Device for detecting oxygen with oxidase

• US 5,804,401 A
• Filed: 01/15/1997
• Issued: 09/08/1998
• Est. Priority Date: 01/09/1995
• Status: Expired due to Fees

• Current Assignee: The Board of Trustees of Michigan State University (Michigan State University)
• Original Assignee: The Board of Trustees of Michigan State University (Michigan State University)
• Inventors: Harte, Bruce R., Gardiol, Alicia E., Hernandez, Ruben J.
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US08/784,088
• Time in Patent Office: 601 Days
• Field of Search: 435/25, 435/190, 435/286.6, 435/287.1, 435/287.5, 435/288.1, 435/288.7, 436/810, 436/904, 204/403, 204/415
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/26 involving oxidoreductase

Abstract

A method and device (10) for removing oxygen using a reduced oxidase enzyme and for providing a calorimetrically detectable signal of the presence of the oxygen. A reduced laccase or ascorbate oxidase is preferably provided with a substrate which reduces the oxidase. Color is generated which can be detected visually or by other means such as a spectrophotometer. The method and device are particularly useful in the food, pharmaceutical and biological industries.

Claims

1. A device for detecting the presence of oxygen gas in a confined space and which provides a calorimetrically detectable indication of the presence of the oxygen gas which comprises:

(a) a confined space free of oxygen gas, wherein the confined space is provided with a means which allows introduction of the oxygen gas from outside of the confined space;

(b) a reduced oxidase in an aqueous solution in the confined space in an amount which reacts with oxygen introduced into the confined space from outside of the confined space through the means for introduction to provide a colorimetrically detectable indication of the presence of the oxygen at a predetermined concentration due to a change of light absorbance of an oxidized oxidase, which oxidase is directly measured for the light absorbance, which light absorbance is different from the reduced oxidase in the aqueous solution; and

(c) means for correlating the light absorbance of the oxidized enzyme to the presence of oxygen in the confined space.

2. The device of claim 1 wherein the oxidase is a laccase.

3. The device of claim 1 wherein the oxidase is a laccase from Rhus vernicifera and the oxidized oxidase in the aqueous solution is blue in color when exposed to light at 610 nm.

4. The device of any one of claims 1, 2 or 3 wherein the confined space is confined by a transparent container.

5. The device of any one of claims or 1, 2 or 3 wherein the confined space is a thin transparent plastic pouch which allows the oxygen to be transported through the plastic.

6. A test device for determining the presence of oxygen gas in a surrounding fluid containing a material which is deleteriously affected by the oxygen gas and which provides a calorimetrically detectable indication of the presence of the oxygen which comprises:

(a) a container with an atmosphere free of oxygen gas wherein the container is provided with a means which allows permeation of the oxygen gas when present outside of the container into the container from the surrounding fluid;

(b) a reduced oxidase in an aqueous solution in the container in an amount which reacts with oxygen introduced into container at a predetermined concentration to provide a calorimetrically detectable indication of the presence of the oxygen due to a chance of light absorbance of an oxidized oxidase, which is directly measured for the change of the light absorbance, from that of the reduced oxidase in the aqueous solution; and

(c) means for correlating the light absorbance of the oxidized oxidase to determine the presence of oxygen in the surrounding fluid.

7. The device of claim 6 wherein the oxidase is a laccase.

8. The device of claim 6 wherein the oxidase is a laccase from Rhus vernicifera and the oxidized oxidase in the aqueous solution is blue in color when exposed to light at 610 nm.

9. The device of any one of claims 6, 7 or 8 wherein the container allows a visualization of the oxidized oxidase.

10. The device of any one of claims 6, 7 or 8 wherein the container is a thin plastic pouch which allows the oxygen to be transported through the plastic and allows visualization of the oxidized oxidase.

11. The device of any one of claims 6 to 8 wherein the reduced oxidase is present at a concentration between about 0.001 and 1 μ

m.

12. The device of claim 6 at a temperature between about 0°

and 30°

C.

13. The device of claim 12 wherein the reduced oxidase contains a substrate which reduces an oxidized oxidase to the reduced oxidase.

14. The device of claim 13 wherein the solution containing the reduced oxidase also contains a chelating agent.

15. The device of claim 14 wherein the chelating agent is ethylenediaminetetraacetic acid.

16. The device of claim 13 wherein the substrate is an ascorbate salt.

17. The device of claim 16 wherein the ascorbate salt is sodium ascorbate.