Amperometric method for the quantitative determination of 1,4-dihydronicotinamide adenine dinucleotide (NADH) in solution
First Claim
1. A method for the quantitative amperometric determination of NADH in solution in a liquid amenable to measurable electrochemical action of NADH therein, which comprises:
- (a) contacting a sample of said liquid with an electrode, said electrode comprising in contact with the sample a porous layer of finely divided activated carbon or graphite particles having finely divided platinum or palladium adsorbed thereonto, said particles being bonded together in said layer by a resin;
(b) maintaining said electrode at a potential effective to cause oxidation of NADH in the sample; and
(c) measuring the current produced.
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Abstract
A method is disclosed for the quantitative determination of 1,4-dihydronicotinamide adenisne dinucleotide (NADH) in solution. The method comprises contacting the NADH-containing solution with an activated carbon electrode, maintaining the carbon electrode at a controlled, fixed potential effective to cause oxidation of NADH at the electrode surface, and measuring the current output from the carbon electrode, wherein there is used a noble metal containing preferably a platinized or palladized activated carbon electrode comprising a porous, heterogeneous, resin-bonded layer of activated carbon or graphite particles comprising the finely divided noble metal preadsorbed thereon and bonded together with a natural or synthetic resin binder, preferably a hydrophobic resin such as polytetrafluoroethylene.
15 Citations
24 Claims
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1. A method for the quantitative amperometric determination of NADH in solution in a liquid amenable to measurable electrochemical action of NADH therein, which comprises:
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(a) contacting a sample of said liquid with an electrode, said electrode comprising in contact with the sample a porous layer of finely divided activated carbon or graphite particles having finely divided platinum or palladium adsorbed thereonto, said particles being bonded together in said layer by a resin; (b) maintaining said electrode at a potential effective to cause oxidation of NADH in the sample; and (c) measuring the current produced. - View Dependent Claims (4, 5, 7, 8, 9, 13, 14)
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2. A method for the quantitative amperometric determination of a compound selected from the group consisting of NADH, oxidized NADH (NAD), phosphorylated NADH (NADPH) and oxidized NADPH (NADP) in solution in a liquid amenable to measurable electrochemical action of said compound therein, which comprises:
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(a) contacting a sample of said liquid with an electrode, said electrode comprising in contact with the sample a porous layer of finely divided activated carbon or graphite particles having finely divided noble metal or noble metal oxide adsorbed thereonto, said particles being bonded together in said layer by a resin; (b) maintaining said electrode at a potential effective to cause change of the state of oxidation of said compound in the sample; and (c) measuring the current produced. - View Dependent Claims (3, 6)
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10. A method for amperometric determination of the quantity in solution in a liquid of a substance that will undergo enzymatic reaction in the liquid in the presence of an enzyme capable of catalyzing said reaction when NADH is present in the solution as a cofactor to be oxidized electrochemically concomitantly with the enzymatic reaction, which method comprises:
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(a) contacting a sample of the liquid, in the presence of both said enzyme and said cofactor, with an electrode maintained at a fixed potential effective to cause oxidation of NADH in the sample, said electrode comprising in contact with the sample a porous layer of activated carbon or graphite particles having finely divided platinum or palladium adsorbed thereonto, said particles being bonded together in said layer by a resin, (b) thereby initiating the enzymatic reaction of said substance with concomitant oxidation of NADH in the sample; (c) measuring the current produced; and (d) determining from the measured current the concentration, or change of concentration, of NADH in the sample as a result of the enzymatic reaction, whereby to determine indirectly the amount of said substance present in the sample. - View Dependent Claims (15, 16, 17, 18, 19)
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11. A method for amperometric determination of the quantity in solution in a liquid of a substance that will undergo enzymatic reaction in the liquid in the presence of an enzyme capable of catalyzing said reaction when a compound selected from the group consisting of NADH, oxidized NADH (NAD), phosphorylated NADH (NADPH) and oxidized NADPH (NADP) is present in the solution as a cofactor to be reacted electrochemically concomitantly with the enzymatic reaction, which method comprises:
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(a) contacting a sample of the liquid, in the presence of both said enzyme and a said cofactor, with an electrode maintained at a fixed potential effective to cause electrochemical change of the state of oxidation of said cofactor in the sample, said electrode comprising in contact with the sample a porous layer of activated carbon or graphite particles having finely divided noble metal or noble metal oxide adsorbed thereonto, said particles being bonded together in said layer by a resin, (b) thereby initiating the enzymatic reaction of said substance with concomitant change of the state of oxidation of said cofactor in the sample; (c) measuring the current produced; and
(d) determining from the measured current the concentration, or change of concentration, of cofactor in the sample as a result of the enzymatic reaction, whereby to determine indirectly the amount of said substance present in the sample. - View Dependent Claims (12)
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20. An enzyme electrode for use in amperometric determination of the quantity in a liquid sample of a substance that will undergo enzymatic reaction in the sample in the presence of an enzyme capable of catalyzing said reaction when also in the presence of NADH in solution as a cofactor to be oxidized electrochemically concomitantly with the enzymatic reaction, said electrode comprising
(a) a porous layer of finely divided activated carbon or graphite particles having finely divided platinum or palladium adsorbed thereonto, said particles being bonded together in said layer by a resin; -
(b) a said enzyme adsorbed or immobilized in said layer; and (c) NADH adsorbed in said layer so as to be supplied therefrom into solution in the sample, for oxidation in the sample concomitantly with the enzymatic reaction of said substance therein, when the sample is in contact with said layer and an electrical potential is applied through said electrode, whereby electrochemical change of the state of oxidation of said cofactor may be sensed via the electrode to indicate indirectly the concentration of said substance in the sample. - View Dependent Claims (22, 23)
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21. An enzyme electrode for use in the aperometric determination of the quantity in a liquid sample of a substance that will undergo enzymatic reaction in the sample in the presence of an enzyme capable of catalyzing said reaction when also in the presence in solution of a cofactor, selected from the group consisting of NADH, oxidized NADH (NAD), phosphorylated NADH (NADPH) and oxidized NADPH (NADP), to be oxidized or reduced electrochemically concomitantly with the enzymatic reaction, said enzyme electrode comprising
(a) a porous layer of finely divided activated carbon or graphite particles having finely divided noble metal or noble metal oxide adsorbed thereonto, said particles being bonded together in said layer by a resin; -
(b) a said enzyme adsorbed or immobilized in said layer; and (c) a said cofactor adsorbed in said layer so as to be supplied therefrom into solution in the sample for electrochemical reaction in the sample concomitantly with the enzymatic reaction of said substance therein when the sample is in contact with said layer and an electrical potential is applied through said electrode, whereby electrochemical change of the state of oxidation of said cofactor may be sensed via the electrode to indicate indirectly the concentration of said substance in the sample. - View Dependent Claims (24)
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Specification