Redox reversible bipyridyl-osmium complex conjugates

• US 20020090632A1
• Filed: 11/01/2001
• Published: 07/11/2002
• Est. Priority Date: 06/01/1998
• Status: Active Grant

• Current Assignee: Roche Diabetes Care Inc. (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Buck, Harvey B. Jr., Deng, Zhi David
• Granted Patent: US 7,045,310 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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 bipyridyl-osmium complex conjugates 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. 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 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,

R₅ is 4-substituted-2,2′

-bipyridyl or 4,4′

-disubstituted-2,2′

-bipyridyl wherein the 4-substituent is the group B-(L)_k-Q(CH₂)_i- and the 4′

-substituent is a methyl, ethyl or phenyl group;

R, R₁ and R₅ are coordinated to Os through their nitrogen atoms;

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;

B comprises a ligand that binds to the specific binding partner and is selected so that the detectable compound binds competitively with the analyte to said binding partner;

d is +2 or +3;

X and Y are anions selected from monovalent anions chloride, bromide, iodide, fluoride, tetrafluoroborate, perchlorate, and nitrate and divalent anions sulfate, carbonate or sulfite wherein x and y are independently 0, 1, 2, or 3 so that the net charge of X_xY_y is -2 or -3; and

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

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

Gluc-Val-His-Leu-Thr-

3. The method of claim 2 further including the step of determining the concentration of total hemoglobin or unglycosylated hemoglobin in the liquid sample, and calculating the ratio of the concentration of glycosylated hemoglobin to unglycosylated hemoglobin.

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