Apparatus and method for assaying optical isomers

An apparatus for assaying the L- and D-optical isomers in a sample by the flow technique which comprises:

• (I)
• (I-i) means for injecting the sample into a carrier being fed from upstream of a reactor;
• (I-ii) a reactor containing said dehydrogenase for the D-optical isomer and a dehydrogenase for the L-optical isomer in immobilized form;
• and
• (I-iii) an immobilized oxidase for the L-optical isomer and an electrode for detecting the change in the concentration of an electrochemically active substance as resulting from the oxidation of the L-optical isomer in the sample by the action of the immobilized oxidase for said L-optical isomer and said immobilized oxidase being disposed downstream from said reactor;
• or
• (II)
• (II-i) means for injecting the sample into a carrier being fed from upstream of a reactor;
• or
• (II-ii) a reactor containing said dehydrogenase for the D-optical isomer and a dehydrogenase for the L-optical isomer in immobilized form;
• and
• (II-iii) an immobilized oxidase for the D-optical isomer and an electrode for detecting the change in the concentration of an electrochemically active substance as resulting from the oxidation of the D-optical isomer in the sample by the action of the immobilized oxidase for said D-optical isomer and said immobilized oxidase being disposed downstream from said reactor.

In a method of assaying the D- and L-optical isomers in a sample wherein the method comprises the steps of:

• (I)
• (I-1) contacting the sample and an oxidized coenzyme with an enzyme system containing a D-dehydrogenase and a L-dehydrogenase to convert the D-optical isomer in the sample to L-optical isomer and/or the L-optical isomer in said sample to D-optical isomer and
• (I-2) detecting electrochemically the change in the concentration of an electrochemically active substance as resulting from the oxidation of the L-optical isomer in the sample by the action of an oxidase for the L-optical isomer, and
• (I-a) in step (I-2), obtaining a first working curve showing the relation between the concentration of the L-optical isomer and the output value in electrochemical detection using standard solutions of the L-optical isomer as samples,
• (I-b) performing step (I-1) using standard solutions of the L-optical isomer as samples and subsequently performing step (I-2) to obtain a second working curve showing the relation between the concentration of the L-optical isomer and the output value in electrochemical detection,
• (I-c) performing step (I-1) using standard solutions of the D-optical isomer as samples and subsequently performing step (I-2) to obtain a third working curve showing the relation between the concentration of the D-optical isomer and the output value in electrochemical detection,
• (I-d) further performing step (I-2) using the assay sample to obtain a first output value,
• (I-e) performing step (I-1) suing the assay sample and subsequently performing step (I-2) to obtain a second output value, and calculating the content of the D- and L-optical isomers in the assay sample based on the first and second output values and the first, second and third working curves;
• or
• (II)
• (II-1) contacting the sample and an oxidized coenzyme with an enzyme system containing a D-dehydrogenase and a L-dehydrogenase to convert the D-optical isomer in the sample to L-optical isomer and/or the L-optical isomer in said sample to D-optical isomer and
• (II-2) detecting electrochemically the change in the concentration of an electrochemically active substance as resulting from the oxidation of the D-optical isomer in the sample by the action of an oxidase for the D-optical isomer, and
• (II-a) in step (II-2), obtaining a first working curve showing the relation between the concentration of the D-optical isomer and the output value in electrochemical detection using standard solutions of the D-optical isomer as samples,
• (II-b) performing step (II-1) using standard solutions of the D-optical isomer as samples and subsequently performing step (II-2) to obtain a second working curve showing the relation between the concentration of the D-optical isomer and the output value in electrochemical detection,
• (II-c) performing step (II-1) using standard solutions of the D-optical isomer as samples and subsequently performing step (II-2) to obtain a third working curve showing the relation between the concentration of the D-optical isomer and the output value in electrochemical detection,
• (II-d) further performing step (II-2) using the assay sample to obtain a first output value,
• (II-e) performing step (II-1) using the assay sample and subsequently performing step (II-2) to obtain a second output value, and calculating the content of the D- and L-optical isomers in the assay sample based on the first and second output values and the first, second and third working curves.

A method of assaying 3 components of the L-optical isomer, D-optical isomer and the oxidation product derived from the L-optical isomer or D-optical isomer as occurring in a sample which comprises the steps of:

• (I)
• (I-1) assaying the L-optical isomer in said sample,
• (I-2) subjecting the sample to an enzymatic reaction in the presence of a L-dehydrogenase and a D-dehydrogenase of said optical isomers and an oxidized coenzyme and converting the L-optical isomers in the sample to D-optical isomer and/or the D-optical isomer in the sample to L-optical isomer and subsequently assaying the L-optical isomer after said enzymatic reaction, and
• (I-3) contacting the sample with a L-dehydrogenase in the presence of a reduced coenzyme to convert an oxidation product in the sample to the L-optical isomer and assaying the L-optical isomer and calculating the concentration of L-optical isomer and D-optical isomer from the results of steps (I-1), (I-2), and calculating the concentration of the oxidation product derived from the L-optical isomer or D-optical isomer from the results of steps (I-1), (I-3);
• or
• (II)
• (II-1) assaying the D-optical isomer in said sample,
• (II-2) subjecting the sample to an enzymatic reaction in the presence of a L-dehydrogenase and a D-dehydrogenase of said optical isomers and an oxidized coenzyme and converting the L-optical isomer in the sample to D-optical isomer and/or the D-optical isomer in the sample to L-optical isomer and subsequently assaying the D-optical isomer after said enzymatic reaction, and
• (II-3) contacting the sample with a D-dehydrogenase in the presence of a reduced coenzyme to convert an oxidation product in the sample to the D-optical isomer and assaying the D-optical isomer and calculating the concentration of L-optical isomer and D-optical isomer from the results of steps (II-1), (II-2), and calculating the concentration of the oxidation product derived from the L-optical isomer or the D-optical isomer from the results of steps (II-1), (II-3).

A kit comprising:

• (I-1) a first container containing a reagent comprising L-dehydrogenase and D-dehydrogenase of optical isomers and an oxidized coenzyme to convert L-optical isomer in a sample to D-optical isomer and/or D-optical isomer in a sample to L-optical isomer, or
• (I-2) a second container containing a reagent comprising L-dehydrogenase of the optical isomers and a reduced coenzyme to convert an oxidized-form of the optical isomer in a sample to L-optical isomer;
• or
• (II)
• (II-1) a first container containing a reagent comprising L-dehydrogenase and D-dehydrogenase of optical isomers and an oxidized coenzyme to convert L-optical isomer in a sample to D-optical isomer and/or D-optical isomer in a sample to L-optical isomer, or
• (II-2) a second container containing a reagent comprising D-dehydrogenase of the optical isomers and a reduced coenzyme to convert an oxidized-form of the optical isomer in a sample into D-optical isomer.