Method of using enzyme electrode

• US 5,352,348 A
• Filed: 11/03/1992
• Issued: 10/04/1994
• Est. Priority Date: 04/09/1987
• Status: Expired due to Term

• Current Assignee: Nova Biomedical Corporation (Nova Chemicals)
• Original Assignee: Nova Biomedical Corporation (Nova Chemicals)
• Inventors: Winarta, Handani, Young, Chung Chang
• Primary Examiner(s): TUNG, TA HSUNG
• Application Number:
  US07/970,944
• Time in Patent Office: 700 Days
• Field of Search: 204/153.17, 204/153.12, 204/415, 204/403
• US Class Current: 205/778
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: C12Q 1/006 for glucose

Abstract

Disclosed is a method of assaying a high concentration of a substance in a liquid sample with a polarographic cell. The cell contains an electrode assembly that includes a reference electrode and a hydrogen peroxide sensor electrode having a laminated membrane covering the liquid sample contacting face of the sensor electrode. The laminated membrane includes an outer membrane permeable to the substance and oxygen, an inner membrane permeable to hydrogen peroxide and located adjacent the face of the sensor electrode, and an enzyme layer between the inner and outer membrane; the enzyme in the enzyme layer can oxidize the substance to generate hydrogen peroxide. The method includes contacting the outer membrane with the liquid sample; permitting the substance and oxygen in the liquid sample to pass through the outer membrane to contact the enzyme layer so that where the substance is oxidized to generate hydrogen peroxide; permitting the generated hydrogen peroxide to pass through the inner membrane to contact the sensor electrode; and ensuring that the supply of oxygen in the enzyme layer relative to the supply of glucose is sufficient to produce an equilibrium concentration of hydrogen peroxide, which generates a steady state response at the sensor electrode proportional to the concentration of the substance in the liquid sample.

Claims

1. A method of assaying the concentration of a substance in an undiluted whole blood or undiluted plasma sample, which includes catalase, comprising

providing a polarographic cell that includes an electrode assembly comprising a reference electrode and a hydrogen peroxide sensor electrode having a laminated membrane covering a sample-contacting face of said sensor electrode, said laminated membrane comprising an outer membrane permeable to said substance and oxygen while preventing the passage of proteins and red blood cells, an inner membrane permeable to hydrogen peroxide and located adjacent the face of said sensor electrode, and an enzyme layer between said inner membrane and said outer membrane, said enzyme layer comprising an enzyme that can oxidize said substance to generate hydrogen peroxide,

said outer membrane having pores that allow passage of said substance and oxygen from said undiluted sample to said enzyme layer, the thickness of said outer membrane and the size of said pores being selected so that said passage of said substrate is sufficiently hindered relative to said passage of said oxygen that a steady state response proportional to the substance concentration with said electrode assembly can be obtained at substance concentrations in an undiluted whole blood sample of at least 300 mg/dl where the oxygen concentration is equivalent to the oxygen concentration in a plasma sample derived from a normal human subject, and

said outer membrane having a thickness of between 20 and 100μ

to impair the passage of hydrogen peroxide from said enzyme layer to said undiluted sample to an extent great enough to permit said steady state to be reached;

contacting said outer membrane with said undiluted sample comprising said substrate to permit said substance and oxygen in said undiluted sample to pass through said outer membrane to said enzyme layer so that said enzyme oxidizes said substance to generate hydrogen peroxide, said generated hydrogen peroxide passing through said inner layer to contact said face of said sensor electrode;

maintaining said contact between said outer membrane and said undiluted sample for a sufficient period of time to generate a steady state responses proportional to the substrate concentration; and

measuring said steady state response to determine said substrate concentration.

2. The method of claim 1 wherein said outer membrane has a thickness of between 18 and 100μ

.

3. The method of claim 1 wherein said substance is glucose and said enzyme is glucose oxidase.

4. The method of claim 1 wherein said substance is lactate and said enzyme is lactate oxidase.

5. The method of claim 1 wherein said substance is alcohol and said enzyme is alcohol oxidase.

6. The method of claim 1 wherein said substance is cholesterol and said enzyme is cholesterol oxidase.

7. The method of claim 1 wherein said sample is undiluted whole blood.

8. The method of claim 1 wherein said sample is undiluted plasma.