Device and methods of using device for detection of aminoacidopathies

• US 10,392,646 B2
• Filed: 10/17/2013
• Issued: 08/27/2019
• Est. Priority Date: 10/17/2012
• Status: Active Grant

• Current Assignee: Children's National Medical Center, United States Department Of Health And Human Services, University of Maryland, College Park
• Original Assignee: University of Maryland, College Park
• Inventors: Ayyub, Omar Bilal, Behrens, Adam Michael, Kofinas, Peter, Summar, Marshall Lynn, Cabrera-Luque, Juan Manuel, Cunningham, Gary, Simeonov, Anton, Marugan, Juan
• Primary Examiner(s): Kosar, Aaron J
• Application Number:
  US14/436,844
• Publication Number:
  US 20160168613A1
• Time in Patent Office: 2,140 Days
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 11/10 the carrier being a carbohydrate
• CPC Class Codes: C12N 9/0018 Phenylalanine dehydrogenase (1.4.1.20)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• 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 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48
• CPC Class Codes: G01N 2800/04 Endocrine or metabolic disorders
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/6812 Assays for specific amino acids

Abstract

The present invention relates to a biosensor capable of measuring the total concentration of one or a plurality of amino acids with the use of a reagentless system comprising an electrode modified by hydrogel that comprises at least one enzyme that oxidizes at least one substrate that is at least one amino acid. In some embodiments, the biosensor comprises a hydrogel comprising alginate. In some embodiments, the biosensor comprises use of a thermophilic bacterial metabolic enzyme immobilized or attached to the hydrogel.

Claims

1. A biosensor comprising:

at least one electrically conductive support, the at least one electrically conductive support attached to a hydrogel, the hydrogel comprising at least one electron mediator, at least one reduction agent, and at least one metabolic enzyme from a thermophilic bacterial cell or functional fragment thereof, wherein the hydrogel comprises alginate; and

an amperometer and/or voltmeter operably connected to the at least one electrically conductive support;

wherein the at least one electrically conductive support comprises a phenylalanine dehydrogenase or functional fragments thereof; and

wherein the phenylalanine dehydrogenase or functional fragment thereof comprises at least about 70% sequence identity to SEQ ID NO;

1 or SEQ ID NO;

2.

2. The biosensor of claim 1, wherein the biosensor comprises at least three electrically conductive supports, wherein at least one electrically conductive support is a silver and silver chloride wire.

3. The biosensor of claim 1, wherein the biosensor comprises at least a first and a second electrically conductive support, wherein the first electrically conductive support is attached to the hydrogel, and wherein said first and second electrically conductive supports are operably connected to said voltmeter and/or amperometer to apply a voltage therebetween.

4. The biosensor of claim 1, wherein the at least one electrically conductive support comprises an electronegative or anionic chemical component.

5. The biosensor of claim 1, wherein the at least one hydrogel comprises trehalose.

6. The biosensor of claim 1, wherein the biosensor does not comprise one or more of the following:

(i) uricase or a functional fragment thereof;

(ii) a hydrogel comprising dextran or a derivative thereof;

(iii) a bacterial cell;

(iv) an electronic dipole configured for electrophoresis; and

(v)3,4-dihydroxybenzoic acid (3,4-DHB).

7. The biosensor of claim 1, wherein the biosensor is at least 70% biologically active after about thirty days in storage at 4 degrees Celsius.

8. The biosensor of claim 1, wherein the biosensor is not functionally dependent upon exposure to UV light.

9. The biosensor of claim 1, wherein the at least one enzyme or functional fragment thereof comprises at least about 80% sequence identity to SEQ ID NO:

1 or SEQ ID NO;

2.

10. The biosensor of claim 1, wherein the at least one metabolic enzyme or functional fragment thereof comprises at least about 90% sequence identity to SEQ ID NO:

1.

11. The biosensor of claim 1, wherein the hydrogel comprises trehalose, wherein the hydrogel comprises an alginate concentration from about 1% to about 3% weight to volume of the total volume attached to the at least one electrically conductive support;

and wherein the electrically conductive support comprises a wire comprising silver and silver chloride in operable connection to the voltmeter and/or amperometer.

12. The biosensor of claim 1, wherein the hydrogel comprises an alginate comprising a block polymer with a formula:

13. The biosensor of claim 1, wherein the at least one reduction agent is chosen from:

NAD+or FAD+.

14. A system comprising the biosensor of claim 1 in operable connection to at least one computer storage memory, and a computer processor in operable connection with the at least one computer storage memory.

15. The system of claim 14, further comprising a digital display in operable connection to the at least one electrically conductive support by an electrical circuit capable of carrying an a electrical signal corresponding to a measurement of current and/or voltage differential from the voltmeter and/or amperometer to the digital display, wherein the digital display is configured to display concentration value of an amino acid in a sample when the at least one electrically conductive support is in contact with the sample for a time period sufficient for the at least one metabolic enzyme to catalyze the oxidation of its amino acid substrate.

16. The system of claim 15, wherein the metabolic enzyme is a phenylalanine dehydrogenase immobilized within the hydrogel and wherein the hydrogel comprises an alginate concentration from about 1% to about 3% weight to volume of the total volume attached to the at least one electrically conductive support.

17. A biosensor comprising:

at least one electrically conductive support, the at least one electrically conductive support attached to at least one hydrogel, the hydrogel comprising at least one electron mediator, at least one reduction agent, and at least one metabolic enzyme or functional fragment thereof;

wherein the at least one enzyme or functional fragment thereof has an amino acid sequence at least 70% homologous to SEQ ID NO;

1 or at least 70% homologous to a functional fragment of SEQ ID NO;

1; and

an amperometer and/or voltmeter operably connected to the at least one electrically conductive support.

18. The biosensor of claim 17, wherein the enzyme or functional fragment thereof has an amino acid sequence at least 80% homologous to SEQ ID NO:

1 or at least 80% homologous to a functional fragment of SEQ ID NO;

1.

19. The biosensor of claim 17, wherein the enzyme or functional fragment thereof has an amino acid sequence at least 90% homologous to SEQ ID NO:

1 or at least 90% homologous to a functional fragment of SEQ ID NO;

1.