Use of nernstein voltage sensitive dyes in measuring transmembrane voltage

• US 6,759,191 B2
• Filed: 01/21/2003
• Issued: 07/06/2004
• 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: Wada, H. Garrett, Farinas, Javier Anibal
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US10/349,396
• Publication Number:
  US 20040009545A1
• Time in Patent Office: 532 Days
• Field of Search: 435/29, 435/283.1, 435/4, 435/968
• US Class Current: 435/4
• 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 composition, comprising:

a first component comprising a membrane;

a cationic membrane permeable dye; and

,

an anionic membrane permeable redistributing dye.

2. The composition of claim 1, the cationic membrane permeable dye comprising a cationic nucleic acid staining dye.

3. The composition of claim 1, wherein the first component comprises one or more of:

a cell, a mitochondria, a chloroplast, a cell vesicle, and an artificial membrane.

4. The composition of claim 3, wherein the cell comprises one or more of:

an intact cell, an animal cell, a plant cell, a fungal cell, a bacterial cell, a mammalian cell, a primate cell, a rodent cell, a canine cell, a feline cell, and a livestock cell, a cultured cell, a THP-1 cell, a COS cell, a CHO cell, a HEK cell, a HeLA cell, an NIH 3T3 cell, a primary cell, an endoderm cell, an ectoderm cell, a mesoderm cell, a cell derived from a differentiated tissue, a cell derived from an undifferentiated tissue, a blood cell, a peripheral blood cell, a nerve cell, a muscle cell, a skin cell and a bone cell.

5. The composition of claim 1, wherein the cationic membrane permeable dye comprises one or more of:

a Blue-fluorescent SYTO dye, a Green-fluorescent SYTO Dye, an Orange-fluorescent SYTO dye, a Red-fluorescent SYTO dye, SYTO 62, Pur-1, thiazol, aryl, 2DS-7J1, Hoechst 33258, Hoechst 33342 and hexidium iodide;

or wherein the anionic membrane permeable redistributing dye comprises one or more of;

an anionic bis-isoxazolone oxonol dye, a bis-oxonol dye, Oxonol V, Oxonol VI, DiBAC₄(3) DiBAC₄(5), and DiBAC₂(3).

6. A container or microfluidic processor comprising the composition of claim 1.

7. A microfluidic device for monitoring transmembrane potential, the microfluidic device comprising:

a body structure having at least one microscale cavity disposed therein;

a target source of a first composition comprising at least one membrane, which target source is fluidly coupled to the at least one microscale cavity; and

,

a source of one or more voltage sensitive dyes which source is fluidly coupled to the at least one microscale cavity, wherein, during operation of the device, the first composition is contacted to the one or more voltage sensitive dyes in the at least one microscale channel.

8. The device of claim 7, wherein the one or more voltage sensitive dyes comprise one or more of:

a cationic membrane permeable staining dye source comprising one or more cationic membrane permeable dyes, which cationic membrane permeable dye source is fluidly coupled to the at least one microscale cavity, and an anionic membrane permeable redistributing dye source comprising one or more anionic redistributing dye, which anionic redistributing dye source is fluidly coupled to the at least one microscale cavity.

9. The device of claim 7, wherein the cationic dye source is a nucleic acid staining dye.

10. The device of claim 7, wherein the device comprises both the cationic membrane permeable staining dye source and the anionic membrane permeable redistributing dye source, and wherein, during operation of the device, the first composition is contacted, in the presence of the cationic membrane permeable dye and the anionic membrane permeable redistributing dye, to at least one transmembrane potential modulatory composition.

11. The device of claim 7, the device further comprising a source of at least one potential transmembrane potential modulatory composition, which source is fluidly coupled to the at least one microscale cavity.

12. The device of claim 7, further comprising a source of at least one transmembrane potential modulatory composition, wherein, during operation of the device, at least one transmembrane potential modulatory composition is contacted to one or more of:

the first composition, the cationic membrane permeable dye, or the anionic membrane permeable redistributing dye.

13. The device of claim 7, the device further comprising a plurality of sources of at least one potential membrane modulatory composition.

16. The device of claim 11, wherein the at least one potential membrane modulatory composition comprises one or more of:

a membrane hyperpolarization buffer, a membrane depolarization buffer, a compound which alters membrane permeability and a compound which alters transport of an ion across the cell membrane.

17. The device of claim 7, further comprising a signal detector located proximal to or within the microscale cavity, which signal detector detects the a signal.

18. The device of claim 17, wherein the detector detects the detectable signal for a selected length of time (t), or a selected time point (t_p).

19. The device of claim 7, wherein the at least one microscale cavity is a first microscale channel, and wherein, during operation of the device:

the first composition comprising the at least one membrane is flowed from the target source into the microchannel;

the cationic membrane permeable dye or the an anionic membrane permeable redistributing dye is flowed into contact with the first composition; and

,

the detectable signal is monitored at one or more selected time points after contact of the first composition with the cationic membrane permeable dye or the an anionic membrane permeable redistributing dye.

20. The device of claim 19, wherein at least one potential membrane modulatory composition is flowed from the target source into contact with the first composition.

21. The device of claim 7, wherein the membrane is a component of an intact cell.

14. The device of claim 14, wherein the plurality of sources comprise one or more microtiter trays, each of the one or more trays comprising at least one potential membrane modulatory composition.

15. The device of claim 14, wherein the microtiter trays are movably mounted proximal to the body structure, wherein the body structure comprises one or more pipettor channels which are structurally configured to access the trays, which one or more pipettor channels are fluidly coupled to the at least one microscale cavity.