Analytical process and means for measuring the amount of hydrogen peroxide in aqueous media and of organic substrates generating hydrogen peroxide by enzymatic oxidation

Claim 1

A method of quantatively determining a constituent selected from the group which consists of hydrogen peroxide and peroxidase in an aqueous sample, said method comprising the steps of: (a) mixing said sample with a reaction system selectively containing a known amount of peroxidase or hydrogen peroxide, respectively, and in excess of an organic fluorine compound in the form of a fluoroaniline, a fluoroanisole or a fluorophenol having at least one C--F bond cleaving on a peroxidase catalyzed oxidation by hydrogen peroxide whereby F - ions are formed in the system; (b) exposing the reaction system to an F - selective electrode; and (c) electrochemically measuring by said electrode the rate of formation of F - ions liberated by the peroxidase catalyzed cleavage of said C--F bond.

Claim 2

A method of quantatively determining a constituent selected from the group which consists of hydrogen peroxide and peroxidase in an aqueous sample, said method comprising the steps of: (a) mixing said sample with a reaction system selectively containing a known amount of peroxidase or hydrogen peroxide, respectively, and in excess of an organic fluorine compound in the form of a fluoroaniline, a fluoroanisole or a fluorophenol having at least one C--F bond cleaving on a peroxidase catalyzed oxidation by hydrogen peroxide whereby F - ions are formed in the system; (b) exposing the reaction system to an F - selective electrode; and (c) electrochemically measuring by said electrode the rate of formation of F - ions liberated by the peroxidase catalyzed cleavage of said C--F bond.

Claim 7

A method of quantitatively determining in an aqueous medium, an organic substrate sample, which comprises the steps of: (a) preparing an aqueous reagent solution containing at least a buffer, a conjugate oxidase specific to said organic substrate and capable of oxidizing said organic substrate to yield H 2 O 2 as an oxidation product, a peroxidase, and an organic fluoro compound selected from the group of a fluoroaniline, a fluorophenol, and a fluoroanisole, the C--F bond of which is cleavable with quantitative liberation of F - ion by peroxidase-catalyzed oxidation with the H 2 O 2 produced during the oxidase-catalyzed oxidation, the rate of the peroxidase-catalyzed oxidation being much greater than the rate of the oxidase-catalyzed oxidation, and the amount of the organic fluoro compound present being in excess of the rate limiting amount thereof for the peroxidase-catalyzed oxidation; (b) mixing said aqueous reagent solution in excess of the rate limiting amount thereof for the oxidase-catalyzed oxidation with the organic substrate sample to be analyzed to oxidize the organic substrate to form hydrogen peroxide, said hydrogen peroxide then oxidizing the organic fluoro compound in the presence of the peroxidase to quantitatively split off F - ion; (c) measuring, in said mixture formed in step (b), with an F - ion selective electrode the rate variation of the potential of said electrode with time during the peroxidase splitting reaction of said F - ion; (d) comparing said potential rate data thus obtained with standard rate potentials resulting from calibration analysis carried out previously on samples containing known concentrations of the same organic substrate being analyzed; and (e) correlating the value measured in step (c) with the standard data corresponding to the known concentration of said substrate expressed in step (d) so as to determine the concentration of the organic substrate to be analyzed.

Claim 8

A method of quantitatively determining in an aqueous medium, an organic substrate sample, which comprises the steps of: (a) preparing an aqueous reagent solution containing at least a buffer, a conjugate oxidase specific to said organic substrate and capable of oxidizing said organic substrate to yield H 2 O 2 as an oxidation product, a peroxidase, and an organic fluoro compound selected from the group of a fluoroaniline, a fluorophenol, and a fluoroanisole, the C--F bond of which is cleavable with quantitative liberation of F - ion by peroxidase-catalyzed oxidation with the H 2 O 2 produced during the oxidase-catalyzed oxidation, the rate of the peroxidase-catalyzed oxidation being much greater than the rate of the oxidase-catalyzed oxidation, and the amount of the organic fluoro compound present being in excess of the rate limiting amount thereof for the peroxidase-catalyzed oxidation; (b) mixing said aqueous reagent solution in excess of the rate limiting amount thereof for the oxidase-catalyzed oxidation with the organic substrate sample to be analyzed to oxidize the organic substrate to form hydrogen peroxide, said hydrogen peroxide then oxidizing the organic fluoro compound in the presence of the peroxidase to quantitatively split off F - ion; (c) measuring, in said mixture formed in step (b), with an F - ion selective electrode the rate variation of the potential of said electrode with time during the peroxidase splitting reaction of said F - ion; (d) comparing said potential rate data thus obtained with standard rate potentials resulting from calibration analysis carried out previously on samples containing known concentrations of the same organic substrate being analyzed; and (e) correlating the value measured in step (c) with the standard data corresponding to the known concentration of said substrate expressed in step (d) so as to determine the concentration of the organic substrate to be analyzed.

Claim 12

A method of quantitatively determining in an aqueous medium, a conjugate oxidase of an organic substrate, which comprises the steps of: (a) preparing an aqueous reagent containing at least a buffer, a peroxidase, an organic substrate for which the conjugate oxidase is specific, said organic substrate being capable of oxidation by said conjugate oxidase to yield H 2 O 2 and said organic substrate being present in excess of the rate limiting amount for the oxidase-catalyzed oxidation, and an organic fluoro compound selected from the group consisting of a fluoroaniline, a fluorophenol, and a fluoroanisole, the C--F bond of which is cleavable with quantitative liberation of F - ion by peroxidase-catalyzed oxidation with the H 2 O 2 produced during the oxidase-catlyzed oxidation, the rate of the peroxidase-catalyzed oxidation being much faster than the rate of the oxidase-catalyzed oxidation, and the amount of the organic fluoro compound present being in excess of the rate limiting amount for the peroxidase-catalyzed oxidation; (b) immersing in said aqueous reagent solution an F - selective electrode; (c) mixing said aqueous reagent solution with a sample containing the conjugate oxidase to be analyzed in order to oxidize the organic substrate to form hydrogen peroxide, said hydrogen peroxide then oxidizing the organic fluoro compound in the presence of the peroxidase to quantitatively split off F - ion, and measuring the variation of the potential of said F - selective electrode with time in response to change in the [F - ]; and (d) comparing said potential rate data thus obtained, or the corresponding [F - ] data collected from the Nernst equation, with standard rate potentials, or corresponding standard [F - ] data, resulting from calibration analysis carried out earlier on samples containing known concentrations of the conjugate oxidase to be analyzed, and correlating said values measured during step (c) with said standard data to obtain the desired results.

Claim 13

A method of quantitatively determining in an aqueous medium, a conjugate oxidase of an organic substrate, which comprises the steps of: (a) preparing an aqueous reagent containing at least a buffer, a peroxidase, an organic substrate for which the conjugate oxidase is specific, said organic substrate being capable of oxidation by said conjugate oxidase to yield H 2 O 2 and said organic substrate being present in excess of the rate limiting amount for the oxidase-catalyzed oxidation, and an organic fluoro compound selected from the group consisting of a fluoroaniline, a fluorophenol, and a fluoroanisole, the C--F bond of which is cleavable with quantitative liberation of F - ion by peroxidase-catalyzed oxidation with the H 2 O 2 produced during the oxidase-catlyzed oxidation, the rate of the peroxidase-catalyzed oxidation being much faster than the rate of the oxidase-catalyzed oxidation, and the amount of the organic fluoro compound present being in excess of the rate limiting amount for the peroxidase-catalyzed oxidation; (b) immersing in said aqueous reagent solution an F - selective electrode; (c) mixing said aqueous reagent solution with a sample containing the conjugate oxidase to be analyzed in order to oxidize the organic substrate to form hydrogen peroxide, said hydrogen peroxide then oxidizing the organic fluoro compound in the presence of the peroxidase to quantitatively split off F - ion, and measuring the variation of the potential of said F - selective electrode with time in response to change in the [F - ]; and (d) comparing said potential rate data thus obtained, or the corresponding [F - ] data collected from the Nernst equation, with standard rate potentials, or corresponding standard [F - ] data, resulting from calibration analysis carried out earlier on samples containing known concentrations of the conjugate oxidase to be analyzed, and correlating said values measured during step (c) with said standard data to obtain the desired results.