Controlled electroporation and mass transfer across cell membranes

Abstract

Electroporation is performed in a controlled manner in either individual or multiple biological cells or biological tissue by monitoring the electrical impedance, defined herein as the ratio of current to voltage in the electroporation cell. The impedance detects the onset of electroporation in the biological cell(s), and this information is used to control the intensity and duration of the voltage to assure that electroporation has occurred without destroying the cell(s). This is applicable to electroporation in general. In addition, a particular method and apparatus are disclosed in which electroporation and/or mass transfer across a cell membrane are accomplished by securing a cell across an opening in a barrier between two chambers such that the cell closes the opening. The barrier is either electrically insulating, impermeable to the solute, or both, depending on whether pore formation, diffusive transport of the solute across the membrane, or both are sought. Electroporation is achieved by applying a voltage between the two chambers, and diffusive transport is achieved either by a difference in solute concentration between the liquids surrounding the cell and the cell interior or by a differential in concentration between the two chambers themselves. Electric current and diffusive transport are restricted to a flow path that passes through the opening.

What is claimed is:  
     
         1 . A method for performing electroporation in a biological cell in a controlled manner, comprising: 
 (a) placing said biological cell in an electrically conductive medium and applying a voltage across said medium;    (b) continuously detecting the ratio of electric current through the medium to voltage across said medium as an indication of the degree of electroporation of said biological cell; and    (c) adjusting the magnitude of said applied voltage in accordance with changes in the magnitude of said current-to-voltage ratio to achieve a controlled degree of electroporation.    
     
     
         2 . A method in accordance with  claim 1  in which step (b) comprises continuously detecting said current-to-voltage ratio as an indication of the onset of electroporation of said biological cell, and step (c) comprises adjusting the duration of said applied voltage in accordance with said current-to-voltage ratio to achieve a controlled degree of electroporation.  
     
     
         3 . A method in accordance with  claim 1  comprising placing a plurality of said biological cells in said electrically conductive medium, said current-to-voltage ratio thereby serving as an indication of the degree of electroporation averaged over said plurality of biological cells, thereby achieving a controlled averaged degree of electroporation in said plurality of biological cells.  
     
     
         4 . A method in accordance with  claim 1  comprising placing a single biological cell in said electrically conductive medium, said current-to-voltage ratio thereby serving as an indication of the degree of electroporation of said single biological cell, thereby achieving a controlled degree of electroporation in said single biological cell.  
     
     
         5 . A method in accordance with  claim 1  in which said voltage is applied between two microelectrodes positioned with said biological cell in between.  
     
     
         6 . A method in accordance with  claim 1  in which: 
 said voltage is applied between two electrodes in a flow-through channel, said electrodes positioned to apply said voltage in a direction transverse to flow through said channel; 
 step (a) comprises suspending said biological cell in said medium and continuously passing said medium through said channel;  
 step (b) comprises further correlating said current-to-voltage ratio with the presence of said biological cell between said electrodes; and  
 step (c) comprises adjusting the magnitude of said voltage while said biological cell is between said electrodes.  
 
 
     
     
         7 . A method in accordance with  claim 6  comprising suspending a plurality of said biological cells in said electrically conductive medium and continuously passing said medium through said channel such that approximately one cell at a time passes through said electrodes.  
     
     
         8 . A method for performing electroporation in biological tissue in a controlled manner, comprising: 
 (a) placing said biological tissue in an electrically conductive medium and applying a voltage across said medium;    (b) continuously detecting the ratio of electric current through the medium to voltage across said medium as an indication of the degree of electroporation in said biological tissue; and    (c) adjusting the magnitude of said applied voltage in accordance with changes in the magnitude of said current-to-voltage ratio to achieve a controlled degree of electroporation in said biological tissue.    
     
     
         9 . A method for the infusion of a biological cell with a chemical substance by electroporation in a manner that permits detection of the onset of and control of the electroporation, said method comprising: 
 (a) securing said biological cell in an electrical cell containing a liquid with said chemical substance dissolved therein, said electrical cell containing a barrier to electric current, said barrier arranged such that, when a voltage is imposed across said electrical cell, said barrier restricts electric current flow to a flowpath passing through said biological cell while permitting substantially no electric current to bypass said biological cell;    (b) imposing a voltage across said electrical cell and monitoring the relative values of current passing through said cell and of said imposed voltage as an indication of the occurrence of electroporation in said cell.    
     
     
         10 . A method in accordance with  claim 9  in which said barrier divides first and second electrode chambers in said electrical cell and contains an opening smaller in width than said biological cell, and (a) comprises securing said biological cell over an opening such that said cell closes said opening.  
     
     
         11 . A method in accordance with  claim 10  in which said first electrode chamber contains a first electrically conducting liquid and said second electrode chamber contains a second electrically conducting liquid, and said chemical substance is dissolved in only one of said first and second electrically conducting liquids.  
     
     
         12 . A method in accordance with  claim 10  in which said first electrode chamber contains a first electrically conducting liquid and said second electrode chamber contains a second electrically conducting liquid, and said chemical substance is dissolved in both of said first and second electrically conducting liquids.  
     
     
         13 . A method in accordance with  claim 10  in which (a) is accomplished by imposing a pressure differential across said opening to press said biological cell against one side of said opening.  
     
     
         14 . A method in accordance with  claim 10  in which (a) is accomplished by a coating on an area surrounding said opening, said coating comprised of a substance that binds to said barrier.  
     
     
         15 . A method in accordance with  claim 10  in which said first electrode chamber is constructed and arranged to permit continuous flow of liquid therethrough, said method further comprising effecting continuous flow of a first electrically conducting liquid through said first electrode chamber.  
     
     
         16 . A method in accordance with  claim 10  in which said first and second electrode chambers are constructed and arranged to permit continuous flow of liquid through each such chamber independently, said method further comprising effecting continuous flow of a first electrically conducting liquid through said first electrode chamber and continuous flow of a second electrically conducting liquid through said second electrode chamber.  
     
     
         17 . A method in accordance with  claim 9  comprising securing a plurality of said biological cells in said electrical cell in which said barrier restricts current flow to flowpaths passing through all of said plurality of biological cells while permitting substantially no electric current to bypass said plurality of biological cells.  
     
     
         18 . A method in accordance with  claim 9  comprising securing only one said biological cell in said electrical cell, and in which said barrier restricts current flow to a flowpath through said only one biological cell while permitting substantially no electric current to bypass said only one biological cell.  
     
     
         19 . A method in accordance with  claim 9  in which said electric cell is transparent, and said method further comprises observing changes in said biological cell while said voltage is imposed.  
     
     
         20 . A method for the passage of a chemical species across a membrane of a biological cell in a controlled manner to achieve a selected rate of mass transfer across said membrane or to facilitate the determination of mass transfer properties of said membrane, said method comprising: 
 (a) securing said cell over an opening in a barrier between first and second chambers, said opening being smaller in width than said cell such that said cell closes said opening, and    (b) placing a first liquid in said first chamber and a second liquid in said second chamber, at least one of said first and second liquids containing a solute at a concentration sufficiently higher than in said cell to cause said solute to diffuse into said cell.    
     
     
         21 . A method in accordance with  claim 20  in which securing said cell over said opening is accomplished by imposing a pressure differential across said opening to press said cell against one side of said opening.  
     
     
         22 . A method in accordance with  claim 20  in which securing said cell over said opening is accomplished by a coating on an area surrounding said opening, said coating comprised of a substance that binds to said membrane.  
     
     
         23 . A method in accordance with  claim 20  in which said first and second chambers are constructed and arranged to permit continuous flow of liquid through each such chamber independently, and (b) is accomplished by continuous flow of said first liquid through said first chamber and continuous flow of said second liquid through said second chamber.  
     
     
         24 . A method in accordance with  claim 20  in which the concentration of said solute in said first liquid is sufficiently higher than in said second liquid to cause said solute to travel from said first liquid to said second liquid by diffusive transport, said barrier being sufficiently impermeable to said solute and said cell being sufficiently secured in said opening to restrict said diffusive transport to a diffusion path through said cell.  
     
     
         25 . A method in accordance with  claim 24  in which said solute is selected such that its mass transfer coefficient across said membrane is known, and said method is a method for infusing said cell with said solute and further comprises selecting the concentration of said solute in said first liquid and the period of time during which said diffusive transport is continued, both on the basis of said known mass transfer coefficient, to infuse said cell with a preselected amount of said solute.  
     
     
         26 . A method in accordance with  claim 24  further comprising monitoring changes in said cell while said solute is diffusing into said cell, said changes being representative of the degree of infusion of said cell with said solute as a means of determining the rate of mass transfer of said solute across said membrane.  
     
     
         27 . A method in accordance with,  claim 20  in which said barrier and said chambers are enclosed by a housing that is transparent, said method further comprising observing changes in said biological cell while said solute diffuses into said biological cell.  
     
     
         28 . Apparatus for the infusion of a biological cell with a chemical substance by electroporation, said apparatus comprising: 
 an electric cell containing an internal support to hold a biological cell and an internal barrier of a material substantially impermeable to electric current, said barrier positioned to restrict electric current flow in said electric cell to a flowpath crossing said internal support and through any biological cell held thereby; and    means for imposing a voltage across said electric cell and for monitoring the relative values of current and voltage as an indication of the occurrence and degree of electroporation in any biological cell held thereby.    
     
     
         29 . Apparatus in accordance with  claim 28  in which said barrier divides the interior of said electric cell into first and second electrode chambers and said internal support is an opening in said barrier smaller in width than a biological cell.  
     
     
         30 . Apparatus in accordance with  claim 29  further comprising means for imposing a pressure differential across said opening to lodge a biological cell in said opening.  
     
     
         31 . Apparatus in accordance with  claim 29  in which said first electrode chamber is a flow-through channel.