Sensors, and methods of making and using the same

• US 20040224420A1
• Filed: 05/04/2003
• Published: 11/11/2004
• Est. Priority Date: 05/04/2003
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Lippard, Stephen J., Woodroofe, Carolyn Crystal
• Granted Patent: US 7,399,639 B2
• Time in Patent Office: Days
• US Class Current: 436/74
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C09K 2211/1007 Non-condensed systems
• CPC Class Codes: C09K 2211/1011 Condensed systems
• CPC Class Codes: C09K 2211/1022 bridged by heteroatoms, e.g. N, P, Si or B
• CPC Class Codes: C09K 2211/1077 with oxygen
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH

Abstract

The present invention is directed, in part, to sensors for detecting metal ions, and methods of making and using the same.

Claims

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 first fluorophore is represented by the following generalized structure:

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

or V.

3. The compound of claim 2, wherein V further comprises a second Lewis base that is capable of forming a chelating agent with the first Lewis base upon coordination of the first and second Lewis bases 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 6, wherein the atom of Z directly bonded to the 6′

carbon of the indicated aromatic ring is 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 first and the second Lewis bases are nitrogen atoms.

10. The compound of claim 4, wherein, independently for each occurrence, when V occurs in W or W′

, V is AD, and A is one of the following;

—

CH₂—

, —

C(═

O)—

, —

C(=S)—

, —

CH₂CH₂—

, —

CH₂C(═

O)—

, —

CH₂C(═

S)—

, and optionally J is substituted for any hydrogen atom of said —

CH₂—

, —

CH₂CH₂—

, —

CH₂C(=O)—

, or —

CH₂C(═

S)—

, wherein J is a non-interfering substituent.

11. The compound of claim 9, wherein the first fluorophore has one or more K substituents, with each K independently being one of the following:

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, amino, hydroxy, thio, aryloxy, arylalkoxy, hydrogen, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy, hydrogen, amine, hydroxyl, alkoxyl, carbonyl, acyl, formyl or sulfonyl.

12. A compound comprising (a) a first fluorophore comprising a first 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 the first Lewis base to a metal ion;

(b) a second fluorophore; and

(c) a cleavable linker covalently linking the first fluorophore and the second fluorophore, wherein the cleavable linker is intended to be cleaved during use of the compound.

13. The compound of claim 12, wherein the first fluorophore further comprises a second Lewis base that is capable of forming a chelating agent with the first Lewis base upon coordination of the first and second Lewis bases to a metal ion.

14. The compound of claim 13, wherein the first fluorophore further comprises a third Lewis base that is capable of forming a chelating agent with the first Lewis base and second Lewis base upon coordination of the first, second and third Lewis bases to a metal ion.

15. The compound of claim 13, 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.

16. The compound of claim 13, wherein the cleavable linker is cleavable by a naturally occurring biological activity.

17. The compound of claim 16, wherein the naturally occurring biological activity is enzymatic.

18. The compound of claim 13, wherein the cleavable linker comprises an ester.

19. The compound of claim 13, wherein the first fluorophore comprises a fluorescein-based ligand.

20. The compound of claim 19, wherein the second fluorophore does not contain two nitrogen atoms that are capable of forming a chelating agent for coordination to a metal ion.

21. The compound of claim 19, 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.

22. 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 five times as great as the binding affinity of the second fluorophore to the same type of metal ion.

23. The compound of claim 22, wherein the type of metal ion is Zn²⁺.

24. The compound of claim 22, wherein the first fluorophore and the second fluorophore both fluoresce at about 450 nm or above.

25. The compound of claim 21, wherein the fluorescence of the first and second fluorophore are substantially quenched by one another before the cleavable linker is cleaved.

26. A compound represented by the following generalized structure:

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 one of the compounds claimed above;

(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) one of the compounds claimed above; and

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