# US 7,611,863 B2 · Selective staining of biomembranes using voltage-sensitive dyes

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- Number: US 7,611,863 B2
- Document ID: US7611863B2
- Status: Granted
- Kind: B2
- Application number: 11/030,313
- Filed: 2005-01-07
- Published: 2005-11-24
- Granted: 2009-11-03
- Earliest claimed: 2004-01-08
- Assignees: Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V.
- Inventors: Peter Fromherz; Gerd Huebener; Marlon J. Hinner
- Web page: https://patentgrep.com/patent/US7611863B2

## CPC

- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- C12Q 1/42: involving phosphatase
- G01N 33/58: involving labelled substances (G01N33/53 takes precedence)
- C12Q 2334/00: O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q 2337/00: N-linked chromogens for determinations of peptidases and proteinases
- G01N 2458/30: Electrochemically active labels
- Y10T 436/173845: Amine and quaternary ammonium
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The present invention relates to a method for staining membranes, in particular, a method for selective staining of cells using voltage-sensitive dyes.

## Claims

### Claim 1

A method for the enzymatic activation of an amphiphilic voltage-sensitive dye comprising
(a) providing a precursor to the amphiphilic voltage-sensitive dye with said precursor having an enzymatically cleavable hydrophilic group at a lipophilic tail of the amphiphilic voltage-sensitive dye,
(b) cleaving the precursor to the amphiphilic voltage-sensitive dye using an enzyme expressed by a target cell, thereby forming the amphiphilic voltage-sensitive dye, and
(c) binding the amphiphilic voltage-sensitive dye to a lipophilic interior of a lipid membrane of the target cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the membrane is a lipid membrane of a nerve cell or a cancer cell.

### Claim 3 (depends on 1)

The method according to claim 1, wherein a voltage-sensitive fluorescent dye is used.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the dye precursor is a voltage sensitive dye precursor of the formula (I)
X-A-Z-R1R2 (I)
wherein A denotes a voltage-sensitive chromophore which optionally can contain one or more groups Q selected from —(CH2)mSO2OH, —(CH2)m—N+(CH3)3, —CH2—CHOH—CH2—N+(CH3)2—CH2—CH2—OH, —OH, —O—PO(OH)2, —O—SO2OH, —(CH2)n—OH, —(CH2)n—O—PO(OH)2, —(CH2)n—O—SO2OH, an N- or O-linked (poly)carbohydrate and/or an N- or O-linked (poly)amino acid,
X denotes a polar head group selected from —(CH2)m—SO2OH, —(CH2)m—N+(CH3)3, —CH2—CHOH—CH2—N+(CH3)2—CH2—CH2—OH, m and n independently represent integers from 0 to 20 or X represents a group —R3Y or a group Y, wherein R3 is a hydrocarbon linking group with 1-20 C atoms and wherein Y is selected from —O—PO(OH)2, —O—SO2OH, an N- or O-linked (poly)carbohydrate and/or an N- or O-linked (poly)amino acid,
Z is selected from N, P, As, Sb or Bi, and
R1 and R2 independently at each occurrence represent a C1-C20 hydrocarbon residue which can be substituted by one or more groups Y, with the proviso that the dye precursor contains at least one group Y.

### Claim 5 (depends on 4)

The method according to claim 4, wherein at least one residue R1 and/or R2 contains one or more groups Y.

### Claim 6 (depends on 3)

The method according to claim 3, wherein the voltage-sensitivity and/or fluorescence of the dye is activated by cleaving the dye precursor.

### Claim 7

A method for the enzymatic activation of an amphiphilic voltage-sensitive dye comprising
(a) providing a precursor to the amphiphilic voltage-sensitive dye with said precursor having an enzymatically cleavable hydrophilic group at a lipophilic tail of the amphiphilic voltage-sensitive dye,
(b) cleaving the precursor to the amphiphilic voltage-sensitive dye using an enzyme expressed by a target cell, thereby forming the amphiphilic voltage-sensitive dye, and
(c) binding the amphiphilic voltage-sensitive dye to a lipophilic interior of a lipid membrane of the target cell, wherein the enzyme is overexpressed by a transfected cell and provided with a targeting signal which directs the enzyme to the cell wall.

### Claim 8

A method for the enzymatic activation of an amphiphilic voltage-sensitive dye comprising
(a) providing a precursor to the amphiphilic voltage-sensitive dye with said precursor having an enzymatically cleavable hydrophilic group at a lipophilic tail of the amphiphilic voltage-sensitive dye,
(b) cleaving the precursor to the amphiphilic voltage-sensitive dye using an enzyme expressed by a target cell, thereby forming the amphiphilic voltage-sensitive dye, and
(c) binding the amphiphilic voltage-sensitive dye to a lipophilic interior of a lipid membrane of the target cell, wherein dye not bound to the membrane is reconverted into the dye precursor by a second enzyme.

### Claim 9 (depends on 1)

The method according to claim 1 for intracellular staining.

### Claim 10

A voltage sensitive dye precursor having the formula (I)
X-A-Z-R1R2 (I)
wherein A denotes a voltage sensitive chromophore selected from
X denotes a polar head group selected from —(CH2)m—SO2H, —(CH2)m—N+(CH3)3 or —CH2—CHOH—CH2—N+(CH3)2—CH2—CH2—OH, m is an integer from 0 to 6, Z is selected from N, P, As, Sb or Bi and R1 and R2 independently at each occurrence represent a C10-C20 hydrocarbon residue which is substituted with at least one group selected from a (poly-)phosphate group or a (poly-)carbohydrate group.