Device and methods of using device for detection of aminoacidopathies
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.
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
)
2
+
(
y
ks
-
γ
kss
)
2
,
the step of calculating a shortest distance from the space between the two nearest points to a line between the respective calibration points (d sd ) comprises calculating
d
s
d
=
❘
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(
x
kss
-
x
ks
)
(
y
ks
-
y
m
)
-
(
x
kss
-
x
m
)
(
y
ks
-
y
kss
)
❘
"\[RightBracketingBar]"
(
x
ss
-
x
ks
)
2
+
(
y
kss
-
y
ks
)
2
,
and
the step of 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) comprises calculating
C
m
=
d
p
d
c
d
xy
,
where
d
p
=
d
ks
2
-
d
sd
2
.
17 . A system comprising the biosensor of claim 1 and a computer processor in operable connection to at least one computer storage memory.
18 . The system of claim 17 , further comprising:
a digital display in operable connection to the light detector by an electrical circuit capable of carrying an electrical signal corresponding to a measurement of irradiated light from at least one of the one or more reaction surfaces to the digital display,
wherein the digital display is configured to display concentration value of an amino acid in a sample when the light detector collects irradiated light from the at least one of the one or more reaction surfaces after a time period sufficient for the at least one metabolic enzyme to catalyze the oxidation of its amino acid substrate at the at least one of the one or more reaction surfaces.
19 . The system of claim 17 , wherein the at least one of the one or more metabolic enzymes or functional fragments thereof that is at least 80% homologous to a phenylalanine dehydrogenase from Geobacillus thermoglucosidasius is immobilized to at least one of the one or more reaction surfaces with one or a plurality of sugar molecules.
20 . A kit comprising (i) the biosensor of claim 1 ; and (ii) a set of instructions optionally accessible remotely through an electronic medium;
wherein at least one of the one or more reaction surfaces comprises a test strip or filter paper contained within a removable housing having at least one fluid inlet.
21 . A method of quantifying a concentration of one or more amino acids in sample of bodily fluid of a subject comprising contacting a sample of bodily fluid to the biosensor of claim 1 ; and exposing the sample of bodily fluid to light emitted from the light source of the biosensor prior to detecting the light emitted from the one or more reaction surfaces of the biosensor.
22 . The method of claim 21 , wherein the step of contacting comprises exposing the sample of bodily fluid of a subject to the one or more reaction surfaces of the biosensor for a time period sufficient to allow oxidation of at least one amino acid in the sample of bodily fluid by the one or more metabolic enzymes or functional fragments thereof of the biosensor.
23 . The method of claim 21 , wherein the sample of bodily fluid contains whole blood or serum from a subject.
24 . A method of diagnosing a metabolic disease in a subject comprising: contacting a sample of bodily fluid to the biosensor of claim 1 ;
quantifying one or more concentration values of amino acids in the sample using the biosensor;
comparing the one or more concentration values of amino acids in the sample to a threshold value of amino acid concentration identified as being in a healthy range; and identifying the subject as having a metabolic disease if the one or more concentration values of amino acids in the sample exceed or fall below the threshold value.
25 . The method of claim 24 , wherein the metabolic disease is chosen from at least one or a combination of: phenylketonuria, hyperammonemia, and maple syrup urine disease.