Enzymatic Biosensors With Enhanced Activity Retention For Detection Of Organic Compounds

• US 20090026092A1
• Filed: 04/09/2008
• Published: 01/29/2009
• Est. Priority Date: 06/01/2001
• Status: Active Grant

• Current Assignee: Colorado State University Research Foundation, Texas A&M University System
• Original Assignee: Colorado State University Research Foundation
• Inventors: WOOD, Thomas K., DANDY, David S., FRITZSCHE, Michael, REARDON, Kenneth F.
• Granted Patent: US 9,493,805 B2
• Time in Patent Office: Days
• US Class Current: 205/778
• CPC Class Codes: C12Q 1/002 Electrode membranes
• 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: G01N 2333/90241 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: G01N 2333/90245 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: G01N 2333/914 Hydrolases (3)
• CPC Class Codes: G01N 2333/988 Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase

Abstract

Enzymatic biosensors and methods of producing distal tips for biosensor transducers for use in detecting one or more analytes selected from organic compounds susceptible to dehalogenation, organic compounds susceptible to oxygenation and organophosphate compounds susceptible to hydrolysis are disclosed herein, as well as biosensor arrays, methods of detecting and quantifying analytes within a mixture, and devices and methods for delivering reagents to enzymes disposed within the distal tip of a biosensor.

Claims

1. A distal tip of a biosensor ion-sensing transducer for use in detecting an analyte comprising a halogenated organic compound, the tip comprising:

a biocomponent comprising a dehalogenase for carrying out dehalogenation of the compound, said biocomponent immobilized to a surface of the tip by one or more of (a) entrapment within a hydrogel;

(b) entrapment within a polymeric network;

(c) encapsulation;

(d) covalent bonding; and

(e) adsorption;

wherein said biocomponent has been treated to maintain a period of enzymatic efficacy; and

wherein said biocomponent is stabilized to said tip by one or more of crosslinking a surface of said immobilized biocomponent, crosslinking a polymer layer to said biocomponent, adding a gel-hardening agent to said biocomponent, adding a stabilizing agent to said biocomponent, and modifying a component used to immobilize said biocomponent to said surface of the tip.

2. The distal tip of claim 1, further comprising an ion-sensing transducer selected from the group consisting of a pH optode, a pH electrode, a field-effect transistor (FET) and a halide ion-selective electrode (ISE).

3. The distal tip of claim 1, the dehalogenase selected from hydrolases of subclass EC 3.8 and lyases of subclass EC 4.5.

4. The distal tip of claim 1, wherein said biocomponent is treated according to a treatment selected from the group consisting of applying an inhibitor of protein synthesis, adding a protease inhibitor, freeze drying and dry heating.

5. The distal tip of claim 1, wherein said biocomponent is selected from whole isolated cells, purified enzymes and combinations thereof.

6. A distal tip of a biosensor oxygen-sensing transducer for use in detecting an analyte comprising an organic compound, the tip comprising:

a biocomponent comprising at least one enzyme selected from the group consisting of oxygenases from EC family 1.13 and oxygenases from EC family 1.14 for carrying out an oxidation of the compound, said biocomponent immobilized to a surface of the tip by one or more of (a) entrapment within a hydrogel;

(b) entrapment within a polymeric network;

(c) encapsulation;

(d) covalent-bonding; and

(e) adsorption,

wherein said biocomponent is stabilized to said tip by one or more of crosslinking a surface of said immobilized biocomponent, crosslinking a polymer layer to said biocomponent, adding a gel-hardening agent to said biocomponent, adding a stabilizing agent to said biocomponent, and modifying a component used to immobilize said biocomponent to said surface of the tip.

7. The distal tip of claim 6, wherein the oxygenase is selected from the group consisting of toluene o-monooxygenase (TOM), soluble methane monooxygenase (sMMO), toluene xylene-o-monooxygenase (ToMO), toluene-p-monooxygenase (T4MO), toluene-m-monooxygenase (T3MO), phenol hydroxylase (PH) and TOM-Green.

8. The distal tip of claim 6, wherein the compound is selected from the group consisting of chlorinated ethenes.

9. The distal tip of claim 8, wherein the chlorinated ethene is selected from the group consisting of tetrachloroethene (PCE), trichloroethene (TCE), dichloroethene isomers and vinyl chloride (VC).

10. The distal tip of claim 6, further comprising a device for delivering one or more reagents.

11. The distal tip of claim 10, wherein the reagent is selected from oxygen and hydrogen peroxide.

12. A distal tip of a biosensor transducer for use in detecting organic compounds, the tip comprising:

a biocomponent comprising at least two enzymes selected from the group consisting of oxygenases from EC family 1.13 and oxygenases from EC family 1.14 for carrying out an oxidation of one organic compound, a dehalogenase for carrying out dehalogenation of an organic compound, and a hydrolase from subclass EC 3.1 for carrying out hydrolysis of an organic compound, said biocomponent immobilized to a surface of the tip by one or more of (a) entrapment within a hydrogel;

(b) entrapment within a polymeric network;

(c) encapsulation;

(d) covalent-bonding; and

(e) adsorption,

wherein said biocomponent is stabilized to said tip by one or more of crosslinking a surface of said immobilized biocomponent, crosslinking a polymer layer to said biocomponent, adding a gel-hardening agent to said biocomponent, adding a stabilizing agent to said biocomponent, and modifying a component used to immobilize said biocomponent to said surface of the tip.

13. The distal tip of claim 12, further comprising a device for delivering one or more reagents.

14. The distal tip of claim 13, wherein the reagent is selected from oxygen and hydrogen peroxide.

15. The distal tip of claim 13, said biocomponent stabilized to said tip by crosslinking the polymer layer, wherein said crosslinked polymer layer is selected from the group consisting of poly-L-lysine, polyethylenimine, polyacrylic acid, polyvinyl alcohol, polyacrylamide and polyurethane.

16. An array of biosensors for use in detecting organic compounds within a mixture of organic compounds, comprising:

a plurality of biosensors, each biosensor having a distal tip including a biocomponent for use in detecting the organic compounds, the biocomponent of each biosensor comprising a variant enzyme having a distinct selectivity toward the organic compounds, said biocomponent immobilized to a surface of the tip by one or more of (a) entrapment within a hydrogel;

(b) entrapment within a polymeric network;

(c) encapsulation;

(d) covalent-bonding; and

(e) adsorption,

wherein said biocomponent is stabilized to said tip by one or more of crosslinking a surface of said immobilized biocomponent, crosslinking a polymer layer to said biocomponent, adding a gel-hardening agent to said biocomponent, adding a stabilizing agent to said biocomponent, and modifying a component used to immobilize said biocomponent to said surface of the tip.

17. The distal tip of claim 16, further comprising a device for delivering one or more reagents.

18. The distal tip of claim 17, wherein the reagent is selected from oxygen and hydrogen peroxide.

19. The distal tip of claim 16, said biocomponent stabilized to said tip by crosslinking the polymer layer, wherein said crosslinked polymer layer is selected from the group consisting of poly-L-lysine, polyethylenimine, polyacrylic acid, polyvinyl alcohol, polyacrylamide and polyurethane.

20. A distal tip of a biosensor ion-sensing transducer for use in detecting an analyte comprising an organophosphate compound, the tip comprising:

a biocomponent comprising a hydrolase selected from subclass EC 3.1 for carrying out hydrolysis of the compound, said biocomponent immobilized to a surface of the tip by one or more of (a) entrapment within a hydrogel;

(b) entrapment within a polymeric network;

(c) encapsulation;

(d) covalent bonding; and

(e) adsorption;

wherein said biocomponent has been treated to maintain a period of enzymatic efficacy; and

wherein said biocomponent is stabilized to said tip by one or more of crosslinking a surface of said immobilized biocomponent, crosslinking a polymer layer to said biocomponent, adding a gel-hardening agent to said biocomponent, adding a stabilizing agent to said biocomponent, and modifying a component used to immobilize said biocomponent to said surface of the tip.