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WO2015073952A1 - Dispositifs et microsystèmes microfluidiques intégrés et autonomes, exempts de marqueur et de réactif, pour leucocytémie différentielle - Google Patents

Dispositifs et microsystèmes microfluidiques intégrés et autonomes, exempts de marqueur et de réactif, pour leucocytémie différentielle Download PDF

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Publication number
WO2015073952A1
WO2015073952A1 PCT/US2014/065913 US2014065913W WO2015073952A1 WO 2015073952 A1 WO2015073952 A1 WO 2015073952A1 US 2014065913 W US2014065913 W US 2014065913W WO 2015073952 A1 WO2015073952 A1 WO 2015073952A1
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WIPO (PCT)
Prior art keywords
cell
stream
lysate
white blood
deionized water
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PCT/US2014/065913
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English (en)
Inventor
Thomas E. WINKLER
Hadar Shumel BEN-YOAV
Reza Ghodssi
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University of Maryland Baltimore
University of Maryland College Park
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University of Maryland Baltimore
University of Maryland College Park
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Priority to US15/034,719 priority Critical patent/US20160274020A1/en
Publication of WO2015073952A1 publication Critical patent/WO2015073952A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • 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
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • G01N15/131—Details
    • G01N15/132—Circuits
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/011—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells with lysing, e.g. of erythrocytes
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/016—White blood cells
    • 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
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • G01N15/131—Details
    • G01N2015/133—Flow forming
    • 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
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • G01N2015/135—Electrodes

Definitions

  • the present disclosure relates to the field of detection of substances present in biological fluids. More particularly, the present disclosure relates to devices, systems and methods for detection of analytes and substances in biological fluids such as blood.
  • Plasma acellular
  • a simple cell count is useful, e.g. to diagnose anemia, but the utility of such measurements increases significantly with the ability to also determine size, surface markers, and interior composition.
  • Applications range from CD4 T-cell monitoring in cases of HIV to stem cell characterization in research.
  • the current gold standard for such measurements is bulky benchtop flow cytometers. These rely on a focused stream of blood cells being subjected to multiple analysis methods, involving fluorescent labels for population-specific surface antigens (e.g.
  • Flow cytometers allow for highly accurate analysis, but rely on labels and complex optics. These factors are some of the major barriers in bringing this technology to the point of care (POC), where it would benefit patients as well as physicians by providing immediate results and increasing accessibility, especially in remote locations [2].
  • LOC Lab-on-a-chip
  • this signal can be used to differentiate particles based on their size.
  • This is useful for blood cell differentials, as there are significant differences in geometry - discoid red blood cells with 6-8 m diameters, compared to spherical white blood cells with diameters ranging from 6-20 m for the various sub-populations. Red blood cells, outnumbering white blood cells approximately 1000:1 , all but prohibit an accurate count of different leukocyte sub-types in whole blood. More elaborate implementations of the impedance method can give additional information about the cells: while direct current (DC) or low frequency alternating current (AC) impedance is sensitive to size, higher frequency AC probes the internal structure of the cell.
  • DC direct current
  • AC alternating current
  • Leukocyte sub-populations can be distinguished by combining these modes, especially when the much more numerous erythrocytes are lysed by addition of chemical reagents to reduce interference.
  • an integrated LOC system based on impedance cytometry has been shown to be capable of quantifying the different types of blood cells, including neutrophils [12,13].
  • a notable limitation is the reliance on chemicals to achieve erythrocyte lysis and sufficient cell type differentiation (saponin and formic acid, followed by sodium carbonate after a set exposure time).
  • Multi-frequency impedance cytometry has been presented as an attractive method for multi-dimensional single-cell analysis in LOC systems [4], [5].
  • current implementations still suffer from limited resolution, and employ multi-layer fabrication processes.
  • flow focusing has been utilized to enhance the performance of coulter counter-type devices, to date no systematic study has been conducted on the interplay between flow ratios, particle sizing sensitivity, and throughput [6]. It is only through such studies, both in models and experiments, that optimal utilization of microsystem capabilities becomes possible.
  • the embodiments of the present disclosure provide a novel and non-obvious solution to the problems of mental health treatment as described above by providing a point of care testing (POCT) device that includes a whole blood inlet port in fluidic communication with microchannels extending therefrom.
  • POCT point of care testing
  • the embodiments of the present disclosure provide a point of care testing (POCT) device that limits the amount of required chemicals, as additional reagents complicate LOC packaging.
  • POCT point of care testing
  • the embodiments of the present disclosure provide a point of care testing (POCT) device that eliminates the need for multi-layer fabrication processes that represent a practical drawback in terms of scale-up.
  • POCT point of care testing
  • the embodiments of the present disclosure provide reagent- and label-free assay (only water) in conjunction with impedance cytometry.
  • one embodiment of the present disclosure relates to a method of establishing a differential white blood cell count that includes directing at least one stream of deionized water into a microfluidic device containing a sample of whole blood of a subject or a cell-rich fraction of a whole blood sample or a cell-free fraction of whole blood of a subject or combinations thereof to generate a lysate stream of intact white blood cells; directing at least one stream of deionized water into the lysate stream such that the lysate stream with intact white blood cells is forced to flow in a direction of motion by the at least one stream of deionized water to form a virtual non-conductive aperture in a channel of the microfluidic device; and performing impedance cytometry of the lysate stream in the virtual non-conductive aperture via coplanar electrodes to detect the presence of intact white blood cells in the lysate stream.
  • the method may further include quantitatively differentiating between neutrophils, lymphocytes, monocytes, eosinophils, and basophils in the lysate stream based on the impedance measurements resulting from the performance of the impedance cytometry.
  • the step of directing at least one stream of deionized water into the channel may include symmetrically focusing at least two streams of deionized water orthogonally on opposing sides of the direction of motion of the lysate stream to form the virtual non-conductive aperture.
  • Yet another embodiment of the present disclosure relates to a method of fabricating a microfluidic device that include forming a layer of material on a substrate and adhering a plurality of pairs of co-planar electrodes on the substrate; and forming a plurality of microchannels in the layer of material. At least one of the microchannels is configured and disposed to receive at least one stream of deionized water to effect lysis of a whole blood sample or of a cell-rich fraction of a whole blood sample to produce a lysate stream. At least one of the microchannels is configured and disposed to receive the lysate stream and to receive at least one focusing flow of deionized water to effect a virtual aperture.
  • At least one the pairs of co-planar electrodes is formed under one of the plurality of microchannels in which is generated the virtual aperture such that impedance cytometry of the lysate stream in the virtual aperture is enabled by application of an electric field to at least two pairs of the plurality of pairs of co-planar electrodes.
  • the step of adhering a plurality of pairs of co-planar electrodes on the substrate may include applying a chrome adhesive between the plurality of pairs of co-planar electrodes and the substrate.
  • Still another embodiment of the present disclosure relates to a microfluidic device that includes a layer of material formed over a substrate.
  • a blood separation section is configured and disposed in the layer of material to receive a sample of whole blood of a subject and to separate the whole blood sample into a cell-free fraction and into a cell-rich fraction.
  • An analyte sensor section is configured and disposed in the layer of material to detect an analyte in the cell-free fraction via application of an electrical field and detection of changes in at least one electrical property in the analyte.
  • a cell pre-treatment section is configured and disposed in the layer of material to form a lysate from the cell-rich fraction; and a cell or large particle analyzer section configured and disposed on the layer of material to enable analysis of the lysate from the cell-rich fraction to detect circulating tumor cells or white blood cells including neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
  • the cell or large particle analyzer section may be configured and disposed on the layer of material to enable analysis of the lysate from the cell-rich fraction to enable a differential white blood cell count via coplanar electrodes formed over the substrate that are configured and disposed to enable impedance cytometry of the white blood cells in the cell or large particle analyzer section.
  • a further embodiment of the present disclosure relates to a microfluidic device for establishing a differential white blood cell count that includes a substrate.
  • a layer of material is formed over the substrate and a plurality of microchannels is formed in the layer of material.
  • At least one of the plurality of microchannels is configured and disposed to receive a sample of whole blood of a subject or a cell-rich fraction of a whole blood or combinations thereof.
  • At least one of the plurality of microchannels is configured and disposed to receive at least one stream of deionized water to effect lysis of a whole blood sample or of a cell-rich fraction of a whole blood sample to produce a lysate stream.
  • At least one of the plurality of microchannels is configured and disposed to receive the lysate stream and to receive at least one focusing flow of deionized water to effect a virtual aperture.
  • At least one the pairs of co-planar electrodes is fornned under one of the plurality of microchannels in which is generated the virtual aperture such that impedance cytometry of the lysate stream in the virtual aperture is enabled by application of an electric field to at least two pairs of the plurality of pairs of co-planar electrodes.
  • the plurality of microchannels may include at least two deionized water injection channels and a lysate stream channel such that the at least two deionized water injection channels are configured and disposed to symmetrically focus at least two streams of deionized water orthogonally on opposing sides of a direction of motion of the lysate stream in the lysate stream channel.
  • FIG. 1 illustrates a flow chart of an integrated cell-free and cell-rich fraction microfluidic device and testing interface for implementing a method of testing a cell-free fraction and a cell-rich fraction of a whole blood sample of a patient or subject;
  • FIG. 2A is a perspective view of a microfluidic device that is functionally independent of the integrated microfluidic device of FIG. 1 but is functionally equivalent to the cell-free fraction analysis section of the integrated microfluidic device of FIG. 1 ;
  • FIG. 2B is a plan view of the microfluidic device of FIG. 2A;
  • FIG. 2C is a cross-section view of the microfluidic device of FIG. 2A taken along section line 2C-2C;
  • FIG. 3 illustrates a microfluidic device that is also physically independent of the integrated microfluidic device of FIG. 1 but which is functionally equivalent to the cell-rich fraction analysis section of the integrated microfluidic device of FIG. 1 ;
  • FIG. 4 illustrates an alternate embodiment of the microfluidic device of FIG. 3
  • FIG. 5 illustrates one embodiment of a portion of the whole blood analysis section of the microfluidic devices illustrated in FIG. 3 and FIG. 4;
  • FIG. 6 illustrates a 3-dimensional electrodynamic model that simulates a particle of the lysate stream of FIG. 5 in a section of microfluidic channel which defines first and second vertical channel walls wherein the particle is suspended in the gap between two coplanar electrodes;
  • FIG. 7 is a plot of virtual aperture width and fluid admittance plotted against flow ratio sample to focus
  • FIG. 8 is a plot of the absolute value of the change in impedance ⁇ at 200 kHz as a percentage with respect to the empty channel impedance plotted against virtual aperture width;
  • FIG. 9 is a finite element model simulation of the absolute value of the change in impedance in percent for a cell with given properties represented by a radius, membrane capacitance and cytosol conductivity, and single-parameter variations thereof illustrating three distinct frequency regimes where an increase or decrease significantly alters the signal;
  • FIG. 10 is a plot of impedance at 200 kHz in ohms corresponding to particles passing between the electrodes as a function of time in seconds;
  • FIG. 1 1 is a plot of the average impedance at 200 kHz in percent for separate bead populations as a function of flow ratio sample to focus;
  • FIG. 12 illustrates a perspective view of one embodiment of the integrated microfluidic device described schematically with respect to FIG. 1
  • FIG. 12A is a cross-sectional view of the microfluidic device of FIG. 12 taken along section line 12A-12A;
  • FIG. 12B is a cross-sectional view of the microfluidic device of FIG. 12 taken along section line 12B-12B.
  • a microfluidic device relying solely on impedance measurements to establish a differential white blood cell_count as disclosed herein introduces a number of improvements over previous designs.
  • the design according to embodiments of the present disclosure employs coplanar electrodes, simplifying device assembly as compared to parallel electrodes not least by reducing the number of physical layers from three to two.
  • the flow channels are defined in polydimethylsiloxane (PDMS) fabricated by established molding techniques. This straightforward approach again eliminates complexity over the use of photolithographically patterned polyimide and micromilled polymethyl methylacrylate (PMMA).
  • PDMS polydimethylsiloxane
  • PMMA polymethyl methylacrylate
  • the loss in performance by utilizing coplanar compared to parallel electrodes is about 20%, notably decreasing for increasing cell size [15].
  • the channel height are comparable to the white blood cell diameters at below 20 ⁇ , thus also reducing the impact of vertical cell position in the flow on the measured signal [15].
  • Lateral constraint is provided not by the channel itself, but rather by sheath flow focusing. This phenomenon relies on laminar flow and introduction of fluid streams to either side of the sample stream to force central alignment of cells [17].
  • a microsystem that relies on impedance measurements to establish a differential white blood cell count, introducing a number of improvements over previous designs.
  • the microsystem enables a method for separating whole blood into a cellular component for neutrophil counting and an undiluted acellular component for analyte detection.
  • the overall design, incorporating a main sample flow, pure water lysis flows, focusing flows, and impedance cytometry, is schematically illustrated in FIG. Land is described in more detail below.
  • Gold coplanar electrodes may be photolithographically patterned on glass, via a chrome adhesive therebetween, and SU-8 photoresist may be used to create a negative master structure on silicon.
  • Positive PDMS microfluidics can thus be molded and cured, and subsequently bonded to the glass reversibly by simple application of pressure, or permanently by prior application of oxygen plasma.
  • the impedance measurements rely on four sequential sets of coplanar parallel electrode pairs - one for direct current (DC) measurements, one for high-frequency alternating current (AC) measurements, the other two as respective references.
  • the references serve to account for the impedance from the acellular component at both DC and AC frequencies, assuming a cell density resulting in spacing between individual cells larger than the electrode gap.
  • the different measurements can be correlated for each cell.
  • Sample flow is provided by pressure actuation from external syringe pumps, connected through capillary tubing.
  • the channels are pre-treated with bovine serum albumin (BSA) protein to reduce sticking of blood cells to the highly hydrophobic PDMS.
  • BSA bovine serum albumin
  • Embodiments of the microsystem of the present disclosure enable a decrease in fabrication complexity and in reliance on chemicals through a coplanar electrode design and reliance on pure water to lyse erythrocytes, respectively.
  • Embodiments of the microsystem of the present disclosure incorporate flow focusing of the white blood cell enhanced fraction via hydrodynamic effects of pure water to create a "virtual aperture" to achieve increased, tunable cell characterization performance and throughput.
  • FIG. 1 there is illustrated a flow chart of an integrated cell-free and cell-rich fraction microfluidic device and testing interface 100 for implementing a method of testing a cell-free fraction and a cell-rich fraction of a whole blood sample of a patient or subject. More particularly, the integrated device and testing interface 100 includes a patient or subject 102.
  • cell-free fraction refers to a blood sample from which at least 99% of cellular components such as erythrocytes and leukocytes have been removed from a whole blood sample leaving a plasma of less than 1 % cellular composition. .
  • cell-rich fraction refers to a whole blood sample from which 20% or less of plasma volume has been removed, leaving a sample containing 99% of cellular components such as erythrocytes and leukocytes, potentially concentrated with respect to typical whole blood.
  • white blood cells also referred to as leukocytes, include neutrophils, lymphocytes, monocytes, eosinophils, and basophils, each of which may exist independently in a whole blood sample or cell-rich fraction.
  • the method includes extracting or receiving a whole blood sample 104 from the patient or subject 102 and directing the whole blood sample 104 to a blood separation section 1002 of integrated cell-free fraction analysis and cell-rich fraction analysis microfluidic device 1000.
  • the whole blood sample 104 may be directed to the intake of the blood separation section 1002 via generally one micropump 105 that may be externally positioned with respect to the microfluidic device 1000, as shown schematically in FIG. 1 , or embedded within the microfluidic device 1000 (not shown).
  • the method includes, via the whole blood sample separation section 1002, separating the whole blood sample 104 into a cell-free fraction 1 102 and into a cell-rich fraction 1202.
  • the method includes directing the cell-free fraction 1 102 to a cell-free fraction analysis section 1 100 of microfluidic device 1000 and directing the cell-rich fraction 1202 to a cell-rich fraction analysis section 1200 of the microfluidic device 1000.
  • the method of testing includes sensing in the cell-free fraction analysis section 1 100 an analyte or biomarker 1 125 such as, e.g, a drug or pharmaceutical, metabolites, vitamins, viruses, bacteria, hormones, enzymes, inflammatory mediators, chemokines, immunoglobulin isoiypes, intracellular signaling molecules, apop oiic mediators, adhesion molecules, and antibodies etc.
  • the method of testing also may include directing the cell-rich fraction 1202 to a cell pre-treatment sub- section 1210 of cell-rich analysis section 1200 wherein pre-treatment may include lysis of the cell-rich fraction 1202, directing the lysate with intact white blood cells 1220 to a cell or large particle analyzer sub-section 1230 and directing impedance cytometry results 1240 as all or part of point-of-care information 106 provided by the microfluidic device 1000.
  • the cell or large particle analyzer sub-section 1230 may detect white blood cells (or leukocytes) which include various sub-types such as neutrophils, lymphocytes, monocytes, eosinophils, and basophils, or circulating tumor cells or both white blood cells and circulating tumor cells.
  • white blood cells or leukocytes
  • the cell or large particle analyzer sub-section 1230 excludes detection of red blood cells since such cells do not survive the lysis process that occurs in cell pre-treatment subsection 1210.
  • the integrated device and testing interface 100 may further include directing the part of point-of-care information 106 to a treatment team of medical professionals or researchers 108 who may direct an adjustment in action plan 1 10 for the patient or subject 102.
  • FIGS. 2A, 2B and 2C illustrate in more detail the cell-free fraction analysis section 1 100 of microfluidic device 1000 which includes analyte or biomarker sensor 1 1 10 for detecting an analyte or biomarker in cell-free fraction 1 102.
  • the analyte or biomarker sensor 1 1 10 includes a counter electrode 1 130a, a working electrode 1 130b and a reference electrode 1 130c wherein the analyte or biomarker. 1 125 is sensed or detected on the working electrode 1 130b by impedance cytometry that involves imposition of an alternating current to the counter electrode 1 130a and working electrode 1 130b in the presence of reference or ground electrode 1 130c.
  • Microfluidic device 1 101 illustrated in FIGS. 2A, 2B and 2C is functionally independent of integrated cell-free fraction analysis and cell-rich fraction analysis microfluidic device 1000 illustrated in FIG. 1 but is functionally equivalent to the cell-free fraction analysis section 1 100 of microfluidic device 1000 that includes analyte or biomarker sensor 1 1 10 for detecting an analyte or biomarker in cell-free fraction 1 102, as described above with respect to FIG. 1 , with the exception that the cell-rich fraction 1202 that is skimmed from whole blood sample 104 is rejected at whole blood sample rejection outlet 1202' while cell-free fraction 1 102 is analyzed via the analyte or biomarker sensor 1 1 10.
  • the microfluidic device 1 101 includes a plasma skimming module 1 1 14, illustrated in the example shown in FIGS. 2A-2C, as a plurality of parallel channels according to one embodiment of the present disclosure, from which the cell-free fraction 1 102 is directed as separated plasma 1 1 16 through separated plasma channels 1 1 16' that intersect, in the example shown in FIGS. 2A-2C orthogonally, the plasma skimming channels 1 1 14.
  • the separated plasma 1 1 16 is directed to flow through the separated plasma channels 1 1 16' toward analyte detection microfluidic channel or recess or chamber 1 1 18 (see also FIG.
  • the plasma skimming module 1 1 14 is configured and disposed to separate plasma 1 1 16 from the whole blood sample 104 prior to entry of the whole blood sample 104 into the detection chamber 1 1 18.
  • the 3-electrode electrochemical detector 1 130 includes linear strip electrode 1 130a having an arcuately shaped counter electrode tip 1 130a', linear strip electrode 1 130b having an arcuately shaped reference electrode tip 1 130b' and linear strip electrode 1 130c having a circularly shaped working electrode tip 1 130c' that is disposed in recess 1 1 18 so that the counter electrode tip 1 130a' and the reference electrode tip 1 130c' are concentrically arranged around the working electrode tip 1 130b'.
  • the working electrode tip 1 130b' may be modified with a redox cycling system (not shown) to amplify the electrochemical signal of the analyte or biomarker 1 125 that is present in the whole blood sample 104.
  • the linear strip electrodes 1 130a, 1 130b and 1 130c form connections to external electronics, such as a potentiostat (not shown) for signal detection.
  • the separated plasma 1 1 16 is then drawn out through the separated plasma sample outlet port 1 124 such as by application of a vacuum connection, not shown, at whole blood sample rejection outlet 1202' and at separated plasma sample outlet port 1 124 or other means known in the art, such as by application of positive pressure via the micorpump 105 (see FIG. 1 ) at whole blood sample inlet port 1 1 12 as described above.
  • the cell-rich fraction analysis section 1200 of integrated microfluidic device 100 relies on impedance measurements to establish a differential white blood cell count. While parallel electrodes as utilized in the prior art offer advantages in terms of accuracy, they are a significant factor in fabrication complexity. By employing coplanar electrodes, device assembly is simplified at least by reducing the number of physical layers required from three to two. Flow channels are defined in polydimethylsiloxane (PDMS) fabricated by established molding techniques, thereby reducing complexity over the use of photolithographically patterned polyimide and micromilled polymethyl methylacrylate (PMMA). Thus, the microfluidic device 1000 for differential white blood cell counting enables low-cost, scalable fabrication.
  • PDMS polydimethylsiloxane
  • the loss in performance by utilizing coplanar compared to parallel electrodes is about 20%, notably decreasing for increasing cell size [15].
  • channels with height matched to the size of the particle or cell of interest , and thus smaller equivalent aperture D A are employed [13].
  • the channel height is comparable to the white blood cell diameters at below 20 ⁇ , thus also reducing the impact of vertical cell position in the flow on the measured signal [15].
  • Lateral constraint is provided not by the channel itself, but rather by sheath flow focusing using pure water. This phenomenon relies on laminar flow and, via at least one micropump (not shown) that is generally external to the microfluidic device 1000.
  • the micropump is employed to introduce lysis flow of deionized water to create a lysate stream and to cause flow focusing by introducing fluid streams of deionized water to either side of the sample stream to force central alignment of cells [16,17].
  • the micropump 105 is employed to introduce the whole blood sample 104 into the blood separation section 1002.
  • the introduction of the lysis flow and of the focusing flows may be accomplished by either a single external pump or separate dedicated pumps, one for the lysis flow and one for the focusing flow or flows.
  • microfluidic device 1000 to enhance differential white blood cell detection performance in an impedance-based lab on a chip (LOC) device represents a novel means for differential white blood cell detection.
  • LOC impedance-based lab on a chip
  • FIG. 3 illustrates a microfluidic device 1201 that is also physically independent of integrated cell-free fraction analysis and cell-rich fraction analysis microfluidic device 1000, and is thus a standalone device with respect to microfluidic device 1000, but which is functionally equivalent to the cell-rich fraction analysis section 1200 of microfluidic device 1000. Therefore, microfluidic device 1201 includes cell pre-treatment sub-section 1210 and cellular analysis sub-section 1230 for establishing a differential white blood cell count in the lysate with intact white blood cells 1220, as described above with respect to FIG.
  • the whole blood sample 104 is now pumped, via one or more micropumps such as the generally one micropump 105 that may be externally positioned with respect to the microfluidic device 1201 , as described above schematically in FIG. 1 , or embedded within the microfluidic device 1201 (not shown), directly into the inlet of whole blood receiving channel 104'.
  • one or more micropumps such as the generally one micropump 105 that may be externally positioned with respect to the microfluidic device 1201 , as described above schematically in FIG. 1 , or embedded within the microfluidic device 1201 (not shown), directly into the inlet of whole blood receiving channel 104'.
  • a microfluidic device may receive a whole blood sample and separate the whole blood sample into a cell-free fraction and into a cell-rich fraction and subject both the cell-free fraction and the cell-rich fraction to electrically-based analysis techniques.
  • a microfluidic device may receive a whole blood sample and separate the whole blood sample into a cell-free fraction and into a cell-rich fraction and subject the cell-rich fraction to a means for causing lysis on the cell-rich fraction to form a lysate stream with intact white blood cells.
  • a microfluidic device may receive a whole blood sample and subject the whole blood sample to a means for causing lysis on the whole blood sample to form a lysate stream with intact white blood cells without having first separated the whole blood cells into a cell-free fraction and into a cell-rich fraction.
  • a microfluidic device may receive a whole blood sample and separate the whole blood sample into a cell-free fraction and into a cell-rich fraction and subject only the cell-free fraction to an electrically-based analysis technique or subject only the cell-rich fraction to an electrically-based analysis technique.
  • the method of testing also may include directing the cell-rich fraction 1202, or, in the embodiment of the microfluidic device 1201 of FIG.
  • pre-treatment may include lysis of the whole blood sample 104, directing the resulting lysate with intact white blood cells 1220 to a lysate analysis or impedance cytometry sub-section 1230 and directing impedance cytometry results 1240 as all or part of point-of-care information 106 provided by the microfluidic device 1000.
  • microfluidic device 1201 in FIG. 3 illustrates in more detail the cell-rich fraction analysis section 1200 of microfluidic device 1000 as formed in cell-rich fraction analysis section microfluidic Iayer1201 ' (for illustration purposes) as a whole blood sample analysis section 12001 .
  • Microfluidic device 1201 includes sample flow channel 1040' that is configured and disposed to receive a whole blood sample 1040 that is directed into the sample flow channel 1040' and wherein a first lysis flow channel 1204a' receives a first lysis flow 1204a and a second lysis flow channel 1204b' receives a second lysis flow 1204b' such that the first lysis flow channel 1204a' and the second lysis flow channel 1204b' intersect on opposing sides the sample flow channel 1040' in a quasi-tee or converging forked configuration 1205 to enable mixing of the sample flow 1040 with the first lysis flow 1204a and with the second lysis flow 1204b.
  • the first lysis flow 1204a and the second lysis flow 1204b may be of de-ionized water.
  • the sample mixture 1206 is directed into pre- treatment section 1210 that includes a series of channel loops 1208a...1208n, that are sufficient in number and length to provide sufficient exposure duration time of the sample cells in the sample mixture 1206 to the de-ionized water to cause lysis of the erythrocyte cells such that the sample mixture 1206 emerges from the channel loops 1208a...1208n as a lysate stream 1212 with intact white blood cells which are directed into a lysate channel 1212'.
  • the lysate stream 1212 is directed into lysate flow channel 1212' wherein a first focusing flow channel 1214a' receives one or more focusing flows, e.g., a first focusing flow 1214a and a second focusing flow channel 1214b' receives a second focusing flow 1214b' such that, in a similar manner as with respect to the lysis flow described above, the first focusing flow channel 1214a' and the second focusing flow channel 1214b' intersect on opposing sides the lysate flow channel 1212' in a quasi-tee or forked configuration 1208 to enable the lysate stream 1212 to be directed into a lysate stream channel 1220' that is configured and disposed in the microfluidic layer 1201 ' such that a lysate stream 1212" with intact white blood cells is directed to flow in a direction of motion, as indicated by arrow A, in the lysate stream channel 1220'.
  • a first focusing flow channel 1214a' receives
  • At least two deionized water injection channels 1214a' and 1214b' are configured and disposed in the microfluidic device 1000 such that at least two streams of deionized water 1214a and 1214b are directed into the lysate stream channel 1220' to force the lysate stream 1212" to flow in the direction of motion A between two streams of deionized water 1214a" and 1214b", respectively, to form a virtual non- conductive aperture 1222 in the lysate stream channel 1220'.
  • the one or more deionized water injection channels 1214a' and 1214b' are configured and disposed to symmetrically focus the two or more streams of deionized water 1214a and 1214b orthogonally on opposing sides of the direction of motion A of the lysate stream 1212" in the lysate stream channel 1220'.
  • the microfluidic device 1000 further includes an impedance cytometry section 1230 wherein at least two co-planar electrodes, e.g., electrodes 1230a1 , 1230a2 or 1230b1 , 1230b2 or 1230c1 , 1230c2 or 1230d1 , 1230d2, are configured and disposed on a surface 1203 of the microfluidic layer 1201 ' such that the white blood cells/leukocytes in the lysate stream channel 1220' are exposed to an alternating current at at least one frequency emitted from the at least two co-planar electrodes 1230a1 , 1230a2 or 1230b1 , 1230b2 or 1230c1 , 1230c2 or 1230d1 , 1230d2.
  • at least two co-planar electrodes e.g., electrodes 1230a1 , 1230a2 or 1230b1 , 1230b2 or 1230c1 , 1230c2 or 1230d1 , 12
  • the co-planar electrodes are configured in sequential sets of co-planar parallel electrode pairs 1230a1 , 1230a2 followed by 1230b1 , 1230b2 followed by 1230c1 , 1230c2 followed by 1230d1 , 1230d2 that are each positioned orthogonally on the surface 1201 such that the lysate stream channel 1220' crosses over in an orthogonal manner each of the sequential sets of co-planar parallel electrode pairs.
  • the impedance measurements rely on sequential sets of parallel electrode pairs - one for low-frequency measurements, e.g., 1230a1 and 1230a2, and one for high-frequency measurements, e.g., 1230b1 and 1230b2.
  • Two additional pairs of electrodes, e.g., 1230c1 ,1230c2 and 1230d1 , 1230d2 are included as optional references at the respective frequencies to allow for differential measurements, assuming a cell density resulting in spacing between individual cells larger than the electrode gap (see FIGS. 5 and 6 as described below).
  • Sample flow is provided by pressure actuation from external syringe pumps, e.g., one or more micropumps 105 as shown in FIG. 1 , connected through capillary tubing.
  • External electronics for signal recording connected to a microprocessor such as a personal computer (PC) running LabVIEW (National Instruments, Inc., Austin, Texas, USA), may be employed for data acquisition.
  • the external electronics may include impedance recording equipment such as an impedance analyzer or LCR meter (e.g., IET/QuadTech 1910/1920 1 MHz LCR Meter, IET Labs, Inc., Roslyn Heights, New York, USA). .
  • co-planar parallel electrode pair 1230a1 , 1230a2 and co-planar parallel electrode pair 1230b1 , 1230b2 may each be operated at, for example, 100 kilohertz (kHz) and (absolute values of) impedance measurements Z in ohms ( ⁇ ) or in percent change in (absolute values of) impedance ⁇ may be taken. These measurements may be repeated by co-planar parallel electrode pair 1230c1 , 1230c2 and co-planar parallel electrode pair 1230d1 , 1230d2 when the intact white blood cell traverses into the respective gap between each pair of electrodes.
  • co-planar parallel electrode pair 1230a1 , 1230a2 may be operated at, for example, 100 kilohertz (kHz) and co-planar parallel electrode pair 1230b1 , 1230b2 may be operated at, for example, 500 kilohertz (kHz) and (absolute values of) impedance measurements Z in ohms ( ⁇ ) or in percent change in (absolute values of) impedance ⁇ may be taken.
  • co-planar parallel electrode pair 1230c1 , 1230c2 operating at 100 kHz and co-planar parallel electrode pair 1230d1 , 1230d2 operating at 500 kHz when the intact white blood cell traverses into the respective gap between each pair of electrodes.
  • the method includes quantitatively differentiating between neutrophils, lymphocytes, monocytes, eosinophils, and basophils in the lysate stream 1212" based on impedance measurements resulting from the performance of the impedance cytometry as described above.
  • the lysate stream 1212" is directed to a waste flow outlet 1224.
  • FIG. 4 illustrates an alternate embodiment of microfluidic device 1201 described above with respect to FIG. 3, and is thus another example of a standalone device with respect to microfluidic device 1000 in FIG. 1 .
  • Microfluidic device 1251 illustrates in more detail the cell-rich fraction analysis section 1200 of microfluidic device 1000 as formed in microfluidic layer 1251 ' (for illustration purposes) as a whole blood sample analysis section 12002. More particularly, whole blood sample analysis section 12002 is identical to whole blood sample analysis section 12001 in FIG.
  • whole blood sample analysis section 12002 includes a common water lysis flow inlet 1204 for the first lysis flow channel 1204a' that receives first lysis flow 1204a and for the second lysis flow channel 1204b' that receives second lysis flow 1204b'.
  • whole blood sample analysis section 12002 includes a common water focus flow inlet 1214 for the first focusing flow channel 1214a' that receives first focusing flow 1214a and for the second focusing flow channel 1214b' that receives second focusing flow 1214b'.
  • whole blood sample analysis section 12002 formed in microfluidic layer 1251 ' further includes the sequential sets of co-planar parallel electrode pairs 1230a1 , 1230a2 followed by 1230b1 , 1230b2 followed by 1230c1 , 1230c2 followed by 1230d1 , 1230d2 that are respectively connected to a power supply and impedance recording equipment (not shown), such as an impedance analyzer or LCR meter, (e.g., IET/QuadTech 1910/1920 1 MHz LCR Meter, IET Labs, Inc., Roslyn Heights, New York, USA) via connections and pads 1230a10and 1230a20 for electrodes 1230a1 and 1230a2, respectively, connections and pads 1230b10 and 1230b20 for electrodes 1230b1 and 1230b2, respectively, connections and pads 1230c10 and 1230c20 for electrodes 1230c1 and 1230c2, respectively, and connections and pads 1230d10 and 1230d20 for electrodes 1230d
  • the electrodes 1230a1 , 1230a2, 1230b1 , 1230b2, 1230c1 , 1230c2, 1230d1 , 1230d2 are positioned under lysate stream 1220 in the same sequential manner as displayed in FIG. 3.
  • the connection pads 1230a1 , 1230b1 , 1230c1 and 1230d1 are disposed on the left side of lysate stream 1212" with respect to the downstream direction of flow while connection pads 1230a2, 1230b2, 1230c2 and 1230d2 are disposed on the right side of lysate stream 1212" with respect to the downstream direction of flow.
  • the impedance measurements rely on sequential sets of parallel electrode pairs - one for low-frequency and one for high-frequency measurements. Two additional pairs of electrodes are included as optional references at the respective frequencies to allow for differential measurements, assuming a cell density resulting in spacing between individual cells larger than the electrode gap. At a known flow rate, the sequential measurements can be correlated for each cell. Sample flow is provided by pressure actuation from external syringe pumps, connected through capillary tubing. External electronics are utilized for signal recording, connected to a PC running LabVIEW for data acquisition.
  • the lysate stream 1212" with intact white blood cells is directed to flow in the direction of motion, as indicated by arrow A, in the lysate stream channel 1220'.
  • At least two streams of deionized water 1214a and 1214b are directed into the lysate stream channel 1220' such that the lysate stream 1212"is forced to flow in the direction of motion A between two streams of deionized water 1214a" and 1214b", respectively, to form the virtual non- conductive aperture 1222 in the lysate stream channel 1220'.
  • microfluidic devices 1201 in FIG. 3 and 1251 in FIG. 4 is presented as representative examples of the lysis sub-section 1210 to form lysate stream 1212 and of the lysate analysis section 1230 that enables analysis of the compressed lysate stream 1212" from the cell-rich fraction 1202
  • the integrated microfluidic device 1000 need not be limited to detection of white blood cells but may also be applied to cytometry of other cells such as the known various sub-types of white blood cells and circulating tumor cells and except for red blood cells since the lysis is intended to remove such cells from the lysate stream 1212,
  • Other methods cell detection for the integrated device 1000 may include visual or optical detection via observation of the lysate stream 1212 under a microscope.
  • Gold coplanar electrodes were photolithographically patterned on a glass or silicon oxide substrate as one example, and SU-8 photoresist was used to create a negative master structure on silicon. Positive PDMS microfluidics can thus be molded and cured, and subsequently bonded to the glass reversibly by simple application of pressure, or permanently by prior application of oxygen plasma and thus fabricated by standard microfabrication approaches. Gold could conceivably also be another chemically inert conductor. As described above, the impedance measurements rely on the sequential sets of parallel electrode pairs - one for low-frequency and one for high-frequency measurements.
  • Two additional pairs of electrodes are included as optional references at the respective frequencies to allow for differential measurements, assuming a cell density resulting in spacing between individual cells larger than the electrode gap.
  • the sequential measurements can be correlated for each cell.
  • Sample flow is provided by pressure actuation from external syringe pumps, connected through capillary tubing.
  • external electronics for signal recording such as a potentiostat, impedance analyzer, or LCR meter as described above are connected to a PC running LabVIEW for data acquisition.
  • a mold was created using SU-8 2015 negative photoresist patterned on silicon using contact photolithography. Using this master, channels were cast from poly(dimethylsiloxane) (PDMS). After thermal curing at 60 °C, the PDMS was diced and 2 mm diameter fluidic connections were punched.
  • PDMS poly(dimethylsiloxane)
  • microfluidic devices 1201 and 1251 illustrated in FIGS. 3 and 4, respectively, and as further described below with respect to microfluidic device 1000 in FIGS. 12, 12A and 12B comprise two physical layers.
  • Four pairs of microelectrodes 1230a1 , for impedance measurements 1230a1 , 1230a2 followed by 1230b1 , 1230b2 followed by 1230c1 , 1230c2 followed by 1230d1 , 1230d2 each have a width dimension We with gap G between the two electrodes in each pair and are disposed on a lower or first layer of glass or silicon oxide layer via a chrome adhesive therebetween.
  • the width We is about 25 ⁇ and the gap G is also about 25 ⁇ .
  • the microfluidic channels are formed in an upper or second layer of PDMS wherein the microfluidic channels are formed with a cross-section of 75x20 ⁇ 2 (width ⁇ height).
  • the lysate stream channel 1220' has a channel width W of about 75 ⁇ and a height H of about 20 ⁇ .
  • the upper or second layer of PDMS is formed on both the electrodes and the lower or first layer substrate of glass or silicon oxide.
  • the PDMS is plasma-bonded to the glass and also seals and adheres to the gold coplanar electrodes. [0038] FIG.
  • FIG. 5 illustrates one embodiment of a portion of the whole blood analysis section 1200 of microfluidic device 1201 as illustrated in FIG. 3 and microfluidic device 1251 as illustrated in FIG. 4.
  • Water focusing flows 1214a and 1214b are directed into the lysate stream 1212 in lysate channel 1212'.
  • Lysate channel 1212' intersects between first focusing flow channel 1214a' and second focusing flow channel 1214b' in a crossed intersection 1205 such that when the two or more streams of deionized water 1214a and 1214b are directed into the lysate stream channel 1220', a compressed lysate stream 1212" is forced to flow in the direction of motion A between two streams of deionized water 1214a" and 1214b", respectively, to form virtual non-conductive aperture 1222 in the lysate stream channel 1220'.
  • the lysate stream channel 1220' is positioned over sequential sets of co-planar parallel electrode pairs 1230a1 , 1230a2 followed by 1230b1 , 1230b2 such that a detection region 1231 for white blood cells is formed by the gap G between the set of co-planar parallel electrode pairs 1230a1 , 1230a2 and by the gap G between the set of co-planar parallel electrode pairs 1230b1 , 1230b2. Detection of white blood cells occurs by electric fields from the sequential sets of co-planar parallel electrode pairs 1230a1 , 1230a2 and 1230b1 , 1230b2 propagating through the virtual aperture 1222 in the gaps G.
  • the presence of the sequential sets of co-planar parallel electrode pairs 1230a1 , 1230a2 followed by 1230b1 , 1230b2 followed by 1230c1 , 1230c2 followed by 1230d1 , 1230d2 enables performing impedance cytometry of the white blood cells/leukocytes in the lysate stream 1212" in the lysate stream channel 1220' at the one or more frequencies.
  • co-planar parallel electrode pair 1230a1 , 1230a2 and co-planar parallel electrode pair 1230b1 , 1230b2 may each be operated at, for example, 100 kilohertz (kHz) and (absolute values of) impedance measurements Z in ohms ( ⁇ ) or in percent change in (absolute values of) impedance ⁇ may be taken. These measurements may be repeated by co-planar parallel electrode pair 1230c1 , 1230c2 and co-planar parallel electrode pair 1230d1 , 1230d2 when the intact white blood cell 12120 traverses into the respective gap G between each of those pairs of electrodes.
  • kHz kilohertz
  • co-planar parallel electrode pair 1230a1 , 1230a2 may be operated at, for example, 100 kilohertz (kHz) and co-planar parallel electrode pair 1230b1 , 1230b2 may be operated at, for example, 500 kilohertz (kHz) and (absolute values of) impedance measurements Z in ohms ( ⁇ ) or in percent change in (absolute values of) impedance ⁇ may be taken.
  • co-planar parallel electrode pair 1230c1 , 1230c2 operating at 100 kHz and co-planar parallel electrode pair 1230d1 , 1230d2 operating at 500 kHz when the intact white blood cell traverses into the respective gap between each pair of electrodes. It is assumed that each electrode pair operates at one specific frequency, but as the intact white blood cell 12120 travels through lysate stream channel 1220' the cell will experience the particular operating frequency of each pair of electrodes..
  • the measurements at the coplanar electrode pair 1230b1 , 1230b2 and at coplanar electrode pair 1230d1 , 1230d2 are considered to be "empty channel" readings since the microfluidic devices 1201 and 1251 should be designed such that statistically it is anticipated that while an intact white blood cell 12120 traverses into the gap G between electrode pair 1230a1 , 1230a2, or between electrode pair 1230c1 , 1230c2, no particle is anticipated to be present in the gap G between electrode pair 1230b1 , 1230b2 or electrode pair 1230d1 , 1230d2, respectively while the impedance measurements are being recorded.
  • FEM finite element modeling
  • the 2D (two-dimensional) hydrodynamic model considered a slow-diffusing species (particles) and a fast-diffusing species (ions) introduced through a center sample channel, focused symmetrically by deionized water (DI-H2O) flows.
  • FIG. 5 A representative simulation is shown in FIG. 5.
  • the main model outputs of interest are the cross-sectional concentration profiles downstream from the flow focusing inlets - the respective full width at half maximum (FWHM) for the ions can be considered equivalent to the VA width.
  • FWHM full width at half maximum
  • the distance X between the convergent tee 1208 and the upstream edge 1230a1 ' of the first electrode 1230a1 is also designed to a minimum value so that the impedance measurements occur at a position before significant divergence of the virtual aperture VA occurs downstream of the final electrode 1230d2.
  • the spacing between the coplanar electrodes 1230a1 , 1230a2 followed by 1230b1 , 1230b2 followed by 1230c1 , 1230c2 followed by 1230d1 , 1230d2 generally does not significantly affect the impedance measurements as long as the electrodes are located before any significant divergence of the virtual aperture VA occurs.
  • FIG. 6 illustrates a perspective sample model geometry which represents the detection portion 1231 identified in FIG. 5.
  • the model output is the change in impedance
  • the 3D electrodynamic model simulates particle 12120 of the lysate stream 1212" having radius r, conductivity a, and permittivity ⁇ , suspended in a section of microfluidic channel, i.e., lysate stream channel 1220' which defines first vertical channel wall 1226a and second vertical channel wall 1226b, in the gap G between two coplanar electrodes, e.g., electrodes 1230a1 and 1230a2.
  • the deionized water focus flows 1214a and 1214b define respectively deionized water focus flow width W H 2oa between channel wall 1226a and the compressed lysate stream 1212" and deionized water focus flow width W H 2ob between channel wall 1226b on the opposite side of the compressed lysate stream 1212" and of the lysate stream channel 1220'.
  • the model output is the change in impedance ⁇ AZ ⁇ measured across electrodes between particle and no-particle conditions.
  • the virtual aperture VA represents the cross-sectional area defined by the width W and height H of the lysate stream 1212", excluding the widths W H 20a and W H 20b of the deionized water focus flows 1214a and 1214b in the channel 1220'.
  • the microfluidic devices 1000, 1201 and 1251 are described and illustrated in FIGS. 1 -6 and later in FIGS. 12, 12A, 12B below as configured to receive two deionized water lysis flows 1204a and 1204b, only one lysis flow such as 1204a or 1204b is required, or the microfluidic devices 1000, 1201 and 1251 may be configured to receive additional lysis flow or flows (not shown).
  • microfluidic devices 1000, 1201 and 1251 are described and illustrated in FIGS. 1 -6 and later in FIGS. 12, 12A, 12B below as configured to receive two deionized water focus flows 1214a and 1214b, only one focus flow such as 1214a or 1214b is required, in which case the virtual aperture VA occurs directly between second vertical channel wall 1226b and focus flow 1214a or between first vertical channel wall 1226a and focus flow 1214b.
  • microfluidic devices 1000, 1201 and 1251 may be configured to receive additional focus flow or flows (not shown).
  • the fabricated microfluidic devices were connected to syringes using Tygon tubing (Cole-Parmer; Vernon Hills, Illinois, USA). Constant flow was provided through syringe pumps (KDS230 (KD Scientific, Inc.; Holliston, Massachusetts, USA), Genie Plus (Kent Scientific Corporation; Torrington, Connecticut, USA), NE-300 (New Era Pump Systems, Inc.; Farmingdale, New York, USA)). Admittance measurements for model verification were done using a VSP-300 potentiostat (Bio-Logic; Claix, France).
  • Impedance cytometry data was recorded via LabView utilizing an E4980A Precision LCR Meter (Agilent; Santa Clara, California, USA).
  • the background signal was determined through MATLAB (MathWorks, Inc.; Natick, Massachusetts, USA) robust local regression smoothing of the raw data, the signal peaks using a peak finding algorithm. Population averages were calculated using histogram peak fits in OriginPro (OriginLab Corporation; Northampton, Massachusetts, USA).
  • the LOCs Prior to use, the LOCs were rinsed with Fetal Bovine Serum (FBS; Life Technologies; Carlsbad, California, USA) to reduce PDMS hydrophobicity.
  • FBS Fetal Bovine Serum
  • PBS phosphate-buffered saline
  • sucrose Sigma-Aldrich; St. Louis, Missouri, USA
  • impedance cytometry measurements are generally recorded via application of alternating current (AC)
  • AC alternating current
  • DC direct current
  • the flow rates are the main external parameters to control the VA width, the critical parameter for impedance cytometry performance.
  • hydrodynamic FEM was utilized to determine the ionic FWHM for a range of flow ratios (FR) of phosphate-buffered saline (PBS)-based sample to DI-H2O focus.
  • FR flow ratios
  • PBS phosphate-buffered saline
  • FIG. 7 the left vertical axis is virtual aperture width VA (or Wp) in microns ( ⁇ ) plotted against horizontal axis of flow ratio sample to focus FR (1 :x) where the FEM results are indicated in circles and the experimental results are indicated by crosses.
  • VA virtual aperture width
  • ⁇ microns
  • the virtual aperture width VA decreases drastically over 80 microns at zero FR to approximately 3-4 microns as flow focusing is introduced, with the effect saturating at high flow ratios.
  • the behavior is independent of flow rate, at least in the laminar flow regime.
  • the vertical axis is the absolute value of the change in impedance ⁇ at 200 kHz as a percentage with respect to the empty channel impedance Z plotted against the horizontal axis of virtual aperture width in microns (Mm).
  • the cell radius R is 50 ⁇ and the results are plotted for a 25 ⁇ channel width (crosses) and for a 50 ⁇ channel width (boxes)
  • a frequency of f 200 kHz was found to be most sensitive to resistive properties, and thus r, and this was used throughout this work.
  • the plot shows data for channel widths of 25 ⁇ and 50 ⁇ , revealing the signal is independent of the actual channel width (memory constraints prevented simulations for 75 ⁇ width). Therefore, at the chosen f, the VA is expected to function in a manner identical to a physical constriction.
  • 1 .8% to
  • 18%.
  • Model predictions Electrodynamic finite element modeling (FEM) effectively illustrates the expected utility of flow focusing in impedance cytometry.
  • the relative ⁇ at 200 kHz induced by an R 5 ⁇ cell, plotted in FIG. 9, is increased up to 10-fold through flow focusing, and independent of the actual channel width.
  • FIG. 9 is a plot of FEM simulation of
  • the MATLAB-processed data shows distinct peaks in impedance ⁇ AZ
  • FIG. 1 1 is a plot of the average impedance ⁇ AZ ⁇ at 200 kHz in percent (%) for separate bead populations as a function of flow ratio sample to focus FR.
  • the graph indicates up to 276% enhanced size- based differentiation, from ⁇
  • 0.55% to ⁇
  • 1.52%. Underlying this are overall increases in
  • FIG. 12 illustrates a perspective view of one embodiment of the integrated microfluidic device 1000 that has been described schematically with respect to FIG. 1 above. More particularly, integrated microfluidic device 1000 includes whole blood sample inlet port 104' wherein the whole blood sample 104 from the patient or subject 102 is directed to blood separation section 1002 of microfluidic device 1000.
  • the integrated microfluidic device 1000 includes an upper layer or microfluidic layer of material 1232 that incorporates cell-rich fraction analysis section microfluidic layer 1201 ' described above with respect to FIG. 3.
  • Whole blood sample separation section 1002 is configured and disposed in the microfluidic layer of material 1232 to receive the sample 104 of whole blood of a subject via the whole blood sample inlet port 104' and to separate the whole blood sample 104 into cell- free fraction 1 102 and into cell-rich fraction 1202.
  • analyte sensor sub-section 1 1 10 is configured and disposed in the microfluidic layer of material 1232 to detect an analyte 1 125 in the cell-free fraction 1 100.
  • the analyte sensor sub-section 1 1 10 includes counter electrode 1 130a, a working electrode 1 130b and a reference electrode 1 130c wherein the analyte or biomarker 1 125 is sensed or detected on the working electrode 1 130b by impedance cytometry that involves imposition of an alternating current to the counter electrode 1 130a and working electrode 1 130b in the presence of reference or ground electrode 1 130c. As described above with respect to FIGS.
  • the analyte or biomarker 1 125 may include, e.g, a drug or pharmaceutical, metabolites, vitamins, viruses, bacteria, hormones, enzymes, inflammatory mediators, chemokines, immunoglobulin isotypes, intracellular signaling molecules, apopiotic mediators, adhesion molecules, and antibodies etc.
  • the microfluidic device 1000 includes, as previously described above with respect to FIGS. 1 and 3-6, the lysis sub-section 1210 that is configured and disposed on the microfluidic layer of material 1232 to form lysate stream 1212 from the cell-rich fraction 1202, and also includes the lysate analysis section 1230 that is configured and disposed on the substrate 1010 to enable analysis of the compressed lysate stream 1212" from the cell-rich fraction 1202.
  • FIGS. 12A and 12B are cross-sectional views of the microfluidic device 1000 taken along section line 12A-12A and section line 12B-12B, respectively, wherein the upper layer or microfluidic layer of material 1232 may be fabricated from PDMS or other suitable materials such as moldable plastic. All of the previously described features of the integrated microfluidic device 1000 of FIGS. 1 and 12, or of the microfluidic device 1 101 of FIGS. 2A, 2B, 2C, or of the microfluidic device 1201 of FIG. 3 or microfluidic device 1251 of FIG. 4 and as further described in FIGS. 5 and 6 may be fabricated as described above for microfluidic device 1000.
  • the co-planar electrodes 1 130a, 1 130b, 1 130c and the co-planar electrodes 1230a1 , 1230a2, 1230b1 , 1230b2, 1230c1 , 1230c2, 1230d1 , 1230d2 are disposed on a glass substrate 1236 via a chrome adhesive 1234 applied between the lower surfaces of the co-planar electrodes and the upper surface 1236' of the glass substrate 1236.
  • the microfluidic device 1000 and correspondingly microfluidic devices 1 101 , 1201 and 1251 , is thus a composite 1010 of the glass substrate 1236 and the microfluidic layer 1232 including the coplanar electrodes 1 130 for microfluidic device 1 101 or coplanar electrodes 1230a1 , 1230a2, 1230b1 , 1230b2, 1230c1 , 1230c2, 1230d1 , 1230d2 and connection pads 1230a10, 1230a20, 1230M 0, 1230b20, 1230c10, 1230c20, 1230d10, 1230d20 for microfluidic devices 1201 and 1251 , as appropriate, and the chrome adhesive layer 1234.
  • microfluidic devices 1000, 1201 or 1251 are based upon calibration of the particular device to known cell types that have been verified to be present in the lysate stream 1212 via standard laboratory techniques.

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Abstract

La présente invention concerne un procédé permettant d'établir une leucocytémie différentielle, le procédé comprenant l'envoi d'au moins un courant d'eau désionisée dans un dispositif microfluidique contenant un échantillon de sang entier ou une fraction riche en cellules pour générer un courant de lysat de leucocytes ; l'envoi d'au moins un courant d'eau désionisée dans le courant de lysat pour former une ouverture virtuelle non conductrice dans un canal du dispositif ; et la mise en œuvre d'une cytométrie par impédance du courant de lysat au moyen d'électrodes coplanaires pour détecter la présence de leucocytes intacts. Le dispositif microfluidique comprend une section séparation de sang. Un capteur d'analyte détecte les changements électriques dans une fraction ne contenant pas de cellules. Le lysat provenant d'une fraction riche en cellules est analysé pour détecter des cellules cancéreuses en circulation ou des leucocytes, tels que des neutrophiles, des lymphocytes, des monocytes, des éosinophiles et des basophiles. Un procédé de fabrication et un dispositif microfluidique autonome riche en cellules permettant d'effectuer une leucocytémie différentielle sont décrits.
PCT/US2014/065913 2013-05-09 2014-11-17 Dispositifs et microsystèmes microfluidiques intégrés et autonomes, exempts de marqueur et de réactif, pour leucocytémie différentielle Ceased WO2015073952A1 (fr)

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CN112698024A (zh) * 2020-12-08 2021-04-23 华中农业大学 一种基于差分阻抗颗粒计数的免疫分析方法
CN112730560A (zh) * 2020-12-10 2021-04-30 东南大学 微流控阻抗细胞仪及其制备方法

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