Sensors, and methods of making and using the same

1. A compound comprising a first fluorophore, a second fluorophore and a cleavable linker covalently linking the first fluorophore and the second fluorophore, wherein the fluorescence of the first and second fluorophores are substantially quenched by one another before the cleavable linker is cleaved, and wherein the first fluorophore is represented by the following structure:

or a tautomer thereof, wherein, independently for each occurrence:

Y is O, S, Se, NR, or C(CH 3 ) 2 , wherein R is an alkyl;

Q is O, S or Se;

Z is V, QH, or QU′ wherein U′ is a hydroxyl-protecting group;

W and W′ are each independently K or V;

K is hydrogen, halogen, linear or branched alkyl, alkenyl, linear or branched aminoalkyl, linear or branched acylamino, linear or branched acyloxy, linear or branched alkoxycarbonyl, linear or branched alkoxy, linear or branched alkylaryl, linear or branched hyrdoxyalkyl, linear or branched thioalkyl, acyl, aryloxy, arylalkoxy, alkynyl, cyano, sulihydryl, carbamoyl, trifluoromethyl, lower alkylthio, lower alkylamino, nitro, amine, hydroxyl, carbonyl, formyl or sulfonyl;

L is the cleavable linker covalently bonded to either the 5 or 6 position of the indicated aromatic ring;

L comprises an ester; and

V is a chemical moiety comprising at least one Lewis base capable of coordinating to a metal ion; and

wherein one or more fluorescence properties of the first fluorophore change measurably upon coordination of said Lewis base to a metal ion.

2. The compound of claim 1 , wherein Q is O and Z is OH, OU′ or V.

3. The compound of claim 1 , wherein V further comprises at least two Lewis bases that are together capable of forming a chelating agent upon coordination to a metal ion.

4. The compound of claim 3 , wherein independently for each occurrence:

(a) when V is in W, V occurs only once in the compound or occurs only one other time in the compound in W′ or Z; or

(b) when V is in W′, V occurs only once in the compound or occurs only one other time in the compound in W; or

(c) when V is in Z, V occurs only once in the compound or occurs only one other time in the compound in W.

5. The compound of claim 4 , wherein V occurs only once in the compound in W or W′.

6. The compound of claim 4 , wherein V occurs only once in the compound in Z.

7. The compound of claim 4 , wherein the Z is V, wherein V is directly bonded to the 6′ carbon of the indicated aromatic ring through a nitrogen atom.

8. The compound of claim 4 , wherein V occurs twice in the compound in W or W′ and is the same in W and W′.

9. The compound of claim 4 , wherein the Lewis bases are nitrogen atoms.

10. The compound of claim 1 , wherein Q is O, Z is OH, and the atom of K directly bonded to each the 2′ carbon and the 7′ carbon of the indicated aromatic ring is a Cl atom.

11. The compound of claim 1 , wherein said L is —C(═O)NH—(CH 2 ) n —O—, wherein n is an integer from 2 to 4 inclusive.

12. The compound of claim 1 , wherein said second fluorophore is represented by the following formula:

13. The compound of claim 1 , wherein V is independently for each occurrence selected from the group consisting of:

14. The compound of claim 1 , wherein K is lower alkoxyl, phenoxy, or benzyloxy.

15. A compound comprising (a) a first fluorophore comprising at least one Lewis base capable of forming a coordination bond with a metal ion, wherein one or more fluorescence properties of the first fluorophore changes upon coordination of said Lewis base to a metal ion; (b) a second fluorophore; (c) a cleavable linker covalently linking the first fluorophore and the second fluorophore, wherein the cleavable linker comprises an ester and said ester is intended to be cleaved during use of the compound; and (d) the fluorescence of the first and second fluorophores are substantially quenched by one another before the cleavable linker is cleaved.

16. The compound of claim 15 , wherein the first fluorophore further comprises at least two Lewis bases that are together capable of forming a chelating agent upon coordination to a metal ion.

17. The compound of claim 16 , wherein the first fluorophore further comprises at least one additional Lewis base that is capable of forming a chelating agent together with the other Lewis bases upon coordination of each to a metal ion.

18. The compound of claim 16 , wherein the brightness of the first fluorophore in the presence of a metal ion to which the first fluorophore is capable of coordinating is after cleavage of the cleveable linker at least twice as great as before cleavage.

19. The compound of claim 16 , wherein the cleavable linker is cleavable by a naturally occurring biological activity.

20. The compound of claim 19 , wherein the naturally occurring biological activity is enzymatic.

21. The compound of claim 16 , wherein the first fluorophore comprises a fluorescein-based ligand.

22. The compound of claim 21 , wherein the second fluorophore does not contain two nitrogen atoms that are capable of forming a chelating agent for coordination to a metal ion.

23. The compound of claim 21 , wherein the binding affinity of the first fluorophore to at least one type of transition metal cation is at least two times as great as the binding affinity of the second fluorophore to the same type of metal ion.

24. The compound of claim 23 , wherein the binding affinity of the first fluorophore to at least one type of transition metal cation is at least five times as great as the binding affinity of the second fluorophore to the same type of metal ion.

25. The compound of claim 24 , wherein the type of metal ion is Zn 2+ .

26. The compound of claim 24 , wherein the first fluorophore and the second fluorophore both fluoresce at about 450 nm or above.

27. A method of detecting, and optionally quantifying the concentration of, a metal ion in a sample or a patient, comprising:

(a) adding to a sample or administering to a patient a compound of any one of claims 1 - 9 , 15 - 20 , 21 - 26 , 10 , 11 , 12 , 13 , or 14 ;

(b) measuring fluorescence of the first fluorophore of the compound in the sample or the patient after cleavage of the cleavable linker has occurred for at least some measurable portion of the compound added to the sample or administered to the patient; and

(c) determining whether the metal ion is present in the sample or patient, and optionally the concentration of the metal ion in the sample or patient.

28. The method of claim 27 , further comprising measuring fluorescence of the second fluorophore of the compound in the sample or the patient after cleavage of the cleavable linker has occurred for at least some measurable portion of the compound added to the sample or administered to the patient.

29. The method of claim 28 , further comprising before measuring the fluorescence of the first and second fluorophore, causing the cleavable linker to cleave for at least some measurable portion of the compound added or administered.

30. The method of claim 28 , wherein the concentration of the metal ion is determined.

31. The method of claim 28 , wherein the compound is added to a sample and the sample comprises cells.

32. A diagnostic kit for a metal ion, comprising:

(a) a compound of any one of claims 1 - 9 , 15 - 20 , 21 - 26 , 10 , 11 , 12 , 13 , or 14 ; and

(b) instructions for using the compound to detect a metal ion in a sample or a patient.