Redox reversible imidazole osmium complex conjugates

• US 6,262,264 B1
• Filed: 05/28/1999
• Issued: 07/17/2001
• Est. Priority Date: 06/01/1998
• Status: Expired due to Term

• Current Assignee: Roche Diabetes Care Inc. (Roche Holding AG)
• Original Assignee: Roche Diagnostics Corporation (Roche Holding AG)
• Inventors: Buck, Harvey B. Jr., Deng, Zhi David
• Primary Examiner(s): Lambkin, Deborah C.
• Application Number:
  US09/322,500
• Time in Patent Office: 781 Days
• Field of Search: 546/2, 435/7.1, 205/792
• US Class Current: 546/2
• CPC Class Codes: C07F 15/0026 without a metal-carbon linkage
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C07K 5/101 the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
• CPC Class Codes: C07K 7/02 Linear peptides containing at least one abnormal peptide link
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 9/001 the peptide sequence having less than 12 amino acids and not being part of a ring structure
• CPC Class Codes: G01N 2333/805 Haemoglobins; Myoglobins
• CPC Class Codes: G01N 2458/30 Electrochemically active labels
• CPC Class Codes: G01N 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/536 with immune complex formed in liquid phase
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10T 436/105831 Protein or peptide standard or control [e.g., hemoglobin, etc.]

Abstract

Novel imidazole-osmium complex conjugates, their preparation, and their use in electrochemical assays are described. The redox reversible-osmium complexes can be prepared to exhibit unique reversible redox potentials and can thus be used in combination with other electroactive redox reversible species having redox potentials differing by at least 50 millivolts in electrochemical assays designed for use of multiple electroactive species in the same cell and in the same sample without interference between the two or more redox coupled conjugate systems.

Claims

1. An electrochemically detectable compound of the formula

wherein

R and R₁ are the same or different and are 2,2′

-bipyridyl, 4,4′

-disubstituted-2,2′

-bipyridyl, 5-5′

-disubstituted,-2,2′

-bipyridyl, 1,10-phenanthrolinyl, 4,7-disubstituted-1,10-phenanthrolinyl, or 5,6-disubstituted-1,10-phenanthrolinyl, wherein each substituent is a methyl, ethyl, or phenyl group,

R and R₁ are coordinated to Os through their nitrogen atoms;

q is 1 or 0;

R₇ is B—

(L)_k—

Q(CH₂)_i—

;

R₂ is hydrogen, methyl, or ethyl when q is 1, and R₂ is B—

(L)_k—

Q(CH₂)_i—

when q is 0;

wherein in the group B—

(L)_k—

Q(CH₂)_i—

Q is O, S, or NR₄ wherein R₄ is hydrogen, methyl or ethyl;

—

L—

is a divalent linker;

k is 1 or 0;

i is 1, 2, 3, 4, 5 or 6; and

B is hydrogen or a group comprising a ligand capable of binding to a specific binding partner;

Z is chloro or bromo;

m is +1 or +2;

X is chloride, bromide, iodide, fluoride, tetrafluoroborate, perchlorate, nitrate, sulfate, carbonate, or sulfite;

Y is chloride, bromide, iodide, fluoride, tetrafluoroborate, perchlorate or nitrate; and

n is 1 or zero,

provided that when X is sulfate, carbonate, or sulfite, n is zero,

and when m is 1, n is zero and X is not sulfate, carbonate or sulfite.

2. The detectable compound of claim 1 wherein R and R₁ are the same or different and are 2,2′

-bipyridyl, 4,4′

-disubstituted-2,2′

-bipyridyl, 5,5′

-disubstituted -2,2′

-bipyridyl.

3. The detectable compound of claim 2 wherein R₂ is hydrogen or methyl.

4. The detectable compound of claim 3 wherein Z is chloro.

5. The detectable compound of claim 4 wherein

X is chloride, bromide, or tetrafluoroborate,

Y is chloride, bromide, or tetrafluoroborate, and

when m is 2, n is 1, and when m is 1, n is zero.

6. The detectable compound of claim 1, wherein R and R₁ are each 2,2′

-bipyridyl.

7. The detectable compound of claim 1 wherein

R and R₁ are the same or different and are 2,2′

-bipyridyl, 4,4′

-dimethyl-2,2′

-bipyridyl, 4-4′

-diphenyl-2,2′

-bipyridyl, 1,10-phenanthrolinyl, 4,7-dimethyl-1,10-phenanthrolinyl, 4,7-diphenyl-1,10-phenanthrolinyl, or 5,6-dimethyl-1,10-phenanthrolinyl; and

R and R₁ are each coordinated to Os through their nitrogen atoms.

8. The detectable compound of claim 1, 2, 3, 4, 5, 6, or 7 wherein q is 1, the group R₇ is bonded to C-4, and —

L—

is the divalent residue of a heterofunctional crosslinker of the formula S—

L′

—

T wherein L′

is a divalent linker and S and T are different electrophilic groups capable of reacting with a nucleophilic group to form a covalent bond.

9. The detectable compound of claim 1, 2, 3, 4, 5, 6, or 7 wherein q is 1, the group R₇ is bonded to C-4, and —

L′

—

is the divalent residue of a homofunctional crosslinker of the formula S—

L′

—

T wherein L′

is a divalent linker and S and T are the same electrophilic groups capable of reacting with a nucleophilic group to form a covalent bond.

10. The detectable compound of claim 8 wherein B comprises an epitope recognizable by an antibody capable of specific binding to a biologically significant compound so that the antibody is capable of specific binding to said compound competitively with the biologically significant compound.

11. The detectable compound of claim 9 wherein B comprises an epitope recognizable by an antibody capable of specific binding to a biologically significant compound so that the antibody is capable of specific binding to said compound competitively with the biologically significant compound.

12. The detectable compound of claim 11 wherein B comprises a glycosylated peptide of the formula

13. The detectable compound of claim 11 wherein B comprises a peptide of the formula

14. A method for measuring the concentration of an analyte in a sample of a liquid, said method comprising

contacting said sample with predetermined amounts of (1) a specific binding partner for said analyte, and (2) a detectable compound of claim 1 wherein B comprises a ligand selected so that the detectable compound binds competitively with the analyte to said binding agent, and

determining electrochemically the concentration of the detectable compound of claim 1 not bound to the specific binding agent.

15. The method of claim 14 wherein the liquid sample is diluted blood, the analyte is glycosylated hemoglobin and the group B in the compound of claim 1 comprises a glycosylated peptide of the formula

16. The method of claim 15 further including the step of determining the concentration of total hemoglobin and unglycosylated hemoglobin in the liquid sample, and calculating the ratio of the concentration of glycosylated hemoglobin to unglycosylated hemoglobin.

17. The method of claim 15 wherein the concentration of the unbound compound of claim 1 is determined by dc voltammetry using an interdigitated microarray electrode.