Methods for determining peroxidatively active substances

• US 5,332,662 A
• Filed: 07/31/1992
• Issued: 07/26/1994
• Est. Priority Date: 07/31/1992
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Products GmbH (Siemens AG)
• Original Assignee: Syntec Incorporated (Atos SE)
• Inventors: Ullman, Edwin F.
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): Leary, Louise N.
• Application Number:
  US07/923,080
• Time in Patent Office: 725 Days
• Field of Search: 435/28, 435/19, 435/25, 435/4, 435/7.1, 435/810, 435/188, 436/537, 436/534, 536/4.1, 536/17, 536/17.9
• US Class Current: 435/28
• CPC Class Codes: C07D 219/06 Oxygen atoms
• CPC Class Codes: C12Q 1/28 involving peroxidase
• CPC Class Codes: C12Q 2326/00 Chromogens for determinations of oxidoreductase enzymes
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Methods and compositions are disclosed for determining a peroxidatively active substance (PAS). The methods comprise the step of detecting a fluorescent signal produced upon cleavage of a compound of the formula F-L-Q, wherein F is a fluorester capable of producing the signal, Q is a quencher capable of quenching the signal when linked to F, and L is a bond, or a linking group having a bond, wherein the bond is capable of being cleaved by a reaction of the PAS with a substrate of the PAS and a hydrogen donor wherein the cleavage of the bond substantially reduces the quenching. The methods have application in a wide variety of systems including assays and improved assays for analytes. Also disclosed are kits for conducting the methods and improvements in accordance with the present invention.

Claims

1. A kit for detecting a peroxidatively active substance (PAS) in a sample or medium suspected of containing said PAS, said kit comprising in packaged combination:

a compound of the formula F-L-Q wherein F is a fluorescer capable of producing a fluorescent signal, Q is a quencher capable of quenching by energy transfer said signal when linked to F, and L is a bond or linking group containing a bond wherein said bond is capable of being cleaved by an oxidation-reduction reaction of said PAS with a substrate of said PAS and a hydrogen donor wherein said cleavage substantially reduces said quenching; and

a hydrogen donor.

2. The kit of claim 1 wherein said bond is contained in a functional group selected from the group consisting of ethers, thioethers, disulfides esters, amides, and carbonates.

3. The kit of claim 1 wherein said fluorescer is contained in a moiety selected from the group consisting of fluorescein, rhodamine, acridine, rosamine, naphthylamine, coumarin, benzoxazole, benzoxdiazole, anthracene, merocyanine, and perylene moieties.

4. The kit of claim 1 wherein said peroxidatively active substance is selected from the group consisting of complexed transition metal ions, hemes, hemoproteins, and peroxidases.

5. The kit of claim 1 wherein said quencher is contained in a moiety selected from the group consisting of aniline, phenol, and thiophenol moieties.

6. The kit of claim 1 wherein said F-L-Q is of the formula:

##STR35## wherein;

X is O, S (sulfur atom), or NR₁ wherein R₁ is H, alkyl, or aryl, with the proviso that O or S (sulfur atom) be bound to carbon atoms;

Z is C or N;

b is 0 or 1;

a and c are independently 0, 1 or 2 with the proviso that the ring contain 5 to 7 atoms;

R is independently H or a substituent having from 1 to 50 atoms other than hydrogen, which atoms are independently selected from the group consisting of carbon, oxygen, nitrogen, sulfur, phosphorus, and halogen, or two R'"'"'s can be taken together to form a ring, being substituted or unsubstituted, a single ring or part of a fused ring system, wherein one R is OH, SH (thio), or amino nitrogen bound to a site on the ring, wherein said site is separated from D by an even number of ring atoms excluding X; and

D is O or S (sulfur atom) wherein said O or S (sulfur atom) is bonded to F or Q with the proviso that the two electron oxidation potential of said compound of the formula F-L-Q is greater than that of said hydrogen donor.

7. The kit of claim 1 wherein said hydrogen donor is selected from the group consisting of benzidines, p-aminophenols, 4-aminoantipyrenes, and p-phenylenediamines.

8. The kit of claim 1 wherein said hydrogen donor is of the formula:

##STR36## wherein;

A is a hydroxyl group, an amino group, --NHR₃, or --NR₃ R₄, wherein R₃ and R₄ are independently an alkyl group having 1 to 5 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, a hydroxyalkyl group having 1 to 5 carbon atoms, or an acylaminoalkyl group having 1 to 5 carbon atoms;

or R₃ and R₄ may combine to form a ring which is unfused or fused, substituted or unsubstituted, a single ring, or part of a multi-ring system;

R₁ and R₂ are independently a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, a halogen atom, a carboxyl group, a methanesulfonyl group or a phenyl group;

or R₁ and R₂ may combine together to form a cycloalkene, an aromatic ring or a heterocyclic ring; and

n is 1 to 5.