ES2683698T3 - Methods and systems for microfluidic processing - Google Patents

Abstract

A microfluidic device (110) for processing a microfluidic sample containing cells, comprising: a lysis module (300) configured to receive a microfluidic sample containing cells, wherein the lysis module comprises a lysis zone (302) , in which the microfluidic sample is a liquid; a positioning element (312) configured to inhibit the downstream movement of the microfluidic sample to position the microfluidic sample in a lysis position; a lysis mechanism (308) within the lysis zone (302), to release intracellular content from cells within the lysis zone; and a gas actuator (314) disposed upstream of the positioning element (312) and the lysis zone (302) so that a premium part of the microfluidic sample is disposed upstream of the gas actuator (314) and a second part of the microfluidic sample is disposed downstream of the gas actuator (314) and upstream of the positioning element (312), the gas actuator (314) configured to provide sufficient gas pressure to prepare a micro drop (304) that has a predetermined volume comprising intracellular content released from cells of the microfluidic sample containing cells within the lysis zone (302) by separating the second part of the microfluidic sample from the first part of the microfluidic sample and moving the second part of the microfluidic sample at a location downstream of the lysis mechanism (308) and the positioning element (312).

Description

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DESCRIPTION

Methods and systems for microfilm processing Field of the invention

The present invention relates to methods and systems for processing samples through the use of microfluidic systems.

Background

Microfluidic devices are normally formed of substrates (made of silicon, glass, ceramic, plastic and / or quartz) that include a network of microchannels through which the fluid flows with the control of a propulsion mechanism. The microchannels normally have at least one dimension that is of the order of nanometers to hundreds of micrometers.

Micro-medical devices offer several advantages over traditional macro-scale instrumentation. For example, in general, they require substantially smaller fluid samples, use much less reagent and process these fluids at substantially higher speeds than macro-scale equipment. A microfilm device can use only minimal amounts of sample to determine chemical and physical properties of the sample, such as subjecting the sample to fluid processing.

In many cases, the accuracy of such fluid processing depends on the relative sample amounts and reagent used. For example, when a sample is analyzed for its "genetic fingerprint" of DNA, the results may depend on the concentration of reagents used to increase the DNA present in the sample. Thus, if an inadequate sample ratio with respect to reagent is used, the result may be inaccurate. Since microflmedic devices process samples and reagents in minute quantities, even the slightest uncertainty in the amount of reagent or sample used can present an uncertainty in the results of a microflmedic analysis.

Variations in the amount of samples and reagents processed by a microflmed device may originate from various sources. For example, some microfluidic devices manipulate continuous streams that flow from liquid. Changes in the viscosity of the liquid may 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 microfilm device. Dilution of the sample can occur when a liquid flow stream is used to move components of the sample from one location to another within a microfilm device.

The analysis of cell micro fluids within body fluids is especially problematic due to the relatively small amount of cells available for analysis and the inherent difficulty in handling such materials.

Sample manipulation may involve the movement of a sample between different locations within a microfilm device. For example, electric fields are used as a propulsion mechanism for some microfluidic devices. In such devices, a high voltage, of the order of kilovolts, is applied through electrodes within the device to thereby generate an electric field in the microchannels. The field imposes an ion force within the fluid, thus propelling the ions through the microchannel. The fluid itself can also be propelled by the movement of ions that move within the fluid.

Gas pressure is also used to propel fluid through the microchannels. In some devices, a source of pressurized gas, external to the micro-chemical device, is connected to the micro-medical device to supply a gas pressure, which propels the fluid. The gas pressure can also be generated by a heated chamber inside the microfilm device itself to propagate fluid within a microchannel.

WO98 / 22625 A1 discloses microfabricated isothermal nucleic acid amplification devices and methods. US 6,130,098 discloses micro-scale devices that allow the movement and mixing of microdroplets through microchannels.

Summary of the invention

The scope of protection is defined in the independent claims, to which reference should now be made. Advantageous features are set forth in the dependent claims.

In accordance with one aspect of the claimed invention, a micro-medical device is provided for processing a micro-medical sample containing cells, comprising: a lysis module configured to receive a micro-medical sample containing cells, in which the lysis module comprises a lysis zone, in which the microflmedic sample is a liquid; a positioning element configured to inhibit the current movement

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below the microflmedic sample to position the microflmedical sample in a lysis position; a lysis mechanism within the lysis zone, to release intracellular content from cells within the lysis zone; and a gas actuator disposed upstream of the positioning element and the lysis zone so that a first part of the micro-chemical sample is arranged upstream of the gas actuator and a second part of the micro-medical sample is disposed downstream of the actuator of gas and upstream of the positioning element, the gas actuator configured to provide a sufficient gas pressure to prepare a micro drop having a predetermined volume comprising intracellular content released from cells of the microflmedical sample containing cells within the zone of lysis by separating the second part of the micro-medical sample from the first part of the micro-medical sample and moving the second part of the micro-medical sample to a location downstream of the lysis mechanism and the positioning element.

In accordance with another aspect of the claimed invention, a micro-medical method is provided for processing a micro-medical sample containing cells, comprising: positioning, using a positioning element, of the micro-medical sample containing cells in a lysis position with respect to to a lysis mechanism of a lysis zone of a lysis module of a micro-medical device, the micro-medical sample containing cells comprising a liquid containing cells; actuation of the lysis mechanism to release intracellular material from cells of the microflmedic sample containing cells; the actuation of a gas actuator disposed upstream of the positioning element and the lysis zone to provide a sufficient gas pressure to separate a part of the microflmed sample containing cells that are downstream of the gas actuator and upstream of the positioning element from a part of the microflmedic sample containing cells that is upstream of the gas actuator and moving the downstream part of the microflmedic sample containing cells to a location downstream of the lysis mechanism and the positioning element, the downstream part of the microflmedic sample being a droplet comprising intracellular material released from cells of the microflmedic sample containing cells 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 refers to the use of a plurality of gas actuators to apply pressure at different locations within the microfilm system to thereby supply force to move samples. For example, in one embodiment, a first gas actuator provides sufficient gas pressure to move a first sample from a first location to a second location of the microfilm device. A second gas actuator provides a gas pressure to move another sample from a third location to a fourth location of the microfilm device.

In another example, a plurality of gas actuators cooperate to move the same fluid sample. A first gas actuator provides sufficient gas pressure to move the micro drop between the first and second processing zone of the microfilm device and, a second gas actuator provides a gas pressure to move the micro drop to a third processing zone.

In preferred embodiments, the plurality of actuators are integral with a micro-medical network through which the micro-chemical samples flow. For example, a plurality of gas actuators can be manufactured on the same substrate that forms the microfilm network. One such gas actuator engages the network at a first location to provide gas pressure to move a microflmed sample into the network. Another gas actuator engages the network at a second location to provide gas pressure to additionally move at least a portion of the micro-chemical sample within the network.

In another aspect, the disclosure refers to the use of valves with the plurality of actuators. For example, in one embodiment, a valve is coupled to a micro-medical network so that, when the valve is closed, it substantially loosens the second gas actuator of 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 allowing it to expand in the desired direction. They also extend the range over which an actuator can propel a micro drop, preventing the gas from dissipating in certain areas upstream of the micro drop.

Another aspect of the present disclosure relates to a microfluidic system and method for processing a fluid containing particles, such as, for example, a liquid containing bacterial cells or human cells.

In one aspect, the disclosure relates to the preparation of a sample of particles enriched from the fluid containing particles. For example, a microfluidic system for preparing an enriched sample includes an enrichment zone and a flow circulation member arranged in fluid communication with the enrichment zone. The flow circulating member allows the flow of the fluid containing particles to pass through and out of the enrichment zone, while causing the particles of the fluid containing particles to accumulate within the zone, thereby preparing, a sample of enriched particles in the enrichment zone. The flow circulating member may include a plurality of vias, such as pores,

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that have a sufficient size to allow the passage of the fluid through them but of a size sufficiently small to allow the passage of particles through them. Suitable flow circulation members are composed of, for example, filter elements, such as filter paper, porous glasses and porous gels.

The microfilm system may include an actuator that moves the enriched particle sample from the enrichment zone without essentially dissolving 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 the gas pressure associated with the downstream channel. For example, the gas actuator may include a heat source in thermal contact with a volume of gas. The expansion of the gas when the heat source is operated creates a sufficient gas pressure to move the enriched particle sample from the enrichment zone to another location within the microfilm system, such as a location containing modules for additional fluid processing.

In another aspect, the disclosure refers to a microfluidic system and method for processing an enriched particle sample that includes cells trapped in a liquid. For example, the system includes a lysis zone to receive the sample containing enriched cells and a positioning element to position the sample containing enriched cells in a lysis position in the vicinity of a lysis mechanism. The lysis mechanism releases intracellular material, such as DNA or RNA, from the cells. In one embodiment, the lysis mechanism includes electrodes to generate a sufficient electric field to release intracellular content from the cells. Alternatively, the lysis mechanism can lyse the cells using chemical, thermal and / or ultrasonic techniques or any combination of these techniques.

In one embodiment, the lysis zone releases intracellular content from cells of the fluid containing cells and then prepares from this fluid a micro drop containing intracellular content releasing cells. The micro drop is preferably prepared from only a part of the fluid containing cells. For example, a preferred micro drop includes less than about 90 percent of the cell-containing fluid. In another embodiment, the lysis zone receives a micro drop of the fluid containing cells and releases the intracellular content of the cells within the drop.

In another aspect, the disclosure relates to a micro-chemical substrate to process the intracellular content of cells suspended in fluids. The substrate includes an enrichment zone, a lysis module, a micro drop formation module, mixing module and an amplification module. The enrichment zone that prepares a sample of enriched particles from the fluid that contains cells. The lysis module, which is coupled to the enrichment zone to receive the enriched particle sample, releases intracellular material from cells within the sample to thereby form a lysed sample. The micro-droplet formation module thus forms a first micro-drop of fluid from the lysed sample and sends it to a mixing module for mixing with a reagent micro-drop. The amplification module amplifies intracellular material within the micro drop formed from the mixture.

Another aspect of the disclosure relates to a microfluidic system and method for processing a fluid containing cells, such as, for example, a liquid containing bacterial cells or human cells. For example, the system includes a lysis zone to receive the sample containing cells and a positioning element to position the sample containing cells in a lysis position in the vicinity of a lysis mechanism. The lysis mechanism releases intracellular material, such as DNA or RNA, from the cells. In one embodiment, the lysis mechanism includes electrodes to generate a sufficient electric field to release intracellular content from the cells. Alternatively, the lysis mechanism can lyse the cells using chemical, thermal and / or ultrasonic techniques or any combination of these techniques.

In one embodiment, the lysis zone releases intracellular content from cells of the fluid containing cells and then prepares from this fluid a micro drop containing intracellular content releasing cells. The micro drop is preferably prepared from only a part of the fluid containing cells. For example, a preferred micro drop includes less than about 90 percent of the cell-containing fluid. In another embodiment, the lysis zone receives a micro drop of the fluid containing cells and releases the intracellular content of the cells within the drop.

The positioning elements help to place the sample of fluid containing cells in the vicinity of the lysis mechanism so that the lysis mechanism can release intracellular material from the cells. These elements preferably work differently from a valve, which could completely obstruct the passage of material between upstream and downstream locations adjacent to the valve. Instead, they normally provide resistance to fluid flow at a desired location (the lysis position) to thereby control the fluid location.

In one embodiment, the positioning element is disposed downstream of the lysis mechanism to position a part upstream of a sample containing cells (such as a micro drop) in the lysis position. The positioning element preferably increases a surface tension of a running surface

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below the sample containing cells to thereby inhibit the movement downstream of the sample. For example, the positioning element may include a reduced amount of wetting material, such as hydrophobic material, arranged to come into contact with a part of the surface downstream of the microbead containing cells.

In another embodiment, the positioning element is arranged upstream of the lysis zone to position a downstream part of the micro drop that contains cells in the lysis position. The positioning element includes a ventilation outlet, which substantially equals a gas pressure upstream of the microbead containing cells with a gas pressure downstream of the microbead containing cells to thereby stop the movement downstream of the micro drop that contains cells. When the micro drop is in the lysis position. A valve is preferably arranged to subsequently obstruct the gas passage between the lysis zone and the ventilation outlet to allow an upstream gas pressure to move the drop further downstream for further processing. For example, the microfilm system may include a mixing zone downstream of the enrichment zone and / or lysis zone, to mix the micro drop that emerges from these zones with a predetermined amount of reagent material.

In another aspect, the disclosure relates to a micro-chemical substrate to process the intracellular content of cells suspended in fluids. The substrate includes a lysis module, a micro drop formation module, mixing module and an amplification module. The lysis module releases intracellular material from cells within the sample to thereby form a lysed sample. The micro-droplet formation module thus forms a first micro-drop of fluid from the lysed sample and sends it to a mixing module for mixing with a reagent micro-drop. The amplification module amplifies intracellular material within the micro drop formed from the mixture.

Brief description of the drawings

The present invention is described below in relation to the following drawings, in which:

Fig. 1 shows a microfilm system according to the invention; Fig. 2 is an expanded view of a microflmed device.

Fig. 3 is a schematic of a micro-medical device of the micro-medical system of Fig. 1;

Fig. 4 shows a top view of the micro-medical device of Fig. 3;

Fig. 5 shows a partial cross-sectional view of the micro-medical device of Fig. 4;

Fig. 6 shows a partial cross-sectional view of an upper substrate of the micro-chemical device of

Fig. 2;

Fig. 7 shows a second partial cross-sectional view of an upper substrate of the microflmed device of Fig. 2;

Fig. 8a shows a top view of a micro-drop preparation area of the micro-medical device of Fig. 4 before the preparation of a micro-drop;

Fig. 8b shows a cross-sectional view of the micro-drop preparation area of Fig. 8a;

Fig. 9a shows a top view of a micro-drop preparation zone of the micro-chemical device of the

Fig. 4 after the preparation of a micro drop;

Fig. 9b shows a cross-sectional side view of the micro-drop preparation area of Fig. 9a; Fig. 10a-10c show cross-sectional views of an assisted capillary fluid barrier of the present invention;

Fig. 11a-11c show top views of a fluid barrier comprising a ventilation outlet; Fig. 12a-12b show top views of the lysis module of the microflmed device of Fig. 4, before and after the preparation of a lysed sample;

Figs. 13a and 13b show a second embodiment of a lysis module of the invention; Fig. 14 shows a pulse circuit associated with the lysis module of Fig. 4; and Fig. 15a and 15c show a second module of micro drop preparation of the invention.

Detailed description of a preferred embodiment

The present invention relates to micro-chemical 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 an embodiment described below, the fluid includes particles that tend to move with the fluid. The fluid component of the fluid containing particles is a gas or, preferably, a liquid. The particles of the fluid containing particles are preferably whole particles, such as bacterial cells or cells of an animal, such as a human. However, they can include intracellular material from such cells. For example, a system of the invention can be used to process a sample of bacterial cells to determine if the bacteria are pathogenic.

A. System overview

Fig. 1 depicts a microfilm system 100 that includes a microflm device 110 and cartridge

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corresponding 120, which receives one or more fluid samples and processes the samples with computer control 127 and data acquisition and control panel (DAQ) 126.

The computer 127 preferably performs high-level functions, such as providing a user interface that allows the user to select desired operations, notifying the DAQ 126 for the operations to select and showing the user the results of such operations. These operations include, for example, subjecting a sample to processing steps within various processing zones of the microfilm device. The computer 127 may be a portable computer to facilitate the transport of the micro-medical system.

Computer 127 is connected to DAQ 126 via connection 128, which provides I / O data, electrical connection, ground, restart and other functional connectivity. Alternatively, a wireless link 132 can be provided between the computer 127 and the DAQ 126 for data exchange and control signal through wireless elements 132 (a) and 132 (b). When the data link is a wireless link, for example, DAQ 126 may have a separate energy source, such as a baton.

In general, the DAQ 126 controls the operation of the microflmed device 110 according to the high level of instructions received from the computer 127. More specifically, to implement a desired operation required by the computer 127, the DAQ 126 supplies the control signals electrical fittings to cartridge 120 through contacts 125.

Cartridge 120 provides electrical and optical connections 121 for electrical and optical signals between DAQ 126 and microfilm substrate 110, thereby allowing DAQ 126 to control the operation of the substrate.

The chip carrier cartridge 120 is shown being inserted into (or removed from) a DAQ 126 interface hardware container having standardized electrical and optical contacts 125 to correspond with corresponding contacts 121 of the chip carrier cartridge 120. The mayonnaise of contacts are for electrical signals, while certain ones are for optical signals (IR, visible, UV, etc.) in the case of optically excited or optically controlled micro-chemical processors. Alternatively (not shown), the complete DAQ 126 may be a single ASIC chip that is incorporated into the chip carrier cartridge 120, in which the contacts 121,125 are converted into conductive vias on a printed circuit board.

B. Microfluidic device

Fig. 2 illustrates the general structural of a preferred type of microfilm device. The device includes an upper substrate 130, which is linked to a lower substrate 132 to form a fluid network.

The upper substrate 130 depicted in FIG. 2 is preferably formed of glass and has a microflmed net 134 on its lower surface 136. Those skilled in the art will recognize that substrates composed of silicon, glass, ceramic, plastic and / or quartz are all acceptable at the contact of the present invention. .

The microflmedic network includes a plurality of zone. The number of zones, as well as the complete topology of the micro-medical network, will depend on the particular application for which the micro-medical device is designed to perform. The zones of the microflmedical device may have any form of cross section, such as generally arched or generally polygonal. For example, an area may include channels, cameras or other substantially enclosed spaces. By "substantially enclosed" it is meant that materials enter or leave the zones only through predetermined ways. Examples of such routes include channels, microchannels and the like, which are interconnected with various zones. The zones preferably have at least one micro-scale dimension, such as less than about 250 pm or, more preferably, less than about 75 pm.

The channels and cameras of the microfilm network are etched on the lower surface 136 of the upper substrate 130 using known photolithographic techniques. More specifically, templates or transparent masks containing opaque designs are used to photo-define objects on the substrate surface. The patterns on the templates are generated with design programs with the help of a computer and can delineate structures with stroke widths less than one micrometer. Once a template has been generated, it can be used almost indefinitely to produce identical replica structures. Consequently, even extremely complex microflmedic networks can be reproduced in bulk quantities at a minimal gradual unit cost. Alternatively, if a plastic material is used, the upper substrate can be formed using injection molding techniques, in which microchannels are formed during the molding process.

The lower substrate 132 may include a glass base 138 and an oxide layer 140. Within the oxide layer 140, resistive heaters 142 and electrical conduits 144 are formed using photolithographic techniques. The conduits 144 are connected to terminals 146 that are exposed at the edge of the substrate to allow electrical connection to the cartridge 120, thus allowing the DAQ 126 to control the heaters. More specifically, to activate a heater 142, the DAQ 126 applies a voltage across a pair of terminals 146 (through the cartridge 120) to supply current through the conduits 146 and the heater 142, by heating,

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thus, the resistive heating element 142.

Metal heating elements 142 are positioned so that, when the upper and lower substrates are bonded together, the heaters reside directly below certain regions of the upper substrate fluid network to be able to heat the content of these regions. The silicon oxide layer 140 prevents heating elements 142 from coming into contact directly with material in the microfilm network.

The oxide layer 140, the heating elements 142 and resistive conduits 144 have been manufactured using well known photolithographic techniques, such as those used to etch the microfilm network.

Fig. 3 illustrates a top-down view of the microfilm device 110. As shown, the substrate has a sample input module 150 and a reagent input module 152 to allow the sample and reagent materials, respectively , are introduced into the device 110. Preferably, the input modules 150, 152 are arranged to allow automatic material entry using a computer controlled laboratory robot 154.

The substrate also includes processing module 156, 158, 160, 166 and 162 to process the sample and reagent materials. Within these processing modules, a variety of physical and chemical processing steps can be submitted. For example, enrichment module 156 prepares a sample of fluid having a relatively high concentration of cell particles, lysis module 160 releases intracellular material from cell particles, mixing module 166 mixes the resulting sample with certain reagents As another example, an amplification processing module 162 can be used to amplify and detect minute amounts of DNA within a sample.

Various modules of the microfilm device 110 are connected, such as by channels 164, to allow the materials to move from one location to another within the device 110. The actuators 168, 170, 172 associated with the microflm device provide a driving force, such as a gas pressure, to move the sample and reagent material along with the channels and zones. For example, a first actuator 168 moves material downstream from the processing module 156 to the processing module 158. When the processing is completed within the processing module 158, a second actuator 170 moves material downstream to mix the processing module 160. Subsequently, the actuator 170 or an additional actuator moves the material to the mixing module 166, in which the material is mixed with a reagent moved by the actuator 172. Finally, the actuator 172 or other actuator, moves the mixed material to the module 162.

Since each actuator is preferably responsible for moving materials within only a subset of the modules of the device 110, the 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, of the micro-medical device 110, which include the actuators, are preferably under computer control to allow automatic sample processing and analysis.

C. Multiple actuators

The various actuators of the microfilm device 110 cooperate to move material between different locations of the microflm device 110. For example, the actuator 168 moves material, such as an enriched sample, between an enrichment zone 931 and a micro-drop preparation module 158. actuator 170 prepares a micro drop from the enriched sample and, in doing so, moves the micro drop to the lysis zone 950. The actuator 170 is used to move material from the lysis zone 950 to the mixing module 166. It should be noted, without However, another actuator may be arranged in the intermediate between the lysis zone 950 and the micro-drop preparation zone to move the lysed sample downstream to the mixing module 166.

The actuators of the device 110 can also cooperate in the movement of two quantities of material simultaneously. For example, as described above, actuator 172 and actuator 170 cooperate for reagent mixtures and lysed drops. Such cooperative actuators can be controlled independently of each other to ensure proper mixing. For example, if a material is known to be viscous, the driving force that this material can be increased independently of the driving force that moves the other material.

The multiple actuators and modules of the microfilm device 110 are preferably operatively connectable and insulated by the valves of the microflm device. For example, a closed state of any of the valves 915, 216 operatively affixes the micro-drop preparation module 170 of the enrichment module 156. Thus, one or more actuators can be used to move materials between predetermined locations within a predetermined location. micro-medical device 110, without disturbing or contacting material present in an operatively isolated module. The ability to operatively connect and isolate desired modules is advantageous in microfluidic devices that have many processing functions. Also, these valves also control the direction of the propulsive force of the actuators preventing the expanding gas from traveling in certain directions, while allowing it to expand in the direction

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desired. This also extends the range over which an actuator can propel a micro drop, preventing the gas from dissipating in certain areas upstream of the micro drop.

The following demonstrates that the cooperative operation of such multiple actuators is an illustrative embodiment having a plurality of processing modules, namely an enrichment zone 915, a micro drop preparation module 158, a cell lysate module 160, a mixing module 166 and a DNA manipulation module 167.

1. Enrichment module

to. Structure of enrichment module.

As for Figs. 4 and 5, a microflm device 901 includes an enrichment module 156 to concentrate samples received therein. These samples include fluids that contain particles, such as fluids that contain bacterial cells. In general, the enrichment module 156 receives a flow of fluid containing particles from an input port 180 of the input module 150 and allows the fluid to pass through the area while accumulating particles within the area. In this way, the more fluid flows through the area, the concentration of particles increases within the module. The resulting concentrated fluid sample is referred to herein as an enriched particle sample.

The enrichment module includes an enrichment zone 931 (Fig. 5), a flow circulation member 900, valves 915, 919 and a sample introduction channel 929. The valve 919 is connected between the flow circulation member 900 and the actuator 168 as shown and the valve 915 is connected between the flow circulating member and a downstream channel 937 that leads to the processing module 158. These valves can be of any type suitable for use in a microflmed device, such as thermally actuated valves, as described in the pending application for publication No. 09 / 953.921, filed on September 9, 2001. The valves may be reversible between the open and closed states to allow the reuse of the enrichment module. 931

The flow circulating member is also connected to the sample input module 150 through the sample introduction channel 929 to allow the fluid to flow into the enrichment zone. Valve 913 is connected to this sample input channel to control the inlet flow and the outflow of the inlet port.

Fig. 5 is a cross-sectional view of the enrichment zone showing the flow circulation member in greater detail. As shown, the flow circulating member 900 has first and second surfaces 941, 943. The first surface 941 is preferably adjacent to the enrichment chamber 931. The second surface 941 is preferably separated from the enrichment chamber 931 by the member of flow circulation 900. The flow circulation member 900 is preferably formed of a material having smaller than the diameter of the particles to be enriched, such as porous or less than about 2 micrometers in diameter, for example, approximately 0 , 45 micrometers. Suitable materials for constructing the flow circulating member 900 include, for example, filter media such as paper or fabrics, polymers having a network of glassware and glassy materials, such as glass frits.

Figs. 6 and 7 represent cross-sectional views of the upper substrate 130 illustrating an enrichment zone 931. As shown, the fluid exits the enrichment zone 931 through the surface 941, passes through the member 900 and enters a space 400. The space 400 may include an absorbent material 402 to absorb the leaving fluid. In this way, the space 400 preferably provides a substantially self-contained region in which fluid can be collected leaving the enrichment zone without coming into contact with external parts of the microfilm system 100.

The space 400 is formed during the fabrication of the upper substrate 130. As discussed above, microflamic features, such as zones and channels, are manufactured on the surface 136 of the substrate 130. The space 400, however, is manufactured on a surface 137, which is preferably disposed on the other side of the substrate 130, opposite surface 136. Thus, even when the surface 136 corresponds to the lower substrate 132, the fluid can exit the enrichment zone 931 through the member of 900 flow circulation.

The flow circulation member 900 and the absorbent material 402 do not require adhesives or other fasteners to position themselves within the substrate 130. Instead, the flow circulation member 900 and the absorbent material 402 may be formed in a shape and size that is substantially corresponds to the space 400. The friction thus maintains the flow circulating member 900 and the absorbent material 402 in place once they have been placed in the space 400. Any residual gap in the locations 404 between the member of flow circulation 900 and the substrate 130 must be small enough to prevent particles from leaving the enrichment zone 931 through the gap 404. Naturally, adhesive or other fastening means can be used to secure the flow circulation member 900 or absorbent material 402.

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In an alternative embodiment, a flow circulating member is integrally formed with a substrate through the use of microfabrication techniques, such as chemical attack, that introduce pores or other vias into the substrate. The pores provide the passage of fluid between the enrichment zone 931 and an external part of the substrate.

b. Enrichment module operation

To enrich a sample, the device 901 works as follows. Referring to Fig. 4, the valves 915, 919 are initially closed and the valve 913 is open. A fluid containing particles is introduced into the inlet port 180. Since valve 913 is open, it allows the sample to pass along channel 929 in enrichment zone 931. Alternatively, enrichment zone 931 can be configured to receive samples directly, such as by injection. Since the valves 915 and 919 are closed, the fluid is prevented from escaping into the actuator 977 and the downstream channel 937.

Thus, the flow circulation member 900 provides only one path for the fluid to flow out of the enrichment channel. The fluid passes through the surface 941 and leaves the enrichment zone 931 through the second surface 943, while the particles accumulate within the area. The enrichment zone 931 may, therefore, receive a volume of fluid that is larger than the volume of the enrichment chamber 931. Thus, as the fluid flows through the chamber, the concentration of particles within the chamber increases in relation to the concentration in the fluid containing particles supplied in the sample inlet. When the particles are cells, the concentration or number of cells in zone 931 preferably becomes large 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 a sample of enriched particles from particles of fluids containing particles received therein. The enriched particle sample has a substantially higher ratio of particles per fluid volume (PPVF) than the corresponding ratio of fluid containing particles 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 higher than the PPVF of the particle containing fluid.

After a sufficient volume of fluid containing particles has been received through the enrichment zone 931, the valve 913 closes, thereby blocking the flow of fluid in the enrichment zone and preventing the material in the zone 931 return to the sample introduction port 180. The valves 915, 919 are then opened, preferably when the heat sources associated with them are actuated. When opened, valve 919 allows actuator 168 to push the enriched sample and, valve 915 allows the enriched sample to move downstream.

The actuator 168 provides a driving force that moves the enriched particle sample from the enrichment zone 931. The actuator 168 is preferably a gas actuator, which provides a gas pressure when a heat source 975 is operated, which is in communication. thermally with a volume of gas 977. The actuation of the heat source 975 increases the temperature and, therefore, the pressure, of gas 977. The flow circulating member and the fluid therein substantially prevents the gas from escape from the enrichment zone. Thus, the resulting gas pressure moves the enriched particle sample downstream of the enrichment zone 931.

The gas actuator may include elements to facilitate alternative pressure generation techniques such as chemical pressure generation. In another embodiment, the actuator can decrease a volume of gas associated with a portion upstream of the enrichment zone so as to 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 piston or diagram.

Instead of generating a positive pressure upstream of the enrichment zone, the gas actuator can decrease a pressure downstream of the zone in relation to an upstream pressure. For example, the gas actuator may include a cooling element in thermal contact with a volume of gas associated with a portion downstream of the area. The contraction of the gas when the cooling element is operated creates a difference in gas pressure between the upstream and downstream portions of the enriched zone to move the enriched particle sample from the enrichment zone. Alternatively, a mechanical actuator can be used to increase a volume of gas associated with a portion downstream of the enrichment zone to thereby decrease the gas pressure and move the enriched particle sample from the enrichment zone.

The enriched particle sample preferably moves downstream without essentially dilution thereof, it is dear, the concentration of the enriched particles does not decrease substantially when it is moved from the enrichment zone 931. Thus, the removal of particles from the channel of Enrichment of the present invention does not require diluting or otherwise contacting the particles with a fluid other than the fluid of the fluid containing particles introduced into the enrichment channel. In contrast, in systems

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which concentrate substances by surface adsorption, the removal of adsorbed substances requires an elution fluid, which comes into contact and thus dilutes the substances.

When removed from the enrichment zone of the present invention, the enriched particle sample is preferably received by the downstream channel 937. The downstream channel 937 leads to other processing modules, which perform further processing of the particle sample. enriched In the embodiment of Fig. 3, the enriched particle sample is received by a micro-drop preparation module 158, which prepares a micro-drop sample comprising a part of the enriched particle sample.

2. Microgout preparation module

to. Characteristics of a micro drop

An 802 micro drop is a discrete sample that has a predetermined volume between, for example, about 1.0 picoliter and about 0.5 microliters. Thus, micro drops prepared by the micro drop preparation module provides a known amount of sample for further processing. The volume of the micro-drop prepared by the micro-drop preparation module is preferably essentially independent of the viscosity, electrical conductivity and osmotic force of the micro-drop fluid.

The micro drop 802 is preferably defined by upstream and downstream limits each formed by a respective liquid gas interface 804, 806. The liquid from the interface is formed by a surface of a liquid that forms the microdrop. The gas of the interface is gas present in the microfluidic channels of the microflmed device 901.

b. Structure and operation of the micro drop preparation module

Referring to Figs. 8a-8b and 9a-9b, the micro-drop preparation module 158 prepares a micro-drop 802 from a micro-medical sample received therein. This module includes a micro-drop preparation zone 800, a positioning element 979, a gas actuator 170 and a valve 216 that cooperates to prepare the micro-drop 800 from micro-chemical samples received from the enrichment zone.

As explained above, the actuator 168 of the enriched zone pushes the enriched sample in the micro-drop preparation zone 800. The enriched sample moves until it reaches the positioning element 979. In general, a positioning element inhibits the current progress below a microflmed sample to position the sample in a desired location. However, as explained in more detail below, the positioning element does not permanently inhibit the progress of the sample. Instead, it allows the microfilmed sample to continue downstream at a predetermined later time.

The leading edge of the microflmed sample 808 reaching the positioning element 979 is positioned downstream from an opening 820 of the gas actuator 170. Consequently, a first part 821 of the microflmed sample 808 is arranged upstream from the opening 820 and a second part 822 of microfilm sample 808 is disposed downstream of the opening 820.

Referring to Fig. 8a-8b, the gas actuator 170 is operated, such as by DAQ 126, to thereby generate a sufficient gas pressure to separate the microdroplet 802 from the second part 822 of the microflmed sample. 808. The gas pressure is preferably provided by actuating a heat source 958, which heats a volume of gas associated with a gas actuator 957. As the pressure increases, the gas expands, thereby separating a micro drop 802 of the rest of the sample 808. The micro drop 802 may comprise only a portion, such as less than about 75% or less than about 50%, of the microflmed sample 808 received by the micro drop drop zone 800. Dimensions of the micro drop 802 are determined by the volume of the channel between the fluid barrier 979 and the opening 820. For example, for a channel having a uniform cross-sectional area, a length h of the micro drop 802 corresponds to a distance d4 between the positioning element 979 and the opening 820. In this way, a microfilm device can be configured to prepare microdrops of any volume by varying the length between the fluid barrier and the corresponding actuator opening.

The continued actuation of the gas actuator 170 exceeds the inhibitory effect of the positioning element 979, thereby leading the micro drop 802 to a location downstream of the micro drop drop zone 800 while the second part 822 of the microflmed sample is moves upstream from the microdrop 802 to the cell lysis module 160.

3. Cell lysis module

Referring again to Fig. 3, a lysis module 160 receives the microdroplet 802 prepared by the microgout preparation zone 800. In general, the lysis module 160 releases material from inside the particles, such

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as by the release of intracellular material from cells.

As shown in Figs. 4 and 12, lysis module 160 includes a lysis zone 950, a lysis mechanism within the lysis zone (such as electrodes 954) and a ventilated positioning element 200 positioned upstream from the lysis zone. The lysis mechanism preferably includes a set of electrodes or other structures to generate electric fields within the lysis zone. The ventilated positioning element preferably includes a ventilation outlet 202, a valve 204 and a second positioning element 206 to inhibit fluid flowing in the ventilation outlet.

As explained above, the actuator 170 of the micro drop preparation module 158 conducts a micro drop within the cell lysis module 160. As the micro drop moves into module 160, the ventilated positioning element 200 positions the micro drop 802 in a position of lysis with respect to electrodes 954. More specifically, as the micro drop reaches a lysis module 160 passes the opening of the positioning element 200, since the second positioning element 206 prevents the micro drop from flowing into the outlet of ventilation 202. When the rear end of the micro drop passes the opening of the barrier 200, the propulsion gas from the actuator 170 is dissipated through the ventilation outlet 202, thereby substantially equalizing the upstream gas pressure of the micro-drop 802 with a pressure downstream of the micro-drop 802. In this way, the micro-drop stops the movement in a position of right lysis below the barrier 200. Preferably, in the lysis position, substantially all of the micro drop 802 is disposed between an upstream edge 212 and a downstream edge 214 of the electrodes 954.

After placing the micro-drop 802 in the cell lysis position, a pulse circuit of DAQ 126 supplies a pulsed voltage signal through electrodes 954. In response, electrodes 954 generate a pulsed electric field in the vicinity of the electrodes Since the micro drop is positioned in its vicinity, the cells within the micro drop are subjected to the pulsed field. Preferably, substantially all cells, such as greater than about 75%, of the micro drop are subjected to an electric field sufficient to release intracellular material therefrom. The lysis module thus prepares a lysed micro drop 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 provides a corresponding sequence of electric field pulses in the vicinity of electrodes 954 having an amplitude and duration sufficient for release a desired amount of intracellular material from cells within the micro drop.

Intracellular material present in the lysed drop is accessible for additional processing steps. For example, DNA and / or RNA released from cells is accessible for amplification by polymerase chain reaction. As used herein, the term "lysis" does not require that the cells be completely broken. Instead, lysis refers to the release of intracellular material. For example, instead of breaking the cells, the electric field can increase the porosity of the cell membranes by an amount that allows the release of material without a permanent rupture of the membranes.

Other lysis mechanisms can also be used to release intracellular material from cells. For example, material can be released by subjecting cells to other forces that include, for example, shock or osmotic pressure. Chemicals, selected from the group of surfactants, solvents and antibiotics, can be contacted with the cells. mechanical shearing methods can also be used to release intracellular materials.

The lysed drop can be moved downstream to the mixing module 160 for further processing. To move the lysed microbead downstream, the valve 216, which is deposited upstream of the lysis zone 950, is closed. The valve 204 also closes to prevent gas from leaving the lysis zone 950 through the ventilation outlet. The actuator 170 is then operated, as described above, to provide a sufficient gas pressure to move the lysed micro drop down the lysis zone 950.

In an alternative embodiment, a lysis module 300, as shown in Figs. 13a, 13b, includes a lysis zone 302 that is configured to prepare a lysed microdrock 304 of predetermined volume from a microflmed sample 306, which can have an undetermined volume The lysis zone 302 preferably includes a lysis mechanism such as electrodes 308. The electrical conduits 310 provide a connection to a pulse circuit of DAQ 126, through contacts 112, chip carrier 120 and contacts 125. A positioning element 312 is disposed downstream of the lysis zone 302. An actuator 314 is disposed upstream of the lysis zone. The actuator 314 preferably includes a second positioning element 316 to prevent fluid from the microfluidic sample from entering it.

Lysis zone 302 works as follows. The microflmed sample 306 enters the lysis zone 302 and moves downstream until a downstream interface 316 of the microflmed sample 306 meets the positioning element 312. The positioning element 312 preferably increases a surface tension of the downstream interface of the microflmed sample 306, thereby inhibiting a movement

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further downstream and positioning a part of the microflmedical sample in a lysis position with respect to electrodes 308. The lysis position is defined as the location of the part of the microflmedical sample disposed downstream of the actuator 314 and upstream of the element of positioning 312. Preferably, the actuator 314 and the positioning element 312 are arranged adjacent to the electrodes 308 so that all the material present in the lysis position is subjected to the electric hood when the electrodes 308 are operated.

The electrode drive 308 in the embodiment described above, provides a sufficient electric field to release intracellular material from cells present in part of the micro-medical sample in the lysis position. Once a sufficient amount of intracellular material has been released, the actuator 314 is actuated to prepare the lysed micro drop 304 from the microflmed sample 306. The actuator 314 preferably provides a sufficient gas pressure to move the lysed micro drop 304 to a part. downstream of a microfluidic device such as a mixing module 166.

4. Mixing module and reagent input module

Referring again to Fig. 4, a lysed sample prepared by the lysis module 160 is received by the mixing module 166. The mixing module 166 includes a mixing zone 958. In this area, the lysed cell sample it is contacted, such as by mixing, with an amount of reagent received from reagent source module 152. Reagent source module 152 includes a reagent microbead preparation zone (ZPMR) 434, which preferably works for Prepare a micro drop that has a predetermined volume of reagent.

to. Reagent input module

The reagent input module 152 is essentially the same as the micro drop formation module 158, however, it is specifically designed for the formation of a reagent micro drop that has a predetermined volume that will provide a desired ratio of reagent to sample when mixed with the micro drop of the cell lysis module 160. The module 152 includes an entry post 420, a valve 422 and an actuator 172, each of which links a reagent source channel 428. An overflow channel 424, which also links reagent source channel 428, can be provided. The actuator 172 may include a second positioning element 432 to prevent liquid from entering it.

The reactive materials, which preferably comprise at least one liquid, are introduced through the inlet port 420, such as with a pipette or a syringe. Examples of suitable reactive 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 reactive material moves downstream into the reagent source channel 428 until a downstream portion of the reactive material comes into contact with a positioning element 426. Any additional reactive material that continues to be received within the reagent source module preferably enters in the overflow channel 424. When the reagent introduction is completed, the valve 422 is closed to prevent the reagent from leaving the reagent source channel through the reagent source port 420.

b. Mixing module

The mixing zone 958 of the mixing module includes attached first and second channels 410, 412. Materials that move downstream towards the mixing zone 958 come into contact with each other and preferably are mixed therein. Due to the micro-scale dimensions of the mixing zone 958, the sample and the reactive materials are preferably mixed by diffusion even in the absence of other mass transport sources, such as mechanical agitation. However, it should be understood that, stirring forces, such as acoustic waves, can be applied to improve mixing within the mixing zone 958.

C. Mixing module operation and reagent input module

Reagent source module 152 and mixing module 166 preferably operates as follows. When a lysed sample from lysis zone 950 is ready to be mixed with a reagent material, actuator 172 is actuated to prepare a reagent micro drop. The reagent micro drop is prepared from the reagent material part downstream of an 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 micro drop is determined by the distance between the positioning element 426 and the opening of the actuator 430.

The reagent microbead moves downstream to channel 412 of the reagent mixing zone. Meanwhile, a sample of lysate material, such as a lysed micro drop, moves downstream from the lysis zone 950 to the channel 410 of the mixing zone 958. The actuator 170 can provide the driving force to move the current lysed micro drop. down. Alternatively, as discussed above, another one may be provided.

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actuator upstream of lysis zone 950 but downstream of actuator 170 to provide the necessary driving force.

The sample and reactive material enter a downstream channel 438 of the mixing zone 958, when the materials come into contact and mix. Since both the lysed sample and the reactive material are mixed in the form of micro-drops, the mixing zone 958 prepares an amount of mixed material that has a predetermined ratio of sample to reagent. The volumes of microdroplets prepared within the microfilm device 110 are preferably independent of the physical properties, such as the viscosity, electrical conductivity and osmotic force, of the microdroplets. Thus, the mixing zone 958 prepares an amount of mixed material that has a sample with respect to reactive material that is also independent of the physical and chemical properties of the mixed materials. A vent outlet 440, which is downstream of the various areas of the microfilm device 110 ensures that the accumulation of downstream pressure does not inhibit the downstream movement of the sample within the microflimetric device 110.

5. DNA manipulation module

The mixed and reactive lysed cell sample is received within a DNA manipulation zone 971 of the DNA manipulation module 162. The module 162 can perform, for example, the restriction, digestion, ligation, hybridization and amplification of DNA material. . In one embodiment, the DNA manipulation zone 971 is configured to perform the PCR amplification of nucleic acids present within the lysed cell sample. The vent outlet 440 prevents the pressure inside the zone 971 from increasing according to the lysed cell sample and the reagent being introduced thereto. Valves 972 and 973 of the DNA manipulation module 162 can be closed to prevent substrates within the zone from leaving, such as by evaporation, during PCR amplification. The DNA manipulation zone is configured with heat sources under computer control 127 to allow the thermal cycle of the DNA manipulation zone during amplification, as is understood by one skilled in the art.

System 901 also includes a detector 981 to detect the presence of amplified polynucleotides produced by PCR. The detector 981 is preferably an optical detector in optical communication, such as by an optical fiber 981, with zone 971. A light source, such as a laser diode, introduces light to the DNA manipulation zone 971 to generate indicator fluorescence of the amount of amplified polynucleotides present therein. Fluorescence appears from fluorescent labels, included in the reagent and associated with polynucleotides when amplified.

D. Preferred positioning elements

Next, preferred positioning elements are discussed.

1. Non-wetting positioning elements

A positioning element 979 can be formed by a non-wetting material arranged to come into contact with a microflmed sample. The physical-chemical properties of the non-wetting material are chosen in consideration of the type of liquid that forms the microflmed sample. For example, when the sample is a microflmed sample in 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 the microfilm device 901. For microflimed samples formed of organic solvents, the non-wetting material may comprise a hydrophilic material.

When a microflmed sample 808 meets the positioning element 979, the microflmed sample fluid experiences an increased surface tension at the downstream interface 810, whose increased surface tension inhibits the continued downstream movement of the microflmed sample 808. Increase The difference in gas pressure between the upstream and downstream parts of the microfilmed sample exceeds the resistance and moves the microflmed sample downstream.

2. Assisted capillary positioning elements

Referring to Fig. 10a-10c, another type of positioning element can be formed by modifying the dimensions of the microflmed channel to form an assisted capillary positioning element (EPCA) 700. An EPCA comprises a zone fed upstream 702, a zone of load 704 and a stop zone 704. A microflmed sample 720 that is found in the EPCA moves downstream until a downstream interface 710 of the microflmed sample comes into contact with surfaces upstream 714 of the load zone 706. In this point, the capillary action causes the microflmed sample to move downstream until the downstream sample interface 710 meets the opening 712 between the load zone 704 and the stop zone 706. The surface tension resists that the tendency of the microflmed sample continue downstream past the opening 714. In this way, the microflmed sample 720 is positioned at a predetermined location. rolled along the axis of the channel with respect to the positioning element 700.

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The volume of the microflmed sample that meets the EPCA preferably has a larger volume than the volume of the loading zone 704 to ensure that the microflmed sample will advance completely to the opening. For fluids that have similar surface tensions and interface properties such as water, the depth d1 of the loading zone 704 is preferably about 50% or less of the respective depths d2, d3 of the stop and feed zones.

The tendency of a micro-medical sample to move in a given direction is governed by the relationship between the mean radius of curvature (RCM) of the front part of the micro-medical sample and the RCM of the rear part of the micro-medical sample. The curvatures depend on the contact angle of the sample fluid and the dimensions of the area in which the micro drop is moving. An RCM ri of a micro-drop interface in the loading zone is preferably lower than the RCM r2 of a micro-drop interface within the feed zone or an RCM r3 of a drop interface within the stop zone. The RCM r2 is preferably larger than the RCM r3. Thus, the radius of curvature of the downstream micro drop interface increases when it encounters the stopping dinner, thereby inhibiting additional downstream movement. Preferably, the angle of contact of the fluid with the wall is substantially constant throughout the area of assisted capillary loading.

3. D. Ventilated positioning elements

Referring to Fig. 11a-11c, a positioning element 500 functions to position a microfilm sample 502 by reducing the gas pressure acting with an upstream portion 504 of the microfilm sample in relation to the gas pressure acting with a downstream part 506 of the microflmedic sample. The positioning element 500 includes a ventilation outlet 508 arranged in gaseous communication with an area 510 along which micro-chemical sample 502 moves. The ventilation outlet 508 preferably communicates with the area 510 through a passage 526. The zone can be, for example, a channel or a conduit. The positioning element 500 may also include a second positioning element 516, such as a non-wetting material, to substantially prevent fluid from the microfluidic sample from coming into contact with the ventilation outlet.

An open state of a vent 512 allows the passage of gas between zone 510 and the vent 508. A closed state of the valve 512 prevents such a passage of gas. The valve 514 is preferably thermally actuated and includes a mass 514 of TRS.

An actuator 518 is arranged upstream of the positioning element 500. The actuator 518 is preferably a gas actuator and may include a heat source 520 for heating gas associated with an actuator 518. The actuator 518 may include a positioning element 522, such as a non-wetting material, to substantially prevent fluid from the microfluidic sample from entering it.

The positioning element 500 preferably operates as follows. Referring to Fig. 11a, the microflmed sample 502 moves downstream in the direction of arrow 524. The microflmed sample is preferably moved by a gas pressure provided from an upstream actuator, which is not shown in the Fig. 9a-9c. The gas pressure acts with the upstream part 504.

Referring to Fig. 11b, when the upstream portion 504 passes the opening of the vent outlet 508, the upstream gas dissipates through the vent outlet 508, thereby reducing the upstream pressure. The pressure reduction, which preferably equalizes the downstream and upstream pressures, reduces or eliminates the driving force tending to drive the micro-chemical sample downstream.

Referring to Fig. 11c, the vent outlet 512 is enclosed to prevent the passage of gas between zone 510 and vent outlet 508. Preferably, the TRS 514 moves in the passage 526. When the valve 512 is closed, the actuator drive 518 provides a driving force to move the microfilm sample 502 downstream in the direction of arrow 528 for further processing.

4. Active fluid positioning elements

Referring to Fig. 15a-15c, a micro-drop preparation module 652 has a micro-drop preparation zone 650, an active fluid positioning element 654, an actuator 656 and a valve 658. A second actuator 660 is operatively associated with the active positioning element 654 for introducing a micro-medical sample 666 to the micro-drop preparation zone 650. A second actuator 660 is preferably located upstream of the valve 658. A micro-drop preparation module 652 prepares a micro-drop 668, which has a predetermined volume from the microflmed sample 666 received therein.

During operation, the microfilm preparation module 652 receives the microflmed sample 666, which moves downstream due to the driving force provided by the second actuator 660. The driving force is preferably an upstream gas pressure, which is larger than a downstream gas pressure acting with the microflmed sample 666. The microflmed sample moves downstream until a downstream portion thereof 670 meets the active positioning element 654, which comprises

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preferably a sensor 672 having electrical conduits 674. The conduits 674 are in electrical communication with I / O pins of the microfluidic device to allow the signals of the sensor 672 to be received by a DAQ.

The sensor element 672 is preferably a pair of electrical contacts. To detect the presence of the liquid, the DAQ 126 applies a small voltage across the conduits 674 and measures the resulting current. According to the liquid of the microflmed sample, it comes into contact with the first and second contacts, the current passing between them changes, thus indicating to DAQ 126 that the liquid has reached sensor 672.

When it is recognized that the liquid has reached the sensor 672, the DAQ instructs the second actuator 660 to decrease a downstream driving force that acts with the microflmed sample 666. For example, the DAQ can reduce a current flowing through a source of heat 676 associated with a second actuator 660 thereby reducing a temperature of a gas therein. The temperature reduction reduces the pressure of gas acting with an upstream portion 678 of the microflmed sample, thereby inhibiting the downstream movement of the microflmed sample 666. The microflmed sample is positioned so that the first part 680 is located downstream of the actuator 656 and a second part 682 is located upstream of the actuator 656.

To prepare the micro drop 668, the DAQ 126 drives the actuator to provide a driving force that prepares the micro drop 668 from the first part 680 of the microflmed sample 666. The micro drop 668 moves downstream while the second part 682 of the Micro-medical sample 666 moves upstream of actuator 656. During the micro-drop preparation, valve 658 may be closed to substantially isolate actuator 656 from second actuator 660 and other parts upstream of the microflmed device.

The active positioning element preferably functions as a closed loop that provides feedback from sensor 672 to the DAQ. Feedback is indicated when a microfilm sample has reached a predetermined position within the microfilm device. When feedback is received, the DAQ changes the state of the actuator that provides the driving force to move the micro drop.

While the previous invention has been described with reference to certain preferred embodiments, it should be noted that the scope of the present invention is not limited thereto. Thus, one skilled in the art can find variations of these preferred embodiments, which, however, are within the scope of the claims.

Claims (9)

5 10 fifteen twenty 25 30 35 40 Four. Five fifty 55 60 65 1. A micro-medical device (110) for processing a micro-medical sample containing cells, comprising: a lysis module (300) configured to receive a micro-medical sample containing cells, in which the lysis module comprises a lysis zone (302), in which the micro-medical sample is a liquid; a positioning element (312) configured to inhibit the movement downstream of the microflmed sample to position the microflmed sample in a lysis position; a lysis mechanism (308) within the lysis zone (302), to release intracellular content from cells within the lysis zone; Y a gas actuator (314) disposed upstream of the positioning element (312) and the lysis zone (302) so that a premium part of the microflmed sample is arranged upstream of the gas actuator (314) and a second part of the micro-medical sample, downstream of the gas actuator (314) and upstream of the positioning element (312), the gas actuator (314) is configured to provide sufficient gas pressure to prepare a micro drop (304) having a predetermined volume comprising intracellular content released from cells of the microflmedic sample containing cells within the lysis zone (302) by separating the second part of the microflmedic sample from the first part of the microflmedic sample and moving the second part of the micro-medical sample at a location downstream of the lysis mechanism (308) and the positioning element (312). 2. The micro-medical device (110) according to claim 1, wherein the device comprises a substrate (130, 132) and in which the lysis mechanism (308) and the gas actuator (314) are integral with the substrate. 3. The microfilm device (110) according to claim 1 or 2, in the gas actuator (314) comprises a heat source to heat an amount of gas thereby increasing a gas pressure. 4. The microfilm device (110) according to any one of the preceding claims, wherein the positioning element (312) comprises a ventilation outlet to substantially equalize a gas pressure upstream of the microflmed sample containing cells with a pressure of downstream gas of the microflmed sample containing cells when the microflmed sample containing cells is in the lysis position to thereby inhibit the downstream movement of the microflmed sample containing cells downstream from the position of lysis 5. A micro-medical method for processing a micro-medical sample containing cells, comprising: the positioning, using a positioning element (312), of the micro-medical sample containing cells in a lysis position with respect to a lysis mechanism (308) of a lysis zone (302) of a lysis module (300) of a microfluidic device (110), the microflmedical sample containing cells comprising a liquid which contains cells; the actuation of the lysis mechanism (308) to release intracellular material from cells of the microflmedical sample containing cells; the actuation of a gas actuator (314) disposed upstream of the positioning element (312) and the lysis zone (302) to provide a sufficient gas pressure to separate a part of the microflmed sample containing cells that are current below the gas actuator (314) and upstream of the positioning element (312) from a part of the microflmed sample containing cells that are located upstream of the gas actuator (314) and move the downstream part of the microflmed sample which contains cells at a location downstream of the lysis mechanism (308) and the positioning element (312), the downstream part of the micro-medical sample being a micro drop (304) comprising intracellular material released from sample cells microfilm that contains cells and has a predetermined volume. 6. The microflmed method according to claim 5, wherein the positioning step comprises contacting the downstream surface of the microflmed sample with a hydrophobic material. 7. The microflmed method according to any one of claims 5 or 6, wherein the positioning step comprises increasing a radius of curvature of the microflmed sample. 8. The microfluidic method according to any one of claims 5 to 7, wherein the positioning step comprises substantially equalizing a gas pressure upstream of the microflmed sample with a gas pressure downstream of the microflmed sample. 9. The microfluidic method according to any one of claims 5 to 8, wherein the second part of the microflmedic sample comprises less than about 90 percent of the microflmedic sample containing cells.