Method for quantitatively determining a particular substrate catalyzed by a multisubstrate enzyme

• US 4,404,066 A
• Filed: 05/13/1982
• Issued: 09/13/1983
• Est. Priority Date: 08/25/1980
• Status: Expired due to Term

• Current Assignee: YSI Incorporated (Xylem, Inc.)
• Original Assignee: The Yellow Springs Instrument Company Incorporated
• Inventors: Johnson, Jay M.
• Primary Examiner(s): Kaplan, G. L.
• Application Number:
  US06/377,765
• Time in Patent Office: 488 Days
• Field of Search: 204/1 E, 204/195 B, 204/195 P, 204/403, 435/190, 435/817
• US Class Current: 205/778
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

A method for control of relative enzyme activity for analytical purposes. The activity of a multisubstrate enzyme may be controlled as a function of the electrical potential applied to the enzyme. The enzyme is preferrably incorporated into a thin layer electrochemical cell laminate having exterior membrane layers and an interior enzyme layer. A control electrode located within the enzyme layer applies an electrical potential to the enzyme. An intermediate electron transfer agent may be used to transfer electrons to and from the enzyme and the control electrode.

Claims

1. In a method for quantitatively determining the amount of a particular substrate, catalyzed by a multisubstrate enzyme (having a direct redox dependent activity) to react with oxygen and form hydrogen peroxide as a reaction product, wherein the improvement comprises the steps of:

applying an electrical potential to said enzyme, said electrical potential being sufficient to control the activity of said multisubstrate enzyme, contacting said enzyme with a solution containing at least said particular substrate, and measuring for an electroactive reactant or product of the reaction mechanism whereby a rapid, direct quantitative analysis is obtained.

2. The method of claim 1 wherein a single substrate is present and said electrical potential is maintained at a predetermined level in order to boost and stabilize the activity of the substrate.

3. The method of claim 1 wherein more than one substrate is present and said electrical potential is selected to eliminate activity toward all but one of the substrates.

7. The method of claim 1, 4 or 6 wherein hydrogen peroxide is the electroactive product being measured.

8. The method of claim 1 or 6 wherein galactose oxidase is the multisubstrate enzyme.

9. The method of claims 1, 4 or 6 wherein said multisubstrate enzyme is positioned between a first outer membrane layer and a second inner membrane layer,

said first outer membrane layer adapted to be adjacent said solution and allowing passage of low molecular weight materials therethrough but excluding high molecular weight materials contained in said solution,

said second inner membrane layer adapted to be adjacent the means for measuring said electroactive reactant or product of the reaction mechanism and excluding passage of interfering low molecular weight materials and any mediator but permitting passage of said electroactive reactant or product of the reaction mechanism,

and said electrical potential is applied to said enzyme by an electrically conductive layer positioned between said first and second membrane layers and in contact with said enzyme.

4. In a method for quantitatively determining the amounts of particular substrates, catalyzed by a multisubstrate enzyme (having a direct redox dependent activity) to react with oxygen and form hydrogen peroxide as a reaction product, wherein the improvement comprises the steps of:

applying at least two different electrical potentials to said enzyme, said electrical potentials being sufficient to render different the relative sensitivity of the enzyme for at least two of the substrates present, contacting said enzyme with a solution containing at least two substrates, and measuring for an electroactive reactant or product of the reaction mechanism whereby a rapid, direct quantitative analysis is obtained.

5. The method of claim 4 wherein galactose oxidase is the multisubstrate enzyme and said method is conducted under essentially diffusion limiting conditions.

6. In a method of measuring for the presence of substrates, catalyzed by a multisubstrate enzyme (having a direct redox dependent activity) to react with oxygen and form hydrogen peroxide as a reaction product, wherein the improvement comprises the steps of applying an electrical potential to said enzyme, contacting said enzyme with a solution containing at least one substrate catalyzed by said enzyme, continuously varying the electrical potential applied and measuring for an electroactive reactant or product of the reaction mechanism as a function of the potential applied to said enzyme whereby a rapid, direct qualitative analysis is obtained.