Microfluidic assay for rapid optimization of cell electroporation

Abstract

An electroporation device with a volume of varying cross-sectional area that be used as a fast assay device for determining the optimal conditions for plasma membrane electroporation.

What is claimed is: 
     
         1 . An apparatus for cell electroporation, the apparatus comprising:
 a structure, encompassing a volume defining a cross-sectional area, configured to contain cells having a plasma membrane and background media, the background media including an exogenous agent capable of translocating across a plasma membrane in an electroporated state; and   an arrangement of electrodes arranged relative to the structure to produce levels of an electromagnetic field, at least one of the levels sufficient to electroporate at least a subset of the plasma membranes in at least a portion of the volume, the exogenous agent responsively translocating across at least some of the subset of the plasma membranes in the electroporated state into cells to enable detection of cells having the exogenous agent therein, within the volume, the structure enabling observation of a transition of cells without the exogenous agent therein to cells with the exogenous agent therein to enable correlating the transition to a threshold level of the electromagnetic field that causes cell electroporation.   
     
     
         2 . The apparatus of  claim 1 , further comprising an electrical power source coupled to the arrangement of electrodes, the power source configured to stimulate the electrodes to produce the electromagnetic field, the exogenous agent responsively translocating across at least a subset of the plasma membranes in the electroporated state into cells. 
     
     
         3 . The apparatus of  claim 1 , wherein the arrangement of electrodes includes at least one pair of electrodes positioned at opposing ends of the volume. 
     
     
         4 . The apparatus of  claim 1 , wherein the arrangement of electrodes includes at least one pair of electrodes positioned axially along a length of the volume. 
     
     
         5 . The apparatus of  claim 1 , wherein the arrangement of electrodes includes at least one pair of electrodes positioned in a coaxial configuration along a length of the volume. 
     
     
         6 . The apparatus of  claim 1 , wherein the arrangement of electrodes includes at least one pair of electrodes that are isolated from the volume via a solid, fluid, or gas barrier. 
     
     
         7 . The apparatus of  claim 1 , wherein the volume contains a static solution of the plasma membranes and the background media. 
     
     
         8 . The apparatus of  claim 1 , wherein the volume is a flow path. 
     
     
         9 . The apparatus of  claim 8 , wherein the flow path is a channel. 
     
     
         10 . The apparatus of  claim 8 , wherein the volume contains a static solution of the cells and the flow path delivers a background buffer having the exogenous agent therein. 
     
     
         11 . The apparatus of  claim 10 , wherein the static solution of the cells and the background buffer is achieved by porous media located within the volume. 
     
     
         12 . The apparatus of  claim 10 , wherein the static solution of the cells is achieved by an adhesive coating located within the volume. 
     
     
         13 . The apparatus of  claim 1 , wherein the volume is in fluid association with an inlet port and an outlet port that permit a solution of the cells having a plasma membrane and the background media to flow into and out of the volume, respectively. 
     
     
         14 . The apparatus of  claim 1 , wherein the cross-sectional area varies across a dimension of the volume. 
     
     
         15 . The apparatus of  claim 1 , wherein the cross-sectional area is tapered. 
     
     
         16 . The apparatus of  claim 1 , wherein the cross-sectional area is tapered bidirectionally toward a region within the volume. 
     
     
         17 . The apparatus of  claim 1 , wherein the volume defines a cross-sectional area that decreases between an inlet reservoir to an outlet reservoir, the inlet reservoir and the outlet reservoir located on opposing ends of the volume. 
     
     
         18 . The apparatus of  claim 1 , wherein the volume defines a cross-sectional area that constricts with a curved geometry. 
     
     
         19 . The apparatus of  claim 18 , wherein the curved geometry results in a linear electromagnetic field gradient within the volume. 
     
     
         20 . The apparatus of  claim 1 , wherein the volume defines a cross-sectional area that decreases between an inlet reservoir and a length along the volume and increases between the length along the volume and an outlet reservoir, the inlet reservoir and the outlet reservoir located on opposing ends of the volume. 
     
     
         21 . The apparatus of  claim 1 , wherein the cross-sectional area geometry is selected from the group consisting of circular, triangular, square, rectangular, ellipsoidal, trapezoidal, pentagonal, hexagonal, octagonal, and star-shaped. 
     
     
         22 . The apparatus of  claim 1 , wherein at least two arrangements of cross-sectional areas are arranged in series, in parallel, or a combination thereof. 
     
     
         23 . The apparatus of  claim 1 , wherein the cells having a plasma membrane are eukaryotic cells. 
     
     
         24 . The apparatus of  claim 1 , wherein the cells having a plasma membrane are prokaryotic cells. 
     
     
         25 . The apparatus of  claim 23 , wherein the eukaryotic cells are selected from the group consisting of mammalian cells, protozoa, plant cells, yeast cells, and fungi. 
     
     
         26 . The apparatus of  claim 24 , wherein the prokaryotic cells are bacterial cells or archaeal cells. 
     
     
         27 . The apparatus of  claim 1 , wherein the electromagnetic field has a distribution within the volume that results in one or more pulses. 
     
     
         28 . The apparatus of  claim 1 , wherein the electromagnetic field has a distribution within the volume that varies spatially within the volume. 
     
     
         29 . The apparatus of  claim 1 , wherein the electromagnetic field has a distribution within the volume that includes a mathematically-shaped function. 
     
     
         30 . The apparatus of  claim 1 , wherein the exogenous agent includes a dye. 
     
     
         31 . The apparatus of  claim 1 , wherein correlating the transition to a threshold level of the electromagnetic field is a function of location of the transition within the structure. 
     
     
         32 . The apparatus of  claim 1 , wherein correlating the transition to a threshold level of the electromagnetic field is a function of timing of the transition within the structure. 
     
     
         33 . The apparatus of  claim 1 , wherein the exogenous agent includes a reporting probe that can be detected after contact with intracellular substances. 
     
     
         34 . The apparatus of  claim 1 , wherein the electromagnetic field has a pulse duration selected from the group consisting of about 10 ns to about 0.25 ms, about 0.25 ms to about 5.0 ms, about 0.01 ms to about 0.50 ms, about 0.1 ms to about 2 ms, about 1 ms to about 10 ms, about 3 ms to about 50 ms, and about 0.25 ms to about 50 ms. 
     
     
         35 . The apparatus of  claim 1 , wherein the electromagnetic field has a magnitude of at least about 0.5 kV/cm. 
     
     
         36 . The apparatus of  claim 1 , wherein the electromagnetic field has a magnitude selected from the group consisting of from about 0.25 kV/cm to about 10 kV/cm, from about 1 kV/cm to about 20 kV/cm, from about 5 kV/cm to about 50 kV/cm, from about 7.5 kV/cm to about 15 kV/cm, from about 0.25 kV/cm to about 50 kV/cm; or from about 50 kV/cm to about 250 kv/cm. 
     
     
         37 . A method for performing cell electroporation, the method comprising:
 determining a threshold level of an electromagnetic field that causes electroporation of at least a subset of plasma membranes of cells within a portion of a volume of a structure, the volume of the structure defining a cross-sectional area with a capacity to accept an electromagnetic field having an electromagnetic field strength, the volume including background media having an exogenous agent capable of translocating across a plasma membrane in an electroporated state induced at least in part as a function of the electromagnetic field strength, the determining including observing, within the volume, a transition of cells without the exogenous agent therein to cells with the exogenous agent therein, a parameter associated with the transition correlating to the threshold level.   
     
     
         38 . The method of  claim 37 , further comprising applying the threshold level of the electrical field to the volume in a manner enabling electroporation of the plasma membranes and enabling the exogenous agent to translocate across the plasma membranes in the electroporated state into cells. 
     
     
         39 . The method of  claim 37 , further comprising using an electrical power source to drive at least one arrangement of electrodes arranged relative to the volume to produce the electromagnetic field, the exogenous agent responsively translocating across at least some of the subset of plasma membranes in the electroporated state. 
     
     
         40 . An apparatus for performing cell electroporation, comprising:
 means for encompassing a volume, the volume defining a cross-sectional area with a capacity to accept an electromagnetic field having an electromagnetic field strength, the volume including cells having a plasma membrane and background media having an exogenous agent capable of translocating across a plasma membrane in an electroporated state induced at least in part as a function of the electromagnetic field strength;   means for applying the electromagnetic field to the volume to cause electroporation of at least a subset of the plasma membranes in at least a portion of the volume; and   means for enabling observation in the volume of a transition of cells without the exogenous agent therein to cells with the exogenous agent therein, the transition correlating to a threshold level of the electromagnetic field that causes cell electroporation.   
     
     
         41 . The apparatus of  claim 1 , wherein the arrangement of electrodes includes at least one electrode positioned at an inlet port of the volume and at least another electrode positioned at an outlet port of the volume.