Enzyme-based system and sensor for measuring acetone

• US 20050084921A1
• Filed: 05/05/2004
• Published: 04/21/2005
• Est. Priority Date: 11/09/2001
• Status: Active Grant

• Current Assignee: Invoy Holdings, Inc.
• Original Assignee: Kemeta, LLC
• Inventors: Danowski, Kristine L, Strickland, Alan D, McIntyre, James A, Miller, Theodore E Jr., Rosner, Bettina M, Cranley, Paul E, Allen, Jeffrey R, Subramanian, Venkiteswaran, Sun, Larry
• Granted Patent: US 7,794,994 B2
• Time in Patent Office: Days
• US Class Current: 435/25
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/04 acyclic
• CPC Class Codes: C12P 7/28 Acetone-containing products
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence

Abstract

Described are enzyme systems specific for acetone and methods of using these enzyme systems to detect acetone in biological or environmental samples. Biosensors containing these enzyme systems are disclosed, in which detection of acetone may be achieved by linking electrochemical, photometric, or other detection means to one or more acetone-specific enzyme reactions or pathways. Methods of using such acetone-specific biosensors include subject management of weight loss, disease detection, and bioavailability monitoring of therapeutics.

Claims

1. An acetone-specific enzyme system including an enzyme that selectively targets acetone as a substrate, coupled to a detectable signal mediator.

2. The acetone-specific enzyme system according to claim 1, wherein the acetone-specific enzyme is any one of an acetone mono-oxygenase, an acetone carboxylase, or a secondary alcohol dehydrogenase.

3. The acetone-specific enzyme system according to claim 1, wherein the acetone-specific enzyme system is coupled to a signal mediator detectable by electrochemical or photometric means.

4. The acetone-specific enzyme system according to claim 3, wherein the acetone-specific enzyme system is any one of acetone carboxylase product formation coupled to NAD(P)H oxidation, acetone carboxylase ATP-hydrolysis coupled to NAD(P)H oxidation, acetone carboxylase ATP hydrolysis coupled to H₂O₂ formation, secondary alcohol dehydrogenase (S-ADH) with concomitant NAD(P)H oxidation, acetone carboxylase coupled to β

-hydroxybutyrate dehydrogenase catalyzed NAD(P)⁺ formation coupled to H₂O₂ production, S-ADH catalyzed NAD(P)⁺ formation coupled to H₂O₂ production, acetone mono-oxygenase coupled to NAD(P)H oxidation, or acetone mono-oxygenase coupled to H₂O₂ production, or acetone monooxygenase-catalyzed NAD(P)+ formation coupled to H2O2 formation.

5. The acetone-specific enzyme system according to claim 4, wherein the secondary alcohol dehydrogenase is obtained from a mammal or from a microorganism that is any one of the aerobic bacteria, anaerobic bacteria, yeasts, fungi, or methanogenic Archaea.

6. The acetone-specific enzyme system according to claim 5, wherein the secondary alcohol dehydrogenase is isolated from a species of any one of Thermoanaerobium, Xanthobacter, Pseudomonas, Rhodococcus, or Mycobacterium.

7. The acetone-specific enzyme system according to claim 5, wherein the secondary alcohol dehydrogenase is isolated from Xanthobacter autotrophicus strain Py2.

8. The acetone-specific enzyme system according to claim 4, wherein the acetone carboxylase is isolated from a species of any one of Xanthobacter, Rhodobacter, or Rhodococcus.

9. The acetone-specific enzyme system according claim 4, wherein the signal mediator is a member of any one of organic cofactors, inorganic cofactors, indicators, electron transfer mediators, photometric mediators, enzyme reaction by-products, or combinations thereof.

10. The acetone-specific enzyme system according to claim 9, wherein a signal from the electrochemically detectable signal mediator is linearly or exponentially amplified by recycling enzyme substrates to magnify electrochemical signal output.

11. A method for using an acetone-specific enzyme system according to claim 1, wherein said method involves

a) combining

i) at least one pharmaceutical compound, with

ii) at least one tag selected from the group consisting of acetoacetate and pharmaceutically acceptable acetoacetate salts, esters, and amides, to form a tagged pharmaceutical compound;

b) administering the tagged compound to a subject; and

c) thereafter, detecting release of the tag into the subject'"'"'s bloodstream via measurement, using the acetone-specific enzyme system, of the acetone concentration in a biological sample from the subject, wherein the acetone is present in the subject'"'"'s biological sample due to degradation of the tag, forming the acetone;

d) correlating the acetone concentration measured from the biological sample with release of the pharmaceutical compound from the administered tagged compound; and

e) optionally, repeating steps c) and d) to determine a rate of release of the pharmaceutical compound from the administered tagged compound.

12. A method for using an acetone-specific enzyme system according to claim 1, wherein said method involves

a) combining

i) at least one tag selected from the group consisting of acetoacetate and pharmaceutically acceptable acetoacetate salts, esters, and amides, with

ii) a biocompatible composition, to form a biocompatible implant or device;

b) placing the biocompatible implant or device in a subject;

c) thereafter, detecting release of acetoacetate into the subject'"'"'s bloodstream via measurement, using the acetone-specific enzyme system, of the acetone concentration in a biological sample from the subject, wherein the acetone is present in the subject'"'"'s biological sample due to degradation of the tag, forming the acetone;

d) correlating the acetone concentration measured from the biological sample with degradation or release of the biocompatible composition from the biocompatible implant or device; and

e) optionally, repeating steps c) and d) to determine a rate of degradation or release of the biocompatible implant or device.

13. A method for using an acetone-specific enzyme system according to claim 1, wherein said method involves

tagging a biocompatible implant or device by combining at least one member selected from the group consisting of acetoacetate, an acetoacetate salt, an acetoacetate ester and an acetoacetate amide with a biocompatible composition to form a biocompatible implant or device;

placing the biocompatible implant or device in a subject; and

measuring release of acetoacetate from the biocompatible implant or device.

40. A polypeptide molecule obtained from the protein according to claim 37, (1) said polypeptide molecule having (a) a molecular mass of approximately 37.1 kDa as determined by mass spectrometry, (b) a pI of approximately 7.4 as determined by isofocusing electrophoresis, and (c) a tetradecameric N-terminal amino acid sequence of SEQ ID NO:

7, and (2) said polypeptide molecule being capable of being degraded to form fragments having the amino acid sequences of SEQ ID NO;

8 to SEQ ID NO;

19.

14. A method for detecting acetone in a biological sample, said method involving:

introducing a biological containing acetone to a biosensor comprising at least one acetone-specific enzyme system that utilizes acetone as a substrate; and

detecting products produced upon interaction between the acetone and acetone-specific enzyme system.

15. The method according to claim 14, wherein detection is achieved by electrochemical, photometric, or calorimetric means.

16. The method according to claim 14, wherein said method further includes

facilitating electrochemical transduction of the at least one acetone-specific enzyme system and the acetone in the biological sample via an electrochemically treated electrode;

and

electrochemically detecting a product resulting from a reaction between the at least one acetone-specific enzyme system and the acetone in the biological sample.

17. The method according to claim 14, wherein the at least one acetone-specific enzyme system includes an enzyme that is any one of acetone mono-oxygenase, acetone carboxylase, or secondary alcohol dehydrogenase.

18. The method according to claim 17, wherein the at least one acetone-specific enzyme system includes a member that is any one of acetone carboxylase product formation coupled to NAD(P)H oxidation, acetone carboxylase ATP-hydrolysis coupled to NAD(P)H oxidation, acetone carboxylase ATP hydrolysis coupled to H₂O₂ formation, secondary alcohol dehydrogenase (S-ADH) with concomitant NAD(P)H oxidation, acetone carboxylase coupled to β

-hydroxybutyrate dehydrogenase catalyzed NAD(P)⁺ formation coupled to H₂O₂ production, S-ADH catalyzed NAD(P)⁺ formation coupled to H₂O₂ production, or acetone mono-oxygenase coupled to NAD(P)H oxidation.

19. The method according to claim 18, wherein the biological sample is in vapor form.

20. The method according to claim 19, wherein said method involves electrochemically detecting acetone in the vapor sample at a level of 0.2 ppm to 10 ppm.

31. A method of using the biosensor according to claim 20 to monitor a subject'"'"'s medical condition, wherein the method involves

a) obtaining at least one biological sample from the subject and introducing the biological sample to the biosensor;

b) measuring acetone concentration in the biological sample(s) via detecting acetone interactions with the acetone-specific enzyme system of the biosensor;

c) optionally, interfacing the biosensor with a computer network to transmit the measurement(s); and

d) correlating the acetone concentration measurement(s) with the subject'"'"'s medical condition.

32. The method according to claim 31, wherein the medical condition being monitored is diabetes or weight loss.

21. A biosensor for detecting acetone in a biological sample containing:

at least one acetone-specific enzyme system comprising an enzyme that selectively targets acetone as a substrate, coupled to a detectable signal mediator; and

a means for detecting a product resulting from a reaction between the at least one acetone-specific enzyme system and acetone in the biological sample.

22. The biosensor according to claim 21, further including a housing having a port for introducing the biological sample to the acetone-specific enzyme system.

23. The biosensor according to claim 21, wherein the acetone-specific enzyme system is present in a dried or lyophilized form until contacting the biological sample.

24. The biosensor according to claim 21, wherein the detection is achieved via electrochemical, photometric, or calorimetric means.

25. An electrochemical biosensor according to claim 24, wherein the at least one acetone-specific enzyme system includes an enzyme that is any one of acetone mono-oxygenase, acetone carboxylase, or secondary alcohol dehydrogenase.

26. The electrochemical biosensor according to claim 25, wherein the at least one acetone-specific enzyme system is a member that is any one of acetone carboxylase product formation coupled to NAD(P)H oxidation, acetone carboxylase ATP-hydrolysis coupled to NAD(P)H oxidation, acetone carboxylase ATP hydrolysis coupled to H₂O₂ formation, secondary alcohol dehydrogenase (S-ADH) with concomitant NAD(P)H oxidation, acetone carboxylase coupled to β

-hydroxybutyrate dehydrogenase catalyzed NAD(P)⁺ formation coupled to H₂O₂ production, S-ADH catalyzed NAD(P)⁺ formation coupled to H₂O₂ production, acetone mono-oxygenase coupled to NAD(P)H oxidation, acetone mono-oxygenase coupled to H₂O₂ production, or acetone monooxygenase-catalyzed NAD(P)+ formation coupled to H₂O₂ formation.

27. An electrochemical biosensor according to claim 25, wherein the acetone-specific enzyme system is storage stabilized by the presence of trehalose.

28. An electrochemical biosensor according to claim 25, wherein the biological sample is either in liquid or vapor form.

29. An electrochemical biosensor according to claim 25, further including either means for generating, from the electrochemical response that occurs during acetone detection, a qualitative or quantitative result, or means for interfacing said biosensor with said means for generating a qualitative or quantitative result.

30. An electrochemical biosensor according to claim 25, having at least two analyte detection electrodes that are grouped in a sequentially arranged cluster along a sample detection pathway, wherein at least one of said analyte detection electrodes is an acetone-specific electrode operably comprising an acetone-specific enzyme system.

33. A kit for detecting acetone in a sample, the kit comprising at least one acetone-specific enzyme system which includes an enzyme that selectively targets acetone as a substrate, coupled to a detectable signal mediator;

and a housing for the at least one acetone-specific enzyme system.

34. The kit according to claim 33 further containing a disposable test strip upon which the acetone-specific enzyme system is disposed.

35. The kit according to claim 34, wherein the strip is separate from the housing such that the strip may be inserted into the housing, the housing having a port into which the sample may be introduced.

36. The kit according to claim 34, wherein the strip and the housing for the strip are fashioned as a single, disposable unit, the housing having a port into which the sample may be introduced.

37. A disposable test strip upon which an acetone-specific enzyme or acetone-specific enzyme system is disposed.

38. The strip according to claim 37, wherein the acetone-specific enzyme is any one of an acetone mono-oxygenase, an acetone carboxylase, or a secondary alcohol dehydrogenase.

39. A protein obtained from Xanthobacter autotrophicus Py2 (ATCC PTA-4779), having NAD⁺-dependent secondary alcohol dehydrogenase activity, having the ability to reduce acetone to isopropanol, and having specific activity for ketones and secondary alcohols;

(A) said protein (1) having, for the oxidation of isopropanol to acetone, a pH optimum of approximately 7.8, and (2) having, for the oxidation of alcohols, an average specific activity ratio for secondary-to-primary alcohols of at least 50;

1 when tested at pH 7.8 under equivalent conditions individually with C3-C5 straight chain secondary alcohols and with C2-C5 straight chain primary alcohols;

(B) said protein having, for the reduction of acetone to isopropanol, (1) a pH optimum of approximately 6.2, (2) an apparent K_m of approximately 144±

18 μ

M, (3) an apparent V_max of approximately 43.4±

1.2 μ

mol acetone reduced·

min<sup>−

1</sup>·

mg<sup>−

1</sup> protein, (4) an apparent k_cat of approximately 30.4 sec<sup>−

1</sup>, (5) an apparent k_cat/K_m of approximately 2.1×

10⁵, and (6) a K_m for NADH of approximately 5.1±

0.4 μ

M; and

(C) said protein comprising at least one polypeptide molecule, (1) said polypeptide molecule having (a) a molecular mass of approximately 37.1 kDa as determined by mass spectrometry, (b) a pI of approximately 7.4 as determined by isofocusing electrophoresis, and (c) a tetradecameric N-terminal amino acid sequence of SEQ ID NO;

7, and (2) said polypeptide molecule being capable of being degraded to form fragments having the amino acid sequences of SEQ ID NO;

8 to SEQ ID NO;

19.