WO2021185599A1 - Trieuse microfabriquée avec étage de tri magnétique et distributeur de gouttelettes - Google Patents

Trieuse microfabriquée avec étage de tri magnétique et distributeur de gouttelettes Download PDF

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Publication number
WO2021185599A1
WO2021185599A1 PCT/EP2021/055583 EP2021055583W WO2021185599A1 WO 2021185599 A1 WO2021185599 A1 WO 2021185599A1 EP 2021055583 W EP2021055583 W EP 2021055583W WO 2021185599 A1 WO2021185599 A1 WO 2021185599A1
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Prior art keywords
droplet
fluid
bead
microfabricated
target
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PCT/EP2021/055583
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English (en)
Inventor
Rockenbach ALEXANDER
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Miltenyi Biotec B.V. & Co. KG
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Publication of WO2021185599A1 publication Critical patent/WO2021185599A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
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    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
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    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/30—Combinations with other devices, not otherwise provided for
    • G01N15/1023—
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    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/061—Counting droplets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0622—Valves, specific forms thereof distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
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    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical applications
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    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G01N2015/1028—

Definitions

  • the present invention is directed to a system for the manipulation of particles and biological materials, and forming droplets containing these particles.
  • nucleotides added to a template strand during sequencing-by-synthesis typically are labeled, or are intended to generate a label, upon incorporation into the growing strand. The presence of the label allows detection of the incorporated nucleotide. Effective labeling techniques are desirable in order to improve diagnostic and therapeutic results.
  • microfluidic devices [0005] At the same time, precision manipulation of streams of fluids with microfluidic devices is revolutionizing many fluid-based technologies. Networks of small channels are a flexible platform for the precision manipulation of small amounts of fluids.
  • the utility of such microfluidic devices depends critically on enabling technologies such as the microfluidic pumps and valves, electrokinetic pumping, dielectrophoretic pump or electrowetting driven flow. The assembly of such modules into complete systems provides a convenient and robust way to construct micro fluidic devices.
  • the structures and methods involve positioning fluid samples, e.g., in the form of droplets, in a carrier fluid (e.g., an oil, which may be immiscible with the fluid sample) in predetermined regions in a micro fluidic network.
  • a carrier fluid e.g., an oil, which may be immiscible with the fluid sample
  • positioning of the droplets can take place in the order in which they are introduced into the microfluidic network (e.g., sequentially) without significant physical contact between the droplets. Because of the little or no contact between the droplets, there may be little or no coalescence between the droplets. Accordingly, in some such embodiments, surfactants are not required in either the fluid sample or the carrier fluid to prevent coalescence of the droplets.
  • USP 9,410,151 provides microfluidic devices and methods that are useful for performing high-throughput screening assays and combinatorial chemistry.
  • This patent provides for aqueous based emulsions containing uniquely labeled cells, enzymes, nucleic acids, etc., wherein the emulsions further comprise primers, labels, probes, and other reactants.
  • An oil based carrier-fluid envelopes the emulsion library on a microfluidic device. Such that a continuous channel provides for flow of the immiscible fluids, to accomplish pooling, coalescing, mixing, Sorting, detection, etc., of the emulsion library.
  • USP 9,399,797 relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same.
  • a method for determining the nucleic acid make-up of a sample is provided.
  • USP 9,150,852 describes barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct
  • the object of the invention to provide a microfabricated system that can separate target particles from non-target material, also separate a labelled bead, and combine the two particles in a single droplet.
  • the droplet may comprise a first aqueous fluid, such as a saline or buffer fluid.
  • the droplet may be dispensed into a stream of a second fluid, immiscible with the first fluid.
  • the droplet may maintain its integrity as a single, discrete, well defined unit because the fluids are immiscible and the droplets do not touch or coalesce.
  • the target particle is a biological material such as a cell, with antigens located on its outer surface
  • the target particle may become attached to the bead by conjugation of these antigens with antibodies disposed on the bead.
  • the bead may further be labelled by an identifying fluorescent signature, which may be a plurality of fluorescent tags affixed to the bead. Accordingly, each target cell, now bound to an identifiable, labelled fluorescent bead, may be essentially barcoded for its own identification. This may allow a large number of experiments to be performed on a large population of such droplets, encased in the immiscible fluid, because the particles are all identifiable and distinguishable.
  • a magnetic separation chamber may be included to enrich or concentrate the population of target cells, by removing or separating other particles.
  • a microfluidic channel formed in a substrate and at least one particle and non-target material suspended in a first fluid, wherein the first fluid is flowing in the microfluidic fluid channel, and wherein the at least one particle is labeled with a magnetic bead, a micro fabricated MEMS fluidic valve, configured to open and close the microfluidic channel to form a droplet.
  • the device may also include a magnetic source which generates a magnetic field; which interact with the magnetic bead a droplet comprising a first fluid dispensed at an end of the microfluidic channel, wherein a dimension of the droplet is determined by a timing of opening and closing of the microfabricated microfluidic valve, and a source of a second fluid immiscible with the first fluid wherein the droplet is dispensed from the microfluidic channel into, and immersed in, the second immiscible fluid.
  • a magnetic source which generates a magnetic field
  • the system may use either or both of a magnetic and/or fluorescent moiety bound to a target particle.
  • the system may further comprise a fluid sample stream flowing in the microfluidic channel, wherein the fluid sample stream comprises target particles and non target material, and an interrogation region in the microfluidic channel.
  • the target particle may be identified among non-target material, and the microfabricated MEMS fluidic valve may separate the target particle from the non-target material in response to a signal from the interrogation region, and direct the target particle into the droplet.
  • the system may also make use of a bead attached to a plurality of fluorescent tags, wherein the fluorescent tags specify the identity of the bead with a fluorescent signal, and wherein the microfabricated MEMS fluidic valve is configured to separate the bead and direct the bead into the droplet, wherein the bead and a target particle, are located within the same droplet.
  • FIG. 1 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid with the microfabricated MEMS fluidic valve in the closed position;
  • FIG. 2 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid with the microfabricated MEMS fluidic valve in the open (sort) position;
  • Fig. 3 is a chart showing the functional dependence of the water droplet size on the duration that the microfabricated MEMS fluidic valve is open;
  • FIG. 4 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid generating an empty droplet in oil;
  • FIG. 5 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid generating a droplet, wherein the droplet contains both a particle and a bead;
  • FIG. 6 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid in a butt junction
  • FIG. 7 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with a laser assisted droplet coalescence
  • Fig. 8 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with a variable channel cross section.
  • Fig. 9 illustrates an embodiment wherein a magnetic stage is provided in addition to the microfabricated droplet sorter.
  • Fig. 10 illustrates further details of the magnetic stage of the microfabricated droplet dispensing device, wherein the droplet is submerged in an immiscible fluid
  • Fig. 11 illustrates further details of the magnetic stage embodiment of the microfabricated droplet dispensing device, wherein the droplet is submerged in an immiscible fluid and may be dispensed into an optically transparent viewing areas.
  • the system includes a microfabricated droplet dispenser that dispenses the droplets into an immiscible fluid.
  • the system may be applied to a fluid sample stream, which may include target particles as well as non-target material.
  • the target particles may be biological in nature, such as biological cells like T-cells, tumor cells, stem cells, for example.
  • the non-target material might be plasma, platelets, buffer solutions, or nutrients, for example.
  • the microfabricated MEMS valve may be, for example, the device shown generally in Figs. 1 and 2. It should be understood that this design is exemplary only, and that other sorts of MEMS valves may be used in place of that depicted in Figs. 1 and 2.
  • Fig. 1 is an plan view illustration of the novel microfabricated fluidic MEMS droplet dispensing device 10 in the quiescent (un-actuated) position.
  • the MEMS droplet dispensing device 10 may include a microfabricated fluidic valve or movable member 110 and a number of microfabricated fluidic channels 120, 122 and 140.
  • the fluidic valve 110 and microfabricated fluidic channels 120, 122 and 140 may be formed in a suitable substrate, such as a silicon substrate, using MEMS lithographic fabrication techniques as described in greater detail below.
  • the fabrication substrate may have a fabrication plane in which the device is formed and in which the movable member 110 moves. Details as to the fabrication of the valve 110 may be found in US Patent 9,372,144 (the ‘144 patent) issued June 21, 2016 and incorporated by reference in its entirety.
  • a fluid sample stream may be introduced to the microfabricated fluidic valve 110 by a sample inlet channel 120.
  • the sample stream may contain a mixture of particles, including at least one desired, target particle and a number of other undesired, nontarget materials.
  • the particles may be suspended in a fluid, which is generally an aqueous fluid, such as saline.
  • this aqueous fluid may be the first fluid, and this first fluid may be immiscible in a second fluid, as described below.
  • the target particle may be a biological material such as a stem cell, a cancer cell, a zygote, a protein, a T-cell, a bacteria, a component of blood, a DNA fragment, for example, suspended in a buffer fluid such as saline.
  • the fluid inlet channel 120 may be formed in the same fabrication plane as the valve 110, such that the flow of the fluid is substantially in that plane. The motion of the valve 110 may also be within this fabrication plane.
  • the decision to sort/save or dispose/waste a given particle may be based on any number of distinguishing signals.
  • the fluid sample stream may pass through an interrogation region 170, which may be a laser interrogation region, wherein an excitation laser excites fluorescent tag affixed to a target particle.
  • the fluorescent tag may emit fluorescent radiation as a result of the excitation, and this radiation may be detected by a nearby detector, and thus a target particle or cell may be identified.
  • the microfabricated MEMS valve may be actuated, as described below, and the flow directed from the nonsort (waste) channel 145 to the sort channel 122, as illustrated in Fig. 2.
  • the actuation means may be electromagnetic, for example.
  • the analysis of the fluorescent signal, the decision to sort or discard a particle, and the actuation of the valve may be under the control of a microprocessor or computer.
  • the actuation may occur by energizing an external electromagnetic coil and core in the vicinity of the valve 110.
  • the valve 110 may include an inlaid magnetically permeable material, which is drawn into areas of changing magnetic flux density, wherein the flux is generated by the external electromagnetic coil and core.
  • other actuation mechanisms may be used, including electrostatic and piezoelectric. Additional details as to the construction and operation of such a valve may be found in the incorporated ‘144 patent.
  • the decision is based on a fluorescence signal emitted by the particle, based on a fluorescent tag affixed to the particle and excited by an illuminating laser.
  • these fluorescent tags may be identifiers or a barcoding system.
  • other sorts of distinguishing signals may be anticipated, including scattered light or side scattered light which may be based on the morphology of a particle, or any number of mechanical, chemical, electric or magnetic effects that can identify a particle as being either a target particle, and thus sorted or saved, or an nontarget particle and thus rejected or otherwise disposed of.
  • This system may also be used to sort the labelled or barcoded bead.
  • the “target particle” may be either a cell and/or a labelled bead.
  • the microfabricated MEMS fluidic valve 110 With the valve 110 in the position shown in Fig. 1, the microfabricated MEMS fluidic valve 110 is shown in the closed position, wherein the fluid sample stream, target particles and non-target materials flow directly in to the waste channel 140. Accordingly, the input stream passes unimpeded to an output orifice and channel 140 which may be out of the plane of the inlet channel 120, and thus out of the fabrication plane of the device 10. That is, the flow is from the inlet channel 120 to the output orifice 140, from which it flows substantially vertically, and thus orthogonally to the inlet channel 120.
  • This output orifice 140 leads to an out-of-plane channel that may be perpendicular to the plane of the paper showing Fig. 1. More generally, the output channel 140 is not parallel to the plane of the inlet channel 120 or sort channel 122, or the fabrication plane of the movable member 110.
  • the output orifice 140 may be a hole formed in the fabrication substrate, or in a covering substrate that is bonded to the fabrication substrate. Further, the valve 110 may have a curved diverting surface 112 which can redirect the flow of the input stream into a sort output stream, as described next with respect to Fig. 2.
  • the contour of the orifice 140 may be such that it overlaps some, but not all, of the inlet channel 120 and sort channel 122. By having the contour 140 overlap the inlet channel, and with relieved areas described above, a route exists for the input stream to flow directly into the waste orifice 140 when the movable member or valve 110 is in the un-actuated waste position.
  • Fig. 2 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid with the microfabricated MEMS device 10.
  • the MEMS device 10 may include a MEMS fluidic valve 110 in the open (sort) position. In this open (sort) position, a target cell 5 as detected in the laser interrogation region 170 may be deflected into the sort channel 122, along with a quantity of the suspending (buffering) fluid.
  • the movable member or valve 110 is deflected upward into the position shown in Fig. 2.
  • the diverting surface 112 is a sorting contour which redirects the flow of the inlet channel 120 into the sort output channel 122.
  • the sort output channel 122 may lie in substantially the same plane as the inlet channel 120, such that the flow within the sort channel 122 is also in substantially the same plane as the flow within the inlet channel 120. Actuation of movable member 110 may arise from a force from force-generating apparatus (not shown).
  • force-generating apparatus may be an electromagnet, however, it should be understood that force-generating apparatus may also be electrostatic, piezoelectric, or some other means to exert a force on movable member 110, causing it to move from a first position (Fig. 1) to a second position (Fig. 2).
  • the micromechanical particle manipulation device shown in Figs. 1 and 2 may be formed on a surface of a fabrication substrate, wherein the micromechanical particle manipulation device may include a microfabricated, movable member 110, wherein the movable member 110 moves from a first position to a second position in response to a force applied to the movable member, wherein the motion is substantially in a plane parallel to the surface, a fluid sample inlet channel 120 formed in the substrate and through which a fluid flows, the fluid including at least one target particle and non-target material, wherein the flow in the fluid sample inlet channel is substantially parallel to the surface, and a plurality of output channels 122, 140 into which the microfabricated member diverts the fluid, and wherein the flow in at least one of the output channels 140 is not parallel to the plane, and wherein at least one output channel 140 is located directly below at least a portion of the movable member 110 over at least a portion of its motion.
  • channel 122 is referred to as the “sort channel” and orifice 140 is referred to as the “waste orifice”, these terms can be interchanged such that the sort stream is directed into the waste orifice 140 and the waste stream is directed into channel 122, without any loss of generality.
  • the “inlet channel” 120 and “sort channel” 122 may be reversed.
  • the terms used to designate the three channels are arbitrary, but the inlet stream may be diverted by the valve 110 into either of two separate directions, at least one of which does not lie in the same plane as the other two.
  • the term “substantially” when used in reference to an angular direction, i.e. substantially tangent or substantially vertical, should be understood to mean within 15 degrees of the referenced direction.
  • substantially orthogonal to a line should be understood to mean from about 75 degrees to about 105 degrees from the line.
  • the suspending aqueous fluid may flow into the sort channel 122, and from there to the edge of the fabrication substrate.
  • the fluid that was flowing in the fluid sample inlet channel 120 may then form a droplet at the edge of the fabrication substrate.
  • the generated droplet might flow to and accumulate in the sort chamber.
  • Various structures may be used in this region to promote the formation of the droplet. These structures may be, for example, rounded comers or sharp edges which may influence or manipulate the strength or shape of the meniscus forces, wetting angle or surface tension of the first fluid droplet. These structures may be generally referred to as a “nozzle” indicating the region where the droplet is formed. At this nozzle point where the droplet is formed, an additional manifold may deliver an immiscible second fluid to the aqueous droplet, suspending the aqueous droplet in the fluid and preserving its general contours and boundary layers.
  • the valve 110 may be used to sort both a target cell and a bead.
  • Laser induced fluorescence may be the distinguishing feature for either or both particles. These particles may both be delivered into a single droplet. These particles may be suspended in, and surrounded by, an aqueous first fluid, such as saline. Accordingly, the droplet may comprise primarily this first fluid, as well as the chosen particle(s), a target cell and/or a bead. The bead may be “barcoded”, that is, it may carry identifying markers. The droplet may then be surrounded by an immiscible second fluid that is provided by a source of the second fluid,
  • droplets may be formed at the intersection with the immiscible fluid. These droplets may be encased in an immiscible second fluid, such as a lepidic fluid or oil 200, as shown in Figs. 1 and 2.
  • an immiscible second fluid such as a lepidic fluid or oil 200
  • the oil 200 may be applied symmetrically by oil input 220 and oil input 240.
  • the immiscible fluid may serve to maintain the separation between droplets, so that they do not coalesce, and each droplet generally contains only one target particle and only one bead.
  • the stream of oil may exit the sort outlet via 260.
  • the lipidic fluid may be a petroleum based lipidic fluid, or a vegetable based lipidic fluid, or an animal based lipidic fluid.
  • the pace, quality and rate of droplet formation may be controlled primarily by the dynamics of the MEMS valve 110. That is, the quantity of fluid contained in the droplet, and thus the size of the droplet, may be a function of the amount of time that the MEMS valve 110 is in the open or sort position shown in Fig. 2.
  • the functional dependence of the size of the droplet on the valve open time is illustrated in Fig. 3. As can be seen in Fig. 3, the diameter of the droplet is proportional to the valve open time, over a broad range of values. Only at exceedingly large droplets and long open times (greater than about 100 psecs and 60 microns diameter) does the functional dependence vary from its linear behaviour.
  • the length of the sort pulse can determine the size of the generated droplet. If the pulse is too short, the oil meniscus may remain intact and no water droplet is formed. If the sort pulse is sufficiently long, a droplet may be formed at the exit and released into the stream of the second immiscible fluid.
  • a target cell 5 is sorted within this time frame, the target cell 5 may be enclosed in the aqueous droplet. If the target particle is not sorted within this time frame, an empty aqueous droplet, that is, a droplet without an enclosed particle 5, may be formed. The situation is shown in Fig. 4.
  • the MEMS valve 110 may be made on the fabrication surface of at least one semiconductor substrate. More generally, a multi-substrate stack may be used to fabricate the MEMS valve 110. As detailed in the ‘144 patent, the multilayer stack may include at least one semiconductor substrate, such as a silicon substrate, and a transparent glass substrate. The transparent substrate may be required to allow the excitation laser to be applied in the laser interrogation region 170.
  • the droplet 300 may be formed at the edge of the semiconductor substrate, or more particularly, at the edge of the multilayer stack.
  • the droplet 300 may be formed at the exit of the sort channel 122 from this multilayer stack.
  • the droplet is not formed at the edge of the multilayer stack, but instead may be formed at the intersection of the sort flow and oil input, within the semiconductor substrate.
  • a structure may be formed that promotes the formation of the droplet. This structure may include sharply rounded corners so as to manipulate surface tension forces, and the formation of meniscus and wetting angles.
  • the structure designed to promote droplet formation may be referred to herein as a nozzle 150, and the term “nozzle” may refer generally to the location at which the droplet may be formed.
  • a flow junction with the immiscible second fluid.
  • the sort channel downstream of the valve, there may be a flow junction with oil (as a carrier for water droplets) flowing from the sides towards the sort channel 122.
  • This flow junction may have an inlet 220 and 240 on either end of the sort channel 122, forming an oil stream 200 downstream of the nozzle 150 and sort channel 122.
  • the method for forming a droplet in oil may be as follows.
  • a target cell is first detected in the laser interrogation region 170.
  • a computer or controller may monitor the signals from the laser interrogation region.
  • the computer or controller may send a signal to open the MEMS valve 110 by energizing the electromagnet. Magnetic interactions then move the MEMS valve as shown in Fig. 2.
  • a target cell 5 may be deflected into the sort channel, along with a quantity of the suspended fluid.
  • a bead is then sorted to accompany the sorted cell as a unique barcode.
  • a second sort pulse is long enough to cause an instability in the oil-water interface and form a water droplet in oil containing the cell and the bead.
  • the effluent may be directed into a waste receptacle, until a target particle is detected. It may also be the case that continued leakage of the fluid sample stream through the gaps around the MEMS valve 110, may eventually cause a water droplet to form. Because no target cell has been detected, and the MEMS valve 110 has not been opened, this aqueous droplet may be empty.
  • Fig. 4 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid generating an empty first fluid droplet 300 in oil 200. This situation may occur if no target particle is present in the fluid sample stream.
  • the MEMS valve 110 may leak slightly, causing an aqueous droplet to form but without an enclosed target particle. In this case, the droplet may be allowed to flow into a waste area of a holding receptacle.
  • the MEMS valve 110 may sort both a target particle 5 (here, a target cell 320) and a bead 310, as shown in Fig. 5.
  • the bead may be a biologically inert material coated with a biologically active material, and additional compounds.
  • the biologically active materials may be antibodies that can become conjugated to antigens appearing on a target cell surface 320.
  • the bead may further be coupled to a plurality of fluorescent tags, that is, compound which fluoresces when irradiated by an excitation laser of the proper wavelength and intensity. This plurality of fluorescent tags may be different for each bead 310, and may therefore act as a signature or identifier for the bead.
  • a bead 310 When a bead 310 is in proximity to a target cell 320, and the antibodies of the bead 310 may become conjugated with the antigens of the cell, the bead, along with its identifying fluorescent tags, may become affixed to the cell 320.
  • the bead 310 provides an identifying marker for the cell 320, or a “barcode” which identifies the cell.
  • a computer or controller may associate this particular barcode with the particular cell. Accordingly, a large number of such droplets may be placed in a small volume of fluid, each containing a target cell and identifying barcode and all within a field of view of a single detector. This may allow a very large number of biological assays or polymerase chain reactions, to be undertaken in parallel, and under a single detection system.
  • Fig. 5 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid generating a droplet in oil, wherein the droplet contains both a particle or cell 320 and a bead 310.
  • the MEMS valve 110 may first sort a particle 320, enclosing the particle 320 in an aqueous droplet as described above.
  • the MEMS valve 110 may then also sort a barcoded bead 310 , and both particle 320 and the bead 310 may be enclosed in the same aqueous droplet, as shown in Fig. 5.
  • Fig. 6 is a schematic illustration of another embodiment of a microfabricated droplet dispenser with an immiscible fluid in a butt junction.
  • the application of the surrounding second immiscible fluid is asymmetrical. Instead of coming both from the right and the left of the nozzle region, the oil 200, the oil junction is applied in parallel to the sort channel 122 and may exit downstream 260 of the sort channel 122.. The second immiscible fluid may flow from right to left. The aqueous fluid droplet may break the oil meniscus from the side channel, as shown.
  • each droplet 300 in oil 200 may contain both a target cell 320 and an identifying bead 310.
  • Fig. 7 is a schematic illustration of another embodiment of a microfabricated droplet dispenser with a laser assisted droplet coalescence.
  • the two particles the target cell 320 and the bead 310 are sorted separately and placed into two separate aqueous droplets in the oil stream 200.
  • the sort pulse is long enough to cause an instability in the oil-water interface and form a water droplet in oil containing the cell.
  • the two separate droplets are then merged by application of laser light 400 on to oil channel containing the aqueous droplets.
  • a pulsed CO2 laser may be directed onto the channel as shown in Fig. 7, to heat the droplets.
  • the application of energy causes the fluids to heat, which weakens meniscus and membrane forces, allowing the droplets to merge.
  • the microfabricated droplet dispenser in Fig. 7 may have a symmetric (or asymmetric) oil input configuration.
  • the droplets 300 may be encased in an immiscible second fluid, such as a lepidic fluid or oil 200.
  • the oil 200 may be applied symmetrically by oil input 220 and oil input 240.
  • the stream of oil may exit the sort outlet via 260.
  • the embodiment shown in Fig. 7 may have a flow channel which is capable of sorting two aqueous droplets, and then merging them into a single larger droplet.
  • the sort pulse is long enough to cause an instability in the oil-water interface and form a water droplet in oil containing the cell. Then a bead is sorted and a separate droplet is formed.
  • the first droplet may contain a target cell 320, and the second aqueous droplet may contain a bead 310 as previously described.
  • a merging area is a portion of the sort flow channel 122 wherein the laser 400 is directed. The laser light may be focused to increase its peak intensity.
  • the applied laser light may heat the droplet as well as the surrounding fluid, and allow the two droplets to merge.
  • the merging may be caused by the laser-induced heating and consequent weakening of surface tension of the fluid droplet.
  • the first droplet may contain the bead 310
  • the second aqueous droplet may contain the target cell 320.
  • the application of heat onto the channel in the laser 400 may serve to heat the fluids and allow the two droplets to merge.
  • at the output of the microfabricated droplet dispenser may emerge an aqueous droplet encased in oil wherein the droplet contains both a target cell 320 and a bead 310.
  • the bead 310 may have a fluorescent barcode affixed to it, and the bead may be conjugated to the target cell 320.
  • Fig. 8 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with a variable channel cross section.
  • the microfabricated droplet dispenser in Fig. 8 may have a symmetric (or asymmetric) oil input configuration.
  • the droplets may be encased in an immiscible second fluid, such as a lepidic fluid or oil 200.
  • the oil 200 may be applied symmetrically by oil input 220 and oil input 240.
  • the stream of oil may exit the sort outlet via 260.
  • the embodiment shown in Fig. 8 may have a flow channel which is capable of sorting two aqueous droplets, and then merging them into a single larger droplet.
  • the sort pulse is long enough to cause an instability in the oil-water interface and form a water droplet 300 in oil containing the cell. Then a bead 310 is sorted and a separate droplet is formed.
  • the first droplet may contain a target cell 320, and the second aqueous droplet may contain a bead 310 as previously described.
  • a merging area 500 is a portion of the sort channel 122 having a variable cross section 500. The sudden widening of the channel in the merging area 500 may serve to slow the flow down within the merging area, allowing the two droplets to merge. In other words, the sudden widening may produce geometry-induced flow slowdown, which allows the droplets to merge.
  • the first droplet may contain the bead 310
  • the second aqueous droplet may contain the target cell 320.
  • the sudden widening of the channel in the merging area 500 may serve to slow the flow down within the merging area, allowing the two droplets to merge.
  • at the output of the microfabricated droplet dispenser may emerge an aqueous droplet 300 encased in oil 200 wherein the droplet 300 contains a target cell 320 and a bead 310.
  • the bead 310 may have a fluorescent barcode affixed to it, and the bead may be conjugated to the target cell 320.
  • a fluid with magnetic beads 20 for labelling is pumped from a magnetic bead input chamber 22.
  • the magnetic bead input chamber 22 may containing a plurality of magnetic beads 20.
  • the beads may have an antigen binding structure that allows it to binder to an antibody on the membrane of a target sell 10.
  • the target cells 10, may be held in a sec at the chamber 12.
  • the beads are then combined with a plurality of target cells 10 stored or input into in the sample chamber 12.
  • a sample consisting of different cell types are provided to the sample input chamber 10.
  • a volume of a fluid with magnetic beads for labelling is pumped from a magnetic bead input chamber 22, into the sample input chamber 10.
  • the magnetic beads 20 may become bound with the targe cell 10, to form a conjugated bead-cell combination 10-20.
  • They bead-cell combination 10-20 may then proceed to a magnetic separation column 500.
  • the magnetic separation column 500 may include a magnetic source 550 which may generate a magnetic field which may interact with magnetic beads 20 coupled to the target cells 10. Accordingly, the magnetic source may attract the beads coupled to the target cells, and confine them to the magnetic separation chamber 50. And this way, the flow may be depleted of the species which are bound to the magnetic beads 20.
  • the magnetic source 550 may include either a permanent magnet or an electromagnet, for example.
  • the magnetically labelled target cells 10-20 may be held back by the magnetic force generated by a magnetic source 550.
  • Unlabelled (non magnetic) cells 10 may be pumped back into the input chamber 12, or they may proceed to the label release chamber 800. Accordingly, unlabelled cells 10 may emerge first from the magnetic separation column 500, followed by bead-cell combinations 10-20.
  • the bead-cell combinations 10-20 may then proceed to the label release chamber 800, where the magnetic bead 20 may be removed from the target cell 10. Accordingly, the flow may first include unlabelled cells 10, followed by labelled bead-cell combinations 10-20.
  • a second option is the release of cells directly from the magnetic separation column 500 in a way that the binding between target cell and bead is released.
  • the magnetically labelled cells may be released from the label release chamber without removing the magnetic bead. As with the magnetic separation column 500, this may be accomplished, for example, by removing the current or voltage through an electromagnet, or by moving the permanent magnet. The labeled bead may then follow the unlabeled cells in time, and can be separated temporally.
  • a solvent or enzyme may be applied to the target cell 10 and label 20, thus removing the magnetic label 20 from the target cell 10. Accordingly, the label release chamber 800 may hold back the magnetic beads, which are no longer used for separation. These now unlabelled cells 10 may now again can be led back to the input chamber. Accordingly, Fig. 9 also illustrates a fluid feedback loop which may reduce the amount of reagent necessary to process the sample.
  • the cells not routed back to the sample input chamber 12 may proceed into the fluorescent labeling chamber 90.
  • the target cells 10 may be bound to a fluorescent label 950, which fluoresces upon radiation by an appropriate wavelength.
  • the target sell 10 may exit the apparatus by either directed into the waste channel shown, or by being encapsulated in a droplet. This may happen in the encapsulation region 120. As shown in Fig. 9.
  • the fluorescent labelling chamber may also include bead re-labelling, as shown in Fig. 9. Accordingly, in the fluorescent labelling chamber 900, cells, dye and barcoded beads may be mixed, such that the fluorescent moiety binds to the target cells 10.
  • the target cells may be labelled with a fluorescent dye and optionally another bead.
  • the cells Downstream of the fluorescent labelling chamber 900, the cells may traverse an interrogation zone 100, wherein they may be illuminated with laser light of an appropriate wavelength. Upon irradiation, the fluorescent moiety may emit a fluorescent photon, which may be detected by the optical detector described in considerable detail above. One or more lasers may illuminate the cell such that when the photon is detected, the valve 110 is triggered to open, depending on the criteria implemented in the software and described above. Machine learning techniques may be applied to this recognition and triggering event.
  • the fluorescent label on the cell which identifies the cell may be excited by the laser, and the resulting fluorescence detected, thus identifying the cell.
  • Target cells 10 along with their associated bead or fluorescent moiety may be directed into a sort channel whereas non-target material may be directed into a waste channel.
  • this waste channel may be perpendicular to the input channel and the sort channel, and thus not depicted explicitly in the figures using this valve design.
  • Sorted cells and sorted beads may finally be encapsulated either with a passive droplet forming intersection 1200 or with a encapsulation valve described below with respect to Fig. 11.
  • the next Fig. 10 shows the microfabricated sorter with magnetic separation and droplet dispenser in a second embodiment.
  • a number of auxiliary devices are applied to the system, including mixers, valves, and pumps. In the embodiment illustrated in Fig. 10, these additional supporting devices are shown disposed in convenient locations.
  • These devices may include mixers which stir, mix or agitate the contents of a fluid reservoir in order to thoroughly mix the suspended particles contained therein.
  • the mixer described may be a bubble jet, wall jet, ciliated mixer, magnetically driven rotor, a ultrasonic mixer, an alternating magnetic field moving magnetic particles or fluids.
  • the binding of a bead to a cell may take intimate contact between the particles.
  • a plurality of mixers depicted in Fig. 10 may provide this function. Accordingly, the mixers 14, 24, 840 and 940 may provide this opportunity for particle-particle binding, to sample input chamber 10, bead chamber 10, label release chamber 800 and fluorescent labelling chamber 900, respectively.
  • the decision to allow a particle to proceed through the process, or to be returned to the input, or to be directed into a waste channel may be determined by a set of switching valves.
  • These structures are labelled 570 and 870.
  • a switching valve 570 may be disposed at the output of the magnetic separation chamber 500 to direct fluid either downstream to the label release chamber 800 or it may direct the fluid into the feedback loop to rejoin the input stream at the fluid transport pump 16.
  • Another switching valve 870 may be disposed at the output of the label release chamber 800 to direct fluid either downstream to the fluorescent labelling chamber 900 or it may direct the fluid into the feedback loop to rejoin the input stream at the fluid transport pump 860.
  • a plurality of pumps may provide the fluid pressure that forces the fluid though the depicted manifold. These pumps may be deployed in certain areas.
  • the volume of a fluid with magnetic beads for labelling may be pumped from a magnet bead input chamber 22 by a micro fluidic pump 26 into the sample input chamber 12.
  • the volume of a fluid with magnetic bead-cell combinations 10-20 may then be pumped from the sample chamber 12 to the magnetic separation column 500 by the fluid transport pump 16.
  • the volume of a fluid with magnetic bead-cell combinations 10-20 may then be pumped from the label release chamber 800 to the fluorescent labelling chamber 900 by fluid transporting pump 860.
  • the volume of a fluid with magnetic bead-cell combinations 10-20 may then be pumped from the fluorescent labelling chamber 900 to the microfabricated droplet sorting vale 110 by fluid transporting pump 960.
  • fluid transporting pump 960 may then be pumped from the fluorescent labelling chamber 900 to the microfabricated droplet sorting vale 110 by fluid transporting pump 960.
  • this is only one particular embodiment, and that these structures may be in disposed in different places, or may come in different numbers. Accordingly, it should be understood that a variety of fluid paths and manifolds may be designed with the components shown in Fig. 10. In particular, using careful placement of pumps 16, 26, etc and valves 14, 24, etc. any arbitrary fluid paths may be constructed to direct the target cells, beads, fluorescent markers, etc. to the appropriate channels. It should also be understood that auxiliary channels to, for example, conduct waste material away may exist even though not depicted in these figures. One of ordinary skill is assumed to understand the required placement of such elements.
  • the target cells may be labelled with fluorescent dye. After that the fluorescently labelled cells are pumped into the interrogation zone 100.
  • Cells and beads sorted after each other may be encapsulated either with a passive droplet forming intersection 120 (embodiment 1) or with a encapsulation valve described in (embodiment 2). Exemplary embodiments follow.
  • the fluid flow as determined by the hydrostatic water pressure in the pressurized microchannels shown, may simply be allowed to leak at a rate that droplets are formed at the desired rate.
  • the droplet will form when the surface energy to form a droplet is lower than the energy to form the surface of the capillary reaching into the hydrophobic liquid.
  • various structures may be used in this region to promote the formation of the droplet. These structures may be, for example, rounded corners or sharp edges which may influence or manipulate the strength or shape of the meniscus forces, wetting angle or surface tension of the first fluid droplet. These structures may be generally referred to as a “nozzle” indicating the region where the droplet is formed. At this nozzle point where the droplet is formed, an additional manifold may deliver an immiscible second fluid to the aqueous droplet, suspending the aqueous droplet in the fluid and preserving its general contours and boundary layers.
  • Multiple magnetically driven valves on one chip may led to interference of the magnetic pulses between both valves.
  • This can be reduced by using shielding 1050 of the valves by magnetic material.
  • the shielding may use a permeable magnetic material.
  • Ni/Fe “permalloy” consists of 80% nickel and 20% iron. The deposition by plating or sputtering of 80/20 permalloy is well known in the art. Accordingly, permeable structures 1050 may be deposited around the structures to shield them from stray magnetic fields- The shielding may only be applied on 3 sides.
  • the magnetic flux may be led from the flux-generating solenoid tip into the actuator and into the shielding favouring the shielding as path back to close the flux line. The flux density may thereby be reduced outside the shielding and therefore also the interference with other magnetic fields which may be driving the other actuators.
  • the magnetic separation stage may also be included in a plural actuator embodiment, wherein at least two actuators are fabricated on a substrate.
  • a first actuator may sort a bead
  • a second actuator may sort a cell.
  • the sorted bead may then be combined with the sorted cell to obtain a clearly identified bead-cell combination.
  • This bead-cell combination may then be dispensed in a water droplet that may be encased in a surrounding immiscible fluid.
  • the droplet may be an aqueous suspension of cells and beads, which is surrounded by a fluid which is immiscible with the droplet.
  • a second microfabricated fluid valve or another valve sorting small liquid volumes may also be used to create the droplets.
  • the volumes may be on the order of a picoliter or smaller.
  • the first microfabricated fluid valve may be used to form the droplet under active control.
  • a volume of between 0.5 pL up to 300 pi may be used, with a typical volume of about lOpL,”
  • the pace, quality and rate of droplet formation may be controlled primarily by the dynamics of the MEMS valve 110. In other embodiments, other fluid control devices may be used. Further contributing to the droplet formation are leakage rate of the valve, pressure of the liquids, size and geometry of the oil input channels. That is, the quantity of fluid contained in the droplet, and thus the size of the droplet, may be a function of the amount of time that the MEMS valve 110 is in the open or sort position shown in Fig. 2. The functional dependence of the size of the droplet on the valve open time is illustrated in Fig. 3. As can be seen in Fig. 3, the diameter of the droplet is proportional to the valve open time, over a broad range of values. Only at exceedingly large droplets and long open times (greater than about 100 psecs and 60 microns diameter) does the functional dependence vary from its linear behaviour.
  • the length of the sort pulse can determine the size of the generated droplet. If the pulse is too short, the oil meniscus may remain intact and no water droplet is formed. If the sort pulse is sufficiently long, a droplet may be formed at the exit and released into the stream of the second immiscible fluid.
  • Fig. 11 shows this embodiment. This method and device are similar to those illustrated in Fig. 10, except for the dispensing of the droplet. Accordingly, structures that are repeated from Fig. 10 are simply labelled “A” and the changing components are labelled “B”. For example, B in Fig. 10 reference the passive droplet formation, whereas B in Fig. 11 depicts the active droplet formation using a microfabricated second valve 110’.
  • the second microfabricated sorting valve 110’ may be used to meter the droplet size, as described above.
  • the second valve 110’ is not used to sort particles but instead may be used to meter a certain volume of fluid, and thereby release droplets of known and controlled timing and dimension.
  • the droplet may be dispensed under the control of a second microfabricated valve, as shown.
  • this second valve 110’ may also be used to dispense the droplet enclosing the target particle in a particular well of an optically transparent multiwell observation plate. In this arrangement, the contents of the wells may be viewed by for example, an optical microscope.
  • the fluid may be directed to the input of the second microfabricated sorting valve 110’.
  • the cell may have been lysed in the already formed droplet.
  • the material may be lysed, so as to free the intracellular material. This material may be sorted by secondary sorting valve 110’.
  • sorted cells and beads may be combined in a collection zone 160, where cells and beads can intermingle.
  • the encapsulation valve 170 may be triggered.
  • Cell-bead combinations may be encapsulated in an hydrophobic liquid.
  • the sorted portion of buffer with cell and bead may be led as droplets to the encapsulation output 180 channel.
  • lysis may start as soon as the cell and enzyme are combined into one water droplet. But lysis is generally slower than the droplet formation. Accordingly, the velocity of the droplet formation may need to be adjusted to give lysis sufficient time to proceed.
  • the identified particle or droplet may then be dispensed into a receptacle appropriately positioned.
  • a biological object is any material having a biological aspect or function.
  • biological objects may include beads, bioparticles, cells, gels or gel beads, protein, DNA (or natural product), DNA, RNA and the like.
  • Cells, beads and lysed fragments may be recovered and led to a second detection or interrogation zone 1300. Here, only those cells, beads or cell fragments are chosen which are of interest for an further optical analysis. Those particles of interest are sorted with a second valve 110’ . The remaining material is lead to a waste channel 70.
  • the cell traps or micro cavities 750 may separate the cells from each other by defining a liquid compartment of a certain size.
  • the size of each compartment is chosen in a way to separate cells from each other and hold them back also during washing steps.
  • the compartment should be adapted to the size of the examined cells but smaller than 1 mm. Typically, the size is slightly bigger than the cells but smaller than two cells. Except the device is designed to collect more than one cell in one compartment. In case of traps, 750, the cells are hold by a pressure drop between input and trap side of a channel smaller than the cells. These separation steps help to analyse cells over time and separate from each other.
  • An optical microscope (not shown) may be used to inspect the contents of each well or trap 750.
  • a microfabricated droplet dispenser comprising a microfluidic channel formed in a substrate and at least one target particle and non-target material suspended in a first fluid, wherein the first fluid is flowing in the microfluidic fluid channel, and wherein the at least one particle is labeled with a magnetic bead, a microfabricated MEMS fluidic valve, configured to open and close the microfluidic channel to form a droplet.
  • the device may also include a magnetic source which generates a magnetic field; which interact with the magnetic bead a droplet comprising a first fluid dispensed at an end of the microfluidic channel, wherein a dimension of the droplet is determined by a timing of opening and closing of the microfabricated microfluidic valve, and a source of a second fluid immiscible with the first fluid wherein the droplet is dispensed from the microfluidic channel into, and immersed in, the second immiscible fluid.
  • a magnetic source which generates a magnetic field
  • the droplet dispenser may further comprise a fluid sample stream flowing in the microfluidic channel, wherein the fluid sample stream comprises target particles and non target material, an interrogation region in the microfluidic channel, wherein a target particle is identified among non-target material; and wherein the microfabricated MEMS fluidic valve is configured to separate the target particle from the non-target material in response to a signal from the interrogation region, and direct the target particle into the droplet.
  • It may also include a bead attached to a plurality of fluorescent tags, wherein the fluorescent tags specify the identity of the bead with a fluorescent signal, and wherein the microfabricated MEMS fluidic valve is configured to separate the bead and direct the bead into the droplet, wherein the bead and a target particle, are located within the same droplet.
  • the bead may comprise a plurality of fluorescent tags, such that the bead has an identifying fluorescent signature.
  • the bead may also have at least one antibody, that binds to an antigen on the target particle.
  • the microfabricated MEMS valve may move in a single plane when opening and closing, and wherein that plane is parallel to a surface of the substrate.
  • the droplet may be dispensed at a nozzle structure formed in the microfluidic channel in the substrate.
  • the source of immiscible fluid is disposed symmetrically about the nozzle. Surfactant may be added to the fluid stream.
  • the droplet dispenser may further comprise a laser focused on the microfluidic channel upstream of the nozzle, heating the droplet to assist in severing the droplet from the fluid in the micro fluidic channel, or to heat the droplet to coalesce adjacent droplets in the microfluidic channel.
  • the microfluidic channel may have a channel widened area, wherein the cross section of the channel increases and then decreases.
  • the microchannel may intersect the source of immiscible fluid in a butt junction.
  • the target particles are at least one of T- cells, stem cells, cancer cells, tumor cells, proteins and DNA strands.
  • a method for dispensing droplets may include forming a first fluidic channel on a substrate and forming a microfluidic channel on a substrate, providing a first fluid flowing in the first microfluidic fluid channel, and wherein the first fluid comprises at least one particle is labeled with a magnetic bead with identifiers disposed thereon.
  • the method may also include separating the at least one particle with the magnetic bead using a magnetic source.
  • the method may include opening and closing a microfabricated MEMS fluidic valve, The method may further comprise opening and closing a microfabricated MEMS fluidic valve, to open and close the microfluidic channel, forming a droplet containing the least one particle and bead and the first fluid; and providing a source of an immiscible second fluid, immiscible with the first fluid, and dispensing a droplet of the first, wherein a dimension of the droplet is determined by a timing of opening and closing of the microfabricated microfluidic valve, and wherein the droplet encloses at least one of the bead and the target particle.
  • the fluid flowing in the microfluidic channel may include target particles and non-target material.
  • the method may further include identifying a target particle among non target material in a laser interrogation region, opening and closing the microfabricated MEMS fluidic valve to separate the identified target particle from the non-target material in response to a signal from the interrogation region, and directing the target particle into the droplet.
  • the method may also include providing a bead attached to a plurality of fluorescent tags, wherein the fluorescent tags specify the identity of the bead with a fluorescent signal, separating the bead using the microfabricated MEMS fluidic valve, and directing the bead into the droplet, wherein the bead and the target particle, are located within the same droplet.
  • the droplet may be formed at a nozzle structure formed in the substrate.
  • the method may further include heating the fluid with a laser focused just upstream of the nozzle.

Abstract

L'invention concerne une structure de distribution de gouttelettes microfabriquée, qui peut comprendre une vanne fluidique microfluidique MEMS, conçue pour ouvrir et fermer un canal microfluidique. L'ouverture et la fermeture de la vanne peuvent séparer une particule cible et une bille d'un flux d'échantillon, et diriger ces deux particules en une seule gouttelette formée au niveau bord du substrat. La gouttelette peut ensuite être enfermée dans un écoulement de gaine d'un fluide non miscible.
PCT/EP2021/055583 2020-03-16 2021-03-05 Trieuse microfabriquée avec étage de tri magnétique et distributeur de gouttelettes WO2021185599A1 (fr)

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