Actimed Laboratories, Inc.

Alexei Sukhotin

David James Gibboni

Alexander G Ignotenko

Viktor G Kartsev

A color-forming coupler is provided which can be used in test compositions, as well as methods and apparatus for detection of hydrogen peroxide. The color-forming coupler compounds react with a hydrazone or an aminoantypyrine in the presence of peroxidase and hydrogen peroxide to produce a darkly colored compound. In the presence of peroxidase and hydrogen peroxide, the couplers of the present invention react with a hydrazone or an aminoantipyrine to produce a deeply colored dye, which can be used to give a visual indication of the analyte present in the sample. These couplers can be used in diagnostic tests in which the analyte is converted to hydrogen peroxide. The color-forming couplers are N,N-disubstituted anilines of the formula shown in FIG. 1 , wherein R.sup.1 and/or R.sup.2 =H, C.sub.1 -C.sub.9 alkyl, C.sub.1 -C.sub.9 -C.sub.9 alkoxy, NR.sup.3 R.sup.4 (where R.sup.3 and/or R.sup.4 =H, C.sub.1 -C.sub.9 alkyl, aryl, or heteroryl), F, Cl, Br, I, COOR.sup.5 (where R.sup.5 =H, C.sub.1 -C.sub.9 alkyl, aryl, or heteroaryl), CN, CONR.sup.6 R.sup.7 (where R.sup.6 and/or R.sup.7 =H, C.sub.1 -C.sub.9 alkyl, aryl, or heteraryl), aryl, aryloxy, heteroaryl, heteroaryloxy; and R.sup.11, R.sup.12, R.sup.13, and/or R.sup.14 =H, C.sub.1 -C.sub.9 alkyl, C.sub.1 -C.sub.9 alkoxy, or NR.sup.15 R.sup.16 wherein at least one of R.sup.11 and R.sup.14 is NR.sup.15 R.sup.16 wherein only one of R.sup.11 and R.sup.14 is H or C.sub.1 -C.sub.2 alkyl; where R.sup.15 and/or R.sup.16 =H, C.sub.1 -C.sub.9 alkyl, aryl, or heteroaryl, F, Cl, Br, I, COOR.sup.17, where R.sup.17 =H, C.sub.1 -C.sub.9 alkyl, aryl, or heteroaryl, CN, CONR.sup.18 R.sup.19 (where R.sup.18 and/or R.sup.19 =H, C.sub.1 -C.sub.9 alkyl, aryl, or heteroaryl, aryl, aryloxy, heteroaryl, heteroaryloxy; and n=0-10 and m=0-10 and Z and/or y=H, OH, SH, COOR.sup.8 (where R.sup.8 =H, C.sub.1 -C.sub.9 alkyl, aryl, or heteroaryl), CN, NR.sup.9 R.sup.10 (where R.sup.9 and/or R.sup.10 =H, C.sub.1 -C.sub.9 alkyl, aryl, or heteroaryl), NR.sup.20 NHR.sup.21 (where R.sup.20 and/or R.sup.21 =H, C.sub.1 -C.sub.9 alkyl, aryl, or heteroaryl), where at least one of Z or Y is COOR.sup.8, CN NR.sup.9 R.sup.10, or NR.sup.20 NHR.sup.21 or 4-amino antipyrines.

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Chernogolovka, Russia

Alexei Sukhotin