Integrated and standalone label and reagent-free microfluidic devices and microsystems for differential white blood cell counts

Abstract

A method of establishing a differential white blood cell count includes directing at least one stream of deionized water into a microfluidic device containing a sample of whole blood or a cell-rich fraction to generate a lysate stream of intact white blood cells; directing at least one stream of deionized water into the lysate stream to form a virtual non-conductive aperture in a channel of the device; and performing impedance cytometry of the lysate stream via coplanar electrodes to detect the presence of intact white blood cells. A microfluidic device includes a blood separation section. An analyte sensor detects electrical changes in a cell-free fraction. Lysate from a cell-rich fraction is analyzed to detect circulating tumor cells or white blood cells including neutrophils, lymphocytes, monocytes, eosinophils, and basophils. A method of fabricating and a standalone cell-rich microfluidic device are disclosed for differential white blood cell counts.

What is claimed is: 
     
         1 . A method of establishing a differential white blood cell count comprising:
 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.   
     
     
         2 . The method according to  claim 1 , further comprising
 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.   
     
     
         3 . The method according to  claim 1 , wherein the step of directing at least ONE stream of deionized water into the channel includes 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. 
     
     
         4 . A method of fabricating a microfluidic device comprising:
 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,   wherein 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,   wherein 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 and   wherein 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.   
     
     
         5 . The method of fabricating according to  claim 4 , wherein the step of adhering a plurality of pairs of co-planar electrodes on the substrate includes applying a chrome adhesive between the plurality of pairs of co-planar electrodes and the substrate. 
     
     
         6 . A microfluidic device comprising:
 a layer of material formed over a substrate;   a blood separation section 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 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 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.   
     
     
         7 . The microfluidic device according to  claim 6 , wherein
 the cell or large particle analyzer section is 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.   
     
     
         8 . A microfluidic device for establishing a differential white blood cell count comprising:
 a substrate:   a layer of material formed over the substrate: and   a plurality of microchannels formed in the layer of material,   at least one of the plurality of microchannels 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,   wherein 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,   wherein 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 and   wherein 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.   
     
     
         9 . The microfluidic device according to  claim 8 , wherein with respect to the at least one of the plurality of microchannels that 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,
 the plurality of microchannels comprises 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.