Fluorescent metal ion indicators with large stokes shifts

Patent metadata

Field	Value
Publication number	US-9846168-B2
Application number	US-201615152346-A
Country	US
Kind code	B2
Filing date	May 11, 2016
Priority date	Jul 27, 2004
Publication date	Dec 19, 2017
Grant date	Dec 19, 2017

Abstract

The present invention provides fluorogenic compounds for the detection of target metal ions wherein the compounds exhibit a Stokes shift greater than 50 nm and the detectable signal is modulated by photoinduced electron transfer (PET). The present compounds consist of three functional elements, the ion sensing moiety (chelating moiety), the reporter moiety (fluorophore or fluorescent protein) and spacer or linker between the sensing and reporter moieties of the present compound that allows for PET upon binding of a metal ion and excitation by an appropriate wavelength.

First claim

What is claimed is:

1. A compound for the detection of metal ions wherein a detectable response is modulated by photoinduced electron transfer (PET), wherein the compound comprises a fluorescent protein that is covalently bonded to the metal chelating moiety by a linker,
—(CR 2 ) n NR′—, wherein R and R′ are independently hydrogen, alkyl, or substituted alkyl and n is 1-10;
or —(CR 2 ) n — wherein R is alkyl or substituted alkyl and n=1-10;
or (CH 2 ) p wherein p is 2-10.

2. A composition comprising:
a) the compound of claim 1 ; and,
b) a metal ion that is capable of being chelated by the compound.

3. The composition of claim 2 , wherein the metal ion is selected from the group consisting of Ca 2+ , Zn 2+ , Mg 2+ , Ga 3+ , Tb 3+ , La 3+ , Pb 2+ , Hg 2+ , Cd 2+ , Cu 2+ , Ni 2+ , Co 2+ , Fe 2+ , Mn 2+ , Ba 2+ , and Sr 2+ .

4. A method for binding a target metal ion in a sample, comprising steps of:
a. contacting the sample with the metal ion binding compound of claim 1 to form a contacted sample; and,
b) incubating the contacted sample for sufficient time to allow the metal ion binding compound to chelate the target metal ion whereby the metal ion is bound.

5. The method of claim 4 , wherein the sample comprises live cells, intracellular fluids, extracellular fluids, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions or biological fluids or chemical reactors.

6. The method of claim 4 , wherein the sample comprises blood cells, immune cells, cultured cells, muscle tissue, neurons, extracellular vesicles; vascular tissue, blood fluids, saliva, urine; water, soil, waste water, sea water; pharmaceuticals, foodstuffs or beverages.

7. The method of claim 4 , wherein the method further comprises detecting a target metal ion wherein the sample is illuminated with an appropriate wavelength whereby the target metal ion is detected.

8. The method of claim 4 , wherein the metal ion is Ca 2+ , Zn 2+ , Mg 2+ , Ga 3+ , Tb 3+ , La 3+ , Pb 2+ , Hg 2+ , Cd 2+ , Cu 2+ , Ni 2+ , Co 2+ , Fe 2+ , Mn 2+ , Ba 2+ , or Sr 2+ .

9. A kit for binding a metal ion in a sample, comprising at least one metal binding compound of claim 1 and instructions for use thereof.

10. The kit of claim 9 , wherein the kit independently further comprises one or more components selected from the group consisting of a calibration standard of a metal ion, an ionophore, a metal ion indicator other than for calcium ions, a detectable signal standard, an aqueous buffer solution, an antibody or fragment thereof, a reference dye standard and an organic solvent.

11. A compound for the detection of metal ions, wherein the compound comprises:
a metal chelating BAPTA moiety; and
a fluorescent protein that is covalently bonded to the metal chelating moiety by a linker,
—(CR 2 ) n NR′—, wherein R and R′ are independently hydrogen, alkyl, or substituted alkyl and n is 1-10;
or —(CR 2 ) n — wherein R is alkyl or substituted alkyl and n=1-10;
or (CH 2 ) p wherein p is 2-10.

12. The compound of claim 11 wherein the metal ion is a calcium ion.

Assignees

• Life Technologies Corp

Inventors

• Gee Kyle
• Martin Vladimir

Classifications

• C07K14/195

  from bacteria · CPC title

• C09K11/06

  containing organic luminescent materials · CPC title

• C07D413/04

  directly linked by a ring-member-to-ring-member bond · CPC title

• C09B11/08

  Phthaleins; {Phenolphthaleins; Fluorescein} · CPC title

• C09B5/62

  Cyclic imides or amidines of peri-dicarboxylic acids of the anthracene, benzanthrene, or perylene series · CPC title