Method for measuring substrate concentrations

• US 4,129,478 A
• Filed: 10/18/1976
• Issued: 12/12/1978
• Est. Priority Date: 08/01/1974
• Status: Expired due to Term

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Higelin, Jean-Claude, Racine, Philippe, Mindt, Wolfgang, Engelhardt, Roland
• Primary Examiner(s): Tung, T.
• Application Number:
  US05/733,570
• Time in Patent Office: 785 Days
• Field of Search: 204/1 E, 204/195 B, 204/195 P, 195/103.5 R, 195/103.5 C
• US Class Current: 205/778
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

The concentration of substrates of enzyme reactions is measured by determining the current produced at an platinum or gold electrode by oxidizing an acceptor utilizing a measuring device comprising a measuring cell, an enzyme electrode in contact with the fluid sample and containing an platinum or gold electrode, an enzyme layer and a reference electrode in contact with the fluid sample. In this manner important physiological parameters, such as the concentration of lactate and glucose in fluid biological samples, can be measured quickly and accurately.

Claims

1. A method of measuring the concentration of substrates of enzyme reactions occurring during intermediate metabolism, more particularly the concentration of lactate and glucose in fluid biological samples, wherein the substrate concentration is measured by measuring a current produced by oxidation of an acceptor at an enzyme electrode with a measuring device substantially comprising a measuring cell for receiving the fluid sample, the enzyme electrode and a reference electrode, both electrodes being in contact with the fluid sample, and the enzyme electrode containing a platinum or gold electrode, an enzyme layer and a semi-permeable membrane covering the enzyme layer, the temperature at which the enzymatic reaction takes place being regulated at a pre-determined value and a direct voltage being applied between the enzyme electrode and the reference electrode, which method is characterized by performing a local temperature regulation of the enzyme layer within the enzyme electrode by means of a temperature control of the gold or platinum electrode thereof, filling the measuring cell with a buffer solution containing an acceptor prior to filling the cell with a fluid sample and keeping the cell filled with said buffer solution over a time interval long enough to enable diffusion of a quantity of the acceptor through the semi-permeable membrane of the enzyme electrode into the enzyme-containing layer, said quantity of acceptor being sufficiently high for assuring a linear response of the enzyme electrode, sucking the buffer solution from the measuring cell at the end of said time interval, filling the measuring cell with a fluid sample and measuring the concentration of the substrate contained in the fluid sample by determining the peak value of the current flowing through the enzyme electrode with an electronic circuit.

2. A method according to claim 1, wherein potassium hexacyanoferrate-(III) is used as an acceptor for measuring the concentration of glucose in fluid biological samples.

3. A method according to claim 2, wherein the fluid samples are diluted biological fluids.

4. A method according to claim 3, wherein the dilution ratio is between 1:

5 and 1;

20.