EP3022544A1 - Puce microfluidique - Google Patents

Puce microfluidique

Info

Publication number
EP3022544A1
EP3022544A1 EP13889551.1A EP13889551A EP3022544A1 EP 3022544 A1 EP3022544 A1 EP 3022544A1 EP 13889551 A EP13889551 A EP 13889551A EP 3022544 A1 EP3022544 A1 EP 3022544A1
Authority
EP
European Patent Office
Prior art keywords
microfluidic chip
sheath
components
input channel
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13889551.1A
Other languages
German (de)
English (en)
Other versions
EP3022544A4 (fr
Inventor
Zheng Xia
Yu Zhou
John Larsen
Guocheng SHAO
Shane Peterson
Marjorie FAUST
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ABS Global Inc
Original Assignee
Premium Genetics UK Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Premium Genetics UK Ltd filed Critical Premium Genetics UK Ltd
Publication of EP3022544A1 publication Critical patent/EP3022544A1/fr
Publication of EP3022544A4 publication Critical patent/EP3022544A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • B01L3/502776—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 focusing or laminating flows
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • 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
    • B01L2200/0636—Focussing flows, e.g. to laminate flows
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • 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
    • B01L2300/0874—Three dimensional network
    • 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
    • B01L2300/0887—Laminated structure
    • 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
    • B01L2400/0433—Moving fluids with specific forces or mechanical means specific forces vibrational forces
    • B01L2400/0439—Moving fluids with specific forces or mechanical means specific forces vibrational forces ultrasonic vibrations, vibrating piezo elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the present invention relates to a microfluidic chip design which is used to isolate particles or cellular materials into various components and fractions, using laminar flows.
  • the present invention relates to a microfluidic chip design which is used to isolate particles or cellular materials into various components and fractions, using laminar flows.
  • the present invention relates to a microfluidic chip system, which includes a microfluidic chip loaded on a microfluidic chip cassette which is mounted on a microfluidic chip holder.
  • the microfluidic chip includes a plurality of layers in which are disposed a plurality of channels including: a sample input channel into which a sample fluid mixture of components to be isolated is inputted; a first plurality of sheath fluid channels into which sheath fluids are inputted, the first plurality of sheath fluid channels which intersect the sample input channel at a first intersection, such that the sheath fluids compress the sample fluid mixture on at least two sides, such that the sample fluid mixture becomes a relatively smaller, narrower stream, bounded by the sheath fluids, while maintaining laminar flow in the sample input channel; a second plurality of sheath fluid channels, substantially of the same dimensions as the first plurality of sheath fluid channels, into which sheath fluids are inputted, the second plurality of sheath fluid channels which intersect the sample input channel at a second intersection downstream from the first intersection, in a second direction substantially 90 degrees above and below the sample input channel, such that the sheath fluids from the second plurality of sheath fluid channels
  • the microfluidic chip includes an interrogation apparatus which interrogates and identifies said components in said sample fluid mixture in said sample input channel, in an interrogation chamber disposed downstream from said second intersection.
  • the microfluidic chip includes an isolating mechanism which isolates selected of said components in said sample fluid mixture downstream from said interrogation chamber, by displacing a trajectory of a stream of said sample fluid mixture in said sample input channel, and pushing said selected components in said displaced stream of sample fluid mixture into one of said plurality of output channels which lead from said interrogation chamber.
  • the microfluidic chip further includes at least one jet chamber containing sheath fluids introduced into said jet chamber by at least one air vent; and at least one jet channel which is connected to said at least one jet chamber, said at least one jet channel which enters said sample input channel to said interrogation chamber.
  • the isolating mechanism includes at least one piezoelectric actuator assembly disposed on at least one side of said sample input channel.
  • the piezoelectric actuator assembly is an external, stacked piezoelectric actuator assembly.
  • the microfluidic chip further includes a diaphragm which covers each said jet chamber; and wherein said external, stacked piezoelectric actuator assembly aligns with and displaces said diaphragm, to drive said sheath fluids in said jet chamber into said sample input channel, to displace said trajectory of said stream of said sample fluid mixture in said sample input channel into one of said plurality of output channels.
  • the external, stacked piezoelectric actuator assembly is disposed in a microfluidic chip holder.
  • the microfluidic chip further includes an electronic circuit connected to the piezoelectric actuator assembly, the electronic circuit which amplifies an electronic signal generated by a resistance force from the piezoelectric actuator being in contact with the diaphragm.
  • an electric signal from the piezoelectric film shows how much strain is generated by the external, stacked piezoelectric actuator assembly.
  • an indicator of contact is turned on automatically when contact between the piezoelectric actuator and the diaphragm is made.
  • the electronic signal exceeds a set threshold, and the piezoelectric actuator assembly compresses the jet chamber to jet sheath fluids from the jet chamber into the sample fluid channel.
  • the indicator of contact includes a light, a sound, a haptic, or any combination thereof.
  • the piezoelectric actuator assembly includes a flexible diaphragm which covers said jet chamber; and a piezoelectric material bonded on a top surface of said diaphragm by an adhering mechanism.
  • the flexible diaphragm when voltage is applied across electrodes of the piezoelectric actuator assembly, the flexible diaphragm bends into the jet chamber and squeezes the sheath fluids from the jet chamber into the sample input channel to deflect the selected components into one of the plurality of output channels.
  • the jet channel is tapered when it connects to the sample input channel.
  • the microfluidic chip further includes a plurality of outputs disposed at ends of said plurality of output channels. In one embodiment, the plurality of output channels increase in dimension from the sample input channel.
  • the microfluidic chip further includes a plurality of notches disposed at a bottom edge of the microfluidic chip to isolate the plurality of outputs.
  • the sample input channel and the plurality of sheath channels are disposed in one or more planes of the microfluidic chip.
  • the sample input channel and the plurality of sheath channels are disposed in one or more structural layers, or in-between structural layers of the microfluidic chip.
  • At least one of the plurality of sheath channels is disposed in a different plane than a plane in which the sample input channel is disposed.
  • At least one of the plurality of sheath channels is disposed in a different structural layer than a structural layer in which the sample input channel is disposed.
  • the sample input channel tapers at an entry point into the first intersection with said plurality of sheath channels.
  • the sample input channel tapers into said interrogation chamber.
  • the plurality of sheath fluid channels taper at entry points into the sample input channel at least one of the first intersection or the second intersection.
  • the interrogation chamber includes an opening cut through the structural layers in the microfluidic chip; and a top window is configured to receive a first covering in an opening in at least one layer of the structural layers; and a bottom window is configured to receive a second covering in an opening in at least one layer of the structural layers.
  • the interrogation chamber includes an opening cut through the planes in the microfluidic chip; and a top window is configured to receive a first covering in an opening in at least one plane of the planes of the microfluidic chip; and a bottom window is configured to receive a second covering in an opening in at least one plane of the planes of the microfluidic chip.
  • the interrogation apparatus includes a light source configured to emit a beam through the first covering, to illuminate and excite the components in said sample fluid mixture; and wherein emitted light induced by the beam passes through said second covering and is received by an objective lens.
  • the interrogation apparatus includes a light source configured to emit a beam through structural layers of the microfluidic chip, to illuminate and excite the
  • the interrogation apparatus includes a light source configured to emit a beam through the planes of the microfluidic chip, to illuminate and excite said components in said sample fluid mixture; and wherein emitted light induced by said beam is received by an objective lens.
  • the emitted light received by the objective lens is converted into an electronic signal which triggers said piezoelectric actuator assembly.
  • one of the sample fluid mixture or the sheath fluids is pumped into the microfluidic chip by a pumping apparatus.
  • the external tubing communicates fluids to the microfluidic chip.
  • the components are cells.
  • the cells to be isolated include at least one of viable and motile sperm from non- viable or non-motile sperm; sperm isolated by gender and other sex sorting variations; stem cells isolated from cells in a population; one or more labeled cells isolated from un-labeled cells including sperm cells; cells, including sperm cells, distinguished by desirable or undesirable traits; genes isolated in nuclear DNA according to a specified characteristic; cells isolated based on surface markers; cells isolated based on membrane integrity or viability; cells isolated based on potential or predicted reproductive status; cells isolated based on an ability to survive freezing; cells isolated from contaminants or debris;
  • healthy cells isolated from damaged cells red blood cells isolated from white blood cells and platelets in a plasma mixture; or any cells isolated from any other cellular components into corresponding fractions.
  • the isolated components are moved into one of the plurality of output channels, and unselected components flow out through another of the plurality of output channels.
  • the microfluidic chip further includes a computer which controls the pumping of one of the sample fluid mixture or the sheath fluids into the microfluidic chip.
  • the microfluidic chip further includes a computer which displays the components in a field of view acquired by a CCD camera disposed over the opening in the microfluidic chip.
  • the microfluidic chip system includes: a microfluidic chip loaded on a microfluidic chip cassette which is mounted on a microfluidic chip holder, the microfluidic chip having a sample input for introducing sample fluid into the microfluidic chip, and sheath inputs for introducing sheath fluid into the microfluidic chip; and a pumping mechanism which pumps said sample fluid from a reservoir into the sample input of the microfluidic chip, and pumps the sheath fluids into the sheath inputs of the microfluidic chip.
  • a method of orienting and isolating components in a fluid mixture includes: inputting a sample fluid mixture containing components into a sample input channel of a microfluidic chip; inputting sheath fluids into a plurality of first sheath fluid channels of the microfluidic chip, the sheath fluids from the first sheath fluid channels which join the sample fluid mixture in the sample input channel at a first intersection of the plurality of first sheath fluid channels and the sample input channel; wherein the sheath fluids from the first sheath fluid channels compress the sample fluid mixture in one direction in the sample input channel to focus the components in the sample fluid mixture around a center of the sample input channel; and inputting sheath fluids into a plurality of second sheath fluid channels of the microfluidic chip, the sheath fluids from the plurality of second sheath channels which join the sample fluid mixture in the sample input channel at a second intersection of the plurality of second sheath fluid channels and the sample input channel, downstream from the first intersection;
  • FIG. 1 shows an exploded perspective view of an illustrative embodiment of a microfluidic chip according to one embodiment consistent with the present invention.
  • FIGS. 2A-2C show top views of the assembled microfhiidic chip of FIG. 1, according to variant embodiments consistent with the present invention.
  • FIG. 3 shows a cross-sectional view of an interrogation chamber of the microfhiidic chip of FIGS. 1-2, according to one embodiment consistent with the present invention.
  • FIG. 4 shows a cross-sectional internal view of an illustrative interrogation by a light source of components flowing in a fluid mixture, through the microfhiidic chip of FIGS. 1-2, and an illustrative action of one of two (mirrored) piezoelectric actuator assemblies, according to one embodiment consistent with the present invention.
  • FIG. 5A shows a perspective internal, and oblique view of components flowing through the microfhiidic chip of FIGS. 1-2, and an illustrative operation of two-step focusing, according to one embodiment consistent with the present invention.
  • FIG. 5B shows a perspective oblique view of the channels and interrogation chamber disposed in the microfhiidic chip of FIGS. 1-2C, according to one embodiment consistent with the present invention.
  • FIG. 6 shows a schematic illustration of a front view of a main body of a microfhiidic chip holder, according to one embodiment consistent with the present invention.
  • FIG. 7 shows a schematic illustration of a side view of a piezoelectric actuator assembly of the microfhiidic chip holder of FIG. 6, according to one embodiment consistent with the present invention.
  • FIG. 8 shows a schematic illustration of a front view of a microfhiidic chip holder, according to one embodiment consistent with the present invention.
  • FIG. 9 shows a pumping mechanism which pumps sample fluid and sheath or buffer fluids into the microfluidic chip, according to one embodiment consistent with the present invention.
  • the present disclosure relates to a microfluidic chip design, which is used to isolate particles or cellular materials, such as sperm, and other particles or cells, into various components and fractions, using laminar flows.
  • the various embodiments of the present invention provide for isolating components in a mixture, such as, for example: isolating viable and motile sperm from non-viable or non-motile sperm; isolating sperm by gender, and other sex sorting variations; isolating stems cells from cells in a population; isolating one or more labeled cells from un-labeled cells distinguishing desirable/undesirable traits; isolating genes in nuclear DNA according to a specified
  • isolating cells based on surface markers isolating cells based on membrane integrity (viability), potential or predicted reproductive status (fertility), ability to survive freezing, etc.; isolating cells from contaminants or debris; isolating healthy cells from damaged cells (i.e., cancerous cells) (as in bone marrow extractions); red blood cells from white blood cells and platelets in a plasma mixture; and isolating any cells from any other cellular components, into corresponding fractions.
  • the subject matter of the present disclosure is also suitable for other medical applications as well.
  • the various laminar flows discussed below may be utilized as part of a kidney dialysis process, in which whole blood is cleansed of waste products and returned to the patient.
  • the various embodiments of the present disclosure may have further applicability to other biological or medical areas, such as for separations of cells, viruses, bacteria, cellular organelles or subparts, globular structures, colloidal suspensions, lipids and lipid globules, gels, immiscible particles, blastomeres, aggregations of cells, microorganisms, and other biological materials.
  • the component separation in accordance with the present disclosure may include cell "washing", in which contaminants (such as bacteria) are removed from cellular suspensions, which may be particularly useful in medical and food industry applications.
  • cell "washing" in which contaminants (such as bacteria) are removed from cellular suspensions, which may be particularly useful in medical and food industry applications.
  • prior art flow-based techniques have not recognized any applicability to separation of non-motile cellular components, as have the present invention.
  • the subject matter of the present disclosure may also be utilized to move a species from one solution to another solution where separation by filtering or centrifugation is not practical or desirable.
  • additional applications include isolating colloids of a given size from colloids of other sizes (for research or commercial applications), and washing particles such as cells, egg cells, etc. (effectively replacing the medium in which they are contained and removing contaminants), or washing particles such as nanotubes from a solution of salts and surfactants with a different salt concentration or without surfactants, for example.
  • the action of isolating species may rely on a number of physical properties of the objects or components including self-motility, self-diffusivity, free-fall velocity, or action under an external force, such as an actuator, an electromagnetic field or a holographic optical trap.
  • the properties which may be sorted upon include, for example, cell motility, cell viability, object size, object mass, object density, the tendency of objects to attract or repel one another or other objects in the flow, object charge, object surface chemistry, and the tendency of certain other objects (i.e., molecules) to adhere to the object.
  • the various embodiments of the microfluidics chip utilize one or more flow channels, having a plurality of substantially laminar flows, allowing one or more components to be interrogated for identification, and to be isolated into flows that exit into one or more outputs.
  • the various components in the mixture may be isolated on-chip by using further isolation mechanisms, such as, for example, flow mechanisms, or optical tweezing or holographic optical trapping, or by magnetics (i.e., using magnetic beads).
  • the various embodiments of the present invention thereby provide separation of components on a continuous basis, such as, within a continuous, closed system without the potential damage and
  • the continuous process of the present invention also provides significant time savings in isolating components.
  • the apparatus, methods and systems of the present invention may be extended to other types of particulate, biological or cellular matter, which are capable of being interrogated by fluorescence techniques within a fluid flow, or which are capable of being manipulated between different fluid flows into different outputs.
  • FIG. 1 is an illustrative embodiment of a microfluidic chip 100.
  • the microfluidic chip 100 is manufactured of a suitable thermoplastic (e.g., low auto-fluorescing polymer etc.) through an embossing process or injection molding process, as well known to one of ordinary skill in the art, and is of suitable size.
  • a suitable thermoplastic e.g., low auto-fluorescing polymer etc.
  • the microfluidic chip 100 includes a plurality of structural layers in which are disposed micro-channels which serve as sample input channel(s), sheath or buffer fluid channel(s), output channel(s), etc.
  • the micro-channels are of suitable size to accommodate a particulate laminar flow, and may be disposed in any of the layers of the chip 100 in the appropriate length, as long as the object of the present invention is realized.
  • the desired flow rate through the microfluidic chip 100 may be controlled by a predetermined introduction flow rate into the chip 100, maintaining the appropriate micro-channel dimensions within the chip 100, by pumping mechanisms, providing narrowing of the micro-channels at various locations, and/or by providing obstacles or dividers within the micro-channels.
  • a plurality of inputs is provided into the microfluidic chip 100, which provide access to the micro-channels/channels.
  • a sample input 106 is used for introducing a sample of components 160 in a sample fluid mixture 120(see FIGS. 4-5) into a sample input channel 164A of the microfluidic chip 100 from a reservoir source (see FIG. 9).
  • the microfluidic chip 100 also includes at least one sheath/buffer input (in one embodiment, sheath/buffer inputs 107, 108) for the introduction of sheath or buffer fluids. In one
  • sheath/buffer inputs in the microfluidic chip 100 which include a sheath/buffer input 107 and sheath/buffer input 108, both disposed proximate to the sample input 106, and which both introduce sheath or buffer fluids into the microfluidic chip 100.
  • the sheath or buffer fluids are well known in the art of microfluidics, and in one embodiment, may contain nutrients well known in the art to maintain the viability of the components 160 (i.e., sperm cells) in the fluid mixture.
  • the location of the sheath/buffer inputs 107, 108 may vary, and they may access micro-channels in the chip 100 which are in the same or different structural layers.
  • fill holes or air vents 121, 122 - if not sealed - can be used to introduce sheath or buffer fluids into jet chambers 130, 131 (described later).
  • a plurality of output channels stemming from main channel 164 is provided for removal of fluid which has flowed through the microfluidic chip 100, including the isolated components 160 and/or sheath or buffer fluids.
  • the left side output channel 140 ends at a first output 111
  • the center output channel 141 ends at a second output 112
  • the right side output channel 142 ends at a third output 113.
  • the number of outputs may be less or more depending on the number of components 160 to be isolated from the fluid mixture 120.
  • a plurality of notches or recesses 146 are disposed at a bottom edge of the microfluidic chip 100 to separate the outputs (i.e., outputs 111-113) and for attachment of external tubing etc.
  • the first output 111, the second output 112 and the third output 113 are reached via output channels 140-142 which originate from interrogation chamber 129 (see FIGS.2A- 4).
  • the microfluidic chip 100 has a plurality of structural layers in which the micro-channels are disposed.
  • the channels may be disposed in one or more layers or in- between layers.
  • four structural plastic layers 101-104 are shown to comprise the microfluidic chip 100.
  • the channels may be disposed in any of the layers as long as the object of the present invention is achieved.
  • a gasket of any desired shape, or O-rings, may be provided to maintain a tight seal between the microfluidic chip 100 and the microfluidic chip holder 200 (see FIG. 6).
  • a gasket it may be a single sheet or a plurality of components, in any configuration, or material (i.e., rubber, silicone, etc.) as desired.
  • a first gasket 105 is disposed at one end of the microfluidic chip 100 and interfaces, or is bonded (using an epoxy) with layer 104.
  • a plurality of holes 144 are provided in the first gasket 105 and are configured to align with the sample input 106, sheath/buffer input 107, sheath/buffer input 108, and air vents 121, 122, to provide access thereto.
  • a second gasket 143 is disposed at another end of the microfluidic chip 100 opposite to the first gasket 105, and interfaces or is bonded with (using epoxy) the top structural layer 104.
  • the second gasket 143 is configured to assist sealing, as well as stabilizing or balancing the microfluidic chip 100 in the microfluidic chip holder 200 (see FIG. 6).
  • holes and posts 145 are disposed at various convenient positions in the microfluidic chip 100 to fix and align the multiple layers (i.e., layers 101-104) during chip fabrication.
  • a sample fluid mixture 120 including components 160 is introduced into sample input 106, and the fluid mixture 120 flows through main channel 164 toward interrogation chamber 129 (see FIGS. 2A, 4, and 5).
  • the sheath or buffer fluids 163 are introduced into sheath/buffer inputs 107, 108, and flow through channels 114, 115 and 116, 117, respectively, into the main channel 164, and towards the interrogation chamber 129 before flowing out through output channels 140-142.
  • sheath or buffer fluids 163 can be introduced into jet chambers 130, 131 through air vents 121, 122 to fill the chambers 130, 131 after manufacture of the
  • microfluidic chip 110 if the chambers 130, 131 are not filled with sheath or buffer fluids 163during manufacture.
  • sheath or buffer fluids 163 used are well known to one of ordinary skill in the art of micro fluidics.
  • the fluid mixture 120 from main channel 164 joins with the sheath or buffer fluids 163 from channels 114, 115 at intersection 161 in the same plane of the
  • buffer fluids 163 from channels 116, 117 join the combined fluid mixture 120 and sheath or buffer fluids 163from first intersection 161, downstream at second intersection 162.
  • channels 114, 115 are substantially the same dimensions as channels 116, 117, as long as the desired flow rate(s) is achieved to accomplish the object of the present invention.
  • channels 114-117, 123, 124, 140-142, 125a, 125b, 126a, 126b, 127, 128 may have substantially the same dimensions, however, one of ordinary skill in the art would know that the size of any or all of the channels in the microfluidic chip 100 may vary in dimension (i.e., between 50 and 500 microns), as long as the desired flow rate(s) is achieved to accomplish the object of the present invention.
  • the channels 114-117, 123, 124, 140-142, 125a, 125b, 126a, 126b, 127, 128, of the microfluidic chip 100 may not just vary in dimension, but may have tapered shapes at entry points to other channels in the chip 100 in order to control the flow of fluid through the channels.
  • main channel 164 may taper at the entry point into intersection 161 (see FIG.
  • the sample fluid mixture 120 becomes a relatively smaller, narrower stream, bounded or surrounded by sheath or buffer fluids 163, while maintaining laminar flow in channel 164A.
  • the main channel 164 entering into intersection 161 could be of any physical arrangement, such as a rectangular or circular- shaped channel, as long as the object of the present invention is obtained.
  • At least one of the channels 116, 117 is disposed in a different structural layer of the microfluidic chip 100, than the layer in which the channel 164 is disposed.
  • channel 116 may be disposed in layer 103 and channel 117 may be disposed in layer 101 (see FIG. 1), such that channels 116, 117 are at different planes from the other channels 164 and 114, 115 (in layer 102), when the sheath or buffer fluids 163 join the fluid mixture 120 at intersection 162.
  • main channel 164 is disposed between layers 102, 103 (see FIG.
  • channels 114-117, 164, 123, 124, 140-142, 125a, 125b, 126a, 126b, 127, 128 etc. can be disposed in any layer or between any two layers. Further, although the channels 114-117, 164, 123, 124, 140-142, 125a, 125b, 126a, 126b, 127, 128 etc. are described in exemplary
  • the sheath or buffer fluids in channels 116, 117 join the fluid mixture via holes cut in the layers 101-103 at substantially vertical positions above and below the intersection 162.
  • the sheath or buffer fluids from channels 116, 117 compress the fluid mixture 120 flow in a perpendicular manner with respect to channel 164B, such that the components 160 in the fluid mixture 120 are compressed or flattened, and oriented in the selected or desired direction (see below), while still maintaining laminar flow in channel 164B.
  • channels 114, 115 and 116, 117 are depicted as partially coaxial to one another with a center point defined by the sample input 106.
  • channels 114, 115 and 116, 117 are disposed in a substantially parallel arrangement, with the channels 114, 115 and 116, 117 being equidistant to main channel 164.
  • the depicted configuration may be different as long as it achieves the desired features of the present invention.
  • channels 114, 115 preferably join intersection 161 in the same plane, at an angle of 45 degrees or less, whereas channels 116, 117, which parallel sample input channel 164A, join intersection 162 from different layers, at an angle of substantially 90 degrees.
  • channels 114, 115 preferably join intersection 161 in the same plane, at an angle of 45 degrees or less
  • channels 116, 117 which parallel sample input channel 164A, join intersection 162 from different layers, at an angle of substantially 90 degrees.
  • the components 160 in the fluid mixture 120 flow through channel 164B into an interrogation chamber 129, where the components 160 are interrogated.
  • a flexible diaphragm 170, 171 (see FIG. 1) made from a suitable material, such as one of stainless steel, brass, titanium, nickel alloy, polymer, or other suitable material with desired elastic response, covers jet chambers 130, 131.
  • an actuator is disposed on at least one side of channel 164B and the interrogation chamber 129 (see FIGS. 2A and 2B), in order to cause mechanical displacement of the diaphragm 170, 171, in order to jet or push sheath or buffer fluids 163 from one of the jet chambers 130, 131 on that side of channel 146B, to push components 160 from channel 164C into one of the output channels 140, 142 on the other side of the channel 164B.
  • the actuator would jet sheath or buffer fluids 163 from jet chamber 130 into channel 164C, and push target components 160 in channel 164C into output channel 142 to isolate the target components from the fluid mixture 120.
  • This embodiment is useful when only one type of target components 160 are isolated (which may require only two output channels 141, 142, for example, instead of three output channels 140-142) (see FIG. 2B).
  • the actuator may be one of a piezoelectric, magnetic, electrostatic, hydraulic, or pneumatic type actuator. Although a disc-shaped actuator assembly (i.e., 109, 110) is shown in FIGS. 1-2, one of ordinary skill in the art would know that any type or shape of actuator which performs the needed function could be used. In other embodiments, the actuator is disposed on either side of channel 164B (as shown in FIG. 2A), but in other embodiments, more than one actuator (of a relatively smaller size) may be disposed on one or more sides of channel 164B and connected to channel 164B via jet channels (see FIG. 2C).
  • two external, stacked piezoelectric actuator assemblies 209, 210 are provided (see FIGS. 6 and 7) which align with and actuate the diaphragms 170, 171.
  • the external, stacked piezoelectric actuator assemblies 209, 210 are disposed in a microfluidic chip holder 200.
  • the stacked piezoelectric actuator assemblies 209, 210 each include a piezoelectric actuator 219, 220, respectively, which have a high resonant frequency, and which each are disposed at a position in a center of, and in contact with the diaphragm 170, 171, to squeeze the sheath or buffer fluids 163 from the chamber 130, 131 into channel 164C.
  • the microfluidic chip holder 200 may be of any type known to one of ordinary skill in the art, and is configured to precisely position piezoelectric actuators 219, 220, such that the piezoelectric actuators 219, 220 may maintain constant contact with diaphragms 170, 171 of the microfluidic chip 100. For example, in one embodiment, this is accomplished by each piezoelectric actuator assembly 209, 210 being mounted (or adhered using a suitable epoxy) on lockable adjustment screws 201 which move the piezoelectric actuators 219, 220 into position against the diaphragms 170, 171, respectively; and thumb screws 202 with threaded bodies that act to move the screws 202 against the diaphragms 170, 171 for stabilization.
  • a spacer 203 attached to the piezoelectric actuator 219, 220 allows a feasible contact to be made between it and a diaphragm 170, 171 of the microfluidic chip 100.
  • the adjustment screws 201 allow a user to adjust the position of the piezoelectric actuators 209, 210 relative to the microfluidic chip 100 for both coarse and fine adjustment.
  • the thumb screws 202 may be tightened to secure the piezoelectric assemblies 209, 210 to the main chip body 100 or loosened to remove the piezoelectric actuator assemblies 209, 210 from the main chip body 100.
  • At least one piezoelectric actuator (209 or 210) is mounted on a plate (not shown) which can be translated in direction normal to the diaphragm (170 or 171) of the microfluidic chip 100.
  • An adjustment screw 201 is mounted on the holder 200 and can be extended and retracted by turning the screw 201. The tip of the adjustment screw 201 is against the plate. As the screw 201 is extended, the plate along with the piezoelectric actuator 209, 210, is pushed toward the diaphragm 170, 171 in a translation motion, such that a feasible contact is made between the piezoelectric actuator 209, 210 and the diaphragm 170, 171.
  • the positioning of the piezoelectric actuators 209, 210 is adjusted through translation of the piezoelectric actuators 209, 210 only, while in the previous embodiment where the piezoelectric actuator 209, 210 is mounted directly on the adjustment screw 201, the positioning of the piezoelectric actuator 209, 210 is a combination of translation and rotation of the piezoelectric actuator 209, 210, during which damage can be caused on the delicate piezoelectric actuator 209, 210.
  • an electronic circuit is connected to the stacked piezoelectric actuator assembly 209, 210 before driving it.
  • the resistance force from the diaphragm 170, 171 causes the strain on the stacked piezoelectric actuator 219, 220, which generates an electronic signal.
  • the electronic circuit is able to amplify the electronic signal to a predetermined value to trigger an LED (light emitting diode).
  • piezoelectric actuator 219, 220 is in contact with the diaphragm 170, 171 the LED is turned on automatically, which indicates contact between the stacked piezoelectric actuator 219, 220 and the diaphragm 170, 171 is made. This contact sensing allows enough force for the actuators 219, 220 to compress the chambers 130, 131 to jet fluid 163 into the channel 164B.
  • the LED is one example of an indicator of contact.
  • a feedback is generated for the user, which can be in any of the following forms: a light (i.e., LED), a sound (i.e., buzzer), a haptic (i.e., vibrator), or any combination thereof.
  • a light i.e., LED
  • a sound i.e., buzzer
  • a haptic i.e., vibrator
  • a thin film of piezoelectric material (well-known to one of ordinary skill in the art) is directly deposited on the top surface of at least one diaphragms 170, 171, to form at least one piezoelectric actuator assembly 109, 110 (see FIGS. 2A and 4) to displace (bend) the respective diaphragm 170, 171 and drive the fluids in the respective jet chamber 130, 131 into channel 164C, respectively.
  • the piezoelectric material is permanently bonded with the previously described flexible diaphragm 170, 171 by an adhering mechanism.
  • the whole diaphragm 170, 171 bends into the chamber 130, 131 and squeezes the fluid 163 therein, into the channel 164C to deflect the target or selected components 160 into a side output channel 140, 142.
  • only one piezoelectric actuator assembly may be required to jet sheath or buffer fluids 163 from jet chamber 130 into channel 164C, and push target components 160 in channel 164C into output channel 142 to isolate the target components from the fluid mixture 120, as shown in FIG. 2B.
  • the piezoelectric actuator assemblies 109, 110 are used to seal the jet chambers 130, 131, respectively, at layer 103, for example - but one of ordinary skill in the art would know that it could be in any structural layer - after the chambers 130, 131 are filled with sheath or buffer fluids 163, to make the microfluidic chip 100 impervious to fluid leakage.
  • the piezoelectric actuator assemblies 109, 110 satisfy the requirement of low flow rates considering the relatively small bend displacement of the diaphragms 170, 171, and low force thereon, in contrast to the large displacement and strong force applied by the external, stacked piezoelectric actuator assemblies 209, 210 which are able to work at very high flow rates.
  • the actuator assemblies 109, 110, 209, 210 can be chosen independently for use in the microfluidic chip 100 based upon the different operation speeds and flow rate requirements.
  • a thin piezoelectric film disposed on top of the diaphragm 170, 171 works as a strain sensor to determine how much strain or displacement the external, stacked piezoelectric actuator assemblies 209, 210 generate as they are triggered by the electronic signal to displace the respective diaphragms 170, 171.
  • the diameter and thickness of the piezoelectric thin film depends on the cross-section of the external, stacked piezoelectric actuator 219, 220 and the force generated on the diaphragm 170, 171.
  • the piezoelectric thin film and diaphragm 170, 171 may be different from the one discussed above in the alternative embodiment.
  • air vents 121, 122 are provided to remove air from jet chambers 130, 131 respectively (see FIG. 2A), after manufacturing when the chambers 130, 131 are filled with sheath or buffer fluids 163 - forcing air out through the air vents 121, 122 - and before the chambers 130, 131 are sealed with sheath or buffer fluids 163 therein.
  • sheath or buffer fluids 163 may be introduced through the vents 121, 122 into the chambers 130, 131 if this is not done during manufacturing.
  • the sheath of buffer, or other fluids 163 disposed in the jet chambers 130, 131 may be the same or different from the sheath or buffer fluids 163 inputted through channels 114, 115, 116, or 117.
  • sheath or buffer fluids 163 are used to fill up the jet chambers 130, 131, they may be inputted through inputs 121, 122 and flowed through channels 123, 124 respectively, to enter jet chamber 130 via channels 125a and 125b, and jet chamber 131 via channels 126a and 126b.
  • jet channel 127 leaves jet chamber 130, and jet channel 128 leaves jet chamber 131, and both jet channels 127, 128 enter the interrogation chamber 129 (see FIG. 2A).
  • the jet channels 127, 128 may be disposed in any layer of the chip 100 and enter the channel 164C at any angle in the same plane.
  • the jet channels 127, 128 may be tapered when they connect to the main channel 164C.
  • the jet channels 127, 128 may have a particular angle, or be of a different structure, as long as they achieve the described features of the present invention.
  • the jet channels 127, 128 work to displace or bend the diaphragms 170, 171, respectively, and jet or squeeze sheath or buffer fluids 163 into the channel 164C.
  • the jet channels 127, 128 work to displace or bend the diaphragms 170, 171, respectively, and jet or squeeze sheath or buffer fluids 163 into the channel 164C.
  • the jet channels 127, 128 work to displace or bend the diaphragms 170, 171, respectively, and jet or squeeze sheath or buffer fluids 163 into the channel 164C.
  • the jet channels 127, 171 return to a neutral (unbent) position
  • 127, 128 which issue from jet chambers 130, 131, work as diffusers to ensure that a net fluid volume from the jet chambers 130, 131 to the channel 164C is maintained, and that it is easy to refill the chambers 130, 131 with sheath or buffer fluids 163.
  • output channels 140-142 depart from channel 164C within interrogation chamber 129 to outputs 111-113.
  • more than one on-chip piezoelectric actuator assembly 109, 110, or external, stacked piezoelectric actuator assembly 209, 210 may be used to connect to each of jet channels 127,
  • the distance from each of the jet channels 127, 128 entries into channel 164C to each of the output channels 140-142 should be shorter than the distance between components 160, to avoid target components 160 mixing with undesired components 160 (further described below).
  • the cross-section and the length of the output channels 140-142 should be maintained at a predetermined volume ratio (i.e., 2: 1:2, or 1:2: 1 etc.) to obtain the desired hydraulic resistance of the output channels 140-142.
  • an interrogation apparatus is disposed downstream from where channels 116, 117 enter into channel 164B.
  • channel 164B tapers into the interrogation chamber 129, which speeds up the flow of the fluid mixture through the
  • interrogation chamber 129 includes an opening or window 149 (see FIG. 3) cut into the microfluidic chip 100 in at least the uppermost layer (i.e., layer 104 or other), and another opening or window 152 is cut into the chip 110 in at least the lowermost layer (i.e., layer 101 or other).
  • an opening 150 is cut into the microfluidic chip through layers 101- 104.
  • the top window 149 is configured to receive a first covering 133 and the bottom window 152 is configured to receive a second covering 132.
  • the windows 149, 152 may be located in any suitable layer and need not be in the uppermost/lowermost layers.
  • the coverings 133, 132 may be made of any material with the desired transmission requirements, such as plastic, glass, or may even be a lens. Note that although the relative diameters of the windows 149, 152 and opening 150 are shown in FIG. 3, these may vary according to manufacturing considerations.
  • the above-mentioned first and second coverings 133, 132 are configured to enclose the interrogation chamber 129.
  • the windows 149, 152 and coverings 133, 132 (see FIG. 3), allow the components 160 flowing in the fluid mixture 120 in channel 164B (see FIG. 5A) through the interrogation chamber 129, to be viewed through opening 150, and acted upon by a suitable light source 147 configured to emit a high intensity beam 148 with any wavelength that matches excitable components in the fluid mixture 120.
  • a suitable light source 147 configured to emit a high intensity beam 148 with any wavelength that matches excitable components in the fluid mixture 120.
  • a laser 147 is shown, any suitable other light sources may be used, such as a light emitting diode ( LED), arc lamp, etc. to emit a beam which excites the components.
  • a high intensity laser beam 148 from a suitable laser 147 of a preselected wavelength- such as a 355 nm continuous wave (CW) (or quasi-CW) laser 147 - is required to excite the components 160 in the fluid mixture (i.e., sperm cells).
  • the laser 147 (see FIG. 3) emits a laser beam 148 through window 149 in layer 104, through the covering 133 at an uppermost portion of the chip 100, through opening 150, and through covering 132 and window 152 in layer 101 of the chip 100, to illuminate the components 160 flowing through channel 164B in interrogation region 129 of the chip 100.
  • the light beam 148 can be delivered to the components 160 by an optical fiber that is embedded in the microfluidic chip 100 at opening 150.
  • the high intensity beam 148 interacts with the components 160 (see detailed explanation below), and passes through the first and second coverings 133, 132, to exit from bottom window 152, such that the emitted light 151, which is induced by the beam 148, is received by an objective lens 153.
  • the objective lens 153 may be disposed in any suitable position with respect to the microfluidic chip 100. Because the interrogation chamber 129 is sealed by the first and second coverings 133, 132, the high intensity beam 148 does not impinge on the microfluidic chip 100 and damage the layers 101-104. Thus, the first and second coverings 133, 132 help prevent damage to the microfluidic chip 100 from the high intensity beam 148 and photonic noise induced from the microfluidic chip material (i.e., plastic).
  • the emitted light 151 received by the objective lens 153 is converted into an electronic signal by an optical sensor 154, such as a photomultiplier tube (PMT) or photodiode, etc.
  • the electronic signal can be digitized by an analog-to-digital converter (ADC)
  • DSP digital signal processor
  • the piezoelectric drivers and piezoelectric actuators are part of two piezoelectric actuator assemblies (109, 110, or 209, 210) respectively, disposed on either side of the interrogation chamber 129.
  • the trigger signal sent to the piezoelectric actuators (109, 110, or 219, 220) is determined by the sensor raw signal, to activate the particular piezoelectric actuator assembly (109, 110, 209, 210) when the selected component is detected.
  • the thickness of the diaphragm 170, 171 may be different and is dependent upon the voltage applied via electrical wires through the actuator assemblyl09, 110 on the chip 100.
  • the diaphragms 170, 171 bend and change (increase) the pressure in the chambers 130, 131.
  • the at least one of the piezoelectric actuator assemblies (109, 110, or 209, 210) is used to act upon the desired components 160 in the fluid mixture in channel 164C, as the components 160 leave the opening 150 for interrogation area 129 after interrogation.
  • actuator driver 157b and piezoelectric actuator assembly 110 are not illustrated in FIG. 4, the operation and configuration of actuator driver 157b and piezoelectric actuator assembly 110 are the same as that of the actuator driver 157a and the piezoelectric actuator assembly 109.
  • piezoelectric actuator 157b acts to deflect components 160 in the flow stream in channel 164C to the right output channel 142, and to the third output 113.
  • the piezoelectric actuator assembly 110 which jets sheath or buffer fluid 163 from the jet chamber 131 via jet channel 128, and deflects target or selected components 160 to the left output channel 140and the third output 113.
  • a piezoelectric actuator assembly 106A i.e., a piezoelectric disc similar to the piezoelectric actuator assemblies 109, 110, and of a suitable size - see FIG. 2C), or a suitable pumping system (see FIG. 9, for example - discussed later), is used to pump sample fluid 120 in channel 164 toward intersection 161.
  • the sample piezoelectric actuator assembly 106A would be disposed at sample input 106.
  • a measure of control can be made over the spacing of the components 160 therein, such that a more controlled relationship may be made between the components 160 as they enter the main channel 164.
  • the (target) components 160 proceed from main channel 164 to the center output channel 141, and to the second output 112, and the sheath or buffer fluids 163 proceed through output channels 140, 142, to outputs 110, 112, respectively.
  • the output channels 140-142 increase in dimension from the channel 164C, leaving the interrogation chamber 129, such that the output ratio for enrichment of the isolated component 160, is increased through the relevant channel(s).
  • the microfluidic chip 100 is provided in a sterile state, and may be primed with one or more solutions (i.e., sheath or buffer fluids 163), or purged of any fluids or materials by either draining the microfluidic chip 100 or by flowing sheath or buffer fluids 153 or other solutions through the microfluidic chip 100, according to known methods.
  • the air vents 121, 122 are sealed.
  • the air vents 121, 122 may be left open for additional sheath or buffer fluids 163 to be added to the chambers 130, 131 during operation.
  • the components 160 that are to be isolated include, for example: isolating viable and motile sperm from non- viable or non-motile sperm; isolating sperm by gender, and other sex sorting variations; isolating stems cells from cells in a
  • the components 160 may be cells or beads treated or coated with, linker molecules, or embedded with a fluorescent or luminescent label molecule(s).
  • the components 160 may have a variety of physical or chemical attributes, such as size, shape, materials, texture, etc.
  • a heterogeneous population of components 160 may be measured simultaneously, with each component 160 being examined for different quantities or regimes in similar quantities (e.g., multiplexed measurements), or the components 160 may be examined and distinguished based on a label (e.g., fluorescent), image (due to size, shape, different absorption, scattering, fluorescence, luminescence characteristics, fluorescence or luminescence emission profiles, fluorescent or luminescent decay lifetime), and/or particle position etc.
  • a label e.g., fluorescent
  • image due to size, shape, different absorption, scattering, fluorescence, luminescence characteristics, fluorescence or luminescence emission profiles, fluorescent or luminescent decay lifetime
  • a two-step focusing method of a component sorting system consistent with the present invention may be used, as illustrated in FIG. 5A, in order to position the components 160 in channel 164B for interrogation in the interrogation chamber 129.
  • the first focusing step of the present invention is accomplished by inputting a fluid sample 120 containing components 160, such as sperm cells etc., through sample input 106, and inputting sheath or buffer fluids 163 through sheath or buffer inputs 107, 108.
  • the components 160 are pre-stained with dye (e.g., Hoechst dye), in order to allow fluorescence, and for imaging to be detected.
  • sheath or buffer fluids 163 are disposed in jet chambers 130, 131, and inputs 121, 122 sealed.
  • components 160 in the sample fluid mixture 120 flow through main channel 164, and have random orientation and position (see inset A).
  • the sample mixture 120 flowing in main channel 164 is compressed by the sheath or buffer fluids 163 from channels 114, 115, in a first direction (i.e., at least horizontally, on at least both sides of the flow, if not all sides depending on where the main channel 164 enters the intersection 161), when the sheath or buffer fluids 163 meet with the sample mixture 120.
  • the components 160 are focused around the center of the channel 164, and may be compressed into a thin strip across the depth of the channel 164A.
  • the intersection 161 leading into channel 164A is the focusing region.
  • the components 160 i.e., sperm cells move toward the center of the channel 164 width.
  • the present invention includes a second focusing step, where the sample mixture 120 containing components 160, is further compressed by sheath or buffer fluids 163 from a second direction (i.e., the vertical direction, from the top and the bottom) entering from channels 116, 117 at intersection 162.
  • the intersection 162 leading into channel 164B is the second focusing region. Note that although the entrances into intersection 162 from channels 116, 117 are shown as rectangular, one of ordinary skill in the art would appreciate that any other suitable configuration (i.e., tapered, circular) may be used.
  • the sheath or buffer fluids 163 in the channels 116, 117 enter at different planes into the channel 164A-B, to align the components 160 in the center of the channel 164B by both width and depth (i.e., horizontally and vertically) as they flow along channel 164B.
  • the sample mixture 120 is again compressed by the vertical sheath or buffer fluids 163 entering at channels 116, 117, and the sample 120 stream is focused at the center of the channel 164B depth, as illustrated in FIG. 5 A, and the components 160 flow along the center of the channel 164B in approximately single file formation.
  • the components 160 are sperm cells 160, and because of their pancake-type or flattened teardrop shaped head, the sperm cells 160 will re-orient themselves in a predetermined direction as they undergo the second focusing step - i.e., with their flat surfaces perpendicular to the direction of light beam 148 (see FIG. 5A).
  • the sperm cells 160 develop a preference on their body orientation while passing through the two-step focusing process.
  • the sperm cells 160 tend to be more stable with their flat bodies perpendicular to the direction of the compression.
  • the sperm cells 160 which start with random orientation, now achieve uniform orientation.
  • the sperm cells 160 not only make a single file formation at the center of the channel 164B, but they also achieve a uniform orientation with their flat surface normal to the direction of compression in the second focusing step.
  • all components 160 introduced into sample input 106 which may be other types of cells or other materials as described above, etc., undergo the two-step focusing steps, which allow the components 160 to move through the channel 164B in a single file formation, in a more uniform orientation (depending on the type of components 160), which allows for easier interrogation of the components 160.
  • the components 160 are detected in the interrogation chamber 129 at opening 150 through coverings 132, 133, using the light source 147.
  • Light source 147 emits a light beam 148 (which may be via an optical fiber) which is focused at the center of the channel 164C at opening 150.
  • the components 160 such as sperm cells 160, are oriented by the focusing streams (i.e., sheath or buffer fluid 163 streams which act on sample stream 120) such that the flat surfaces of the components 160 are facing toward the beam 148.
  • all components 160 are moved into single file formation by focusing as they pass under beam 148.
  • the components 160 pass under light source 147 and are acted upon by beam 148, the components 160 emit the fluorescence which indicates the desired components 160.
  • the components 160 For example, with respect to sperm cells, X chromosome cells fluoresce at a different intensity from Y chromosome cells; or cells carrying one trait may fluoresce in a different intensity or wavelength from cells carrying a different set of traits.
  • the components 160 can be viewed for shape, size, or any other distinguishing indicators.
  • the emitted light beam 151 (in FIG. 3) is then collected by the objective lens 153, and subsequently converted to an electronic signal by the optical sensor 154.
  • the electronic signal is then digitized by an analog-digital converter (ADC) 155 and sent to an electronic controller 156 for signal processing.
  • ADC analog-digital converter
  • the electronic controller can be any electronic processer with adequate processing power, such as a DSP, a Micro Controller Unit (MCU), a Field Programmable Gate Array (FPGA), or even a Central Processing Unit (CPU).
  • the DSP-based controller 156 monitors the electronic signal and may then trigger at least one actuator driver (i.e., 157a or 157b), to drive one of the two piezoelectric actuator assemblies (109, 110, or 219, 220 - part of the respective piezoelectric actuator assemblies 109, 110, 209, 210) when a desired component 160 is detected.
  • at least one actuator driver i.e., 157a or 157b
  • the FPGA-based controller monitors the electronic signal and then either communicates with the DSP controller or acts independently to trigger at least one actuator driver (i.e., 157a or 157b), to drive one of the two piezoelectric actuator assemblies (109, 110, or 219, 220 - part of the respective piezoelectric actuator assemblies 109,110, 209, 210) when a desired component 160 is detected.
  • at least one actuator driver i.e., 157a or 157b
  • selected or desired components 160 in channel 164C in the interrogation chamber 129 are isolated by a jet stream of buffer or sheath fluids 163 from one of the jet channels 127, 128, depending on which output channel 140, 142 is desired for the selected component 160.
  • the electronic signal activates the driver to trigger external stacked piezoelectric actuator 219 (or activates driver 157 a to trigger actuator 109), at the moment when the target or selected component 160 arrives at the cross-section point of the jet channels 127, 128 and the main channel 164C.
  • sheath or buffer fluids 163 jetted from one of the jet channels 127, 128 divert target or selected components 160 from their ordinary paths in channel 164C, toward one of the selected or desired, respective output channels 140, 142, isolating those target components 160, and enriching the flows in those output channels 140, 142, and depleting the flow in the sample fluid 120 which continues straight out through output channel 141 with unselected components, if any.
  • no triggering of the piezoelectric actuator assemblies 109, 110 means that the unselected components 160 in the fluid mixture 120 continue straight out through output channel 141.
  • the isolated components 160 are collected from one of the first output 111, or the third output 113, using known methods in the art, for storing, for further separation, or for processing, such as cryopreservation.
  • components 160 that were not isolated into outputs 111, 113 may also be collected from second output 112.
  • Portions of the first, second, and third outputs 111-113 may be characterized electronically, to detect
  • concentrations of components pH measuring, cell counts, electrolyte concentration, etc.
  • interrogation of the sample 120 containing components 160 is accomplished by other methods.
  • portions of, or outputs from, the microfluidic chip 100 may be inspected optically or visually.
  • methods for interrogation may include direct visual imaging, such as with a camera, and may utilize direct bright-light imaging or fluorescent imaging; or, more sophisticated techniques may be used such as spectroscopy, transmission spectroscopy, spectral imaging, or scattering such as dynamic light scattering or diffusive wave spectroscopy.
  • the optical interrogation region 129 may be used in conjunction with additives, such as chemicals which bind to or affect components of the sample mixture 120 or beads which are functionalized to bind and/or fluoresce in the presence of certain materials or diseases. These techniques may be used to measure cell concentrations, to detect disease, or to detect other parameters which characterize the components 160.
  • polarized light back scattering methods may also be used.
  • the components 160 are interrogated as described above.
  • the spectrum of those components 160 which had positive results and fluoresced i.e., those components 160 which reacted with a label) are identified for separation by the activation of the piezoelectric assemblies 109, 110, 209, 210.
  • the components 160 may be identified based on the reaction or binding of the components with additives or sheath or buffer fluids 163, or by using the natural fluorescence of the components 160, or the fluorescence of a substance associated with the component 160, as an identity tag or background tag, or met a selected size, dimension, or surface feature, etc., are selected for separation.
  • selection may be made, via computer 182 (which monitors the electronic signal and triggers the piezoelectric assemblies 109, 110, 209, 210) and/or operator, of which components 160 to discard and which to collect.
  • the user interface of the computer system 182 includes a computer screen which displays the components 160 in a field of view acquired by a CCD camera 183 over the microfluidic chip 100.
  • the computer 182 controls any external devices such as pumps (i.e., pumping mechanism of FIG. 9), if used, to pump any sample fluids 120, sheath or buffer fluids 163 into the microfluidic chip 100, and also controls any heating devices which set the temperature of the fluids 120, 163 being inputted into the microfluidic chip 100.
  • pumps i.e., pumping mechanism of FIG. 9
  • the microfluidic chip 100 is loaded on a chip cassette 212, which is mounted on chip holder 200.
  • the chip holder 200 is mounted to a translation stage (not shown) to allow fine positioning of the holder 200.
  • the microfluidic chip holder 200 is configured to hold the microfluidic chip 100 in a position such that the light beam 148 may intercept the components 160 in the above described manner, at opening 150.
  • a gasket layer 105 forms a substantially leak- free seal between the main body 211 and the microfluidic chip 100.
  • a microfluidic chip holder 200 is made of a suitable material, such as aluminum alloy, or other suitable metallic/polymer material, and includes a main body 211, and at least one stacked external piezoelectric actuator 209, 210.
  • the main body 211 of the holder 200 may be any suitable shape, but its configuration depends on the layout of the chip 100.
  • the stacked external piezoelectric actuators 209, 210 must be placed over the diaphragm(s) 170, 171, such that contact is made between a tip of the piezoelectric actuator 219, 220 and the diaphragm 170, 171 of the microfluidic chip 100.
  • the main body 211 of the holder 200 is configured to receive and engage with external tubing (see FIG. 9) for communicating fluids/samples to the microfluidic chip 100.
  • cassette 212 and holder 200 The details of these cassette 212 and holder 200 and the mechanisms for attachment of the chip 100 to the cassette 212 and holder 200, are not described in any detail, as one of ordinary skill in the art would know that these devices are well-known and may be of any configuration to accommodate the microfluidic chip 100, as long as the objectives of the present invention are met.
  • a pumping mechanism includes a system having a pressurized gas 235 which provides pressure for pumping sample fluid mixture 120 from reservoir 233 (i.e., sample tube) into sample input 106 of the chip 100.
  • a pressure regulator 234 regulates the pressure of gas 235 within the reservoir 233, and a pressure regulator 239 regulates the pressure of gas 235 within the vessel 236.
  • a mass flow regulator 232a, 232b controls the fluid 163 pumped via tubing 231a, 231b, respectively, into the sheath or buffer inputs 107, 108, respectively.
  • tubing 230, 231a, 231b is used in the initial loading of the fluids 120 into the chip 100, and may be used throughout chip 100 to load sample fluid 120 into sample input 106, or sheath or buffer inputs 107, 108.
  • tubing (not shown) can provide fluid 163 from manifold 238 into air vents 121, 122 to fill chambers 130, 131, for example.
  • any of the operations, steps, control options, etc. may be implemented by instructions that are stored on a computer-readable medium such as a computer memory, database, etc. Upon execution of the instructions stored on the computer-readable medium, the instructions can cause a computing device to perform any of the operations, steps, control options, etc. described herein.
  • the operations described in this specification may be implemented as operations performed by a data processing apparatus or processing circuit on data stored on one or more computer-readable storage devices or received from other sources.
  • a computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages, and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, object, or other unit suitable for use in a computing environment.
  • a computer program may, but need not, correspond to a file in a file system.
  • a program can be stored in a portion of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, subprograms, or portions of code).
  • a computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a communication network.
  • Processing circuits suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer.
  • microfluidic chip as shown in the various illustrative embodiments, are illustrative only. Although only a few embodiments have been described in detail in this disclosure, many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter described herein. Some elements shown as integrally formed may be constructed of multiple parts or elements, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied.

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Abstract

La présente invention porte sur une puce microfluidique qui oriente et isole des composants dans un mélange de fluide d'échantillon par focalisation en deux étapes, des fluides de gaine comprimant le mélange de fluide d'échantillon dans un canal d'entrée d'échantillon dans une direction, de telle sorte que le mélange de fluide d'échantillon devient un courant plus étroit lié par les fluides de gaine, et en ayant les fluides de gaine qui compriment le mélange de fluide d'échantillon dans une seconde direction davantage vers l'aval, de telle sorte que les composants sont comprimés et orientés dans une direction sélectionnée pour traverser une chambre d'interrogation dans une formation en file simple pour identification et séparation par différents procédés. Le mécanisme d'isolation utilise des ensembles actionneurs piézoélectriques empilés, externes disposés sur un organe de maintien de puce microfluidique, ou des ensembles actionneurs piézoélectriques sur puce, de telle sorte que les ensembles actionneurs sont déclenchés par un signal électrique pour actionner des chambres d'éjection sur chaque côté du canal d'entrée d'échantillon, pour éjecter des composants sélectionnés dans le canal d'entrée d'échantillon dans l'un des canaux de sortie.
EP13889551.1A 2013-07-16 2013-07-16 Puce microfluidique Withdrawn EP3022544A4 (fr)

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CN105556279A (zh) 2016-05-04
WO2015009284A1 (fr) 2015-01-22
IL243630A0 (en) 2016-02-29
CN110975947A (zh) 2020-04-10
EP3022544A4 (fr) 2017-06-21
CN105556279B (zh) 2019-11-26
HK1224377A1 (zh) 2017-08-18
JP6205055B2 (ja) 2017-09-27
JP2016527502A (ja) 2016-09-08

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