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WO2008036083A1 - Cytomètre de flux microfluidique et ses applications - Google Patents

Cytomètre de flux microfluidique et ses applications Download PDF

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Publication number
WO2008036083A1
WO2008036083A1 PCT/US2006/036457 US2006036457W WO2008036083A1 WO 2008036083 A1 WO2008036083 A1 WO 2008036083A1 US 2006036457 W US2006036457 W US 2006036457W WO 2008036083 A1 WO2008036083 A1 WO 2008036083A1
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Prior art keywords
microchannel
branch
flow
cells
particle
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Ceased
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English (en)
Inventor
Dongqing Li
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Vanderbilt University
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Vanderbilt University
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Priority to PCT/US2006/036457 priority Critical patent/WO2008036083A1/fr
Publication of WO2008036083A1 publication Critical patent/WO2008036083A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • 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
    • B01L3/502746—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 the means for controlling flow resistance, e.g. flow controllers, baffles
    • 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
    • B01L3/502776—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
    • 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
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
    • 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
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • 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
    • 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
    • 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
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • 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
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0424—Dielectrophoretic 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
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0346—Capillary cells; Microcells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/02—Mechanical
    • G01N2201/022—Casings
    • G01N2201/0221—Portable; cableless; compact; hand-held

Definitions

  • [n] represents the nth reference cited in the reference list.
  • [25] represents the 25th reference cited in the reference list, namely, Ye C, Xuan X, Li D.
  • the present invention relates generally to a flow cytometer, and more particularly to a microfluidic flow cytometer and applications of same.
  • Flow cytometry provides a method of detecting and analyzing particles contained in a sample, for example, blood cells in blood such as red blood cells (erythrocytes), white blood cells (leukocytes) and blood platelets (thrombocytes), or material components in urine such as bacteria, blood cells, white blood cells, epithelial cells or casts. These cells or material components may increase or decrease in number in accordance with a disease. Accordingly, a disease can be diagnosed by detecting the status of each cell or material component on the basis of information about granules or particles in the sample. For example, in the field of HIV treatments, a single most important parameter for disease staging is the number of the CD4+ T cell (in the unit of cells/mm 3 ) in peripheral blood.
  • CD4+ T cell numbers can be cumbersome and expensive.
  • the total lymphocyte count is determined by a routine CBC (complete blood count) assay, the percentage of CD4+ T lymphocytes as a function of total lymphocytes is determined by a flow cytometry, and these values are multiplied to determine an absolute CD4+ T cell number.
  • a benchtop flow cytometry is very expensive and needs to be careful maintained.
  • the operation of such a benchtop flow cytometer requires specially trained personnel.
  • the sample volumes consumed by the benchtop flow cytometry are very large, typically in a range of hundred microliters to several hundred microliters.
  • Tung et al. [1] presented a flow cytometer chip using polydimethylsiloxane (PDMS) for fluorescence-labeled particle detection using a two-color, multi-angle detection system via embedded fibers.
  • PDMS polydimethylsiloxane
  • the size of the flow cytometer is significantly reduced.
  • the flow cytometer chip lacks portability as it requires a manually operated external liquid handling system, e.g., two syringe pumps and tubing, to focus a cell-carrying stream in a detection channel.
  • [2] reported a flow cytometer chip using electrokinetically microfluidic flow focusing mechanism.
  • the flow cytometer chip has a glass plate with a pair of embedded optical fibers for counting particles moving through a microchannel. All these flow cytometers have only one function, i.e. counting the number of single-sized particles.
  • a practical flow cytometer must be able to handle mixtures of diverse cells that must be differentiated and counted by size and by their fluorescent dye tags.
  • using embedded waveguides or optical fibers on each side of the detection channel of a flow cytometer requires large lateral space and thus prevents such a chip from having multiple parallel channels, which increases the throughput of the device.
  • a microchip based CD4 counting system was also reported in [3]. However, it requires a large external liquid delivery system including a pump, tubing and valves, an external membrane filter for cell separation, and a conventional optical detection system. The system is manually operated with complicated procedures, and is not portable. Therefore, a heretofore unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies.
  • the present invention in one aspect, relates to a flow cytometer that can be used for counting and differentiating particles in a liquid medium of interest.
  • the liquid medium of interest may comprise a biological fluid of a living subject.
  • the biological fluid includes blood or urine.
  • the blood or urine comprises one or more types of particles or cells.
  • the one or more types of cells are differentiatable by their sizes, functions or a combination of them.
  • the one or more types of cells may comprise CD4+ cells, and/or CD3+ cells.
  • the CD4+ cells and CD3+ cells are labeled with a first and second antibodies, respectively, where the first and second antibodies are excited with light of different wavelengths.
  • the one or more types of cells are associated with a disease.
  • the flow cytometer comprises a first substrate having a first surface and an opposite, second surface defining a body portion therebetween.
  • the flow cytometer further comprises a microchannel structure formed in the body portion of the first substrate for differentiating particles in a liquid medium of interest.
  • the microchannel structure includes a first particle separation unit, a second particle separation unit, and a flow focusing unit.
  • each of the first and second particle separation units has a first and second inlet ports, a first and second outlet ports, and a first, second and third microchannels.
  • Each of the first, second and third microchannels is formed with a first open end, an opposite, second open end, and a first side wall and an opposite, second side wall defining a corresponding channel width, wl, w2, w3, therebetween, respectively.
  • Each channel width is in a range of about 0.1-1,000 ⁇ m, preferable in a range of about 1- 500 ⁇ m.
  • the first microchannel is in fluid communication with the first inlet port and the second microchannel through the first and second open ends, respectively, thereby forming a first junction of the first and second microchannels.
  • the first junction divides the second microchannel into a first branch and a second branch, wherein the first branch is between the first open end of the second microchannel and the first junction, and the second branch is between the first junction and the second open end of the second microchannel.
  • the second microchannel is in fluid communication with the second inlet port and the third microchannel through the first and second open ends, respectively, thereby forming a second junction of the second and third microchannels.
  • the second junction divides the third microchannel into a first branch and a second branch, wherein the first branch is between the first open end of the third microchannel and the first junction, and the second branch is between the first junction and the second open end of the third microchannel.
  • the third microchannel is in fluid communication with the first and second outlet ports through the first and second open ends, respectively.
  • the liquid medium of interest is input to the first inlet port of the first particle separation units.
  • each of the first and second particle separation units further has a hurdle protruded inwards from the first side wall of the second microchannel in the second branch.
  • the hurdle has a cross-sectional geometric shape with a height, h.
  • the cross-sectional geometric shape is selected from the group consisted of a triangle, a square, a rectangle, a semi-circle and a polygon.
  • the height h is less than the width, w2, of the second microchannel so as to allow particles of the liquid medium of interest to pass through the second branch of the second microchannel.
  • the hurdle is formed of a dielectric material.
  • the flow focusing unit has a first, second and third inlet ports, an outlet port, and a first and second microchannel, each of the first and second microchannels formed with a first open end, an opposite, second open end, and a first side wall and an opposite, second side wall defining a corresponding channel width therebetween.
  • Each channel width is in a range of about 0.1-1,000 ⁇ m, preferable in a range of about 1-500 urn.
  • the first microchannel is in fluid communication with the first inlet port and the outlet port through its first and second open ends, respectively.
  • the second microchannel is in fluid communication with the second and third inlet ports through its first and second open ends, respectively.
  • the first and second microchannels are in fluid communication with each other through a junction formed therein.
  • the junction divides each of the first and second microchannels into a first branch and a second branch, wherein the first branch of each of the first and second microchannels is between the first open end of the corresponding microchannel and the junction, and the second branch of each of the first and second microchannels is between the junction and the second open end of the corresponding microchannel.
  • the first particle separation unit, the second particle separation unit, and the flow focusing unit are adapted such that the first inlet port of the second particle separation unit coincides with one of the first and second outlet ports of the first particle separation unit, and the first inlet port of the flow focusing unit coincides with one of the first and second outlet ports of the second particle separation unit.
  • the flow cytometer comprises a fluid control member configured to control flow of the liquid medium in the microchannel structure.
  • the fluid control member includes a plurality of electrodes, each electrode placed in a corresponding port of the first and second particle separation units and the flow focusing unit; and a power source electrically coupled with the plurality of electrodes for individually applying voltages to each of the plurality of electrodes so as to generate desired electrokinetically microfiuidic flows in the first and second particle separation units and the flow focusing unit for separating and transporting the particles in the liquid medium.
  • the fluid control member further comprises a controller in communication with the power source and the plurality of electrodes for regulating voltages applied to each of the plurality of electrodes.
  • the flow cytometer comprises a second substrate having a first surface and an opposite, second surface.
  • the second substrate is bonded to the first substrate such that the first surface of the second substrate is substantially in contact with the second surface of the first substrate, thereby sealing the microchannel structure formed in the body portion of the first substrate.
  • each of the first and second substrates is formed of a corresponding dielectric material, wherein the first substrate is formed of polydimethylsiloxane (PDMS), and the second substrate is formed of glass, respectively.
  • PDMS polydimethylsiloxane
  • the voltages are applied to the electrodes placed in the first and second inlet ports and the first and second outlet ports of each of the first and second particle separation units, respectively, such that the generated electrokinetically microfiuidic flows cause (i) a liquid medium of interest introduced to the first inlet port and a buffer solution introduced to the second inlet port to move along the first microchannel and the first branch of the second microchannel, respectively, towards the first junction, and to merge into a stream of fluid therein; (ii) the merged stream of fluid to move along the second branch of the second microchannel towards and through the hurdle and towards the second junction, and to separate into a first and second streams of fluid therein; and (ii) the separated first and second streams of fluid to move along the first and second branches of the third microchannels towards the first and second outlet ports, respectively, of the corresponding particle separation unit, wherein the separated first stream of fluid contains particles that are substantially different from these contained in the separated second stream of fluid.
  • the voltages are applied to the electrodes placed in the first, second and third inlet ports and the outlet ports of the flow focusing unit such that the generated electrokinetically microfluidic flows cause a particle-carrying flow from the first inlet port, a first buffer solution flow from the second inlet port, a second buffer solution flow from the third inlet port to move towards and meet at the junction, and to move towards the outlet port; and the first buffer solution flow and the second buffer solution flow to squeeze the particle-carrying flow to a desired size in the second branch of the first microchannel, thereby focusing the particle- carrying flow such that each particle moves singly along the second branch of the first microchannel towards the outlet port.
  • the flow cytometer comprises an optical detection unit configured for counting and differentiating particles in the liquid medium
  • the optical detection unit includes one or more input optical fibers. Each input optical fiber is positioned over the second branch of the first microchannel of the flow focusing unit from the first substrate for delivering a corresponding beam of laser thereto to illumine the particles in the focused stream of fluid passing therethrough.
  • the optical detection unit also includes one or more output optical fibers. Each output optical fiber is positioned opposite to a corresponding input optical fiber from the second substrate such that when a particle passes through a position to which a beam of laser is delivered from the corresponding input optical fiber, the output optical fiber receives a signal associated with the particle.
  • each of the one or more input optical fibers and the one or more output optical fibers comprises a multimode optical fiber that has a diameter in a range of about 10-200 ⁇ m.
  • the signal associated with the particle comprises a fluorescent signal emitted from the particle in response to the illumination of the beam of laser.
  • the optical detection unit further includes a plurality of detectors coupled with the one or more output optical fibers for recording signals received from the one or more output optical fibers. The recorded signals are usable for counting and differentiating the particles passing through the second branch of the first microchannel of the flow focusing unit.
  • the optical detection unit may also include a plurality of filters. Each filter is coupled between the one or more output optical fibers and one of the plurality of detectors, respectively.
  • the present invention relates to a flow cytometer.
  • the flow cytometer comprises a microchannel structure adapted for transporting a fluid medium containing one or more types of particles; means for generating electrokinetically microfluidic flows to transport the fluid medium in the microchannel structure so as to differentiate the one or more types of particles in the fluid medium therein; and an optical detection system configured to detect the differentiated one or more types of particles of the fluid medium.
  • the microchannel structure includes at least one particle separation unit, wherein the at least one particle separation unit comprises at least one inlet port, a first and second outlet forts, and at least one channel in fluid communication with the at least one inlet port and the first and second outlet ports, wherein the at least one microchannel is formed with at least one side wall and a hurdle protruded inwards from the at least one sidewall such that when the fluid medium is introduced into the at least one microchannel and passes through the hurdle, the one or more types of particles are dielectrophoretically differentiated into a first and second groups of particles in accordance with their sizes, wherein the first and second groups of particles move towards the first and second outlet ports, respectively.
  • the hurdle has a cross-sectional geometric shape selected from the group consisted of a triangle, a square, a rectangle, a semi-circle and a polygon.
  • the microchannel structure further comprises a flow focusing unit in fluid communication with the at least one particle separation unit, wherein the flow focusing unit comprises at least one inlet port, an outlet port and at least one microchannel in fluid communication with the at least one inlet port and the outlet port, and wherein when one of the first and second groups of particles received in a corresponding outlet port of the at least one particle separation unit is introduced to the at least one microchannel from the at least one input port, each particle moves singly along the at least one microchannel towards the outlet port.
  • the optical detection system includes one or more input optical fibers, each input optical fiber positioned over the at least one microchannel of the flow focusing unit for delivering a corresponding beam of laser thereto to illumine the particles passing therethrough; one or more output optical fibers, each output optical fiber positioned opposite to a corresponding input optical fiber such that when a particle passes through a position to which a beam of laser is delivered from the corresponding input optical fiber, the output optical fiber receives a signal associated with the particle; and a plurality of detectors coupled with the one or more output optical fibers for recording signals received from the one or more output optical fibers, wherein the recorded signals are usable for counting and differentiating the particles passing through the second branch of the first microchannel of the flow focusing unit.
  • the liquid medium of interest comprises a biological fluid of a living subject, wherein the biological fluid comprises blood or urine, and wherein the blood or urine comprises one or more types of particles or cells, wherein the one or more types of cells are differentiatable by their sizes, functions or a combination of them.
  • the present invention relate to a method for counting and differentiating particles in a liquid medium of interest, where the liquid medium of interest contains one or types of particles.
  • the method includes the steps of providing a microchannel structure on a first substrate; generating electroldnetically microfluidic flows to transport the liquid medium in the microchannel structure so as to differentiate the one or more types of particles in the liquid medium therein; and detecting the differentiated one or more types of particles in the liquid medium.
  • the microchannel structure comprises at least one particle separation unit.
  • the at least one particle separation unit comprises a first and second inlet ports, a first and second outlet ports, and a first to third microchannels, each of the first to third microchannels formed with a first open end, an opposite, second open end, and a first side wall and an opposite, second side wall defining a corresponding width therebetween.
  • the first microchannel is in fluid communication with the first inlet port and the second microchannel through the first and second open ends, respectively, thereby forming a first junction that divides the second microchannel into a first branch and a second branch, wherein the first branch is between the first open end and the first junction, and the second branch is between the first junction and the second open end.
  • the second microchannel is in fluid communication with the second inlet port and the third microchannel through its first and second open ends, respectively, thereby forming a second junction that divides the third microchannel into a first branch and a second branch, wherein the first branch is between the first open end and the second junction, and the second branch is between the second junction and the second open end.
  • the third microchannel is in fluid communication with the first and second outlet ports through its first and second open ends, respectively.
  • the at least one particle separation units further has a hurdle protruded inwards from the first side wall of the second branch of the second microchannel.
  • the hurdle has a cross-sectional geometric shape with a height, h, wherein the cross-sectional geometric shape is selected from the group consisted of a triangle, a square, a rectangle, a semi-circle and a polygon, and the height h is less than the width, w2, of the second microchannel so as to allow one or more types of particles of the liquid medium to pass through the second branch of the second microchannel.
  • the microchannel structure further comprises a flow focusing unit in fluid communication with the at least one particle separation unit, wherein the flow focusing unit further has a first, second and third inlet ports, an outlet port, and a first and second microchannels, each of the first and second microchannels formed with a first open end, an opposite, second open end, and a first side wall and an opposite, second side walls defining a width therebetween.
  • the first microchannel is in fluid communication with the first inlet port and the outlet port through its first and second open ends, respectively.
  • the second microchannel is in fluid communication with the second and third inlet ports through its first and second open ends, respectively.
  • the first and second microchannels are in fluid communication with each other through a junction formed therein, the junction divides each of the first and second microchannels into a first branch and a second branch.
  • the first branch of each of the first and second microchannels is between the first open end of the corresponding microchannel and the junction, and wherein the second branch of each of the first and second microchannels is between the junction and the second open end of the corresponding microchannel.
  • the step of generating electrokinetically microfluidic flows comprises the steps of placing an electrode into a corresponding port for each of the first and second inlet ports and the first and second outlet ports of the at least one particle separation unit and the first, second and third inlet ports and the outlet port of the flow focusing unit; and individually applying voltages to each of the placed electrodes to generate electrokinetically microfluidic flows in the at least one particle separation unit and the flow focusing unit.
  • the generated electrokinetically microfluidic flows in the at least one particle separation unit cause (1) a liquid medium of interest introduced to the first inlet port and a buffer solution introduced to the second inlet port to move along the first microchannel and the first branch of the second microchannel, respectively, towards the first junction, and to merge into a stream of fluid therein; (2) the merged stream of fluid to move along the second branch of the second microchannel towards and through the hurdle and towards the second junction, and to separate into a first and second streams of fluid therein; and (3) the separated first and second streams of fluid to move along the first and second branches of the third microchannels towards the first and second outlet ports, respectively, of the corresponding particle separation unit, wherein the separated first stream of fluid contains particles that are substantially different from these contained in the separated second stream of fluid.
  • the generated electrokinetically microfluidic flows in the flow focusing unit cause a particle-carrying flow from the first inlet port, a first buffer solution flow from the second inlet port, a second buffer solution flow from the third inlet port to move towards and meet at the junction, and to move towards the outlet port; and the first buffer solution flow and the second buffer solution flow to squeeze the particle-carrying flow to a desired size in the second branch of the first microchannel, thereby focusing the particle-carrying flow such that each particle moves singly along the second branch of the first microchannel towards the outlet port.
  • the detecting step comprises the steps of delivering at least one beam of laser to the second branch of the first microchannel of the flow focusing unit at a position to illumine a particle passing through the position; collecting signals for a period of time, each signal associated with a particle passing through the position; and analyzing the collected signals to determine the number and type of the particles passing through the second branch of the first microchannel of the flow focusing unit.
  • the signal associated with the particle comprises a fluorescent signal emitted from the particle in response to the illumination of the at least beam of laser.
  • the liquid medium of interest may comprise a biological fluid of a living subject.
  • the biological fluid includes blood or urine.
  • the blood or urine comprises one or more types of particles or cells.
  • the one or more types of cells are differentiatable by their sizes, functions or a combination of them.
  • the one or more types of cells may comprise CD4+ cells, and/or CD3+ cells.
  • the CD4+ cells and CD3+ cells are labeled with a first and second antibodies, respectively, where the first and second antibodies are excited with light of different wavelengths.
  • the one or more types of cells are associated with a disease, which may be then detected and/or treated through the cells.
  • Fig. 1 shows schematically (a) an electrokinetically microfluidic flow cytometer lab-on-a-chip device and (b) a particle separation unit of the flow cytometer lab-on-a-chip device according to one embodiment of the present invention
  • Fig. 2 shows schematically (a) a contour of a DC electric field around an insulating hurdle in a microchannel of a particle separation unit and (b) an enlarged view of a particle moving around the edge region of the insulating hurdle according to one embodiment of the present invention, where the darkness level indicates the magnitude of the DC electric field, the x-direction is alone the microchannel length (flow), and the y- direction is along the channel width, and the x-y coordinates are normalized by the channel width;
  • Fig. 3 shows schematically a vertical optical detection system according to one embodiment of the present invention
  • Fig.4 shows schematically a perspective view of a flow cytometer lab-on-a-chip device according to one embodiment of the present invention
  • Fig. 5 shows superimposed sequential microscope images of the separation of polystyrene particles having sizes of about 6 ⁇ m and about 15 ⁇ m by an induced DC- DEP force according to one embodiment of the present invention
  • Fig. 6 shows (a) schematically an electrokinetically controlled flow focusing system and (b) an image of a focused fluorescent particles stream according to one embodiment of the present invention, where the arrows indicate the flow directions;
  • Fig. 7 shows (a) partially optical fibers embedded in a PDMS chip, wherein the thinner fiber introduces the laser beam, the thicker fiber detects the laser, and a particle is detected once it passes through the laser beam, and (b) the detected optical signal strength, where each peak represents one particle;
  • Fig. 8 shows a comparison of CD4 and CD8 percentages from whole blood staining vs. ficoll-isolated PBMC, where whole blood was stained with antibodies, followed by lysis of RBC, and then run directly without washes, and subjects 1, 2, and 3 are HIV-uninfected, subjects 4 and 5 are HIV-infected, note lower CD4+ T cell percentages;
  • Fig. 9 shows representative flow cytometry plots of whole blood stained with CD3 (APC) and CD4 (FITC) antibodies, (a) forward and side scatter differentiates lymphocytes, monocytes, and Polymorphonuclear cells (PMNs), where horizontal lines represent relative size demarcations (based on forward scatter) that preferentially include lymphocytes (approximately 4-10 micron size), (b): gate on CD3+ cells (T cells), and (c) dots 910 represent CD3+ CD4+ T cells, note monocytes 920, very few of which are in this size gate, which whey stain dimly with anti-CD4 and do not express CD3; and Fig. 10 shows the use of "TruCount" beads to evaluate absolute T cell numbers,
  • CD45 a marker for all white blood cells, vs. side scatter, PMNs 1010; monocytes 1020; CD3+ lymphocytes 1030; CD3+CD4+ lymphoctes 1040, where size beads are at upper left, and calibration (“TruCount”) beads for counting are bright green 1050 at far right, and
  • CD3 and CD4 expression on CD45+ lymphocytes Since the number of beads per tube and the volume of added blood are known, the absolute CD4+ T cell count can be calculated.
  • LOC lab-on-a-chip
  • the LOC is capable of handling substantially small fluid volumes down to less than picoliters to perform desired biological and/or chemical analysis.
  • microchannel refers to a channel structure having a cross-sectional dimension, e.g., a width, a depth or a diameter, in a microscale range from about 0.1 ⁇ m to about 1 mm.
  • the microchannels preferably have a cross-sectional dimension between about 0.1 ⁇ m and 500 ⁇ m, more preferably between about 0.1 ⁇ m and 300 ⁇ m.
  • a device referred to as being microscale includes at least one structural element or feature having such a dimension.
  • microfluidics refers to the science of designing, manufacturing, and formulating devices and processes that deal with volumes of fluid on the order of nanoliters (nl) or picoliters (pi).
  • a microfluidic device has one or more channels with a cross-sectional dimension less than 1 mm.
  • Common fluids used in microfluidic devices include whole blood samples, bacterial cell suspensions, protein or antibody solutions and various buffers.
  • microfluidic devices include, but not limited to, capillary electrophoresis, isoelectric focusing, immunoassays, flow cytometry, sample injection of proteins for analysis via mass spectrometry, PCR (polymerase chain reaction) amplification, DNA (deoxyribonucleic acid) analysis, cell manipulation, cell separation, cell patterning and chemical gradient formation. Many of these applications have utility for clinical diagnostics.
  • electrokinetics refers to the science of electrical charges in moving substances, such as water or blood, which studies particle motion that is the direct result of applied electric fields. Electrokinetics includes electroosmosis, electrophoresis, dielectrophoresis and electrorotation.
  • Electroosmosis also called electroendosmosis, is the motion of polar liquid through a membrane or other porous structure (generally, along charged surfaces of any shape and also through non-macroporous materials which have ionic sites and allow for water uptake, the latter sometimes referred to as "chemical porosity" under the influence of an applied electric field.
  • electrostatic charge will be established at the surface.
  • These surface charges in turn attract the counter ions in the liquid to the region close to the solid-liquid interface to form the electrical double layer. In the electrical double layer region, there are excess counter ions (net charge). If the solid surface is negatively charged, the counter ions are the positive ions.
  • electroosmosis electrophoresis
  • electroosmosis electrophoresis
  • electrophoresis electrophoresis
  • an external electrical field is applied tangentially to the solid surface
  • the excess counter ions will move under the influence of the applied electrical field, pulling the liquid with them and resulting in electroosmotic flow.
  • the liquid movement is carried through to the rest of the liquid in the microchannel by the viscous effect.
  • electroosmotic flow is preferred over pressure driven flow.
  • One of the reasons is the plug-like velocity profile of electroosmotic flow. This means that fluid samples can be transported without dispersion caused by flow shear.
  • pumping a liquid through a small microchannel requires applying a very large pressure difference depending on the flow rate.
  • Electroosmotic flow can generate the required flow rate in very small microchannels without any applied pressure difference cross the channel. Additionally, using electroosmotic flow to transport liquids in complicated microchannel networks does not require any external mechanical pump or moving parts, it can be easily realized by controlling the applied electrical fields via electrodes.
  • Electrophoresis is the motion of a charged particle relative to the surrounding liquid under an applied electrical field.
  • the net velocity of a charged particle is determined by the electroosmotic velocity of the liquid and the electrophoretic velocity of the particle. If the surface charge of the particle is not strong or the ionic concentration of the liquid (e.g., typical buffer solutions) is high, the particle will move with the liquid.
  • Using electrical fields to manipulate and transport particles and biological cells in microchannels is particularly suitable for LOC applications. It should be noted that the applied electrical field has negligible effects on the cells, other than generating the flow and the cell motion.
  • EDL electrical double layer
  • Dielectrophoresis or its acronym "DEP” refers to a phenomenon in which a force is exerted on a dielectric particle when it is subjected to a non-uniform electric field. This force does not require the particle to be charged. All particles exhibit dielectrophoretic activity in the presence of electric fields. However, the strength of the force depends strongly on the medium and particles' electrical properties, on the particles' shape and size, as well as on the frequency of the electric field. Consequently, fields of a particular frequency can manipulate particles with great selectivity. This has allowed, for example, the separation of cells or the orientation and manipulation of nanoparticles.
  • the present invention provides an electrokinetically microfluidic flow cytometer LOC device that integrates multiple laboratory functions and/or processes on a single, small sized chip. By detecting different fluorescent signals carried by the cells, the flow cytometer LOC device can count and differentiate the cells.
  • the electrokinetically microfluidic flow cytometer LOC device possesses unique features absent in the conventional benchtop flow cytometers.
  • the flow cytometor LOC device is a fully automatic, stand-alone, and portable device.
  • the flow cytometor LOC device has wide applications in biomedical diagnosis of infectious diseases (e.g., HIV), cancers (e.g., leukemia), and other diseases that can be diagnosed by analyzing cells in blood and in body fluids.
  • infectious diseases e.g., HIV
  • cancers e.g., leukemia
  • the flow cytometor LOC device is particularly useful in field applications or point-of-testing applications where only very small amount of samples are available and immediate diagnostics is required (e.g., diagnostics of HIV and leukemia).
  • an electrokinetically microfluidic flow cytometer 100 is schematically shown according to one embodiment to the present invention, which is an LOC device.
  • the flow cytometer 100 is adapted for counting and differentiating particles in a liquid medium of interest.
  • the liquid medium of interest can be a biological fluid of a living subject, such as blood or urine.
  • the blood or urine has one or more types of particles or cells.
  • the one or more types of cells are differentiatable by their sizes, functions or a combination of them.
  • the change or percentage of the one or more types of cells in the blood or urine may be associated with a disease.
  • the flow cytonieter 100 includes a first substrate 110 having a first surface 112 and an opposite, second surface 114 defining a body portion 116 therebetween, and a microchannel structure 120 that is formed in the body portion 116 of the first substrate 110.
  • the microchannel structure 120 includes a first particle separation unit 130, a second particle separation unit 140, and a flow focusing unit 150.
  • the first and second particle separation units 130 and 140 are structurally and functional similar to each other, as shown in Fig. Ia.
  • Each of the first and second particle separation units 130 (140) has a first and second inlet ports (wells) 131 (141) and 133 (143), a first and second outlet ports 135 (145) and 137 (147), and a first to third microchannels 132 (142), 134 (144) and 136 (146).
  • first particle separation units 130 according to the embodiment of the present invention is illustrated and described in further details as follows.
  • each of the first, second and third microchannels 132, 134 or 136 is formed with a first open end 132a, 134a or 136a, an opposite, second open end 132b, 134b or 136b, with a first side wall 132f, 134f or 136f and an opposite, second side wall 132g, 134g or 136g defining a corresponding channel width, wl, w2 or w3, therebetween, respectively.
  • Each microchannel 132, 134 or 136 has at least one cross- sectional dimension in a microscale.
  • the first, second and third microchannels 132, 134 or 136 can have the same cross-sectional dimension or substantially different cross- sectional dimensions.
  • each channel width, wl, w2 or w3 is in a range of about 0.1-1,000 ⁇ m, preferable in a range of about 1-500 ⁇ m.
  • the first microchannel 132 is in fluid communication with the first inlet port 131 and the second microchannel 134 through the first and second open ends 132a and 132b, respectively, thereby forming a first junction 134c of the first and second microchannels 132 and 134.
  • the first junction 134c is a T-like junction that divides the second microchannel 134 into a first branch 134d and a second branch 134e.
  • the first branch 134d is between the first open end 134a of the second microchannel 134 and the first junction 134c
  • the second branch 134e is between the first junction 134c and the second open end 134b of the second microchannel 134.
  • the second microchannel 134 is in fluid communication with the second inlet port 133 and the third microchannel 136 through the first and second open ends 134a and 134b, respectively, thereby forming a second junction 136c of the second and third microchannels 134 and 136.
  • the second junction 136c is a T-like junction that divides the third microchannel 136 into a first branch 136d and a second branch 136e.
  • the first branch 136d is between the first open end 136a of the third microchannel 136 and the first junction 136c
  • the second branch 134e is between the first junction 134c and the second open end 134b of the third microchannel 136.
  • the third microchannel 136 is in fluid communication with the first and second outlet ports 135 and 137 through the first and second open ends 136a and 136b, respectively.
  • the first particle separation units 130 further has a hurdle 138 protruded inwards from the first side wall 134f of the second branch 134e of the second microchannel 134.
  • the hurdle 138 has a cross-sectional geometric shape of rectangle with a height, h.
  • the cross-sectional geometric shape can also be a triangle, a square, a semi-circle or a polygon.
  • the height h of the hurdle 138 is less than the width, w2, of the second microchannel 134, thereby allowing particles of the liquid medium of interest to pass through the second branch 134e of the second microchannel 134.
  • the hurdle 138 is formed of a dielectric material.
  • the separation of particles in the liquid medium of interest is performed by a DC-dielectrophoresis (DEP) force.
  • DEP DC-dielectrophoresis
  • a non-uniform local electric field 290 at the area of a hurdle 230 in a fluidic microchannel 234 and an induced DEP force, F DEP> on a particle 280 moving along the electric filed 290 are schematically shown.
  • the non-uniform local electric field 290 at the hurdle 238 is corresponding to an applied DC field disturbed by the hurdle 238.
  • the hurdle 238 is attached on or protruded from one side of the microchannel to form an abruptly narrow section 234m in the microchannel 234. Since only the liquid (an aqueous solution) conducts the electrical field, the narrow section 234m of the microchannel 234 generates a spatially non-uniform DC electrical field 290 in the liquid near the hurdle 238.
  • FIG. 2b An enlarged view of the local electrical field 290 near the up-stream corner 238a of the hurdle 238 is shown in Fig. 2b.
  • EEF electroosmotic flow
  • EP electrophoresis
  • a particle 280 moves towards the entrance region 234ml of the narrow section 234m of the microchannel 234.
  • the electric field 290 is stronger in the region close to a corner 238a of the hurdle 238 than that in the region far from to the corner 238a of the hurdle 238.
  • the negative DEP force, FQ EP directs to the region of lower electric-field strength, the particle 280 experiences a repulsive force from the corner 238a of the hurdle 238.
  • the magnitude of the repulsive DEP force is proportional to the volume of the particle 238 and the local value of (E • V)E , as indicated by equation (1).
  • the repulsive DEP force on a 15 ⁇ m particle is 27 times of that on a 5 ⁇ m particle under the same conditions. Therefore, a larger particle is subject to a stronger DEP force and tends to be pushed further away from the corner compared with a smaller particle.
  • the similar DEP repulsion occurs when the particle passes by the other corner 238b of the hurdle 238.
  • the trajectory shift in y-direction is different for particles of different sizes and hence particles are separatable by size.
  • a particle 180a is smaller than a particle 180b in a liquid medium of interest.
  • a hurdle 138 is formed on one side wall 134f of the microchannel 134 to form an abruptly narrow section 134m. Since only the liquid medium of interest conducts the electrical field, the narrow section 134m of the microchannel 134 generates a spatially non-uniform DC electrical field in the liquid medium near the hurdle 138.
  • the liquid medium having a mixture of large and small cells 180b and 180a is introduced into the particle separation unit 130 from the first microchannel 132.
  • the negative DC-DEP force at the corners 138a and 138b of the hurdle 138 pushes the larger cells 180b further from the corner 138b of the hurdle 138 than the smaller cells 180a is, and thus generates different trajectories for smaller and larger cells 180a and 180b once they pass the hurdle 138.
  • a T-shaped channel structure 136c is used so that the separated small cells 180a and the separated large cells 180b are drawn into the first outlet port (well) 136a and the second outlet port (well) 136b, respectively, by electroldnetically microfluidic flows.
  • the design parameters, e.g., the hurdle size and position and the controlling parameters, e.g., applied voltages, of the flow cytometer LOC device need to be optimized. This can be done theoretically and experimentally. Theoretically, the influences of different parameters on the particle (cell) trajectory are simulated using a complicated theoretical model and numerical simulation [12, 15-26], so as to obtain the optimal design parameters and controlling parameters. Experimentally, fixed human blood cells are used.
  • fluorescent (carboxylate-modified) polystyrene particles of different sizes: 1 ⁇ m, 3 ⁇ m, 4 ⁇ m, 6 ⁇ m, 10 ⁇ m, 12 ⁇ m, and 15 ⁇ m in diameter (Bangs Laboratory Inc.) are used as sample particles for evaluation. These particle sizes are similar to the size of typical blood cells and the small components involved in the samples.
  • all the microchannels 132, 134 and 136 and all the wells (inlet and outlet ports) 131, 133, 135 and 137 are primed with about 1 mM sodium carbonate buffer solution. Then, the cells or particle mixture (a liquid medium of interest) and a buffer solution are introduced into the first and second inlet ports (wells) 131 and 133 with a syringe.
  • Other tools can also be used to practice the present invention.
  • a high-voltage DC power supply (Labsmith HVS448) is used to supply voltages to the four platinum electrodes submerged in the wells 131, 133, 135 and 137 as so to generate desired electrokinetically microfluidic flows to drive the liquid medium through the particle separation unit 130.
  • a voltage controller coupled with the high- voltage DC power supply (not shown) is used to adjust independently the voltage applied to each of the four electrodes. Following the results of the numerical simulations as the guidance, the voltages applied to the four electrodes are adjusted such that in operation, the liquid medium of interest introduced into the first inlet port 131 and the buffer solution introduced into the second inlet port 133 move along the first microchannel 132 and the first branch 134d of the second microchannel 134, respectively, towards the first junction 134c, and merge into a stream of fluid therein. The merged stream of fluid then moves along the second branch 134e of the second microchannel 134 towards the hurdle 138 and passes through the hurdle 138.
  • an induced DC-DEP force at the corners 138a and 138b of the hurdle 138 pushes the larger cells 180b in the liquid medium further from the corner 138b of the hurdle 138 than the smaller cells 180a in the liquid medium are, thereby, separating the cells into two groups according to the cell sizes.
  • the group of the separated cells in small sizes and the group of the separated cells in large sizes move along the first and second branches 136d and 136e of the third microchannels 136 towards the first and second outlet ports (wells) 135 and 137, respectively.
  • the whole separation process is completed within about 60 seconds, and the EOF flow rate in the microchannels is small, the effect of the pressure-driven flow is minimized by using sufficiently large well size and by carefully balancing the liquid level in four wells.
  • the cell/particle motion is monitored by a fluorescent microscope (MBA801, Nikon, Inc., Japan) and recorded by a progressive CCD camera (Qlmaging, Inc., British Columbia, Canada).
  • the flow focusing unit 150 has a first, second and third inlet ports 151, 153 and 155, an outlet port 157, and a first and second microchannel 152 and
  • Each of the first and second microchannels 152 is formed with a first open end 152a (154a), an opposite, second open end 152b (154b), a first side wall 152f (154f) and an opposite, second side wall 152g (154g) defining a corresponding channel width therebetween.
  • Each channel width is in a range of about 0.1-1,000 ⁇ m, preferable in a range of about 1-500 ⁇ m.
  • the first microchannel 152 is in fluid communication with the first inlet port 151 and the outlet port 157 through its first and second open ends 152a and 152b, respectively.
  • the second microchannel 154 is in fluid communication with the second and third inlet ports 153 and 155 through its first and second open ends 154a and 154b, respectively.
  • the first and second microchannels 152 and 154 are in fluid communication with each other through a junction 152c formed therein. As shown in Fig. Ia, the junction 152c divides each of the first and second microchannels 152 and 154 into a first branch 152d (154d) and a second branch 152e (154e).
  • the first branch of each of the first and second microchannels 152 and 154 is between the first open end of the corresponding microchannel 152 and 154 and the junction, and the second branch of each of the first and second microchannels 152 and 154 is between the junction and the second open end of the corresponding microchannel 152 and 154.
  • the outlet port 157 is provided with a corresponding electrode (not shown).
  • These electrodes are electrically coupled with a high- voltage DC power supply (not shown) and a voltage controller (not shown) for applying voltages thereto to generate electrokinetically microfluidic flows in the flow focusing unit 150.
  • the voltages are applied such that the generated electrokinetically microfluidic flows cause a corresponding group of the separated cells in the first inlet port 151 and the buffer solution introduced to the second and third inlet ports 153 and 155 to move towards the junction 152c, to meet at the junction 152c, and to move towards the outlet port 157 along the second branch 152e of the first microchannel 152.
  • the flows of the corresponding group of the separated cells from the first inlet port 151 and the buffer solution from the second and third inlet ports 153 and 155 are laminar flows and do not mix when they move along the second branch 152e of the first microchannel 152, 654d and 654e.
  • the two side flows (buffer solution) squeeze the central cell-carrying flow to a desired size, thereby focusing the corresponding group of the separated cells in the second branch 152e of the first microchannel 152.
  • particles (cells) 680a and 680b singly pass through the detecting point.
  • the merged stream of fluid is focused by the electrokinetically microfluidic flows moving towards the junction 152c, from the first branch and second branch 153d and 153e of the second microchannel 153, such that each particle in the merged stream of fluid moves singly along the second branch 152e of the first microchannel 152 towards the outlet port 157.
  • the first inlet port 141 of the second particle separation unit 140 coincides with one of the first and second outlet ports 135 and 137 of the first particle separation unit 130
  • the first inlet port 151 of the flow focusing unit 150 coincides with one of the first and second outlet ports 145 and 147 of the second particle separation unit 140, such that the first particle separation unit 130, the second particle separation unit 140, and the flow focusing unit 150 are in fluid communication with each another.
  • the flow cytometer 100 may have a second substrate having a first surface and an opposite, second surface.
  • each of the first and second substrates is formed of a corresponding dielectric material, wherein the first substrate is formed of polydimethylsiloxane (PDMS), and the second substrate is formed of glass, respectively.
  • PDMS polydimethylsiloxane
  • the microchannel structure 120 in the PDMS substrate in one embodiment, is fabricated following the soft lithography protocol [13]. A detailed fabrication procedure is described in reference [14].
  • the microfluidic flow cytometer of the present invention includes a microchannel structure having several distinctive functional units: a first DC-DEP separation unit, a second DC-DEP separation unit, and a flow focusing unit. These units are operated in a time sequence. Since all microchannels are connected and there are no mechanical valves, it is critical to control the flow of liquid in the microchannel network structure, i.e., control the flow directions in certain microchannels while keeping liquid in other channels stationary. This is realized by controlling the applied electrical field, i.e., different voltages at different electrodes in different wells (ports).
  • the automatic, electroldnetically microfluidic flow is controlled, not only with spatial precision but also with temporal precision.
  • These flow controls include the flow direction, flow switching and reagent holding in the wells (reservoirs).
  • a flow cytometer 300 also includes an optical detection unit 370 for counting and differentiating the particles in the liquid medium.
  • an optical detection unit 370 for counting and differentiating the particles in the liquid medium.
  • a vertical detection method is employed, which reduces the complexity of making the lab-on-a-chip (device) and the cost, and thus makes the lab-on-a-chip disposable.
  • the optical detection unit 370 includes one or more input optical fibers.
  • two optical fibers 371 and 373 are utilized to practice the present invention.
  • Each optical fiber 371 (273) has a first end 371a (373a) and an opposite, second end 371b (373b) coupled to two lasers 341 and 342, respectively.
  • two lOO ⁇ m fiber-coupled lasers one emits light in red (650nm) and the other in blue (488nm). They are small, simple and inexpensive.
  • the red laser used to practice the present invention is made from Lasermate Group, Inc., California. Other types of lasers can also be used to practice the present invention.
  • each optical fiber 371 (273) is positioned over the second branch 352e of the first microchannel 352 of the flow focusing unit 350 from the first substrate 310 for delivering a corresponding beam of laser thereto to illumine the particles, for example, 380a and 380b, in the focused stream of fluid 351a when they pass the positions underneath the two optical fibers 371 and 373.
  • the optical detection unit 370 also includes one or more output optical fibers. As shown in Fig. 3, two optical fibers 372 and 374 are employed in the embodiment of the present invention, each optical fiber 372 (374) having a working end 372a (374a). The working end 372a (374a) of each optical fiber 372 (274) is positioned opposite to a corresponding input optical fiber 371 (273) from the second substrate 360 such that when a particle (cell) 380a (380b) passes through a position to which a beam of laser is delivered from the corresponding input optical fiber 371 (373), the output optical fiber 372 (374) receives a signal associated with the particle (cell) 380a (380b).
  • the signal associated with the particle (cell) 380a (380b) comprises a fluorescent signal emitted from the particle in response to the illumination of the beam of laser.
  • each of the one or more input optical fibers and the one or more output optical fibers comprises a multimode optical fiber that has a diameter in a range of about 10-200 ⁇ m.
  • the optical detection unit 370 further includes detectors 378a-378d coupled with the two optical fibers 372 and 374 for recording signals received from the one or more output optical fibers 372 and 374. After electronic amplification, each recorded signal is fed to the data acquisition card inside a computer for processing as so to count and differentiate the particles passing through the second branch 352e of the first microchannel 352 of the flow focusing unit 350.
  • a FITC filter 375 is used for the blue laser 341, while a Cy5 filter 376 is used for the red laser 342.
  • a silicon photodiode array (Hamamatsu, USA) is also employed.
  • the Si photodiode array includes 10 Si PIN photo-detectors and each of them is coupled with a fiber of lOO ⁇ m in diameter.
  • the detection microchannel is 100 ⁇ m in width and 50 ⁇ m in depth. As illustrated in Fig. 3, the output sensing (photo-detecting) fibers 372 and 374 approach the microchannel 352 and the cells 380a and 380b from the bottom surface 364 of the glass substrate 360.
  • the excitation lights are introduced by optical fibers from the top surface 312 of the PDMA plate 310.
  • the fiber ends 371a, 373a and 372a, 374a touch the bottom glass plate 360 and the top PDMS plate 310, respectively.
  • a fiber positioner is adapted for holding and aligning the fibers 371-374 with the fluidic channel 352.
  • refractive index matching oil is applied between fiber ends 371 a, 373 a and 372a, 374a and the top surfaces 312 of the PDMA plate 310 and the bottom surface 364 of the glass plate 360 to reduce both excitation power and fluorescent emission loss.
  • photo detectors D1-D4 378a-378d are deployed at two locations 365a and 365b opposite to the two excitation laser beams delivered by the fibers 371 and 373, as shown in Fig. 3.
  • a specific excitation laser 341 (343) is introduced from the top surface 312 of the LOC device to the liquid medium (sample) 351a through an optical fiber 371 (373) and an optical fiber 372 (374) underneath the LOC device collects the light signal emitted from the cell 380a (38Ob) responsive of the excitation of the corresponding laser.
  • the collected light signal is split into two branches 372b (374b) and 372c (374c).
  • One 372c (374c) goes directly into the photo diode detector D2 378b (D3 378c) and the other 372b (374b) goes through a filter 375 (376) first and then reaches another photo diode detector Dl 378a (D4 378c).
  • the filter 375 (376) is adapted for passing the specific emission wavelength for the specific dye tagged on CD4+ or CD3+ cells.
  • CD4+ cells are labeled with AlexaFluor-488-conjugated antibodies (only excited with the 488nm wavelength laser) and CD3+ cells are labeled with AlexaFluor-647-conjugated antibodies (only excited with the 650nm wavelength laser) (Becton Dickenson, San Jose, CA).
  • the peak emission wavelength is 665 nm for AlexaFluo-647, and 520 nm for AlexaFluo-488. Since the emission spectra of these fluorochromes do not overlap, compensation of the detector system is not necessary.
  • the small and portable optical detection system 370 is capable of detecting these two emission wavelengths.
  • a Cy5 filter 376 is used to detect the AlexaFluor-647 emission
  • a FITC filter 375 is used to detect AlexaFluor-488 emission.
  • the signals collected by the photo detector Dl 378a and the photo detector D4 378d in Fig. 3 can be used to determine the number of CD4 and CD3 cells, respectively.
  • a cell passes through a laser beam, it blockes the light path and generate a signal.
  • the signal collected at the photo detector D2 (D3) without going through the optical filter indicates whether there is a cell passing the laser beam.
  • the signals collected at the photo detector D2 (D3) shows the total number of cells passing through the system.
  • the fluorochromes are selected to have different excitation wavelengths and non-overlapping emission wavelengths, cells can have a low level of expression of markers that can confound their discrimination.
  • small granulocytes can overlap in size with larger lymphocytes, they are CD3(-) and CD4(-) and readily differentiated from T lymphocytes.
  • Monocytes can overlap lymphocytes by size, and have a low level CD4 expression, but are CD3 (-). Therefore, it is necessary to distinguish the false signals detected at photo diode detectors Dl and D4 that are generated by monocytes.
  • a cell that shows a detectable AlexaFluor-488 emission (CD4) but weak or absent AlexaFluor-647 (CD3) emission would be considered as a monocyte.
  • a relatively strong AlexaFluor-488 emission signal detected at the photo detector Dl and a weak AlexaFluo-647 emission signal detected at the photo detector D4 are from the same cell.
  • the signals collected at the photo diode detector D3 enable one to distinguish this kind of false signal by comparing the signals collected at the four photo detectors.
  • all the signals collected at the four detectors Dl -D4 are recorded with a timer in a microprocessor chip in the flow cytometer. Since the signals detected at the detectors Dl and D2 are from the same physical position, the signals simultaneously detected by Dl and D2 are from the same cell; Dl counts CD4 cells, while D2 counts events. Similarly the signals simultaneously detected by D3 and D4 are from the same cell; D3 counts events, while D4 counts CD3 cells.
  • cell subsets are sorted to high purity with the FACSaria sorter, and the sorted subpopulations are precisely counted with the GUAVA counter.
  • the GUAVA counter is specifically adapted to provide accurate cell counts of cells in suspension, and is used in cell processing laboratories to minimize variation from manual cell counting.
  • all isolated cells that is analyzed by the described methods is first quantified by the GUAVA.
  • the purified cell subsets is mixed at defined ratios and simultaneously evaluated by the flow cytometer LOC device of the present invention and the FACSaria.
  • a control device is utilized to control the multiple steps of electrokinetic microfluidic processes, synchronize the microfluidics and optical detection, and collecting data and computing the results.
  • the control device may have at least four (4) analogue inputs, twelve (12) digital outputs, and one timer. Using the signal from the digital output, the voltages applied at different wells are controlled to achieve the desired flows at the different functional units in the flow cytometer lab-on-a-chip.
  • outputs of the four photodiode detectors are collected through the four analogue input channels continuously with the time references. To maximize the ratio of signal to noise of the optical signal detection, a lock-in amplification technique is used in the control device. The information collected at the four detectors is further analyzed to provide complete information (the total number and the percentage) of the CD4 and CD3 cells in the sample.
  • a handheld flow cytometer LOC device 400 according to one embodiment of the present invention is shown schematically.
  • the optical fibers 471 and 473 introducing the excitation lasers and the electrodes 450 are built into the cover lid 410 (only dot 471 and dot 473 are shown to indicate the fiber heads' positions. The remaining fibers are not shown for clarity).
  • the tip of the detection optical fibers 471 and 473 are fixed at the surface of the chip-holding stage 418.
  • the microfluidic flow cytometer chip 415 is placed on the chip-holding stage 418 which ensures the precise alignment between the optical fibers and the detection microchannel 442, and between the electrodes 440 and wells 430 of the microchannel structure 420.
  • the sample and the buffer solution are loaded to the specific wells 430 by using a pipette.
  • the operator just needs to close the cover lid 410 and to press the button 482 to start the operation program.
  • the chip 415 can be disposed after the test.
  • the operation program is stored in a microprocessor chip (not shown) in the handheld LOC device 400.
  • the status of the operation is shown on the LCD screen 470 of the handheld flow cytometer LOC device 400.
  • the operation can be stopped by pushing the button 484 if necessary.
  • Essential test results are shown on the screen 470 and the completed test results can be either displayed on the screen 470 or printed out.
  • the complete testing data is temporarily saved in the device 400 and can be download to a computer or memory card for further analysis.
  • Another aspect of the present invention provides a method for counting and differentiating particles in a liquid medium of interest, where the liquid medium of interest contains one or more types of particles.
  • the method includes the steps of providing a microchannel structure on a first substrate; generating electrokinetically microfluidic flows to transport the liquid medium in the microchannel structure so as to differentiate the one or more types of particles of the liquid medium therein; and detecting the differentiated one or more types of particles of the liquid medium.
  • the microchannel structure is disclosed as above.
  • the step of generating electrokinetically microfluidic flows comprises the steps of placing an electrode into a corresponding port for each port of the microchannel structure; and individually applying voltages to each of the placed electrodes to generate desired electrokinetically microfluidic flows in the microchannel structure.
  • the detecting step comprises the steps of delivering at least one beam of laser to a microchannel at a position to illumine a particle passing through the position; collecting signals for a period of time, each signal associated with a particle passing through the position; and analyzing the collected signals to determine the number and type of the particles passing through the microchannel.
  • the signal associated with the particle comprises a fluorescent signal emitted from the particle in response to the illumination of the at least beam of laser.
  • the flow cytometer lab-on-a-chip device is capable of detecting and/or treating a large number of different cells as required in clinical applications, and minimizes the total number of cells and particles to be counted. Minimizing the total number of to-be-counted events reduces the analysis time and the complexity of the optical detection system while increasing the accuracy.
  • the flow cytometer lab-on-a-chip device in operation removes large cells such as granulocytes and monocytes, and small components such as platelets and the lysed red cells' debris, prior to counting CD4 and CD3 cells.
  • Another feature of such a flow cytometer lab-on-a-chip device is to provide the total number of CD4+ T lymphocytes, in addition to their percentages, in the sample of interest. Because monocytes can overlap lymphocytes in size and can also express low levels of CD4, they must be identified to avoid falsely elevated counts of CD4+ lymphocytes.
  • the flow cytometer lab-on-a-chip device includes no external pump, no tubing and valves, no bulky optical detection instruments, and a low-cost disposable chip. Electrokinetic-microfluidic means to transport liquid and cells in microchannels requiring only the application of electrical fields via electrodes inserted in different wells. A portable multiple wavelength detection system is utilized by small diode lasers, Si-PEST detectors and optical fibers. Additionally, the flow cytometer lab- on-a-chip is made of PDMS and glass plates by a soft photolithography technique, no embedded waveguides or optical fibers is embedded into the chip, thereby, making the chip inexpensive and disposable. These and other aspects of the present invention are further described below.
  • Fig. 5 shows an image of trajectories 510 and 520 of polystyrene particles having sizes of about 6 ⁇ m and about 15 ⁇ m, separated by particle separation unit 500.
  • the trajectories 510 and 520 of polystyrene particles are obtained by superimposing a series of sequential microscopy images.
  • the microchannel 534 in this embodiment is about 300 ⁇ m in width and about 40 ⁇ m in depth (perpendicular to the paper).
  • the narrow section 534m of the microchannel 534 is about 60 ⁇ m in width.
  • the voltages applied to the first and second inlet ports and a first and second outlet ports are about 245 V, 500 V 5 55 V and 0 V, respectively.
  • a flow cytometer is capable of focusing a cell-carrying stream so that only single cells are allowed to pass the sensing (detecting) point, and optically detecting a specific type of cell by detecting the fluorescent signal carried by each cell.
  • a stream (flow) focusing system 650 according to one embodiment of the present invention is shown.
  • the stream focusing system 650 has a cross-shaped microchannel structure having a horizontal microchannel 652 and a vertical microchannel 654 in fluid communication with the horizontal microchannel 652 through a junction 655 formed therein.
  • the microchannel structure is filled with a buffer solution.
  • One end 652a of the horizontal channel 652 of the microchannel structure is in fluid communication with a sample well filled with a buffer solution containing the cells to be detected, the other end 652b of the horizontal channel 652 is in fluid communication with a waste collection well.
  • the ends 654a and 654b of the vertical microchannel 654 are respectively in fluid communication with two wells filled with a buffer solution.
  • Four electrodes are inserted in these wells. When different voltages are applied to the four wells via the electrodes, the electrical fields generate electroosmotic flows in the microchannel structure.
  • the electrical fields are applied in such a way that the three liquid streams 652d, 654d and 654e from the sample well and the two buffer wells flow towards the waste well, and they meet at the cross intersection (junction) 655.
  • the electroosmotic flows in the microchannel structure are laminar flows and do not mix streams 652d, 654d and 654e.
  • the two side flows (buffer solution) 654d and 654e squeeze the central cell- carrying flow 652d to a desired size, thereby focusing the stream 652d.
  • particles (cells) 680a and 680b singly pass through the detecting point.
  • a set of four electrical potential values applied to the four wells is dependent from a specified main flow (the cell-carrying solution) rate and a specified cell size (the focused stream size).
  • Controlling the flow field in the intersection region of the cross microchannel also depends on the liquid properties (e.g., viscosity and ionic concentration), the shape and the size of the intersection and the applied electrical fields. In one embodiment, this is achieved by developing a theoretical model that simulates accurately the flows and the focusing process. Such an experimentally verified model is then used to control the lab-on-a-chip flow cytometer operation via a computer program.
  • a fluorescent image analysis system is used to visualize the flow focusing process near the intersection. The profile of the focused flow stream is measured. The prediction (the numerical simulation results) of such a flow focusing is verified by the experimental results [5-9].
  • Fig. 6 shows the flow focusing images demonstrating the online counting of particles in a flow cytometer chip by using embedded optical fibers in the PDMS flow cytometer chip.
  • a small size semiconductor laser and a Si-PIN detector are used for optical detection.
  • the detection system allows an easy switch between two-fiber detection mode and one-fiber detection mode, and is capable of counting particles, measuring particle velocity and identifying particle sizes.
  • Fig. 7 shows a pair of the embedded optical fibers on the opposite sides of a microchannel, and the particle counting data. By simply adding additional lasers of a different wavelength and additional Si-PIN photo detectors, this device can detect different wavelengths carried by different particles or cells.
  • the process is performed as follows: about 50 ⁇ l volumes of blood are mixed with about 50 ⁇ l of a red blood cell lysis buffer (Caltag, Burlingame, CA) to lyse the red blood cells, and then diluted with about 500 ⁇ l of de-ionized water, which this protocol fixes WBC in the sample, and lyses RBC. About 10 ⁇ l of this sample solution is loaded to the sample well (S in Fig. 1) on the chip by a micro-pipette. About 10 ⁇ l of the sample solution contains approximately 8,000 cells (granulocytes, monocytes, and lymphocytes) and approximately 100,000 small components (platelets, RBC debris, etc).
  • On-chip processes (1) Removing cells larger than 10 ⁇ m by a DC-DEP technique. This is conducted by applying predetermined voltages to wells Bl, S, Cl and C2, as shown in Fig. 1. This process reduces the total number of cells to be counted and thus reduces the time, the number of detection microchannels and the complexity of the optical detection system. It is noted that the sample solution contains approximately a total of 8,000 cells, and T lymphocytes are smaller than 10 ⁇ m. By removing the cells larger than 10 ⁇ m, the total number of to-be-counted cells is reduced by 2/3, to about 3,000 cells. (2) Removing components smaller than 4 ⁇ m (platelets, RBC debris, etc) by the DC-DEP technique.
  • This separation is for two purposes. First, it is to reduce the total number of particles to be counted and hence reduce the time, reduce the number of detection microchannels and the complexity of the optical detection system. T lymphoctes are larger than 4 ⁇ m, and virtually all the debris components in a typical sample are less than 4 ⁇ m. By removing the small components (smaller than 4 ⁇ m), the total number of to-be-counted particles is dramatically reduced to 3,000 total cells, which is predominantly lymphocytes. Secondly, because some of these small components could carry the dyes by non-specific adsorption, removing these small components improves the reliability of the CD4 and CD3 counting.
  • the separated cells (with a size range from 4 ⁇ m to 10 ⁇ m) are electrokinetically transported from well C4 to the flow focusing channel.
  • a vertical optical detection method is used, i.e., using two optical fibers from the top of the PDMS to introduce the exciting laser beams, and two optical fibers underneath the glass plate to receive the emission light signals.
  • the DC-DEP separation of larger cells takes approximately one minute to complete.
  • the typical speed of particle electrokinetic motion in the microchannels is about 1000 ⁇ m/s.
  • the approximate 108,000 particles (cells and small components) in the 10 ⁇ l sample 100,000 of them are smaller than 4 ⁇ m.
  • the narrowest section of the microchannel in the DC- DEP part is approximately 50 ⁇ m, considering that multiple particles are moving in parallel through the microchannel, approximately 2,000 ⁇ 3,000 particles/second or 120,000-180,000 particles/min are processed.
  • the approximately 3,000 larger cells (>10 ⁇ M) out of the 108,000 particles (cells and the small components) can therefore be separated within one minute.
  • the DC-DEP separation of small components takes approximately one minute to complete.
  • the typical speed of particle electrokinetic motion in the microchannels is about 1000 ⁇ m/s.
  • About 97% of the particles are the small components with size smaller than 4 ⁇ m. Since multiple particles are moving in parallel through the channel, approximately 2,000 ⁇ 3,000 particles/second or 120,000-180,000 particles/min are processed. Therefore approximately 100,000 small particles can therefore be separated within one minute.
  • This lysis step preserves the WBC in the sample, and in three subjects there is no difference in the % values of CD4 or CD8 lymphocytes comparing whole blood staining to staining of peripheral blood mononuclear cells (PBMC) obtained after ficoll density centrifugation ( Figure 4).
  • PBMC peripheral blood mononuclear cells
  • any device that requires “counting” of individual cells needs to discriminate between an actual cell, and debris.
  • debris should be filtered prior to reaching the laser to minimize potential noise, e.g. autofluorescence from dead cell debris.
  • noise e.g. autofluorescence from dead cell debris.
  • Fig. 9 traditional gating strategies are used to illustrate this point.
  • the total number of "events" recorded by the cytometer in the figure below is 85,026. Of those events, 12,431 events are larger than the lower line (approximately 4 microns, but this is not a precise value).
  • the upper threshold (approximately 10 microns) is used to exclude the larger monocytes and polymorphonuclear leukocytes (PMNs).
  • the size exclusion criteria remove 90% of PMNs and 70% of monocytes from the final analysis. This leaves approximately 4,873 events in the proper size range to include all the lymphocytes. In the proposed device, only particles of this size range will reach the lasers.
  • Fig. 9b shows all events in the "4-10 micron" size range, and their expression of the CD3+ and CD4+ cell markers. These two colors easily discriminate the T cells from the monocytes (minimal CD3 staining, and CD4 dim) and PMNs (CD3 and CD4 negative).
  • the miniature flow cytometer provides s a "single platform" method of counting T cells, a standard need to be designed for comparison.
  • Fig. 8 it demonstrates the high concordance in the % of CD4 T cells derived from whole blood staining and PBMC isolated over ficoll, where the absolute CD4+ T cell number in whole blood samples is evaluated.
  • the current standard for analysis is a "dual platform method”.
  • Whole blood is run on a Coulter counter for evaluation of total lymphocytes (part of a CBC panel). The percentage of CD4+ T cells is evaluated by flow, and these two numbers are multiplied to give the total CD4+ T cell number.
  • the present invention provides a new standard that uses a "single platform" method. This is achieved by running a known number of standard beads in the sample. With this method, about 50 microliters of blood are added to a standardized tube with a known number of beads (49, 944 in this case).
  • the absolute CD4 count is determined by the following formula: (number of CD3+CD4+ events / number of beads counted) x (number of beads per tube / sample volume). Referring to Fig. 10, a relatively healthy HIV(+) individual is shown. The CD4+ T cell count was 449/mm 3 (normal 400-1600). The actual value from the reference laboratory was 446/mm 3 .
  • the present invention discloses an electrokinetic microfluidic flow cytometer lab-on-a-chip device that realizes multiple functions and/or processes for flow cytometry.
  • the device utilizes a miniature laser-optical fiber based multiple wavelength detection system to count and differentiate particles in a liquid medium of interest.
  • the electrokinetic microfluidic LOC device of the present invention can find many applications in a wide spectrum of fields including, but not limited to, counting CD4 cells, proteomics and DNA analysis, drug development, chemical development, and so on.
  • Sinton D, Ren L, Li D A dynamic loading method for controlling on-chip microfluidic sample injection. J Colloid Interface Sci. 2003;266:448-56. [9]. Sinton D, Ren L, Li D. Visualization and numerical modelling of microfluidic on- chip injection processes. J Colloid Interface Sci. 2003 ;260:431-39.
  • Xiang Q Hu G, Gao Y, Li D. Miniaturized immunoassay microfluidic system with electrokinetic control. Biosens Bioelectron. 2005. [18].
  • Xuan X Raghibizadeh S, Li D. Wall effects on electrophoretic motion of spherical polystyrene particles in a rectangular poly(dimethylsiloxane) microchannel. J Colloid Interface Sci. 2005. [19].
  • Xuan X Ye C 5 Li D. Near-wall electrophoretic motion of spherical particles in cylindrical capillaries. J Colloid Interface Sci. 2005;289:286-90.

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Abstract

Dans un mode de réalisation, le cytomètre de l'invention concerne: une microstructure à canal (352) conçue pour transporter un milieu liquide contenant un ou plusieurs types de particules (380a, 380b); des moyens de génération électrocinétique de flux microfluidique pour le transport du milieu liquide dans la structure à microcanal (352) de manière à différentier un ou plusieurs types de particules (380a, 380b) présentes dans ce milieu liquide; et un système de détection optique (378a, 378b, 378c, 378d) couplé à la microstructure en vue de la détection d'un ou plusieurs types différentiés de particules dans le milieu liquide.
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WO2010099118A1 (fr) * 2009-02-27 2010-09-02 Beckman Coulter, Inc. Système optique stabilisé pour cytométrie de flux
EP2406007A4 (fr) * 2009-03-10 2013-03-06 Univ Monash Agrégation plaquettaire utilisant un dispositif microfluidique
JP2015512615A (ja) * 2012-02-04 2015-04-30 センター フォー セルラー アンドモレキュラー プラットフォームズ（シー−シーエーエムピー）Ｃｅｎｔｒｅ Ｆｏｒ Ｃｅｌｌｕｌａｒ Ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｐｌａｔｆｏｒｍｓ（Ｃ−Ｃａｍｐ） マイクロ流体ベースのフローアナライザ
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WO2016095902A1 (fr) * 2014-12-18 2016-06-23 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Dispositif d'exposition
EP3234549A4 (fr) * 2015-01-30 2018-08-08 Hewlett-Packard Development Company, L.P. Puce de diagnostic
JP2018508766A (ja) * 2015-01-30 2018-03-29 ヒューレット−パッカード デベロップメント カンパニー エル．ピー．Ｈｅｗｌｅｔｔ‐Ｐａｃｋａｒｄ Ｄｅｖｅｌｏｐｍｅｎｔ Ｃｏｍｐａｎｙ， Ｌ．Ｐ． 診断チップ
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JP2020076736A (ja) * 2018-09-10 2020-05-21 ソニー株式会社 微小粒子分取装置、細胞治療薬製造装置、微小粒子分取方法、及びプログラム
JP7338336B2 (ja) 2018-09-10 2023-09-05 ソニーグループ株式会社 微小粒子分取装置、細胞治療薬製造装置、微小粒子分取方法、及びプログラム
JP2023144136A (ja) * 2018-09-10 2023-10-06 ソニーグループ株式会社 微小粒子分取装置、細胞治療薬製造装置、微小粒子分取方法、及びプログラム
JP7589775B2 (ja) 2018-09-10 2024-11-26 ソニーグループ株式会社 微小粒子分取装置、細胞治療薬製造装置、微小粒子分取方法、及びプログラム
EP3994449A4 (fr) * 2019-07-03 2023-08-16 Centre for Cellular and Molecular Platforms Analyseur microfluidique
CN115248320A (zh) * 2021-04-26 2022-10-28 深圳市帝迈生物技术有限公司 Poct血细胞分析仪及其使用方法
WO2024068291A1 (fr) * 2022-09-28 2024-04-04 Leibniz-Institut Für Festkörper- Und Werkstoffforschung Dresden E. V. Composant microfluidique

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