Device and methods of using device for detection of aminoacidopathies

Patent metadata

Field	Value
Publication number	US-12540348-B2
Application number	US-201515304852-A
Country	US
Kind code	B2
Filing date	Apr 17, 2015
Priority date	Apr 17, 2014
Publication date	Feb 3, 2026
Grant date	Feb 3, 2026

Abstract

The present disclosure relates to a biosensor capable of measuring the total concentration of one or a plurality of amino acids with the use of a reaction surface comprising one or a plurality of metabolic enzymes or functional fragments thereof, but wherein the reaction surface does not comprise an electrode or electrically conductive support. In some embodiments, the biosensor comprises use of a thermophilic bacterial metabolic enzyme immobilized or attached to the reaction surface.

First claim

The invention claimed is:

1 . A biosensor comprising:
a light source; a light detector comprising a complementary metal-oxide semiconductor (CMOS) or a charged-coupled device camera; and one or more reaction surfaces comprising at least one oxidizing agent lyophilized or desiccated onto the one or more reaction surfaces, and one or more metabolic enzymes or functional fragments thereof; wherein at least one of the one or more metabolic enzymes or functional fragments thereof is at least 80% homologous to a phenylalanine dehydrogenase from Geobacillus thermoglucosidasius; wherein the one or more reaction surfaces do not comprise and are not attached to an electrically conductive support; and wherein the one or more reaction surfaces are free of an electron mediator; and wherein the light source is positioned at a distance from at least one of the one or more reaction surfaces sufficient to irradiate the at least one of the one or more reaction surfaces and the light detector is positioned at a distance from the one or more reaction surfaces sufficient to collect irradiated light from the one or more reaction surfaces, and wherein the biosensor further comprises a housing that contains at least a first fluid opening adjacent to and in fluid communication with a filter paper immediately adjacent to a microfluidic chamber that comprises at least one of the one or more reaction surfaces.

2 . The biosensor of claim 1 , wherein at least one of the one or more reaction surfaces further comprises at least one circuit connecting the light detector to a controller.

3 . The biosensor of claim 1 , wherein at least one of the one or more reaction surfaces is a filter paper that comprises the one or more metabolic enzymes.

4 . The biosensor of claim 1 , wherein the one or more reaction surfaces are free of 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).

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

6 . The biosensor of claim 1 , wherein at least one of the one or more reaction surfaces comprises a volume from about 10 uL to about 100 μL of fluid.

7 . The biosensor of claim 1 , wherein the biosensor is free of an electron mediator selected from: thionine, o-phenylenediamine, methylene blue, and toluidine blue.

8 . The biosensor of claim 1 , wherein the at least one oxidizing agent is chosen from: NAD+ or FAD+.

9 . The biosensor of claim 1 , wherein at least one of the one or more reaction surfaces consists of a filter paper comprising a mixture of at least one lyophilized metabolic enzyme or functional fragment thereof and a sugar at a concentration from about 100 mM to about 400 mM.

10 . The biosensor of claim 1 further comprising a hydrogel comprising alginate.

11 . The biosensor of claim 10 , wherein the alginate comprises a block polymer with a formula:

wherein m and n each are any positive integer.

12 . The biosensor of claim 1 , wherein the biosensor directly detects a reduction of the at least one oxidizing agent.

13 . The biosensor of claim 1 further comprising whole blood at the one or more reaction surfaces.

14 . The biosensor of claim 1 further comprising a Tris or glycine buffer in contact with the one or more metabolic enzymes or functional fragments thereof.

15 . The biosensor of claim 1 , wherein the biosensor is configured to compute a concentration of phenylalanine in a sample by comparing target data values to a calibration curve, the target data values captured by the light detector upon exposure of the sample to light by the light source, and the computation comprising:
determining a shortest distance from each target data value to a respective calibration point of the calibration curve, identifying two nearest points (d ks and d kss ) from the shortest distance from each target value to the respective calibration point, determining a concentration range (d c ) between the two nearest points, calculating a distance between x and y coordinates (d xy ) on a chromaticity space between the two nearest points, calculating a shortest distance from the space between the two nearest points to a line between the respective calibration points (d sd ), identifying d m as the largest of d ks , d kss , and d sd , calculating a variation (v+/−) as a ratio of (d m d c )/d xy , and determining a distance from one corresponding calibration point on the calibration curve to a point on a line where d sd is measured to arrive at the concentration of phenylalanine (Cm).

16 . The biosensor of claim 15 , wherein
the step of determining a shortest distance from each target data value to a respective calibration point of the calibration curve comprises calculating

d
k

=

(

x
k

-
x

)

2

+

(

y
k

-
y

)

2

,

where k is an integer from 1 to the number of stored x and y pairs (points) in the calibration curve, the step of determining a concentration range (de) between the two nearest points comprises calculating d C =|C ks −C kss , where C ks and C kss are the concentrations of the points corresponding to d ks and d kss ,
the step of calculating a distance between x and y coordinates (d xy ) on a chromaticity space between the two nearest points comprises calculating

d

xy

=

(

x

ks

-

x

kss

)

Assignees

• Univ Maryland
• Childrens Nat Medical Ct

Inventors

• Ayyub Omar Bilal
• Heligman Brian Theodore
• Kofinas Peter
• Summar Marshall Lynn
• Cabrera-Luque Juan Manuel
• Cunningham Gary

Classifications

• G01N2800/52

  Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis · CPC title

• G01N2800/04

  Endocrine or metabolic disorders · CPC title

• G01N2333/90622

  Phenylalanine dehydrogenase (1.4.1.20) · CPC title

• G01N2333/904

  acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1) · CPC title

• G01N2333/902

  Oxidoreductases (1.) · CPC title