Use of Nernstein voltage sensitive dyes in measuring transmembrane voltage

• US 6,979,553 B2
• Filed: 09/05/2003
• Issued: 12/27/2005
• Est. Priority Date: 10/08/1999
• Status: Expired due to Term

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Inventors: Farinas, Javier Anibal, Wada, H. Garrett
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US10/655,697
• Publication Number:
  US 20040048239A1
• Time in Patent Office: 844 Days
• Field of Search: 435/29, 435/4, 435/7.2, 435/968
• US Class Current: 435/29
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 2001/302 Stain compositions
• CPC Class Codes: G01N 2458/00 Labels used in chemical analysis of biological material
• CPC Class Codes: G01N 31/22 using chemical indicators G01N31/02 takes precedence
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Abstract

Transmembrane potential measurement methods using cationic dyes, and anionic dyes are provided. Compositions of the cationic and anionic dyes and microfluidic systems which include the dyes and membranes are provided in conjunction with processing elements for transmembrane potential measurements.

Claims

1. A cell-based assay method for determining a permeability of a cell membrane, the method comprising:

flowing a suspension of cells in a microchannel;

flowing a membrane permeable dye into the resulting flowing cell suspension, thereby contacting the cells and the dye;

monitoring a detectable signal from the dye after the dye contacts the cells, and

incubating the cells and the dye in the microchannel for a time period sufficient for the detectable signal from the dye to reach an equilibrium level, whereby the equilibrium level of the detectable signal is indicative of the permeability of the cell membrane.

2. The method of claim 1, wherein said flowing comprises:

electroosmotic flow or pressure-based flow.

3. The method of claim 1, wherein the microchannel comprises a microfluidic device.

4. The method of claim 1, wherein the cells are selected from the group consisting of:

bacteria, plant cells, animal cells, fungi, yeast, cardiac cells, and nerve cells.

5. The method of claim 1, wherein said flowing a dye comprises introduction of the dye into the microchannel through a side channel intersection.

6. The method of claim 1, wherein a time from contact of the cells and the dye to said detecting comprises about 100 seconds or less.

7. The method of claim 1, wherein the time from contact of the cells and the dye to said detecting ranges from about 75 seconds to about 10 seconds.

8. The method of claim 1, wherein the cells uptake the dye.

9. The method of claim 1, wherein the cell suspension comprises from about 200 cells to about 50 cells.

10. The method of claim 1, wherein the dye is selected from the group consisting of:

a fluorescent dye, a cation indicating dye, a calcium indicating dye, a membrane potential indicating dye, and a Nernstian dye.

11. The method of claim 1, further comprising contacting the cell suspension with a modulatory composition.

12. The method of claim 11, wherein the modulatory composition is selected from the group consisting of:

a neurotoxin, a neurotransmitter, a protein, a peptide, a lipid, a carbohydrate, an organic molecule, a drug, a receptor ligand, an antibody, a cytokine, a chemokine, a hormone, and a cell.

13. The method of claim 10, wherein the detectable signal comprises detecting a change in the dye associated with a cell response to the modulatory composition, wherein the change in the dye is selected from the group comprising:

a changed fluorescence, a changed light absorbance, and a change in a luminesce.

14. The method of claim 12, wherein the cell response is selected from the group consisting of:

a changed cation concentration, a changed membrane potential, and a changed calcium ion concentration.