred microdroplet includes less than about 90 percent of the cell-containing fluid. In another embodiment, the lysing zone receives a microdroplet of cell-containing fluid and releases the intracellular contents of the cells within the droplet.
- The positioning elements assist in placing the cell containing fluid sample in the vicinity of the lysing mechanism so that the lysing mechanism can release intracellular material from the cells. These elements preferably operate differently from a valve, which would completely obstruct passage of material between upstream and downstream locations adjacent the valve. Rather, they typically provide resistance to fluid flow at a desired location (the lysing position) to thereby control fluid placement.
- In one embodiment, the positioning element is disposed downstream of the lysing mechanism to position an upstream portion of a cell-containing sample (such as a microdroplet) in the lysing position. The positioning element preferably increases a surface tension of a downstream surface of the cell-containing sample to thereby inhibit downstream movement of the sample. For example, the positioning element may include an amount of reduced-wetting material, such as a hydrophobic material, disposed to contact a portion of the downstream surface of the cell-containing microdroplet.
- In another embodiment, the positioning element is disposed upstream of the lysing zone to position a downstream portion of the cell-containing microdroplet in the lysing position. The positioning element includes a vent, which substantially equalizes a gas pressure upstream of the cell-containing microdroplet with a gas pressure downstream of the cell-containing microdroplet to thereby stop downstream movement of the cell-containing microdroplet. When the microdroplet is in the lysing position. A valve is preferably disposed to subsequent obstruct passage of gas between the lysing zone and the vent to allow an upstream gas pressure to once again move the droplet further downstream for additional processing. For example, the microfluidic system may include a mixing zone downstream of the enrichment zone and/ or lysing zone, to mix the microdroplet which emerges from these zones with a predetermined amount of reagent material.
- In another aspect, the disclosure relates to a microfluidic substrate for processing the intracellular contents of cells suspended in fluids. The substrate includes a lysing module, a microdroplet formation module, mixing module and an amplification module. The lysing module releases intracellular material from cells within the sample to thereby form a lysed sample. The microdroplet formation module then forms a first microdroplet of fluid from the lysed sample and forwards it to a mixing module for mixing with a microdroplet of reagent. The amplification module amplifies intercellular material within the microdroplet formed from the mixture.
- The present invention is described below in reference to the following drawings, in which:
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Fig. 1 shows a microfluidic system according to the invention; -
Fig. 2 shows an expanded view of a microfluidic device. -
Fig. 3 shows a schematic of a microfluidic device of the microfluidic system ofFig. 1 ; -
Fig. 4 , shows a top view of the microfluidic device ofFig. 3 ; -
Fig. 5 shows a partial cross-sectional view of the microfluidic device ofFig. 4 ; -
Fig. 6 shows a partial cross-sectional view of an upper substrate from the microfluidic device ofFig. 2 ; -
Fig. 7 shows a second partial cross-sectional view of an upper substrate from the microfluidic device ofFig. 2 ; -
Fig. 8a shows a top view of a microdroplet preparation zone of the microfluidic device ofFig. 4 before preparation of a microdroplet; -
Fig. 8b shows cross sectional view of the microdroplet preparation zone ofFig. 8a ; -
Fig. 9a shows a top view of a microdroplet preparation zone of the micro fluidic device ofFig.4 after preparation of a microdroplet; -
Fig. 9b shows a cross sectional side view of the microdroplet preparation zone ofFig. 9a ; -
Figs. 10a-10c show cross sectional side views of a capillary assisted fluid barrier of the present invention; -
Figs. 11a-11c show top views of a fluid barrier comprising a vent; -
Figs. 12a and 12b show top views of the lysing module of the microfluidic device ofFig. 4 , before and after preparation of a lysed sample; -
Figs. 13a and 13b show a second embodiment of a lysing module of the invention; -
Fig. 14 shows a pulsing circuit associated with the lysing module ofFig. 4 ; and -
Figs. 15a-15c show a second microdroplet preparation module of the invention. - The present invention relates to microfluidic systems and methods for processing materials, such as samples and reagents. More specifically, one aspect of the invention relates to microfluidic systems and methods for moving fluids within a microfluidic system. In one embodiment described below, the fluid includes particles which tend 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 cells of an animal, such as a human. However, they may include intracellular material from such cells. For example, a system of the invention may be used to process a sample of bacterial cells to determine whether the bacteria are pathogenic.
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Fig. 1 depicts amicrofluidic system 100 that includes amicrofluidic device 110 andcorresponding cartridge 120, which receive one or more fluid samples and process the samples under the control ofcomputer 127 and data acquisition and control board (DAQ) 126. -
Computer 127 preferably performs high level functions, such as supplying a user interface that allows a user to select desired operations, notifying theDAQ 126 as to the selected operations, and displaying for the user the results of such operations. These operations include, for example, subjecting a sample to process steps within the various process zones of the microfluidic device. Thecomputer 127 may be a portable computer to facilitate transport of the microfluidic system. -
Computer 127 is connected to DAQ 126 viaconnection 128, which provides data I/O, power, ground, reset, and other functional connectivity. Alternatively, awireless link 132 between thecomputer 127 and theDAQ 126 may be provided for data and control signal exchange via wireless elements 132(a) and 132(b). Where the data link is a wireless link, for example, theDAQ 126 may have separate power source, such as a battery. - In general,
DAQ 126 controls the operation ofmicrofluidic device 110 in accordance with the high level instructions received fromcomputer 127. More specifically, to implement a desired operation requested bycomputer 127,DAQ 126 supplies the appropriate electrical control signals tocartridge 120 viacontacts 125. -
Cartridge 120 provides electrical andoptical connections 121 for electrical and optical signals between theDAQ 126 and themicrofluidic substrate 110, thereby allowingDAQ 126 to control the operation of the substrate. - The
chip carrier cartridge 120 is shown being inserted into (or removed from) an interface hardware receptacle of theDAQ 126 having electrical andoptical contacts 125 standardized to mate with a correspondingcontacts 121 of thechip carrier cartridge 120. Most contacts are for electrical signals, while certain ones are for optical signals (IR, visible, UV, etc.) in the case of optically-monitored or optically-excited microfluidic processors. Alternatively (not shown), theentire DAQ 126 may be a single ASIC chip that is incorporated into theChip Carrier Cartridge 120, wherein contacts 121,125 would become conductive pathways on a printed circuit board. -
Fig. 2 illustrates the general structure of a preferred type of microfluidic device. The device includes anupper substrate 130, which is bonded to alower substrate 132 to form a fluid network. - The
upper substrate 130 depicted inFIG. 2 is preferably formed of glass and has amicrofluidic network 134 in itsbottom surface 136. Those skilled in the art will recognize that substrates composed of silicon, glass, ceramic, plastic, and/or quartz are all acceptable in the context of the present invention. - The microfluidic network includes a plurality of zones. The number of zones, as well as the overall topology of the microfluidic network, will depend upon the particular application which the microfluidic device is designed to perform. The zones of the microfluidic device may have any cross-sectional shape, such as generally arcuate or generally polygonal. For example, a zone may include channels, chambers or other substantially enclosed spaces. By "substantially enclosed" it is meant that materials enter or exit the zones only through predetermined pathways. Examples of such pathways include channels, microchannels and the like, which interconnect the various zones. The zones preferably have at least one micro-scale 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 in the
bottom surface 136 of theupper substrate 130 using known photolithographic techniques. More specifically, transparent templates or masks containing opaque designs are used to photo-define objects on the surface of the substrate. The patterns on the templates are generated with computer-aided-design programs and can delineate structures with line-widths of less than one micron. Once a template is generated, it can be used almost indefinitely to produce identical replicate structures. Consequently, even extremely complex microfluidic networks can be reproduced in mass quantities and at low incremental unit cost. Alternatively, if a plastic material is used, the upper substrate may be formed using injection molding techniques, wherein the micro-channels are formed during the molding process. - The
lower substrate 132 may include aglass base 138 and anoxide layer 140. Withinoxide layer 140,resistive heaters 142 andelectric leads 144 are formed using photolithographic techniques. The leads 144 connect toterminals 146 which are exposed at the edge of the substrate to permit electrical connection tocartridge 120, thereby permitting DAQ126 to control the heaters. More specifically, to activate aheater 142,DAQ 126 applies a voltage across a pair of terminals 146 (via cartridge 120) to supply current throughleads 146 andheater 142, thereby heating theresistive heater element 142. -
Metal heater elements 142 are positioned so that, when the upper and lower substrates are bonded together, the heaters reside directly beneath certain regions of the fluid network of the upper substrate so as to be able to heat the contents of these regions. Thesilicon oxide layer 140 prevents theheating elements 142 from directly contacting with material in the microfluidic network. - The
oxide layer 140,heating elements 142, and resistive leads 144 are fabricated using well-known photolithographic techniques, such as those used to etch microfluidic network. -
Fig. 3 illustrates a top-down view ofmicrofluidic device 110. As shown, the substrate has asample input module 150 andreagent input module 152 to allow sample and reagent materials, respectively, to be input todevice 110. Preferably,input modules laboratory robot 154. - The substrate also includes
process modules enrichment module 156 prepares a fluid sample having a relatively high concentration of cell particles, lysingmodule 160 releases intracellular material from the cell particles, and mixingmodule 166 mixes the resultant sample with certain reagents. As another example, anamplification process module 162 maybe used to amplify and detect minute quantities of DNA within a sample. - Various modules of
microfluidic device 110 are connected, such as bychannels 164, to allow materials to be moved from one location to another within thedevice 110.Actuators first actuator 168 moves material downstream fromprocess module 156 to processmodule 158. Upon completion of processing withinprocess module 158, asecond actuator 170 moves material downstream to mixingprocess module 160. Subsequently,actuator 170 or an additional actuator moves the material to mixingmodule 166, where the material mixes with a reagent moved byactuator 172. Finally,actuator 172, or another actuator, moves the mixed material tomodule 162. - Because each actuator is preferably responsible for moving materials within only a subset of the modules of
device 110, sample materials can be controlled more precisely than if a single actuator were responsible for moving material throughout the entire device. The various functional elements, ofmicrofluidic device 110, including the actuators, are preferably under computer control to allow automatic sample processing and analysis. - The various actuators of
microfluidic device 110 cooperate to move material between different locations ofmicrofluidic device 110. For example,actuator 168 moves material, such as an enriched sample, between anenrichment zone 931 and amicrodroplet preparation module 158.Actuator 170 prepares a microdroplet from the enriched sample and, in so doing, moves the microdroplet to a lysing zone 950.Actuator 170 is used to move material from the lysing zone 950 to mixingmodule 166. It should be noted, however, that another actuator may be disposed intermediate between lysing zone 950 and microdroplet preparation zone to move the lysed sample downstream to themixing module 166. - Actuators of
device 110 may also cooperate in moving two amounts of material simultaneously. For example, as described above,actuator 172 andactuator 170 cooperate to mix reagent and lysed microdroplets. Such cooperative actuators can be controlled independently of one another to ensure proper mixing. For example, if one material is known to be more viscous, the motive force moving that material can be increased independently of the motive force moving the other material. - The multiple actuators and modules of
microfluidic device 110 are preferably operatively connectable and isolatable by the valves of microfluidic device. For example, a closed state of either ofvalves microdroplet preparation module 170 fromenrichment module 156. Thus, one or more actuators can be used to move materials between predetermined locations withinmicrofluidic device 110, without perturbing or contacting material present in an operatively isolated module. The ability to operatively connect and isolate desired modules is advantageous in microfluidic devices having many process functions. Further, these valves also control the direction of the propulsive force of the actuators by preventing the expanding gas from traveling in certain directions, while permitting it to expand in the desired direction. This also extends the range over which an actuator can propel a microdroplet, by preventing the gas from dissipating in certain in areas upstream from the microdroplet. - The following demonstrates the cooperative operation of such multiple actuators in an example embodiment having a plurality of processing modules, namely an
enrichment zone 915, amicrodroplet preparation module 158, acell lysing module 160, amixing module 166 and a DNA manipulation module 167. - Referring to
Figs. 4 and5 , a microfluidic device 901 includes anenrichment module 156 for concentrating samples received therein. These samples include particle-containing fluids, such as bacterial cell-containing fluids. In general,enrichment module 156 receives a flow of particle-containing fluid from aninput port 180 ofinput module 150, and allows the fluid to pass through the zone while accumulating particles within the zone. Thus, as more fluid flows through the zone, the particle concentration increases within the module. The resultant concentrated fluid sample is referred to herein as an enriched particle sample. - The enrichment module includes an enrichment zone 931 (
Fig. 5 ), a flow throughmember 900,valves sample introduction channel 929.Valve 919 is connected between the flow throughmember 900 andactuator 168 as shown, andvalve 915 is connected between the flow through member and adown stream channel 937 which leads toprocess module 158. These valves may be of any type suitable for use in a microfluidic device, such as thermally actuated valves, as discussed in co-pending application No.09/953,921, filed September 9, 2001 enrichment module 931. - The flow through member is also connected to the
sample input module 150 via thesample introduction channel 929 to allow fluid to flow into the enrichment zone.Valve 913 is connected to this sample introduction channel to control the in-flow and outflow of fluid from the input port. -
Fig. 5 is a cross-sectional view of the enrichment zone which shows the flow through member in greater detail. As shown, flow throughmember 900 has first andsecond surfaces First surface 941 is preferablyadjacent enrichment chamber 931.Second surface 941 is preferably spaced apart from theenrichment chamber 931 by flow throughmember 900. Flow throughmember 900 is preferably formed of a material having pathways smaller than the diameter of the particles to be enriched, such as pores of less than about 2 microns in diameter, for example, about 0.45 microns. Suitable materials for constructing flow throughmember 900 include, for example, filter media such as paper or textiles, polymers having a network of pathways, and glassy materials, such as glass frits. -
Figs. 6 and 7 depict cross sectional views ofupper substrate 130 that illustrate anenrichment zone 931. As shown, fluid exitsenrichment zone 931 throughsurface 941, passes throughmember 900 and enters aspace 400.Space 400 may include anabsorbent material 402 to absorb the exiting fluid. Thus,space 400 preferably provides a substantially self-contained region in which fluid exiting the enrichment zone can collect without contacting exterior portions of themicrofluidic system 100. -
Space 400 is formed during the fabrication ofupper substrate 130. As discussed above, microfluidic features, such as zones and channels, are fabricated atsurface 136 ofsubstrate 130.Space 400, however, is fabricated at asurface 137, which is preferably disposed on the other side ofsubstrate 130, oppositesurface 136. Thus, even whensurface 136 is mated withlower substrate 132, fluid can exitenrichment zone 931 via flow throughmember 900. - Flow through
member 900 andabsorbent material 402 do not require adhesives or other fasteners for positioning withinsubstrate 130. Rather flow throughmember 900 andabsorbent material 402 may be formed of a shape and size that substantially corresponds tospace 400. Friction then holds flow throughmember 900 andabsorbent material 402 in place once they are positioned inspace 400. Any residual gap atlocations 404 between flow throughmember 900 andsubstrate 130 should be small enough to prevent particles from exitingenrichment zone 931 through thegap 404. Naturally, adhesive or other fastening means may be used to secure flow throughmember 900 orabsorbent material 402. - In an alternative embodiment, a flow through member is formed integrally with a substrate by using microfabrication techniques, such as chemical etching, that introduce pores or other pathways into the substrate. The pores provide fluid passage between
enrichment zone 931 and an outer portion of the substrate. - To enrich a sample, the device 901 operates as follows. Referring to
Fig. 4 ,valves valve 913 is open. A particle-containing fluid is introduced intoinput port 180. Sincevalve 913 is open, it allows the sample to pass alongchannel 929 intoenrichment zone 931. Alternatively,enrichment zone 931 can be configured to receive samples directly, such as by injection. Sincevalves actuator 977 anddownstream channel 937. - Thus, flow through
member 900 provides the only path for fluid to exit the enrichment channel. Fluid passes throughsurface 941 and exitsenrichment zone 931 viasecond surface 943, while particles accumulate within the zone.Enrichment zone 931 can therefore receive a volume of fluid that is larger than the volume of theenrichment chamber 931. Thus, as fluid flows through the chamber, the concentration of particles within the chamber increases relative to the concentration in the particle-containing fluid supplied at the sample input. Where the particles are cells, the concentration or number of cells inzone 931 preferably becomes great enough to perform a polymerase chain reaction (PCR) analysis of polynucleotides released from the cells in a downstream processing module. -
Enrichment zone 931 thus prepares an enriched particle sample from particles of particle-containing fluids received therein. The enriched particle sample has a substantially higher ratio of particles per volume of fluid (PPVF) than the corresponding ratio of the particle-containing fluid received by the enrichment zone. The PPVF of the enriched particle sample is preferably at least about 25 times, preferably about 250 times, more preferably about 1,000 times greater than the PPVF of the particle-containing fluid. - After a sufficient volume of particle containing fluid has been received by
enrichment zone 931,valve 913 is closed thereby blocking further flow of fluid into the enrichment zone, and preventing material inzone 931 from returning to thesample introduction port 180.Valves valve 919 allowsactuator 168 to push enriched sample, andvalve 915 allows the enriched sample to move downstream. -
Actuator 168 provides a motive force that moves the enriched particle sample fromenrichment zone 931.Actuator 168 is preferably a gas actuator, which provides a gas pressure upon actuation of aheat source 975, which is in thermal communication with a volume ofgas 977. Actuation ofheat source 975 raises the temperature and, therefore the pressure, ofgas 977. The flow through member and the fluid therein substantially prevents gas from escaping the enrichment zone. Thus, the resulting gas pressure moves the enriched particle sample downstream from theenrichment zone 931. - The gas actuator may include elements to facilitate alternative pressure generation techniques such as chemical pressure generation. In another embodiment, the actuator may decrease a volume of gas associated with an upstream portion of the enrichment zone to thereby create a pressure differential across the sample that moves the sample from the enrichment zone. An example of such an element is a mechanical actuator, such as a plunger or diagram.
- Rather than generating a positive pressure upstream from the enrichment zone, the gas actuator may decrease a pressure downstream from the zone relative to a pressure upstream. For example, the gas actuator may include a cooling element in thermal contact with a volume of gas associated with a downstream portion of the zone. Contraction of the gas upon actuating the cooling element creates a gas pressure difference between the upstream and downstream portions of the enrichment zone to move the enriched particle sample from the enrichment zone. Alternatively, a mechanical actuator may be used increase a volume of gas associated with a downstream portion of the enrichment zone to thereby decrease the pressure of the gas and move the enriched particle sample from the enrichment zone.
- The enriched particle sample is preferably moved downstream with essentially no dilution thereof, i.e., the concentration of the enriched particles is not substantially decreased upon movement from the
enrichment zone 931. Thus, removal of particles from the enrichment channel of the present invention does not require diluting or otherwise contacting the particles with a fluid different from the fluid of the particle-containing fluid introduced to the enrichment channel. In contrast, in systems that concentrate substances by surface adsorption, removal of the adsorbed substances requires an elution fluid, which contacts and thereby dilutes the substances. - Upon removal from the enrichment zone of the present invention, the enriched particle sample is preferably received by
downstream channel 937.Downstream channel 937 leads to other processing modules, which perform further processing of the enriched particle sample. In the embodiment ofFig. 3 , the enriched particle sample is received by amicrodroplet preparation module 158, which prepares a microdroplet sample comprising a portion of the enriched particle sample. - A
microdroplet 802 is a discrete sample having a predetermined volume between, for example, about 1.0 picoliter and about 0.5 microliters. Thus, microdroplets prepared by microdroplet preparation module provide 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, electrical conductivity, and osmotic strength of the fluid of the microdroplet. -
Microdroplet 802 is preferably defined by upstream and downstream boundaries each formed by a respectivegas liquid interface - Referring to
Figs. 8a-8b and9a-9b ,microdroplet preparation module 158 prepares amicrodroplet 802 from a microfluidic sample received therein. This module includes amicrodroplet preparation zone 800, apositioning element 979, agas actuator 170, and avalve 216 which cooperate to preparemicrodroplet 800 from microfluidic samples received from the enrichment zone. - As explained above,
actuator 168 of the enriched zone pushes the enriched sample into themicrodroplet preparation zone 800. The enriched sample moves until reachingpositioning element 979. In general, a positioning element inhibits the downstream progress of a microfluidic sample to thereby position the sample at a desired location. However, as explained more fully below, the positioning element does not permanently inhibit progress of the sample. Rather, it allows the microfluidic sample to continue downstream at a predetermined later time. - The leading edge of
microfluidic sample 808 that reachespositioning element 979 is positioned downstream from anopening 820 ofgas actuator 170. Accordingly, afirst portion 821 ofmicrofluidic sample 808 is disposed upstream from opening 820 and asecond portion 822 ofmicrofluidic sample 808 is disposed downstream fromopening 820. - Referring to
Figs. 8a-8b ,gas actuator 170 is actuated, such as byDAQ 126, to thereby generate a gas pressure sufficient toseparate microdroplet 802 from thesecond portion 822 ofmicrofluidic sample 808. The gas pressure is preferably provided by the actuation of aheat source 958, which heats a volume of gas associated with gas actuator 957. As the pressure increases, the gas expands, thereby separating amicrodroplet 802 from the rest ofsample 808.Microdroplet 802 may comprise only a portion, such as less than about 75%, or less than about 50%, ofmicrofluidic sample 808 received bymicrodroplet preparation zone 800. The dimensions ofmicrodroplet 802 are determined by the volume of the channel betweenfluid barrier 979 andopening 820. For example, for a channel having a uniform cross-sectional area, a length l1 ofmicrodroplet 802 corresponds to a distance d4 betweenpositioning element 979 andopening 820. Thus, a microfluidic device can be configured to prepare microdroplets of any volume by varying the length between the fluid barrier and corresponding actuator opening. - Continued actuation of
gas actuator 170 overcomes the inhibitory effect ofpositioning element 979, thereby drivingmicrodroplet 802 to a location downstream ofmicrodroplet preparation zone 800 while thesecond portion 822 of the microfluidics sample moves upstream frommicrodroplet 802 tocell lysis module 160. - Referring back to
Fig. 3 , alysing module 160 receives themicrodroplet 802 prepared bymicrodroplet preparation zone 800. In general, lysingmodule 160 releases material from inside the particles, such as by releasing intracellular material from cells. - As shown in
Figs. 4 and12 , lysingmodule 160 includes a lysing zone 950, a lysing mechanism within the lysing zone (such as electrodes 954), and a ventedpositioning element 200 positioned upstream from the lysing zone. The lysing mechanism preferably includes a set of electrodes or other structures for generating electric fields within the lysing zone. The vented positioning element preferably includes avent 202, avalve 204, and asecond positioning element 206 for inhibiting fluid from flowing into the vent. - As explained above,
actuator 170 of themicrodroplet preparation module 158 drives a microdroplet intocell lysis module 160. As the microdroplet moves intomodule 160, ventedpositioning element 200 positions microdroplet 802 in a lysing position with respect toelectrodes 954. More specifically, as the microdroplet arrives in lysingmodule 160 it passes the opening ofpositioning element 200, becausesecond positioning element 206 inhibits the microdroplet from flowing intovent 202. When the rear end of the microdroplet passes the opening ofbarrier 200, the propulsion gas fromactuator 170 dissipates throughvent 202, thereby substantially equalizing gas pressure upstream ofmicrodroplet 802 with a pressure downstream ofmicrodroplet 802. Thus, the microdroplet stops movement at a lysing position just downstream frombarrier 200. Preferably, in the lysing position, substantially all ofmicrodroplet 802 is disposed between anupstream edge 212 and adownstream edge 214 ofelectrodes 954. - After microdroplet 802 is placed in the cell lysing position, a pulse circuit of
DAQ 126 supplies a pulsed voltage signal acrosselectrodes 954. In response,electrodes 954 generate a pulsed electric field in the vicinity of the electrodes. Because the microdroplet is position in this vicinity, cells within the microdroplet are subjected to the pulsed field. Preferably, substantially all of the cells, such as greater than about 75%, of the microdroplet are subjected to an electric field sufficient to release intracellular material therefrom. The lysing module thus prepares a lysed microdroplet comprising a predetermined amount of sample. - A preferred pulse circuit is shown in
Fig. 14 . In general, this circuit generates a sequence of voltage pulses that yields a corresponding sequence of electrical field pulses in the vicinity ofelectrodes 954 having an amplitude and duration sufficient to release a desired amount of intracellular material from cells within the microdroplet. - Intracellular material present in lysed microdroplet is accessible to further process steps. For example, DNA and/or RNA released from cells is accessible for amplification by a polymerase chain reaction. As used herein, the term lysing does not require that the cells be completely ruptured. Rather, lysing refers to the release of intracellular material. For example, rather than rupturing the cells, the electric field may increase the porosity of cell membranes by an amount that allows release of intracellular material without permanent rupture of the membranes.
- Other lysing mechanisms may also be employed to release intracellular material from cells. For example, material may be released by subjecting cells to other forces including for example osmotic shock or pressure. Chemicals, selected from the group of surfactants, solvents, and antibiotics may be contacted with the cells. Mechanical shear methods may also be used to release intracellular materials.
- The lysed microdroplet may be moved downstream to mixing
module 160 for further processing. To move lysed microdroplet downstream,valve 216, which is disposed upstream of lysing zone 950, is closed.Valve 204 is also closed to prevent gas from exiting lysing zone 950 via vent.Actuator 170 is then actuated, as described above, to provide a gas pressure sufficient to move lysed microdroplet downstream of lysing zone 950. - In an alternative embodiment, a
lysing module 300, as shown inFigs. 13a, 13b , includes a lysingzone 302 which is configured to prepare a lysedmicrodroplet 304 of predetermined volume from amicrofluidic sample 306, which may have an indeterminate volume.Lysing zone 302 preferably includes a lysing mechanism such aselectrodes 308. Electrical leads 310 provide a connection to a pulse circuit ofDAQ 126, viacontacts 112,chip carrier 120, andcontacts 125. Apositioning element 312 is disposed downstream of lysingzone 302. Anactuator 314 is disposed upstream from lysing zone.Actuator 314 preferably includes asecond positioning element 316 to prevent fluid from the microfluidic sample from entering therein. -
Lysing zone 302 operates as follows. Themicrofluidic sample 306 enters lysingzone 302 and moves downstream until adownstream interface 316 of themicrofluidic sample 306encounters positioning element 312. Thepositioning element 312 preferably increases a surface tension of the downstream interface of themicrofluidic sample 306, thereby inhibiting further downstream movement and positioning a portion of the microfluidic sample in a lysing position with respect toelectrodes 308. The lysing position is defined as the location of the portion of the microfluidic sample disposed downstream ofactuator 314 and upstream ofpositioning element 312. Preferably,actuator 314 andpositioning element 312 are disposedadjacent electrodes 308 such that substantially all of the material present in the lysing position is subjected to the electric field upon actuatingelectrodes 308. - Actuation of
electrodes 308 in the embodiment described above, provides an electrical field sufficient to release intracellular material from cells present in the portion of the microfluidic sample in the lysing position. Once a sufficient amount of intracellular material has been released,actuator 314 is actuated to prepare lysedmicrodroplet 304 from themicrofluidic sample 306.Actuator 314 preferably provides a gas pressure sufficient to move the lysedmicrodroplet 304 to a downstream portion of a microfluidic device such asmixing module 166. - Referring back to
Fig. 4 , a lysed sample prepared by lysingmodule 160 is received by mixingmodule 166. Mixingmodule 166 includes amixing zone 958. In this zone, the lysed cell sample is contacted, such as by mixing, with an amount of reagent received from thereagent source module 152.Reagent source module 152 includes a reagent microdroplet preparation zone (RMPZ) 434, which preferably operates to prepare a microdroplet having a predetermined volume of reagent. -
Reagent input module 152 is essentially the same asmicrodroplet formation module 158, however, it is specifically designed for formation of a microdroplet of reagent having a predetermined volume which will yield a desired ratio of reagent to sample when mixed with the microdroplet fromcell lysing module 160.Module 152 includes an input port 420, avalve 422, and anactuator 172, each of which joins areagent source channel 428. Anoverflow channel 424, which also joinsreagents source channel 428, may also be provided.Actuator 172 may include asecond positioning element 432 to prevent liquid from entering therein. - Reagent materials, which preferably comprise at least one liquid, are introduced via input port 420, such as with a pipette or syringe. Examples of suitable reagent materials include substances to facilitate further processing of the lysed cell sample, such as enzymes and other materials for amplifying DNA therein by polymerase chain reaction (PCR). The reagent material moves downstream within
reagent source channel 428 until a downstream portion of the reagent material contacts apositioning element 426. Any additional reagent material that continues to be received within reagent source module preferably entersoverflow channel 424. When the introduction of reagent is complete,valve 422 is closed to prevent reagent from exiting reagent source channel via reagent source port 420. - Mixing
zone 958 of the mixing module includes adjoined first andsecond channels 410, 412. Materials moving downstream toward mixingzone 958 contact one another and preferably mix therein. Because of the micro-scale dimensions of mixingzone 958, the sample and reagent materials preferably mix by diffusion even in the absence of other sources of mass transport, such as mechanical agitation. It should be understood however, that agitation forces, such as acoustic waves may be applied to enhance mixing within mixingzone 958. -
Reagent source module 152 and mixingmodule 166 preferably operate as follows. When a lysed sample from lysing zone 950 is ready to be mixed with reagent material,actuator 172 is actuated to prepare a microdroplet of reagent. The microdroplet of reagent is prepared from the portion of reagent material downstream of an opening 430 ofactuator 172 and upstream of positioning element 427. Thus, assuming that the dimensions of thereagent source channel 428 are constant, the volume of the microdroplet of reagent is determined by the distance between thepositioning element 426 and the actuator opening 430. - The microdroplet of reagent moves downstream toward channel 412 of reagent mixing zone. Meanwhile, a sample of lysed material, such as a lysed microdroplet, is moved downstream from lysing zone 950 toward
channel 410 of mixingzone 958.Actuator 170 may provide the motive force to move the lysed microdroplet downstream. Alternatively, as discussed above, another actuator may be disposed upstream of lysing zone 950 but downstream ofactuator 170 to provide the necessary motive force. - The sample and reagent material enter a
downstream channel 438 of mixingzone 958, where the materials contact and mix. Because both the lysed sample and reagent material are mixed in the form of microdroplets, mixingzone 958 prepares an amount of mixed material having a predetermined ratio of sample to reagent. The volumes of microdroplets prepared withinmicrofluidic device 110 are preferably independent of physical properties, such as viscosity, electrical conductivity, and osmotic strength, of the microdroplets. Thus, mixingzone 958 prepares an amount of mixed material having a sample to reagent material that is also independent of the physical and chemical properties of the mixed materials. Avent 440, which is downstream of the various zones of the microfluidic device 110 ensures that downstream pressure buildup does not inhibit downstream movement of samples withinmicrofluidic device 110. - The mixed lysed cell sample and reagent are received within a
DNA manipulation zone 971 ofDNA manipulation module 162.Module 162 can perform, for example, restriction, digestion, ligation, hybridization and amplification of DNA material. In one embodiment,DNA manipulation zone 971 is configured to perform PCR amplification of nucleic acids present within the lysed cell sample.Vent 440 prevents pressure from increasing withinzone 971 as the lysed cell sample and reagent are being introduced thereto. Valves 972 and 973 ofDNA manipulation module 162 may be closed to prevent substances therein zone from exiting, such as by evaporation, during PCR amplification. The DNA manipulation zone is configured with heat sources under control ofcomputer 127 to allow thermal cycling of DNA manipulation zone during amplification, as understood by one of skill in the art. - System 901 includes also includes a detector 981 to detect the presence of amplified polynucleotides produced by PCR. Detector 981 is preferably an optical detector in optical communication, such as by a fiber optic 981, with
zone 971. A light source, such as a laser diode, introduces light toDNA Manipulation zone 971 to generate fluorescence indicative of the amount of amplified polynucleotides present therein. The fluorescence arises from fluorescent tags, included in the reagent and associated with the polynucleotides upon amplification. - Preferred positioning elements are discussed below.
- A
positioning element 979 may be formed by a non-wetting material disposed to contact a microfluidic sample. The physio-chemical properties of the non-wetting material are chosen upon considering the type of liquid forming the microfluidic sample. For example, where the microfluidic sample is an aqueous sample, the positioning element preferably comprises a hydrophobic material. An exemplary hydrophobic material includes a non-polar organic compound, such as an aliphatic silane, which can be formed by modifying an internal surface of microfluidic device 901. For microfluidic samples formed of organic solvents, the non-wetting material may comprise a hydrophilic material. - When
microfluidic sample 808encounters positioning element 979, the liquid of the microfluidic sample experiences an increased surface tension atdownstream interface 810, which increased surface tension inhibits continued downstream motion ofmicrofluidic sample 808. Increasing the gas pressure difference between upstream and downstream portions of the microfluidic sample overcomes the resistance and moves the microfluidic sample downstream. - Referring to
Figs. 10a-10c , another type of positioning element may be formed by modifying the dimensions of the microfluidic channel to form a capillary assisted positioning element (CAFB) 700. A CAFB comprises anupstream feed zone 702, aloading zone 704, and astop zone 704. Amicrofluidic sample 720 encountering the CAFB moves downstream until adownstream interface 710 of the microfluidic sample contactsupstream surfaces 714 of theloading zone 706. At this point, capillary action causes the microfluidic sample to move downstream until thedownstream sample interface 710 encounters theopening 712 between theloading zone 704 and thestop zone 706. Surface tension resists the tendency of the microfluidic sample to continue downstreampast opening 714. Thus, themicrofluidic sample 720 is positioned at a predetermined location along the channel axis with respect topositioning element 700. - The volume of the microfluidic sample encountering the CAFB preferably has a larger volume than a volume of the
loading zone 704 to ensure that the microfluidic sample will advance fully to opening. For fluids that have similar surface tensions and interface properties as water, the depth d1 of theloading zone 704 is preferably about 50% or less of the respective depths d2, d3 of the stop and feed zones. - The tendency of a microfluidic sample to move in a given direction is governed by the ratio between the mean radius of curvature (MRC) of the front of the microfluidic sample and the MRC of the back of the microfluidic sample. These curvatures depend upon the contact angle of the fluid of the sample and the dimensions of the zone in which the microdroplet is moving. A MRC r1 of a microdroplet interface in the loading zone is preferably smaller than a MRC r2 of a droplet interface within the feed zone or a MRC r3 of a droplet interface within the stop zone. The MRC r2 is preferably larger than the MRC r3. Thus, the radius of curvature of the downstream microdroplet interface increases upon encountering the stop zone thereby inhibiting further downstream movement. Preferably, the contact angle of the fluid with the wall is substantially constant throughout the capillary assisted loading zone.
- Referring to
Figs. 11a-11c , apositioning element 500 operates to position amicrofluidic sample 502 by reducing the gas pressure acting upon anupstream portion 504 of the microfluidic sample relative to the gas pressure acting upon adownstream portion 506 of the microfluidic sample.Positioning element 500 includes avent 508 disposed in gaseous communication with azone 510 along whichmicrofluidic sample 502 moves. Vent 508 preferably communicates withzone 510 via a passage 526. The zone may be for example, a channel or conduit.Positioning element 500 may also include asecond positioning element 516, such as a non-wetting material, to substantially prevent fluid from the microfluidic sample from contacting the vent. - An open state of a
valve 512 allows passage of gas betweenzone 510 and vent 508. A closed state ofvalve 512 prevents such passage of gas.Valve 514 is preferably thermally actuated and includes amass 514 of TRS. - An
actuator 518 is disposed upstream ofpositioning element 500.Actuator 518 is preferably a gas actuator and may include aheat source 520 to heat a gas associated withactuator 518.Actuator 518 may include apositioning element 522, such as non-wetting material, to substantially prevent fluid from the microfluidic sample from entering therein. -
Positioning element 500 preferably operates as follows. Referring toFig. 11a ,microfluidic sample 502 moves downstream in the direction ofarrow 524. Microfluidic sample is preferably moved by a gas pressure provided from an upstream actuator, which is not shown inFigs. 9a-9c . The gas pressure acts uponupstream portion 504. - Referring to
Fig. 11b , whenupstream portion 504 passes the opening ofvent 508, the upstream gas dissipates throughvent 508, thereby reducing the upstream pressure. The pressure reduction, which preferably equalizes the downstream and upstream pressures, reduces or eliminates the motive force tending to urge the microfluidic sample downstream. - Referring to
Fig. 11c ,valve 512 is closed to prevent passage of gas betweenzone 510 and vent 508. Preferably,TRS 514 moves into passage 526. Upon closingvalve 512, the actuation ofactuator 518 provides a motive force to movemicrofluidic sample 502 downstream in the direction ofarrow 528 for further processing. - Referring to
Figs. 15a-15c , amicrodroplet preparation module 652 has amicrodroplet preparation zone 650, an activefluid positioning element 654, anactuator 656, and avalve 658. Asecond actuator 660 is operatively associated with theactive positioning element 654 to introduce amicrofluidic sample 666 to themicrodroplet preparation zone 650.Second actuator 660 is preferably located upstream fromvalve 658.Microdroplet preparation module 652 prepares amicrodroplet 668, which has a predetermined volume from themicrofluidic sample 666 received therein. - In operation,
microfluidic preparation module 652 receives themicrofluidic sample 666, which moves downstream because of a motive force provided by thesecond actuator 660 . The motive force is preferably an upstream gas pressure, which is greater than a downstream gas pressure acting upon themicrofluidic sample 666. The microfluidic sample moves downstream until adownstream portion 670 thereof encountersactive positioning element 654, which preferably comprises asensor 672 having electrical leads 674. The leads 674 are in electrical communication with I/O pins of the microfluidic device to allow signals fromsensor 672 to be received by a DAQ. -
Sensing element 672 is preferably a pair of electrical contacts. To sense the presence of the liquid,DAQ 126 applies a small voltage across leads 674 and measures the resultant current. As the liquid of the microfluidic sample contacts the first and second contacts, the current passing therebetween changes, thereby indicating to DAQ 126 that the liquid has arrived atsensor 672. - Upon recognition that the liquid has arrived at
sensor 672, the DAQ instructssecond actuator 660 to decrease a downstream motive force acting upon themicrofluidic sample 666. For example, DAQ may reduce a current flowing through aheat source 676 associated withsecond actuator 660 thereby reducing a temperature of a gas therein. The temperature reduction reduces the gas pressure acting upon aupstream portion 678 of microfluidic sample thereby inhibiting the downstream motion of themicrofluidic sample 666. The microfluidic sample is positioned such that afirst portion 680 is located downstream ofactuator 656 and a second portion 682 is located upstream ofactuator 656. - To prepare
microdroplet 668,DAQ 126 actuates actuator to provide a motive force which prepares themicrodroplet 668 from thefirst portion 680 ofmicrofluidic sample 666.Microdroplet 668 moves downstream while the second portion 682 of themicrofluidic sample 666 moves upstream fromactuator 656. During microdroplet preparation,valve 658 may be closed to substantially isolate the actuator 656 fromsecond actuator 660 and other upstream portions of the microfluidic device. - The active positioning element preferably operates as a closed loop element that provides feedback from
sensor 672 to the DAQ. The feedback is indicated when a microfluidic sample has reached a predetermined position within the microfluidic device. Upon receiving the feedback, the DAQ changes the state of the actuator providing the motive force to move the microdroplet. - While the above invention has been described with reference to certain preferred embodiments, it should be kept in mind that the scope of the present invention is not limited to these. Thus, one skilled in the art may find variations of these preferred embodiments which, nevertheless, fall within the scope of the claims.
Claims (9)
- A microfluidic device (110) for processing a cell-containing microfluidic sample, comprising:a lysing module (300) configured to receive a cell-containing microfluidic sample, wherein the lysing module comprises a lysing zone (302), wherein the microfluidic sample is a liquid;a positioning element (312) configured to inhibit downstream movement of the microfluidic sample to position the microfluidic sample in a lysing position;a lysing mechanism (308) within the lysing zone (302), to release intracellular contents from cells within the lysing zone; anda gas actuator (314) disposed upstream from the positioning element (312) and the lysing zone (302) such that a first portion of the microfluidic sample is disposed upstream from the gas actuator (314) and a second portion of the microfluidic sample is disposed downstream from the gas actuator (314) and upstream of the positioning element (312), the gas actuator (314) configured to provide a gas pressure sufficient to prepare a microdroplet (304) having a predetermined volume comprising intracellular contents released from cells of the cell-containing microfluidic sample within the lysing zone (302) by separating the second portion of the microfluidic sample from the first portion of the microfluidic sample and moving the second portion of the microfluidic sample to a location downstream of the lysing mechanism (308) and the positioning element (312).
- The microfluidic device (110) of claim 1, wherein the device comprises a substrate (130,132), and wherein the lysing mechanism (308) and gas actuator (314) are integral with the substrate.
- The microfluidic device (110) of claim 1 or claim 2, wherein the gas actuator (314) comprises a heat source to heat an amount of gas thereby increasing a pressure of the gas.
- The microfluidic device (110) of any one of the preceding claims, wherein the positioning element (312) comprises a vent to substantially equalize a gas pressure upstream of the cell-containing microfluidic sample with a gas pressure downstream of the cell-containing microfluidic sample when the cell-containing microfluidic sample is in the lysing position to thereby inhibit downstream movement of the cell-containing microfluidic sample downstream from the lysing position.
- A microfluidic method for processing a cell-containing microfluidic sample, comprising:positioning, using a positioning element (312), the cell-containing microfluidic sample in a lysing position with respect to a lysing mechanism (308) of a lysing zone (302) of a lysing module (300) of a microfluidic device (110), the cell-containing microfluidic sample comprising a cell-containing liquid;actuating the lysing mechanism (308) to release intracellular material from cells of the cell-containing microfluidic sample;actuating a gas actuator (314) disposed upstream of the positioning element (312) and the lysing zone (302) to provide a gas pressure sufficient to separate a portion of the cell-containing microfluidic sample that is downstream of the gas actuator (314) and upstream of the positioning element (312) from a portion of the cell-containing microfluidic sample that is upstream of the gas actuator (314) and move the downstream portion of the cell-containing microfluidic sample to a location downstream of the lysing mechanism (308) and the positioning element (312), the downstream portion of the microfluidic sample being a microdroplet (304) comprising intracellular material released from cells of the cell containing microfluidic sample and having a predetermined volume.
- The microfluidic method of claim 5, wherein the positioning step comprises contacting the downstream surface of the microfluidic sample with a hydrophobic material.
- The microfluidic method of any of claims 5 or 6, wherein the positioning step comprises increasing a radius of curvature of the microfluidic sample.
- The microfluidic method of any of claims 5 to 7, wherein the positioning step comprises substantially equalizing a gas pressure upstream of the microfluidic sample with a gas pressure downstream of the microfluidic sample.
- The microfluidic method of any one of claims 5 to 8, wherein the second portion of the microfluidic sample comprises less than about 90 percent of the cell-containing microfluidic sample.
Priority Applications (1)
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EP18185265.8A EP3427834A1 (en) | 2001-07-26 | 2002-03-27 | Methods and systems for microfluidic processing |
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US14519 | 1998-01-28 | ||
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US307638P | 2001-07-26 | ||
US10/014,520 US7270786B2 (en) | 2001-03-28 | 2001-12-14 | Methods and systems for processing microfluidic samples of particle containing fluids |
US14520 | 2001-12-14 | ||
US10/014,519 US7192557B2 (en) | 2001-03-28 | 2001-12-14 | Methods and systems for releasing intracellular material from cells within microfluidic samples of fluids |
US75371 | 2002-02-15 | ||
US10/075,371 US7323140B2 (en) | 2001-03-28 | 2002-02-15 | Moving microdroplets in a microfluidic device |
PCT/US2002/009440 WO2003012406A1 (en) | 2001-07-26 | 2002-03-27 | Methods and systems for microfluidic processing |
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EP18185265.8A Division EP3427834A1 (en) | 2001-07-26 | 2002-03-27 | Methods and systems for microfluidic processing |
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EP1438567A1 EP1438567A1 (en) | 2004-07-21 |
EP1438567A4 EP1438567A4 (en) | 2010-06-02 |
EP1438567B1 true EP1438567B1 (en) | 2018-07-25 |
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EP18185265.8A Withdrawn EP3427834A1 (en) | 2001-07-26 | 2002-03-27 | Methods and systems for microfluidic processing |
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EP18185265.8A Withdrawn EP3427834A1 (en) | 2001-07-26 | 2002-03-27 | Methods and systems for microfluidic processing |
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JP (1) | JP4596776B2 (en) |
ES (1) | ES2683698T3 (en) |
WO (1) | WO2003012406A1 (en) |
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US6048734A (en) | 1995-09-15 | 2000-04-11 | The Regents Of The University Of Michigan | Thermal microvalves in a fluid flow method |
US6692700B2 (en) | 2001-02-14 | 2004-02-17 | Handylab, Inc. | Heat-reduction methods and systems related to microfluidic devices |
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US7323140B2 (en) | 2001-03-28 | 2008-01-29 | Handylab, Inc. | Moving microdroplets in a microfluidic device |
US7010391B2 (en) | 2001-03-28 | 2006-03-07 | Handylab, Inc. | Methods and systems for control of microfluidic devices |
US7829025B2 (en) | 2001-03-28 | 2010-11-09 | Venture Lending & Leasing Iv, Inc. | Systems and methods for thermal actuation of microfluidic devices |
US8895311B1 (en) | 2001-03-28 | 2014-11-25 | Handylab, Inc. | Methods and systems for control of general purpose microfluidic devices |
US7731906B2 (en) | 2003-07-31 | 2010-06-08 | Handylab, Inc. | Processing particle-containing samples |
US7059352B2 (en) | 2004-03-31 | 2006-06-13 | Lifescan Scotland | Triggerable passive valve for use in controlling the flow of fluid |
US7156117B2 (en) | 2004-03-31 | 2007-01-02 | Lifescan Scotland Limited | Method of controlling the movement of fluid through a microfluidic circuit using an array of triggerable passive valves |
US7665303B2 (en) | 2004-03-31 | 2010-02-23 | Lifescan Scotland, Ltd. | Method of segregating a bolus of fluid using a pneumatic actuator in a fluid handling circuit |
CA3198754A1 (en) | 2004-05-03 | 2005-11-17 | Handylab, Inc. | A microfluidic device and methods for processing polynucleotide-containing samples |
US8852862B2 (en) | 2004-05-03 | 2014-10-07 | Handylab, Inc. | Method for processing polynucleotide-containing samples |
US10900066B2 (en) | 2006-03-24 | 2021-01-26 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US11806718B2 (en) | 2006-03-24 | 2023-11-07 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US7998708B2 (en) | 2006-03-24 | 2011-08-16 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US8883490B2 (en) | 2006-03-24 | 2014-11-11 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
JP5415253B2 (en) | 2006-03-24 | 2014-02-12 | ハンディラブ・インコーポレーテッド | Integrated system for processing microfluidic samples and methods of use thereof |
US8765076B2 (en) | 2006-11-14 | 2014-07-01 | Handylab, Inc. | Microfluidic valve and method of making same |
US8105783B2 (en) | 2007-07-13 | 2012-01-31 | Handylab, Inc. | Microfluidic cartridge |
US9186677B2 (en) | 2007-07-13 | 2015-11-17 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US9618139B2 (en) | 2007-07-13 | 2017-04-11 | Handylab, Inc. | Integrated heater and magnetic separator |
US8182763B2 (en) | 2007-07-13 | 2012-05-22 | Handylab, Inc. | Rack for sample tubes and reagent holders |
US8287820B2 (en) | 2007-07-13 | 2012-10-16 | Handylab, Inc. | Automated pipetting apparatus having a combined liquid pump and pipette head system |
US20090136385A1 (en) | 2007-07-13 | 2009-05-28 | Handylab, Inc. | Reagent Tube |
CA2693654C (en) | 2007-07-13 | 2018-02-13 | Handylab, Inc. | Polynucleotide capture materials, and methods of using same |
US8133671B2 (en) | 2007-07-13 | 2012-03-13 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
USD787087S1 (en) | 2008-07-14 | 2017-05-16 | Handylab, Inc. | Housing |
BR112013026451B1 (en) | 2011-04-15 | 2021-02-09 | Becton, Dickinson And Company | system and method to perform molecular diagnostic tests on several samples in parallel and simultaneously amplification in real time in plurality of amplification reaction chambers |
ES2645966T3 (en) | 2011-09-30 | 2017-12-11 | Becton, Dickinson And Company | Unified test strip |
USD692162S1 (en) | 2011-09-30 | 2013-10-22 | Becton, Dickinson And Company | Single piece reagent holder |
WO2013067202A1 (en) | 2011-11-04 | 2013-05-10 | Handylab, Inc. | Polynucleotide sample preparation device |
BR112014018995B1 (en) | 2012-02-03 | 2021-01-19 | Becton, Dickson And Company | systems to perform automated testing |
US11666910B2 (en) | 2018-11-26 | 2023-06-06 | Hewlett-Packard Development Company, L.P. | Microfluidic devices |
CN112023990B (en) * | 2019-06-03 | 2023-06-23 | 利多(香港)有限公司 | Microfluidic detection chip and manufacturing method |
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US5304487A (en) * | 1992-05-01 | 1994-04-19 | Trustees Of The University Of Pennsylvania | Fluid handling in mesoscale analytical devices |
US5639423A (en) * | 1992-08-31 | 1997-06-17 | The Regents Of The University Of Calfornia | Microfabricated reactor |
US5856174A (en) * | 1995-06-29 | 1999-01-05 | Affymetrix, Inc. | Integrated nucleic acid diagnostic device |
US6168948B1 (en) * | 1995-06-29 | 2001-01-02 | Affymetrix, Inc. | Miniaturized genetic analysis systems and methods |
US5863502A (en) * | 1996-01-24 | 1999-01-26 | Sarnoff Corporation | Parallel reaction cassette and associated devices |
JPH10108666A (en) * | 1996-10-04 | 1998-04-28 | Asahi Medical Co Ltd | Hollow fiber type incubator |
US6379929B1 (en) * | 1996-11-20 | 2002-04-30 | The Regents Of The University Of Michigan | Chip-based isothermal amplification devices and methods |
DE69934449T2 (en) * | 1998-03-12 | 2007-09-27 | Miltenyi Biotech Gmbh | Microcolumn system for magnetic separation |
JP4074713B2 (en) * | 1998-07-29 | 2008-04-09 | 財団法人川村理化学研究所 | Liquid feeding device and manufacturing method thereof |
JP2000166535A (en) * | 1998-12-10 | 2000-06-20 | Nippon Laser Denshi Kk | Divided microinjector |
DE19948473A1 (en) * | 1999-10-08 | 2001-04-12 | Nmi Univ Tuebingen | Method and device for measuring cells in a liquid environment |
US6272939B1 (en) * | 1999-10-15 | 2001-08-14 | Applera Corporation | System and method for filling a substrate with a liquid sample |
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2002
- 2002-03-27 WO PCT/US2002/009440 patent/WO2003012406A1/en active Application Filing
- 2002-03-27 EP EP02715213.1A patent/EP1438567B1/en not_active Expired - Lifetime
- 2002-03-27 ES ES02715213.1T patent/ES2683698T3/en not_active Expired - Lifetime
- 2002-03-27 EP EP18185265.8A patent/EP3427834A1/en not_active Withdrawn
- 2002-03-27 JP JP2003517550A patent/JP4596776B2/en not_active Expired - Lifetime
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US6130098A (en) * | 1995-09-15 | 2000-10-10 | The Regents Of The University Of Michigan | Moving microdroplets |
Also Published As
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WO2003012406A1 (en) | 2003-02-13 |
EP1438567A4 (en) | 2010-06-02 |
WO2003012406A9 (en) | 2003-03-20 |
EP1438567A1 (en) | 2004-07-21 |
JP4596776B2 (en) | 2010-12-15 |
EP3427834A1 (en) | 2019-01-16 |
JP2005514718A (en) | 2005-05-19 |
ES2683698T3 (en) | 2018-09-27 |
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