Combination of Single-Cell Electroporation and Electrical Recording Using the Same Electrode

Abstract

Methods for stimulating exocytosis from a cell are provided where the same electrochemical microelectrode is used to electroporate an adjacent cell and then measure quantal exocytosis from the adjacent cell. Also provided are methods for stimulating and measuring exocytosis from a select cell population arrayed on a chip comprising addressable electrodes. Calcium independent stimulation of exocytosis with inorganic anions such as chloride ions is also provided. These methods can provide for specific stimulation of a desired subset of cells without exposing other nearby cells to the stimulus.

What is claimed is: 
     
         1 . A method for stimulating and measuring exocytosis from a cell the method comprising:
 electrically stimulating said cell with one or more voltage pulses while said cell resides in a drop of solution to provide a voltage gradient in solution surrounding the cell, said solution drop being confined within a well such that said solution drop is sitting on a surface of an electrode located at the bottom of said well and the cell being adjacent to the electrode surface, wherein said electrical stimulation is induced by passing a transient current through said electrode with a potentiostat, the potentiostat modified to include a diode network in parallel configuration with a feedback resistor of the potentiostat to allow for the passage of the transient current; and   measuring, by said same electrode, a release of an electrochemically active substance from said stimulated cell while said solution drop is sitting on said electrode surface the potentiostat configured to pass a pico-amp level current through said electrode.   
     
     
         2 . The method of  claim 1 , further comprising:
 arraying a plurality of cells on a chip, the chip comprising a plurality of addressable electrodes in an array, each electrode of the array having a surface, the plurality of cells residing in a plurality of the solution drops, each of the plurality of solution drops including at least one of the cells, and wherein the arraying step comprises depositing a plurality of the solution drops in a plurality of wells to confine said deposited solution drops such that each deposited solution drop is sitting on a surface of a different electrode of the array; and   performing the electrically stimulating step and the measuring step for one or more of the deposited solution drops with respect to the electrodes on which those deposited solution drops sit.   
     
     
         3 . The method of  claim 2 , wherein said stimulating step and the measuring step are performed when the cells are adjacent to surfaces of essentially all of said addressable electrodes. 
     
     
         4 . The method of  claim 2 , wherein said electrically stimulating step comprises addressing a subset of said electrodes with a voltage pulse to limit the electrical stimulation to a subset of said cells, said cell subset being those one or more cells that are adjacent to surfaces of said addressed electrode subset. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein said electrically stimulating step comprises electroporating said cell. 
     
     
         7 . The method of  claim 6 , further comprising introducing a membrane-impermeable substance into said cell by electroporation. 
     
     
         8 . The method of  claim 7 , wherein said membrane-impermeable substance comprises a peptide, an antibody, a DNA molecule, or an RNA molecule. 
     
     
         9 . The method of  claim 8 , further comprising using said DNA molecule or said RNA molecule to express a gene of interest or to inhibit expression of a gene of interest in said select cell population. 
     
     
         10 . The method of  claim 9 , further comprising incubating said cell, wherein said cell is incubated after said electrically stimulating step and before said measuring step. 
     
     
         11 . The method of  claim 4 , further comprising applying an agent to said stimulated cells. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . A method for stimulating and measuring exocytosis from a cell, comprising:
 applying, through an electrode located at the bottom of a well, a voltage pulse to an intact cell while said cell resides in a solution to provide a voltage gradient in solution surrounding the cell, the solution being confined within said well and sitting on a surface of said electrode such that said cell is adjacent to the electrode surface, the applied voltage being sufficient to cause a stimulation of said cell, stimulation of said cell being induced by passing a transient current through said electrode with a potentiostat, the potentiostat modified to include a diode network to allow for the passage of the transient current; and   recording, through said same electrode while the intact cell remains adjacent to the electrode, a current signal indicative of a release of an electrochemically active substance from said stimulated cell, the potentiostat passing a pico-amp level current through said electrode.   
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 14 , wherein said applied voltage pulse comprises a first pulse portion and a second pulse portion, wherein said first pulse portion causes said cell stimulation, and wherein said recording step is performed during said second pulse portion. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 14 , wherein said current signal comprises an amperometric spike. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 14 , wherein said stimulation comprises a triggering of an action potential in said cell. 
     
     
         23 . The method of  claim 14 , wherein said stimulation comprises electroporation of said cell. 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 14 , further comprising:
 confining a plurality of cells in a plurality of said wells, each of said wells having a different electrode at its second end such that said different electrodes define a microchip electrode array; and   performing said applying step and said recording step for a plurality of said cells, wherein said applying step comprises selectively addressing a subset of said electrodes with said voltage pulse to stimulate a desired subset of said cells without exposing other nearby cells to stimulation.   
     
     
         27 . The method of  claim 14  wherein said electrode comprises an electro-chemical electrode. 
     
     
         28 . (canceled) 
     
     
         29 . The method of  claim 14 , wherein said cell is a neuroendocrine cell and wherein said recorded current signal is indicative of quantal exocytosis. 
     
     
         30 . The method of  claim 14  wherein said applying and recording steps are performed while said cell is contacting said electrode surface. 
     
     
         31 . The method of  claim 14  wherein said applying and recordings step are performed while said cell is within about 3 micrometers of said electrode surface. 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . (canceled)