Use of nernstein voltage sensitive dyes in measuring transmembrane voltage

• US 6,537,771 B1
• Filed: 10/06/2000
• Issued: 03/25/2003
• Est. Priority Date: 10/08/1999
• Status: Expired due to Term

• Current Assignee: Caliper Holdings Incorporated
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Wada, H. Garrett, Farinas, Javier Anibal
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/684,313
• Time in Patent Office: 900 Days
• Field of Search: 435/29, 435/4, 435/968, 435/33, 435/7.2
• 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 method of generating a signal output which is sensitive to membrane potential, the method comprising:

providing a first component comprising one or more membranes;

adding a cationic membrane permeable nucleic acid staining dye to the first component; and

,

monitoring a first signal output from the cationic membrane permeable nucleic acid staining dye, wherein the first signal output is correlated with the transmembrane potential across the one or more membranes.

2. The method of claim 1, the method comprising monitoring a change in transmembrane potential, the monitoring of the first signal output comprising monitoring a change in the first signal output over time and the correlating the first signal output with the transmembrane potential across the one or more membrane comprises determining the rate of change of the first signal over time.

3. The method of claim 1, the providing step comprising flowing the first component through a first microfluidic channel, which microfluidic channel intersects a second microfluidic channel, wherein the cationic membrane permeable nucleic acid staining dye is flowed through the first or second microchannel and into contact with the at least one membrane.

4. The method of claim 3, the method comprising flowing an anionic membrane permeable redistributing dye or a neutral dye through the first or second channel and into contact with the at least one membrane.

5. The method of claim 1, further comprising exposing the one or more membranes to one or more transmembrane potential modulatory compositions and monitoring an effect of the one or more transmembrane potential modulatory compositions on the first signal output, thereby monitoring an effect of the one or more transmembrane potential modulatory compositions on the transmembrane potential.

6. The method of claim 5, the one or more transmembrane potential modulatory compositions comprising one or more compositions selected from the group consisting of:

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

14. The method of claim 1, 7, 8 or 13, comprising monitoring the first or second signal outputs over a selected period of time (t), which period is less than about 100 seconds.

15. The method of claim 14, wherein t is between about 0.1 and about 80 seconds.

16. The method of claim 1, 7 or 13, wherein the first or second signal output is monitored at one or more time points, which one or more time points are before equilibration of the first voltage sensitive membrane permeable dyes, the at least second voltage sensitive membrane permeable dyes or the cationic membrane permeable nucleic acid staining dye across the one or more membranes.

22. The method of claim 1, 7, 8 or 17, wherein the first mixture comprises an anionic membrane permeable redistributing dye, or wherein the method comprises adding an anionic membrane permeable redistributing dye to the first component and measuring a second signal output from the anionic membrane permeable redistributing dye, thereby providing a further indication of changes in the transmembrane potential.

23. The method of claim 22, further comprising determining a ratio of the first and second signal.

24. The method of claim 22, 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).

25. The method of claim 22, wherein the cationic membrane permeable nucleic acid staining dye is SYTO 62, at a concentration of between about 0.01 and about 50 μ

M and the anionic dye is DiBAC₄(3), at a concentration of between about 0.01 and about 50 μ

M.

26. The method of claim 1, 7, or 8 wherein the one or more membrane is a component of an intact cell, which cell is suspended in a fluid comprising one or more ion selected from:

NA⁺, K⁺, Cl⁻

, H⁺, Ca²⁺, and HCO₃⁻

.

27. The method of claim 1, 7, or 8 wherein the one or more membrane is a cell membrane.

28. The method of claim 27, wherein the cell membrane is present in an intact or live cell, or, wherein the cell is selected from:

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, 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 primary cell derived from differentiated tissue, a primary cell derived from undifferentiated tissue, a primary cell derived from blood, a primary cell derived from peripheral blood, a primary cell derived from nerve, a primary cell derived from muscle, a primary cell derived from skin and a primary cell derived from bone.

29. The method of claim 28, wherein the intact or live cell has a transmembrane potential of about −

100 mV to about 10 mV.

30. The method of claim 1, 7, or 8 wherein the first signal is detected optically.

31. The method of claim 1, 7, or 8 wherein the first signal is detected at between about 20°

C. and 40°

C.

32. The method of claim 1, 7, or 8 wherein monitoring the first signal output comprises detecting one or more fluorescent emission produced by the cationic membrane permeable nucleic acid staining dye or at least one of the membrane permeable voltage sensitive dye(s).

33. The method of claim 1, 7, or 8, further comprising contacting the first component or the first mixture with a transmembrane potential modulator and monitoring an effect of the transmembrane potential modulator by monitoring the first signal output.

34. The method of claim 33, wherein the transmembrane potential modulator is a control modulator or a test modulator.

35. The method of claim 34, wherein the control modulator is selected from:

a molecule, a neurotoxin, a set of neurotoxins, a neurotransmitter, a set of neurotransmitters, a protein, a set of proteins, a peptide, a set of peptides, a lipid, a set of lipids, a carbohydrate, a set of carbohydrates, an organic molecule, a set of organic molecules, a drug, a set of drugs, a receptor ligand, a set of receptor ligands, an antibody, a set of antibodies, a cytokine, a set of cytokines, a chemokine, a set of chemokines, a hormone, a set of hormones, a cell, a set of cells, a protein attached to a cell, and a protein attached to a set of cells.

58. The method of claim 1, wherein the first signal output is monitored prior to equilibration of the cationic membrane permeable nucleic acid staining dye across at least one of the one or more membranes.

7. A method of producing a signal which is dependent on transmembrane potential, the method comprising:

flowing a first mixture comprising one or more membranes and at least two voltage sensitive membrane permeable dyes through a first channel region; and

,

monitoring at least a first signal output from at least one of the at least two voltage sensitive membrane permeable dyes, thereby producing a signal which is dependent on the transmembrane potential across the one or more membranes.

9. The method of claim 7 or 8, wherein the first mixture comprises one or more components selected from the group consisting of:

a cationic dye, a cationic membrane permeable nucleic acid staining dye, an anionic dye and a neutral dye.

10. The method of claim 9, wherein the voltage sensitive dyes comprises an anionic dye, a cationic dye, or a cationic membrane permeable nucleic acid staining dye and one or more additional dye selected from the group consisting of:

an anionic dye, Oxonol V, Oxonol VI, DiBAC₄(3), DiBAC₄(5), DiBAC₂(3), WW781, RGA-30, a cationic dye, an indo-carbocyanine dye with a short alkyl tail, a thio-carbocyanine dye with a short alkyl tail, an oxa-carbocyanine dye with a short alkyl tail, an amino napthyletheny pyridinium dye, a dialkyl amino phenylpolyenyl pyridinium dye, a cationic membrane permeable nucleic acid staining dye, a SYTO dye, SYTO 62, and a neutral dye.

11. The method of claim 7 or 8, wherein the first mixture is provided to the first channel by flowing a first component comprising one or more membrane from a source to a first channel region and flowing a labeling composition comprising the one or more voltage sensitive dyes into contact with the membrane.

12. The method of claim 7 or 8, further comprising:

hyperpolarizing or depolarizing the membrane, or changing a permeability property of the membrane, and monitoring flow of the at least one voltage sensitive dye across the membrane by monitoring the first signal output, thereby measuring changes in the transmembrane potential.

13. The method of claim 7, further comprising monitoring flow of at least a second of the at least two voltage sensitive membrane permeable dyes across the membrane by monitoring at least a second signal output from the second voltage sensitive dyes.

17. The method of claim 7 wherein at least one of the voltage sensitive membrane permeable dyes is a cationic membrane permeable nucleic acid staining dye.

18. The method of claim 7, 13, 14 or 17, wherein a rate of dye translocation across the membrane depends on the transmembrane potential across the membrane.

19. The method of claim 7, 13, 14 or 17, wherein the cationic membrane permeable nucleic acid staining dye is a cyanine dye, or a cyclic-substituted unsymmetrical cyanine dye.

20. The method of claim 7 or 17, wherein the cationic membrane permeable nucleic acid staining dye is a dye selected from:

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

21. The method of claim 7, 8 or 17, wherein at least one of the voltage sensitive membrane permeable dyes comprises SYTO 62.

8. A method of producing a signal which is dependent on transmembrane potential, the method comprising:

flowing a first mixture comprising one or more membranes and one or more voltage sensitive dyes through a first channel region; and

,

monitoring at least a first signal output from at least one of the voltage sensitive dyes, thereby producing a signal which is dependent on the transmembrane potential across the one or more membranes,

wherein the voltage sensitive dyes comprise one or more membrane permeable redistributing dyes, which one or more membrane permeable dyes comprise one or more ionic dye wherein flow of the membrane permeable redistributing dyes across the membrane is detected by monitoring the one or more signal outputs from the membrane permeable redistributing dyes before an equilibrium distribution of the membrane permeable redistributing dyes is reached.

57. The method of claim 8 wherein the ionic dye is a cationic membrane permeable nucleic acid staining dye.

36. A method of generating an optical signal which is sensitive to transmembrane potential, the method comprising:

providing a first component comprising one or more membrane;

adding a cationic membrane permeable redistributing dye to the first component;

adding an anionic membrane permeable redistributing dye to the first component; and

,

measuring a first signal output from the cationic dye and a second signal output from the anionic dye, wherein one or more of the first and second signal outputs comprises an optical signal output, thereby generating the optical signal which is sensitive to the transmembrane potential.

37. The method of claim 36, wherein the cationic dye is a membrane permeable nucleic acid staining dye or a cationic rhodamine, an indo-carbocyanine dye, a thio-carbocyanine dye, an oxa-carbocyanine dye, an amino napthylethylenyl pyridinium dye, a dialkyl amino phenyl polyphenyl pyridinium dye, or wherein the anionic membrane permeable redistributing dye comprises one or more of:

Oxonol V, Oxonol VI, and DiBAC₄(3) DiBAC₄(5), DiBAC₂(3).

38. The method of claim 36, further comprising adding a neutral dye to the first component.

39. The method of claim 36, further comprising adding a neutral dye to the first component, wherein the neutral dye produces a control signal output which is dependent on one or more of:

temperature, incubation time and overall membrane permeability.

51. The method of claim 36, wherein the cationic redistributing dye comprises Syto 62 and the anionic dye comprises DiBAC₄(3).

40. A method of generating an optical signal which is dependent on transmembrane potential, the method comprising:

providing a first component comprising one or more membrane;

adding at least a first membrane permeable redistributing dye to the first component, wherein the first membrane permeable redistributing dye comprises an ion;

measuring one or more signal output from the first redistributing dye before an equilibrium dye distribution is established, which one or more signal output comprises at least one optical signal output, thereby providing the optical signal which is dependent on transmembrane potential.

41. The method of claim 40, further comprising correlating the one or more signal output to a change in transmembrane potential.

42. The method of claim 40, comprising adding at least a second membrane permeable redistributing dye to the one or more component and measuring one or more signal outputs from the second membrane permeable redistributing dye before an equilibrium dye distribution is established.

43. The method of claim 42, wherein the first and second dyes are added to the first component at approximately the same time and the signal outputs from the first and second membrane permeable redistributing dyes are measured at approximately the same time.

44. The method of claim 42, wherein the first and second redistributing dyes comprise an anionic dye and a cationic dye.

45. The method of claim 42 or 44, wherein the first and second redistributing dyes comprise one or more of:

an anionic dye, a cationic dye, a cationic membrane permeable nucleic acid staining dye, and a neutral dye.

46. The method of claim 42, further comprising adding at least a third membrane permeable redistributing dye to the one or more component and measuring one or more signal outputs from the third membrane permeable redistributing dye before an equilibrium dye distribution is established.

47. The method of claim 46, wherein the first and second redistributing dyes comprise one or more of:

an anionic dye and a cationic dye and wherein the third membrane permeable redistributing dye comprises a neutral dye.

48. The method of claim 47, wherein the signal output for the neutral dye is correlated to a temperature-dependent change in membrane permeability or a time-dependent change in membrane permeability.

49. The method of claim 45, wherein the cationic dye is a nucleic acid staining dye.

50. The method of claim 45, wherein the cationic dye comprises a nucleic acid staining dye selected from:

a Blue-fluorescent SYTO dye, a Green-fluorescent SYTO Dye, an Orange-fluorescent SYTO dye, a Red-fluorescent SYTO dye, Pur-1, thiazol, aryl, 2DS-7J1, Hoechst 33258, Hoechst 33342 and hexidium iodide or wherein the anionic redistributing dye comprises one or more of Oxonol V, Oxonol VI, and DiBAC₄(3) DiBAC₄(5), DiBAC₂(3).

52. A method of generating a signal output which is sensitive to membrane potential, the method comprising:

providing a first component comprising one or more membranes;

adding a cationic membrane permeable nucleic acid staining dye to the first component, wherein the dye is Red-fluorescent dye SYTO 62; and

,

monitoring a first signal output from the cationic membrane permeable nucleic acid staining dye, wherein the first signal output is correlated with the transmembrane potential across the one or more membranes.

53. A method of producing a signal which is dependent on transmembrane potential, the method comprising:

flowing a first mixture comprising one or more membranes and one or more voltage sensitive dyes through a first channel region, wherein the one or more voltage sensitive dyes comprise SYTO 62; and

,

monitoring at least a first signal output from at least one of the voltage sensitive dyes, thereby producing a signal which is dependent on the transmembrane potential across the one or more membranes.

54. A method of generating a signal output which is sensitive to membrane potential, the method comprising:

providing a first component comprising one or more membranes;

adding a cationic membrane permeable nucleic acid staining dye to the first component;

adding an anionic membrane permeable redistributing dye to the first component;

monitoring a first signal output from the cationic membrane permeable nucleic acid staining dye, wherein the first signal output is correlated with the transmembrane potential across the one or more membranes; and

,

measuring a second signal output from the anionic membrane permeable redistributing dye, thereby providing a further indication of changes in the transmembrane potential;

wherein the cationic membrane permeable nucleic acid staining dye is SYTO 62, at a concentration of between about 0.01 and about 50 μ

M and the anionic dye is DiBAC₄(3), at a concentration of between about 0.01 and about 50 μ

M.

55. A method of producing a signal which is dependent on transmembrane potential, the method comprising:

flowing a first mixture comprising one or more membranes, one or more voltage sensitive dyes and an anionic membrane permeable redistributing dye through a first channel region;

monitoring at least a first signal output from at least one of the voltage sensitive dyes, thereby producing a signal which is dependent on the transmembrane potential across the one or more membranes; and

measuring a second signal output from the anionic membrane permeable redistributing dye, thereby providing a further indication of changes in the transmembrane potential;

wherein the cationic membrane permeable nucleic acid staining dye is SYTO 62, at a concentration of between about 0.01 and about 50 μ

M and the anionic dye is DiBAC₄(3), at a concentration of between about 0.01 and about 50 μ

M.

56. A method of generating an optical signal which is sensitive to transmembrane potential, the method comprising:

providing a first component comprising one or more membrane;

adding a cationic membrane permeable redistributing dye comprising Syto 62 to the first component;

adding an anionic membrane permeable redistributing dye comprising DiBAC₄(3) to the first component; and

,

measuring a first signal output from the cationic dye and a second signal output from the anionic dye, wherein one or more of the first and second signal outputs comprises an optical signal output, thereby generating the optical signal which is sensitive to the transmembrane potential.