Enzyme-based system and sensor for measuring acetone
First Claim
1. An acetone-specific enzyme system including an enzyme that selectively targets acetone as a substrate, coupled to a detectable signal mediator.
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Accused Products
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.
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Citations
40 Claims
- 1. An acetone-specific enzyme system including an enzyme that selectively targets acetone as a substrate, coupled to a detectable signal mediator.
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14. A method for detecting acetone in a biological sample, said method involving:
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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. - View Dependent Claims (15, 16, 17, 18, 19, 20, 31, 32)
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21. A biosensor for detecting acetone in a biological sample containing:
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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. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30)
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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.
- View Dependent Claims (34, 35, 36)
- 37. A disposable test strip upon which an acetone-specific enzyme or acetone-specific enzyme system is disposed.
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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;
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(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 Km of approximately 144±
18 μ
M, (3) an apparent Vmax of approximately 43.4±
1.2 μ
mol acetone reduced·
min−
1·
mg−
1 protein, (4) an apparent kcat of approximately 30.4 sec−
1, (5) an apparent kcat/Km of approximately 2.1×
105, and (6) a Km 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.
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Specification