JP2016516562A - Method and system for microfluidic processing - Google Patents

Abstract

Methods and systems are provided for microfluidic cartridges that include high performance actuators useful for analyte detection, labeling and analysis. The microfluidic processing system is for performing chemical or biochemical reactions or series of reactions with small amounts (typically between 1 microliter and 10 milliliters) of reactants and products. Microfluidic processing systems can include a network of tubes that are associated with discrete components such as valves and sensors, or integrated devices made from plastic, glass, metal or other materials or combinations of materials. Here, components such as valves and sensors are built into the device and connected by flow passages formed in the material.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of US Provisional Application No. 61 / 771,708, filed Mar. 1, 2013, which is incorporated herein by reference in its entirety.

  This application is related to US Provisional Application No. 61 / 771,694, filed Mar. 1, 2013, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was achieved with government support under NIH contract number HHSN272200900029C and NIH grant number 2R44AI073221 awarded by the National Institutes of Health It was. The government has certain rights in the invention.

The present invention relates to methods and systems for moving and processing fluids through an assay system.

2. Description of Related Art A microfluidic processing system is for carrying out a chemical or biochemical reaction or series of reactions with small amounts (typically between 1 microliter and 10 milliliters) of reactants and products. is there. Microfluidic processing systems can include a network of tubes that are associated with discrete components such as valves and sensors, or integrated devices made from plastic, glass, metal or other materials or combinations of materials. Here, components such as valves and sensors are built into the device and connected by flow passages formed in the material.

  Conventional microfluidic processing systems are used to perform chemical or biochemical reactions or series of reactions, reciprocating displacement pumps, peristaltic effects, syringe pumps, surface tension effects, volume forces on magnetic beads from external or internal magnetic field sources Use a vacuum manifold, electrokinetic effect, electrochemical effect or a combination thereof.

Flow within a microfluidic processing system is typically associated with the predominance of viscous effects over inertial effects, referred to as the low Reynolds number region. Many applications of microfluidic processing systems involve one or more high molecular weight reactants that have correspondingly low binary diffusivities. For example, molecular mechanics simulations have shown that a ribonucleic acid strand of approximately 9800 bases constituting the human immunodeficiency virus (HIV) genomic material with a molecular weight of 3.1 × 10 ⁶ daltons is approximately D = 2 × 10 ⁻¹² m It shows that underwater diffusivity of ² s ^-1 , so in 10 minutes, one-dimensional diffusion is associated with a displacement of only 50 microns. The combination of the viscous effect over the inertial effect and the relatively slow diffusivity of the reactants of interest is a fluid mechanics for macroscopic mixing of two or more solutions in a microfluidic system. Imposes the need for a mechanism.

  A small amount of gas introduced as part of the process being performed or unintentionally occurring as when expansion or contraction of the fluid passage in the direction of flow tends to trap bubbles during filling Often found in microfluidic systems. A volume of gas in a microfluidic system can act as a low pass filter for a mechanical forcing function acting on the system. This is sometimes referred to as fluid capacitance. Tubing can also be a source of fluid capacitance.

  The tendency of the trapped air to act as a low pass filter creates an incentive to position the fluid actuator in close physical proximity to the fluid volume where the fluid actuator exerts a force and acts mechanically.

  In some applications of microfluidic systems, there is a need to carry out the reaction in a fluid pathway that can be discarded after a single use. For example, in the diagnosis of infectious disease, a microfluidic system used to process a body fluid sample can be considered a biohazard waste after the assay is complete. The very high negative impact of contamination during the production process creates an incentive that allows the microfluidic system used for antibody purification to be completely discarded after a single use. Many types of microfluidic actuators, such as piezoelectric actuators and electromagnetic actuators, are too expensive to be included in a single-use microfluidic cartridge. Piezoelectric and electromagnetic actuators require mechanical energy transfer into the cartridge and can be prone to failures associated with actuator and cartridge misalignment. Actuation mechanisms such as electrochemical gas generation and surface tension based actuation can be economically built into the cartridge, but are associated with slow response times, low power, lack of range and other limitations.

  There is a need for a microfluidic system that can perform macroscopic rapid mixing of one or more reactants. The fast response time and high output of the fluid actuator is important for mixing two or more fluids or reacting two or more species in the mixture within the cartridge. Current microfluidic actuators have limitations such as low fluid power generation capability, sustained power and slow response time. Electroosmotic flow generation can be associated with high power and fast response time, but in some cases, particles are transported through the EO microfluidic device because particles in the sample can block the EO microfluidic device. Cannot be done and the fluid will be adversely affected by the high electric field inside the EO device.

  The present invention addresses these and other prior art shortcomings.

The present invention relates to a microfluidic process comprising a plurality of fluid passages, at least one junction that connects the plurality of fluid passages, and at least two fluid transport mechanisms including at least one high performance fluid actuator. Includes system. High performance fluid actuators have a fluid power generation capability of at least ^10-8 watts, can sustain power for at least 30 seconds, and have a response time for fluid power generation of less than 10 seconds.

  In some embodiments, the microfluidic processing system is an integrated system referred to as a cartridge. In some embodiments, the cartridge has a drainage volume of 500 cubic centimeters or less, or 50 cubic centimeters or less.

  In some aspects, the high performance fluid actuator can convert electrical power directly into fluid power. In some embodiments, the operation of the high performance fluid actuator does not include the transfer of mechanical energy from the external device to the at least one high performance fluid actuator.

  In some embodiments, the response time for power generation is less than 2 seconds, less than 0.2 seconds, or less than 0.04 seconds. In one aspect, the actuator can act on at least 10 microliters of liquid so that the liquid flows through a fluid resistance associated with a pressure drop of at least 1 kPa at a flow rate of at least 0.1 mL per minute.

  In another aspect, the high performance actuator is coupled to a pulse generator or other controlled time varying voltage source. In some aspects, the high performance fluid actuator can generate fluid power through electrokinetic effects. In some embodiments, the electrokinetic effect is electroosmotic flow. The electroosmotic flow can be generated in the gap of the slat structure within the slit capillary or within each of the at least one fluid actuator.

  In another aspect, electroosmotic flow is generated within each of the fluid actuators within a bed of packed beads, an integral porous structure or an array of cylindrical channels.

  In some embodiments, the microfluidic cartridge includes an opening for receiving a starting material within a network of fluid passages. The opening can be closed with a plug or capping element. The plug or capping element can receive the fluid conduit and can be hermetically sealed when the fluid conduit is withdrawn. In other embodiments, the fluid conduit can be received by a plug or capping element and can include a needle, tube, rigid fluid conduit, or semi-rigid fluid conduit. The plug or capping element can include an elastomeric material. In another aspect, the plug or capping element has a closure mechanism.

  In another aspect, the cartridge includes a controller that can control power delivery from the power source to the high performance fluid actuator. The cartridge can include a power source operably coupled to the at least one high performance fluid actuator. The power source can be located in the external device and coupled to the cartridge by electrical connection. In some embodiments, the power source is electric or pneumatic. The power source can be a battery that can be located inside the cartridge. In other aspects, the battery can be located in an external device and coupled to the cartridge by electrical connection.

  The cartridge can include a second opening for receiving a processing fluid coupled to a network of fluid passages. The processing fluid can be contained within a network of fluid passages. The processing fluid can include a first reagent that can lyse cells or organelles. The first reagent includes a surfactant or other surfactant. In another embodiment, the first reagent comprises an enzyme such as lysozyme.

  In some embodiments, the processing fluid comprises a homogenization solution that can homogenize a tissue sample or other heterogeneous biological material.

  In other embodiments, the processing fluid comprises a solution that can reduce or eliminate the biological activity of a living cell, tissue or organism. The processing fluid can include glass beads or other solid material that can cause mechanical fracture of the starting material. In some embodiments, the processing fluid can include glycogen or other polysaccharides. The processing fluid can include carrier RNA.

  In some embodiments, the cartridge includes a third opening for receiving the actuator fluid coupled to the high performance fluid actuator. The actuator working fluid can be positioned within at least one high performance fluid actuator.

  In another embodiment, the portion of the network of fluid passages includes a second reagent. The second reagent can include silica beads, particles or paramagnetic beads. The second reagent can also be a fluorescent bead or fluorescent molecule. The second reagent can be an alkaline phosphatase substrate or a chemiluminescent molecule such as a lanthanide or a lanthanide chelate. In other embodiments, the second reagent comprises a monoclonal or polyclonal antibody, and the monoclonal or polyclonal antibody can be linked to a signaling molecule.

  The second reagent can be an oligonucleotide probe or primer, or a combination of probes or a combination of primers. Oligonucleotide probes can be used for human immunodeficiency virus, hepatitis C virus, hepatitis B virus, M. tuberculosis bacteria, C. trachomatis bacteria, influenza viruses, respiratory organs It can specifically bind to a defined region of genetic material of the endoplasmic reticulum virus or another virus of the human respiratory tract. The oligonucleotide probe can bind to a defined region of oncogene DNA or RNA. In some embodiments, the oligonucleotide probe is labeled and the label is a fluorescent or luminescent signaling molecule or quencher, aptamer, photosensitizer molecule, photoactive indicator precursor molecule, or photosensitizer. It can be a sensitizer molecule and a photoactive indicator precursor molecule.

  In some embodiments, the photosensitizer molecule and the photoactive indicator precursor molecule comprise: one or more sensitizers, one or more sensitizer oligonucleotides, and such sensitizers And at least one sensitizer labeled particle comprising a matrix for co-locating the sensitizer oligonucleotide; and one or more emitter agents, one or more sensitizer oligonucleotides, and At least one emitter-labeled particle comprising a matrix for co-locating such emitter agent and emitter oligonucleotide. The photosensitizer molecule can be in an excited state that generates singlet oxygen molecules. The photoactive indicator precursor molecule can react with singlet oxygen molecules to form a photoactive indicator.

  In other embodiments, the second reagent can be a quantum dot or other crystalline semiconductor particle. The second reagent can be a nucleic acid specific fluorescent or luminescent dye for sequence independent measurement of the nucleic acid. The second reagent can be a molecule that can participate in Forster resonance energy transfer (FRET) or other resonance energy transfer processes. In another embodiment, the second reagent comprises a labeled protein, labeled nucleic acid, or labeled carbohydrate species for measurement of specific cellular compounds.

  The second reagent can include a solution having a dye for specific or non-specific labeling of cells. The second reagent may be a primer, a probe, a combination of primer and probe, or polymerase chain reaction, transcription-mediated amplification, amplification based on a nucleic acid sequence, or another for amplifying at least one specified nucleic acid sequence Enzymes can be included that can catalyze chemical reactions. The enzyme can be a DNA polymerase, reverse transcriptase, RNA polymerase, ribonuclease H (RNAse H), DNA helicase, or recombinase.

  In another embodiment, the starting material comprises a fluid phase, a fluid-containing matrix or a solid phase. The starting material can be blood, sputum or other body fluid. Starting materials can include biological tissue, raw materials or intermediates for drugs or vaccines, agricultural products, soil, or another environmental sample.

  In one aspect, the cartridge includes a first fluid passage containing a first material and a second fluid passage containing a second material, wherein the first fluid passage and the second fluid passage are Forming a confluence in the microfluidic cartridge. In another embodiment, the meeting point is a T-shaped meeting point or a Y-shaped meeting point. In yet another embodiment, the confluence point allows formation of one or more microfluidic droplets resulting from the fusion of the first and second materials from the first and second fluid passages. In other embodiments, each of the one or more droplets contains an analyte or reagent. In another embodiment, each of the one or more droplets is at least one primer and a polymerase chain reaction, transcription-mediated amplification, nucleic acid sequence-based amplification, or another for amplifying at least one target nucleic acid sequence. An enzyme capable of catalyzing the chemical reaction of In some embodiments, each of the one or more droplets includes a label. In other embodiments, the first or second material comprises a processing fluid. In another embodiment, each of the one or more droplets comprises a cell.

  In another embodiment, the cartridge includes a plurality of fluid passages including different temperature zones for performing the stages of the amplification reaction. In one embodiment, a plurality of fluids are combined within the plurality of fluid passages to induce a labeling or hybridization reaction.

  The present invention includes a system that includes a microfluidic cartridge as described above and a device that includes a power source and in some aspects is adapted to supply power to the microfluidic cartridge. In other embodiments, the microfluidic cartridge has an on-board power source. The device is further adapted to sense an indicator of the assay result. The sensor can sense visible light or another type of electromagnetic radiation generated inside the cartridge. In some embodiments, the device is further adapted to sense the location or distribution of paramagnetic beads within the cartridge. The apparatus can be adapted to sense electron spin nuclear magnetic resonance or other physical properties of species inside the cartridge.

Another aspect includes a method comprising providing a first fluid to a channel in a microfluidic cartridge connected to a plurality of fluid passages and including at least one confluence between the fluid passages. The microfluidic cartridge includes at least one high-speed microfluidic actuator having a fluid power generation capability of at least ^10-8 watts capable of sustaining power for at least 30 seconds and a response time for power generation of less than 10 seconds . The method includes operating a microfluidic actuator in a time-varying manner, so that the first fluid and the second fluid are introduced into a network of fluid passages to generate alternate plugs of fluid, where The length of each plug volume is less than 5 times the smallest average diameter of such fluid passages. High-speed microfluidic actuators can generate fluid power by electrokinetic effects. The electrokinetic effect can be generated by an electroosmotic flow generated within an array of slits, a packed bead bed, or an integral porous structure.

  The method includes labeling a subset of cells within the first fluid with a labeled molecule or labeled particle within the second fluid that is specific for at least one type of molecule within the cell membrane. The method can include the step of coloring the cells in the first fluid using a cell penetrating dye contained in the second fluid.

  In other embodiments, the method uses DNA or RNA contained within the first fluid using a photosensitizer molecule or photoactive indicator precursor molecule contained in the second fluid, or a combination thereof. Labeling a subset of. The method can also include labeling a subset of DNA or RNA contained within the first fluid using a lanthanide chelate contained in the second fluid. The method includes lysing cells or other biological material within the first fluid with a surfactant or other surfactant contained in the second fluid. The surfactant can be sodium lauryl sulfate, hexadecyltrimethylammonium bromide or another cationic surfactant.

  In another embodiment, the method includes lysing cells or other biological material within the first fluid using an enzyme. The enzyme can be lysozyme. The method further includes homogenizing a tissue sample or other heterogeneous biological material from the first fluid. The method also includes reducing the biological activity of the living cell, tissue or organism in the first fluid. The step of reducing biological activity can include using a highly basic solution such as sodium hydroxide or sodium hypochlorite.

  The method further includes lysing cells or other biological material in the first fluid using glass beads or other solid material for mechanical disruption in the second fluid. The method includes mixing the swab or porous matrix with a first fluid and releasing the soil or other environmental sample bound within the swab or porous matrix.

  In one embodiment, the first fluid comprises dendritic cells and the method comprises pulsing the dendritic cells to induce an element of an immune response to the attack.

  The method can include generating a pharmacological substance or vaccine. The method includes increasing the biological activity of the pharmacological agent. The method can also include binding DNA or RNA molecules contained within the first fluid to glycogen or silica. The method also includes purifying glycogen complex or co-precipitated DNA and RNA, or purifying DNA or RNA molecules bound to silica beads or silica-containing structures. The method includes eluting DNA and RNA from glycogen or silica beads or silica-containing structures.

  The method also includes detecting the presence or absence of the analyte in the first fluid. The detecting step includes sensing visible light or another type of electromagnetic radiation from chemiluminescent or fluorescent molecules bound to the analyte. The detecting step can include sensing the location or distribution of paramagnetic beads bound to the analyte, or sensing the nuclear magnetic resonance or other physical property of the species bound to the analyte. .

In one aspect, the method also includes a step for generating a plurality of microdroplets in the plurality of fluid passages. In another embodiment, the plurality of microdroplets are formed by pulsating at least two fluids, wherein the pulsation occurs by a plurality of high speed microfluidic actuators within the microfluidic cartridge. The method can also include detecting the presence or absence of an analyte in each of the plurality of microdroplets. In another aspect, the method includes performing an amplification reaction in each of the plurality of microdroplets by moving the plurality of microdroplets through a plurality of temperature zones in the microfluidic cartridge. In yet another embodiment, the method includes detecting the presence of a target amplicon in each of the plurality of microdroplets. The method also includes measuring the melting temperature (T _m ) of the target nucleic acid molecule in each of the plurality of microdroplets. In one embodiment, the method comprises performing a melting temperature ( _Tm ) analysis of genetic differences in viral RNA from a reference strain.

  The figures illustrate various aspects of the present invention for purposes of illustration only. Those skilled in the art will readily appreciate from the following description that alternative embodiments of the structures and methods illustrated herein may be used without departing from the principles of the invention described herein. right.

2 is an example of a cutaway view looking down from above the interior of a microfluidic cartridge, in accordance with an aspect of the present invention. Treatment of fluids each containing a single lysate according to one embodiment of the invention, and two lysates spatially averaged over a short channel section of the fluid path downstream of the confluence and plotted as a function of time The concentration of each of the substances is illustrated. FIG. 6 illustrates a graph illustrating a functional relationship between maximum voltage and plug width for two fluids downstream of a junction according to one embodiment of the present invention. FIG. 4 illustrates various flow path confluence geometry according to one aspect of the present invention. FIG. 4 illustrates a graph showing plug width versus microactuator voltage for a microfluidic cartridge, according to one aspect of the invention. FIG. 6 illustrates a graph showing plug width versus microactuator voltage for various neck down diffuser junction design in a short plug width region, according to one aspect of the present invention. 2 is an example of a side cutaway view of a microfluidic cartridge, according to one embodiment of the invention. 2 is an example of a side cutaway view of a microfluidic cartridge including an opening, according to one embodiment of the invention. FIG. 2 is an example of a side cutaway view of a microfluidic cartridge including a viewing window, according to one aspect of the invention. FIG. 2 is an example of a side cutaway view of a microfluidic cartridge including a microfluidic actuator and electrodes, according to one aspect of the invention. FIG. 2 is an example of a fluid stopper in an internal channel of a microfluidic cartridge, according to one aspect of the invention. 2 is an example of an instrument docked to a microfluidic cartridge according to an aspect of the present invention. 3 illustrates an example of a fluid plug generated within a fluid passage of a microfluidic cartridge, according to one aspect of the present invention. 2 is a photograph of a microfluidic cartridge and instrument for improved microfluidic processing in accordance with an aspect of the present invention. 2 is a photograph of a microfluidic cartridge for improved microfluidic processing in accordance with an aspect of the present invention. 1 is an isometric view of an exemplary microfluidic cartridge for performing a process on a sample, according to one aspect of the invention. FIG. 1 is a top view of a microfluidic cartridge according to an aspect of the present invention. FIG. FIG. 4 illustrates the high uniformity of microfluidic cartridges for use in biochemical processes at different times under the same defined conditions, according to one aspect of the invention. A series of bead binding experiments were performed with oligonucleotide targets present in the starting solution at concentrations of 1 × 10 ⁻¹³ M, 1 × 10 ⁻¹² M and 1 × 10 ⁻¹¹ M. Under the control of a high-performance actuator, the target-containing solution is mixed with a solution containing two types of beads, using singlet oxygen as an intermediate so that the emitted light persists after the excitation source is extinguished. Fluorescence was measured at approximately 610 nanometers. The plotted value is an indication of the target starting concentration. At least 10 assays were performed at each concentration. FIG. 3 illustrates the amount of time required for a biochemical reaction to reach the desired endpoint using the microfluidic cartridge of the present invention. An assay similar to that described for FIG. 18 was performed. 2 is an example of a potential waveform applied to a pair of high performance actuators to achieve rapid pulsatile flow at a microfluidic junction, according to one aspect of the invention. FIG. 4 illustrates an exemplary confluence geometry for synchronizing fluid mixing in accordance with an aspect of the present invention. 2 is a process flow diagram for a quantitative real-time polymerase chain reaction assay using a microfluidic cartridge of the present invention for applications such as quantification of HIV genetic material, according to embodiments of the present invention. FIG. 4 illustrates an exemplary configuration for using the microfluidic cartridge of the present invention in the treatment of a compartment of fluid according to aspects of the present invention, wherein each compartment or each set of compartments is of that compartment or set of compartments. Can go through a process that is selected for. FIG. 4 illustrates individual processing of a fluid compartment or set of compartments using a microfluidic cartridge, according to aspects of the present invention. FIG. 4 illustrates an example of individual processing of a fluid compartment or set of compartments using a microfluidic cartridge, according to aspects of the present invention.

Detailed Description of the Invention
The flow in the schematic microfluidic processing system, typically referred to as the low Reynolds number region, associated with dominance of viscous effects on inertial effects [1], [2]. Many applications of microfluidic processing systems involve one or more high molecular weight reactants with correspondingly low binary diffusivities [3], [4]. For example, molecular dynamics simulation [5] shows that a ribonucleic acid chain of approximately 9800 bases constituting the human immunodeficiency virus (HIV) genomic material having a molecular weight of 3.1 × 10 ⁶ daltons is approximately D = 2 × 10 ⁻ It shows that it has an underwater diffusivity of ¹² m ² s ⁻¹ , so that one-dimensional diffusion in 10 minutes is associated with a displacement of only 50 microns. The combination of the viscous effect over the inertial effect and the relatively slow diffusivity of the reactants of interest is a fluid mechanics for macroscopic mixing of two or more solutions in a microfluidic system. Imposes the need for a mechanism.

  When an aqueous solution contacts a surface such as glass or silica, the surface becomes negatively charged due to depronation of surface silanol groups. As a result of depronation, an electric double layer results. The surface charge attracts dissolved counter ions and repels co-ions, resulting in charge separation. The Debye length is the characteristic thickness of the double layer. Mobile ions in the diffusion counterion layer are driven by an externally applied electric field, and the moving ions pull the bulk liquid by viscous force interaction.

式中、aは、2つのポンピング表面の間の分離距離の二分の一であり、μは、流体粘性であり、dp/dxは、流れに反する圧力勾配であり、εは、流体誘電率であり、ζは、ゼータ電位であり、αは、イオンエネルギーパラメータであり、Gは、二重層の厚みのための補正項である。幅広平行表面が帯電し、対イオンを引きつけ、共イオンに反発して、電荷二重層を形成する。二重層のイオンの外層は、可動性である。軸方向電場を加えることは、可動イオンに力を及ぼし、可動イオンのエレクトロマイグレーションは、粘性相互作用によってバルク流体を引っ張る。ゼータ電位は、電気浸透流上の表面状態の効果を特徴付ける。ゼータ電位は、表面/流体界面の近くの正味過量の表面電荷平衡イオンと関連する経験的なパラメータである。 The average velocity of electroosmotic flow generated between two wide parallel surfaces by applying an axial electric field Ex is:

Where a is half the separation distance between two pumping surfaces, μ is the fluid viscosity, dp / dx is the pressure gradient against the flow, and ε is the fluid dielectric constant. Yes, ζ is the zeta potential, α is the ion energy parameter, and G is the correction term for the bilayer thickness. The wide parallel surface is charged, attracts counterions and repels coions to form a charge double layer. The outer layer of bilayer ions is mobile. Applying an axial electric field exerts a force on the mobile ions, and the electromigration of the mobile ions pulls the bulk fluid by viscous interaction. Zeta potential characterizes the effect of surface conditions on electroosmotic flow. Zeta potential is an empirical parameter associated with a net excess of surface charge equilibrium ions near the surface / fluid interface.

Definitions Terms used in the claims and specification are defined as set forth below unless otherwise specified.

  “Electroosmotic flow” refers to the movement of a liquid induced by an applied voltage across a fluid conduit. The fluid conduit can be any porous material, capillary tube, membrane, substrate, microchannel or passage to allow the flow of liquid. An electric potential can be applied between any two parallel surfaces.

  “Microfluidic actuator” or “fluid actuator” refers to a component that converts power or another form of easily stored or generated energy into fluid power and applies a force to the mass of fluid to create a pressure gradient Means to transport the mass of fluid [6].

  “Taylor dispersion” refers to transporting and propagating a solute mass in a laminar flow through a long, straight tube or other similar flow passage, which initially is a solute mass within the flow. Enclosed in a plug (or plugs), such plug has an axial dimension comparable to the tube cross-section [2].

  “Zeta potential” refers to an empirical or semi-empirical parameter that is included in many mathematical models of electroosmotic flow, and when other factors are equal, a higher absolute value of zeta potential is generally higher flow rate and [7], [8] associated with higher back pressure.

  It should be noted that as used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is.

Microfluidic System Overview The present invention includes microfluidic systems such as cartridges or similar sealed fluid processing devices. In some embodiments, the microfluidic cartridge has no mechanical moving parts and eliminates failure modes associated with sliding contact, fluidic fitting, and the like. In one aspect, the microfluidic cartridge runs on battery power and incorporates EO fluid activation without the need for an external syringe pump or any other means of fluid activation. In another aspect, the microfluidic cartridge includes an internal mechanism for moving fluid pressurized by the microfluidic actuator.

In some embodiments, the microfluidic cartridge is small in size and can be used with handheld portable devices. For example, the cartridge can be less than 40 cm ³ in volume (2 cm × 2 cm × 10 cm = 40 cm ³ ). In addition, the cartridge can have a drainage of 50-500 cc. For example, the cartridge is small enough to fit in a human hand and can be dimensioned for mass production at low cost.

  The microfluidic system includes a network of fluid passages. The passageway can include pipes, tubes, sealed channels or other sealed structures to hold the fluid and allow fluid transport. The fluid passage can be loaded with a small amount of at least two different fluids. The fluids can have a volume of, for example, less than 10 milliliters each. In one embodiment, at least one of the fluids is loaded into the cartridge through the port during or around operation. In other embodiments, the fluid is preloaded in the cartridge.

  The network of fluid passages can be connected by one or more junctions. Each confluence can be configured in various arrangements and designs, combining two or more fluid passages.

The microfluidic cartridge includes at least two microfluidic actuators, and the at least one microfluidic actuator is a high performance microfluidic actuator. At least one high performance microfluidic actuator has a fluid power generation capability of at least ^10-8 watts, can sustain power for at least 30 seconds, and has a response time for power generation of less than 10 seconds . The network of fluid passages is in fluid communication with the microfluidic actuator.

  The microfluidic system can be a cartridge made from plastic, glass or other material. Fluid passages and other features inside the cartridge can be generated by machining, hot embossing, injection molding or other means. The cartridge can be assembled from multiple parts by thermal bonding, laser welding, ultrasonic welding, or by use of an epoxy resin or pressure sensitive adhesive or other adhesive means.

  The microfluidic actuator of the present invention can operate through the generation of electroosmotic flow.

  The microfluidic actuators of the present invention can be made from silicon, glass, plastic or other materials. In some embodiments, the microfluidic actuator is made from a single crystal silicon wafer coated with multiple layers of silicon oxide and silicon nitride, and an opening in the single crystal silicon wafer is formed by a photolithographic feature characterization process. definition process, followed by time-multiplexed inductively coupled plasma (TM), also known as deep-reactive ion enhanced (DRIE) etching [9] -ICP) Made by etching. Microfluidic actuators can be generated from single crystal silicon wafers by a simple one-step photolithography process. These microfluidic actuators are economical to integrate into disposable microfluidic cartridges for various applications.

  In some embodiments, the microfluidic cartridge is designed to dock or bind to an instrument for analyzing or processing the fluid or sample inside the cartridge. The instrument can include various detection or monitoring components for analyzing a fluid or sample, and can include a power supply or an electrical circuit for providing energy to the cartridge.

  In some aspects, the power source and associated electrical circuitry are contained within the cartridge, which operates without connection to external hardware.

  In FIG. 1, an example of a microfluidic cartridge 100 is shown from the perspective of a cutaway view from the top, inside the cartridge 100. The microfluidic cartridge includes a first fluid passage 101, a second fluid passage 102, a third fluid passage 103, and a junction 104 connecting the first, second and third fluid passages. The microfluidic cartridge includes a first pressure source and a second pressure source. Each of the pressure sources can be a microfluidic actuator 105b, 106b, at least one of which is a high performance microfluidic actuator. In some embodiments, the actuator may also include one or more pistons or piston-like elements 105a and 106a. In some embodiments, the piston-like elements 105a and 105b can be solid material plugs that form a perimeter seal with the interior of the fluid passageway through which the plugs travel.

  In one aspect, the microfluidic actuators 105b, 106b act on the processing fluid contained within the fluid passages 101, 102 via pistons or piston-like elements 105a and 106a. For example, the operation of the first microfluidic actuator 105b pushes the piston 105a of the actuator forward. The movement of the piston 105a pressurizes the fluid in the fluid passage 101 and advances the fluid toward the confluence 104. Similarly, the operation of the second microfluidic actuator 106b pushes the second piston 106a forward. The movement of the piston 106a pressurizes the fluid inside the fluid passage 102 and advances the fluid toward the confluence 104. The two processing fluids are combined and mixed at the junction 104.

  In other embodiments, the pistons 105a, 106a are not present as solid elements in the cartridge. The actuator fluid is contained within the microfluidic actuators 105b, 106b or is in fluid contact with the microfluidic actuators 105b, 106b and is separated from the processing fluid in the fluid path by a barrier fluid plug. In some embodiments, the barrier fluid is air or another gas. Due to the fluid movement of the actuator fluid, the air plug is pressurized and moved forward, which in turn pressurizes the processing fluid in the fluid passage to cause fluid movement of the processing fluid. The function of the air plug as a piston is enhanced by the surface tension effect. In some embodiments, the inner surface of the fluid passageway through which the air plug passes is hydrophobic and lacks a distinct axial feature that facilitates the flow of the actuator working fluid along the wall of the air passage past the air plug. In some embodiments, a plug of immiscible fluid functions as a piston. In some embodiments, there is no fluid plug that separates the actuator fluid from the processing fluid, and the actuator fluid is in direct contact with the processing fluid within the fluid path, but the processing fluid (eg, two immiscible fluids) and Do not mix. Actuator fluid movement causes corresponding pressurization and movement of the processing fluid.

  Moving fluid through the fluid passage toward the junction 104 results in at least one fluid passing through the junction 104 and proceeding into the third fluid passage 103. For fluid passages having a cross-sectional dimension of less than 10 mm and containing a liquid phase fluid, the fluid flow within passages 101, 102 and confluence 104 can be characteristically laminar.

  The first and second fluid actuators 105b and 106b are such that the velocity and flow rate of the fluid within the first and second fluid passages 101 and 102 are substantially unchanged and constant over time and immediately beyond the junction 104. Can be actuated to provide a semi-individual fluid laminar flow in the region of the fluid passageway 103. If there is a series of fluid passage cross sections over a distance several millimeters beyond the confluence 104 in the direction of flow (referred to as the axial direction), the concentration of the first fluid is approximately 100% in a region of the cross section. And the concentration of the second fluid is approximately 100% in another region. The persistence of such spatial localization as a function of axial distance from the confluence 104 is approximately inversely proportional to the diffusivity of the species in the processing fluid.

  Deviations from the laminar flow operation described above, such as alternate plugs of processing fluid, may result from one or more time-varying actions of the microfluidic actuator, and one or more corresponding time-varying pressurization and flow of processing fluid. Can occur. In one example, the first microfluidic actuator 105b is operated with a square wave voltage input at a given frequency and less than 100% duty cycle, and the second microfluidic actuator 106b is less than 100% at the same frequency. Actuated with a square wave voltage input at a duty cycle, the first actuator square wave is out of phase with the second actuator square wave.

  FIG. 2 shows that the processing fluid is an aqueous solution each containing a single dissolved substance, and the concentration of each of the two dissolved substances is spatially averaged over a short channel area of the fluid passage 103 downstream of the confluence 104. And is plotted as a function of time. The out-of-phase motion of the actuator results in sequential injection of alternating plugs of fluid contained within the fluid passages 101 and 102. Due to the predominance of viscous forces over inertial forces, molecular diffusion can be a primary mechanism where the chemistry and biochemical components of such fluids mix when the two fluids are combined inside a microfluidic cartridge. A spatially non-uniform distribution of fluid can shorten the distance at which such diffusion occurs and accelerate chemical and biochemical reactions.

  FIG. 3 is an example of a functional relationship between the maximum voltage, duty cycle and duration of microactuator operation and the two types of plug width downstream of the junction 104. The data plotted in FIG. 3 was collected with a cartridge of the present invention in which the fluid passage is cylindrical with a diameter of approximately 1 mm. Microfluidic actuators convert electrical power into fluid power by the generation of electroosmotic flow in gaps inside slat structures containing silicon coated with silicon nitride and silicon oxide thin films. One of the two solutions contains a fluorescent species, so the plug width can also be monitored by an epifluorescence microscope equipped with a CCD camera. A voltage in the range of 75V to 175V was applied to two actuators operating with a phase shift, with 50% duty cycle, and on-state durations of 100 and 200 milliseconds. As shown, an axial 2 mm fluid stopper can also be created. Short plug downstream mixing can occur by Taylor dispersion.

  The minimum axial dimension of the fluid plug can be constrained by the cross-sectional dimension of the flow passage at the junction. FIG. 4 is an example of the geometry of the flow path merge point where the flow path necks down in the region immediately adjacent to the merge point or the cross-sectional dimension decreases.

  FIG. 5 shows that the combination of neck-down geometry and fast microactuator response can produce a very short plug of fluid.

  FIG. 6 illustrates a graph showing plug width versus microactuator voltage for various neck-down diffuser junction design in the short plug width region of FIG. At the confluence where the channel necked down from 1 mm diameter to 0.25 mm diameter, a 50 millisecond on-state duration was produced that was axially less than 50 microns. With this size plug, the first solution containing a relatively slowly diffusing species such as 200 nm diameter beads will mix thoroughly with the second solution in less than 10 minutes.

  For better control over differential fluid transport and / or for mixing multiple fluids, multiple microfluidic actuators use multiple channels and junctions to move and combine fluids Can be used. Each microfluidic actuator 105b, 106b is fluidly connected to the actuator fluid and generates a flow of processing fluid. For example, two microfluidic actuators 105b, 106b can produce a mixture of two processing fluids. The mixture can then be combined with a third fluid in another fluid passage using the fluid pressure of two additional microfluidic actuators.

  In some embodiments, the microfluidic cartridge 100 is loaded with two fluids: one fluid in the first fluid passage 101 and the other fluid in the second fluid passage 102. The In some embodiments, the fluid is loaded at or about the time of manufacture of the microfluidic cartridge. The microfluidic cartridge 100 can include an actuator fluid in fluid connection with each of the microfluidic actuators 105b, 106b.

  In other embodiments, the microfluidic cartridge 100 is loaded with reagents within the fluid passages 101,102. The reagent can be in the form of a fluid phase, a dried reagent, or attached to the surface or wall of the fluid passage (eg, a bead or particle). In some embodiments, the reagent is present in the processing fluid and includes a surfactant or other surfactant to lyse the cell or organelle. The reagent can be an enzyme such as lysozyme. In other embodiments, the reagent is an antibody, protein, peptide, oligonucleotide or particle for binding, hybridizing, or interacting with an analyte in a sample or processing fluid. Other examples of reagents are described in detail below.

  FIG. 7 is an example of the microfluidic cartridge 100 of FIG. 1, and shows the inside of the cartridge 100 from a side perspective view. As shown in FIG. 1, the microfluidic cartridge includes a first fluid passage 101, a second fluid passage 102, a third fluid passage 103, and a junction 104 where the first and second fluid passages meet. The microfluidic cartridge includes a first fluid actuator 105b and a second fluid actuator 106b. The cartridge also includes one or more pistons or piston-like elements 105a and 106a that are pushed forward by the first and second fluid actuators 105b, 106b.

  Referring now to FIG. 8, an opening 801 is shown on the top of the microfluidic cartridge 100 that can be used to contain a starting material, sample, or fluid for subsequent processing. it can. The opening is connected to a network of fluid passages. The opening can be connected to a fluid passage for processing the starting material. To prevent fluid from flowing out of opening 801 during operation of microfluidic cartridge 100, opening 801 can have a cap, capping element, plug or other type of closure 802. In some aspects, the opening 801 can be hermetically closed by a mechanism such as a pneumatic valve. The opening 801 can be self-sealed by a passive mechanism such as a perforated elastomeric structure that can elastically deform when acted upon by a narrow conduit such as a syringe. In other embodiments, the plug or capping element 802 can receive a fluid conduit and can be hermetically sealed when the fluid conduit is withdrawn. The fluid conduit can be a needle, tube, rigid fluid conduit or semi-rigid fluid conduit. The opening can also be closed by a thermophoric effect, an electromagnetic effect or an electrostatic effect.

  In one aspect, the microfluidic cartridge 100 includes at least one component or module to facilitate monitoring of fluid processing or to analyze the output of a fluid process. In FIG. 9, the microfluidic cartridge 100 is optically transparent, allowing observation or monitoring of fluid in the third fluid passageway 103, such as fluid color, opacity and other such physical properties. Including area 901. The transparent region 901 can allow analysis of the fluid inside the fluid passage 103 using techniques such as fluorescence, chemiluminescence or other analysis methods such as those described herein.

  In FIG. 10, a first microfluidic actuator 1001 is shown, having a perforated structure 1001a, a fluid passage having at least one cross section within 3 digits of the characteristic thickness of the electric double layer, and a perforated structure A perforated structure 1001a having at least one electrode on each side thereof. The electrodes are electrically connected to the metal contacts and are placed on both sides of the microfluidic actuator 1001. An electric field is applied across the electrodes. In one embodiment, the electric field is applied across the electrodes by a trace or wire 1002 that runs through or along a portion of the microfluidic cartridge and terminates at a contact 1003.

  In other embodiments, the microfluidic actuator 1001 is coupled to a pulse generator or other controlled time varying voltage source and at least a pair of electrodes. A pulse generator or controlled time-varying voltage source can produce a pattern of voltage pulses or a time-differential voltage pulse on the microfluidic actuator 1001.

  In some embodiments, electroosmotic flow is generated within a plurality of slit capillaries within microfluidic actuator 1001. The electroosmotic flow can also be generated within a bed of packed beads within the microfluidic actuator 1001, within an integral porous structure, or within an array of cylindrical channels.

  In other embodiments, the microfluidic actuator 1001 is filled with an actuator fluid having chemical properties that facilitate formation at the fluid solid interface of the electric double layer with a very effective zeta potential (eg, primarily oxygen and silicon). An aqueous solution for perforated structures having an internally perforated surface containing). The application of an electric field generates an electroosmotic flow within the microfluidic actuator 1001 perforations. For perforated structures of insulating silicon with slit-like perforations having a smaller cross-sectional dimension between 1 and 10 microns, such electroosmotic flow can be induced by fluid resistance and / or for pressure heads of 10 kPa or more. Can be driven into the passage 101. The pressure associated with electroosmotic flow can occur within a few microseconds, and the first fundamental limitation is the rate of momentum diffusion from the wall of each slit-like perforation to the center plane of each perforation .

In one aspect, the microfluidic actuator 1001 has a fluid power generation capability of at least ^10-8 watts, can sustain power for at least 30 seconds, and the response time for power generation is, for example, less than 10 seconds Less than 2 seconds, less than 0.2 seconds or less than 0.04 seconds. The microfluidic actuator can also pressurize at least 10 microliters of liquid so that the liquid flows through a fluid resistance associated with a pressure drop of at least 1 kPa at a flow rate of at least 0.1 mL per minute.

  The microfluidic actuators of the present invention are superior by being small enough to fit a defined size cartridge, by extracting relatively small power, and by fast response times. Each microfluidic actuator circulates on and off (or transitions between different fluid power generation states) at 0.1 hertz or faster, preferably 1 hertz or faster, more preferably 10 hertz or faster )can do. Equivalently, a microfluidic actuator has a rise time of 10 seconds or less, or a rise time of 1 second or less, or a rise time of 0.1 seconds or less.

  Fast response time and high output are important. The reason is that the reaction rates of the two species initially contained within separate fluid phases are such that when the two fluid phases are introduced into the reaction channel with short individual plugs, the two fluids are continuously Or one dimension that is much larger than the other two dimensions, such as when the two fluids are replaced by a well (ie, pipe or sealed channel) instead of being introduced into the reaction channel with a long plug This is because it is significantly faster than when introduced into a container having an internal dimension aspect ratio of approximately 1 (unity).

式中、Uは、平均速度であり、rは、動径座標であり、aは、円筒形通路の半径である。栓が流体通路を下って移動するにつれて、放物線状流れプロファイルは、対応する栓ひずみ1101、1102を引き起こす。栓と共に収容されている粒子は、ひずんだ栓1103から半径方向に拡散することができる。粒子は、流体通路中心線近くの栓前部から半径方向外側1103aと、壁近くの栓後部から半径方向内側1103bに、拡散する。この現象は、テイラー分散として知られており、それは2つまたはそれ以上の流体の効率的な混合を生じる。同様の拡散効果が、非円筒形流体通路内で生じることができる。 Referring now to FIG. 11, a schematic diagram of the interior of the passage in the microfluidic cartridge 100 is shown. Spatial inhomogeneity can facilitate the reaction of two fluid phases by sequential injection of alternating plugs of fluid followed by a series of plug pressure driven flows through the fluid passageway 1100. Fluid flow in the low Reynolds number region can be well modeled by assuming that the flow velocity at the fluid passage 1100 wall is zero (no slip boundary condition). For cylindrical passages, the radial flow velocity profile is parabolic, described by the following equation:

Where U is the average velocity, r is the radial coordinate, and a is the radius of the cylindrical passage. As the plug moves down the fluid path, the parabolic flow profile causes a corresponding plug strain 1101, 1102. Particles contained with the plug can diffuse radially from the distorted plug 1103. Particles diffuse from the plug front near the fluid path centerline radially outward 1103a and from the plug back near the wall radially inward 1103b. This phenomenon is known as Taylor dispersion, which results in efficient mixing of two or more fluids. Similar diffusion effects can occur in non-cylindrical fluid passages.

  In FIG. 12, the micro docks to instrument 1200 useful for facilitating improved fluid processing, for monitoring processing, for analyzing process output, or for other processing steps. A fluid cartridge 100 is shown. The microfluidic cartridge 100 can include a sensor that senses visible light or another type of electromagnetic radiation generated within the cartridge. In one embodiment, the instrument 1200 includes an optical detector or other sensor 1201, such as a CCD imager or photomultiplier tube. In another embodiment, the microfluidic cartridge 100 includes a detector for detecting fluorescence emission from fluorescently labeled molecules.

  In one aspect, the instrument 1200 houses a power source and an electrical circuit 1202 for supplying a time varying voltage or other input to the microfluidic actuator 1001. In another embodiment, the control voltage is supplied via a pin-based interconnect 1203 connected to a power source / controller by a ribbon cable 1204. In some embodiments, the power supply is a battery.

  In other embodiments, the microfluidic cartridge 100 is coupled to an external power source. An external power source can be coupled to the microfluidic cartridge 100 by electrical connection. The microfluidic cartridge 100 can include a controller that can control the power supply from the power source. The power source can be operably coupled to the microfluidic actuator 1001. In some embodiments, the power source is electric, pneumatic, or a battery. The battery can be located inside the external device or can be coupled to the microfluidic cartridge 100 by electrical connection.

  In some embodiments, the cartridge components are produced from specialized polystyrene and / or ABS plastic resin by injection molding. Cartridge component bonding is by die-cut pressure sensitive adhesive, by thermal bonding, by ultrasonic welding, by laser welding, by epoxy resin, by a combination of these means, or by other means Can do.

  FIG. 13 is an example of alternating fluid stoppers generated within the fluid passages of a microfluidic cartridge according to one embodiment of the present invention.

  FIG. 14 illustrates an exemplary microfluidic cartridge 1400 and instrument 1401 for improved microfluidic processing, according to embodiments of the present invention. The outer housing of instrument 1401 has been removed to show the internal configuration. Microfluidic cartridge 1400 includes microfluidic actuator 1401 (four actuators outlined in black inside the cartridge). FIG. 14 shows four microfluidic actuators 1401 inside the cartridge 1400. The microchannel network 1405 is formed of the plastic material of the cartridge 1400. The microchannel network 1405 includes channels that connect to each of the two fluid ports of the microfluidic actuator 1401 and each of the two fluid ports of the other three microfluidic actuators. Circuit board 1404 includes electrical contacts for each of the microfluidic actuator electrodes. Electrical contacts are routed to instrument 1401 via cable 1406 using an interconnector. Instrument 1401 includes a microprocessor, power management hardware, and other components for controlling the voltage applied across the electrode pair of actuator 1401 and across the other three actuator electrode pairs. The instrument functions include obtaining independently controlled potentials of 100 volts, 200 volts, 400 volts or other voltages, such potentials being switchable under microprocessor control at frequencies above 10 Hz. .

  FIG. 15 illustrates a microfluidic cartridge 1400 for improved microfluidic processing according to an embodiment of the present invention. The bottom plate 1500 and the top plate 1501 of the cartridge are shown separated from each other in this view to show the internal configuration. When the two plates are fitted together, they form a microfluidic cartridge 1400 similar to that shown in FIG. The cartridge is configured for four high performance microfluidic actuators, each in a different stage of assembly in this photo. The cartridge 1400 includes a bottom electrode 1502 and a semiconductor chip 1503, includes a slat structure for generating an electroosmotic flow positioned on the bottom electrode, and includes an intervening chip sealing gasket. Another semiconductor chip 1504 is similar to 1503 and includes an additional gasket placed over the slat structure semiconductor chip. The cartridge 1400 also includes an upper electrode 1505 having an additional gasket.

  FIG. 16 is an isometric view (mechanical drawing) of an exemplary microfluidic cartridge 1600 for performing a process on a sample, according to an embodiment of the invention. Cartridge 1600 includes inlet ports 1601 and 1602 that can fluidly interact with a module containing at least one high performance fluid actuator. Each of the fluid passages 1604 and 1605 can hold a volume of fluid. In one example, one of the fluids can be butanol or another precipitating agent. In another example, one of the fluids can contain a complex that tends to precipitate, such as a polysaccharide-binding nucleic acid. The geometry inside the fluid passages 1604 and 1605 can be designed such that a defined fluid exhibits a defined flow characteristic within the passage. For example, a fluid passage carrying butanol has a smaller cross-sectional dimension (compared to a fluid passage to hold an aqueous solution) to better maintain butanol flow head integrity during transport driven by a microfluidic actuator ). The cartridge 1600 can include a chamber for receiving a reactant. The cartridge 1600 can be made from a cyclic olefin polymer or other polymer. The cartridge 1600 can include elements formed from two or more materials so that the cartridge area intended to store the solvent can withstand degradation over time and achieve other design goals. . The cartridge 1600 can include a chamber 1606 into which two solutions are transported by the action of one or more microfluidic actuators, at least one of which is a high performance microfluidic actuator. Chamber 1606 can be configured such that buoyancy effects associated with different densities of two solutions or phases facilitate mixing of the two solutions or phases. The two solutions can be a solvent and a nucleic acid containing solution. Mixing of the solvent and nucleic acid-containing solution requires that the fluid be transported into the chamber 1606 where the withdrawal of one or more liquid phases from the chamber can result from surface tension effects, buoyancy effects, or a combination of these effects. , Bubbles are held in such a chamber. The cartridge 1600 includes a component 1607 that incorporates a porous structure. Nucleic acid-containing solutions or other solutions can be passed through a porous structure, for example. This passage can be followed by a flow of a solvent such as ethanol through the porous structure to wash away unbound material such as proteins. Nucleic acids can be eluted into channel 1608 by passing water through the porous structure.

  FIG. 17 is a top view of an exemplary microfluidic cartridge according to an embodiment of the present invention.

FIG. 18 shows data from experiments performed using microfluidic cartridges or the present invention. The data shows that biochemical processes and other processes with high uniformity in the results of multiple processes are performed at different times under the same defined conditions. A series of experiments were performed with oligonucleotide targets present in the starting solution at concentrations of 1 × 10 ⁻¹³ M, 1 × 10 ⁻¹² M, and 1 × 10 ⁻¹¹ M. Under the control of a high performance actuator, the target-containing solution was mixed with a solution containing two types of beads. Since two types of beads have been functionalized with two types of probes, each oligonucleotide target will tend to bind to one of each bead. The beads were colored such that excitation with light at approximately 680 nanometers resulted in emission at approximately 610 nanometers, with singlet oxygen as an intermediate. Therefore, the emitted light persists after extinguishing the excitation source. The plotted value is an indication of the target starting concentration. At least 10 assays were performed at each concentration. The data was jittered for clarity.

  FIG. 19 shows data from a microfluidic cartridge or experiment performed using the present invention. The data indicates that microfluidic cartridges can be used to reduce the time required to reach a desired endpoint, such as a signal where a biochemical reaction or other process exceeds a minimum threshold . An assay similar to that described with respect to FIG. 18 was performed. The target-containing solution and bead-containing solution were mixed at the confluence by flow driven by a high performance microfluidic actuator. The assay was performed under the following two conditions: 1) using a rapid pulsating flow of fluid while the fluid meets at the confluence, and 2) using a continuous flow while the fluid meets at the confluence. The combined solution was then incubated for 10 or 20 minutes before reading. As a control, the assay was also run without target in the target solution. As shown in FIG. 19, the luminescence signal for a 10 minute incubation with rapid pulsatile flow (approximately proportional to the number of bead pair-target complexes formed) is for the 20 minute incubation with laminar flow. Comparable to the signal.

  FIG. 20 is an example of a potential waveform applied to a pair of high performance actuators to achieve rapid pulsating flow at a microfluidic junction.

  FIG. 21 illustrates a confluence geometry for synchronizing fluid mixing using the present invention. The first flow path 2101 and the second flow path 2102 enter the junction 2100. The cross-sectional area of the second flow passage 2102 immediately adjacent to the merge point is smaller than the cross-section of the first flow passage 2101. The first flow path 2101 passes through the confluence along a substantially straight line and transitions to the third flow path 2103. The second flow passage 2102 forms an angle with both the first and third flow passages 2101, 2103. The reduction in the cross-section of the second flow passage 2102 immediately adjacent to the meeting point causes the hydrophobic solution to tend to form a meniscus at the meeting point. Under a pulsating flow driven by a high performance actuator, the flow front 2104 in the second flow passage 2102 is an alternating convex and concave meniscus despite the application of net positive fluid power to the fluid volume by the actuator. With formation, it can be held at the junction. This stall effect can be maintained until the flow head 2005 moving forward from the first flow path 2101 reaches the confluence and contact occurs between the two flow heads. This effect, and similar such effects, can be used to synchronize fluid mixing. Synchronized fluid mixing may be associated with better reproducibility between runs and other favorable assay performance characteristics.

  FIG. 22 is a process flow diagram for a quantitative real-time polymerase chain reaction assay using the present invention for applications such as quantification of HIV genetic material. Sample 2200, which may contain genetic material of interest, such as bacterial DNA or messenger RNA, or viral RNA, includes a microfluidic channel and at least two fluid actuators 2204, at least one of which is a high performance fluid actuator Installed in the system. The fluid actuator facilitates the polymerase chain reaction involving the genetic material in the sample in the polymerase chain reaction module 2203. The polymerase chain reaction process can be preceded by a reverse transcription process in reverse transcription module 2203. The fluid actuator facilitates reverse transcription of RNA contained in the sample in the reverse transcription module 2202. Such reverse transcription can be preceded by a sample preparation process in the sample preparation module 2201. Such a sample preparation process can be facilitated by the action of at least one high performance microfluidic actuator.

  FIG. 23 shows a configuration for using the microfluidic cartridge of the present invention in the processing of a compartment of fluid, wherein each compartment or set of compartments is selected for that compartment or set of compartments. You can go through the process. A cartridge or other fluid network 2300 houses the main channel 2301. In fluid connection with the main channel is an array 2302 of at least two secondary channels. The secondary channel including the secondary channel array is fluidly connected to at least one microfluidic actuator 2304. The main channel is fluidly connected to the microfluidic actuator 2303. At least one of the microfluidic actuators 2303 and 2304 is a high performance microfluidic actuator.

  FIG. 24 shows the individual processing of a fluid compartment or set of compartments using the microfluidic cartridge of the present invention. A volume of the first fluid 2400 contained within the channel 2301 can be pressurized and transported by the action of the microfluidic actuator 2303. A volume of second fluid 2401 in the secondary channel within secondary channel array 2302 can be pressurized and transported by the action of microfluidic actuator 2304. Additional secondary channels within the secondary channel array are pressurized by microfluidic actuators and other means to inject the volume of fluid in the secondary channel into the volume of fluid in the first channel at the junction 2402 Can do. A volume of immiscible fluid or air or other gas 2403 can be injected to partition the fluid in the main channel, such a partition corresponds to a particular secondary channel, and therefore the fluid in a particular fluid compartment. Mainly comprises a first fluid combined with a defined second fluid injected from a particular secondary channel. Such a mixture of a first fluid and a defined second fluid can travel into a downstream channel, chamber or other fluid container 2405.

  In some embodiments, the microfluidic cartridge of the present invention is an inlet for a purified sample (sample preparation can be incorporated as needed) added via a pipette; to drive the assay process Integrated charging slit actuator; amplification module; sample is first reconstituted PCR primer and then circulated through three different temperature zones for reverse transcriptase process and amplification Droplet module that generates a series of droplets containing beacons; a melting temperature scanning zone, where reassortant resolution, where the main actuator reciprocates the droplets back and forth through the detection zone At the same time, a unique photochemical thermosensitive method uses a temperature of each drop at each point between the lamps. The used to determine accurately, including the melting temperature scanning zones.

  FIG. 25 shows an example of a droplet design in a microfluidic cartridge that uses separate processing of fluid compartments according to an embodiment of the invention. Various subchannels under the control of individual microfluidic actuators (eg, an array of 24 charged slit actuators) turn the solution containing the lyophilized beacon probe into an amplicon solution (output from the amplification module). Sequential injections can generate a series of droplets that travel down the primary reaction channel or fluid path toward a detection region (eg, a melt temperature analysis zone or a fluorescence detector). In some embodiments, rapid pulsatile flow driven by individual charged slit microactuators can be used to accelerate probe binding to a large amount of slowly amplifying target amplicons. In one aspect, the charged slit actuator used for droplet generation operates temporarily and continuously. One high voltage signal will be applied to the cartridge for the droplet generating charged slit actuator and will be transmitted to the appropriate actuator by the high voltage on the cartridge that demultiplexes on the printed circuit board of the cartridge.

The inventive method microfluidic cartridge can be produced from individual plastic components and individual microfluidic actuators. The components can be assembled by various means.

  A description of making a microfluidic actuator is described in US Provisional Patent Application No. 61 / 771,694, filed Mar. 1, 2013, which is incorporated herein by reference in its entirety.

A. Introduction and transport of reactants including starting materials In some embodiments, reactants or solutions containing reactants are added to a microfluidic cartridge for processing and subsequent analysis. The reactant may include blood, sputum, tissue, body fluid, cell, cellular component, extracellular fluid, protein, DNA, RNA, and the like. The starting material may also include dry reagents or biological materials for addition to the processing fluid. The starting material can be a fluid phase, a fluid-containing matrix or a solid phase. Starting materials can include intermediates for drugs or vaccines. In some cases, the starting material includes agricultural products, soil or environmental samples.

  The starting material is mixed with the first fluid within the cartridge passage. The first fluid can be mixed with a swab or porous matrix that includes soil or other environmental sample bound within the swab or porous matrix.

  The starting material can be processed by adding a surfactant to lyse the cells or cell membrane. Surfactants disrupt cell membranes and include sodium lauryl sulfate, hexadecyltrimethylammonium bromide or other cationic or zwitterionic surfactants. Examples of surfactants include Triton X-100, Triton X-114, NP-40, Tween 20, Tween 80, SDS (sodium dodecyl sulfate) and CHAPS.

  Enzymes can be used to lyse cells, remove cell walls, or process cells or cellular components in a sample. Examples of enzymes include lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanase, protease or mannase.

  Processing of the starting material can be carried out by mixing the sample with a homogenization solution. For example, the solution can homogenize a tissue sample or other heterogeneous biological sample. The homogenization solution can contain N-acetyl-L-cysteine or hypertonic saline. The homogenization solution can contain a reducing agent such as thioredoxin.

  The homogenization solution can also include DNAse or other proteins for disruption of DNA and cell debris. The solution can make it possible to reduce or eliminate the biological activity of a living cell, tissue or organism. The homogenization solution can be highly basic and can include sodium hydroxide or sodium hypochlorite.

  In some embodiments, the solution comprises glass beads, steel beads, zirconium silicate beads, zirconium oxide beads, or other solid material used for mechanical disruption of sample material. Beads or solid materials are used to destroy cells or cellular material in a process called beadbeating. The solution can also contain glycogen or polysaccharide.

  In other embodiments, the solution comprises carrier RNA for DNA extraction from the sample. A solvent such as acetone can also be used to extract cellular proteins.

  In some embodiments, the starting material includes dendritic cells, can be mixed with the first fluid in the cartridge, and pulsed to induce an element of the immune response to the attack.

  The starting material can be processed and then analyzed using the methods described herein. In some cases, the processed starting material is combined with other reagents or fluids in a microfluidic cartridge as a fluid.

B. Analyte Labeling A method is provided for labeling an analyte in a processing fluid. Examples of analytes include proteins, DNA, RNA, antibodies, peptides, or other compounds produced by the host. Analytes can include DNA, RNA, antibodies, peptides, or proteins produced outside the host, such as proteins released by pathogens during the course of infection.

  In one aspect, a process for labeling an analyte using a microfluidic cartridge is provided. In one aspect, the microfluidic actuator pressurizes a pump that drives a processing fluid that includes a fluid that includes an analyte and a labeled molecule into a common fluid path, and the fluid is adapted for labeling to occur. In some embodiments, the microfluidic actuator generates a Taylor dispersion of alternating plugs of fluid to mix the solution for labeling.

  Exemplary labeling reagents include chemiluminescent species such as luminal, isoluminol, acridinium ester, thioester, sulfonamide and phenanthridium ester, alkaline phosphatase; phycoerythrin, colloidal gold or other colloidal metals, etc. Fluorescent species; or including quantum dots. Other fluorescent reagents include lanthanides or lanthanide chelates (such as europium, samarium, terbium, dysprosium, etc.) and can be used in SOCLE assays as described below.

  Quantum dots are crystalline semiconductor particles whose electronic characteristics are closely related to the size and shape of individual crystals. In general, the smaller the crystal size, the larger the band gap and the greater the energy difference between the highest valence band and the lowest conduction band, so more energy excites the dots. At the same time, more energy is released when the crystal returns to its quiescent state. For example, in the application of fluorescent dyes, this is equivalent to a higher frequency of light emitted after dot excitation when the crystal size is smaller, resulting in red to blue in the emitted light. Brings a color shift to. In addition to such tuning, the main advantage with quantum dots is that they have a very precise control over the conductivity of the material, since a high level of control over the size of the crystals produced is possible. Is possible.

  Fluorescence, chemiluminescence and phosphorescence are three different types of luminescence properties (light emission from a substance). Fluorescence is a property where light is absorbed and re-emitted within a few nanoseconds (approximately 10 nanoseconds) with lower energy (longer wavelengths), whereas bioluminescence is a property where light is a substrate Biological chemiluminescence, characterized in that it is generated by an enzymatic chemical reaction above. Phosphorescence is a property of a material that absorbs light and emits energy for a few milliseconds or more (due to a forbidden transition to the ground state of the triplet state, while fluorescence is an excited singlet state To occur).

  Fluorescent labeling is the process of covalently attaching a fluorophore to another molecule, such as a protein, nucleic acid molecule, lipid or other small molecule. Reactive derivatives of fluorophores can be used to selectively bind to functional groups within the target molecule. Common reactive groups are labeled with isothiocyanate derivatives such as FITC and TRITC, succinimidyl esters such as NHS-fluorescein, maleimide active fluorophores such as fluorescein-5-maleimide, or fluorophore labels such as 6-FAM phosphoramidite. Oligonucleotides. The fluorescent protein or fluorophore can also be non-specifically or non-covalently bound to the protein. The fluorescently labeled molecule is excited by light (excitation source) and emits fluorescence, which can be detected by the naked eye or by a fluorescence detector. Various light sources can be used as excitation sources, including lasers, photodiodes and lamps, especially xenon arc lamps and mercury lamps.

  The analyte can be labeled with a fluorophore for detection by FRET (Forster (fluorescence) resonance energy transfer)), resonance energy transfer (RET) or electron energy transfer (EET). FRET is a mechanism that explains the energy transfer between two chromophores. A donor chromophore that is initially in its electronically excited state can transfer energy to the acceptor chromophore via nonradiative dipole-dipole coupling. For example, an analyte labeled with cyan fluorescent protein (CFP) can transfer the energy after excitation to yellow fluorescent protein (YFP) that emits a fluorescent signal for detection.

  Analytes can be labeled and detected using silica beads, particles or paramagnetic beads. The beads or particles can be hybridized or bound to the target analyte and purified or separated from the fluid by magnetic separation, affinity purification, or the like.

  Analytes can also be labeled and detected using oligonucleotide probes. The probe can be RNA or DNA, or a modified version thereof. Oligonucleotide probes can be designed to target specific nucleic acid sequences specific to viruses, bacteria, infectious organisms or human genes. Examples of target nucleic acid sequences include HIV, hepatitis B, hepatitis C, Mycobacterium tuberculosis, Chlamydia trachomatis, influenza virus, respiratory syncytial virus, human respiratory tract virus, or cancer-related genes (eg, ERRB2) contains a specific sequence. Oligonucleotide probes can be labeled with fluorescent molecules, luminescent signaling molecules, or quencher molecules.

  Other examples of labeling reagents include labeled carbohydrates, labeled nucleic acids or labeled proteins for the measurement of specific cellular compounds.

  In some embodiments, the fluid in the fluid passage comprises a dye for specific or non-specific labeling of cells.

  In other embodiments, the fluid in the fluid pathway comprises a primer, a probe, or a combination of primer and probe, and polymerase chain reaction, transcription-mediated amplification, amplification based on a nucleic acid sequence or at least one specified nucleic acid sequence. Includes enzymes that can catalyze another chemical reaction to amplify. The enzyme can include DNA polymerase, reverse transcriptase, RNA polymerase, ribonuclease H, DNA helicase, or recombinase.

  In another embodiment, the labeling reagent is attached to, coupled to or coupled to the walls of the fluid passage. As the fluid containing the target analyte passes through the passage in the cartridge, the target analyte associates or binds to the bound reagent.

  The labeling of the analyte can be followed by detection of the analyte present in the sample or measurement of the amount of analyte. For example, species labeled with fluorescent particles can be detected by irradiating with light at the excitation frequency of the fluorescent label and measuring the emitted light.

  Labeling can also be followed by separation of the analyte. For example, analytes can be separated by labeling the species with magnetic particles and imposing a magnetic field through which the labeled species are differentially transported.

C. Labeling a protein with an antibody A sample or starting material can be combined with a first solution containing, for example, a first set of antibodies that specifically bind to a target protein in the sample. The combined fluid can be moved by electroosmotic flow in the passage that passes by the region of the wall of the fluid passage. The wall is bound to a second set of antibodies that specifically bind to different epitopes of the target protein. Target proteins that bind to the wall region and form a sandwich with the first set of antibodies can be detected by a spectrometer or other instrument that measures fluorescence.

  By using antibodies specific for two or more targets and providing more than two different antibodies, for example, each antibody bound to a distinct region of the wall, specifically target multiple targets It can be detected and measured. The use of multiple fluorescent labels further expands the utility.

  Many other assays similar to the basic antibody sandwich assay can be performed, such as detection of genomic material using pairs of oligonucleotide probes. Among the many suitable non-optical assay means are electrochemical assay methods and assay methods using paramagnetic beads.

D. SOCLE Detection Assay An exemplary labeling and detection method for analytes used in microfluidic cartridges is singlet oxygen catalyzed luminescence (SOCLE). SOCLE is a variant of luminescence oxygen channeling (Ullman et al., Luminescent oxygen channeling assay (LOCI): sensitive, broadly applicable homogeneous immunoassay method. (1996). Clin. Chem 42, 1518-1526; Ullman et al , Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. (1994). Proceedings of the National Academy of Sciences 91, 5426-5430), widely used in well-known commercial immunoassay systems.

  The two-part SOCLE assay incorporates a probe-bound photosensitizer and chemiluminescent / fluorescent emitter beads. Excitation of sensitizer beads with light generates a fluorescent signal only when hybridization to the target brings the sensitizer and emitter beads very close (<200 nm). Singlet oxygen acts as an energy transport intermediate. Since the diffusion of singlet oxygen between the sensitizer and the emitter bead requires a finite time, there is a temporal separation of the excitation and photon counting steps. Thus, background fluorescence is reduced by several orders of magnitude because the excitation radiation source is turned off before reading. The SOCLE assay is non-enzymatic (ie, no heat labile protein).

  In one example, the microfluidic cartridge includes a fluid that includes a first target nucleic acid and a second fluid that includes a sensitizer oligonucleotide bound to a sensitizer bead. The sensitizer oligonucleotide comprises a sequence that is complementary to the first target nucleic acid. The first target nucleic acid molecule hybridizes with the sensitizer oligonucleotide bound to the bead by combining the two fluids together using the methods described above. For example, the two fluids are pressurized and brought together by electroosmotic flow to a junction where the two fluids join in the passage. The third fluid can include a second target nucleic acid molecule that is complementary to the first target nucleic acid molecule and that is also complementary to the emitter oligonucleotide bound to the emitter bead. The third fluid can be mixed with the fourth fluid such that the emitter oligonucleotide-beads hybridize with the second target nucleic acid. Sensitizer-bead complex molecules and emitter-bead complex molecules are mixed at a separate junction that joins the two fluid paths by generating alternating fluid plugs using the microfluidic actuator described above. (A) a first target nucleic acid and sensitizer oligonucleotide-bead complex, and (b) a second target nucleic acid and emitter oligonucleotide-bead complex). Hybridization of the first and second target nucleic acids produces a signal when the two complexes are hybridized and the emitter and sensitizer beads are in close proximity to each other (eg, at <200 nm spacing). be able to. In some examples, the sensitizer-bead complex and the emitter-bead complex both hybridize to a bridge probe (oligonucleotide) that is complementary to the first target nucleic acid and the second target nucleic acid. The bridge probe helps to form an oligonucleotide complex between the emitter oligonucleotide-bead complex and the sensitizer oligonucleotide-bead complex.

  In other examples, the fluid pathway includes a matrix for co-localizing with the sensitizer oligonucleotide-bead complex or a matrix for co-localizing with the emitter oligonucleotide-bead complex. The fluid containing the target nucleic acid molecule can hybridize to the sensitizer oligonucleotide-bead or to the emitter oligonucleotide-bead complex.

  From inside the cartridge, the sample may require excitation at 680 nm and detection at 615 nm. Reading from the sample can be detected from the detection window of the cartridge. The excitation phase can occur one after another with a transition period of approximately a few milliseconds or less. The excitation source can be high intensity and the detector can be highly sensitive. The optical module should be designed to meet these requirements. The excitation source is a light emitting diode (LED), which is an efficient light source that emits over a relatively narrow range of wavelengths. The light emitted by the assay is detected by a photomultiplier tube (PMT). The lens, bandpass filter, and dichroic beam splitter direct light from the LED into the cartridge and from the cartridge into the PMT.

  A typical read cycle sequence consists of setting the PMT control voltage low (typically 0.3V), turning the LED on for 0.5 seconds, turning the LED off, and finally increasing the PMT control. It involves reading the result of SOCLE signaling in the readout well (typically up to 0.8V). Custom analog hardware was built to provide current-voltage conversion, filtering and amplification or attenuation. Five analog-to-digital converter channels (three signal channels with different amplification, temperature, and PMT control voltages) are read out (typically 0.7 seconds) for the duration of the experiment. Data from the five channels is streamed to the microprocessor in real time, which integrates the signal and compares it to a standard curve or lookup table to determine the starting sample concentration. The dynamic range over which the starting sample concentration is determined can be increased by lowering the PMT control voltage after the saturation signal is measured.

E. Droplet-Based Assay The microfluidic cartridge of the present invention divides a sample or starting material into a plurality of compartmentalized assay mixtures that are isolated from each other in each droplet by an intervening immiscible carrier fluid Can be used to detect and quantify multiple analytes in a sample or starting material. This splitting can take place anywhere inside the cartridge and can occur at any stage of sample or starting material processing. Examples of the stages where splitting can occur are immediately after introduction of the sample or starting material into the cartridge; after the filtration process; after the nucleic acid extraction process; segments of genomic material in all species of a particular category (eg, bacterial target 16S region for) after the process amplified by the polymerase chain reaction or another method; and after labeling with fluorescent beads or other signaling particles.

  The microfluidic cartridge of the present invention can include a plurality of fluid passages that meet at a junction in the cartridge. In some embodiments, this is a T-shaped junction or a Y-shaped junction. Two immiscible fluids from two separate fluid passages meet at the confluence in the cartridge and can form droplets at the confluence when the two fluids collide (e.g., oil-in-water droplets, Water-in-oil droplets). The junction and fluid passage can be sufficiently narrow so that when two immiscible fluids meet at the junction, a single droplet is formed. As the fluid flows forward at the confluence, multiple droplets can be formed that are merged in a single passage.

  In some embodiments, the microfluidic cartridges of the present invention accurately generate multiple compartmentalized assay mixtures in arbitrarily selected volumes without fluid transport or mechanical energy transfer from external components into the cartridge. Can be used to make. The ability to arbitrarily select the volume of the mixture to be compartmentalized differentiates droplet formation using the present invention; in contrast, in droplet formation with a droplet generator that does not include a high performance actuator, the geometry of the confluence Geometric shape and fluid properties are the primary determinants of compartment volumes and limit the ability to arbitrarily select these volumes. The at least one microfluidic actuator acts on a processing fluid contained within the fluid passage so that such fluid can travel toward the confluence. Similarly, the second microfluidic actuator acts on an immiscible carrier fluid within the second fluid passage so that such carrier fluid can travel toward the junction. If at least one of the actuators in the microfluidic cartridge is a high performance actuator, pressurization of either the first fluid or the carrier fluid, or both, can be rapidly pulsed or otherwise time-varying. Can be pressurized in a manner, so that a fluid compartment having the desired compartment volume is formed by the merging of two fluids at the junction.

  The microfluidic cartridge of the present invention is also used for droplet-based assays where droplet formation facilitates the detection and quantification of multiple analytes within a single starting sample known as multiplexing Can be done. A double stopper of immiscible carrier fluid and reagent can be sequentially injected into the stopper of the sample to form multiple fluid compartments, and the reagents are selected such that different reactions occur within a particular compartment. Each of these reactions corresponds to the detection of a specific analyte of interest. Each reaction can take place within a single droplet, and a detector within the microfluidic cartridge can detect the radiation or signal from each reaction in the droplet.

  In another aspect, the microfluidic cartridge of the present invention can be used to generate droplets and to perform PCR amplification on multiple droplets within the cartridge. In some embodiments, each droplet includes a target nucleic acid molecule, an enzyme, and a PCR primer mixture for an amplification module of nucleic acid molecules in the sample. Each droplet can contain PCR reagents and can be circulated through three different temperature zones (eg, for reverse transcriptase reaction and amplification) within the microfluidic cartridge. In some embodiments, the microfluidic cartridge can generate droplets within one region of the cartridge (eg, the confluence of at least two fluid passages generates a series of at least 24 droplets) Used for). PCR amplification can occur by movement of a droplet (via a microfluidic actuator) through a preset temperature zone in the cartridge.

  More particularly, amplification can be achieved by reciprocating a fluid containing droplets between three temperature zones in a cartridge for reverse transcription and amplification. The low thermal mass of the fluid stopper allows the fluid temperature to equilibrate in seconds in each zone, resulting in a rapid amplification cycle. The fast transient response time of the charged slit actuator further improves the amplification process by reducing the time required to reciprocate the solution between zones. In addition to the three zones, the amplification module can include a reagent reconstitution zone. A single relatively large (eg, 4 mm × 6 mm) charged slit microactuator can be used to perform these steps. For amplification module thermal engineering, thermal analysis can be performed within COMSOL to establish mandatory cartridge thermal mass assignments to keep the zone temperature nominally +/- 1 ° C as the solution moves between zones. it can.

  In other embodiments, the cartridge includes a melting temperature scanning zone (where the rear sortant analysis is performed using a main actuator that reciprocates the droplets back and forth through the detection zone) and the photochemical thermal method comprises the cartridge Is used to accurately determine the temperature of each droplet at various points in time.

  In one aspect, the droplet generator in the cartridge (as shown in FIGS. 23-25) is positioned downstream of the amplification module. The droplet generator can perform individual reconstitution of many lyophilized reagent plaques (eg, 24 plaques containing 24 beacons) used in melting temperature scanning assays, First, a series of droplets are generated by sequentially pulsing each beacon solution volume into the amplicon solution. The flow in the main channel can be driven by a large charged slit actuator used in the amplification stage. In one aspect, multiple small actuators (eg, 24 small actuators) can include both dry beacon reconstitution (in hybridization buffer) and injection, each driving one side of the channel. The secondary channel actuator and the primary actuator can work together to reciprocate a series of droplets back and forth through the detector during melt temperature analysis.

  In some embodiments, the microfluidic cartridge comprises a charged slit actuator drive electronics, cartridge temperature control, sensing optical components, instrument control electronics, a touch screen user interface controlled by a separate microcontroller, power electronics As well as appliances, including communication hardware including RFID, WiFi and Ethernet connectivity.

F. Confluence geometry to improve mixing In addition to mixing fluids at simple T or Y junctions, the present invention provides a confluence geometry that particularly facilitates rapid mixing. It can be used to mix fluids at a junction having a shape. A confluence configuration (sometimes referred to as a neck-down confluence) in which one or more channel cross-sections immediately adjacent to the confluence are smaller than a cross-section farther from the confluence is combined with the present invention. More rapid macro-mixing can be promoted than either the non-neck-down confluence in the present invention or the non-invention confluence (with or without neck-down). The smaller adjacent channel cross-section corresponds to a smaller minimum fluid plug volume for individual plug injection using the present invention. After a series of pulsations has moved beyond the neck-down region of the reaction channel that is in close proximity to the confluence, the conservation of mass is a small dimension of the fluid stopper, compared to the dimensions inside the neck-down channel region. Need to expand to shorter plugs. The contribution of Taylor dispersion to mixing is correspondingly increased compared to the confluence process with larger volume plugs, such as non-necked down geometry.

G. Use surface tension effects to improve control over the fluid in the cartridge, reduce bubbles, synchronize fluid for mixing at the junction, or otherwise improve assay performance Channel and Junction Features to Do Channel features proximate to the junction can be used to improve the performance of mixing using the present invention. For fluids that are hydrophobic with respect to the material that makes up the fluid path, the inclusion of cavity-like features proximate the junction can be used with the present invention to align the fluids prior to mixing. For a cavity feature, where the axial length along which the expansion and contraction occurs is short compared to the channel diameter, which is a substantially uniform radial expansion and contraction of the generally cylindrical channel, Retained inside the cavity when pressurized by a high performance actuator operating at a given duty cycle and average power due to energy storage associated with surface tension mediated flow front deformation at the entrance to the cavity Can do. The increase in duty cycle and / or average power of high performance actuators can overcome the meniscus energy storage effect and move the fluid flow head beyond the cavity inlet. If the fluid flow characteristics (eg, flow rate) inside the cartridge are subject to uncertainty due to, for example, patient-to-patient variations in hematocrit for whole blood samples, for example, fluid at the low flow extreme in the parameter space By maintaining the performance of high performance actuators at low duty cycle / low power conditions until the time intended for the design has elapsed, the low in the cavity (eg, using hematocrit at the upper end of the physiological range) Duty cycle / low power stall effects can be used to reduce the impact of such uncertainties on assay performance. With a combination of similar effects, using the present invention, the cavity can function as a trap for air or other gas bubbles entrained in the fluid.

  The present invention can also be used to improve assay performance by inclusion of cavity-like features in the T-junction secondary channel immediately adjacent to the junction. For a cavity feature where the axial length along which the expansion occurs is short compared to the channel diameter, which is a substantially uniform radial extension of the substantially cylindrical channel, the first fluid volume flow front is Due to the energy storage associated with surface tension mediated flow front deformation at the entrance to the cavity, being held inside the cavity when pressurized by a high performance actuator operating at a given duty cycle and average power Can do. On the condition that the axial length of the cavity is short compared to the channel diameter, the second fluid passing through the substantially straight T-junction passage overcomes the meniscus energy storage effect and Can enter the confluence.

H. Reaction with Solid Phase to Promote the Detection of Components The present invention, in combination with known methods, facilitates the reaction between the components of the solution and the solid phase to detect or detect and quantify such components. Can be promoted. The solution can be flowed into a chamber containing one or more inner surface regions to which the probe is bound. Such surface bound probes can be oligonucleotide probes, antibody probes or other probes. The surface binding probe may be positioned relative to a sensing element or sensing system that facilitates identification of binding events between the surface binding probe and the solution phase reactant. Such reactants can be oligonucleotides, proteins, sugars, cells or other reactants. Surface-coupled probes can be configured in one-dimensional, two-dimensional or three-dimensional arrays. A sensing element or sensing system can be configured to measure parameters of interest of individual elements of the array. The sensing element can be designed to measure parameters including temperature, pH and electromagnetic radiation. Two microfluidic actuators, at least one of which is a high performance actuator, can be used to induce time-varying flows proximate some or all of the probe functionalized surface area. Time-varying flow can result in the exchange of a volume of solution in close proximity to such a surface area, so that a fluid volume containing a relatively high concentration of unbound reactants takes up the reactants. Proximate to the surface so that it can bind to the surface binding probe.

I. Reactant Metering The present invention provides a cartridge or solution containing a defined volume of a fluid phase reactant, such as blood, plasma, urine or another biological fluid, or a component for chemical or biochemical synthesis processes. It can be drawn into other microfluidic networks for subsequent processing or analysis. A volume of reactant flows by pipetting, by pouring from another container, or by direct flow from a source (eg, flowing directly through an opening in the skin created by the action of a mechanical lancet Blood), or by other means, can be loaded into the chamber. The loading process can be inaccurate, for example, when a nurse or other medical professional visually confirms that the volume of the reactant exceeds the minimum volume indicated by the filling line on the chamber. is there. Thus, the first microfluidic actuator can draw a volume of reactant into a fluid path or a chamber different from the intake chamber by operating for a specified period of time and at a specified power level. Is predetermined by the characterization of the microactuator and associated microfluidic network to correspond to the preferred volume for subsequent processing. This is referred to herein as open loop metering. Alternatively, the first microfluidic actuator causes the volume of reactant to be fluidized by operating until a time when the sensor indicates that a volume of reactant within the fluid path has reached a specified value. Can be pulled into the aisle. Such a sensor can be a capacitive sensor that indicates a change in capacitance as the reaction stream head advances through the channel to a predetermined position. This is referred to herein as a closed loop metric.

  The volume of reactant drawn into the first fluid passage can be mixed with other fluids at the junction, and such mixing is driven by the combined action of the first and second microfluidic actuators. One of which is a high performance actuator as described herein. The reactants drawn into the first fluid passage can reconstitute the dried or lyophilized material as soon as it is drawn into the first passage or chamber, or at a later stage in processing. .

J. Lysis of cells The present invention can be used to efficiently lyse cells contained within a biological matrix. Whole blood or plasma can be drawn into the fluid passage by the first microactuator. Contains one or more compounds that tend to break down cell walls and membranes, such as guanidine thiocyanate or another protein denaturant, polysorbate 20 or another detergent / emulsifier, and proteinase K or another serine proteinase A solution can be loaded into the second fluid passage. Loading the second fluid passage with the lysis solution can be preceded by reconstitution of one or more of the lysis solution components from a dry or lyophilized state. The dried or lyophilized material can be in the form of pellets, can be a plaque-like composition that covers a portion of the interior surface of the fluid passageway, or is porous within the passageway or chamber. Can be distributed in the quality material.

K. Use with Temperature Controller The present invention provides resistance heaters and thermoelectric coolers to facilitate the reaction and other to increase, decrease or adjust the temperature of the fluid volume Can be used with elements and systems.

L. Extraction of DNA and RNA from Complex Starting Samples The present invention can be combined with known methods for reversible binding of DNA, RNA and other nucleic acids from starting samples. The solution containing the nucleic acid can be flowed through the porous structure by the action of a microfluidic actuator. The porous structure can be a packed silica bead bed, which is known to reversibly bind to nucleic acids. The porous structure has a pore size distribution and can be otherwise configured to capture precipitated nucleic acid, eg, nucleic acid precipitated from solution by binding to glycogen. Prior to the flow of the nucleic acid-containing solution through the porous structure, the efficiency of binding to the nucleic acid originally exposed inside the cell, where the cell is exposed to a compound that tends to destroy the cell walls and membranes Can improve the process. Prior to the nucleic acid containing solution flowing through the porous structure, some or all of the nucleic acid contained therein tends to bind to the polysaccharide or precipitate such sugar-nucleic acid complexes. The process of binding with other substances can be performed. Prior to the nucleic acid-containing solution flowing through the porous structure, sugar-nucleic acid complexes or other precipitating complexes precipitate from the solution and are retained near or within the porous structure. The first microfluidic actuator and the second fluidic actuator, at least one of which is a high performance fluid actuator, can perform a process of mixing the nucleic acid-containing solution with butanol or another solvent. Mixing the solvent and the nucleic acid-containing solution can involve transporting the fluid into the chamber, where buoyancy effects associated with the different densities of the solvent and the nucleic acid-containing solution facilitate the mixing of the two phases. Mixing the solvent with the nucleic acid-containing solution can involve transporting the fluid into the chamber, where one or more liquid phases are withdrawn from the chamber and the surface tension effect, buoyancy effect or of these effects. By the combination, bubbles are held inside such a chamber. The passage of the nucleic acid-containing solution through the porous structure can be followed by flowing through a porous structure of a solvent such as ethanol to wash away non-binding material such as proteins. There can be more than one such washing step. The passage of the nucleic acid-containing solution through the porous structure can be followed by passing water or another solution that is prone to reversible binding of the nucleic acid so that the nucleic acid can pass through such water or other solution. When transported out of the porous structure, it will tend to elute from the porous structure.

  The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should, of course, be taken into account.

  The practice of the present invention uses conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the skill of the art unless otherwise noted. Such techniques are explained fully in the literature. For example, TE Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition , 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd See Ed. (Plenum Press) Vols A and B (1992).

Example 1: Application to HIV testing The methods described above can be used to detect or analyze HIV-1 RNA genetic material or viral proteins. Even in the most severe HIV patients, the amount of virus in their bloodstream is relatively small, so direct detection of the target species requires sophisticated methods.

  Starting materials, including patient blood or body samples, are provided in a microfluidic cartridge. The sample can be processed using a homogenization solution, beads or enzymes to lyse the cells in the sample within the fluid pathway. Homogenization solution and sample can be mixed using a microfluidic actuator in the cartridge.

  The processed fluid sample can then be mixed with the second fluid by pulsating the fluid together using two or more microfluidic actuators in the microfluidic cartridge. it can. The second fluid can contain reagents such as antibodies, oligonucleotide probes, or labeled molecules that specifically bind to proteins, DNA, RNA or other molecules that are specific for the HIV virus.

  Detection of HIV virus can be performed using any of the detection methods provided above.

Example 2: Detection of Dengue Virus The method described above can be used to detect or analyze Dengue virus genomic material. Dengue virus (DENV) is a potential biological weapon defense pathogen and has been classified as a significant international public health concern by the World Health Organization (WHO). Using the microfluidic cartridge and method described above, dengue virus can be detected in a sample.

  Starting materials, including patient blood or body samples, are provided in a microfluidic cartridge. The sample can be processed using a homogenization solution, beads or enzymes to lyse the cells in the sample within the fluid pathway. The homogenized solution and sample are mixed using the Taylor dispersion of fluid stoppers generated by the microfluidic actuator in the cartridge.

  The optimized performance of this assay requires operation within the microfluidic cartridge described above. Cartridge-integrated microfluidic actuators drive sample and reagent transport with millisecond time resolution, substantially accelerating bead-primer reactions for diffusion-limited well-format reactions while simultaneously integrating cartridges The mold heater controls the temperature of the reaction chamber and readout well with an accuracy of 1 degree.

Example 3: Detection of Mycobacterium tuberculosis (MTB) and analysis of MTB genomic material to identify drug resistant strains
MTB can be identified from a sample or starting material using the methods described above.

  Some strains of MTB are resistant to certain antibiotics that are widely used to treat MTB. The ability to identify drug resistance allows different drugs to be selected to treat individuals infected with resistant strains. Examples of MTB resistance that are clinically important include resistance to rifampicin, isoniazid, fluoroquinolone and pyrazinamide.

  Samples such as sputum samples from patients who are known to be infected with MTB or suspected of being infected with MTB are homogenized in the cartridge of the present invention, and genomic material comprising DNA or ribosomal RNA is It can be released from bacteria. The sample can then go through a series of steps that include the addition of primers to produce large copies of the region of interest, such as the rpoB gene, in a process called isothermal amplification. The action of the microfluidic actuator in the cartridge can rapidly mix the probe and primer to advance the isothermal amplification process quickly and efficiently. The amplified target can then be labeled using, for example, molecular beacons.

Example 4: Quantification of HIV virus by polymerase chain reaction (PCR) The microfluidic cartridge of the present invention is the amount of HIV genetic material in a sample of material known or suspected of containing HIV genetic material. Can be used to determine accurately. Such a substance can be a whole blood sample, a plasma sample or other sample. A quantity of sample can be combined with reverse transcriptase to facilitate reverse transcription of viral RNA into cDNA. This combination is preceded by one or more processes to lyse the viral coat, remove potential interfering substances, or otherwise prepare the sample for reverse transcription. Can do. Such sample preparation can be performed within the same cartridge or other microfluidic channel network module as the reverse transcription process, or can be performed completely or partially outside the cartridge or microfluidic network module. Combining a reverse transcriptase with a material known or suspected of containing HIV genetic material can occur at a confluence when the reverse transcription genetic material is in solution form or in lyophilized form Can occur by passage of the material into a chamber containing reverse transcriptase, which is in dry form or in another form. Reconstitution of reverse transcriptase, which is in dry form, lyophilized form or otherwise, requiring reconstitution can be facilitated by rapid pulsatile flow driven by one or more high performance actuators.

  Reverse transfer can occur in the cartridge chamber using an associated element to control the temperature of the volume of material where the reverse transfer occurs. Such elements are resistance heaters, elements for raising or lowering the temperature of a mass of material by the thermoelectric effect, resistance temperature sensors, automatic or manual adjustment of heat production by heating elements as a function of the output of the temperature sensor Electrical circuit configurations and other elements can be included. The solution that has undergone the reverse transcription reaction can be combined with primers, enzymes, and other reagents for the polymerase chain reaction. Combining with primers, enzymes and other reagents is through fluid transport of primers, enzymes and other reagents into the reaction chamber in which the reverse transcribed DNA was generated, driven by at least one high performance actuator. Alternatively, it can occur by fluid transport of a solution containing reverse transcribed DNA, driven by at least one high performance actuator, into another chamber or two or more other chambers. Amplicons generated by PCR can be detected while the PCR reaction is being performed, or by endpoint methods. Amplicons generated by PCR are probes specific to amplicons with fluor or other luminescent particles, quencher particles, and hairpin structures, which emit luminescence when excited when bound to amplicons. By including the probe shown, it can be analyzed. PCR reactions can be analyzed by changing the temperature of the solution and monitoring the binding with the labeled probe. PCR reactions can be performed in multiple fluid compartments. At least one high performance actuator can facilitate fluid compartmentalization. The fluid compartment can constitute an emulsion. The volume and number of compartment elements are selected to facilitate quantification through analysis of compartment element fractions found to contain at least one copy of DNA reverse transcribed prior to PCR initiation. be able to.

Example 5: Detection of influenza and other respiratory pathogens and distinction between such pathogens The present invention is for melting temperature ( _Tm ) analysis of genetic differences in influenza A virus (IAV) RNA from reference strains Can be used to. Targeted amplification can be performed based on possible target identification information. Targeted amplification can be concentrated on HA and NA antigens. Targeted amplification can rapidly classify IAVs against reference strains over some or all of the eight genomic segments. Targeted amplification can incorporate the predicted target sequence or serve as an unbiased search for rearrangement at the genomic level.

  Although the invention has been particularly shown and described with reference to preferred embodiments and various alternative embodiments, it will be understood that various changes in form and detail may be made thereto without departing from the spirit and scope of the invention. It will be understood by those skilled in the relevant art.

  All references, issued patents and patent applications cited within the text of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims (142)

With multiple fluid passages;
At least one junction that connects the plurality of fluid passageways;
At least two fluid transport means comprising at least one high performance fluid actuator comprising:
The at least one high performance fluid actuator comprises:
Fluid power generation capacity of at least ^10-8 watts, and power can last for at least 30 seconds, and
A microfluidic cartridge comprising: said at least two fluid transport means that are individual components within the cartridge having a response time for generating said fluid power of less than 10 seconds.   The cartridge of claim 0 having a drainage volume of 500 cubic centimeters or less.   3. The cartridge according to claim 2, having a drainage amount of 50 cubic centimeters or less.   The cartridge of claim 0, wherein the at least one high performance fluid actuator is capable of converting electrical power into fluid power.   The cartridge of claim 4, wherein the conversion of electrical power to fluid power occurs without intermediate energy conditions.   The cartridge of claim 0, wherein operation of the at least one high performance fluid actuator does not include transfer of mechanical energy from an external device to the at least one high performance fluid actuator.   The cartridge according to claim 0, wherein a response time for generating power is less than 2 seconds.   8. The cartridge according to claim 7, wherein a response time for power generation is less than 0.2 seconds.   9. The cartridge according to claim 8, wherein the response time for power generation is less than 0.04 seconds.   The actuator of claim 0, wherein the actuator is capable of pressurizing at least 10 microliters of liquid such that the liquid flows through a fluid resistance associated with a pressure drop of at least 1 kPa at a flow rate of at least 0.1 mL per minute. cartridge.   The cartridge of claim 0, wherein the high performance actuator is coupled to a pulse generator or other controlled time-varying voltage source and at least one electrode.   The cartridge of claim 0, wherein the at least one high performance fluid actuator is capable of generating fluid power by electrokinetic effects.   13. A cartridge according to claim 12, wherein the electrokinetic effect comprises electroosmotic flow.   14. The cartridge of claim 13, wherein the electroosmotic flow is generated within a plurality of slit capillaries within each of the at least one fluid actuator.   14. The cartridge of claim 13, wherein the electroosmotic flow is generated within a bed of filled beads within each of the at least one fluid actuator.   14. The cartridge of claim 13, wherein the electroosmotic flow is generated within an integral porous structure within each of the at least one fluid actuator.   14. The cartridge of claim 13, wherein the electroosmotic flow is generated within an array of cylindrical channels within each of the at least one fluid actuator.   The cartridge of claim 0, comprising an opening for receiving a starting material within a network of fluid passages.   19. A cartridge according to claim 18, wherein the opening is closed with a plug or capping element.   19. A cartridge according to claim 18, wherein the plug or capping element can receive a fluid conduit and can be hermetically sealed when the fluid conduit is withdrawn.   21. The cartridge of claim 20, wherein the fluid conduit that can be received by the stopper or capping element comprises a needle, tube, rigid fluid conduit or semi-rigid fluid conduit.   21. The cartridge of claim 20, wherein the plug or capping element comprises an elastomeric material.   21. The cartridge of claim 20, wherein the plug or capping element includes a closure mechanism.   The cartridge of claim 1, further comprising a controller capable of controlling power delivery from the power source to the at least one high performance fluid actuator.   The cartridge of claim 0, further comprising a power source operably coupled to the at least one high performance fluid actuator.   26. The cartridge of claim 25, wherein the power source is located within the external device and is coupled to the cartridge by electrical connection.   26. The cartridge of claim 25, wherein the power source is electrical.   26. The cartridge of claim 25, wherein the power source is pneumatic.   26. The cartridge of claim 25, wherein the power source includes a battery.   30. The cartridge of claim 29, wherein the battery is located inside the cartridge.   30. The cartridge of claim 29, wherein the battery is located inside the external device and is coupled to the cartridge by electrical connection.   The cartridge of claim 1, further comprising a second opening for receiving a processing fluid and coupled to a network of fluid passages.   The cartridge of claim 1, further comprising a processing fluid contained within the network of fluid passages.   34. The cartridge of claim 33, wherein the processing fluid comprises a first reagent capable of lysing cells or organelles.   35. The cartridge of claim 34, wherein the first reagent comprises a surfactant or other surfactant.   35. The cartridge of claim 34, wherein the first reagent comprises an enzyme.   40. The cartridge of claim 36, wherein the enzyme is lysozyme.   34. The cartridge of claim 33, wherein the processing fluid comprises a homogenizing solution capable of homogenizing a tissue sample or other heterogeneous biological material.   34. The cartridge of claim 33, wherein the processing fluid comprises a solution that can reduce or eliminate the biological activity of a living cell, tissue or organism.   34. The cartridge of claim 33, wherein the processing fluid comprises glass beads or other solid material that can cause mechanical disruption of the starting material.   34. The cartridge of claim 33, wherein the processing fluid comprises glycogen or other polysaccharide.   34. The cartridge of claim 33, wherein the processing fluid comprises carrier RNA.   The cartridge of claim 1, further comprising a third opening for receiving the actuator fluid and coupled to the at least one high performance fluid actuator.   The cartridge of claim 1, further comprising an actuator working fluid within the at least one high performance fluid actuator.   The cartridge of claim 1, wherein a portion of the network of fluid passages includes a second reagent.   46. The cartridge of claim 45, wherein the second reagent comprises silica beads or particles.   46. The cartridge of claim 45, wherein the second reagent comprises paramagnetic beads.   46. The cartridge of claim 45, wherein the second reagent comprises fluorescent beads or fluorescent molecules.   46. The cartridge of claim 45, wherein the second reagent comprises a chemiluminescent molecule.   50. The cartridge of claim 49, wherein the chemiluminescent molecule comprises an alkaline phosphatase substrate.   46. The cartridge of claim 45, wherein the second reagent comprises a lanthanide or a lanthanide chelate.   46. The cartridge of claim 45, wherein the second reagent comprises a monoclonal or polyclonal antibody.   53. The cartridge of claim 52, wherein the monoclonal or polyclonal antibody is linked to a signaling molecule.   46. The cartridge of claim 45, wherein the second reagent comprises an oligonucleotide probe or primer, a combination of probes, or a combination of primers.   55. The cartridge of claim 54, wherein the oligonucleotide probe specifically binds to a defined region of human immunodeficiency virus genetic material.   55. The cartridge of claim 54, wherein the oligonucleotide probe specifically binds to a defined region of hepatitis C virus genetic material.   55. The cartridge of claim 54, wherein the oligonucleotide probe specifically binds to a defined region of hepatitis B virus genetic material.   55. The cartridge of claim 54, wherein the oligonucleotide probe specifically binds to a defined region of genetic material of Mycobacterium tuberculosis bacteria.   55. The cartridge of claim 54, wherein the oligonucleotide probe specifically binds to a defined region of genetic material of Chlamydia trachomatis bacteria.   55. The cartridge of claim 54, wherein the oligonucleotide probe specifically binds to a defined region of genetic material of influenza virus, respiratory syncytial virus or another virus of the human respiratory tract.   55. The cartridge of claim 54, wherein the oligonucleotide probe specifically binds to a defined region of oncogene DNA or RNA.   55. The cartridge of claim 54, wherein the oligonucleotide probe is labeled.   64. The cartridge of claim 62, wherein the label comprises a fluorescent or luminescent signaling molecule or quencher thereof.   55. The cartridge of claim 54, wherein the oligonucleotide probe comprises an aptamer.   46. The cartridge of claim 45, wherein the second reagent comprises a photosensitizer molecule.   46. The cartridge of claim 45, wherein the second reagent comprises a photoactive indicator precursor molecule.   46. The cartridge of claim 45, wherein the second reagent comprises a photosensitizer molecule and a photoactive indicator precursor molecule. A photosensitizer molecule and a photoactive indicator precursor molecule are
a. At least one sensitizer comprising one or more sensitizers, one or more sensitizer oligonucleotides, and a matrix for co-locating such sensitizers and sensitizer oligonucleotides Labeled particles; and b. Comprising at least one emitter-labeled particle comprising one or more emitter agents, one or more sensitizer oligonucleotides, and a matrix for co-locating such emitter agents and emitter oligonucleotides. Item 67. The cartridge according to Item 67.   69. The cartridge of claim 68, wherein the photosensitizer molecule can be in an excited state that generates singlet oxygen molecules.   69. The cartridge of claim 68, wherein the photoactive indicator precursor molecule is capable of reacting with singlet oxygen molecules to form a photoactive indicator.   46. The cartridge of claim 45, wherein the second reagent comprises quantum dots or other crystalline semiconductor particles.   46. The cartridge of claim 45, wherein the second reagent comprises a nucleic acid specific fluorescent or luminescent dye for sequence independent measurement of the nucleic acid.   48. The cartridge of claim 45, wherein the second reagent comprises a molecule that can participate in Forster resonance energy transfer (FRET) or other resonance energy transfer processes.   46. The cartridge of claim 45, wherein the second reagent comprises a labeled protein, labeled nucleic acid or labeled carbohydrate species for measurement of specific cellular compounds.   46. The cartridge of claim 45, wherein the second reagent comprises a solution containing a dye for specific or non-specific labeling of cells.   46. The cartridge of claim 45, wherein the second reagent comprises a primer, a probe, or a combination of primer and probe.   77. The cartridge of claim 76, wherein the enzyme can catalyze a polymerase chain reaction, transcription-mediated amplification, amplification based on a nucleic acid sequence, or another chemical reaction to amplify at least one specified nucleic acid sequence. .   77. The cartridge of claim 76, wherein the enzyme comprises DNA polymerase, reverse transcriptase, RNA polymerase, ribonuclease H, DNA helicase or recombinase.   The cartridge of claim 0, wherein the starting material comprises a fluid phase, a fluid-containing matrix or a solid phase.   The cartridge of claim 0, wherein the starting material comprises blood, sputum or other body fluid.   The cartridge of claim 0, wherein the starting material comprises biological tissue.   The cartridge of claim 0, wherein the starting material is a raw material or intermediate for a drug or vaccine.   The cartridge of claim 0, wherein the starting material is an agricultural product.   The cartridge of claim 0, wherein the starting material is soil or another environmental sample.   The cartridge of claim 0, further comprising a first fluid passage containing a first material and a second fluid passage containing a second material, wherein the first fluid passage and the second fluid passage. Forming a confluence in the microfluidic cartridge.   86. The cartridge according to claim 85, wherein the joining point is a T-shaped joining point or a Y-shaped joining point.   86. The cartridge of claim 85, wherein the confluence point enables formation of one or more microfluidic droplets resulting from the fusion of the first and second materials from the first and second fluid passages.   90. The cartridge of claim 86, wherein the one or more droplets each contain an analyte or reagent.   One or more droplets each catalyze at least one primer and a polymerase chain reaction, transcription-mediated amplification, nucleic acid sequence-based amplification, or another chemical reaction to amplify at least one target nucleic acid sequence 90. The cartridge of claim 86, comprising an enzyme that can be made.   90. The cartridge of claim 86, wherein the one or more droplets each comprise a label.   86. The cartridge of claim 85, wherein the first or second material comprises a processing fluid.   90. The cartridge of claim 86, wherein the one or more droplets each contain a cell.   The cartridge of claim 1, wherein the plurality of fluid passages comprise different temperature zones for performing the stages of the amplification reaction.   The cartridge of claim 1, wherein a plurality of fluids are combined in a plurality of fluid passages to induce a labeling or hybridization reaction. 95. A microfluidic cartridge according to any one of claims 1 to 94; and a system comprising a power source and adapted to supply power to the microfluidic cartridge.   96. The system of claim 95, wherein the device is further adapted to sense an indicator of assay results.   96. The system of claim 95, wherein the sensor senses visible light or another type of electromagnetic radiation generated within the cartridge.   96. The system of claim 95, wherein the device is further adapted to sense the location or distribution of paramagnetic beads within the cartridge.   96. The system of claim 95, wherein the device is further adapted to sense an electron spin nuclear magnetic resonance or other physical property of a species inside the cartridge. Providing a first fluid in a microfluidic cartridge to a channel connected to a plurality of fluid passages and including at least one junction between the fluid passages, the microfluidic cartridge comprising at least one A high-speed microfluidic actuator, wherein the at least one high-performance fluid actuator is a separate component within the cartridge, and the at least one high-performance fluid actuator is at least 10 ^-8 watts and powered Having a fluid power generation capability capable of lasting at least 30 seconds and a response time for power generation of less than 10 seconds; and
Operating the microfluidic actuator in a time-varying manner such that the first fluid and the second fluid are introduced into the network of fluid passages to generate alternating plugs of fluid, A method wherein the length of each plug volume is less than 5 times the smallest average diameter of such fluid passages.   101. The method of claim 100, wherein the high-speed microfluidic actuator generates fluid power through electrokinetic effects.   102. The method of claim 101, wherein the electrokinetic effect is generated by electroosmotic flow.   105. The method of claim 102, wherein the electroosmotic flow is generated within an array of slits.   105. The method of claim 102, wherein the electroosmotic flow is generated inside a packed bead bed.   105. The method of claim 102, wherein the electroosmotic flow is generated within an integral porous structure.   101. The method of claim 100, further comprising labeling a subset of cells within the first fluid with a labeled molecule or labeled particle within the second fluid that is specific for at least one type of molecule within the cell membrane. Method.   101. The method of claim 100, further comprising the step of coloring the cells in the first fluid with a cell penetrating dye contained in the second fluid.   Further labeling a subset of DNA or RNA contained within the first fluid with a photosensitizer molecule or photoactive indicator precursor molecule contained in the second fluid or a combination thereof. 101. The method of claim 100, comprising.   101. The method of claim 100, further comprising labeling a subset of DNA or RNA contained within the first fluid with a lanthanide chelate contained in the second fluid.   101. The method of claim 100, further comprising lysing cells or other biological material within the first fluid with a surfactant or other surfactant contained in the second fluid.   111. The method of claim 110, wherein the surfactant comprises sodium lauryl sulfate.   111. The method of claim 110, wherein the surfactant comprises hexadecyltrimethylammonium bromide or another cationic surfactant.   101. The method of claim 100, further comprising the step of lysing the cells or other biological material within the first fluid with an enzyme.   114. The method of claim 113, wherein the enzyme comprises lysozyme.   101. The method of claim 100, further comprising homogenizing a tissue sample or other heterogeneous biological material from the first fluid.   101. The method of claim 100, further comprising reducing the biological activity of a living cell, tissue or organism in the first fluid.   117. The method of claim 116, wherein reducing the biological activity comprises using a highly basic solution.   118. The method of claim 117, wherein the highly basic solution comprises sodium hydroxide.   118. The method of claim 117, wherein the highly basic solution comprises sodium hypochlorite.   101. The method of claim 100, further comprising lysing cells or other biological material in the first fluid with glass beads or other solid material for mechanical disruption in the second fluid.   101. The method of claim 100, further comprising mixing the swab or porous matrix with the first fluid.   122. The method of claim 121, further comprising releasing a soil or other environmental sample that is bound within the swab or porous matrix.   101. The method of claim 100, wherein the first fluid comprises dendritic cells.   124. The method of claim 123, further comprising the step of pulsing dendritic cells to induce an element of an immune response to the attack.   101. The method of claim 100, further comprising producing a pharmacological agent or vaccine.   101. The method of claim 100, further comprising increasing the biological activity of the pharmacological agent.   101. The method of claim 100, further comprising binding DNA or RNA molecules contained within the first fluid to glycogen.   101. The method of claim 100, further comprising binding DNA or RNA molecules contained within the first fluid to silica.   128. The method of claim 127, further comprising purifying glycogen complex or co-precipitated DNA and RNA.   129. The method of claim 128, further comprising purifying the DNA or RNA molecule bound to the silica beads or silica-containing structure.   131. The method of claim 129 or 130, further comprising eluting DNA and RNA from glycogen or silica beads or silica-containing structures.   132. The method of any one of claims 100-131, further comprising detecting the presence or absence of an analyte in the first fluid.   135. The method of claim 132, wherein the detecting comprises sensing visible light or another type of electromagnetic radiation from chemiluminescent or fluorescent molecules bound to the analyte.   135. The method of claim 132, wherein the detecting step comprises sensing the location or distribution of paramagnetic beads that are bound to the analyte.   135. The method of claim 132, wherein the detecting step comprises sensing a nuclear magnetic resonance or other physical property of the species that is bound to the analyte.   102. The method of claim 100, further comprising generating a plurality of microdroplets in the plurality of fluid passages.   137. The method of claim 136, wherein the plurality of microdroplets are formed by pulsing at least two fluids, the pulsation being caused by a plurality of high speed microfluidic actuators in the microfluidic cartridge.   137. The method of claim 136, further comprising detecting the presence or absence of an analyte in each of the plurality of microdroplets.   138. The method of claim 136, further comprising performing an amplification reaction in each of the plurality of microdroplets by moving the plurality of microdroplets through the plurality of temperature zones in the microfluidic cartridge.   140. The method of claim 139, further comprising detecting the presence of a target amplicon in each of the plurality of microdroplets.   137. The method of claim 136, further comprising measuring a melting temperature of the target nucleic acid molecule in each of the plurality of microdroplets.   142. The method of claim 141, further comprising performing a melting temperature analysis of genetic differences in viral RNA from the reference strain.

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