Incorporation of enzymes into biosensors for detecting, measuring, and / or monitoring amino acid concentration

Abstract

The present disclosure relates to enzymes, sensors, kits, and methods of detecting, measuring, and / or monitoring amino add concentration. In aspects, sensors described herein include: a substrate; a first electrode formed on the substrate, wherein the first electrode is operable as a working electrode; an entrapment layer formed on the first electrode, wherein the entrapment layer comprises at least one oxidase, or at least one oxidase and at least one racemase; and a second electrode formed on the substrate, wherein the second electrode is operable as a counter electrode and / or a reference electrode. The disclosure further provides for methods of manufacturing sensors described herein; and for use of sensors described herein, for example, in test strips, devices, and systems for detecting and measuring amino add concentration.

Description

Attorney Docket No. 081906-1535050-256510PCIncorporation of Enzymes Into Biosensors for Detecting, Measuring, and / or Monitoring Amino Acid ConcentrationCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U S- Provisional Application No.63 / 737,000, filed December 20, 2024, US. Provisional Application No. 63 / 737,262, filed December 20, 2024, and U. S. Provisional Application No. 63 / 798,156, filed May 1, 2025. The content of each is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] A Sequence Listing in XML format, submitted under 37 C. F. R. §§ 1.831- 1.835, entitled 3000146-000004 Sequence Listing.xml, 123,462 bytes in size, generated on December 9, 2025, and filed electronically via Patent Center, is provided in lieu of a paper copy. This Sequence Listing is incorporated by reference into the specification for its disclosures,TECHNICAL FIELD

[0003] The present disclosure relates to enzymes, sensors, kits, and methods for detecting, measuring, and / or monitoring amino acid concentration in samples. In an aspect, the present disclosure relates to using enzymes described herein for various applications, such as use in test strips and additional detection platforms, sensors, and systems for detecting and measuring amino acid concentration.BACKGROUND

[0004] Free amino acids may be present anywhere in a human body, such as in a body fluid or in an organ tissue. The presence of these amino acids can be an indicator of possible disease or abnormality. For example, fluctuation in branched- chain amino acids (BCAA) concentration, such as leucine, isoleucine, and valine, is associated with change of muscle health; fluctuation in alanine concentration is associated with muscle health, gluconeogenesis activity and muscle catabolism; and fluctuation in glutamine concentration is associated with metabolic diseases and / or certain types of cancer. It has been reported that elevated phenylalanine in blood has been linked to undiagnosed galactosaemia, it was also suggested that in patients with chronic kidney disease (CKD) may experience a decrease in tyrosine level and tyrosine / phenylaianine ratio in plasma and muscle, as well as a slightAttorney Docket No. 081906-1535050-256510PCincrease in phenylalanine level in plasma and muscle, Garibotto et al, Clinics / Nutrition 29 (2010) 424-433.

[0005] Free amino acids present in an organ tissue may also be a useful indicator. For example, it was reported that the branched-chain amino acids (BCAA) profile of kidney tissue may indicate the performance of the kidney in organ transplantation. Ahmadi et al., Kidney International (2024) 106, 712-722.

[0006] Traditionally, High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) is being applied to detecting the presence and concentrations of amino acids in biological samples. While this method is accurate, it requires expensive, specialized equipment, labor-intensive sample preparation, and lengthy processing times, rendering it impractical for real-time clinical or patient use. Thus, the monitoring and / or controlling of the level of certain types of amino acids in the body can be beneficial, especially if it may be performed in a simple, cost- effective, and real-time manner.BRIEF SUMMARY OF THE DISCLOSURE

[0007] The disclosure relates to enzymes, sensors, kits, and methods of detecting, measuring, and / or monitoring amino acid concentration.

[0008] In an embodiment, sensors described herein comprise: a substrate; a first electrode formed on the substrate; an entrapment layer formed on the first electrode, wherein the entrapment layer comprises at least one D-amino acid oxidase and at least one racemase, wherein the at least one racemase comprises a racemase that is active against leucine and / or alanine; and a second electrode formed on the substrate, wherein the second electrode is operable as a counter electrode and / or a reference electrode.

[0009] In some embodiments, the sensor is a wearable or a nonwearable sensor.

[0010] In some embodiments, the sensor is a blood test strip, a sweat sensor, or a microneedle-based sensor.

[0011] In an embodiment, sensors described herein comprise: a first sensor capable of detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, and a second sensor capable ofAttorney Docket No. 081906-1535050-256510PCdetecting, measuring, and / or monitoring a concentration of an analyte other than an amino acid.

[0012] In some embodiments, the analyte other than an amino acid is glucose and / or a ketone. In some embodiments, the term “ketone s)’' includes ketones, ketone bodies, and / or precursors of ketone bodies, such as acetone, acetoacetic acid, beta-hydroxybutyrate (BHB), beta-ketopentanoate, beta-hydroxypentanoate, 1,3-butanediol, and medium chain triglycerides (MCT) containing fatty acids with hydrocarbon side chains in the length of 6-12 carbons. Other additional analytes are described in further details below.

[0013] In some embodiments, the entrapment layer comprises at least one additional racemase selected from the group consisting of: an arginine racemase, a histidine racemase, a lysine racemase, an aspartate racemase, a glutamate racemase, a serine racemase, a threonine racemase, an asparagine racemase, a glutamine racemase, a cysteine racemase, a proline racemase, an alanine racemase, a valine racemase, an isoleucine racemase, a leucine racemase, a methionine racemase, a phenylalanine racemase, a tyrosine racemase, and / or a tryptophan racemase, and / or a combination thereof.

[0014] In some embodiments, the at least one D-amino acid oxidase is active against one or more of D~arginine, D-histidine, D-iysine, D-aspartic acid, D-glutamic acid, D-serine, D-threonine, D-asparagine, D-glutamine, D-cysteine, glycine, D- proline, D-alanine, D-valine, D-isoleucine, D-leucine, D-methionine, D-phenylalanine, D-tyrosine, and / or D~tryptophan.

[0015] In some embodiments, the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 3, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81.

[0016] In some embodiments, the at least one racemase comprises an amino acid sequence of one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81, wherein said amino acid sequence comprises one, at most two, at most three, atAttorney Docket No. 081906-1535050-256510PCmost four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions,

[0017] In some embodiments, the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 33 comprising, wherein SEQ ID NO: 33 comprises an alanine at position 24,

[0018] In some embodiments, the at least one racemase comprises an amino acid sequence of SEQ ID NO: 33, wherein said amine acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions and wherein SEQ ID NO: 33 comprises an alanine at position 24.

[0019] In some embodiments, the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%. at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 4.

[0020] In some embodiments, the at least one D-amino acid oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 34.

[0021] In some embodiments, the at least one D-amino acid oxidase comprises an amino acid sequence of SEQ ID NO: 34, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

[0022] In some embodiments, the at least one D-amino acid oxidase is active against D-leucine and inactive against D~methionine.

[0023] In some embodiments, the at least one D-amino acid oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 89, 94, or 97.

[0024] In some embodiments, the first electrode is operable as a working electrode (WE), and the second electrode is operable as the counter electrode (CE) and the reference electrode (RE).

[0025] In some embodiments, the sensor further comprises a third electrode formed on the substrate, the first electrode is operable as a working electrode, theAttorney Docket No. 081906-1535050-256510PCsecond electrode is operable as the counter electrode, and the third electrode is operable as the reference electrode.

[0026] In some embodiments, the sensor further comprises one or more coatings and / or spacers made of ion-exchange resins, polyesters, polyethylene terephthalate (PET), PVC, PTFE, and / or polyolefin, the one or more coatings and / or spacers are formed on the first electrode, the second electrode, and / or the third electrode, and facilitates capillary-driven filling of a sample. In some embodiments, the one or more coatings may be made of hydrogels, such as polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyacrylate, polyvinylpyrrolidone (PVP), hyaluronic acid, chitosan, heparin, alginate, gelatin, fibrin, and zwitterionic materials. In some embodiments, the one or more spacers may be made of micro- and nano-patterned structures, such as embossed features, printed spacer elements, microchannel-forming layers, and three-dimensional (3D) printed or additively manufactured patterns.

[0027] In some embodiments, the sensor is capable of detecting, measuring, and / or monitoring the amino acid concentration or concentrations of more than one amino acids of a body fluid, wherein the body fluid comprises one or more of whole blood, serum, plasma, a blood fraction other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal discharge, milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, glandular secretions, exudate, contents of cysts, and / or ascites. In some embodiments, the body fluid may comprise any biological fluid, tissue-derived material, secreted substance, organ- associated medium, artificial or engineered fluid, or cellular environment capable of containing or transporting amino acids.

[0028] In some embodiments, the sensor may be used for detecting, measuring, and / or monitoring the amino acid concentration or concentrations of more than one amino acids in urine. For example, amino acids and / or amino acid metabolites such as phenylalanine, tyrosine, a-aminoadipic acid, cystathionine, and lysine may be presented at a higher level in urine of overweight patients. See, e.g., Mariana Doce Passadore et al., Front Physiol., April 29, 2025, 16:1524939, the content of which is hereby incorporated by reference in its entirety. In some embodiments, the sensor may be used in conjunction with weight controlling programs and treatments, such as administration of GLP-1 agonists and GlucagonAttorney Docket No. 081906-1535050-256510PCreceptor agonists, GIP receptor agonists, 5-HTzc receptor agonists, cannabinoid receptor antagonists, melanocortin 4 receptor agonists, adrenergic agonists, fibroblast growth factor-21 receptor agonists, thyroid hormones, lipase inhibitors, serotonin-norepinephrine reuptake inhibitors, and SGLT2 inhibitors. In some embodiments, the weight controlling programs and treatments include administration of Dulaglutide (Trullcity®), Exenatide (Byetta®)* Exenatide extended-release (Bydureon®), Liraglutide (Victoza®), Lixisenatide (Adlyxin®), Semaglutide injection (Ozempic®), Semaglutide tablets (Rybelsus®), and / or tirzepatide (Mounjaro®).

[0029] In some embodiments, when the sensor is used in conjunction with weight controlling programs and treatments, the amino acid monitoring is performed at least once every day, at least twice every day, at least 3 times a day, at least 4 times a day, at least 5 times a day, at least once every 2 days, at least once every 3 days, at least once every 4 days, at least once every 5 days, at least once every 6 days, or at least once every 7 days, or at least once every 2 weeks, at least once every 3 weeks, or at least once every 4 weeks. In some embodiments, when the sensor is used in conjunction with weight controlling programs and treatments, the amino acid monitoring is performed for at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months.

[0030] In an embodiment, sensors described herein comprise: a substrate; a first electrode formed on the substrate; an entrapment layer formed on the first electrode; and a second electrode formed on the substrate, wherein the second electrode is operable as a counter electrode and / or a reference electrode.

[0031] The disclosure further relates to enzymes, sensors, kits, and methods of detecting, measuring, and / or monitoring amino acid concentration. In an embodiment, sensors described herein comprise: a substrate; a first electrode formed on the substrate, wherein the first electrode is operable as a working electrode; an entrapment layer formed on the first electrode, wherein the entrapment layer comprises at least one oxidase and at least one racemase, wherein the at least one racemase comprises a racemase that is active against leucine; and a second electrode formed on the substrate, wherein the second electrode is operable as a counter electrode and / or a reference electrode. In another embodiment, sensors described herein comprise: a substrate; a first electrode formed on the substrate,Attorney Docket No. 081906-1535050-256510PCwherein the first electrode is operable as a working electrode; an entrapment layer formed on the first electrode, wherein the entrapment layer comprises at least one oxidase; and a second electrode formed on the substrate, wherein the second electrode is operable as a counter electrode and / or a reference electrode.

[0032] In some embodiments, the entrapment layer comprises at least one additional racemase selected from the group consisting of: an arginine racemase, a histidine racemase, a lysine racemase, an aspartate racemase, a glutamate racemase, a serine racemase, a threonine racemase, an asparagine racemase, a glutamine racemase, a cysteine racemase, a proline racemase, an alanine racemase, a valine racemase, an isoleucine racemase, a leucine racemase, a methionine racemase, a phenylalanine racemase, a tyrosine racemase, and / or a tryptophan racemase, and / or a combination thereof. In some embodiments, the at least one additional racemase is selected from the group consisting of: an arginine racemase, a lysine racemase, an alanine racemase, an isoleucine racemase, a leucine racemase, an aspartate racemase, a glutamate racemase, a proline racemase, and / or a serine racemase, and / or a combination thereof. In some embodiments, the at least one additional racemase is active against one or more of alanine, methionine, phenylalanine, serine, proline, arginine, leucine, isoleucine, and / or valine.

[0033] In some embodiments, the sensors, kits, and methods of the present disclosure utilize a combination of racemases active on branched-chain amino acids (BCAAs) and Arginine, thereby providing dual BCAA and arginine sensors.

[0034] In some embodiments, the at least one oxidase comprises an arginine oxidase, a histidine oxidase, a lysine oxidase, an aspartate oxidase, a glutamate oxidase, a serine oxidase, a threonine oxidase, an asparagine oxidase, a glutamine oxidase, a cysteine oxidase, a glycine oxidase, a proline oxidase, an alanine oxidase, a valine oxidase, an isoleucine oxidase, a leucine oxidase, a methionine oxidase, a phenylalanine oxidase, a tyrosine oxidase, and / or a tryptophan oxidase, and / or a combination thereof.

[0035] An oxidase may exhibit activity against a specific amino acid, or against more than one amino acids (besides the specific amino acid identified by the name of the oxidase). As used herein, an “arginine oxidase” refers to an oxidase that exhibits activity against arginine, but said oxidase may also exhibit activity against one or more amino acids of other types. Likewise, as used herein, a “histidineAttorney Docket No. 081906-1535050-256510PCoxidase" refers to an oxidase that exhibits activity against histidine, but said oxidase may also exhibit activity against one or more amino acids of other types.

[0036] in some embodiments, the at least one oxidase is active against one or more of D-arginine, D-histidine, D-lysine, D~aspartic acid, D-glutamic acid, D-serine, D-threonine, D-asparagine, D-glutamine, D-cysteine, glycine, D-proline, D-alanine, D-valine, D-isoleucine, D-leucine, D-methionine, D-phenylalanine, D-tyrosine, and / or D-tryptophan. In some embodiments, the at feast one oxidase is active against one or mere of D-alanine, D-proline, D-arginine, D-leucine, D-isoleucine, and / or D~valine.

[0037] In some embodiments, the at least one oxidase comprises at least one L-amino acid oxidase. In some embodiments, the at least one L-amino acid oxidase is active against one or more of L-arginine, L-histidine, L-lysine, L-aspartic acid, L- glutamic acid, L-serine, L-threonine, L-asparagine, L-glutamine, L-cysteine, glycine, L-proline, L-alanine, L-valine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-tyrosine, and / or L-tryptophan. In some embodiments, the at least one L-amino acid oxidase is active against one or more of L-alanine, L-proline, L-arginine, L-leucine, L-isoleucine, and / or L-valine.

[0038] In some embodiments, the at least one oxidase comprises at least one hydroxy acid oxidase. In some embodiments, the at least one hydroxy acid oxidase has cryptic activity against one or more of alanine, proline, arginine, leucine, isoleucine, and / or valine,

[0039] In some embodiments, the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81. In some embodiments, the at least one racemase comprises an amino acid sequence of one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. 27, 28, 29, 30, 31. 32, 33, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

[0040] In some embodiments, the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,Attorney Docket No. 081906-1535050-256510PCat least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 33.

[0041] In some embodiments, the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 33 comprising, wherein SEQ ID NO: 33 comprises an alanine at position 24. In some embodiments, the at least one racemase comprises an amino acid sequence of SEQ ID NO: 33, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions and wherein SEQ ID NO: 33 comprises an alanine at position 24.

[0042] In some embodiments, the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 4.

[0043] In some embodiments, the at least one oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 34. In some embodiments, the at least one oxidase comprises an amino acid sequence of SEQ ID NO: 34, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

[0044] In some embodiments, the at least one oxidase comprises at least one D-amino acid oxidase. In some embodiments, the at least one D-amino acid oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 34. In some embodiments, the at least one D-amino acid oxidase comprises an amino acid sequence of SEQ ID NO: 34, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

[0045] In some embodiments, the at least one L-amino acid oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 1, 2, or 3. In some embodiments, the at least one L-Attorney Docket No. 081906-1535050-256510PCamino acid oxidase comprises an amino acid sequence of one of SEQ ID NOs: 1, 2, or 3, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

[0046] In some embodiments, the at feast one hydroxy acid oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54. In some embodiments, the at least one hydroxy acid oxidase comprises an amino acid sequence of one of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

[0047] In some embodiments, the entrapment layer further comprises at least one redox mediator, wherein the at least one redox mediator comprises one or more of Prussian blue, ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complexes, and / or organometallic ruthenium complexes.

[0048] In some embodiments, the entrapment layer further comprises a co¬ factor. In some embodiments, the co-factor includes flavin adenine dinucleotide (FAD) and / or pyridoxal 5'-phosphate (PLP).

[0049] In some embodiments, the entrapment layer comprises: a transducer layer, wherein the transducer layer comprises the at least one redox mediator; and a receptor layer, wherein the receptor layer comprises the at least one oxidase and the at least one racemase.

[0050] In some embodiments, the sensor further comprises a protective layer formed on the entrapment layer, wherein the protective layer comprises an inner protective layer and / or an outer protective layer. The protective layer may be formed by one or more polymeric materials. For example, the protective layer may be formed by one or more of polyurethane (PU), polyethylene glycol (PEG) and PEG- related polymers (such as Macrogol and polyethylene glycol-polyvinyl alcohol (PEG-PVA)), polyvinyl chloride (PVC), Nafion, silicone and silicone-based polymers (such as polydimethylsiloxane (PDMS), polymethylhydrosiloxane (PMHS), and polyvinylAttorney Docket No. 081906-1535050-256510PCsiloxane (PVS)), polyaniline (PANI), poly(methyl methacrylate) (PMMA), polypyrrole (PPy), cellulose, cellulose acetate, polycarbonates (PC), polyacetylene (PAc).Poly(p-phenylene vinylene) (PPV), Rayon, chitosan, and / or Polythiophenes (PTs).

[0051] In some embodiments, the inner protective layer and the outer protective layer may be formed by the same material(s). For example, in some embodiments, the inner protective layer and the outer protective layer may be formed by Nafion.

[0052] In some embodiments, the inner protective layer and the outer protective layer may be formed by different materials. For example, in some embodiments, the inner protective layer may be formed by polyethylene glycol (PEG), and the outer protective layer may be formed by Nafion. In some other embodiments, the inner protective layer may be formed by chitosan, and the outer protective layer may be formed by PVC.

[0053] In some embodiments, the at least one oxidase and / or the at least one racemase are immobilized to the entrapment layer. In some embodiments, the entrapment layer comprises a conductive polymer film, and the at least one oxidase and / or the at least one racemase are immobilized to the conductive polymer film. In some embodiments, the conductive polymer film comprises one or more of poly(thionine), polyphenylene vinylene, polypyrrole, polythiophene, polyaniline, and / or polyphenylene sulfide, and / or a combination thereof.

[0054] In some embodiments, the at least one oxidase and / or the at least one racemase are cross-linked to the conductive polymer film via a cross-linking agent. In some embodiments, the cross-linking agent includes one or more of glutaraldehyde, diisocyanates, carbodiimides, acyl azide, genipin, polyepoxides, chitosan, epoxy silanes, PEG derivatives, thiols (such as methanethiol, ethanethiol, 1 -propanethiol, and 2-propanethiol), and / or diazonium salts, and / or a combination thereof.

[0055] In some embodiments, the substrate is formed of an insulative material. In some embodiments, a material of the substrate comprises one or more of a composite material, a fibrous material, a woven textile, a non-woven textile, a polymer, an adhesive, a film, a gel, PTFE, and / or silicone.

[0056] In some embodiments, the first electrode is operable as the working electrode, and the second electrode is operable as the counter electrode and the reference electrode.Attorney Docket No. 081906-1535050-256510PC

[0057] In some embodiments, the first electrode is made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, and the second electrode is an Ag / AgCl electrode, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper-copper(II) sulfate electrode (CSE), a Pd / Hs electrode, or a mercury-mercurous sulfate electrode (MSE).[00581 In some embodiments, the sensor further comprises a third electrode formed on the substrate, wherein the first electrode is operable as the working electrode, wherein the second electrode is operable as the counter electrode, and wherein the third electrode is operable as the reference electrode.

[0059] As used herein, “stencil” when referring to screen-printed electrochemical biosensor fabrication is a patterned mesh screen that permits conductive or functional inks to pass through designated open regions while blocking ink in others, thereby defining the electrode geometry, thickness, and spatial layout on the substrate during printing. The stencil governs print resolution, layer uniformity, and deposited film thickness. As such, it is a critical component for producing reproducible working, counter, and reference electrodes in screen-printed electrodes (SPEs) and related biosensor platforms.

[0060] As used herein, “drop casting” refers to surface-modification techniques in which a defined volume of a solution or suspension (e.g., containing enzymes, polymers, or other functional materials) is deposited onto the electrode surface and subsequently dried under ambient or controlled conditions. As the solvent evaporates, a thin film or coating is formed, enabling uniform deposition of the materials onto the substrate.

[0061] As used herein, “working electrode" or " WE" refer to an electrode at which the electrochemical reaction of interest occurs, typically involving the oxidation or reduction of the target analyte or an associated mediator. The working electrode converts chemical information into an electrical signal through faradaic processes. It serves as the primary site of electron transfer, mass transport, and reaction-coupled phenomena relevant to the sensing mechanism. The potential of the working electrode is actively controlled by the potentiostat relative to the reference electrode (RE). A recognition layer, such as an immobilized enzyme or other biorecognition element, is deposited onto the working electrode surface to confer analyte specificity.Attorney Docket No. 081906-1535050-256510PCAdditional layers may be applied over the recognition layer as modification or protective strategies to enhance sensor performance, including but not limited to improvements in stability, selectivity, antifouling behavior, and / or overall analytical capability.

[0062] As used herein, “protective layer” refers to material layers applied to shield underlying sensing components from external physical, chemical, or mechanical damage while permitting selective passage of desired analytes. Such layers often function as semipermeable membranes such as PVC, PU, or biopolymer coatings such as chitosan.

[0063] As used herein, “entrapment layer” refers to a polymeric matrix or matrices used to physically immobilize active components such as enzymes, nanoparticles, or other biorecognition elements, while preserving their biological or catalytic activity. Entrapment materials commonly include chitosan, glutaraldehyde-crosslinked polymers, and other hydrogels that provide a stable microenvironment for the immobilized species. In some embodiments, the entrapment layer may be made of a material comprising one or more of poly(thionine), polyphenylene vinylene, polypyrrole, polythiophene, polyaniline, and / or polyphenylene sulfide, and / or a combination thereof.

[0064] As used herein, “spacer", “device spacer’', or “spacer (device context)” refer to engineered layers or physical structures incorporated into a sensing device to cover or protect the sensing interface and to assist with the capillary-driven transport of liquid samples, In many lateral-flow or microfluidic sensor formats, spacers also regulate sample volume and ensure consistent filling and flow dynamics across the sensor strip.

[0065] In addition to the spacer layer, one or more separation or filtration layers comprising blood-cell filtration membranes may be applied between the sensing surface and the spacer layer to reduce or prevent interference from blood cells during analyte detection.

[6066] As used herein, “device substrate” or substrate (device)” refer to structural platforms or base materials upon which a sensing device is fabricated or assembled. Typical substrates include PET sheet, glass, silicon, and ceramic materials, selected based on mechanical stability, chemical compatibility, and fabrication requirements.Attorney Docket No. 081906-1535050-256510PC

[0067] As used herein, “biological substrate" or “substrate (biological)” refer to target analytes or reactants that interacts with a biological recognition element or enzyme. In biosensing, the substrate is the molecule whose presence or concentration the sensor is designed to detect.

[0068] Generally, the fabrication of screen-printed electrodes (SPEs) begins with printing the conductive connection layer (e.g., Ag / AgCI ink), which also serves as the reference electrode layer, onto a substrate such as a PET sheet using a screen-printing machine and a pre-designed stencil. This initial layer is subsequently dried at a controlled temperature (e.g., about 70 °C for about 20 min). After drying, the working electrode (WE) and counter electrode (CE) layers are printed using the appropriate functional inks (e.g,, carbon ink or mediator-modified carbon ink) and then dried underspecified conditions. Depending on the device design, an insulating or dielectric layer may be applied thereafter to define the active electrode area and to protect the underlying conductive tracks.

[0069] Following completion of the printing and drying processes, the SPEs are fully fabricated and prepared for subsequent modification (e.g., drop casting), functionalization, or electrochemical characterization.

[0070] In some embodiments, the reaction enzymes, such as the at least one D-amino acid oxidase and the at least one racemase, are immobilized on the surface of the sensor electrodes or substrate. Various designs for immobilizing the reaction enzymes, such as the designs depicted in FIGs.57A-57C, may be applied.

[0071] In some embodiments, the reaction enzymes may be immobilized or entrapped within an entrapment material (entrapment layer), as shown in RG. 57A.The entrapment material comprises a polymeric matrix that physically confines active components, such as enzymes, nanoparticles, cofactors, mediators, or other biorecognition elements, while preserving their biological or catalytic activity.Exemplary entrapment materials include, but are not limited to, chitosan, glutaraldehyde-crosslinked polymers, hydrogels, and related polymeric networks that provide a stable microenvironment for the immobilized species. From a macroscopic perspective, the entrapment layer contains and retains the enzyme(s). In certain embodiments, the enzyme(s) may be chemically or physically cross-linked to the polymeric molecules of the entrapment matrix. Thus, the enzyme(s) are entrapped within the polymeric matrix that forms the first functional layer on the electrode surface. In some embodiments, the entrapment layer formed from polymeric orAttorney Docket No. 081906-1535050-256510PCpolymer-related matrices is not limited to containing enzymes but may additionally include proteins, cofactors, mediators, stabilizers, conductive additives, structural additives, nanoparticles, and / or other functional materials that enhance sensor performance, stability, and / or sensitivity.

[0072] The entrapment layer or entrapment material may further be protected by one or more coating or protective layers. Such layers often function as semipermeable membranes such as PVC, PU, or biopolymer coatings such as chitosan. FIG. 57 A depicts a configuration comprising a Protective layer I (2nd layer) and a Protective layer II (3rd layer). These layers function to shield the underlying sensing components from physical, chemical, or mechanical degradation while permitting selective diffusion of target analytes. Suitable coating materials include semipermeable membranes such as PVC, PU, and biopolymer coatings such as chitosan. Additional materials suitable for forming the protective layer(s) are further described below in detail. A single protective layer or multiple synergistic layers may be employed, for example to impart antifouling properties, selective permeability, or enhanced mechanical stability.

[0073] Alternatively, as shown in FIG 57B, the reaction enzymes may be directly deposited onto the surface of the working electrode or substrate and immobilized, forming an enzyme layer. This enzyme layer is subsequently covered by an additional entrapment material layer. When this second layer does not directly interact with or incorporate the enzyme layer, it may alternatively function as a coating or protective layer (e., as the Protective layer I).

[0074] The second and any subsequent layers (e.g., Protective layer I and Protective layer II) serve as coating layers applied to protect the underlying sensing architecture from physical, chemical, or mechanical damage while allowing selective diffusion of analytes. Similar to the design depicted in FIG. 57A, these protective layers may be fabricated from materials such as PVC, PU, or biopolymer coatings (e.g., chitosan) and may consist of a single layer or multiple layers tailored for antifouling, selective permeability, mechanical reinforcement, or other functional enhancements. Additional materials suitable for forming the protective layer(s) are further described below in detail.[0075J The reaction enzymes may also be immobilized onto the surface of the working electrode by the design depicted in FIG. 57C. As shown in FIG. 57C, the enzyme layer (1st layer) is formed and immobilized directly onto the surface of theAttorney Docket No. 081906-1535050-256510PCworking electrode or substrate, and subsequently being entrapped by an overlying entrapment materia!. As previously discussed, exemplary entrapment materials include, but are not limited to, chitosan, glutaraldehyde-crosslinked polymers, hydrogels, and related polymeric networks that provide a stable microenvironment for the immobilized species. In some embodiments, the entrapment layer may be made of a material comprising one or more of poly(thionine), polyphenylene vinylene, polypyrrole, polythiophene, polyaniline, and / or polyphenylene sulfide, and / or a combination thereof,

[0076] As shown in FIG. 57C, a second layer and a third layer may be further formed on the entrapment material to afford further protection. The second layer (Protective layer |) may also serve as an entrapment material on the side facing the enzyme layer. For example, a chitosan layer acting as the second layer may simultaneously function as (!) an entrapment matrix that partially or fully interacts with the enzyme(s), and (ii) a coating or protective layer on Its outward-facing side. The third layer (Protective layer II) comprises an additional coating or protective layer designed to shield the underlying sensing components while permitting selective analyte diffusion. Suitable materials include PVC, PU, biopolymer coatings such as chitosan, and related semi-permeable membranes. One or more coating layers may be employed to achieve application-specific performance such as antifouling behavior or enhanced stability. Additional materials suitable for forming the protective layer(s) are further described below in detail.

[0077] In the designs shown in FIG, 57B and FIG 57C, the first (enzyme) layer that becomes entrapped by the subsequent layer is not limited to enzymes but may incorporate proteins, cofactors, mediators, stabilizers, conductive additives, structural additives, and nanopartictes or related materials intended to improve stability, sensitivity, or overall sensor functionality. These components may be coentrapped within the polymer matrix.

[0078] Certain materials, such as chitosan, may function either as entrapment materials, protective coatings, or both, depending on formulation, thickness, cross-linking, modification, or application requirements.[00791 The enzyme layer may be entrapped either by a subsequent layer or within a polymeric entrapment matrix applied directly onto the surface of the working electrode or substrate.Attorney Docket No. 081906-1535050-256510PC

[0080] Coating or protective layers may consist of a single layer or multiple layers.

[0081] Any of the layers described herein (enzyme / immobilization layers, entrapment layers, and coating / protective layers) may be further modified with additional materials, including but not limited to mediators, stabilizers, conductive additives, nanoparticles, structural additives, or other performance-enhancing agents.[00821 In some embodiments, one or more coatings and / or spacers may be formed on the entrapment layer and / or the protective layer. The one or more coatings and / or spacers may create a three-dimensional space / chamber between the entrapment layer and the one or more coatings and / or spacers, so as to facilitate the filling and / or delivery of the sample fluid.

[0083] In some embodiments, in addition to the spacer layer, one or more separation or filtration layers comprising blood-cell filtration membranes may be applied between the sensing surface and the spacer layer to reduce or prevent interference from blood cells during analyte detection. Exemplary designs of coatings, spacers, and / or filtration layers are further described in detail in the example section below.[0084J As used herein, “reference electrode” refers to an electrode that has a stable and well-defined electrochemical potential that remains effectively constant during measurement. The reference electrode provides a fixed reference point (“zero potential'’) against which the working electrode potential can be precisely controlled and measured. In other words, the reference electrode establishes an absolute potential standard that allows high-precision control of the interfacial potential at the working electrode. Ideally, the reference electrode maintains a stable potential across various media or biofluids relevant to the intended application.

[0085] As used herein, “counter electrode” or “auxiliary electrode” refers to an electrode that completes the electrochemical circuit by permitting current to flow to or from the working electrode. The counter electrode balances the current in the system so that the potential of the working electrode can be accurately regulated. The counter electrode conducts current without imposing constraints on the working electrode’s potential and therefore enables controlled electrochemical reactions at the working electrode interface.Attorney Docket No. 081906-1535050-256510PC

[0086] In a two-electrode system, the system comprises only a working electrode and a counter electrode, and the counter electrode must serve simultaneously as both the counter electrode and the reference electrode. The applied potential is therefore defined between the working electrode and the counter electrode. Two-electrode configurations can be advantageous when redox kinetics are slow or when simplicity, minimal component count, or a compact form factor is required. A two-electrode system may be preferable in specific contexts, such as when fabrication simplicity is essential, when the electrolyte volume is extremely small or confined, when symmetric electrode materials are employed, or when the working electrode and the counter electrode are identical (e.g., platinum-platinum). It also offers advantages in applications that rely on relative current changes rather than absolute potential control, as well as in scenarios where minimizing power consumption is critical.

[0087] Alternatively, in a three-electrode system, the reference electrode and the counter electrode are two distinct and separate electrodes. By including an independent reference electrode, the three-electrode system generally provides superior electroanalytical sensitivity, stability, and reproducibility across most applications. Detailed explanation of two-electrode and three-electrode systems may also be found in, e.g., US 2007 / 0213611 and US 11,633,133, the content of each of which is incorporated herein by reference in its entirety.

[0088] In some embodiments, the first electrode is made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, the second electrode is made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, and the third electrode is an Ag / AgCl electrode, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper--copper(ll) sulfate electrode (CSE), a Pd / Hs electrode, or a mercury-mercurous sulfate electrode (MSE).

[0089] In some embodiments, the first electrode is connected to a first connection, the second electrode is connected to a second connection, and the third electrode is connected to a third connection, wherein the first connection, the second connection, and the third connection are electrically conductive.Attorney Docket No. 081906-1535050-256510PC

[0090] In some embodiments, the sensor may comprise more than three electrodes. For exampie, in some embodiments, the sensor further comprises a fourth electrode, wherein the first electrode is operable as a first working electrode, the second electrode is operable as the counter electrode, the third electrode is operable as the reference electrode, and the fourth electrode is operable as a second working electrode. The first electrode (first working electrode) and the fourth electrode (second working electrode) may be coated with entrapment layers comprising enzymes targeting different amino acids. In some embodiments, the first electrode is coated with an entrapment layer comprising leucine racemase and D- leucine oxidase, and the fourth electrode is coated with an entrapment layer comprising alanine racemase and D-alanine oxidase, such that the first electrode has specificity against leucine (L-leucine), and the fourth electrode has specificity against alanine (L-alanine). In some embodiments, the sensor may comprise arrays of electrodes comprising one, two, three, four, or five, or more than five working electrodes with specificities against different amino acids. In some embodiments, the sensor may comprise arrays of two or three electrode contingents. For example, the sensor may comprise a first three electrode contingent with its working electrode coated with an entrapment layer comprising alanine racemase and D-alanine oxidase, and a second three electrode contingent with Its working electrode coated with an entrapment layer comprising leucine racemase and D-leucine oxidase, such that the first three electrode contingent has specificity against alanine (L-alanine), and the second three electrode contingent has specificity against leucine (L-leucine), In some other embodiments, the two or three electrode contingents may also be electrode contingents with specificity against other analytes of interest, such as glucose and / or ketone. In some embodiments, the term “ketone(s)” includes ketones, ketone bodies, and / or precursors of ketone bodies, such as acetone, acetoacetic acid, beta-hydroxybutyrate (BHB), beta~ketopentanoate, beta-hydroxypentanoate, 1,3-butanediol, and medium chain triglycerides (MCT) containing fatty acids with hydrocarbon side chains in the length of 6-12 carbons. Other additional analytes are described in further details below.

[0091] In some embodiments, a cover layer is formed on the first connection, the second connection, and / or the third connection, wherein the cover layer at least partially covers the first connection, the second connection, and / or the third connection, and wherein the cover layer is made of an electrically insulative material.Attorney Docket No. 081906-1535050-256510PC

[0092] In an embodiment, the disclosure provides a system for detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, comprising: the sensor described herein; and a portable device connected to the sensor. In some embodiments, the sensor is configured to measure an amino acid concentration or concentrations of more than one amino acids contained within a sample and output a data stream to the portable device. In some embodiments, the portable device comprises: a processor configured to process the data stream from the sensor; and an interface configured to display and / or communicate measured amino acid concentration values

[0093] In some embodiments, the sensor is coupled to the portable device via the first connection, the second connection, and / or the third connection.(0094} In some embodiments, the sensor is configured on a disposable test strip. In some embodiments, the sensor is a fingertip sweat sensor. In some embodiments, the sensor is a fingertip capillary blood sensor.

[0095] In some embodiments, the portable device further comprises a signal conditioning circuit to amplify electrical signals detected by the first electrode.

[0096] In some embodiments, the portable device further comprises a memory.

[0097] In some embodiments, the portable device further comprises a wireless communications unit to wirelessly transmit the detected electrical signals and / or the data stream to an external computing device.

[0098] In some embodiments, the sensor is a wearable or a nonwearable sensor. In some embodiments, the sensor is a blood test strip, a sweat sensor, or a microneedle-based sensor.

[0099] In an embodiment, the sensor comprises a first sensor capable of detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, and a second sensor capable of detecting, measuring, and / or monitoring a concentration of an analyte other than an amino acid.

[0100] In some embodiments, the first and second sensors may operate independently, concurrently, sequentially, or cooperatively within the same device, substrate, platform, or array.

[0101] In some embodiments, one or more additional microneedle sensor arrays may be incorporated, each of which can be configured and / or adjusted toAttorney Docket No. 081906-1535050-256510PCdetect, measure, and / or monitor an additional amino acid, an analyte other than an amino acid, or both, and may further include additional counter and / or reference microneedle electrodes as needed.

[0102] In some embodiments, the analyte other than an amino acid is glucose and / or a ketone. In some embodiments, the term “ketone(s)” includes ketones, ketone bodies, and / or precursors of ketone bodies, such as acetone, acetoacetic acid, beta-hydroxybutyrate (BHB), beta-ketopentanoate, beta-hydraxypentanoate, 1,3-butanediol, and medium chain triglycerides (MCI) containing fatty acids with hydrocarbon side chains in the length of 6-12 carbons. Other additional analytes are described in further details below.

[0103] In an embodiment, the disclosure provides a method of manufacturing a sensor for detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, comprising: providing a substrate; forming a first electrode on the substrate, wherein the first electrode is operable as a working electrode: forming an entrapment layer on the first electrode; forming a second electrode on the substrate, wherein the second electrode is operable as the counter electrode; and forming a third electrode on the substrate, wherein the third electrode is operable as the reference electrode.

[0104] In some embodiments, the step of forming the entrapment layer comprises: depositing a conductive polymer material on the first electrode, such that a conductive polymer film is formed on the first electrode; and depositing at least one oxidase and a cross-linking agent to the conductive polymer film, such that the at least one oxidase is immobilized to the conductive polymer film.

[0105] In some embodiments, the method further comprises depositing at least one racemase to the conductive polymer film, such that the at least one racemase is immobilized to the conductive polymer film.

[0106] In some embodiments, the method further comprises depositing at least one redox mediator to the conductive polymer film, such that the at least one redox mediator is immobilized to the conductive polymer film.

[0107] In some embodiments, the method further comprises forming a protective layer on the entrapment layer.

[0108] In some embodiments, the protective layer is formed by depositing a solution containing Nafion on the entrapment layer, and drying the solution.Attorney Docket No. 081906-1535050-256510PCIn some embodiments, the method further comprises: forming a first connection electronically connected to the first electrode; forming a second connection electronically connected to the second electrode; forming a third connection electronically connected to the third electrode; and forming a cover layer on the first connection, the second connection, and / or the third connection, wherein the cover layer at least partially covers the first connection, the second connection, and / or the third connection, and wherein the cover layer is made of an electrically insulative material.

[0109] In an embodiment, methods described herein comprise: subjecting a sample to at least one racemase, wherein the at least one racemase comprises a racemase that is active against leucine; subjecting the sample to at least one oxidase; and detecting and / or measuring for a presence of hydrogen peroxide. In some embodiments, the sample is obtained from a subject, for example, a human.

[0110] In some embodiments, the detecting and / or measuring the presence of hydrogen peroxide comprises: subjecting the sample to one or more redox indicator.

[0111] In some embodiments, the one or more redox indicator includes (e.g,, comprises, consists essentially of or consists of) 2,2'-bipyridine (Ru complex), Nitrophenanthroline (Fe complex), N-Phenylanthraniiic acid, 1,10-Phenanthroline iron(II) sulfate complex, N-Ethoxychrysoidine, 2,2‘-Bipyridine (Fe complex), 5,6- Dimethylphenanthroline (Fe complex), o-Dianisidine, Sodium diphenylamine sulfonate, Diphenylbenzidine, Diphenylamine, Viologen, N, N!~diphenyl~1,4~ diphenyldiamine, Lissamine Green B, N-phenyl~1,4~phenylenediamine, Sodium 2,6- Dibromophenol-indophenol, Sodium o-Cresol indophenol, Thionine, Methylene blue, Indigotetrasulfonic acid, Indigotrisulfonic acid, Indigo carmine, Indigomono sulfonic acid, Phenosafranin, Safranin T, and / or Neutral red, and / or a combination thereof.

[0112] In an embodiment, the disclosure provides a method of detecting, measuring, and / or monitoring amino acid concentration, comprising: subjecting a sample to at least one oxidase; and detecting and / or measuring a presence of hydrogen peroxide. In some embodiments, the sample is obtained from a subject,

[0113] In an embodiment, the disclosure provides a method of detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, comprising: subjecting a sample to the sensor described herein; and detecting and / or measuring a presence of hydrogen peroxide.Attorney Docket No. 081906-1535050-256510PC

[0114] In some embodiments, the sensor can be used to detect or measure free amino acids in any sample. In some embodiments, the sample is obtained from a subject, for example, a human. For example, the sample includes one or more of whole blood, serum, plasma, a blood fraction other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal discharge, milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, contents of cysts, tissue, and / or ascites. In some embodiments, the sample can be obtained through non-invasive or invasive means, optionally via finger stick, blood draw, biopsy, spinal tap, sweat and / or saliva collection.

[0115] In additional embodiments, the enzymes, kits, devices (e.g., sensors), and / or apparatus described herein are used in methods of improving health and wellness in a subject in need thereof.

[0116] In some embodiments, the method of improving health and wellness is used in conjunction with a food item or supplement comprising a high amino acid composition.

[0117] In some embodiments, the food item or supplement contains more than 1g, more than 2.5g, more than 5g, more than 7.5g, more than 10g, more than 12.5g, more than 15g, more than 17.5g, more than 20g, more than 22.5g, more than 25g, more than 27.5g, more than 30g, more than 32.5g, more than 35g, more than 37.5g, more than 40g, more than 42.5g, more than 45g, more than 47.5g, or more than 50g of branched-chain amino acids (BCAA) per unit. In some embodiments, the food item or supplement contains more than 1, more than 2.5g, more than 5g, more than 7.5g, more than 10g, more than 12.5g, more than 15g, more than 17.5g, more than 20g, more than 22.5g, more than 25g, more than 27.5g, more than 30g, more than 32.5g, more than 35g, more than 37.5g, more than 40g, more than 42.5g, more than 45g, more than 47.5g, or more than 50g of leucine per unit. In some embodiments, the food item or supplement contains more than 1g, more than 2.5g, more than 5g, more than 7.5g, more than 10g, more than 12.5g, more than 15g, more than 17.5g, more than 20g, more than 22.5g, more than 25g, more than 27.5g, more than 30g, more than 32.5g, more than 35g, more than 37.5g, more than 40g, more than 42.5g, more than 45g, more than 47.5g, or more than 50g of alanine per unit.

[0118] In additional embodiments, a protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 33 is provided, wherein said proteinAttorney Docket No. 081906-1535050-256510PCcomprises an alanine at position 24. In some embodiments, the protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 33. In some embodiments, the protein comprises an amino acid sequence at least 98% identical to SEQ ID NO: 33. In some embodiments, the protein comprises an amino acid sequence at least 99% identical to SEQ ID NO: 33.

[0119] In some embodiments, the method described herein covers using a test kit, wherein the test kit utilizes the racemase and / or the oxidase described herein.BRIEF DESCRIPTION OF THE FIGURES

[0120] Figure 1: Visual display of the design of SEQ ID NO: 32 (5WYA_Mut1), an engineered isoleucine racemase as predicted by the Rosetta Molecular Modeling Suite.

[0121] Figure 2: Visual display of the design of SEQ ID NO: 33 (5WYA_Mut2), an engineered enzyme that exhibits specific activity against leucine as predicted by the Rosetta Molecular Modeling Suite.

[0122] Figure 3: SDS-PAGE image showing the expression level of SEQ ID NO: 31 (5WYA: “WT”), SEQ ID NO: 32 (5WYA_ ut1: “Mut 1”), and SEQ ID NO: 33 (5WYA_Mut2: “Mut 2”).

[0123] Figures 4A-4E; Detection results of hydrogen peroxide produced by applying the enzyme of SEQ ID NO: 31 (5WYA: “WT 2”) as the racemase in Strategy 2.

[0124] Figures 5A-5E: Detection results of hydrogen peroxide produced by applying the enzyme of SEQ ID NO: 32 (5WYA_Mut1: “Mut 1") as the racemase in Strategy 2.

[0125] Figures 6A-6E: Detection results of hydrogen peroxide produced by applying the enzyme of SEQ ID NO: 33 (5WYA_Mut2: “Mut 22”) as the racemase in Strategy 2.

[0126] Figure 7: Initial screening of sequence similarity networks (SSNs composed of 2,000 AHAO sequences.

[0127] Figures 8A-8C: The top view (FIG. 8A) and cross-sectional views (FIG. 8B and FIG. 8C) of an exemplary sensor according to an embodiment of the present invention.

[0128] Figures 9A-9C: The top view (FIG. 9A) and cross-sectional views (FIG. 9B and FIG.9C) of another exemplary sensor according to an embodiment of the present invention.Attorney Docket No. 081906-1535050-256510PC

[0129] Figure 10: The top view of yet another exemplary sensor according to an embodiment of the present invention.

[0130] Figure 11: The cross-sectional view of an exemplary first electrode, with an exemplary entrapment layer and exemplary protective layers formed thereon according to an embodiment of the present invention.

[0131] Figure 12: An overview of the configuration of the reagent layer and electrochemical reaction according to an embodiment of the present invention.

[0132] Figures 13A-13B: Investigation of the transducer for detecting hydrogen peroxide byproduct according to an embodiment of the present invention.

[0133] Figures 14A-14B: Preliminary detecting results of leucine in PBS according to an embodiment of the present invention.

[0134] Figures 15A-15B: Preliminary detecting results of alanine in PBS according to an embodiment of the present invention.

[0135] Figures 16A-16C: Detecting results of alanine in PBS under different incubation time according to an embodiment of the present invention.

[0136] Figures 17A-17B: Detecting results of L-alanine in PBS according to an embodiment of the present invention.

[0137] Figures 18A-18B: Detecting results of L-alanine in PBS with proteins according to an embodiment of the present invention.

[0138] Figures 19A-19B: Detecting results of L-alanine in PBS according to an embodiment of the present invention.

[0139] Figures 20A-20B: Detecting results of L-alanine in PBS according to an embodiment of the present invention.

[0140] Figures 21A-21B: Detecting results of L-alanine in PBS according to an embodiment of the present invention.

[0141] Figure 22: Relative current responses upon the addition of various potential interferents according to an embodiment of the present invention.

[0142] Figures 23A-23B: Detecting results of L-leucine In PBS according to an embodiment of the present invention.

[0143] Figures 24A-24B: Detecting results of L-leucine in PBS according to an embodiment of the present invention.

[0144] Figures 25A-25B: Detecting results of L-leucine in PBS according to an embodiment of the present invention.Attorney Docket No. 081906-1535050-256510PC

[0145] Figures 26A-26B: Detecting results of L-leucine in PBS according to an embodiment of the present invention.

[0146] Figures 27A-27B: Detecting results of L-leucine in PBS according to an embodiment of the present invention.

[0147] Figure 28: Reproducibility testing results of L-alanine detection according to an embodiment of the present invention.

[0148] Figures 29A-29B: Detecting results of L-alanine in capillary blood according to an embodiment of the present invention.

[0149] Figures 30A-30K: FIGs. 30A-3OC are schematic illustrations of the developed biosensor strip featuring a three-electrode system and the underlying principle electrochemical reaction. FIG. 30D is a schematic illustration of the optimized configuration layers of the biosensor according to an embodiment of the present invention. FIG. 30E-30G are detecting results of L-alanine according to an embodiment of the present invention. FIG, 30H is a schematic illustration of the optimized configuration layers of the biosensor according to an embodiment of the present invention. FIG. 30I-30K are detecting results of L-leucine according to an embodiment of the present invention.

[0150] Figures 31A-31E: Chronoamperometric responses and corresponding current signal profiles for L-alanine level changes in healthy subjects following protein drink consumption over a 2-hour period.

[0151] Figures 32A-32B: Chronoamperometric response of the L-leucine biosensor in capillary blood spiked with different L-leucine concentrations.

[0152] Figures 33A-33B: Schematic illustrations of two exemplary sensors according to embodiments of the present invention.

[0153] Figures 34A-34D: Detecting results of L-alanine and L-leucine in whole blood samples following intake of whey protein (60g). The biosensing strips were manufactured according to typical commercial strip standards (2 mm diameter working electrode and 20pL sample volume). The configuration of the L-alanine and L-leucine sensors are shown in FIG. 30D and FIG. 30H.

[0154] Figures 35A-35C: Evaluation of storage life of L-alanine and L-leucine sensors when storing in a refrigerator (about 1,5~5°C). The configuration of the L- alanine and L-leucine sensors are shown in FIG. 30D and FIG. 30H.Attorney Docket No. 081906-1535050-256510PC

[0155] Figures 36A-36B: Evaluation of storage life of L-alanine and L-leucine sensors under room temperature (about 15-30’C). The configuration of the L- alanine and L-leucine sensors are shown In FIG. 30D and FIG. 30H.

[0156] Figures 37A-37D: Evaluation of storage life of L-leucine sensors coated with stabilizer (trehalose). The configuration of the L-leucine sensor is shown in FIG. 30H FIGs. 37A-37B show the results of storing in a refrigerator (about 1.5-5oC): FIGs. 37C-37D show the results of storing under room temperature (about 15-30 ).

[0157] Figures 38A-38F: Custom-designed sensor strips and storage box.

[0158] Figures 39A-39B: Custom-designed sensor strip with the electrodes attached with an additional layer to define and control the sample fluid’s coverage of the sensing surface area. An additional spacer, which may be transparent or nontransparent, is incorporated to facilitate capillary-driven sample filling.

[0159] Figures 40A-40B: Detecting results of L-alanine by touch-based sweat sensors following intake of whey protein (60g).

[0160] Figures 41A-41B: Schematic illustrations of a blood strip sensor (FIG. 41 A) and a touch-based sweat sensor (FIG. 41B) according to embodiments of the present invention.

[0161] Figures 42A-42B: Detecting results of L-alanine and L-leucine in whole blood samples following intake of whey protein (60 g). The biosensing strips were manufactured according to typical commercial strip standards (2 mm diameter working electrode and 20pL sample volume). The configuration of the L-alanine and L-leucine sensors are shown in FIG, 30D and FIG. 30H

[0162] Figures 43A-43B: Evaluation of storage life of L-alanine and L-leucine sensors when storing in a refrigerator (about 1. S-S’C). The configuration of the L~ alanine and L-leucine sensors are shown in FIG. 30D and FIG. 30H.

[0163] Figures 44A-44D: Detecting results of L-alanine and L-leucine in whole blood samples following beef consumption. FIGs.44A-44B are detecting results of L- alanine following beef consumption (400g); FIGs 44C-44D are detecting results of L- leucine following intake of a full meal (50g beef). The biosensing strips were manufactured according to typical commercial strip standards (2 mm diameter working electrode and 20pL sample volume). The configuration of the L-alanine and L-leucine sensors are shown in FIG. 30D and FIG. 30H

[0164] Figures 45A-45D: Detecting results of L-alanine and L-leucine in whole blood samples following intake of whey protein (60g) and collagen peptides (60g). TheAttorney Docket No. 081906-1535050-256510PCbiosensing strips were customed-manufactured (2 mm diameter working electrode and 5pL sample volume). The configuration of the L-alanine and L-leucine sensors are shown in FIG. 30D and FIG.30H.

[0165] Figures 46A-46J: Detecting results of L-alanine (FIGs.46A-46B) and L- leucine (FIGs.46C-46D) in PBS; and detecting results of L-alanine and L-leucine in whole blood samples following intake of whey protein (60g) (FIGs.46E-46J) The biosensing strips had 3 mm diameter working electrode and 20pL sample volume. The configuration of the L-alanine and L-leucine sensors are shown in FIG.30 D and FIG. 30H.

[0166] Figures 47A-47D: Detecting results of L-alanine and L-leucine in whole blood samples following intake of whey protein (60g). The biosensing strips were manufactured according to typical commercial strip standards (3 mm diameter working electrode and 20pL sample volume). The configuration of the L-alanine and L-leucine sensors are shown in FIG.30D and FIG.30H.

[0167] Figures 48A-48L: Systematic evaluation of sensor design adjustments.FIGs. 48A-48C show various sensor designs with different dimensions according to embodiments of the present invention. FIG.48D compares the dimension of an exemplary sensor with a thumb. FIGs.48E-48J show the electrochemical performance assessment of each electrode design toward H2O2. FIG.48 K shows detection of L-alanine concentrations by a miniaturized sensor strip with a 1 x 2.5 mm WE. FIG.48L shows chronoamperometric responses of the sensor strip with a 1 x 2.5 mm WE in whole-blood samples.

[0168] Figures 49A-49D: Custom-engineered D-amino acid oxidase for L-alanine and L-leucine biosensor systems. Chronoamperometric responses and corresponding current signal profiles for L-alanine (FIGs.49A-49B) and L-leucine (FIGs. 49C-49D).

[0169] Figures 50A-50O: Detection of L-alanine and L-leucine in diluted and whole blood using optimized biosensor systems. FIGs. 50A-50C are schematic illustrations of the optimized configuration layers of the L-alanine sensor (FIG.50A)sthe L-leucine sensor (FIG. 50B):and the L-leucine sensor incorporating the optimized custom-engineered D-amino acid oxidase (FIG.50C). FIG. 50D shows examples of sensor-platform designs with modifiable WE surface that can accommodate the configuration-layer architectures of FIGs. 50A-50C FIGs. 50E-50L are chronoamperometric responses and corresponding current signal profiles forAttorney Docket No. 081906-1535050-256510PCthe L-leucine sensor in diluted blood samples (FIGs.50E-5QF), the L-leucine sensor in whole blood samples (FIGs. 50G-50H), the L-alanine sensor in whole blood samples (FIGs. 50I-50J), and the L-leucine sensor in whole blood samples (FIGs. 50K-50L). FIG. 50M shows simultaneous detection of L-alanine and L-leucine in diluted blood samples. FIG. 50N shows detection of L-alanine in whole-blood samples. FIG. 500 shows detection of L-leucine in whole-blood samples.

[0170] Figures 51A-51F: Chronoamperometric responses and corresponding current signal profiles for different target analytes: L-leucine (FIGs. 51A-51B), L-methionine (FIGs. 51C-51D), and L-arginine (FIGs. 51E-51F).

[0171] Figures 52A-52B: Detecting results of L-alanine by touch-based sweat sensors following intake of whey protein (60g).

[0172] Figures 53A-53B: Simultaneous detection of L-alanine and glucose in capillary blood samples by dual sensors following intake of whey protein.

[0173] Figures 54A-54B: Schematic illustrations of the configuration layers of a microneedle (MN) sensor (FIG. 54A); and real-time continuous detection of L-alanine and L-leucine in human interstitial fluid (ISF) (FIG. 54B).

[0174] Figure 55: A dual-biosensing approach for the simultaneous detection of L- alanine and glucose in capillary blood samples, alongside the detection of L-alanine in human fingertip sweat using touch-based biosensing.

[0175] Figures 56A-56C: A spacer-integrated sensor strip design according to an embodiment of the present invention.

[0176] Figures 57A-57C: Various designs for immobilizing the reaction enzymes (e.g., the racemases and the oxidases).Attorney Docket No. 081906-1535050-256510PCDETAILED DESCRIPTION OF THE DISCLOSURE

[0177] The disclosure provides for enzymes, sensors, kits, and methods of detecting, measuring, and / or monitoring amino acids. In aspects, the disclosure provides for sensors for detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids using enzymes described herein.

[0178] In aspects, the disclosure provides for sensor templates as well as alternative sensor form factors that can be adapted for the detection, measurement, and / or monitoring of any desired specific amino acid or combinations of different amino acids.

[0179] In aspects, the disclosure provides for variations of the sensor templates as well as alternative sensor form factors that require and / or use different sample volumes, including but not limited to sensor modifications and / or adaptations that effectively require and / or use reduced sample volumes (e.g., reduced blood volumes). The required sample volumes for use with the sensors described herein vary depending on the type of sample being collected, the type of device used to assist in its collection, and the device that is used to analyze the sample.

[0180] For example, for blood samples the volume of blood collected to use with a sensor for detecting amino acids according to the present disclosure may vary from 0.1 pL to 20,000 pL (i.e., 0.0001 ml to 20.0 ml), wherein some portion or all of the collected blood sample may be placed on or in the sensors for analysis. In some embodiments, the amount of blood that is collected ranges from 1.0 pL to 100.0 pL, or 5.0 pL to 50.0 pL, or 5.0 µL to 20.0 pL. In some embodiments, the amount of blood that is collected is 0.1 pL, 0.2 pL, 0.3 pL, 0.4 pL, 0.5 pL, 0.6 pL, 0.7 pL, 0.8 pL, 0.9 pL, 1.0 pL, 2.0 pL, 3.0 pL, 4.0 pl, 5.0 pL, 6.0 pL, 7.0 pL, 8.0 pL, 9.0 pL, 10.0 pL, 11.0 pL, 12.0 pL, 13.0 pL, 14.0 pL, 15.0 pL, 16.0 pL, 17.0 pL, 18.0 pL, 19.0 pL, 20.0 pL, 21.0 pL, 22.0 pL, 23.0 pL, 24.0 pL, 25.0 pL, 26.0 pL, 27.0 pL, 28.0 pL, 29.0 pL, 30.0 pL, 31.0 pL, 32.0 pL, 33.0 pL, 34.0 pL, 35.0 pL, 36.0 pL, 37.0 pL, 38.0 pL, 39.0 pL, 40.0 pL, 41.0 pL, 42.0 pL, 43.0 pL, 44.0 pL, 45.0 pL, 46.0 pL, 47.0 pL, 48.0 pL, 49.0 pL, 50.0 pL, 60.0 pL, 70.0 pL, 80.0 pL, 90.0 pL, 100.0 pL, 150.0 pL, 200.0 pL, 250.0 pL, 300.0 pL, 350.0 pL, 400.0 pL, 450.0 pL, 500.0 pL, 550.0 pL, 600.0 pL, 650 pL, 700.0, 750.0 pL, 800.0 pL, 850.0 pL, 900.0 pL, 950.0 pL, 1,000.0 pL, 2,000.0 pL, 3,000.0 pL,Attorney Docket No. 081906-1535050-256510PC4,000.0 pL, 5,000.0 pL, 6,000.0 pL, 7,000.0 pL, 8,000.0 pL, 9,000.0 pL, 10,000.0 pL, 15,000 pL, or 20,000 pL

[0181] The device that can used to detect amino acids in blood according to the present disclosure include any suitable device for blood analysis, including but not limited to microfluidic lab-on-a-chip devices / microfluidic platforms / microfluidic sensors; handheld, portable, point-of-care (POC) devices (e.g., glucose meters, lactate analyzers, hemoglobin testing devices, blood testing strips, etc.); benchtop POC devices; clinical laboratory instruments; clinical chemistry analyzers (e.g,, flow injection analysis systems, flow injection biosensors); blood gas analyzers; complete blood count (CBC) analyzers; immunoassay analyzers; serological testing devices; plasma testing devices; serum testing devices, etc. In some embodiments, the sensors of the present disclosure can be used for in-line detection of amine acid levels in blood flowing through a hemodialysis machine / dlalysis device. See, e.g., Gotch et al., July-September 1990, An on-line monitor of dialyzer Na and K flux in hemodialysis, ASAIO Trans., 36(3): 359-361, PMID; 2252699.

[0182] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine, Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.

[0183] There are various methods in the literature that permit the incorporation of an unnatural amino acid derivative or analog into a polypeptide chain in a sitespecific manner, see, for example, WO 2002 / 086075, US 8,114,648, US 9,163,271, US 7,045,337, US 7,713,721, US 7,354,761, US 8,173,364, US 8,012,739, US 7,368,275, US 8,183,012, US 8,030,074, US 8,173,392, US 7,083,970, US 7,638,300, and US 7,915,025, the content of each of which is incorporated by reference in its entirety.Attorney Docket No. 081906-1535050-256510PC

[0184] Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission, Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0185] “Polypeptide,’’ “peptide,’’ and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0186] In some contexts of the present disclosure, the term “protein” can refer to one or more enzymes used in the sensors provided herein. In contrast, in other contexts of the present disclosure, the term “protein” can refer to a food and / or energy source (e.g., whey or collagen) consumed by a human or animal

[0187] Whey protein is a high-quality protein that comes from whey, the liquid portion of milk that separates during cheese production. Whey is known as a complete protein source, which means it contains all 9 essential amino acids. Whey is rich in BCAAs, especially leucine, which is important for muscle protein synthesis. Whey is absorbed quickly compared to other proteins (i.e„ exhibits fast digestion).

[0188] Collagen is a structural protein that makes up much of the body’s connective tissue. It is the most abundant protein in humans and animals. Collagen is a major component of skin, tendons, ligaments, bones, cartilage, blood vessels, hair, and nails. Collagen is rich in the amino acids glycine, proline, and hydroxyproline, together which form a triple-helix structure that gives collagen its strength.

[0189] In some embodiments, the sensor signals of the leucine sensors described herein increase after the consumption of whey (a leucine rich protein) but not after the consumption of collagen. This demonstrates that the leucine sensors provided herein respond specifically to leucine instead of to protein intake in general (e.g., collagen).

[0090] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, (e.g., two proteases of the disclosure and polynucleotides that encode them) refer to two or more sequences or subsequencesAttorney Docket No. 081906-1535050-256510PCthat are the same or have a specified percentage of amine acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.[0191 J For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0192] In the context of the present application, a sequence that is “at least 80% identical to a reference sequence* is a sequence having, over its entire length, 80%, or more, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% sequence identity with the entire length of a reference sequence. Proteins consisting of an amino acid sequence “at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.6%, or at least 99.7%, or at least 99.8% identical" to a reference sequence may comprise mutations such as deletions, insertions and / or substitutions compared to the reference sequence. In case of substitutions, the protein consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.6%, or at least 99.7%, or at least 99.8% identical to a reference sequence may correspond to a homologous sequence derived from another species than the reference sequence.

[0193] In the context of the present application, the “percentage of identity* can be calculated using a global pairwise alignment ( / .e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. For example, the “needle" program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may be used. The needle program is for example available on the ebi.ac.uk World Wide Web site and is further described in the following publication (EMBOSS: The European Molecular BiologyAttorney Docket No. 081906-1535050-256510PCOpen Software Suite (2000) Rice, P. Longden, L and Bleasby, A. Trends in Genetics 16, (6) pp. 276 — 277). The percentage of identity between two polypeptides, in accordance with the invention, is calculated using the EMBOSS: needle (global) program with a " Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix. Other algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul etaL, J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977, respectively. Software for performing BLAST analyses is publicly available through the Nafional Center for Biotechnology Information. Each of these references are herein incorporated by reference in their entireties.

[0194] “At least one” herein refers to one or more of the specified objects such as 1, 2, 3, 4, 5 or 6 or more of the specified objects. For example, at least one amino acid substitution herein refers to 1, 2, 3, 4, 5 or 6 or more amino acid substitutions.

[0195] “Amino acid substitutions” may be conservative or non-conservative. In an aspect, substitutions are conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and / or chemical properties. In an aspect the conservative substitution leads to the same or similar functional properties.

[0196] In an embodiment, conservative substitutions may include those, which are described by Dayhoff in “The Atlas of Protein Sequence and Structure. Vol. 5s, Natl Biomedical Research, the contents of which are incorporated by reference in their entirety. For example, in an aspect, amino acids, which belong to one of the following groups, can be exchanged for one another, thus, constituting a conservative exchange: Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine (S), threonine (T): Group 2: cysteine (C), serine (S), tyrosine ( Y), threonine (T); Group 3: valine (V), isoleucine (I), leucine (L), methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K), arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H); and Group 6: aspartic acid (P), glutamic acid (E). In an aspect, a conservative amino acid substitution may be selected from the following of T— > A, G—> A, A~*l, T~»V, T— -*!, A~*V, T~*G, and / or T — *S.

[0197] In a further embodiment, a conservative amino acid substitution may include the substitution of an amino acid by another amino acid of the same class,Attorney Docket No. 081906-1535050-256510PCfor example, (1) nonpolar: Ala, Vai, Leu, He, Pro, Met, Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gin; (3) acidic,' Asp, Glu; and (4) basic: Lys, Arg, His, Other conservative amino acid substitutions may also be made as follows: 1 ) aromatic: Phe, Tyr, His: (2) proton donor: Asn, Gin, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gin (see, for example, U. S. Patent No.10,106,805, the contents of which are incorporated by reference in their entirety).

[0198] In another embodiment, conservative substitutions may be made in accordance with Table 1. Methods for predicting tolerance to protein modification may be found in, for example, Guo et al.. Proc. Natl. Acad. Sci., USA, 101 (25):9205- 9210 (2004), the contents of which are incorporated by reference in their entirety.Table 1: Representative Conservative Amino Acid substitutions.Conservative Amino Acid SubstitutionsAmino Acid Substitutions (others are known in the ait) Ala Ser, Gly, CysLys, Gin, HisAsn Gin, His, Glu, AspAsp Glu, Asn, GinCys Ser, Met, ThrGin Asn, Lys, Glu, Asp, ArgGlu Asp, Asn, GinGly Pro, Ala, SerHis Asn, Gin, LysHe Leu, Vai, Met, AlaLeu He, Vai, Met, AlaLys Arg, Gin, HisMet Leu, lie, Vai, Ala, PhePhe Met, Leu, Tyr, Trp, HisSer Thr. Cys, AlaThr Ser, Vai, AlaTrp Tyr, PheTyr Trp, Phe, HisVai He, Leu, Met, Ala. Thr

[0199] In another embodiment, conservative substitutions may be those shown in Table 2 under the heading of “conservative substitutions.” If such substitutions result in a change in biological activity, then more substantial changes, denominatedAttorney Docket No. 081906-1535050-256510PC“exemplary substitutions” in Table 2, may be introduced and the products screened if needed.Table 2: Amino Acid substitutions.Amino Acid SubstitutionsOriginal Residue(naturallyoccurring amino Conservativeacid) Substitutions Exemplary Substitutions Ala (A) Vai Vai; Leu; HeArg (R) Lys Lys; Gin; AsnAsn (N) Gin Gin; His; Asp, Lys; Arg Asp (D) Glu Glu; AsnCys (C) Ser Ser; AlaGin (Q) Asn Asn; GluGlu (E) Asp Asp; GinGly (G) Ala AlaHis (H) Arg Asn; Gin; Lys; ArgHe (I) Leu Leu; Vai; Met; Ala; Phe;NorleucineLeu (L) He Norleucine; He; Vai; Met;Ala; PheLys (K) Arg Arg; Gin; AsnMet (M) Leu Leu; Phe; liePhe (F) Tyr Leu; Vai; lie; Ala; Tyr Pro (P) Ala AlaSer (S) Thr ThrThr (T) Ser SerTrp (W) Tyr Tyr; PheTyr (Y) Phe Trp; Phe; Thr; SerVai (V) Leu He; Leu; Met; Phe; Ala;NorleucineMethods of detecting and measuring amino acid concentration

[0200] Free amino acids may be oxidized by amino acid oxidases. This reaction may convert an amine acid into an imino acid, generating hydrogen peroxide (H2O2) as by-product. An exemplary reaction equation is illustrated below:Attorney Docket No. 081906-1535050-256510PCL- Ami no Acid Oxid cseL-AA

[0201] Therefore, by oxidizing free amino acids and detecting / measuring the hydrogen peroxide concentration, the amount of free amino acids contained in a sample fluid can be calculated. As further discussed below, inventors of the present invention proposed various methods of detecting and measuring free amino acids based on this concept.Strategy 1: Utilization of L Amino Acid Oxidases

[0202] All amino acids can be divided into two classes of stereoisomers: the L configuration and the D configuration. L-amino acids are the dominant form utilized by eukaryotes. Therefore, the free amino acids exist in body fluids such as blood or sweat are typically L-amino acids,

[0203] Amino acid oxidases that specifically catalyzes the oxidation of L-amino acids are L-amino acid oxidases. Because L-amino acids are the dominant form existing in body fluids, L-amino acid oxidases may be applied for directly catalyzing the oxidation of these free L-amino acids. An exemplary reaction equation is illustrated below:L- Amine Acid OxidaseRHg©2L-AA IminoAcid

[0204] This method produces hydrogen peroxide by a direct oxidation of free L-amino acids. This provides a simplified process with an improved specificity.Attorney Docket No. 081906-1535050-256510PCStrategy 2: Utilization of Combination of Racemases with D^Amino AcidOxidases

[0205] Alternatively, L-amino acids may be converted into D-amino acids through racemization, catalyzed by a racemase. D-amino acids may then be oxidized by D-amino acid oxidases, a type of amino acid oxidases that specifically catalyzes the oxidation of D-amino acids. By combining the two reactions, free L- amino acids contained in a sample fluid can be oxidized into imino acids. An exemplary reaction equation is illustrated below:

[0205] This method includes a racemization catalyzed by a racemase.Racemase may have specific reactivity with respect to different types of amino acids. Thus, the inclusion of this additional racemization step may further improve the specificity of the detection / measurement process with respect to certain types of amino acids.Strategy 3: Utilization of Alpha-Hydroxy Acid Oxidases

[0207] For example, alpha-hydroxy acids are structurally similar to amino acids-they differ from amino acids in that their alpha position contains a hydroxy group instead of an amine group. Alpha-hydroxy acid oxidases may catalyze the oxidation of alpha-hydroxy acids, thereby producing hydrogen peroxide. Thus, it is likely that some alpha-hydroxy acid oxidases may also possess cryptic function of catalyzing the oxidation of amino acids. An exemplary reaction equation is illustrated below:Attorney Docket No. 081906-1535050-256510PCNative Function:oHydrs&y AcidOxldssesOHHydroxy Acid OsH& 2-Oxo Acid Cryptic Function:Hydroxy AcidOxidasesOaH; O?IminoL-AAAci

[0208] This method relies upon novel enzyme activities that have not previously been found or developed yet. Thus, it is possible that enzymes with unique specificity may be discovered or developed.Detection of Hydrogen Peroxide (H2O2)

[0209] The amount of free amino acids contained in a fluid sample may be calculated by detecting and measuring the amount of hydrogen peroxide (H2O2) generated by the oxidation of the free amino acids.

[0210] Various ways of detecting and measuring hydrogen peroxide may be applied to the present invention. For example, commercially available test strips, such as the Bartovation hydrogen peroxide strips (https: / / bartovation.com), may be used. These strips may be calibrated at the 0, 1, 3, 10, 50, and 00 ppm. The test strip turns blue if hydrogen peroxide is presented, and bluer color indicates higher concentration of hydrogen peroxide.

[0211] Alternatively, hydrogen peroxide may be detected and measured by adding one or more redox indicators. Redox indicators are compounds that change color when being oxidized or reduced. In the present invention, during or after the oxidization is completed, one or more redox indicators may be added to the sample fluid. The oxidation of the one or more redox indicator by the hydrogen peroxide will trigger a color change, indicating that hydrogen peroxide is produced and existed in the sample fluid. Non-limiting examples of the one or more redox indicators include: 2,2 -bipyridine (Ru complex), Nitrophenanthroline (Fe complex), N-PhenylanthranilicAttorney Docket No. 081906-1535050-256510PCa d, 1,10-Phenanthroline iron(ll) sulfate complex, N-Ethoxychrysoidine, 2,2'-Bipyridine (Fe complex), 5,6-DimethyIphenanthroline (Fe complex), o-Dianlsidine, Sodium diphenylamine sulfonate, Diphenylbenzidine, Diphenylamine, Viologen, N. N Iphenyl-1,4~diphenyldiamine, Lissamine Green B, N~phenyl~1,4- phenylenediamine, Sodium 2,6-Dibromophenol-indophenol, Sodium o~Cresol indophenol, Thionine, Methylene blue, Indigotetrasulfonic acid, Indigotrisulfonic acid, Indigo carmine, Indigomono sulfonic acid, Phenosafranin, Safranin T, and / or Neutral red, and / or a combination thereof.Enzymes

[0212] Enzymes or proteins described herein may be engineered, isolated, or purified. In an aspect, enzymes or proteins described herein may be non-naturally occurring. In other aspects, enzymes or proteins described herein may be modified via amino acid substitution, deletion, and / or truncation. In other aspects, enzymes described herein are optimized to detect or analyze free amine acids. For example, enzymes described herein may be able to facilitate racemization or oxidation of free amino acids. In other aspects, enzymes described herein are designed for use with the systems, devices, kits, and methods described herein.Racemase

[0213] Racemases are enzymes that are able to facilitate racemization of biological molecules, such as free amino acids. Racemases may possess specificity, or specific reactivity, against certain amino acids. For example, alanine racemases are racemases having specificity against alanine. Thus, by utilizing racemases with specific reactivity, one may be able to detect the specific type of free amino acids that is of particular interest in a sample fluid.

[0214] Depending on the type of amino acids to be targeted, various racemases may be used. For example, the racemase may be an arginine racemase, a histidine racemase, a lysine racemase, an aspartate racemase, a glutamate racemase, a serine racemase, a threonine racemase, an asparagine racemase, a glutamine racemase, a cysteine racemase, a proline racemase, an alanine racemase, a valine racemase, an isoleucine racemase, a leucine racemase, a methionine racemase, a phenylalanine racemase, a tyrosine racemase, and / or aAttorney Docket No. 081906-1535050-256510PCtryptophan racemase, and / or a combination thereof. In some embodiments, the racemase may be active against one or more of alanine, methionine, phenylalanine, serine, proline, arginine, leucine, isoleucine, and / or valine.

[0215] In some embodiments, the racemase may include an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99,7%, or at least 99.8% identical to one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. 24, 25, 26, 27, 28, 29, 30, 31, 32. 33, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73. 74, 75, 76, 77, 78, 79, 80, or 81.

[0216] In some embodiments, the racemase may include an amino acid sequence of one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 55. 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73. 74, 75, 76, 77, 78, 79, 80, or 81, wherein said amine acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.Amino Acid Oxidases

[0217] Amino acid oxidases are enzymes that are able to facilitate oxidation of free amino acids. Amino acid oxidases may be divided into two classes: L~amino acid oxidases and D-amino acid oxidases. Amino acid oxidases may also possess specificity, or specific reactivity, against certain amino acids.

[0218] Depending on the type of amino acids to be targeted, various amino acid oxidases may be used. In some embodiments, the amino acid oxidase may be an arginine oxidase, a histidine oxidase, a lysine oxidase, an aspartate oxidase, a glutamate oxidase, a serine oxidase, a threonine oxidase, an asparagine oxidase, a glutamine oxidase, a cysteine oxidase, a glycine oxidase, a proline oxidase, an alanine oxidase, a valine oxidase, an isoleucine oxidase, a leucine oxidase, a methionine oxidase, a phenylalanine oxidase, a tyrosine oxidase, and / or a tryptophan oxidase, and / or a combination thereof.

[0219] In some embodiments, the amino acid oxidase may be active against one or more of D-arginine, D-histidine, D-lysine, D-aspartic acid, D-glutamic acid, D~ serine, D-threonine, D-asparagine, D-glutamine, D-cysteine, glycine, D-proline, D-Attorney Docket No. 081906-1535050-256510PCalanine, D-valine, D-isoleucine, D-leucine, D-methionine, D-phenylalanine, D-tyrosine, and / or D-tryptophan. For example, in some embodiments, the amino acid oxidase may be active against one or more of D-alanine, D-proline, D-arginine, D-leucine, D-isoleucine, and / or D-valine.

[0220] In some embodiments, the amino acid oxidase may be active against one or more of L-arginine, L-histidine, L-lysine, L-aspartic acid, L-glutamic acid. L- serine, L-threonine, L-asparagine, L-glutamine, L-cysteine, glycine, L-proline, L~ alanine, L-valine, L-isoleucine, L~leucine, L-methionine, L-phenylalanine, L-tyrosine, and / or L-tryptophan. For example, in some embodiments, the amino acid oxidase may be active against one or more of L-alanine, L-proline, L-arginine, L-leucine, L-isoleucine, and / or L-valine.

[0221] In some embodiments, the amino acid oxidase may include an amino acid sequence at least 80%, at least 85%, at least 90%. at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% identical to SEQ ID NO: 34.

[0222] In some embodiments, the amino acid oxidase may include an amino acid sequence of SEQ ID NO: 34, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

[0223] In some embodiments, the amino acid oxidase may include a D-amino acid oxidase.

[0224] In some embodiments, the D-amino acid oxidase is active against D- leucine and inactive against D-methionine.

[0225] In some embodiments, the D-amino acid oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 89, 94, or 97.Alpha-Hydroxy Acid Oxidases

[0226] Alpha-hydroxy acid oxidases are enzymes that are able to facilitate oxidation of alpha-hydroxy acids. The alpha-hydroxy acid oxidase may possess cryptic function of catalyzing the oxidation of certain amino acids. In some embodiments, the alpha-hydroxy acid oxidases may possess cryptic activity against one or more of alanine, proline, arginine, leucine, isoleucine, and / or valine.Attorney Docket No. 081906-1535050-256510PC[02271 In some embodiments, the alpha-hydroxy acid oxidase may indude an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% identical to one of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54.

[0228] In some embodiments, the alpha-hydroxy acid oxidase may include an amino acid sequence of one of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.Expression of the Enzymes

[0229] The method of expressing and purifying the enzymes is described in Yoshikuni et al., U. S. Patent Publication No. 2022 / 0348970 Al, the contents of each which are hereby incorporated herein by reference in their entireties.

[0230] Various organisms may be used as the host to express the enzymes of interest. For example, prokaryotic cells such as Escherichia coll cells may be used as the host cell for expressing the enzymes. Non-limiting examples of prokaryotic cells include: Acetobacter aceti, Achromobacter, Acidiphilium, Acinetobacter, Actinomadura, Actinoplanes, Aeropyrum pernix, Agrobacterium, Alcaligenes, Ananas comosus (M), Arthrobacter, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus lentus, Bacillus licheniformis, Bacillus macerans, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium, Brevibacillus brevis, Burkholderia cepacia, Candida cylindracea, Caricapapaya (L), Cellulosimicrobium, Cephalosporium, Chaetomium erraticum, Chaetomium gracile, Clostridium, Clostridium butyricum, Clostridium acetobutylicum, Clostridium thermocellum, Corynebacterium glutamicum,, Corynebacterium efficiens, Escherichia coll, Enterococcus, Erwinia chrysanthemi, Gluconobacter, Gluconacetobacter, Haloarcula, Humicola insolens, Kitasatospora setae, Klebsiella, Klebsiella oxytoca, Kocuria, Lactococcus lactis, Lactobacillus, Lactobacillus fermentum, Lactobacillus sake, Lactococcus, Lactococcus lactis, Leuconostoc, Methylocystis, Methanolobus siciliae, Methanogenium organophilum, Methanobacterium bryantii, Microbacterium imperiale, Micrococcus lysodeikticus, Microlunatus, Mucor javanicus, Mycobacterium, Myrothecium, Nitrobacter, Nitrosomonas, Nocardia, Papaya carica,Attorney Docket No. 081906-1535050-256510PCPediococcus, Pediococcus halophilus, Paracoccus pantotrophus, Propionibacterium, Pseudomonas, Pseudomonas fluorescens. Pseudomonas denitrificans, Pyrococcus, Pyrococcus furiosus, Pyrococcus horikoshii, Rhizobium, Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, Rhizopus delemar, Rhizopus japonicus, Rhizopus niveus, Rhizopus oryzae, Rhizopus oligosporus, Rhodococcus, Sclerotinia libertiana, Sphingobacterium multivorum, Sphingobium, Sphingomonas, Streptococcus, Streptococcus thermophilus Y-1, Streptomyces, Streptomyces griseus, Streptomyces lividans, Streptomyces murinus, Streptomyces rubiginosus, Streptomyces violaceoruber, Streptoverticillium mobaraense, Tetragenococcus, Thermus, Thiosphaera pantotropha, Trametes, Vibrio alginolyticus, Xanthomonas, Zymomonas, and Zymomonas mobilis,

[0231] Likewise, eukaryotic cells may also be used as the host cell for expressing the enzymes. Non-limiting examples of eukaryotic cells include:Aspergillus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus melleus, Aspergillus pulverulentus, Aspergillus saitoi. Aspergillus sojae, Aspergillus terreus, Aspergillus pseudoterreus, Aspergillus usamii, Candida rugosa, Issatchenkia orientalis, Kluyveromyces, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus, Penicillium, Penicillium camembertii, Penicillium citrinum, Penicillium emersonii, Penicillium roqueforti, Penicillium lilacinum, Penicillium multicolor, Pichia, Rhodosporidium toruloides, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Trichosporon penicillatum, Yarrowia lipolytica, and Zygosaccharomyces rouxii.Amino acid sensor

[0232] In other aspects, the disclosure provides for a sensor, such as a wearable or a nonwearable sensor, capable of detecting and / or measuring amino acid concentration including utilizing enzymes described herein. The concentration of a single amino acid and / or concentrations of multiple amino acids of interest in a sample may be detect by the wearable or nonwearable sensor. Such sensors, for example, not-wearable sensors, may be in the format of a test strip or a one-touch fingertip sweat sensor. In other aspects, the enzyme comprises one or more of SEQ ID NO: 1 - 97, fragments, or modified enzymes of SEQ ID NO: 1 - 97 as describedAttorney Docket No. 081906-1535050-256510PCherein. A detailed discussion of suitable applications of the enzyme in various nonwearable and wearable sensors, as well as the structural designs of the sensors, may be found in Yin et al. (US 2024 / 0049994), Wang et al. (US 10,364,452), Tehrani et al. (US 11,877,846), and Wang et al. (US 12,064,264), the content of each of which is hereby incorporated by reference in its entirety.

[0233] The sensors according to embodiments of the present invention may be an electrochemical sensor that includes a multi-electrode contingent, e.g., which can include a two electrode contingent or a three electrode contingent, or arrays of such multi-electrode contingents.

[0234] FIGs. 8A-8C show the top view (FIG, 8A) and cross-sectional views (FIG. 8B and FIG. 8C) of an exemplary sensor according to an embodiment of the present invention. The sensor 100 comprises a first electrode 110 and a second electrode 120 formed on a substrate 101. The substrate 101 supports the electrodes and other components. The substrate 101 may be formed of an electrically insulative material. For example, the material of the substrate 101 may comprise one or more of a composite material, a fibrous material, a woven textile, a non-woven textile, a polymer, an adhesive, a film, a gel, PTFE, and / or silicone. In some embodiments, the substrate 101 may be formed of a silicon layer, a silicon carbide layer, a silicon dioxide (silica) layer, a glass layer, a ceramic layer, a plastic layer (such as polyacrylate, polycarbonate, and polypropylene), and / or a combination thereof. In some embodiments, the substrate 101 may be formed of a transparent material or translucent material. In some other embodiments, the substrate 101 may be formed of an opaque material.

[0235] The first electrode 110 is operable as a working electrode. The working electrode (first electrode 110) is the primary site for electrochemical reactions, where the analyte interacts with the sensing material. The first electrode 110 may be made of an electrically conductive material. For example, the first electrode 110 may be made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof. To improve signal sensitivity, a layer of single-walled or multi-walled carbon nanotubes (CNTs) may be coated on the surface of the first electrode 110. The enzymes used in the present invention are attached to the first electrode 110 to facilitate the redox reaction with the target analyte (e.g., one or more amino acids of interest) contained in a sample, thereby producing one or more products (e.g., hydrogen peroxide) that are redox-active andAttorney Docket No. 081906-1535050-256510PCable to be sensed by the working electrode (first electrode 110). The design details are further discussed below.

[0236] The second electrode 120 may serve as a counter electrode and / or a reference electrode. In a two-electrode system (as shown in FIGs. 8A-8C), the second electrode 120 is operable as the counter electrode and the reference electrode. Alternatively, In a three-electrode system, the second electrode 120 may be operable as the counter electrode. The counter electrode completes the circuit and balances current flow, while the reference electrode provides a stable potential for accurate measurement. When the second electrode 120 is operable as the reference electrode in a two-electrode system, it may be a reference electrode commonly used in the field. For example, when the second electrode 120 is operable as the reference electrode, it may be an Ag / AgCI electrode, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper-copper(ll) sulfate electrode (CSE), a Pd / H₂ electrode, or a mercury-mercurous sulfate electrode (MSE). Alternatively, when the second electrode 120 is operable as the counter electrode in a three-electrode system, it may be made of an electrically conductive material. For example, when the second electrode 120 is operable as the counter electrode, it may be made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof. Details of a three- electrode system are further discussed below.

[0237] The first electrode 110 and the second electrode 120 may further connect to a first connection 112 and a second connection 122, respectively. The first connection 112 and the second connection 122 transfer signals to an external processing unit, enabling efficient detection and analysis. The first connection 112 and the second connection 122 may be made of electrically conductive materials, such as carbon graphite, copper, aluminum, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof.

[0238] The first connection 112 and the second connection 122 may be protected by a cover layer 150. The cover layer 150 may be made of an electrically insulative material, such that a sensing area may be defined, and the first connection 112 and the second connection 122 may be insulated. For example, the cover layer 150 may be made of glass, silicone rubber, porcelain, silicone, epoxy, or plastic materials (such as polyacrylate, polycarbonate, polypropylene, polyvinyl chloride,Attorney Docket No. 081906-1535050-256510PCpolyethylene, and ethylene tetrafluoroethylene). The cover layer 150 may be formed of a transparent material, a translucent material, or an opaque material.

[0239] FIGs. 9A-9C show the top view (FIG. 9A) and cross-sectional views (FIG. 9B and FIG. 9C) of another exemplary sensor according to an embodiment of the present invention. The sensor 200 comprises a first electrode 210, a second electrode 220, and a third electrode 230 formed on a substrate 201. In this three- electrode system, the first electrode 210 is operable as the working electrode, the second electrode 220 is operable as the counter electrode, and the third electrode 230 is operable as the reference electrode. The second electrode 220, functioning as the counter electrode, may be made of an electrically conductive material, such as carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof. The third electrode 230, functioning as the reference electrode, may be a reference electrode commonly used in the field, for example, an Ag / AgCI electrode, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHEj, a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper~copper(ll) sulfate electrode (CSE), a Pd / H₂ electrode, or a mercury-mercurous sulfate electrode (MSE).

[0240] The first electrode 210, the second electrode 220, and the third electrode 230 may further connect to a first connection 212, a second connection 222, and a third connection 232, respectively. The first connection 212, the second connection 222, and the third connection 232 transfer signals to an external processing unit, enabling efficient detection and analysis. The first connection 212, the second connection 222, and the third connection 232 may be protected by a cover layer 250, such that a sensing area may be defined, and the first connection 212, the second connection 222, and the third connection 232 may be insulated.

[0241] The first electrode, the second electrode, and the third electrode may be formed of any suitable shape. For example, the first electrode, the second electrode, and the third electrode may be triangular shapes or rectangular shapes. Alternatively, the first electrode, the second electrode, and the third electrode may have curvature shapes, such as circles, ellipses, ovals, parabolas, or sickle shapes.

[0242] FIG. 10 shows the top view of yet another exemplary sensor according to an embodiment of the present invention. The first electrode 310, the second electrode 320, the third electrode 330, the first connection 312, the second connection 322, and the third connection 332 of the sensor 300 are formed on theAttorney Docket No. 081906-1535050-256510PCsubstrate 301, The cover layer 350 protects the first connection 312, the second connection 322, and the third connection 332, and defines a sensing area. The first electrode 310 is designed to have a circular shape; while the second electrode 320 and the third electrode 330 are designed to have sickle shapes.

[0243] FIG. 11 shows the cross-sectional view of an exemplary first electrode, with an exemplary entrapment layer and exemplary protective layers formed thereon according to an embodiment of the present invention. An entrapment layer 415 is formed on the top surface of the first electrode 410. The entrapment layer 415 comprises one or more enzymes that can react with the target analyte, thereby producing one or more products (e.g., hydrogen peroxide) that are redox-active and able to be sensed by the working electrode (first electrode). For example, in some embodiments, the entrapment layer 415 may comprise at least one oxidase. In some embodiments, the entrapment layer 415 may comprise at least one oxidase and at least one racemase. In some embodiments, the entrapment layer 415 may comprise at least one oxidase and at least one racemase, wherein the at least one racemase comprises a racemase that is active against leucine. In some embodiments, the entrapment layer 415 may comprise at least one oxidase and at least one racemase, wherein the at least one oxidase is active against one or more of D-arginine, D- histidine, D-lysine, D-aspartic acid, D-glutamic acid, D~serine, D-threonine, D~ asparagine, D-glutamine, D-cysteine, glycine, D-proline, D-alanine, D-valine, D- isoleucine, D-leucine, D-methionine, D-phenylalanine, D-tyrosine, and / or D~ tryptophan. In some embodiments, the entrapment layer 415 may comprise at least one oxidase, wherein the comprise at least one oxidase is active against one or more of L-arginine, L~histidine, L-lysine, L-aspartic acid, L~glutamic acid, L-serine, L- threonine, L-asparagine, L-glutamine, L-cysteine, glycine, L-proline, L-alanine, L-valine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-tyrosine, and / or L~ tryptophan. In some embodiments, the entrapment layer 415 may comprise at least one oxidase, wherein the at least one oxidase is at least one hydroxy acid oxidase having cryptic activity against one or more of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and / or tryptophan.

[0244] Mediators or redox mediators are electron transferring agents that can participate in the redox reaction. The entrapment layer 415 may further comprise atAttorney Docket No. 081906-1535050-256510PCfeast one redox mediator, such as one or more of Prussian blue, ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complexes, and / or organometallic ruthenium complexes. For example, the entrapment layer 415 may comprise a transducer layer 414 and a receptor layer 416. The transducer layer 414 comprises the at least one redox mediator, and the receptor layer 416 comprises the enzymes (e.g., the at least one oxidase and the at least one racemase). In some embodiments, the transducer layer 414 comprises Prussian blue, and the receptor layer 416 comprises the enzymes (e.g., the at least one oxidase and the at least one racemase). Under such design, detection of the amino acids of Interest may be facilitated by a coupled reaction mechanism involving production of redox-active products (e.g., hydrogen peroxide) that are then reacted with the redox mediator (e.g., Prussian blue) and being detected chronoamperometrically at a low potential by the mediated reaction (e.g., PB-based mediated reaction). Such coupling reaction mechanism is illustrated in FIG. 12.

[0245] The entrapment layer 415 may be formed of any material that is adapted to capturing or retaining enzymes and / or redox mediators. The entrapment layer 415 may also be formed of any material that is adapted to facilitating the above-discussed coupling reaction. For example, the entrapment layer 415 may be a conductive polymer film, which comprises the at least one oxidase and / or the at least one racemase. In some embodiments, the entrapment layer 415 may be made of a material comprising one or more of poly(thionine), polyphenylene vinylene, polypyrrole, polythiophene, polyaniline, and / or polyphenylene sulfide, and / or a combination thereof. To immobilize the enzymes, the entrapment layer 415 may further be cross-linked with the enzymes (e.g., the at least one oxidase and the at least one racemase). For example, the entrapment layer 415 may be cross-linked with the enzymes (e.g., the at least one oxidase and the at least one racemase) by using a cross-linking agent, such as one or more of glutaraldehyde, diisocyanates, carbodiimides, acyl azide, genipin, and / or polyepoxides, and / or a combination thereof.

[0246] In some embodiments, to improve stability of the enzymes, one or more enzyme stabilizers may be added to the entrapment layer 415 and / or the receptor layer 416. Enzyme stabilizers may include one or more of the following: polyols (such as polyether polyol and polyester polyol), proteins (such as bovine serumAttorney Docket No. 081906-1535050-256510PCalbumin (BSA) ), surfactants (such as Tween 20 (Polysorbate 20), Tween 80 (Polysorbate 80), sugars (such as trehalose, sucrose, glucose, and fructose), Triton X-100, n-Decyl-p~D-maltopyranoside (DM), n~Dodecyl-j3-D~maltopyranoside (DDM), and 6-Cyclohexyl-1’-hexyl-p-D-maltopyranoside (Cymal-6)), nanomaterials, ionic liquids (such as 1-butyl-3-methylimidazolium bromide ([BMIM / Br]), 1-butyl-3-methylimidazolium chloride ([BMIM / Cl]), 1-butyl-3-methylimidazolium iodide ([BMIM / I]), and 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM / TfO])). chitosan, hydrogels (formed by materials such as hyaluronic acid, chitosan, heparin, alginate, gelatin, fibrin, polyvinyl alcohol, polyethylene glycol (PEG), sodium polyacrylate, acrylate polymers, and / or polyvinylpyrrolidone (PVP)), and / or PEG and PEG derivatives (such as Macrogol and polyethylene glycol-poly vinyl alcohol (PEG- PVA)). With the addition of one or more enzyme stabilizers, the storage life and / or the shelf life of the sensor may be further improved.

[0247] The entrapment layer 415 may also be protected by one or more protective layers. For example, in some embodiments, the entrapment layer 415 may be covered by an inner protective layer 417 and / or an outer protective layer 418. In some embodiments, the inner protective layer 417 and / or the outer protective layer 418 may include a charge-exclusion material that allows charge neutral species, positively charged species, and / or negatively charged species to pass through the protective layer(s). In some embodiments, the inner protective layer 417 and / or the outer protective layer 418 may be formed by Nafion or ion-exchange resins (e.g., polystyrene sulfonate and polyacrylate). In this way, the specificity of the sensor may be further improved.

[0248] In some embodiments, the entrapment layer 415 may further comprise a co-factor. In some embodiments, the co-factor includes flavin adenine dinucleotide (FAD) and / or pyridoxal 5’-phosphate (PLP).

[0249] The wearable or nonwearable sensor can be used to detect or measure amino acids in a sample. For example, the wearable or nonwearable sensor can be used to detect a single amino acid and / or multiple amino acids of interest in a sample. The sample may be liquid, such as a body fluid, or solid, such as an organ tissue. For example, the sample comprises one or more of whole blood, serum, plasma, a blood fraction other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal discharge, milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid,Attorney Docket No. 081906-1535050-256510PCsynovial fluid, peritoneal fluid, pericardial fluid, peritoneum, glandular secretions, exudate, contents of cysts, tissue, and / or ascites,

[0250] Textbooks and reference works on biosensors, wearable sensors, and / or electrochemical sensing include but are not limited to Wearable Sensors - Fundamentals, Implementation and Applications, November 13, 2020, Academic Press, Edward Sazonov (ed,); Wearable Biosensing in Medicine and Healthcare, January 5, 2024, Springer Singapore, Kohji Mitsubayashi (edj; Electrochemical Sensors, Biosensors and their Biomedical Applications, November 27, 2007, Academic Press, Zhang et al. (eds ); Biomarkers and Biosensors, December 11, 2014, Elsevier Science, A. Sadana and N. Sadana (authors); Graphene in Wearable Sensors for Health Monitoring, September 26, 2025, Springer Singapore, Debnath et al. (authors); Modern Techniques in Biosensors: Detection Methods and Commercial Aspects, January 6, 2022, Springer, Dutta et al. (authors); Advanced Sensors for Biomedical Applications, June 12, 2022, Springer Cham, O. Kanoun and N. Derbel (eds.): and Biomaterials-Based Sensors, March 2, 2023, Springer Singapore, Kumar et al, (eds.), the contents of each of which are hereby incorporated by reference in their entireties.

[0251] In some embodiments, the sensor is configured on a disposable test strip. In some embodiments, the sensor is a fingertip sweat sensor. In some embodiments, the sensor is a fingertip capillary blood sensor. For example, the sensor may be a fingertip sweat sensor that comprises a plurality of electrodes and a sweat permeation layer that transfers a sweat sample to the plurality of electrodes. In some embodiments, the fingertip sweat sensor may be formed on a rigid substrate, such that a user may press its finger(s) to the fingertip sweat sensor to effectuate the sample collecting process. In some embodiments, the fingertip sweat sensor may be formed on a flexible substrate, such that a user may wrap the fingertip sweat sensor around its finger(s) to effectuate the sample collecting process. In some embodiments, the fingertip sweat sensor may be self-powered. For example, the fingertip sweat sensor may comprise a piezoelectric chip, such that the piezoelectric chip may undergo a non-destructive mechanical deformation upon pressing, thereby generating electrical energy that powers the operation of the fingertip sweat sensor. A detailed discussion of designs of fingertip sweat sensors (i.e., touch-based) suitable for use in the present invention may be found in, e., Yin et at. (US 2024 / 0049994; US 2025 / 0241583; WO 2025 / 010373), Wang et al. (WOAttorney Docket No. 081906-1535050-256510PC2023 / 133530), the contents of each of which are hereby incorporated by reference in their entireties.

[0252] In some embodiments, the test strip includes: a substrate layer; and one or more sensing reagents dispensed upon at least a portion of the substrate layer, wherein at least one of the sensing reagents includes the oxidase.

[0253] In some embodiments, the at least one of the sensing reagents further includes a redox indicator, a co-faotor, a mediator, and / or another adjuvant or excipient.

[0254] In some embodiments, the redox indicator includes one or more of 2,2’- bipyridine (Ru complex). Nitrophenanthroline (Fe complex), N-Phenylanthranilic acid, 1,10-Phenanthro|ine iron(ll) sulfate complex, hPEthoxychrysoidine, 2,2'-Bipyridine (Fe complex), 5,6-Dimethylphenanthroline (Fe complex), o-Dianisidine, Sodium diphenylamine sulfonate, Diphenylbenzidine, Diphenylamine, Viologen, N, N diphenyl-1,4-diphenyldiamine, Lissamine Green B, N~phenyl~1,4-phenylenediamine, Sodium 2,6-Dibromophenol-indophenol, Sodium o~Cresol indophenol, Thionine, Methylene blue, Indigotetrasulfonic acid, Indigotrisulfonic acid, Indigo carmine, Indigomono sulfonic acid, Phenosafranin, Safranin T, and / or Neutral red, and / or a combination thereof.

[0255] In some embodiments, the co-factor includes flavin adenine dinucleotide (FAD).

[0256] Mediators are electron transferring agents that can participate in the redox reaction. In some embodiments, the one or more sensing reagents may further include a mediator to enhance the sensing performance.

[0257] In some embodiments, the mediator includes one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complexes, and / or organometallic ruthenium complexes.

[0258] In other embodiments, the test strip further comprises one or more tetrazolium salts. In some embodiments, wherein one or more of the tetrazolium salts are selected from the group consisting of 2-(p~iodophenyl)'-3~(p~nitrophenyl)-5~ phenyltetrazolium chloride (INT) and S^. S-dimethylthiazolyM^)^^- diphenyltetrazolium bromide (MTT).

[0259] The material of the substrate layer may comprise one or more of a composite material, a fibrous material, a woven textile, a non-woven textile, aAttorney Docket No. 081906-1535050-256510PCpolymer, an adhesive, a film, a gel, PTFE, and / or silicone. The disclosure further provides for a sensor capable of sensing, detecting, and / or measuring amino acid concentration by utilizing enzyme described herein in a continuous, continual, or on- demand manner. In other aspects, the enzyme comprises one or more of SEQ ID NO: 1 ~ 97, fragments, or modified enzymes of SEQ ID NO: 1 - 97 as described herein.

[0260] tn some embodiments, the sensor is configured to be able to detect multiple analytes of interest. This may be achieved by incorporating arrays of (working) electrodes or arrays of multi-electrode (e g., two or three electrode) contingents. For example, FIGs. 33A-33B show biosensor strip designs incorporating multiple biosensor units to enable the simultaneous detection of multiple analytes on a single platform. For example, FIG. 33A shows a dual L-alanine / L-leucine strip, and FIG. 33B shows a four-analyte configuration for the concurrent detection of L~alanine and L-leucine acids, along with metabolites glucose and ketone. " Ketone(s)” includes ketones, ketone bodies, and / or precursors of ketone bodies, such as acetone, acetoacetic acid, beta-hydroxybutyrate (BHB), beta-ketopentanoate, beta-hydroxypentanoate, 1,3-butanediol, and medium chain triglycerides (MCT) containing fatty acids with hydrocarbon side chains in the length of 6-12 carbons. This modular design can be further expanded to accommodate additional analytes beyond four.

[0261] According to the present disclosure, the sensor can be configured to detect one or more additional analytes of interest in addition to the one or more a ino acids being detected, wherein such additional analytes include but are not limited to glucose, ketones (e g., -hydroxybutyrate, acetone), ketone bodies, lactate, pyruvate, cholesterol (total, HOL, LDL), ApoB (Apolipoprotein B), triglycerides, urea, creatine, uric acid, ethanol, acetaldehyde, ascorbic acid (Vitamin C). sodium (NaQ, potassium (K*)fCalcium (Caa+), chromium (Cr), magnesium (Mg2*), chloride (Cl"), ammonium (Nt ), nitrate (NOs), nitrite (NCb”), phosphate PO43', oxygen (SpOa, O2), carbon monoxide (CO), carbon dioxide (CO2), nitric oxide (NO), hydrogen ions, hydrogen sulfide (H2S), hydrogen peroxide (H2O2), chlorinated hydrocarbons, naphthalene, cortisol, insulin, thyroid hormones (e.g., T3, T4), adrenaline, epinephrine, testosterone, estrogen, estradiol, amino acid precursors and derivatives, peptides, C-reactive protein (CRP), troponin l / T, interleukins (e.g., IL-6, IL-8), procalcitonin, ferritin, albumin, neurotransmitters (e.g., dopamine,Attorney Docket No. 081906-1535050-256510PCserotonin), viruses (e.g., SARS-CoV-2, influenza, HIV), bacteria (E. coif, Sa / mone / fe, Listeria), fungi, parasites, mycotoxins (e.g., aflatoxin, ochratoxin), heavy metals (e.g., lead (Pb), mercury (Hg), cadmium (Ca)), pesticides (e.g., atrazine, chlorpyrifos, methyl-parathion, aclonifen), cyanide, phenol, bisphenol A (BPA), estrone, per- and polyfluoroalkyl substances (PFAS), brevetoxin~2, anatoxin-a, antibiotics (e.g., ampicillin, gentamicin, amikacin, tobramycin, penicillin, tetracycline), anti- seizure / anticonvulsants (e.g., phenytoin, phenobarbital, valproic acid, carbamazepine), heart / cardiac medicines (e.g., digoxin, lidocaine, amiodarone), immunosuppressants (e.g,, cyclosporine, tacrolimus, sirolimus), mental health medications (e.g., lithium), chemotherapy drugs (e.g., methotrexate, busulfan), bronchodilators (e.g., theophylline), analgesics (e g,, acetaminophen, ibuprofen), therapeutic drug monitoring targets (e.g., vancomycin), caffeine, theobromine, cocaine, amphetamine, methamphetamine, MDMA (Ecstasy), cathinones, morphine, heroin, nicotine, codeine, fentanyl, methadone, oxycodone, barbiturates, gamma- hydroxybutyrate (GHB), ketamine, lysergic acid diethylamide (LSD), benzodiazepines (BZDs), salicylate, paraoxon, xanthine, and cannabinoids (e.g., CBD, THC, and their metabolites).[0262} In some embodiments, the sensor may be integrated with a system and / or a method that monitors other vital signs. For example, the sensor may be integrated with a system / platform that also monitors a subject’s vital signs, such as body temperature, blood pressure, pulse (heart rate), and respiratory rate (breathing rate). In some embodiments, the sensor may be integrated with touch-based, wearable, and / or implantable sensing platforms.Pretreatment and Activation for the Biosensor Strips / Electrodes[0263} For biosensor strips (such as L-leucine biosensor strips), the working electrode surface may be enhanced prior to modification to achieve effective activation. This process generates micropores and surface roughness, thereby increasing the electroactive surface area, enhancing sensitivity through a greater number of active sites tor analyte interaction, and improving conductivity for efficient electron transfer. Additional activation methods that may be employed include electrochemical activation using a basic solution (such as sodium hydroxide, NaOH) and an acidic solution (such as hydrochloric acid, HCI) under chronoamperometricAttorney Docket No. 081906-1535050-256510PCconditions, chemical activation via acid / base etching, and physical activation methods such as thermal treatment.Surface Chlorination of the Reference Electrode

[0264] The reference electrode may be an Ag / AgCI electrode. To stabilize the silver chloride (AgCI) layer on a screen-printed silver (Ag) or Ag / AgCI reference electrode, additional chlorination may be performed using either: electrochemical chlorination or chemical chlorination with agents such as sodium hypochlorite (NaOCI), ferric chloride (FeCb), or gold chloride (AuCb).

[0265] In some embodiments, the sensor is further designed to afford additional protection. For example, the sensor may comprise one or more coatings and / or spacers made of ion-exchange resins (such as sodium polystyrene sulfonate, polyAPTAC, and polyethylene amine), polyesters, polyethylene terephthalate (PET), PVC, PTFE, and / or polyolefin. Alternatively, in some embodiments, the sensor is packaged. For example, the sensor may be packaged individually (one sensor per pack), or with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 sensors being packaged together.

[0266] In some embodiments, the wearable sensor is configured to continuously, continually, or on-demand measure an amino acid concentration of a subject and output a data stream. In some embodiments, the continuous sensor is an implantable or non-implantable device. In some embodiments, the continuous sensor is an ingestible or injectable device. In some embodiments, the wearable sensor is a needle-based sensor or a microneedle-based sensor. In some embodiments, the wearable sensor may be a reverse iontophoresis sensor, which extracts one or more analytes (e g., one or more amino acids of interest) from the skin by applying an electric field force. In some embodiments, the wearable sensor is configured to measure an amino acid concentration in a body fluid. In some embodiments, the wearable sensor is configured to measure concentrations of multiple amino acids in a body fluid at the same time. In other embodiments, the body fluid comprises one or more of whole blood, serum, plasma, a blood fraction other than serum or plasma, lymph, cerebrospinal fluid (CSF), interstitial fluid (ISF), intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal discharge, milk, mucus, chyme, pus, bite, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid,Attorney Docket No. 081906-1535050-256510PCpericardial fluid, peritoneum, glandular secretions, exudate, contents of cysts, and / or ascites,

[0267] As used herein, “micromole" and “pmol” are equivalent,

[0268] As used herein, “millimole” and “mmol" are equivalent

[0269] As used herein, “pmol / L” and “yMsare equivalent and each refer to micromoles per liter,

[0270] As used herein, “mmol / L” and “mM" are equivalent and each refer to millimoles per liter,

[0271] To convert from mM to pM. multiply by 1000. For example 0.1 mM is equal to 100 yM.

[0272] In some embodiments, the detection of the concentration of an amino acid according to the present disclosure is from about 0.01 mM to about 1.0 mM (equivalent to from about 10 yM to about 1000 pM).

[0273] In some embodiments, the detection of the concentration of an amino acid according to the present disclosure is from about 0,12 mM to about 0,60 M (equivalent to from about 120 pM to about 600 pM),

[0274] In some embodiments, the detection of the concentration of an amino acid according to the present disclosure is from about 0.20 M to about 0.50 mM (equivalent to from about 200 pM to about 500 pM).

[0275] In some embodiments, the detection of the concentration of an amino acid according to the disclosure herein is about 0.01 mM, about 0.02 mM, about 0.03 mM, about 0,04 mM, about 0,05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.09 mM, about 0.10 mM, about 0.15 M, about 0.20 mM, about 0,25 mM, about 0.30 mM, about 0.35 mM, about 0.40 mM, about 0.45 mM, about 0,50 mM, about 0.55 mM, about 0.60 mM, about 0,65 mM, about 0.70 mM, about 0.75 mM, about 0.80 mM, about 0.85 mM, about 0.90 mM, about 0.95 mM, or about 1.0 mM.

[0276] In biological sensors, the electric current (“I") that is applied is measured in the same electrical units used elsewhere: amperes (“A"). However, because the currents generated by biosensors are usually very small, they are usually reported in sub-units of amperes, such as in microamperes pA”), which is equal to 10sA. In some embodiments, the detection of the concentration of an amino acid is based on analyses using before baseline subtraction and after baseline subtraction. Before baseline subtraction refers to the raw electrical current measurements obtained fromAttorney Docket No. 081906-1535050-256510PCthe sensor at each time point before and after e.g. protein intake. In contrast, after baseline subtraction refers to the adjusted electrical current values, where each current measurement is corrected by subtracting the average electrical current recorded prior to e.g., protein uptake (i.e., the baseline). This procedure highlights the true magnitude and direction of change (i.e., increase, decrease, or no change) in electrical current responses following e.g. protein intake by removing background signal variations. See, e.g., FIGs, 45A - 45D.[02771 The disclosure further provides for a system for detecting and measuring amino acid concentration by utilizing enzymes described herein. In some embodiments, the system for detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids comprises a sensor described herein; and a portable device connected to the sensor. The sensor is configured to measure an amino acid concentration or concentrations of more than one amino acids contained within a sample and output a data stream to the portable device. The portable device may comprise: a processor or a processing unit configured to process the data stream from the sensor; and an interface configured to display and / or communicate measured amino acid concentration values. The processor or processing unit may be any unit that is capable of receiving and processing the data stream conveyed from the sensor. For example, the processor or processing unit may be a microcontroller unit (MCU), a central processing unit (CPU), a graphics processing unit (GPU), or a system on a chip (SoC). The processor or processing unit may be installed with application-specific custom firmware containing analyte test algorithms and control commands for all components of the device. For example, any requirements for proper sensor operation are also written in the firmware, including but not limited to, drive voltages for the electrochemical reaction, the method of measurement (one time, real-time, time delay, time average, etc.), variables for temperature compensation, and calibration data. In some embodiments, the processor or processing unit includes a highly-efficient 8-bit AVR-based MCU and contains an embedded potentiostatic firmware designed to execute the pre-programmed amperometric routines (control the potentiostat) and route digitized readings to the wireless transceiver module for transmission. In some embodiments, the processor or processing unit may be programmed to execute a suite of signal-processing algorithms to maximize signal- to-noise ratio as well as an auto-calibration routine.Attorney Docket No. 081906-1535050-256510PC[0278J In some embodiments, self-monitoring amino sense systems or kits (e g., for personal use, including, e.g., at-home use) of the present disclosure comprise a handheld and wireless dual-channel potentiostat, which can be private labeled or contract manufactured. In some embodiments, the systems and kits of the present disclosure include an analyzer (e.g., hardware, software), chemistry (e.g., a testing strip), a mobile device (e.g., a phone or tablet), application software, and / or a lancet. In some embodiments, the self-monitoring amino acid systems or kits of the present disclosure include specialized software (e.g., as applications or programs) which can be accessed via the web (e.g., via cloud storage or direct linkup), or provided directly or indirectly to the user by devices including but not limited to memory chips, floppy discs, USB flash drives, DVDs, or hard disc drives. In some embodiments, the systems and kits of the present disclosure are used as in vitro diagnostic (IVD) medical devices, wherein the software applications do not function as standalone devices but are instead an integral part or component of an IVD system. In some embodiments, the systems or kits further comprise a single¬ use strip (e.g., a sensor strip containing the necessary chemistry as described in detail elsewhere herein) including but not limited to an assay to determine levels of specific amino acids and / or to assess combined levels of select amino acids, wherein the amino acids include but are not limited to leucine, alanine, or combinations of amino acids such as e.g,, alanine / glydne or branched chain amino acids (BCAAs) as provided in detail elsewhere herein. In some embodiments, the self-monitoring amino acid systems and kits of the present disclosure further include a personal device, such as a computer, tablet (e.g., an iPad™), or smartphone, wherein the software for these devices can be made available for download from an application store or service provider to the personal device. In some embodiments, operating instructions are also provided with the systems and kits of the present disclosure, wherein the operating instructions include but are not limited to a description and / or summary of the minimum characteristics necessary for the personal device. In some embodiments, the self-monitoring amino acid systems or kits of the present disclosure further comprise a lancet. In some embodiments, the lancet is an FDA compliant lancet and / or FDA cleared device. In some embodiments, certain stock-keeping unit (SKU) numbers and / or configurations for a suitable lancet to use with the systems and kits of the present disclosure can be provided without a lancet. In some embodiments, the self-monitoring amino senseAttorney Docket No. 081906-1535050-256510PCsystems or kits are intended for seif-testing in the consumer wellness market, whereby users can thereby learn more about their nutrition and / or metabolisms. In some embodiments, the systems and kits of the present disclosure can be used by an individual to generally track their protein absorption and / or metabolism. In some embodiments, the systems and kits of the present disclosure can be used to monitor, optimize, and / or individualize a diet by e g., measuring amino acid levels under different dietary regimes (e.g., alternative protein sources). In some embodiments, the systems and kits of the present disclosure can be used to assess anabolic status such as e.g„ by measuring leucine levels to assess sufficiency to support muscle growth. In some embodiments, the systems and kits of the present disclosure may further include amino acid guidance ranges related to e.g., diet and exercise. In some embodiments, the systems and kits of the present disclosure provide the results of self-testing in various formats including but not limited to amino acid concentrations in pmol / L and / or as an index or indices. In some embodiments, such an individual is interested in sports nutrition, health and wellness, and / or personal nutritional monitoring. In some embodiments, one or more additional testing systems or kits may be used for quality control and / or calibrating of the self¬ monitoring amino sense systems or kits of the present disclosure, wherein such additional testing systems or kits include but not limited to amperometry, cyclic voltammetry, and / or open circuit potentiometry. In some embodiments, the systems and kits of the present disclosure are used for quality control production In supplement, nutritional, and / or drug manufacturing processes, for research use (either exclusively or inclusively to other uses), and / or in laboratory developed tests.

[0279] In some embodiments, the portable device may further comprise a wireless communications unit to wirelessly transmit the detected electrical signals and / or the data stream to an external computing device.

[0280] In some embodiments, an amino acid sensor or sensing system is created by integrating said enzyme into a variety of sensing platforms and / or devices. Platforms and / or devices that can adopt said enzyme include, but are not limited to, pH-change sensors such as the ones described in Chodavarapu et al., US 7,794,584, electrochemical sensors such as the ones described in Simpson et a / ., US 7,081,195, Label etak, US 6,915,147, and Jina et ak, US 2010 / 0049021, optical sensors such as the ones described in Petrich eta / ., US 10,724,943, implantable sensor platforms such as the ones described in Jain, EP 2079358, chip-shapedAttorney Docket No. 081906-1535050-256510PCblood analysis devices such as the ones described in Ogawa eta, US 7,582,259, and monitoring systems where the control terminal is remotely coupled such as the ones described in Karan et a / ., US 2012 / 0245447. The content of each of these patents and applications is hereby incorporated by reference in their entireties.

[0281] The disclosure further provides for methods of manufacturing a sensor for detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids. The method may comprise: providing a substrate; forming a first electrode on the substrate, wherein the first electrode is operable as a working electrode; forming an entrapment layer on the first electrode- forming a second electrode on the substrate, wherein the second electrode is operable as the counter electrode; and forming a third electrode on the substrate, wherein the third electrode is operable as the reference electrode.

[0282] In some embodiments, the step of forming the entrapment layer may comprise: depositing a conductive polymer material on the first electrode, such that a conductive polymer film is formed on the first electrode; and depositing at least one oxidase and a cross-linking agent to the conductive polymer film, such that the at least one oxidase is immobilized to the conductive polymer film.

[0283] In some embodiments, the method may further comprise depositing at least one racemase to the conductive polymer film, such that the at least one racemase is immobilized to the conductive polymer film. In some embodiments, the method may further comprise depositing at least one redox mediator to the conductive polymer film, such that the at least one redox mediator is immobilized to the conductive polymer film. In some embodiments, the method may further comprise forming a protective layer on the entrapment layer.

[0284] The disclosure further provides for methods of detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids by utilizing an enzyme described herein. The method may comprise: subjecting a sample to the sensor described herein; and detecting and / or measuring a presence of hydrogen peroxide.

[0285] In some embodiments, the method of detecting and measuring amino acid concentration comprises: obtaining a sample from a subject; subjecting the sample to a sensor; determining a single point amino acid concentration in the sample; and displaying and / or communicating the single point amino acid concentration on an interface.Attorney Docket No. 081906-1535050-256510PC

[0286] In some embodiments, the method of detecting and measuring amino acid concentration comprises: obtaining a sample from a subject, subjecting the sample to a sensor; determining concentrations of multiple amino acids of interest in the sample; and displaying and / or communicating the concentrations of the multiple amino acids of interest on an interface.Test kits

[0287] The disclosure further relates to test kits suitable for use in, for example, automatic analyzers, systems, and devices.

[0288] In some embodiments, test kits described herein comprises a reagent A and a reagent B. in some embodiments, the test kit is activated by mixing the reagent A with the reagent B.

[0289] In some embodiments, the reagent A comprises an enzyme described herein. In some embodiments, the enzyme described herein includes at least one racemase and / or at least one oxidase. In some embodiments, the enzyme described herein includes at least one L-amino acid oxidase. In some embodiments, the enzyme described herein includes at least one racemase and at least one D-amino acid oxidase. In some embodiments the enzyme described herein includes at least one alpha-hydroxy acid oxidase.

[0290] In some embodiments, the enzyme described herein is dissolved in a buffer solution. In some embodiments, reagent A comprises NaCI and / or KCI. In some embodiments, wherein the concentration of NaCI and / or KCI is 0.1-5. In some embodiments, wherein the reagent A comprises citric acid, acetic acid, KH2PO4, N~cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-(N- morpholino)ethanesulfonic acid (MES), phosphate, and / or tris(hydroxymethyl) aminomethane. In some embodiments, wherein the reagent A has a pH of 3-11, optionally 4-10, optionally 5-9, optionally 6-8.

[6291] In some embodiments, the at least one L-amino acid oxidase comprises, consists essentially of, or consists of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 1, 2, or 3. In some embodiments, the at least one L-amino acid oxidase comprises, consists essentially of, or consists of an amino acid sequence of one of SEQ ID NOs: 1, 2, orAttorney Docket No. 081906-1535050-256510PC3, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

[0292] In some embodiments, the at least one racemase comprises, consists essentially of, or consists of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID Nos: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 5, 16, 17. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81. In some embodiments, the racemase comprises, consists essentially of, or consists of an amino acid sequence of one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 1, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

[0293] In some embodiments, the at least one D-amino acid oxidase includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% Identical to SEQ ID NO: 34. In some embodiments, the at least one D-amino acid oxidase includes an amino acid sequence of SEQ ID NO: 34, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

[0294] In some embodiments, the D-amino acid oxidase is active against D- ieucine and inactive against D~methionine.In some embodiments, the D-amino acid oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 89, 94, or 97.

[0295] In some embodiments, the at least one alpha-hydroxy acid oxidase comprises, consists essentially of, or consists of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 35, 36, 37, 38,82Attorney Docket No. 081906-1535050-256510PC39, 40, 41, 42, 43, 4, 45, 46, 47, 8, 49, 50, 51, 52, 53, or 54. In some embodiments, the at least one alpha-hydroxy acid oxidase comprises, consists essentially of. or consists of an amino acid sequence of one of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.[02961 In some embodiments, the reagent B comprises a redox indicator, a co¬ factor, a mediator, an excipient, an adjuvant, or a carrier, in an embodiment the composition containing the enzyme is dissolved in water. In some embodiments, the redox indicator comprises one or more of 2,2 -bipyridine (Ru complex), Nitrophenanthroline (Fe complex), N-Phenylanthranilic acid, 1,10-Phenanthroline iron(ll) sulfate complex, N~Ethoxychrysoidine, 2,2' -Bipyridine (Fe complex), 5,6- Di methyl phenanthroline (Fe complex), o-Dianisidine, Sodium diphenylamine sulfonate, Diphenylbenzidine, Diphenylamine, Viologen, N, N'-diphenyl-1,4- diphenyldiamine, Lissamine Green B, N-phenyl-1,4~phenylenediamine, Sodium 2,6- Dibromophenol-indophenol, Sodium o-Cresol indophenol, Thionine, Methylene blue, Indigotetrasulfonic acid, Indigotrisulfonic acid, Indigo carmine, Indigomono sulfonic acid, Phenosafranin, Safranin T, and / or Neutral red, and / or a combination thereof. In some embodiments, the co-factor comprises one or more of flavin adenine dinucleotide (FAD), semiquinone form flavin adenine dinucleotide (FADH), quinone form flavin adenine dinucleotide (FADHs), and / or pyridoxal 5'-phosphate (PLP). In some embodiments, wherein the mediator comprises one or more of ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complexes, and / or organometallic ruthenium complexes. In some embodiments, the reagent B comprises NaCI and / or KCI. In some embodiments, the concentration of NaCI and / or KCI is 0.001-5 M. In some embodiments, the reagent B comprises citric acid, acetic acid, KH2PO4, N-cydohexyl-2-aminoethanesulfonic acid (CHES), 2-(N- morpholino)ethanesulfonic acid (MES), phosphate, and / or tris(hydroxymethyl) aminomethane. In other embodiments, the reagent B has a pH of 3-11, optionally 4-10, optionally 5-9, optionally 6-8. In some embodiments, the reagent B comprises one or more tetrazolium salts. In some embodiments, wherein one or more of the tetrazolium salts are selected from the group consisting of 2-(p-tQdophenyl)-3’-(p-Attorney Docket No. 081906-1535050-256510PCnitrophenyl)-5-phenyltetrazolium chloride (INT) and 3-(4)5-dimethylthiazolyl’-1-2)-2,5- diphenyltetrazolium bromide (MTT).Pharmaceutical compositions

[0297] In another embodiment, the invention relates to applications of the enzyme as an ingredient of a composition. In some embodiments, the composition comprises one or more of active ingredients. In some embodiments, the active ingredients further comprise one or more of other enzymes such as glucose oxidase, glucose dehydrogenase, glucosyl transferase, fructosyl transferase, catalase, amylase, lactase, lipase, cholesterol oxidase, creatinase, glutamate oxidase, lactate dehydrogenase* lactate oxidase, penicillinase, tyrosinase, salicylate hydrolase, uricase* xanthine oxidase, and / or protease.

[0298] In some embodiments, the composition may further comprise one or more excipients, one or more adjuvants, and / or one or more carriers. Excipients, adjuvants, and / or carriers commonly known in the field may be found from, for example, Margolin et a / . (US 7,718,169), the content of which is incorporated herein by reference.

[0299] In some embodiments, the one or more excipients comprises one or more of microcrystalline cellulose, Maltrin, Crospovidone, colloidal silicon dioxide, magnesium stearate, talc, sucrose, trehalose, lactose, sorbitol, lactitol, mannitol, inositol, salts of sodium and potassium, such as acetate, phosphates, citrates and borate, glycine, arginine, polyethylene oxide, polyvinyl alcohol, polyethylene glycol, hexylene glycol, methoxy polyethylene glycol, gelatin, hydroxypropyl-p-cyclodextrin, polylysine, polyarginine, amino acids such as glycine, arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, proline, carbohydrates such as glucose, fructose, galactose, mannose, arabinose, xylose, ribose, lactose, trehalose, maltose, sucrose maltodextrins, dextrans, starch, glycogen, alditols such as mannitol, xylitol, lactitol, sorbitol, cyclodextrins such as methyl cyclodextrin, hydroxypropyl- - cyclodextrin and alike, inorganic molecules such as sodium chloride, potassium chloride, magnesium chloride, phosphates of sodium and potassium, boric acid, ammonium carbonate and ammonium phosphate, organic molecules such as acetates, citrate, ascorbate, lactate, glucuronic acid, galacturonic acid, emulsifying or solubilizing / stabilizing agents such as acacia, diethanolamine, glycerylAttorney Docket No. 081906-1535050-256510PCmonostearate, lecithin, monoethanolamine, oleic acid, oleyl alcohol, poloxamer, polysorbates, sodium lauryl sulfate, stearic acid, sorbitan monolaurate, sorbitan monostearate, and other sorbitan derivatives, polyoxyl derivatives, wax, polyoxyethylene derivatives, sorbitan derivatives, viscosity increasing reagents such as agar, alginic acid and its salts, guar gum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose and its derivatives propylene carbonate, polyethylene glycol, hexylene glycol, tyloxapol or salts of such compounds. Additional examples of excipients are described in the Handbook of Pharmaceutical Excipients, published jointly by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain.

[0300] In some embodiments, wherein the one or more adjuvants comprises one or more of water-in-oil and oil-in-water emulsions, aluminum salt adjuvants, liposomes, and / or CpG oligodeoxynucleotide adsorbed to aluminum salt.

[0301] In some embodiments, wherein the one or more carriers comprises one or more of polymers used for encapsulation of protein crystals for delivery of proteins, including controlled release biological delivery. In some embodiments, wherein the polymers include biocompatible and biodegradable polymers, or mixtures thereof. In some embodiments, wherein a rate of dissolution and, therefore, delivery of enzymes will be determined by the particular encapsulation technique, polymer composition, polymer crosslinking, polymer thickness, polymer stability, enzyme crystal geometry and degree, if any, of enzyme crosslinking.

[0302] In some embodiments, wherein the one or more active ingredients) may be present In the composition In association with a polymeric carrier.

[0303] Useful polymeric carriers include, for example, polymers used for encapsulation of protein crystals for delivery of proteins, including controlled release biological delivery. Such polymers include biocompatible and biodegradable polymers, or mixtures thereof. Preferably, the polymeric carrier is a biodegradable polymer. The rate of dissolution and, therefore, delivery of enzymes will be determined by the particular encapsulation technique, polymer composition, polymer crosslinking, polymer thickness, polymer stability, enzyme crystal geometry and degree, if any, of enzyme crosslinking. Other useful carriers include water.Methods of using the sensor or test kit to improve health and / or wellnessAttorney Docket No. 081906-1535050-256510PC

[0304] The disclosure further provides for methods of improving health and / or wellness by using the sensors, devices, systems, or kits integrated with enzymes described herein. Amino acids are essential building blocks for humans. Thus, controlling the intake of certain amino acids may aid in improving one's health and / or wellness.

[0305] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0306] The term “branched-chain amino acids” (BCAA) refers to amino acids having an aliphatic side-chain with a branch. For example, leucine, isoleucine, and valine fall under the category of BCAA.

[0307] The term “aromatic amino adds” refers to amino acids having an aromatic group. For example, tyrosine, tryptophan, and phenylalanine fall under the category of aromatic amino acids.

[0308] As an example, the concentration of plasma branched-chain amino acids (BCAA) and alanine may be an indicator of muscle growth or muscle loss. Mero, Sports Med. 1999 Jun; 27 (6): 347-358; Sas-Nowosielski eta / ., J. Environ. Res. Public He&tth 2021, 18, 5370. For example, an increase in the leucine (a BCAA) concentration may be a signal of muscle growth. Conversely, an increase in the alanine concentration may be a signal of muscle catabolism (waste).

[0309] Another exemplary application of the sensor described herein is for monitoring lean body mass, which is a critical determinant of metabolic health, influencing glucose regulation, insulin sensitivity, and physical performance. For example, GLP-1 receptor agonists have demonstrated notable success in promoting weight loss and reducing prediabetes prevalence in obese adults. Marre et al., Diabet Med, 2009. 26(3): p. 268-78; Garber et al., Lancet, 2009. 373(9662): p. 473- 81; Astrup et al, Lancet, 2009. 374(9701): p. 1606-16. However, it was found that some of the weight loss achieved through GLP-1 receptor agonists results from lean mass reduction. Upon discontinuation of treatment, many patients experience weight regain, further compounded by reduced lean mass, thereby heightening their susceptibility to metabolic issues, including type 2 diabetes. Sargeant et al., Endocrinol Metab (Seoul), 2019. 34(3): p. 247-262; Wilding et al., Diabetes Obes Metab, 2022. 24(8): p. 1553-1564. Thus, in the field of GLP-1 receptor agonists treatments, these findings underscore an urgent need for real-time monitoring of leanAttorney Docket No. 081906-1535050-256510PCmass preservation to support the safer, more effective use of GLP-1 receptor agonists, enabling weight loss without the costly compromise of lean mass reduction.

[0310] In addition, studies show that leucine supplementation during energy deficiency aids in lean mass preservation, underscoring its importance in muscle health management. Su et al., Nutrients, 2022. 14(12); Liu et al., BMC Geriatr, 2024.24(1): p. 541; Pathak et al, Eur J Clin Nutr, 2024. 78(2): p. 155-162, Alanine, a non- essential amino acid produced as a byproduct of branched-chain amino acid breakdown from skeletal muscle and serving as a key gluconeogenic precursor, is a reliable indicator of muscle catabolism. Zhao et al., BMC Geriatr, 2024. 24(1): p. 341; Okun et al., Nat Metab, 2021. 3(3): p. 394-409.

[0311] Accordingly, a food item or supplement that contains one or more amino acids, for example one or more branched-chain amino acids, may be administered to a subject to improve health and / or wellness. For example, a food item or supplement that contains more than 1g, more than 2.5g, more than 5g, more than 7.5g, more than 10g, more than 12.5g, more than 15g, more than 17.5g, more than 20g, more than 22.5g, more than 25g, more than 27,5g, more than 30g, more than 32.5g, more than 35g, more than 37.5g, more than 40 g, more than 42,5 g, more than 45g, more than 47.5g, or more than 50g of branched-chain amino acids (BCAA) per unit may be provided. Alternatively, a food item or supplement that contains more than 1, more than 2.5g, more than 5g, more than 7.5g, more than 10g, more than 12.5g, more than 15g, more than 17.5g, more than 20g, more than 22.5g, more than 25g, more than 27.5g, more than 30g, more than 32.5g, more than 35g, more than 37.5g, more than 40g, more than 42.5g, more than 45g, more than 47.5g, or more than 50g of leucine per unit may be provided. Or, a food item or supplement that contains more than 1g, more than 2.5g, more than 5g, more than 7.5g, more than 10g, more than 12.5g, more than 15g, more than 17.5g, more than 20g, more than 22.5g, more than 25g, more than 27.5g, more than 30g, more than 32.5g, more than 35g, more than 37.5g, more than 40g, more than 42.5g, more than 45g, more than 47.5g, or more than 50g of one or more branched-chain amino acids and / or alanine per unit may be provided.

[0312] One the other hand, under certain conditions, it may be preferable to decrease the intake of one or more amino acids. For exampie, for patients with chronic kidney disease (CKD), it has been reported that a low amino acid diet mayAttorney Docket No. 081906-1535050-256510PCimprove the kidney function end overall health. Barba, et al., Scientific Reports (2021) 11:19184; Cheng et at, Int J. Mol. Sei. 2022, 23, 6047.

[0313] Accordingly, a food item or supplement that contains lesser amount of certain amino acids, for example lesser amount of one or more aromatic amino acids, branched-chain amino acids, and / or alanine, may be administered to a subject to improve health and / or wellness. For example, a food item or supplement that contains less than 1g, less than 2.5g, less than 5g, less than 7.5 g, less than 10g, less than 12.5g, less than 15g, less than 17.5g, less than 20g, less than 22.5g, less than 25g, less than 27.5g, less than 30g, less than 32.5g, less than 35g, less than 37.5g, less than 40g, less than 42.5g, less than 45g, less than 47.5g, or less than 50g of aromatic amino acids per unit may be provided. Alternatively, a food item or supplement that contains less than 1g, less than 2.5g, less than 5g, less than 7.5g, less than 10g, less than 12.5g, less than 15g, less than 17.5g, less than 20g, less than 22.5g, less than 25g, less than 27.5g, less than 30g, less than 32.5g, less than 35g, less than 37.5g, less than 40g, less than 42.5g, less than 45g, less than 47.5g, or less than 50g of branched-chain amino acids (BCAA) per unit may be provided.

[0314] In some other embodiments, the sensor described herein may be applied in monitoring the amino acid profile as well as other analyte of interest of a patient with kidney disease or for the purpose of evaluating the efficacy of kidney disease treatments. For example, it was reported that the branched-chain amino acids (BCAA) profile of kidney tissue may indicate the performance of the kidney in organ transplantation. Ahmadi et at, Kidney International (2024) 106, 712-722, In some embodiments, the sensor described herein may be applied in detecting real time changes of a patient’s amino acid profile as well as his / her blood volume and oxygen saturation in conjunction with or during hemodialysis (dialysis). In some embodiments, the sensor described herein may be applied in detecting and analyzing the amino acid profile in donor kidneys for evaluating their ability to be transplanted.Applications for using the sensor or test kit in fermentation and / or other industrial manufacturing processes

[0315] Because amino acids are critical nutrients for the growth of many microorganisms of industrial importance, monitoring and controlling the amino acidAttorney Docket No. 081906-1535050-256510PClevel in a microbial growth medium is also important in many industrial applications. For example, fermentation (by yeast or other microorganisms) has been applied in many industrial manufacturing processes, including production of wine, beer, ethanol, biofuel, breakdown of biomass and biowaste, production of lactate and lactic acid, and production of diamines.

[0316] In some embodiments, the sensor or test kit of the present invention may be integrated into or applied in conjunction with an industrial manufacturing process, such as with an industrial fermentation process or an industrial fermentation plant. In some embodiments, the sensor or test kit of the present invention is integrated into an in-line system with an industrial fermentation plant. In some embodiments, the fermentation medium or broth stored in an industrial fermentation plant or an industrial fermentation tank is being tested for the levels of one or more amino acids of interest.

[0317] In some embodiments, the testing of one or more amino acid levels is conducted by contacting the sensor or test kit of the present invention with the fermentation medium or broth, in some embodiments, the monitoring and / or controlling of one or more amino acid levels is conducted by testing the one or more amino acid levels once every 1 minute, once every 5 minutes, once every 10 minutes, once every 15 minutes, once every 20 minutes, once every 30 minutes, once every 40 minutes, once every 50 minutes, once every 60 minutes, once every 2 hours, once every 3 hours, once every 4 hours, once every 5 hours, once every 6 hours, once every 7 hours, once every 8 hours, once every 9 hours, once every 10 hours, once every 11 hours, once every 12 hours, once every 24 hours, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 2 weeks, once every 3 weeks, or once every 4 weeks. In some embodiments, the monitoring and / or controlling of one or more amino acid levels is performed for a period of at least 1 day, a period of at least 2 days, a period of at least 3 days, a period of at least 4 days, a period of at least 5 days, a period of at least 6 days, a period of at least 7 days, a period of at least 2 weeks, a period of at least 3 weeks, a period of at least 4 weeks, a period of at least 5 weeks, a period of at least 6 weeks, a period of at least 7 weeks, a period of at least 8 weeks, a period of at least 9 weeks, a period of at least 10 weeks, a period of at least 11 weeks, a period of at least 12 weeks, a period of at least 4 months, a period of at least 5 months, a period of at least 6 months, a period of at least 7Attorney Docket No. 081906-1535050-256510PCmonths, a period of at least 8 months, a period of at least 9 months, a period of at least 10 months, a period of at least 11 months, or a period of at least 12 months. Methods of implementing amino acid supplement and monitoring amino acids by a sensor

[0318] In some embodiments, the food item or supplement is consumed each day for a period of at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks.In some embodiments, amino acid monitoring is performed after 1 day, after 2 days, after 3 days, after 4 days, after 5 days, after 6 days, after 7 days, after 8 days, after 9 days, after 10 days, after 11 days, after 12 days, after 13 days, or after 14 days of following the consumption of the food item or supplement.

[0319] In some embodiments, amino acid monitoring is performed by using a sensor to detect a concentration of an amino acid of interest. In some embodiments, amino acid monitoring is performed by using a sensor to detect concentrations of multiple amino acids of interest at the same time. In some embodiments, the amino acid concentration is a blood amino acid concentration or an interstitial fluid (ISF) amino acid concentration. In some embodiments, the amino acid concentrations are concentrations of the multiple amino acids of interest in blood or in an interstitial fluid (ISF).

[0320] In some embodiments, the amino acid monitoring is performed at least once every day, at least twice every day, at least 3 times a day, at least 4 times a day, at least 5 times a day, at least once every 2 days, at least once every 3 days, at least once every 4 days, at least once every 5 days, at least once every 6 days, or at least once every 7 days, or at least once every 2 weeks, at least once every 3 weeks, or at least once every 4 weeks. In some embodiments, the amino acid monitoring is performed at 7:00 am, at 8:00 am, at 9:00 am, at 10:00 am, at 11:00 am, at 12:00 pm, at 1:00 pm, at 2:00 pm, at 3:00 pm, at 4:00 pm, at 5:00 pm, at 6:00 pm, at 7:00 pm, at 8:00 pm, at 9:00 pm, and / or at 10:00 pm. In some embodiments,Attorney Docket No. 081906-1535050-256510PCthe amino acid monitoring is performed 3 hours before breakfast, 2 hours before breakfast, 1 hour before breakfast, right before breakfast, right after breakfast, 1 hour after breakfast, 2 hours after breakfast, 3 hours after breakfast, 3 hours before iunch, 2 hours before lunch, 1 hour before lunch, right before lunch, right after lunch, 1 hour after lunch, 2 hours after lunch, 3 hours after lunch, 3 hours before dinner, 2 hours before dinner, 1 hour before dinner, right before dinner, right after dinner, 1 hour after dinner, 2 hours after dinner, or 3 hours after dinner.Applications of the enzymes, sensors, kits, and methods for detecting, measuring, and / or monitoring amino acid concentrations(0321] In some embodiments, the enzymes, sensors, kits, and methods of the present disclosure can be utilized for detecting, measuring, and / or monitoring amino acid concentrations according to the representative examples as shown in Table 3.Attorney Docket No 08190*3 •535950-2565'IOPC Table 3: Representative Uses of the Enzymes, Sensors, Kits, anc / or MethodsAmmo Acid Plasma Main Sources Role in Muscle Putative Clinical Relevance of Levels Potential Behavior Type Range in Health Associated Relative Increase Relative Decrease Change Hamans Disease(ymoi / L)Alanine 200-500 Produced from important Liver injury / Elevated alanine Low levels suggest May lead to BCAAs in gluconeogenic gluconeogenic reflects increased severe malnutrition adjustments in muscle or precursor, stress gluconeogenesis or hepatic failure, training load arid meat, dairy, indicator o’ from muscle carbohydrate intake poultry, fish, muscle breakdown, Could indicate or evaluation for nuts, seeds, catabolism Intense exercise, malnutrition and / or overtraining and liver whole grains, or liver stress, severe metabohc disease when fatigue legumes stress^ and tow performance (beans), May reflect muscle appear legumes, fungi, catabolism and / orinsects, algae liver dysfunction Modify exercise intensity. dietary adjustments to balance catabclism / ariabolism, and / or medical intervention if severe breakdownAttorney Docket No 081906- 1535050-236510PC Amiho Acid Plasma Main Sources Role in Muscle Putative Clinical Relevance of Levels Potential Behavior Type Range in Health Associated Relative increase Relative Decrease Change Humans Disease(pirtol / LJArginine 00-100.. Meat, dairy, Supports blood Argininosuccinic Sou ii md cat-; Linked to poor nitric increase dietary poultry, nite, flow and aciduria, mu-auc <, s*ress, oxide synthesis, intake or consider seeds wbcie cardiovascular cardiovascular sepsis, antifor cardiovascular supplements far grams. health by being dysfunction tumors in cancer issues, and for cardiovascular legume®; fiingj;, a precutw for patients malnutrition Support; may indicate insects, algae nitric oxide, need to monitor for aiding in High levels cart tow arginine impairs metabolic shifts in vasodiiaijon appear in sepsis, oltric-oxlde cancer patients tumor metabol isrrt. production and ureaor heavy cycling Gan change exercise Supplementation, tolerance,blppd-preSsure control, and: sleep pualfty, prompting Use or avoidance of arginlne-rich foods or supplements Asparagine 20-60 Meat, dairy, Acute lymphoblastic Asparagine is intentionally depleted in Treatment-related poultry, nite, isutemia therapy; very low levels indicate effective depletion can causeAttorney Docket No 081906- 1535050-236510PC Amiho Acid Plasma Main Sources Role in Mtsscte Putative Clinical Rat e vance of Levels Potential Behavior Type Range in Health Associated Relative increase Relative Decrease Change Humans Disease(jHrtoi / yseeds, whole (asparagirasa asparaginase treahr ent, whereas higher fatigue, nausea, and grains, therapy) levels may reduce fr lerapeutic efficacy, appetite changes, legumes, fungi, leading lo close diet insects, algae counseling and adherence support. Aspartate ‘oC’fO Meat dairy, Aspadyigiucosamiriu Aspartate is usually ow to plasma; In rare defects, may poultry, nuts, ria and other rare sustained elevation suggests ceil present with seeds, whole metetwiic defects breakdown or rare it sbcm errors of developmental delay grains, metabolism, white m inur changes are and cognitive issues, legumes, fungi, often nonspecific influencing teaming insects, algae needs and caregiver support Cysteine 156-300 Meat dairy, Oystinuria and High cystinefeysteine increases kidney Pam and anxiety from poultry, nuts, oxidative stress stone risk and oxidative stress; tow levels recurrent stones can seeds, whole related condition® weaken glutathione- based antioxidant reduce activity levels grains, defenses. and fluid intake legumes, fungi, patterns, white insects, algae antioxidant support may be emphasized.Attorney Docket No 081906- 1535050-236510PC Amin o Acid Plasma Main Sources Role in Mtsscie Putative Clinical Relevance of Levels Potential Behavior Type Range in Health Associated Relative increase Relative Decrease Change Humans Disease(gtnotfLjGlutamine 420-700 Meat, dairy, Critical illness Low glutamine is common in critical Reduced glutamine poultry, nuts, related glutamine Illness, trauma, and prolonged may present as poor seeds, whole depieiion, tumor catabolism, impairing Immune and gut recovery, frequent grains, metabolism function' high levels reflect aggressive infections, and gut legumes, tungi, supplementation or altered tumor discomfort, driving insects, algae metabolism. changes in training, diet, or supplementation Glufemate 18-98 Meat, dairy, Excitotoxi ci ty in Elevated glutamate is linked to exoitotoxle Patients may poufey, nite, stroke, Al.3, neuronal injury in stroke, ACS. and experience anxiety, seeds, whole epilepsy epilepsy; lew levels may reflect poor agitation, cognitive grains, protein intake dr intensive clearance slowing, or seizures, legumes, fithgi, therapies. altering daily function insects, algae and need for neurological care. Glydne 'W-400. Meat, daily, Nonketotic Markedly high glycine indicates Severe defects cause poultry, nite, hyperglycinemia and nonketotic hyperglycinemia or related lethargy, seizures, seeds, whole other metabolic defects, whereas moderate variation and apnea in infants; grains. blocks usually reflects diet or collagen turnover. Iri adults,5.............................Attorney Docket No 081906- 1535050-236510PC AmihoActtl! Plasma Main Sources Rale in Muscle Putative Clinical Relevance of Levels Potential Behavior Type Range in Health Associated Relative increase Relative Decrease Change Human s Disease(jitnot / L)iegumes, fungi, supplemental glycine insects, gigae may modestly improve steep and relaxation.Histsdine 67-120 Meat, dairy, i-iistidinemia Histidine elevations in hi&tidinemia are usually minimal pcnii.iy. nuts:, typically benign, but low levels may impair behavioral impact, seeds, whole hemoglobin buffering and histamine though low levels grains, synthesis. may contribute to legumes, fungi, fatigue and altered insects, algae responseto allergic or Inflammatory triggers.Isoteuclne 40-100 hteat, dairy, Maple Syrup Urine High isoleucine with other BCAAs 6an impair strength, poultry, nuts, Disease (with other appears in MSUD or heavy coordination, and seeds, whole BCAAs) supplementation, white low levels signal recovery, leading to grains, muscle wasting, low protein intake, er reduced physical legumes, fungi, chronic disease. activity or increased insects, algae focus on protein / BCAA intake.Attorney Docket No 081906- 1535050-23651C-PC Amino Acid Plasma Main Sources Role in Muscle Putative Clinical Relevance of Levels Potential Behavior Type Range in Health Associated Relative increase Relative Decrease Change Humans Disease(prrioi / L)Leucine 100-000 Meat, dairy, Strong anabolic Maple Syrup Brine Leucine rises with Leucine fails in Influences decisions (106-190) poultry, fish, matter, Disease, high protein / SCAA sarcopenia, frailty, about protein feeding, nuts, seeds, including far the sarcopehiateging Use or '■dSt.-D and catabolic, illness, 8CAA riming, and whole grains, stimulation of tracking anabolic resistance exercise, legumes muscle protein May indicate high capacity especially in athletes (beans), fungi, synthesis; linked dieiery protein and older aduits insects, algae to muscle mass intake, acute Linked to muscle seeking to preserve and strength starvation, protein wasting, muscle.feeding, diabetes, malnutrition,obesity, and / or recovery needs, Dietary adjustment to inborn errors of prolonged increase protein metabolism {e g„, starvation, low intake; pptenrial maple Syrup urine protein diets, liver supplementation, disease) Cirrhosis, urea cycle especially for elderly disorders, and / or and post-surgery shronia renal testers patients;: preventing or treating the following: malnutrition, burnAttorney Docket No 081906- 1535050-236510PC Amido Acid Plasma Main Sources Rale in Muscle Putative Clinical Relevance of Levels Potential Behavior Type Range in Health Associated Relative increase Relative Decrease Change Humans Disease(ptnol / L}Injury, cancer cachexia,: sarcdperaa, diabetes, obesity, Inborn errors of metabolism, ahd / or overall performance Lysine 90-250 Meat, dairy, Hyperlysinemia, Low lysine Smits protein synthesis, May present as poor poultry, nuts, connective tissue collagen crosslinking, and carnitine wound healing, hair seeds, whole and carnrtfoe issues formation: high levels suggest metabolic and skin changes, or grains, defects or very high infake. fatigue, encouraging: legumes, ftmgi, dietary revision or insects., algae lysine supplementation. Methionine 20-40 Meat, dairy, Homocystinuda: and Elevated methionine often parallels high Can affect mood, poultry, nuts, vascular nsk homocysteine and increased vascular cognition, and seeds, whole risk, While low levels reflect poor intake, cardiovascular health grains, liver disease, or defects In methylation decisions, including legumes, fungi, pathways. 8-vitamlh and insects, algae mathyi-dpHsrAttorney Dackei No 081906- 1535050-236510PC Amiho Acid Plasma Main Sources Rale in Muscle Putative Clinical Relevance of Levels Potential Behavior Type Range in Health Associated Relative increase Relative Decrease Change Humans Disease(pmotfL)strategies and diet modification Piidnyialanine 33-102. Meat, daisy, Phenylketonuria Phenylalanine elevation is diagnostic tor Poor control in PKU poultry, nuts, (PKU) PKU and correlates with newocognitive leads to irritability, seeds, whole injury; tight control keeps levels low and ahention problems, grains, reduces risk. and seaming legumes, fungi, difficulties, strongly insects, algae shaping diet bebsvior and adherence. —Piteiine 13Q-35G Meat, daisy, Collagen and Altered proline tracks collagen turnover, Joint pain, skin lexity, poultry, nuts, connective tissue liver disease, and certain metabolic or slow recovery from seeds, whale disorders defects; high levels may accompany injury can reduce grains, fibrosis or high protein intske. physical activity and legumes, fungi, alter rehabilitation insects, algae plans.Ser ine 80-170 Meat, dairy, Serine biosynthesis Law serine in plasma or CSF is Deficiency leads io poultry, nuts, defects and associated with bidsynthesis defects and seizures and seeds, whole neurodeveiopmenla! neurodeveiopmentat delay; high levels cognitive impairment grains; delay usualiy reflect diet or supplements, tn severe cases; inAttorney Docket No 081906- 1535050-236510PC AmihoAcid Plasma Main Sources Role in Mtsscie Putative Clinical Relevance of Levels Potential Behavior Type Range in Health Associated Relative increase Relative Decrease Change Humans Disease(ptnot / L)legumes, fungi, adults, serine status insects, gigae may modestly influence mental clarify and moon. Threonine 60-200 Meat, daily, Threonine metabolic Threonine shifts mirror protein balance, Symproms such as poultry, nuts, defects, severe gut mucin production, and energy state; low energy, S31 seeds, whole malnutrition extreme levels suggest metabolic errors discomfort, and grains, or severe malnutrition. slower recovery can legumes, fungi, reduce training insects, algae enthusiasm and shift eating patterns. Tryptophan 40-80 Meat, dairy Hartnup disease, Low tryptophan reduces serotonin and Directly affects mood, poultry, nuts, rtiacin deficiency niacin synthesis, linking to depression, steep architecture, seeds, whole states insomnia, and pellagra-iike symptoms: and stress resilience, grains, high: levels follow supptementation or high often driving legumes, fungi, intake. preferences for insects, algae high-Trp foods or supplements.Attorney Dadtet No 081906- 1535050-236510PC Amido Acid Plasma Main Sources Role in Mtsscie Putative Clinical Rat e vance of Levels Potential Behavior Type Range in Health Associated Relative increase Relative Decrease Change Humans Disease(jjirtotfL.}Tyrosine 30-120. Meat, dairy, Tyrosirtemia type High tyrosine in tyro sinemta predicts liver Changes in tyrosine poultry, nite, iiH, PKU failure and neurelog c risk, whereas low may influence seeds whole management levels occur during '-'KU treatment or peer alertness, motivation, grains, intake and body-image via legumes, fungi, pigmentation or insects, algae thyroid-reiated energy shifts.Valine 150-300 Meat, dairy, Mnpie Syrup Urine Valine follows other BCAAs elevated in Alters perceived poultry, nuts, Disease, muscle MSI© or heavy supplementation and stamina and muscle seeds, whale wasting reduced with muscle wasting, low protein endurance. grains, intake, w systemic illness, Influencing protein legumes, fangi, strategies, training insgcte, algae volume, and fatigue managementAttorney Docket No. 081906-1535050-256510PCEXAMPLES

[0322] The following examples illustrate particular aspects of the disclosure and are not intended in any way to limit the disclosure.Example 1: Enzyme Selection / DesignStrategy 1: Utilization of L -Amino Acid Oxidases

[0323] L-amino acids are the dominant form utilized by eukaryotes. In Strategy 1, L-amino acid oxidases are applied to oxidize L-amino acids, thereby producing hydrogen peroxide (H2O2) and imino acids. An exemplary reaction equation is illustrated below.'Add

[0324] Three commercially available L-amino acid oxidases were selected as potentially useful candidates to be applied in Strategy 1: SEQ ID NO: 1, a L-amino- acid oxidase, purchased from Millipore Sigma, Uniport' 093364, derived from Crofafos adamanteus; SEQ ID NO: 2, a L-amino-acid oxidase (apoxin-1), purchased from Millipore Sigma, Uniport: P56742, derived from Crotalus atrox; and SEQ ID NO: 3, a L-amino-acid oxidase (bordonein-L), purchased from Creative Enzymes, Uniport: C0HJE7, derived from Crotalus durissus terrificus. The amino acid sequences of the L-amino acid oxidases are shown in Table 4.Table 4: Amino acid sequences of L-amino anid oxidases.SEQ ID Amino Acid Sequence CommentsSEQ ID NO; 1 MNVFF^ L-amino-acid oxidase RETDYEEFLEIAKNGLTATSNPKRWIVGAG (Uniport; 093364) MAGLSAAYVLAGAGHQVTVLEASERVGGR {Crotalus adamanteus) VRTYRKKDWYANLGPMRLPTKHRIVREYIK KFDLKLNEFSQENENAWYFIKNIRKRVREV KNNPGLLEYPVKPSEEGKSAAQLYVESLR KVVEELRSTNCKYILDKYDTYSTKEYLLKE GNLSPGAVDMIGDLLNEDSGYYVSFIESLK HDDIFGYEKRFDEIVGGMDQLPTSMYEAIKEKVQVHFNARVIEIQQNDREATVTYQTSANAttorney Docket No. 081906-1535050-256510PCEMSSVTADYVIVCTTSRAARRIKFEPPLPP KKAHALRSVHYRSGTKIFLTCTKKFWEDDGlHGGKSTTDLPSRFIYYPNHNFTSGVGVIi AYGIGDDANFFQALDFKDCADIVINDLSLIH ELPKEDIQTFCHPSMIQRWSLDKYAMGGIT TFTPYQFQHFSEALTAPFKRIYFAGEYTAQ FHGWIDSTIKSGLTAARDVNRASENPSGIH LSNDNEF SEQ ID NO; 2 MNVFFMFSLLFLAALGSCAHDRNPLEECF L-amino-acid oxidase RETDYEEFLEIAKNGLTATSNPKRWIVGAG apoxin~1 MAGLSAAYVLAGAGHQVTVLEASERVGGR (Uniport: P56742) VRTYRKKDWYANLGPMRLPTKHRIVREYIK (Crotalus atrox) KFDLKLNEFSQENENAWYFIKNIRKRVREV KNNPGLLEYPVKPSEEGKSAAQLYVESLR KWKELKRTNOKYILDKYDTYSTKEYLLKE GNLSPGAVDMIGDLLNEDSGYYVSFIESLK HDDIFGYEKRFDEIVGGMDQLPTSMYEAIK EKVQVHFNARVIEIQQNDREATVTYQTSAN EMSSVTADYVIVCTTSRAARRIKFEPPLPP KKAHALRSVHYRSGTKIFLTCKKKFWEDD GIRGGKSTTDLPSRFIYYPNHNFTSGVGVtl AYGIGDDANFFQALDFKDCADiVINDLSLIH QLPKEDIQTFCRPSMIQRWSLDKYAMGGIT TFTPYQFQHFSEALTAPFKRIYFAGEYTAQ FHGWIDSTIKSGLTAARDVNRASENPSGIH LSNDNEF SEQ ID NO: 3 MNVFFMFSLLFLAALGSCAHDRNPLEECF L-amino acid oxidase RETDYEEFLEIARNGLTVTSNPKHWIVGA bordonein-L GMAGLSAAYVLAGAGHQVTVLEASERVG (Uniport: C0HJE7) GRVRTYRKKDWYANLGPMRLPTKHRIVRE (Crotaius durissus YIRKFGLQLNEFFQENENAWYFIKNIRKRV temficus) REVKNNPG1LEYPVKPSEEGKSAAQLYVES LRKWKELKRTNCKYILDKYDTYSTKEYLLK EGNLSPGAVDMIGDLLNEDSGYYVSFIESL KHDDIFGYEKRFDEIVGGMDQLPTSMYEAI KEKVQVHFNARVIEIQQNDRETKVTYQTSA NEMSSVTADYVIVCTTSRAARRIKFEPPLP PKKAHALRSVHYRSGTKIFLTCKRKFWED DGIRGGKSTTDLPSRFIYYPNHNFTSGVGVllAYGIGDDANFFQALDFKDCADiVINDLSLI HQLPKEDIQTFCRPSMIQRWSLDKYAMGG ITTFTPYQFQHFSEALTAPFKRIYFAGEYTA QFHGWIDSTIKSGLTAARDVNRASENPSGIHLSNDNEFSt teqy 2: Utilization of Combination of Racemases with D-Amino Acid Oxtoes[0325J Alternatively, L-amino acids may be converted into D-amind acids through racemization, catalyzed by a racemase. D-amino acids may then be oxidized by D-amino acid oxidases, a type of amino acid oxidases that specifically catalyzes the oxidation of D-amino acids. By combining the two reactions, free L-Attorney Docket No. 081906-1535050-256510PCamino acids contained in a sample fluid can be oxidized into imino acids. An exemplary reaction equation is illustrated below:Racemases

[0326] To identify suitable racemase candidates with specificity against certain amino acids of interest for further engineering, a screening of UniProt database was performed. 27 racemases were selected as potentially useful candidates (SEQ ID NOs: 5-31). Among these racemases, 6 of them were identified as potential candidates for possessing alanine specificity (SEQ ID NOs: 4, 5,19-24); 1 of them was identified as a potential candidate for possessing proline specificity (SEQ ID NOs: 11); 1 of them was identified as a potential candidate for serine specificity (SEQ 12); 3 of them were identified as potential candidates for aspartate specificity (SEQ ID NOs: 6-8); 8 of them were identified as potential candidates for leucine (SEQ: 9,10, 25-30): 15 of them were identified as potential candidates for possessing arginine specificity (SEQ ID NOs: 13-18); and 1 of them was identified as potential candidate for possessing specificity against branched-chain amino acids (SEQ ID NO: 31). In addition, a commercially available alanine racemase was purchased from Millipore Sigma (SEQ ID NO: 4). The amino acid sequences of these racemases are shown in Table 5.

[6327] The racemase (SEQ ID NO: 31; Uniport: M1GRN3; derived from LentHactQbacillus buchnen) with specificity against branched-chain amino acids was identified by using the Rosetta Molecular Modeling Suite (Rosetta) protein structure database for structural prediction. The Rosetta Molecular Modeling Suite method isAttorney Docket No. 081906-1535050-256510PCdescribed in detail in Alford etat, J. Ch&m. Theory Comput. 13, 3031-3048 (2017), Leman et al., Nat Methods 17, 665-680 (2020), and Moreti et at, Protein Sei. 27, 259-268 (2017), each of which is incorporated herein by reference,

[0328] in silica design of engineered racemases were then analyzed,, tn SEQ ID NO: 32 (5WYA_Mut1 ), an I24V mutant was introduced; and in SEQ ID NO: 33 (5WYA_Mut2), an I24A mutant was introduced. A visual display of the design of the engineered enzyme SEQ ID NO: 32 (5WYAJVIut1) as predicted by Rosetta is shown in Fig. 1; and a visual display of the design of the engineered enzyme SEQ ID NO: 33 (5WYA__Mut2) as predicted by Rosetta is shown in Fig.2 Fig. 1 shows the binding of isoleucine with pyridoxal 5-phosphate (PLP) cofactor into 5WYA„Mut1 (SEQ ID NO: 32); and Fig.2 shows the binding of leucine with PLP cofactor into 5WYA_Mut2 (SEQ ID NO: 33). As shown in Fig.2, the I24A mutant provides more space to the binding site, thereby decreasing any possible steric hindrance and as a result, improving activity specifically towards leucine. In addition, further screening was conducted, and enzymes with an amino acid sequence of SEQ ID NOs: 55-61 were identified as promising racemase candidates. The amino acid sequences of the engineered enzymes are also shown in Table 5.Table 5: Amino acid sequences of racemases.SEQ ID Amino Acid Sequence Comments AlanineSEQ ID NO: 4 MNDFHRDTWAEVDLDAIYDNVENLRRLLP Alanine racemase DDTHIMAWKANAYGHGDVQVARTALEAG (Uniport: P10724) ASRLAVAFLDEALALREKGIEAPILVLGASR (Geobaciilus PADAALAAQQRIALTVFRSDWLEEASALYS stearoth&rrnaphifus) GPFPIHFHLKMDTGMGRLGVKDEEETKRI VALIERHPHFVLEGLYTHFATADEVNTDYFS YQYTRFLHMLEWLPSRPPLVHCANSAASL RFPDRTFNMVRFGIAMYGLAPSPGIKPLLP YPLKEAFSLHSRLVHVKKLQPGEKVSYGAT YTAQTEEWIGTIPIGYADGWLRRLQHFHVL VDGQKAPIVGRICMDQCMIRLPGPLPVGT KVTLIGRQGDEVISIDDVARHLETINYEVPC TISYRVPRIFFRHKRIMEVRNAIGRGESSA SEQ ID NO: 5 MQAA^ Alanine racemase VAWKANAYGHGLLETARTLPDADAFGVAR (Uniport: P0A6B4) LEEALRLRAGGiTKPVLLLEGFFDARDLPTI (Escherichia coii) SAQHFHTAVHNEEQLAALEEASLDEPVTV WMKLDTGMHRLGVRPEQAEAFYHRLTQC KNVRQPVNIVSHFARADEPKCGATEKQLAI FNTFCEGKPGQRSIAASGGILLVVPQSHFD WVRPGIILYGVSPLEDRSTGADFGCQPVM SLTSSUAVREHKAGEPVGYGGTVWSERDTRLGWAMGYGDGYPRAAPSGTPVLVNGAttorney Docket No. 081906-1535050-256510PCREVPIVGRVAMDMICVDLGPQAQDKAGDP VILWGEGLPVERIAEMTKVSAYELITRLTSR VAMKYVD SEQ ID NO: 6 MKWO Aspartate racemase QEHPKVIIFNNPQIPDRTAYILGKGEDPRPQ (Unsport; 058403) LIWTAKRLEECGADFIIMPCNTAHAFVEDIR (Pyraccccus honkoshii) KAIKIPIISMIEETAKKVKELGFKKAGLLATFG TIVSGVYEKEFSKYGVEIMTPTEDEQKDVM RGIYEGVKAGNLKLGRELLLKTAKILEERGA ECHAGCTEVSWLKQDDLKVPLIDPMDVIA EVAVKVALEK SEQ JD NO: 7 MNSPLAPVGVF^ Glutamate racemase EDIVYVGDTGNGPYGPLTIPEIRAHALAIGD (Unsport: P9WPW9) DLVGRGVKALVIACNSASSACLRDARERY (Mycab&ctemm QVPWEVILPAVRRAVAATRNGRIGVIGTRA tuberculosis) TITSHAYQDAFAAARDTEITAVACPRFVDFV ERGVTSGRQVLGLAQGYLEPLQRAEVDTL VLGCTHYPLLSGLIQLAMGENVTLVSSAEE TAKEWRVLTE1DLLRPHDAPPATRIFEATG DPEAFTKLAARFtGPVLGGVQPVHPSRIHSEQ ID NO. 8 MLEQPIGVIDSGVGGLTVAKEIMRQLPKENI Glutamate racemase 1IYVGDTKRCPYGPRPEEEVLQYTWELTNY (Unlport: P94556) LLENHHIKMLVIACNTATAIALDDIQRSVGIP (Baci / fes subti / is) WGVIQPGARAAIKVTDNQHIGVIGTENTIK SNAYEEALLALNPDLKVENLACPLLVPFVE SGKFLDKTADEIVKTSLYPLKDTSIDSLILGC THYPILKEAIQRYMGEHVNIISSGDETAREV STILSYKGLLNQSPIAPDHQFLTTGARDQFA KIADDWFGHEVGHVECISLQEPIKR SEQ ID NO: 9 iOTE^EfWSQVISRASHWwiVPSRA Leucine / methionine ENALVWDEGRAYIDFLADAAVQNVGHNNP racemase RVVEAVKKTADRLLHFTFIYGFPVEPLLLAE (Unlport: Q5JGG6) KLREIAPLEGAKVAFGLSGSDANDGAIKFA (Thermococcus RAYTGRRSHGYLRSYYGSTYGAMSVTGLD kodakarensis) FEVRSKVGQLSDVHFIPFPNCYRCPFGKE PGKCRMECVSFLKEKFEGEVHAEGTAAUA EAIQGDAGMVVPPENYFKKLKRILDEHGIL LWDEVQSGLGRTGKWFAIEHFGVEPEilTL AKPLGGGLPISAIVGRGEIMDSLPPLGHAF TMSGNPVASAAALAVIEEIEEKELLKRAQIL GERAKRRLEKMKKKHELIGDVRGLGLMLG VDLVKDRETKERAYDEAKKVVWRAYELGLI VAFLQGNVtRIEPPLTIEEEVLDEGLDKLEE AIEDVEEGRVPDEVIEKVQGW SEQ ID NO: 10 MRKEEllER^ Leucine / methionine AKVWDIEEREYIDFLSDAAVQNVGHNNEK racemase VVKAIKEQAERLIHFTFIYGFTLEPLLLAEKL (Unlport: H3ZR39) AEISPIEEPKIAFGLSGSDANDGAIKFARAY (Thermococcus TKRRTLLSYLKSYYGSTYGASSITGLDFHV litorak's) RALVGELSDVHYIPYPDCYRCPFGKERNS CKMECVEYIKAKFEGEVYADGVAALFAEP! QGDAGMWPPEDYFKRVKRILDEHGILLAV DEVQSGLGRTGKWFAIEHFGVKPDHTVAKPLGGGLPISAWGRAEIMDSLPPLGHAFTLIAttorney Docket No. 081906-1535050-256510PCGNPVASRAALAVI EEIEEKDLLKRAEKLGSY AMKRLGKMKEEYELIGDVRGKGLMIGVDL VKDRETKERAYDEAKKWWRAYELGLIVAF LQGNVLRIQPPLTIEKETLDEGLDKLERAIA DVEEGRVGDEALKFVHGWProlineSEQ ID NO: 11 MRKSVCPKQKFFFSAFPFFFFFCVFPUSR Proline racemase A TGQEKLLFDQKYKHKGEKKEKKKNQRANR (Uniport: Q4DA80) REHQQKREIMRFKKSFTCIDMHTEGEAARI (Trypanosoma cruzf) VTSGLPHIPGSNMAEKKAYLQENMDYLRR GIMLEPRGHDDMFGAFLFDPIEEGADLGM VFMDTGGYLNMCGHNSIAAVTAAVETGIVS VPAKATNVPWLDTPAGLVRGTAHLQSGTE SEVSNASilNVPSFLYQQDVWVLPKPYGE VRVDIAFGGNFFAIVPAEQLGIDISVQNLSR LQEAGELLRTEINRSVKVQHPQLPHINTVD CVEIYGPPTNPEANYKNWIFGNRQADRS PCGTGTSAKMATLYAKGQLRIGETFVYESI LGSLFQGRVLGEERIPGVKVPVTKDAEEG MLWTAEITGKAFIMGFNTMLFDPTDPFKN GFTLKQ SEQ I D NO: 12 MSDNLVLPTYDDVASASERIKKFANKTPVL Serine racemase TSSTVNKEFVAEVFFKCENFQKMGAFKFR (Uniport: 059791 ) GALNALSQLNEAQRKAGVLTFSSGNHAQA ( Schizosaccharomyces IALSAKILGIPAKIIMPLDAPEAKVAATKGYG pombe) GQVIMYDRYKDDREKMAKEISEREGLTHPP YDHPHVLAGQGTAAKELFEEVGPLDALFV CLGGGGLLSGSALAARHFAPNCEVYGVEP EAGNDGQQSFRKGSIVHIDTPKTIADGAQT QHLGNYTFSIIKEKVDDILTVSDEELIDCLKF YAARMKIVVEPTGCLSFAAARAMKEKLKNK RIGIIISGGNVDIERYAHFLSQ SEQ ID NO: 13 MSLGIRYLALLPLFVITACQQPVNYNPPATQ Lysine racemase VAQVQPAIVNNSWIEISRSALDFNVKKVQS (Uniport: M4GGR9) LLGKQSSLCAVLKGDAYGHDLSLVARMIEN (Proteus mirabiiis) NVKCIGVTNNQELKEVRDLGFKGRLMRVR NATEQEMAQATNYNVEELiGDLDMAKRLD AIAKQQNKVIPIHLALNSGGMSRNGLEVDN KSGLEKAKQISQLANLKWGIMSHYPEEDA NKVREDLARFKQQSQQVLEVMGLERNNV TLHMANTFATIWPESWLDMVRVGGIFYGD TIASTDYKRVMTFKSNIASINYYPKGNTVGY DRTYTLKRDSVLANIPVGYADGYRRVFSNA GHALIAGQRVPVLGKTSMNTVIVDITSLNNI KPGDEWFFGKQGNSEITAEEIEDISGALFT EMS1LWGATNQRVLVDSEQ ID NO: 14 MSFGIRYLALLPLFV1TACQQPVNHNPPVT Broad specificity QTAQVQPAIVNNSWIEISRSALDFNVRKVQ amino-acid racemase SLLGDKSSLCAVLKGDAYGHDLSLVAPVMI (Uniport:ENNVQCIGVTNNQELKEVRDLGFQGRLMR A0A9X5MRS9) VRNATEQEMAQATSYNVEELIGNLDMAKK (Shigella sp. FC 130) LDAiAKQQNKVIAIHLALNSGGMSRNGLEVNNNAGLEEAKQIAQLTNLKVVGIMSHYPEEDANKVKEDLARFKQQSQKILDVTGLNRKDAttorney Docket No. 081906-1535050-256510PCVTLHMANTFATITVPESWLDMVRVGGIFYG DTlASTDYKRVMTFKSNlASINYYPKGNTV GYDRTYTLKRDSVLANlPVGYADGYRRVF SNAGHALIAGQRVSVLGKTSMNTVMVDIT DLNNIKPGDEWFFGKQGNSEITAEEIEDIS GALFTEMSILWGATNQRVLVD SEQ ID NO: 15 MYPRLLINLKEIEENAKKWEMASRRGIEIV Alanine racemase GVTKVTLGDPRFAETLRKAGIRILGESRIKN domain protein VLRMKKAGIEGPFMLLRLPMMSELVEDVR (Uniport: A0A0F6ALZ8) HFDYIMVSDPDVAKKVDELSREIKRNVKIIY (Thermotoga sp.) MIDVGDLREGVWFEKAVEEIAQCKGANW GIGTNFGCYGGIIPTREKFEILLDIKEKLEKN YGFNIEIVSGGNTPALYALENDEIPEGINQL RIGEAIVLGRDtTNNRVIDWLSQDTFLIEAE VIEVKEKQSVPLGKRGLDVFGRKIEFVDRG IRKRAICALGEQDIDSRGLIPVDKGVEVLHA SSDHIVLDVTDFGDVKVGDVFRFRMTYSC LLKAMTSPFVEKVYEPSIArgtnmeSEQ ID NO. 16 MVEAIHRSTRIEFSKSSLAYNVQYTKQVSG Lysine racemase AKTLWLAVKSNAYGHGLLQVSKIARECGV (Uniport: Q04HB7) DGLAVSVLDEGIAIRQAGIDDFILILGPIDVK (Oenococcus oerw) YAPIASKYHFLTWSSLDWLKSADKILGKEK LSVNLAVDTGMNRIGVRSKKDLKDEIEFLQ EHSDHFSYDGJFTHFASSDNPDDHYFQRQ KNRWELIDGLIMPRYVHVMNSGAAMYHS KELPGCNSIARVGTVVYGVEPSEGVLGPID KLKPVFELKSALTFVKKIPAGEGISYGSKFV TSRDTWIGTLPIGYGDGWLAEYQDFQLUD GQKCRQVGQIAMDQMMVALPHEYPIGTEV TLIGKSGKYENTLYDLHKHSGVPPWKITVA FSDRLKRMWD SEQ ID NO: 17 MVEAIHRSTRIEFSKSSLAYNVQYTKQVSG Alanine racemase AKTLWLAVKSNAYGHGLLQVSKIARECGV (Uniport: D3L711) DGLAVSVLDEGIAIRQAGIDDFILILGPIDVK (O&oococcus oepi) YAPIASKYHFLTWSSLDWLKSADKILGKEK LSVNLAVDTGMNRIGVRSKKDLKDEIEFLQ EHSDHFSYDGIFTHFASSDNPDDHYFQRQ KNRWYELIDGLIMPRYVHVMNSGAAMYHS KELPGCNSIARVGTVVYGVEPSEGVLGPID KLKPVFELKSALTFVKKIPAGEGISYGSKFV TSRDTWIGTLPIGYGDGWLAEYQDFQLUD GQKCRQVGQIAMDQMMVALPHEYPIGTEV TLIGKSGKYENTLYDLHKHSGVPPWKITVA FSDRLKRMWD SEQ ID NO: 18 MVYPRLLINLKEIEENARKWEMASRRGIEI Lysine racemase VGVTKVTLGDPRFAETLRKAGIGILGESRIR (Uniport: Q9X1T3) NVLRMKKAGIEGPFMLLRLPMMSELVEDV (Thermotoga maritima) KHFDYIMVSDPDVAKKVDELSREMKRNVKI IYMIDVGDLREGVWFEKAVEEIAQCRGANI VGIGTNFGCYGGIIPTREKFEILLDIKEKLEK NHGFNIEIVSGGNTPALYALENGEIPEGINQ LRIGEAIVLGRDITNNRVIDWLSQNTFLIEAEVIEVKEKPSVPLGKRGLDVFGRKVDFVDRAttorney Docket No. 081906-1535050-256510PCGIRKRAICALGEQDIDSRGUPVDKGVEVLH ASSDHIVLDVTDFGDVKVGDVFRFRMTYS CLLKAMTSPFVEKVYEPSI SEQ ID NO: 19 MDNF^ Alanine racemase GTQVMAWKANAYGHGDAQVATTALEAGA (Uniport: A0A0Q0ZV01) SYLAVAlLDEALALRQKGlNAPlLVLGASRPA (GeQbaci / / i / 3 sp.) DVELAARHRITLTVFRADWLEEAASIYNGSI PVHLHLKMDTGMGRLGVKDEEETKRITTLI DRHPSFILEGVYTHFATADEVNTDYLSYQY RRFLHMLEWLPSRPPIVHCANSATVLRFPE RAFNMARFGISMYGLAPSPSIKPLLPYELK EAFSLHSRLVHVKRLOPGEKVSYGATYTA QEEEWIGTLPIGYADGWLRRLQHFHVLVN GQKAPIVGRICMDQCMiRLPEPLPIGTKVTL IGRQGDEFISVDDVARYLETINYEVPCTISY RVPRIFFRNKRIMEVRNAVCHG SEQ ID NO: 20 MFYRDTWAEIDLDAIYYNVSQLKNFLPNDV Alanine racemase RIMAVVKANAYGHGDVQVAKTALEAGASYL (Uniport:AVAFLDEALALRKGGIDAPILVLGASRPSDI A0A7U4DMM4) NIAAKHRITLTVFQAEWIEQAADFYSETAPV (G&obacillus sp.) LFHLKMDTGMGRLGVKEETETKRUEQIDR HPYFSLEGVYTHFATADEINTGYFSFQYDN FLRMLEWLPYKPPLIHCGNSATTLRFPDKV FNMARFGISMYGLSPSPEIKQYLPYTLKEA FSLHSRLVHVKKLKPGEKVSYGATYTAETE QWVGTVPIGYADGWLRKLQNFHVLVNGEK APIIGRICMDQLMVRLPKQLPVGTKVTLIGR QGDKYISVDDVAQYLDTINYEIPCTISYRVP RIFFRNKSIMEVRNLVYA SEQ ID NO: 21 MVGSFHRDTWVEVDLDAIFHNVASLRTFL Alanine racemase RKETAIMAWKANAYGHGDVQVAQTALEA (Uniport: A0A7W8ISL9) GASYLAVAFUDEALALRKKGIEAPILVUGAV (Anoxybacillus RPEDIQLAARERITLTVFQQEWLEKAKQW t&pidamans) HQDGPPVALHLKMDTGMGRLGVKEEEET KRIVAMIDEHSAFSLEGVYTHFATADEIDTQ YFSFQYENFLRMLNWLPYQPKWIHCGNSA TSLRFPDKVFNMVRFGIAMYGLTPSLALKP HLPFELKEAFSLHSRIVHVKRLLPGEKVSY GATYTVEKEEWIGTVPIGYADGWIRKLQNF QVLVNGVKAPIIGRICMDQMMIRLPEYVPV GTKVTLIGKQGDAQVTMDDVAQYLGTINYE IPCTISYRVPRIFLKKQSIMEVRNVVLDGNR YV SEQ ID NO: 22 MNSFYRDTWAEIDLDAIHYNVSQLQNFLPN Alanine racemase DTRIMAVVKANAYGHGDAQVAKTALEAGA (Uniport:SYLAVAFLDEALALRKKGITAPILVLGASRPS A0A023CTQ6) DVHLAAKYQITLTVFQPDWVEQAASLYKGT (Parageobacillus EQVRFHUKMDTGMGRLGVKEEAETKRVIE genomosp. T) LIDRHPYFSLEGVYTHFATADEINTDYFSFQ YHHFLQMLEWLPYKPSLIHCGNSATALRFP DKVFNMVRFGISMYGLSPSPAIKPYLPYEL KEAFSLHSRLVHVKKLQPGEKVSYGATYTA ETEQWVGTVPIGYADGWLRKLQNFHVLVNGKKAPIVGRICMDQLMIRLPEPMPVGTKVTAttorney Docket No. 081906-1535050-256510PCLIGRQGDEYISVDDVAQYLGTISYEVPCVIS YRVPRIFFRNKSIMEVRNWLNGNDYV SEQ ID NO: 23 MDEFHRDTWVEIDLDAIYDNVANLRRFLPE Alanine racemase GTQIMAWKANAYGHGDAQVAATALEAGA (Uniport A0A063YVK8) SRLAVAFLDEALALRKKG1DAPILVLGASRP (Geohac / te sp.EDVALAAEHRIALTVFRSDWLEEASSLYNG CAMR5420) STPIHFHLKMDTGMGRLGVKDEEETKRIAA UDRHPPFVLEGVYTHFATADEVNTDYFSY QYARFLHMLDWLPSRPPLVHCANSAAALR FSDRAFNMVRFGISMYGLAPSPDIKPLLPY ELKEAFSLHSRLVHVKKLQPGEKVSYGATY TAQTEEWIGTIPIGYADGWLRRLQHFHVLV GGQRAPIVGRICMDQCMIRLPGPLPVGTK VTIIGRQGDEVISIDDVARHLGTINYEVPCTI SYRVPR1FFRNKRIMGVRNAVGRGSEQ ID NO: 24 MIHSFHRDTWAEVDLDAIFHNVTSLRAFLG Alanine racemase EQTEIMAVVKANAYGHGDVQVARTALEAG (Uniport: A0A167U3S7) ASYLAVAFLDEALALRKKGVGAPILVLGAVR {Anoxybacillus sp. PEDINLAAREQITLTVFQKEWLEKAKNLFTE B7M1) KQPVSFHLKMDTGMGRIGVKEEEETKQIV AMIEEHPFFLLEGVYTHFATADELDTQYFS FQYEKFLRMLEWLPYLPKRVHCGNSATSL RFPDKVFNMVRFGIAMYGLSPSIDMKPLLP FSLKEAFStHSRIVHVKCLRPGEKVSYGAT YTAEEEEWIGTVPIGYADGWIRKLQNFHVL VNGRKVPIVGRICMDQMMIRLPEQVPVGT KVTLIGKQEAAQVTIDDVAKYVGTISYEIPC TISYRVPRIFLKKKSIMEVRNVVLDGNGCS SEQ ID NO: 25 MTTQNVDHDHEVDHRLIQNENKYYAQASR 4-aminobutyrate INYYDLVIESAHDATLVDADGNEYIDLLASA aminotransferase SAINVGHTNEKVVKAIQAQAEKLIHYTPAYF (Uniport: A0A0K2LEB0) HHRPGQELAERLAKSVPGSEKKWFSNSG (Campsnilactobacillus SEANDAIVKFARAYTGRQYIVSYMDAYHGS heitongjiang&nsis) TFGSMALSGVSLNMTRKMGPLMPGVVHV PYPDLYRRYPNETEHDVALRYFDAFKEPFE SFLPPDEVACVLIEPIQGDGGIRKAPEEYM KLVYDYCHEHGILFAVDEINQGMGRTGKM WSYQQFDGIEPDLMSVGKSLASGMPLSAT IGRKEIM ESLDSPAHVFTTAGNPVCCAASL ATLDVLEEEDULEKSTEGGFHAEKRFKDM QKHYPEIGDVRMYGLDGGIELVKDRDSKE PNPDFANKVIYYAFQHGVVMITLKGNILRFQ PPLVISREELDRALDVLDDAFAAAENNQVilPNNEKIGW SEQ ID NO: 26 MEAARVDHNHAKDRRLIERENNYFASAARI 4-aminobutyrate NYYDLVIDSAHGAILKDVDGNEYIDLLASAS aminotransferase ATNIGHTNDLWKAISDQAQKLIHYTPSYFH (Uniport: A0A0R1SEL9) HLPEQEtAEKLASIAPGDSEKEWFGNSGS (Companilactpbacitius DANDAHKFARAYTGRSYIVSYMNAYHGST versmold&nsis) YGSIAVSGVSLNMTRKIGPLMPDWHVPYP DSYRTEKNETEHELSQRYFADFKRPFESFL PADEVACVMVEAIQGDGGLVKAPDEYLHLV YNFCRQHGIUFAVDEVNQGLGRTGHMWSIDNFPGIEPDMISVGKSIASGMPLSAVIGKKEAttorney Docket No. 081906-1535050-256510PCIMESLGAPAHTFTTAGNPVCCAAALATLKVi EQDDLVEKSRVDGQYAKKKFLEMQLRHPN IGDVRMFGLNGGIE1VKDKQSKVADSDFAT KVIYYAFEHGWIITLRGNILRFQPPLVITRS ELNKALEVLDQAFTAVKTGKVSLPQTDTKI GW SEQ ID NO: 27 MIFMSNLEKASRLIDEENNYYARSARINYY 4-aminobutyrate NLVIDHAHDATLVDVDGNKYlDLLASASAiN transaminase VGHTNQRVVKAISDQAQKU HYTPAYFHHV (Uniport:PGIDLAKRLAEIAPGDTPKMVSFGNSGSDA A0A0R1YTN2) NDAHKFSRAYTGRQYWSYMGSYHGSTY (LentHactabaciiius GSQTLSGTSLNMTRKIGPMLPGWHVPYP patafarraginis) DLYRRYEGETEHDVAVRYFDAFKAPFESFL PADETACVLtEAIQGDGGiVKAPEEYMQLV YKFCHDHGILFAIDEVNQGLGRTGKMWGI QQYKDIEPDLMSVGKSLASGMPLSAVIGKK DVMQSLDAPAHLFTTSGNPVCSAAALATL DVIRDDHLVERSATEGAYAKQRFLDMQKR HPMIGDVRMWGLNGGIELVKDPQTKEPDN DGATKVIYYAFAHGVVHTLQGNILRFQPPLViPREQLDQALQVLDDAFTAVENGEVKMPSDTGKiGWSEQ ID NO: 28 MIFINKLTKAKKLVQEESQYYARSARINYYD Acetyiornithine LVIDHAHGATLVDVDDNEYIDLLASASAINV transaminase GHTHENWKAISEQAKKLIHYTPAYFHHVP (Uniport:EIDLAQKLVEiAPGNSEKMVSFGNSGSDAN A0A0R2AKA196) DAHKFSRAYTNRQYIVSYMGSYHGSTYGS (Aprlactcbacfflus QTLSGTSLNMTRNMGPMLPGVVHVPYPD ozensis) LYRRYAGETEHDVAVRCFNEFKKPFESFLP ADETACVLIEPIQGDGGIVKPPEEYMQLVY KFCRENGILFAVDEVNQGLGRTGKMWGiQ QYSDIEPDLMSVGKSLASGLPLSAV1GKKEI iOSLDAPAHLFTTSGNPICAAAALATIKTIED EKLAEKSAVDGAYAKQRFLDMQKRHPMJG DVRMWGLNGGIELVKDSDTKEPDKDAATK VIYYAFNHGWIITLAGNILRFQPPLV1TREQ LDTALQVLDDAFTAVENDEVTLPKSDKKIG W SEQ ID NO: 29 MTTYKVDHDHEVDHRLIKHENKYYASASRI Aspartate NYYDLVIESAHDSTLVDADGNEYIDLLASAS aminotransferase AINVGHTNEKWRAIQEQAEELIHYTPAYFH family protein HRPGQKLAERLAKSVPGVDKQVAFSNSGS (Uniport:EANDAtlKFARAYTERPYIVSFMDSYHGSTF A0A2P4R6Q6) GSMTLSGVSLNMTRKMGPLLPGWHVPY (Companilactobacillus PDLYRKYPHETEHDVALRYFDEFKQPFESF formos&nsis) LPADEVACVLIEPIQGDGGIRKAPEEFMQLV YEFCHQNGILFAVDEINQGMGRTGKMWSY QQFNDIEPDLMSVGKSLASGMPLSATIGKK EIMESLDSPAHTFTTAGNPVCCAASLATLD VLKEENLLEKSTIDGAYAEQKFLEMQKHYP EIGDVRMYGLDGGIELVKDRQTKEPNPDFA NKVIYYAFQHGVVMlTLKGNiLRFQPPLVITRDELDKALNWDAFAAAENNOVliPSNEKiGWAttorney Docket No. 081906-1535050-256510PCSEQ ID NO: 30 MTTQEAHHDHEVDHRLIMHENEAFSKASR Aspartate INYFDLVIESAHNSTLVDADGNEYIDLLASA aminotransferase SSINVGHTNEKWRAIQRQAQKLIHYTSAY family protein FPNRPEQEUERLSQTVPGAPKEVSLANSG (Uniport:SEANDAHKFARGYTGRPYIVSFMDSYHGS A0A5P0ZRE3) TYGSMTLSGVSLNMARKMGPLLPDWHVP (Campanilactobadllus YPDLYRRHKNETEHDVAVRYFEEFKQPFE haladurans) SYLPADEVACILIEPIQGDGGIRKAPEEFMQ LVYEFCHQNGILFAVDEINQGLGRTGKMW SYOQFQDIEPDLMSVGKSLASGMPLSATIG KKEIMESLDSPAHTFTTAGNPICCAASLATL DVLKEQKLLLRSLEKGNYAKKKFLELQNRY PE1GDVRMYGLNGGIELVKDQKTKVPDADF ANKIIYYAFMHGVLMITLKGNILRFQPPLVIT QEELDYAFDVLNDAFKAAENNQVI IPSNAKI GWBranched-Chain Amino Acids (BCAA)SEQ ID NO: 31 MGKLDKASKLIDEENKYYARSARINYYNLV1 Isoleucine 2-epimerase DHAHGATLVDVDGNKYIDLLASASAINVGH (Uniport: M1GRN3) THEKWKAIADQAQKLIHYTPAYFHHVPGM (Lenfy' / actobadf^s ELSEKLAKIAPGNSPKMVSFGNSGSDAND buchneri) AllKFARAYTGRQYiVSYMGSYHGSTYGSQ (5WYA) TLSGSSLNMTRKIGPMLPSVVHVPYPDSY RTYPGETEHDVSLRYFNEFKKPFESFLPAD ETACVLIEPIQGDGGIIKAPEEYMQLVYKFC HEHGILFAIDEVNQGLGRTGKMWAIQQFK DIEPDLMSVGKSLASGMPLSAVIGKKEVMQ SLDAPAHLFTTAGNPVCSAASLATLDVIEYE GLVEKSATDGAYAKQRFLEMQQRHPMIGD VRMWGLNGGIELVKDPKTKEPDSDAATKVI YYAFAHGWIITLAGNILRFQPPLVIPREQLD QALQVLDDAFTAVENGEVTiPKDTGKlGWSEO ID NO: 32 MGKLDKASKLIDEENKYYARSARVNYYNLV Isoleucine 2-epimerase IDHAHGATLVDVDGNKYIDLLASASAINVGH (124V) THEKWKAiADQAQKOHYTPAYFHHVPGM (5WYA„Mut1) ELSEKLAKIAPGNSPKMVSFGNSGSDANDAliKFARAYTGRQYIVSYMGSYHGSTYGSQ TLSGSSLNMTRKIGPMLPSWHVPYPDSY RTYPGETEHDVSLRYFNEFKKPFESFLPAD ETACVLIEPIQGDGGIIKAPEEYMQLVYKFC HEHGILFAIDEVNQGLGRTGKMWAIQQFK DIEPDLMSVGKSLASGMPLSAVIGKKEVMQ SLDAPAHLFTTAGNPVCSAASLATLDVIEYE GLVEKSATDGAYAKQRFLEMQQRHPMIGD VRMWGLNGGIELVKDPKTKEPDSDAATKVI YYAFAHGWIITLAGNILRFQPPLVIPREQLD QALQVLDDAFTAVENGEVTIPKDTGKiGWLeucineSEQ ID NO: 33 MGKLDKASKUDEENKYYARSARANYYNLV Isoleucine 2~epimerase IDHAHGATLVDVDGNKWLLASASAINVGH (I24A) THEKWKAIADQAQKLIHYTPAYFHHVPGM (5WYA_Mut2) ELSEKLAKIAPGNSPKMVSFGNSGSDAND AIIKFARAYTGRQYIVSYMGSYHGSTYGSQTLSGSSLNMTRKIGPMLPSWHVPYPDSYAttorney Docket No. 081906-1535050-256510PCRTYPGETEHDVSLRYFNEFKKPFESFLPAD ETACVUEPiQGDGGIlKAPEEYMQLVYKFC HEHGILFAIDEVNQGLGRTGKMWAIQQFKD1EPDLMSVGKSLASGMPLSAVIGKKEVMQ SLDAPAHLFTTAGNPVCSAASLATLDVIEYE GLVEKSATDGAYAKQRFLEMQQRHPMIGD VRMWGLNGGIELVKDPKTKEPDSDAATKVI YYAFAHGWIITLAGNILRFQPPLVIPREQLD QALQVtDDAFTAVENGEVTIPKDTGKIGWSEQ I D NO: 55 MTTQKLDHDHEVDHRLIVQENKYYAQASRI Aspartate NYYDLVIESAHDSTUDADGNEYIDLLASAS aminotransferase AINVGHTNEKWQAIQEQAEKLIHYTPAYFH family protein (EC HRPGQELAERLAKSVPGAEKQVAFSNSGS 2.6.1.19) EANDAIIKFSRAYTGRPYIVSFMDSYHGST (Uniport.YGSMTLSGVSLNMARKMGPLLPGVVHVP A0A2N7AUX81) YPDVYRRYPNETEHDVALRYFTEFKKPFE (Companr / aceodac^t / s SFLPADEVACVLIEPIQGDGGIRKAPEEFM nuruki) QLVYDFCHQNGILFAVDEINQGMGRTGKM WSYQNFTDIEPDLMSVGKSLASGMPLSAT1 GKKEIMESLDSPAHAFTTAGNPICCAASMA TLDVLEDEDLLERSTVEGAYVEQRFQEMQ KHYQEIGDVRMYGLDGGIELVKDRETKEP NPDFANKVIYYAFQHGWIITLKGNILRFQP PLVITRAELDQALDVLDDAFAAVENNQVIIP SNEKIGW SEQ ID NO: 56 MDTKKVDQNHLADHRLIQRENKYYAKASR Aspartate MDYFDLVIDHAHDATLVDVDGNEYIDVLAS aminotransferase ASAVNVGHTNEKWKAIQDQAEKLVHYTP family protein GYFHHQPEQQLAERLAKLVPGEPKQVTFG (Uniport:NSGSDANDA1IKFSRAYTKRPYIVSFMDSY A0A1P8Q2648) HGSTYGSMTLSGVSLNMTRNMGPMLPDV (Companilactab&ciHus VHVPYPDLYRTEPGESEHDVSLRYFDEFK altii) KPFETFLPVEEVACVLIEPIQGDGGIRKAPE EYMQLLYKFCHDNGILFAVDEVNQGIGRTG KMWSIQQFKDIEPDLMSVGKSLASGMPLS AVIGKKKVMESLDSPAHVFTTSGNPVCCAA ALATLDVLDEKDLLEKSVEDGKYVEEQFLK LKDKYEEIGDVRFVGLDGGIELVTDKKSKT PNPDFANKVIYYAFEHGWIITLKNNILRFQP PLVITREQLDKVLNVLEKAIVAAENDEVDVP DEDSMGW SEQ ID NO: 57 MTLKTHPQAHQLIEREDHYLATAARINYFD 4-aminobuty rate LVIDHAHDAVLTDVDGNEYLDLLASASAINV aminotransferase GHTNAKWKAIQDQAAKLIHYTPAYFHHQP (Uniport:EQQLAERLAKLAPGDDNEVAFGNSGSDAN A0A0R2NYU21) DAHKFARAYTGRQYIVSYMDAYHGSTYGS (Lactiplantibadllus MTLSGVSLNMDRKMGPLLPGWHVPYPD fabifermentans) MYRTLPNETEHDVAQRYFAAFKAPFESYL PADEVACVLIEPIQGDGGIRKAPAEYMQLV YDFCHEHGIUFAVDEVNQGMGRTGKMWSI ERFQNIRPDLMSVGKSLASGLPLSAVIGRR EVMESLGAPAHVFTTAANPICCAASLATLD VLEEEQLLHKSLTDGAYAQQRFLALQAKHPKIGQVRMYGLDGGIELVKDPVTKVADPEFAAttorney Docket No. 081906-1535050-256510PCSOVIYEAFRHGWMITLKGN1LRFQPPLVIT RAQLDFAFNVLDDAMTAAENGQVQRPDG KIGW SEQ ID NO: 58 MADLEKARKLI^ 4-aminobutyrate DHAHGATLVDVDGNHYLDLLASASAINVGH aminotransferase TDERWKAITTQAQKLIHYTPAYFHHVPEIE (Uniport:LAQKLAEIAPGKSPKMVTFGNSGSDANDAi A0A0R1V5L330) IKFARAYTGRQYlVSYMGAYHGSTYGSETL ( Uquarilactobacilius SGVSLNMTRQ1GPMLPGWHVPYPDLYRR satsumensis} LPGESEHEVALRFFAEFKRPFESFLPADET ACVLIEPIQGDGGIVKAPEEYMQLVYNFCH ENGILFAVDEVNQGLGRTGKMWSIQQFKDI EPDLISVGKSLAAGLPLSAVIGKKEHDALGT PAHCFTTSGNPVCAAASLATLNVIEADHLV EKSASDGEYVKKRFLEMQQRHPAIGDVRM WGLNGG1 ELVKDQKTKEPDNETATKVIYYA FAHGVVLITLAGNILRFQPPLVITRAQLDQA LAVLDDAFSAVEQNQVELPQTNEKIGW SEQ ID NO: 59 MATAARiNYYDLVIDHAHGAMLTDVDGNQY 4-arninobutyrate™2- IDLLASASAINVGHTHPRVVKAIQEQAAKU oxoglutarate HYTPAYFHHQPEQRLAERLAKSAPGTDNE transaminase (EC VVFGNSGSDANDAHKFARAYTNRQYiVAYT 2.6.1.19) DAYHGSTYGSMSLSGVSLNMVRKMGPLL (Uniport: A0A2S3U5J9) PGIVHVPYPDCYRTLPHETEHELALRYFEA (Lactipiantibacillus FKAPFDSYLPAEETACVHEPIQGDGGIRKA plantarum) PAEYVQLVYDFCHQHGILFAVDEVNQGMG RTGKMWSIQNFPGIRPDLMSVGKSLASGL PLSAVIGRREVMESLAAPAHTFTTAANPVC CAAALATIDVLADEQLVARSANYGRYAKEQ FLALQQRHPKIGQVRMYGLNGGIELVTDR QSQQPDPDFASDVIYAAFERGWMITLKGN ILRFQPPLViTKTQLDTALTVLDEAMAAAEQGLVKRPQGKIGW SEQ ID NO: 60 MAASKVDHDHVEAKRLIQRENEYYSSATRI Aspartate NYYDLVIDSAHGSILKDVDGNEYIDLLASAS aminotransferase AINVGHTNELWQAIKDOAEKLIHYTPSYFH family protein HRPEQQLAEKLAKLAPGPTPKEVAFGNSG (Uniport: A0A5P0ZK31) SDANDAIIKYARAYTDRPYIVSYMQSYHGS {Compamlactobaclllus TYGSMALSGVSLNMTRKMGPFMPGVTHV mishanenala) PYPDLYRRLPGESEHDVSMRFFNEFLEPF KSYLPAEEVACVLIEPIQGDGGLVKAPEEY MQMVYKFCHDNGILFAVDEVNQGMGRTG HMWGIENYHDIEPDUSVGKSIASGMPLSA VIGKHDIMESLGSPAHTFTTAGNPVCCAAS LATLEVLDKDNLIEKSKVDGEYAKERFEEM QTRHPNIGDVRMFGLNGGIELVKDKESKE ADPDFATKVIYYAFEHGWI1TLRGNILRFQP PLVITRDELDTALNVLDDAFTAAENGEWV PATDEKIGW SEQ ID NO: 61 MDAQKVDHDHEEAHRLiQRENKFYAKSAR 4-aminobutyrate MDYYDLVIDHAHDATLVDVDGNEYIDVLAS aminotransferase ASAINVGHTNEKVVKAIQDQAAKLVHYTPG (Uniport: A0A0R1K5T9) YFHHQPEOELSERLAKLVPGSPKQVTFGN (CompamlactobaclllusSGSDANDAIIKFSRAYTGRPYIVSFMDSYH nodensis)Attorney Docket No. 081906-1535050-256510PCGSTYGSMTLSGVSLNMTRKMGPLLP6VV HVPYPDLYRRLPDETVHDVAMRYFEEFKQ PFETFLPADETACVLiEPiQGDGGlRKAPEE FMQLVYQFCHENGILFAVDEINQGMGRTG KMWSIQQFKDIEPDLMSVGKSLASGMPLS AVIGKKEVMESLDSPAHLFTTSGNPVCCAA ALATLDVLDEKKLLEKSVEDGEYVKKRFLK MQQEFEE1GDVRMYGLDGG1ELVTDKESK TPNTDFASKVIYYAFQHGVVIITLKGNILRFQ PPLVITRAQLDKVLDVLEAAFKAVENDEVTL PEGKIGW SEQ tD NO: 62 MSNLEKASRLIDEENNYYARSARINYYNLVi Aspartate DHAHDATLVDVDGNKYIDLLASASAINVGH aminotransferase TNQRWKAISDQAQKUHYTPAYFHHVPGI family protein DLAKRLAEIAPGDTPKMVSFGNSGSDAND (Uniport:AIIKFSRAYTGRQYWSYMGSYHGSTYGS A0A5R9CV78) QTLSGTSLNMTRKIGPMLPGWHVPYPDL {Lentilactobaciyus YRRYEGETEHDVAVRYFDAFKAPFESFLPA parafarraginis) DETACVLIEAIQGDGGtVKAPEEYMQLVYK FCHDHGILFAIDEVNQGLGRTGKMWGIQQ YKDIEPDLMSVGKSLASGMPLSAVIGKKDV MQSLDAPAHLFTTSGNPVCSAAAUATLDVI RDDHLVERSATEGAYAKQRFLDMQKRHPM IGDVRMWGLNGGIELVKDPQTKEPDNDGA TKVIYYAFAHGWIITLQGNILRFQPPLVIPR EQLDOALQVLDDAFTAVENGEVKMPSDTG KIGW SEO ID NO: 63 MTTQKLAHDHEVDHRLIKNENKYYAQASRI Aspartate NYYDLVIESAHDATLVDADGNEYIDLLASAS aminotransferase AINVGHTNEKVVKAIQEQAEKLIHYTPAYFH family protein HRPAQKLAERLAKLVPGSPKKWFSNSGS (Uniport: A0A386PV56) EANDAVIKFSRAYTGRQYIVSYMDAYHGST (Compani / acfoPacf / tos YGSMALSGVSLNMTRKMGPLMPGWHVP zhachilii) YPDLYRRYPNETEHDVALRYFESFKEPFES YLPADEVACVLIEPIQGDGGfRKAPEEYMQ LVYEFCHKHGILFAVDEVNQGMGRAGKM WSYQQFSDIEPDLMSVGKSLASGMPLSATI GKKEIMESLDSPAHVFTTAGNPVCCAAALA TLDVLEEENLFERSVTEGIYAEQRFQEMKK HYPEIGDVRMYGLDGGIELVKDRESKEPN PDFATKVIYYAFLHGWMITLKGNILRFQPP LVISHEELDQALDVLDDAFAAVENNQVIIPS NEKIGW SEQ ID NO: 64 MSSQNHRLDQQLIEREDHYMATAARINYYD 4~am i nobutyrate-2- LViDHAHGALLTDVDGNQYIDLLASASAINV oxoglutarate GHTHPRWKAIQEQAAKLIHYTPAYFHHQP transaminase (EC EQRLAERLAKSAPGTDNEVVFGNSGSDAN 2.6.1.19) DAHKFARAYTNRQYIVAYTDAYHGSTYGSM (Aminotransferase) SLSGVSLNMVRKMGPLLPGIVHVPYPDCY (Uniport: A0AG9FDC4) RTLPHETEHELALRYFEAFKAPFDSYLPAE (Lactiplantibadllus ETACVnEPIQGDGGIRKAPAEYVQLVYDFC plantarixn) HQHG1LFAVDEVNQGMGRTGKMWSIQNFPGIRPDLMSVGKSLASGLPLSAVIGRREVMESLAAPAHTFTTAANPVCCAAALATIDVLADEAttorney Docket No. 081906-1535050-256510PCQLVARSANYGRYAKEQFLALQQRHPKIGQ VRMYGLNGGIELVTDRQSQQPDPDFASDVlYAAFERGWMITLKGNlLRFQPPLVITKTQL DTALTVLDEAMAAAEQGLVKRPRGKIGW SEQ ID NO: 65 MISNNHRLDQQL1EREDHYMATAARINYYD Aminotransferase LVIDHAHGALLTDVDGNQYIDLLASASAINV (Uniport:GHTHPRWKAIQDQAAKU HYTPAYFHHQP A0A837R8R14) EQQLAERLAKLAPGADNEVI FGNSGSDAN (Lactipfafrtibacillus DAHKFARGYTKRQYIVAYTDAYHGSTYGS p&ntQsus) MSLSGVSLNMTRHMGPLLPGWHVPYPD TYRRLPHETDHQLALRYFDAFKAPFESYLP ADETACVLIEPIQGDGGIRQAPEEYVQLVY DFCHQHG1LFAVDEVNQGMGRTGKMWSI QNFPGIRPDtMSVGKSIASGMPLSAVIGRR EIMESLGAPAHVFTTAANPVCCAAALATLD VLADEELVERSARLGRYAESQFLDLQTRH PKlGQVRMYGLNGGiELVTDQASQQPDPE FASDVIYAAFERGVVMITLKGNILRFQPPLVI TKDQLDTALAAIDDAMTAAEQGRVHRPQG KMGW SEQ ID NO: 66 MTQLRNLEKATGUAQEDQYYARSARIKYF 4-aminobutyrate DLVVDHAQAATLVDVDGNRYIDLLASASAT aminotransferase NVGHSDERWKAITQQAQKLIHYTPAYFHH (Uniport:TPGIELARKLAKLAPGPSTKMVTFGNSGSD A0A0R1M960) ANDAIIKFARAYTGRQNIVSYMGSYHGSTY (UquQrifactobacfflus GSETLSGVSLNMVRKIGPLL. PGWHVPYP o&ni') DLYRKYPGESEHEVAVRYFEDFKRPFSSFL PADETACVLLEAIQGDGGIVKAPEEYLQLV YEFCHEHGILFAVDEVNQGLGRSGKWWSI QHFKGIEPDLMSVGKSLASGLPLSAWGKK AVMESLDAPAHCFTTAGNPVCCAAALATLK IIEDDQLVERSASMGEYVKQRFLDMQKSH PVIGDVRiWGLNGGIELVKDQKTKEPDNEA ATKVIYYAFAHGWLITLSGNILRFQPPLVIP REQLDKALSVLDDAFTAVEQGWALPADNG KIGW SEQ ID NO: 67 MTAE^ Aspartate DLIIDHAHGAVLTDVAGNDYIDLLASASAINV aminotransferase GHTNARWRAIQNQAEKLIHYTPAYFHHQP family protein EQQLAERLSKLAPGDDNEVAFGNSGSDAN (Uniport: A0A660E419) DAHKFSRAYTGRQYIVSYMDAYHGSTYGS (Lactiplantfbacfilus MTLSGVSLNMARKMGPMLPGVVHVPYPD mudanjlangefisis) LYRRLPGESEHELSLRYFAAFKAPFDSYLP VDETACVLIEPIQGDGGIRKAPTEYIQLVYQ FCHDHGILFAVDEVNQGMGRTGKMWSEH FPGVQPDLMSVGKSLASGMPLSAViGRRD VMESLAAPAHVFTTSANPVCCAASLATLDA LAAEDLLHKSIVDGAYVKQRFLALQQRHPK IGDVRLYGLDGG1ELVKDPTTKEPDPEFAS DVIYEAFQHGWM1TLKGNILRFQPPLVITR LQLDRALMVIDHALTAAEQGQVQRPTGKIGwSEQ ID NO: 68 MfiWVbHDHEVDSRLiQNE 4-aminobutyrateNYYDLVIESAHNSTLVDADGNEYIDtLASAS aminotransferaseAttorney Docket No. 081906-1535050-256510PCAINVGHTNEKWKAIQEQAEKLIHYTPAYFH (Uniport:HRPGQELAERLAKLVPGSEKQVVFSNSGS A0A0R1QEU1) EANDAIIKFARGYTGRQYIVSFMDSYHGST ( CompaniiactQb&cillus YGSMTLSGVSLNMTRKMGPMLPGVVHVP miPde / is / s) YPDLYRRYQGETEHDVAVRYFEEFKRPFE SFLPADEVACVLIEPIQGDGGIRKAPEEYM QLVYKFCHDNG1LFAVDEINQGMGRTGQM WSYQQFENIELDLMSVGKSLASGMPLSATI GKKEIMESLDSPAHTFTTAGNPICCAASLAT LDVLEEEDLLEKSATDGIYAEQRFLEMQKQ FPEIGDVRMYGLDGGIELVKDQKTKEPNPD FANKVIYYAFQHGVVMITLKGNILRFQPPLVI THDELEKALDVLEDAFAAAENDQWIPSNE KIGW SEQ ID NO: 69 MVDAKHQMGQRLVQRENHFLASAARINYY aspartate DLVIDHAQGAILTDVDGNDYIDLLASASAVN aminotransferase VGHTHPKVVRAIQQQAEKLIHYTPSYFHHQ family protein PEQQLAERLAALAPGDDREWFGNSGSDA (Uniport:NEAIIKFARAYTGRQYIVSYMDAYHGSTYG ADA2R3JU959) AMTLSGVSLNMTRKMGPLLPGVIHVPYPD (Lactobacillus sp. LYRRLPHESEHAVSMRYFSAFKAPFESYLP CBA3606) ADETAAVLIEPIQGDGGIRQAPAEYLQLVYN FCHAHGILFAVDEVNQGMGRTGKMWSVE RFEHVIPDLMSVGKSLASGMPLSAVIGRRD IMESLAAPAHTFTTGANPICCAASLATLDVL NTEQLLAKSLADGDYVRRQFLKLQQRHPK IGDVRMYGLDGGIEIVKDAVSKTPDPEFAS RVIYAAFQHGWIITLKGNILRFQPPLVITRA QLDTALTVLDAAFTAAKNDDVTLPAGKIGW SEQ ID NO: 70 MNKLEKATALINDESHYNADCTRIDYYNLVI Aspartate DHAHGATLVDVDGNEYIDLLASASAINVGH aminotransferase TPDKWDAITEQAKKLIHYTPAYFHHTPQIE family protein LAERLTKLAPGDSEKMVSFGLTGSDANDAI (Uniport:IKFSRAYTGRQYIVSFMGSYHGSTYGSMSL A0A1S6QHY01) SGTSLNMTRKMGPMLPGVVHVPYPDLYR (Lentilactobaclllus RLPGESEHDVAMRYFDSFKKPFESFLPAD curieae) EVACVLIEPIQGDGGIVKAPEEFMQLVYKF CHENGILFAVDEVNQGLGRTGKMWGIQQY DNIEPDLMSVGKSIAAGMPLSAWGKKEV MQSLEAPANVFTTAANPVCCAASLATLDIL EEEDLVNKSKVDGEYAKQQFLDMQKRHPNiGDVRMWGLNGGIEUVKDRETKEADPDF ATKVIYYAFOHGWIITLCGNILRFQPPLVIT REQLDKAFAVLDDAFTAAENNEVEI PADAG KIGW SEQ ID NO; 71 MLASASAINVGHTNEKWKAIQEQAEKLIH 4~aminobutyrate YTPAYFHHRPGQELAERLAKLVPGTDKQV aminotransferase AFSNSGSEANDAIIKFSRAYTGRQYIVSYM (Uniport:DSYHGSTYGSMSLSGVSLNMTRKMGPLL A0A0R2LA549) PGIVHVPYPDLYRRYPNETEHDVAIRSFKE (Companilactobacillus FKEPFESFLPADEVACVMEPIQGDGGIRKA kimchiensis) PEEYMQLVYKFCHENGILFAVDEINQGMG RTGKMWSYQQFNNIEPDLMSVGKSLASGMPLSATIGKKEIMESLDSPAHTFTTAGNPICAttorney Docket No. 081906-1535050-256510PCCAASMATLDVLEDEELlDRSTSEGAYAEER FLEMQKHYREIGDVRMYGLDGGIELVTDR VSKEPNSDFANKVIYYAFQHGWHTLKGNIL RFQPPLVISRDELDRALNVLDDAFAAAENN QVHPSNEKiGWSEQ ID NO: 72 MKLAKRLAELAPGTSKKLVSFGLSGSDAN Aminotransferase DAIIKFSRGYTGRQYWSFMGSYHGSTYG (Uniport: A0A401FJM8) SMSLSGTSLNMTRKMGPMLPGiVHVPYPD (lenff / acfoit)ac / / fos LYRRLPNESEHDVALRYFDSFKKPFESFLP fcoson / s) SDEVACVLIEPIQGDGGLIKPPAEFMHLIYD FCHEHGILFAVDEVNQGLGRTGQMWGIEQ FPGIEPDLMSVGNHSLLVCRSVL SEQ ID NO: 73 MNAKTNK^ Aminotransferase VVDHAHGATLVDVDGNEYIDLLASASAINV (Uhiport: A0A0D1A881) GHSPDKWQAIQTQAAKLIHYTPAYFHHEP (Paucilactobacillus GQELAQRLAKLAPGNSEKMVAFGNSGAD wasatchensss) VNDAIIKFARAYTGRPYWSYMNSYHGSTY GSQTLSGVSLNMTRKIGSMLPNWHVPFP DTYRHYQNESEHEISLRYFAEFTRPFETDL PADETAVVLIEPIQGDGGIVKAPAEYIHLVYD FCQKNGILFAVDEVNQGLGRTGKMWSIDN FDIEPDLMSVGKSIAAGMPLSAVIGKKKVM AALDAPAHLFTTAGNPICCAAALATLDMIED GLVEKSASDGEFTKQAFKKLQTKYSFIGDV RMYGLSGGIEIVTDKESKTPDALAATKIIYR AFEKGLVLVTLMGNILRFQPPLVITQSQLET SIQKLDEVFNELEQGKIELPAEMNNVGW S EQ I D NO: 74 MGKLDKATKLIDEENQYYARSARINYYNLVI Acetylornithine DHAHGATLVDVDGNKYIDLLASASAINVGH transaminase THEKWKAITDQAQKLIHYTPAYFHHVPGM (Uniport:ELSEKLAKIAPGDSPKMVSFGNSGSDAND A0A0R1L1T708) AIIKFARAYTGRQYSVSYMGSYHGSTYGSQ (LentilactobacWus TLSGSSLNMTRKIGPMLPSVVHVPYPDSY sunkil) RTYPGETEHDISLRYFNEFKKPFESFLPPD ETACVLIEPIQGDGGIVKAPEEYMQLVYKF CHDNGILFATDEVNQGLGRTGKMWSIQQY KDIEPDLMSVGKSLASGMPLSAWGKKEV MQSLDAPAHLFTTAGNPVCSAASLATLDVI EDEGLVEKSAKDGAYTKQRFLDMQKRHP MIGDVRMWGLNGGIELVKDPKTKEPDSDA ATKVIYYAFAHGWIITLAGNILRFQPPLVIPR EQLDTALQVLDDAFTAVENGEVTIPKDTGKiGW SEQ ID NO: 75 MNNLEKASRLIDEENNYYARSARINYYDLVI Aminotransferase DHAHGATLIDVDGNEYIDLLASASAINVGHT (Uniport: A0NL71) NERWKAISRQAQKLIHYTPAYFHHVPGIEL { OenocQccus o&ni) SKRLAELAPGNSPKMVSFGNSGSDANDAII KFSRAYTSRQYIVSYMGSYHGSTYGSQSL SGTSLNMTRKIGPMLPGWHVPYPDLYRR YAGETEHDVAIRYFNAFKAPFESFLPADET ACVLVEPIQGDGGIVKAPEEYMQLVYKFCH DNGILFAVDEVNQGLGRTGKMWGIQQYKD IEPDLMSVGKSLASGMPLSAVVGKKDIMQSLDAPAHIFTTSGNPVCAAASLATLNVIRDDHAttorney Docket No. 081906-1535050-256510PCLVEKSAKDGEYAKQRFLEMQRHHPISGDVR MWGLNGGIELVKDPKTKEAENDAATKVIYY AFAHGVVIITLAGNILRFQPPLVISHKQLDKA LQVLDDAFTAVEQNKVEVPKDTGKIGW SEQ ID NO: 76 MDLKTNKAHAMIAEENRHYAKSARiNYFDL 4-ami nobutyrate VIDHAHGAELVDVDGNKYIDELASASAINVG aminotransferase HTHEKWQAIQDQAAKLIHYTPAYFHHEPG (Uniport:QQLAKKLAEIAPGDSEKMVTFGNSGSDAN A0A0A1GWW4) DAHKFARAYTGRSYWSYMNSYHGSTYGS (Payc / / aotobao / 7 / os QTLSGCSLNMTRKIGPMLPNWHVPFPDA hokkaidonensis) YRTYPNETLHDVSVRYFDEFKRPFETYLPA DETAWLIEPIQGDGGIVKAPDEYLHWVYD FCQEHGILFAVDEVNQGLGRTGKMWSiDN FGiEPDLMSVGKSLASGMPLSAVIGKKEVM ESLDAPAHLFTTSGNPVCCAASLATLQVIE DEHLVEKSATDGAYAKQAFKKMQERYAFIG DVRMYGLNGGlEiVMDKQSKTPDADAATKI IYRAFQKGLVIITLQGNILRFQPPLVITRDQL NEALQKLDDlFAELDQGLlELPDDLGNVGWSEQ I D NO: 77 MEKASELIDEENKYYARSARINYYNLVIDHA Aspartate HGATLVDVSGNKYIDLLASASAVNVGHTHE aminotransferase KVVRAITDQAKKLI H YTPAYFHHVPEIELSK family protein RLAEVAPGNSAKMVSFGNSGSDANDAIIKF (Uniport: A0AAU8IJP) ARAYTGRQYIVSYMGSYHGSTYGSQTLSG (Sporofectobac / / / us sp. TSLNMTRKMGPMLPGWHVPYPDLYRRYP Y61) GESEHDVAMRYFDGFKRPFESFLPADETA CVLIEPVQGDGGIVKAPEEYMQLWKFCHD HGILFAVDEVNQGLGRTGKMWGIQQYKDI EPDLMSVGKSLASGLPLSAIVGKKEVMESL DAPAHLFTTSGNPVCAAAALATLDVIKDEH LVEKSAEDGEYVKQRFKEMQTRHPMIGDi RMWGLNGGIELVKDPETKEPDNDAATKVIY YAFDHGWIITLAGNILRFQPPLVISREQLN QALQVLDDAFTAVENGSVEMPSDTGKIGW SEQ ID NO: 78 MDHRLIKNENKYYAQASRINYYDLVIESAH (S)-3-amino-2- DATLIDADGNEYIDLLASASAINVGHTNEKV methylpropionate VKAIQEQAEKUHYTPAYFHHRPAQELAERL transaminase (EC AKLVPGSPKKWFSNSGSEANDAIIKFSRA 2.6.1,19) (EC 2.6.1.22) YTGRQYIVSYMDAYHGSTYGSMALSGVSL (GABA NMTRKMGPLMPGVVHVPYPDLYRRYPNE aminotransferase) TEHDVALRYFESFKEPFESYLPADEVACVLI (Gamma-amino-N- EPiQGDGGlRKAPEEYMQLVYEFCHEHGIL butyrate transaminase) FAVbEVNQGMGRTGKMWSYQQFQGIEPD (Glutamate:succinic LMSVGKSLASGMPLSATIGKKEIMESLDSP sem (aldehyde AHVFTTAGNPVCCAAALATLDVLDEENLFE transaminase) (L~ RSITEGAYAEQRFQEMQKHYQEIGDVRMY AiBAT) GLDGGIELVKDRESKEPNPDFANKVIYYAF (Uniport:QHGWMITLKGNILRFQPPLVISREELDEAL A0A0R1WSJ4) DVLDNAFAAAENNQVIIPSNEKIGW (Compani / actobac / / tas nant&fjs / s)SEQ ID NO: 79 MDHRLIKHENKYYASASRINYYDLVIESAHD 4-aminobutyrate STLVDADGNEYIDLLASASAINVGHTNEKV aminotransferase VRAIQEQAEELIHYTPAYFHHRPGQKIAER (Uniport: A0A837RKAQ)LAKSVPGVDKQVAFSNSGSEANDAHKFARAttorney Docket No. 081906-1535050-256510PCAYTERPYIVSFMDSYHGSTFGSMTLSGVSL (Companilactobacillus NMTRKMGPLLPGWHVPYPDLYRKYPHET cfustorum) EHDVALRYFDEFKQPFESFLPADEVACVLI EPIQGDGGIRKAPEEFMQLVYEFCHQNGIL FAVDEINQGMGRTGKMWSYQQFNDIEPDL MSVGKSLASGMPLSATIGKKEIMESLDSPA HIM IAGNPVCCAASLATLDVLKEENLLEK STIDGAYAEQKFLEMQKHYPEIGDVRMYGL DGGIELVKDRQTKEPNPDFANKVIYYAFQH GWMITLKGNILRFQPPLVITRDELDKALNIL DDAFAAAENNQVIIPSNEKIGW SEQ ID NO: 80 MEAKKVDHNHEKDKRLIERENQYLASAERt (S)-3-amino-2~NYYDLVIDSAHGAVLKDVDGNEYIDLLASA me thy) propionate SAINVGHTNDLWKAIQDQAEKLIHYTPSYF transaminase (EC HHEPEQQLAEKLAKLAPGPTPKKVIFGNS 2.6.1.19) (EC 2.6.1.22) GSDANDAIIKFARAYTGRQYMVSYMNSYH (GABA GSTYGSMALSGVSLNMTRKMGPLMPGVV aminotransferase) HVPYPDLYRTEPGETEHDVAVRFFEAFKQ (Gamma-amino-N- PFESFLPADEVAAVLIEPIQGDGGLVKAPEE butyrate transaminase) YMQMVYKFCHDNGILFAVDEVNQGMGRT ( G 1 u tamate: su cci n ic GHMWGIENYHDIEPDLISVGKSIASGMPLS semialdehyde AVVGKADVMNSLGSPAHTFTTAGNPVCCA transaminase) (L- AALATLEVLEKDSLIEKSKRDGEYAKERFTE AIBAT) MQSRHPNIGDVRMFGLNGGIELVEDQETK (Uniport:VPDADFATKVITYAFEHGWHTLRGNILRFQ A0A0H4QKL5) PPLVITREELDQALDVLDDAFTAVENGEVS (Compamiactobac / ffijs LPDTDSKIGW ginsenosidfrnutans) SEQ ID NO: 81 MSHLEKASKLVDEENHYYARSARINYYDLV 4-aminobutyrate IDHAHGATLVDVDGNQYIDLLASASAINVG aminotransferase HTHEKVVKAITDQAQKLIHYTPAYFHHVPGJ (Uniport:ELSKKLTEIAPGNSPKMVSFGNSGSDAND A0A0R1SJM2) AHKFARAYTGRQYIVSYMGSYHGSTYGSQ (Lenti / actobadifas TLSGTSLNMTRKIGPMLPGWHVPYPDLYR diolivorans} RYPGESEDDVAKRYFDDFKRPFESFLPAD ETACVLIEPIQGDGGIVKAPEIYMQLVYKFC HDNG1LFAVDEINQGLGRTGKMWSIQQYK DIEPDLMSVGKSLASGMPLSAVIGKKDIMQ SLAAPAHLFTTSGNPICSAASLATLNVIEDE HLVEKSAKDGEYAKHRFLEMQERHPLIGD VRMWGLNGGIELVKDRQTKEPANDEATKV IYYAFAHGWIITLAGNILRFQPPLVIDRQQLDQALQVLDDAFTAVESGNVEIPKNAGKtGWD-amino acid oxidases

[0329] A D-amino acid oxidase was selected as potentially useful candidates to be applied in Strategy 2: SEQ ID NO: 34, a D-amino-acid oxidase, purchased from Millipore Sigma, Uniport: PQ0371, derived from Sus crofa. In addition, further screening was conducted, and enzymes with an amino acid sequence of SEQ ID NOs: 82-97 were identified as promising D-amino acid oxidase candidates. The amino acid sequence of SEQ ID NOs: 34 and 82-97 are shown in Table 6,Attorney Docket No. 081906-1535050-256510PCTable 6: Amino acid sequence of D-amino acid oxidase.SEQ ID Amino Acid Sequence CommentsD-Amino-Acid OxidaseSEQ ID NO: 34 MRWVIGAGVlGLSTALCIHERYHSVtQPLD (Uniport: P00371) VKVYADRFTPFTTTDVAAGLWQPYTSEPS (Sus scrota) NPQEANWNQQTFNYLLSHlGSPNAANMG LTPVSGYNLFREAVPDPYWKDMVLGFRKL TPRELDMFPDYRYGWFNTSLILEGRKYLQ WLTERLTERGVKFFLRKVESFEEVARGGA DVI INCTGVWAGVLQPDPLLQPGRGQilKV DAPWLKNFIITHDLERGIYNSPYUPGLQAVT LGGTFQVGNWNEINNIQDHNTIWEGCCRL EPTLKDAKIVGEYTGFRPVRPQVRLEREQL RFGSSNTEV1HNYGHGGYGLTIHWGCALE VAKLFGKVLEERNLLTMPPSHL SEQ ID NO: 82 MHSQKRWVLGSGVIGLSSALILARKGYSV D-amino-acid oxidase HILARDLPEDVSSQTFASPWAGANWTPFM (DAAO) (DAMOX) TLTDGPRQAKWEESTFKKWVELVPTGHA (DAO) (EC 1.4,3.3) MWLKGTRRFAQNEDGLLGHWYKDITPNY (Uniport: P80324) RPLPSSECPPGAIGVTYDTLSVHAPKYCQ (Rhodotorula YLARELQKLGATFERRTVTSLEQAFDGADL toruloides) WNATGLGAKSIAGIDDQAAEPI RGQTVLV KSPCKRGTMDSSDPASPAYHPRPGGEVIC GGTYGVGDWDLSVNPETVQRILKHCLRLD PTISSDGTIEGiEVLRHNVGLRpARRGGPR VEAERIVLPLDRTKSPLSLGRGSARAAKEK EVTLVHAYGFSSAGYQQSWGAAEDVAQLV DEAFQRYHGAARESKL SEQ ID NO: 83 MRWVIGAGVlGLSTALCIHERYHSVtQPLD D-amino-acid oxidase iKVYADRFTPLTTTDVAAGLWQPYLSDPNN (DAAO) (DAMOX) PQEADWSQQTFDYLLSHVHSPNAENLGLF (DAO) (EC 1, 4,3.3) LISGYNLFHEAIPDPSWKDTVLGFRKLTPR (Uniport: PI 4920) ELDMFPDYGYGWFHTSLILEGKNYLQWLT (Homa sapiens) ERLTERGVKFFQRKVESFEEVAREGADVIV NCTGVWAGALQRDPLLQPGRGQIMKVDA PWMKHFILTHDPERGIYNSPYIIPGTQTVTL GGiFQLGNWSELNNIQDHNTIWEGCCRLE PTLKNARHGERTGFRPVRPQIRLEREQLRT GPSNTEVIHNYGHGGYGLTIHWGCALEAA KLFGRILEEKKLSRMPPSHL SEQ ID NO: 84 MRVAVIGA^ D-amino-acid oxidase VKVYADRFTPFTTTDVAAGLWQPYTSEPS (EC 1.4.3.3) NPQEVNWNQQTFDYLLSHIGSPNAADMG (Uniport: A0A8C3YS98) LTPISGYNLFREAVPDPFWKDIVLGFRKLTP (Catagonus wagneri) RELDMFPDYRYGWFNTSLILEGRKYLKWL TERLTERGVKFFLRKVESFEEVARGGADVIi NCTGVWAGALQPDPLLQPGRGQVIKVDAP WLKNFIITHDLERGiYNSPHVIPGLQAVTLG GIFQLGNWNEMNNIQDHNSIWEGCCRLEP TLKDAKiiGEWTGFRPVRPQVRLEREQLRF GSSNTEVIHNYGHGGYGLTIHWGCALEVA KLFGKVLEERNLLTLPPSHL SEQ ID NO: 85 MRVWIGAGVIGLSTALCIHERYRSVLKSLD D-amino-acid oxidaseVMVYADRFTPLTTTDVAAGLWQPYLSEPS (EC 1.4.3.3)Attorney Docket No. 081906-1535050-256510PCNPQEAHWNQQTFNYLLSLIGSPNAANMGL (Uniport:APVSGYNLFREAVPDPYWKDIVLGFRKLTL A0A4W2F4L0) RELDMFPDYSYGWFNTSLILEGRKYLQWL (Bos indices x Bps TERLTERGVKFFLRKVESFEEWREGADVII taurus (Hybrid cattle)) NCTGVWAGVLQPDPLLQPGRGQHKVDAP WLKHFVITHDPERGIYKSPYIIPGLQAITLG GIFQLGNWSETSNIPDHNTIWEGCCRLEPT LKDAKHGEWSGFRPVRPQVRLEREQLRF GSSNTEVIHNYGHGGFGLTIHWGCAQEVA KLFGKILEERNLLRIPPSHL SEQ ID NO: 86 MRVAIIGAGVIGLSTALCIHEQYHSLVPSLE D-amlno-acid oxidase MEVYADRYTPHTTSDGAAGLWQPYLSDH (EC 14.3.3) SNLQETLWNKETFDYLLGHLGSPAAKEMG (Uniport: A0A663DYS7) tFLISGYNLFTQPVPDPSWKNIVtGFRNLTP (Aqui / a chrysaetos) KELELFPGYSYGWFNTALMLECRNYLPWL TNRLTQRGVKFFHKKVESFQEMFAQGVDV VINCSGVRAGELQPDPELQPGRGQIIKVLA PWVKHFIITHDIESGIYNSPYVIPGSEFTVL GGIYQHGNWNEENSAQDHKTIWENCCRL LPALQKAKIVEEWSGLRPARPCVRLERETV RHGHFQAEVIHNYGHGGFGITIHWGCAMA AARLFGSILQEKQLASPPQPRL SEQ ID NO: 87 MRVCIIGAGVIGLSTAQSIYQHFHSRISPLTI D-amsno-acid oxidase EVYADVFTPLTTSDGAAGLWQPYLYDKGN (EC 14.3.3) VQETKWNKETFDYLLSCLSSPDSVKMGIFL (Uniport: A0A6P6K364) QSGYNLCTETQPDPSFKDIVLGFRQLTKRE (Carass / i / s auratus) LDMFPGYSFGWFHTALMIEGKTYLPWLMD WLGERKVQFYQRKIGSFNELADSGADVIIN CSGVRSGDLQPDPELQPARGQIIKVDAPW LKHFILTHDPSKGLYNSPSVTPGSRFVTVA GVYQEGNWNLQNSSVDHKEIWEAACKLE PSLKHARiVEDWTGLRPTRSKVRLERETIR SGPTSFEViHNYGHGGFGMTIHRGCAEEA ARLFGQILEQKGLLAFSKSHLS SEQ ID NO: 88 MRIAVIGAGVIGLSTALCIHDRFHALVPQLQ D-amino-acid oxidase LEVYADRFTPHTTSDGAAGLWQPYLSDHG (EC 1.4.3.3) NLQETLWNKETFEHLLGYLNSPEAKEMGL (Uniport: A0A8V0ZP68) FLISGYNLFKQPVPDPSWKNIVLGFRNLTP (. Gallus galltis) KELELFPGYSYGWFNTALMLECRSYLPWL TNRLAQRGVKFFHRKVESFEEMFSQGIDV VINCTGI RAGELQPDPALQPARGQilKVLAP WVKHFtiTHDMESGIYSSPYVIPGSEFTVLG GiYQQGNWNEENSAQDHKSIWERCCRLL PMLQKAEIVQEWSGLRPARPTVRLERES1G HGRSRTEVIHNYGHGGFGITIHWGCAMAA ARLLGNILQEKQSQSRLSAAAICQTAEFPP PWPHTT SEQ ID NO: 89 MDQFSPSTTSDVAAGMLIPHTYPDTPIHRQ D-aspartate oxidase KQWFKETFTYLFAISNSAEASEAGIHLVSG (EC 14.3.1) WQVFKSTPKEELPFWSDVVLGFRAMSKA (Uniport:ELLKFPQHRFGQAFTTLKCDCPPYLLWLEK A0A8C0URM1) RLKAAGTQMYTRKVADLWELHSEYDIWN (Cyanistes caeruleus) CTGMGAHQLVGDKQLFPVRGQVLKVHAPWVKQFIRDGDGLTYVYPGIHRVTLGGTREAttorney Docket No. 081906-1535050-256510PCKGSWSLSPDAGTTRDILDRCCSLEPSLQG AQDIEVKVGLRPSRQCVRVQREVLSQGGV KLPVVHNYGHGAGGFSVHRGTAIEAAHLV GECISALQGSSSRAKL SEQ ID NO: 90 MRVWIGAGVIGLSTALCIHERYRSVLPSLD D-amlnoacid oxidase MRVYADRFTPLTNTDVAAGLCQPYLSDPR (EC 1.4.3.3) NPQEADWNQQTFDYLLSRVHSPNAANVG (Uniport: A0A2I3M6S7) LALVSGYNLFHEAIPVARGGADVIINCTGV (Pap / o snub / s) WAGSLQPDPLUQPGRGQIIKVDAPWMKHF HTHDPEKGIYKSPYIIPGIQAVTLGGIFQLGN WSEVNNIQDHNTIWEGCCRLEPTLQDAKi VAELTGLRPVRPQJRLERERLRFASSNTEVi HNYGHGGYGLTJHWGCALEAAKLFGKILEE RKLLKMPPPHL SEQ ID NO: 91 MALSSERLFDSLGHFGRFQACVYFASVFQ D-aspartate oxidase AMSCGIHYLASVFMAVTPNFVCGFPGNVS (EC 1.4.3.1) NILFHNSSASS1QDIWTLWTSTENYWVQL (Uniport: A0A6J0IAK5) ENGEIWEINQCSRSKREVSLDLAYEYKGN {Lepidothrix coranata) KSVFSCSDGFLYDDTKWKSTWTQWDLVC DREWLAKLIQPTFMLGVLIGAV1FGDIADRL GRQRVlWFTSAGQFVFGIAVAFTFDYYSFV TVRFLLAMVSSGYLWAFVYVTEFVGIKAR TWASMHVHAFFAMGIMIVALVGFLVRTWW VYQIFLSIATVPFVLCCWMLPETPFWLLSE ERYEDAQKVINTMARWNKVNTPCKVSELC SVQQDDPVSGRMGENDMSSTKKHNILDLF CNWQiARRTITVWLIWFTGSLGYYVFSLSS VSLGGNEYLNLFUGLQYINYFSKYGWKIFN RCGIWPYISLHSRTVPNNCKITCCWKWKH DVPCRKCGCSFLCISEKCLDFHATWQLKK KGCACPWTAGRLAGCAENHTLFPQLPVLS EKVRFFFLCLCILEMAAPKVAWGAGVIGLS TALCIVEACPSCSVTVLSDQFSPNTTSDVA AGMLIPHTYPGTPIHVQKQWFKETFTYLFAI SNSAEASEAGIHLVSGWQIFKSTPKEELPF WSDAVLDFRPMSEAELQKFPQYRFGQAFT TLKCDCPPYLLWLEKRLKAAGVQTYNRKV SDLWELHSEYDIWNCTGIGAHQLVGDKKL FPVRGQVLKVYAPWVKNFIRDGDGLTYIYP GIQGVTLGGTREKESWSLSPNPGTTKDIF DRCCStEPSLREAQDIKVKVGLRPSRQSV RLQREVLSQGGVKLPWHNYGHGAGGFS VHRGTAKEAARLVGECIAALRGSSSGAKL SEQ ID NO: 92 MRIAVIGAGVIGLSTALCIHDRFHALVPQLQ D-amino-acid oxidase LEVYADRFTPHTTSDGAVGLWQPYLSDHG (EC 14,3.3) NLQETLWNKETFEHLLGYLNSPEAKEMGL (Uniport:FUSGYNLFKQPVPDPSWKNIVLGFRNLTP A0A669PGG2) KELELFPGYSYGWFNTALMLECRNYLPWL (Phasianus colchicus) TNRLAQRGVKFFHRKVESFKEMFSQGIDV VINCTGVRAGELQPDPALQPARGQVIKVLA PWVKHFIITHDTESWIYSSPYVIPGSEFWL GGIYQQGNWNEENSAQDHKSIWERCCRLLPTLQKAEIVQEWSGLRPARPSVRLERESIAttorney Docket No. 081906-1535050-256510PCGHGHSRTEVIHNYGHSGFGITIHWGCAMA AARLLGNILQEKHSQSRL SEQ ID NO: 93 MRVCIIGAGViGLSTAQSIYQHFHSRISPLTI D-amino-acid oxidase EVYADVFTPLTTSDGAAGLWQPYLYDKGN (EC 14.3.3) VQETKWNKETFDYLLSCLSSPDSVKMGIFL (Uniport:QSGYNLCTETAPDPSFKDAVLGFRQLTKR A0A6P6K3M1) ELDMFPGYSFGWFNTALMIEGKTYLPWLM (Carassius auratus) DWLKQRNVNFHQRKIGSFKELADSGADVH NCSGVRSGDUQPDPELQPARGQilKVDAP WIKHWITTHNISSKGDSAYIIPGSRLVTVGG VFQVGNWNtQNSSVDHKNIWEAACKLEP SLQHARIVEDWTGLRPARSKVRLERET1RS GPTSFEVIHNYGHGGFGLTJHRGCAEETAR LFGQJLEQKRPLALSKSRL SEQ ID NO: 94 MAHCNLSSLQAGTMRVWIGAGVVGLSTA D-amino-acid oxidase LCLHERYHPVLPALDVRVYADRFTPFTNSD (EC 1.4.3.3) VAAGLSQAYLSDPSNPQEVHWNQQTFDYL (Uniport: A0A384DFU1) LSHIHSPNAANMGLALISGYNLFREAFPDP (Ursus maritimus) SWKDIVLGFRKLTCRELDMFPDYSYGWFN TSUVEGKRYLPWLTKRLTERGVKFFQRKV ESFEEVARGGADVIINCTGVWSGALQPDP LLKPGRGQHKVDAPWMKHFHTHDPEKGIY QSPYHPGMQAVTLGGIFQLGNWSEVNNN QDHATIWESCCRLEPTLKDAKIVAELTGLR PVRPQiRLEREQLRFGSLNTEVlHNYGHG GYGLTIHWGCAMEAAKLFGNILEERKLLRM PPPHL SEQ ID NO: 95 MRVAIIGAGVIGLSSALCIHQQFHGLLPSLE D-arrnno-acid oxidase LEVYADHFTPHTTSDGAAGLWQPYLSDHG (Uniport: U3K214) NLQETLWNKETFDYLLGQLHSPAAKEMGL (Fic&dula albicollis) FLISGYNLFTEPVPDPSWKNiVLGFRNLTPK ELELFPGYSYGWFNTALMLEGRSYLPWLT NRLTQRGVKFFHKKVESFQELFVQGVDW LNCSGMRAGHLQPDPELQPGRGQIIKVLA PWVKHFIVTHNLKSGIYNSPYHPGSEFTVL GGVYQHGNWSEENSAKDHKSIWDSCCQL LPPLQKAKIVGEWSGLRPARPSVRLERESI RLGNLQGEMALLPPQPAWRKGFQISTNYP WQSAPSAVWDQPGFS SEQ ID NO' 96 MRWVIGAGVIGLSTALaHERYHSVLQSLD D-amino-acid oxidase VKVYAERFTPLTNTDVAAGLWQPYLSEPSN (EC 1.4.3.3) PQEAHWNQQTFNYLLSHIGSPNTADMGLA (Uniport: AOA2Y9FBAO) PVSGYNLFREAVPDPYWKDWLGFRKLTP (Physet&r RELDMFPDYSYGWFNTSLILEGRKYLQWL macrocephaltis) TERLTERGVKFLLRKVESFEEVARGGADVII NCTGVWAGALQPDPLLQPGRGQHKVDAP WMKHFIITHDPERGIYKSPYIIPGIQAVTLGG IFQUGNWSETNN1QDHNTIWEGCCRLEPTL KDAKIVSECTGFRPVRPQVRLEREQLHVG SSNTEVIHNYGHGGYGLTIHWGCALEVAKI FGKILEERNLHRMPPSHL SEQ ID NO: 97 MAGTMRWVIGAGVIGLSTALCI HERYRSV D-amino-acid oxidase LKSLDVMVYADRFTPLTTTDVAAGLWQPYL (EC 1,4.3.3)SEPSNPQEAHWNQQTFNYLLSLIGSPNAAAttorney Docket No. 081906-1535050-256510PCNMGLAPVSGYNLFREAVPDPYWKDIVLGF (Uniport:RKLTLRELDMFPDYSYGWFNTSLILEGRKY A0AAA9SXG3) LQWLTERLTERGVKFFLWKVESFEEVVRE (Bos taunts) GADVIINCTGVWAGALQPDPLLQPGRGQII KVDAPWLKHFVITHDPERGIYKSPYIIPGLQ AITLGGIFQLGNWSETSNIPDHNTIWEGCC RLEPTLKDAKIIGEWSGFRPVRPQVRLERE QLRFGSSNTEViHNYGHGGFGLTIHWGCAQEVAKLFGKILEERNLLRIPPSHLStrategy 3: Utilization of Alpha-Hydroxy Acid Oxidases

[0330] The oxidation of amino acids may also be catalyzed by other types of oxidases. For example, alpha-hydroxy acids are structurally similar to amino acids. Alpha-hydroxy acid oxidases (AHAOs) are enzymes known for their ability to catalyze the oxidation of hydroxy acids into 2-oxo acids, utilizing molecular oxygen (O2) and producing hydrogen peroxide (H2O2) as a byproduct. Due to structural similarities between amino acids and alpha-hydroxy acids, AHAOs may also possess a previously unrecognized, or “cryptic,” activity toward L-amino acids, catalyzing their oxidation and thereby generating hydrogen peroxide. An exemplary reaction equation is illustrated below:Native Function:Hydroxy Acid OxidasesHydroxy Acid2-Oxo AcidCryptic Function:Hy-dr oxy AcidOxidasesIminoAcid

[0331] To identify a suitable candidate AHAO for further investigation and / or engineering, a screening of sequence similarity networks (SSNs) composed of 2,000 AHAO sequences was performed. The SSNs method is described In Atkinson eta / ., PLoS ONE 2009 Feb., 4(2), e4345, and Is incorporated herein by reference. TheAttorney Docket No. 081906-1535050-256510PCinitial screening result is shewn in Fig, 7. 20 enzymes (SEQ ID NOs: 35-54) with an average sequence identity of approximately 65% were selected as potentially useful candidates to be applied in Strategy 3. The amino acid sequences of these alpha¬ hydroxy acid oxidases are shown in Table 7.Table 7: Amino acid sequences of alpha-hydroxy acid oxidases.SEQ ID Amino Acid Sequence CommentsSEQ ID NO: 35 MEKSSCVRALNAVTNPSVYSIFAAVNLTVLV Hydroxyacid oxidase 2PIEEITGPEMPLVCLADFKAHAQKQLSKTS (Uniport: A0A0G2K713) WDFIEGEADDGITYSENIAAFKRIRLRPRYL (Rattus norvegicus) RDMSKVDTRTT1QGQEISAPICISPTAFHSIA WPDGEKSTARAAQEANICYVISSYASYSLE DIVAAAPEGFRWFQLYMKSDWDFNKQMV QRAEALGFKALVITIDTPVLGNRRRDKRNQ LNLEANILLKDLRALKEEKPTQSVPVSFPKA SFCWNDLSLLQSITRLPIILKGILTKEDAELA MKHNVQGIWSNHGGRQLDEVSASIDALR EWAAVKGKI EVYMDGGVRTGTDVLKALAL GARCIFLGRPILWGLACKGEDGVKEVLDILT AELHRCMTLSGCQSVAEISPDLIQFSRL SEQ ID NO: 36 MSESGPEMSLVCLTDFQAQAREHLSKSTR Hydroxyacid oxidase 2DFIEGGADDSVTRDDNIAAFKRIRLRPRYL (Uniport:RDVSEVDTRTTIQGEKVSTPIGIAPTGFHCL A0A2K5DG71) VWPDGEMSTARAAQAAGICYITSTVASCSL (Actus nancymaae) EDIVTAAPEGLRWFQUYVHPDRQLNKQLIH RVESLGFKALVITLDTPVCGNRRYDIQNQL RRNLTLKDLQSPKKGNSLPYFQMASISTSL CWNDLSWFQSITRLPIILKGILTREDAELAV KHNVQGIIVSNHGGRQLDEVLASJDALTEV VAAVKGKIEVYLDGGVRTGNDVLKALALGA KCJFLGRPILWGLACKGEHGVKEVLNILTKE FHTSMALTGCRSVAEINRHLIQFSRL SEQ ID NO 37 MPLACLTDFQAFARERLPKSTWDFIEGGA Hydroxyacid oxidase 2DDSFTRDDNI AAFKRI RLRPRYLKN WNVD (Uniport:TRTTIQGEEITAPICISPTGFHCLVWPDGEM A0A6P6GWR0) STARAAQAAGVCYITSTFATCALEDIAATAP (Puma concolof) RGLRWFQLYVHPDRQLSKQLVQRVESLGF KALWTVDVPKLGNRRHDIRNQLDLKLNLL PKDLRSPKERTSMPYFQMSPIDSSICWND LSWLQSITRLPIILKGILTKEDAELAVKHNVH GHVSNHGGRQLDDVLASIDALAEWAAVK GAMEVYLDGGIRTGNDVLKALALGAKCVF LGRPILWGLAYKGEDGIKELLNIIKNEFHTS MALTGCQSVADINQDMVQFSRL SEQ ID NO: 38 MRNPTNRKSLCCQHRlRLRPRYLKDVSHV FMN hydroxy acid DTRTTIQGAEISAPICIAPTGFHCLAWPDGE dehydrogenase MSTARAAQAAGICYITSTYASCTLEDWATA domain-containing PRGLRWFQLYVQLDRQLNKQLIQKVESLG protein FKALWTVDVPKLGNRRHDIRNQLDLKTNL (Uniport: A0A7J7SXU6) LLKDLRSPKERSSAPYLQMSPIDSSLCWD (Rhinotophus DLSWLQSVTQLPIILKGILTKEDAELAVKHK femmequinum)VHGIIVSNHGGRQLDGVAASiDALTEWAAVAttorney Docket No. 081906-1535050-256510PCKGKIEVYMDGGIRTGSDVtKALALGAKCVF LGRPVLWGLACKGERGVEEVLNILKDEFH TSMALTDGEI SEQ ID NO: 39 MSMVCLTbFEAGARERLSRS Hydroxyacid oxidase 2GEGFTRDDNIAAFKRLRLRPRFLKDVSQV (Uniport:DTRTTIQGEEISAPIGVSPTGFHCLAWPDG A0A7J8CQX0) EMSTARAAQAAGICYITSTYASCTLEDIVTA (Mofossus molossus) APRGFRWFQLYVQPDRQLNKQLIQRVESL GFRALVITVDVPKVGNRRHDIRNQLNLKTN LLLKDLRSPKERNAMPHLQMSPIDGSFCWE0LSWFQSLTQLPIILK6ILTKEDAELAVKH KVHGSIVSNHGGRQLDDVPASVDALPEVVA AVKGRLEVYLDGGVRTGNDVLKALALGAK CVFLGRPILWGLACKGEHGIGEVLNILKDE FHTSMALTGCRSVAEINQDLIQFSRL SEQ ID NO: 40 RIRFRPRMLRDVSTMDIRTKILGSEISFPVG HAOX2 oxidase IAPTGFHQLAWPDGEKSTARAARAMSTCYI (Uniport: A0A7L2A2Y7) ASTYSTCTLEEISAAAPGGLRWFQLYIHRN (Leiathrix lutea) REVSQQLVQRAEALGFRGLVLTADLPYTGK RRDDVRNSFRLPPHMKLKNLEGAFEVCK MSPCFRSLHWRVPHETKTTGLDPSVTWN DIYWLRSLTHLPHVKGILTREDAELAVRYGV QGHVSNHGGRQLDEGPATIDALVEVVEAV RGRVEVYVDGGIRKGSDVLKALALGAKCV FIGRPALWGLAYKGEEGLQDVLRILQDEFR LSMALAGCASVSEIGPHLVQLSKL SEQ ib NO: 41 MPLVCLTDFEAHAREQLSKS Hydroxyacid oxidase 2DDCLTRDDNIAAFKKIRLRPRYLKDVSKVD isoform X1 MRTTIQGEEISTPICIAPTGFHCLAWPDGE (Uniport: A0A8B8Y3U2) MSTARAAQAAGICYITSTYASCSLEDIVATA (Safeenopfora PGGLRWFQLYVHPNRQLNKQLIQKVESLG irMsculus) FKALVIWDAPKIGNRRHNITNQVNEMKNLL LKDLGSPEKGNLMPYLQMSPIDSSICWDD LSWFQSITRLPHLKGILTKEDAELAVKHNVH GIIVSNHGGRQLDEVPASIDALTEWAAVKG KIEVYLDGGIRTGNDVLKALALGAKCVFLG RPILWGLAYKGEHGVEEVLNILKNEFHTSM TLTDGNQLQCERQTRRTPMRPPLTPQF SEQ ID NO: 42 MFPTTSSSEPGFQQLPLNTTLPRDWKTLT Hydroxyacid oxidase 2SGPEIPEMPLVCLTDFQAYAQKHLSKSTWD (Uniport:YEGGADECFTRDDNITAFKRIRLRPRYLKD A0A8C0MFB5) VQEVDTRTTVQGEEITAPICISPTGFHCLV (Can / 's lupus familiaris) WPDGEMSTARAAQAAGICYITSTYASCALE DIVATAPRGLRWFQLYMQSDKQINKQLVQ KVESLGFKALViTVDVPKLGNRRQDIQNQL DLKMNLLLKDLRSTKERNPMPYFQMFPID ASFCWNDLSWLQSITRLPIILKGILTKEDAE LAVKHNVHGIIVSNHGGRQLDDVLASIDAL AEWAAVKGKMEVYLDGGIRTGNDVLKAL ALGAKCVFLGRPiLWGLAYKGEYGVEEVLN IIKNEFHTSMALTGCRSVAEINQDLIQFSRL SEQ ID NO: 43 MAMVCLSDFEAYAKKYLPKIAWDYFAAGA Hydroxyacid oxidase 2DDCHRDDNILAYKRIRFRPRVLRDVSVMD (Uniport:IRTKILGSEISFPVGIAPTGFHQLAWPDGEK A0A8C0UYF9)Attorney Docket No. 081906-1535050-256510PCSTARAARAMNTCYIASTYSTCTLEEISVAAP (Cyanistes caeruteus) GGLRWFQLYiHRNRAASQQLVRRAEALGF QGLVLTADLPYTGKRRDDVRNGFRLPPHM KVKNLERAFEGDDWSEYGLPPNSLDPSVT WNDlYWLRSLTRLPItiKGlLTREDAELAVRH GVQGIiVSNHGGRQLDEGPATIDALVEWE AVRGRVEVYVDGGIRKGSDVLKALALGAK CVFIGRPALWGLAYKAVPVSQRLANTWFS SQSCDQPPAMLTKQRQLKLRSQPE SEQ I D NO: 44 MTMVCLNDFEEYAKQHLSKATWDYYEAG Hydroxy acid oxidase 2ADDCCTRDDNLEAYKRiRLRPRILRDVSIN (Uniport:DTRTSVLGMEISFPVGIAPTGFHCLAWHEG A0A8C1GCG2) ELATARATETVNTCYIASTYSTCSVEEIAAA (Cyprimts c&rpia) APNGYRWFQLYtYRDRKUSEQtVSRVEAL GYKALVLTVDVPYTGKRRNDIRNQFKLPPH LMVKNFEGMFQEQAGSQEEYGIPANTLDP SISWKDVYWLQSLTRLPIOKGILTKEDAELAI EHGVQGIIVSNHGGRQLDGVPATIDCLPESV DAVQGRVEVYMDGGIRTGNDVLKAIALGA KCVFIGRPAIWGLAYKGEEGVREILQILHDE FRLSMALSGCRNVAEINRNLIQFSRL SEQ ID NO: 45 MTLSRRLGGPEMPLVCLADFKAHAQKQLS Hydroxyacid oxidase 2KTSWDFJEGEADDGITYSENIAAFKRiRLRP (Uniport:RYLRDMSKVDTRTTIQGQEISAPICISPTAF A0A8L2UQG3) HSIAWPDGEKSTARAAQEANICYVtSSYAS (Rattus norv&gicus) YSLEDIVAAAPEGFRWFQLYMKSDWDFNK QMVQRAEALGFKALVITIDTPVLGNRRRDK RNQLNLEANILLKDLRALKEVRKEKPTQSV PVSFPKASFCWNDLSLLQSITRLPIILKGILT KEDAELAMKHNVQGIWSNHGGRQLDEVS ASIDALREWAAVKGKIEVYMDGGVRTGTD VLKALALGARCIFLGRPILWGLACKGEDGV KEVLDILTAELHRCMTLSGCQSVAEISPDM QFSRL SEQ ID NO: 46 MNICNREGGRCAEMAMVCLTDFEEYAKEH FMN hydroxy acid LSKATWDYYAAGADECCTRDDNLLAYKRI dehydrogenase RLRPRILRDVSVSDTRTTVQGTEISFPVGiA domain-containing PTAFHCLAWHEGEVATARATEALNTCYITS protein TYSTCSVEEIVAAAPNGYRWFOLYVYRDR (Uniport: A0A9N7YYJ8) KLSEQIVHRVEALGYKALVLTVDVPYTGKR (Pleuranectes platessa) RNDIRNQFKLPPHLKQETAGPEDYGIPANT LDPSISWKDVYWLQSITRLPHIKGILTKEDA ELAVEHGVQGHVSNHGGRQLDGGPASIDA LSEIVDTVQGR1EVYVDGGJRTGSDVLKALA LGAKCVFIGRPAVWGLAYKGEEGVREVLQI LNDEFRL8MALSGCRNVAEINRNUQFAKLSEQ ID NO: 47 MAMVCASDFEDYAKKYLPK^ Hydroxyacid oxidase 2DDCSTRDENIQAYKRIRFRPRMERDVSMM (Uniport: A0A091TC22) DIRTKLLGTEISFPVGIAPTGFHQLAWPDG (PPaethon iepturus) EKSTARAAKAMNTCYIASTYSTCTLEEITAA APGGLRWFOLYI HRNRAVSQQLVQRAEAL GFQGLVLTADLPYTGKRRDDVRNGFRLPP HMKLKNLEGAFEVCKMSPLDPSVTWNDIYWLRSLTHLPHIKGILTKEDAELAVRHGVQGIIAttorney Docket No. 081906-1535050-256510PCVSNHGGRQLDGGPATIDALVEVVEAVQGR VEVYLDGGiRKGSDVLKALALGAKCVFIGR PALWGLAYKGEEGLQDVLRILQDEFRLSM ALAGCASVSEiGRHLVQFSKLSEQ ID NO: 48 MKNRRGGHSHGTQRGQTVILCTSVLSHNL Hydroxyacid oxidase 2HlGPRPLYNMSLGPSQHFEGSLSICSRPVL (Uniport: A0A803K455) GDAEMSLICLADFEAYAKEHLPKATWEYYA (X&napus tropscalis) AGADECCTRDDNLQAFRRIRLRPRMLRDV SVMDTKTTVLGEEISCPIGIAPTAFHCLAWP DGEMSTARAAEALKLLYVASTYATCSVEEIS EAAPEGLRWFQLYVYRDRKLSEQLIRRVE ALGFKALVLTVDVPYTGKRRTDIRNNFRLP PHLKVKNFEGVFEGHGGPDNYGVPLNTLD PSVSWKDICWLRSVTSLPIV1KGILTKEDAELAWYGVQGI IVSNHGGRQLDGELATIDAL AEIVEWQGRIEVYLDGGIRTGSDVLKAIAL GAKCVFLGRPIVWGLTYKGEEGVKG1LQIL TDEFRLSMALSGCRNVSEVNRNLIHVAKL SEQ ID NO: 49 MAMVCLSDFEWAKKYLPKTTWDFFAAGA H AOX2 oxidase DDGTTRDDNILAYKRIRFRPRVLRDASVMD (Uniport: A0A851TFS7) IRTKLLGTEISFPVGIAPTGFHQLACPDGEK {Nothocercus STARAAKAMNVCYIASTYSTSSLEEISAAAP Pigrocapiffus) SGFQWFQLYIHRNRAVSKQLVQQAEALGF QGLVLTADLPYTGKRRDDVRNGFQLPPHM SLKNLEGAFEVCKMMILDPSVTWNDIHWL QSLTHLPIilKGlLTKEDAELAVRHGAQGIIVS NHGGRQLDGGPATIDALVEWEAVQGRVE VYLDGG1RKGSDVLKALALGAKCVFIGRPA LWGLAYKGEEGLQDVLRILRDEFRLSMALT G SEQ ID NO: 50 MPLVCLTDFQAYAQEHLSKSTWDFIEGGA Hydroxyacid oxidase 2DECFTRDDNIAAFKKIRLRPRYLRDVREVD (Uniport: G1MH58) TRTTIQGEE1TVPIC1APTGFHCLVWPDGEM (Ailuropoda STARAAQAAGICYITSTYASCTLEDIVATAP m&lanoteuca) RGLKWFQLYVQSDRQLNKQVVQKAESLG FKALVtTVDTPKIGNRRCDFRNKLDLQMNL LLKDLRSPKERNSMPYFQMCPIDSSFCWN DLSWLQSITRLPIILKGILTKEDAELAVKHNV HGJIVSNHGGRQLDDVPASIDALTEWAAVK GKMEVYLDGGiRTGNDVLKALALGAKCVF LGRPILWGLAYKGEHGVEEVLNLIKNEFHT SMTLTGCRSVAEIHQDLVQFSRtSEQ ID NO: 51 MAMVCLTDFEEYAKEHtSKATWDYYAAGA Hydroxyacid oxidase 2DECCTRDDNLLAYKRIRLRPRILRDVSVSD (Uniport: H3DDZ4) TRTTIQGTEISFPVGIAPTAFHCLAWHEGE (Tetraodon nigroviridis) MATARATEALNTCYITSTYSTCSVEEIVAAA PNGYRWFQLYLYRDRKLSEQIVHRVEALG YKALVLTVDVPYTGKRRNDIRNQFKLPPHL KVKNFDGVFQEAAVTEEYGIPANTLDPSIS WKDVYWLQSITRLPI1IKGILTKEDAELAVEH GVQGIIVSNHGGRQLDGGPASIDALSEIVD TVQGRIEVYLDGGIRTGSDVLKSLALGAKC VFIGRPAVWGLAYKGEEGVREVEQILNDEFRLSMALSGCRNVAEINRNOQFSKFAttorney Docket No. 081906-1535050-256510PCSEQ ID NO: 52 MPLVCtKDFKAHARDRLSKSSWDFIEGVA Hydroxyacid oxidase 2DEGITRDENIAAFKKIRLRPRYLRDVSEVDT (Uniport: I3M3W0) RTTIQGEEISAPICISPTAFHGMFWPDGEVS Uctidomys TARAAOEAGICYITSTFASCTLEDIVASAPK tridecemlindatus) GLRWFQLYVQSDWQLNKQLIQRVESLGFK ALVITVDAPIVGKRREDIRNQLDLMKSLQLK NLRPPTERNSVPYIQSPPISTSFCWDNLS WIQSITQIPIILKGILTKEDAELAVEHNVRGII VSNHGGRQLDEVPASIDALTEVATAVKGKIE VYLDGGVRTGTDVLKALALGAKCIFLGRPIL WGIAYKGEDGIEEVLKILKNEFHTSMTL1G CRSVAEiNRDLIQFSRLSEQ ID NO: 53 MEITNVNEYEAIAKQKLPKMVYDYYASGAE Glycolate oxidase DQWTLAENRNAFSRILFRPRILIDVTNIDMT (Unipod: P05414) TTILGFKISMPIMIAPTAMQKMAHPEGEYAT (Sp / nac / a aleracea) ARAASAAGTIMTLSSWATSSVEEVASTGP GIRFFQLYVYKDRNWAQLVRRAERAGFKAlALTVDTPRLGRREADIKNRFVLPPFLTLKN FEGIDLGKMDKANDSGLSSYVAGQIDRSLS WKDVAWLQTITSLPIEVKGVITAEDARLAVQ HGAAGIIVSNHGARQLDYVPATIMALEEW KAAQGRIPVFLDGGVRRGTDVFKALALGA AGVFIGRPWFSLAAEGEAGVKKVLQMMR DEFELTMALSGCRSLKEISRSHIAADWDGP SSRAVARL SEQ ID NO; 54 MPLVCLADFKAHAQKQLSKTSWDF1EGEA 2~Hydroxyacid oxidase DDGITYSENIAAFKRIRLRPRYLRDMSKVD 2 TRTTIQGQEISAPICISPTAFHS1AWPDGEK (Uniport: Q07523) STARAAQEANICYVISSYASYSLEDIVAAAP (Rattos norvegicus} EGFRWFQLYMKSDWDFNKQMVQRAEAL GFKALVITIDTPVLGNRRRDKRNQLNLEANI LLKDLRALKEEKPTQSVPVSFPKASFCWN DLSLLQSITRLPIILKGILTKEDAELAMKHNV QGIVVSNHGGRQLDEVSASIDALREVVAAV KGKI EVYMDGGVRTGTDVLKALALGARCIF LGRPILWGLACKGEDGVKEVLDILTAELHRCMTLSGCQSVAEISPDUQFSRLExample 2: Enzyme Expression and Purification

[0332] The method of expressing and purifying the engineered enzyme is described in Yoshikuni et al., U. S. Patent Publication No. 2022 / 0348970 A1, the contents of each which are hereby incorporated herein by reference in their entireties.

[0333] In the initial stage, potential enzyme candidates, such as the racemases of interest, were expressed in BLR(DE3) cells to determine the feasibility of mass production. The expression level of each enzyme was evaluated by SDS- PAGE and measuring absorbance at 280 nm using calculated extinction coefficients.Attorney Docket No. 081906-1535050-256510PC

[0334] After initially screening, the selected enzymes and engineered enzymes were mass expressed using the BLR(DE3) cells. Overnight cultures of BLR(DE3) cells suspended in a 2 ml volume were transformed with a pet29b+ plasmid (encoding polypeptides of interest with a C-terminal His-tag) and grown in Terrific Broth with 50 pg / ml kanamycin. Cultures were diluted 1:1000 in 500 ml of Terrific Broth with 1 M MgSO^ 1% glucose and 50 pg / ml antibiotic and then grown at 37° C. for 24 hours. Cultures were pelleted down at 5,000 G for 10 minutes and resuspended in induction (TB broth, 1 mM gS( 1mM IPTG) for induction at 18“ C for 24 hours. At the end of induction, cells were centrifuged, the supernatant was removed and cells were resuspended in 40 ml lysis buffer (50 mM phosphate buffer pH 7.5, 50 M NaCI, 5 mM imidazole) and 1 mM phenylmethylsulphonyl fluoride. The cell lysate suspension was sonicated for 2 min and followed by centrifugation at 5,000 G. The supernatant was loaded onto a gravity flow column with 500 uL Cobalt beads and was washed with 15 ml of lysis buffer five times. Proteins were eluted with 1 ml of elution buffer (50 mM phosphate buffer pH 7.5, 50 mM NaCI, 200 mM imidazole). Protein concentrations were determined using a Synergy H1 spectrophotometer (Biotek) by measuring absorbance at 280 nm using calculated extinction coefficients.Example 3: Results of Enzyme Expression and Purification

[0335] The initial screening results of identifying leucine racemases were shown in Table 8 below.Table 8: A280 results of initial screening.Racemase A280 mg / mL SEQ ID NO: 31 (Uniport: M1GRN3) 1.2(5WYA)SEQ ID NO: 25 (Uniport: A0A0K2LEB0) n.d;SEQ ID NO: 26 (Uniport: A0A0R1SEL9) n.d.SEQ ID NO: 27 (Uniport: A0A0R1 YTN2) n.d.SEQ ID NO: 28 (Unipod: AOAORWAI) n.d.SEQ ID NO: 29 (Uniport: A0A2P4R6Q6) n.d.SEQ ID NO: 30 (Uniport: A0A5P0ZRE3) n.d.SEQ ID NO: 9 (Uniport: Q5JGG6) n.d.SEQ ID NO: 10 (Uniport: H3ZR39) n.d.

[0336] As shown in Table 8 above, SEQ ID NO: 31 (5WYA) exhibited satisfactory level of soluble expression with a A280 of 1.2 mg / mL. Thus, it wasAttorney Docket No. 081906-1535050-256510PCselected for further evaluation and engineering. Two mutants of SEQ ID NO: 31 (5WYA) were created: a I24V mutant (5WYA_ uf1, SEQ iD NO: 32) and a I24A mutant (5WYAJVIut2, SEQ ID NO: 33). The expression level of SEQ ID NO: 31 (5WY ), SEQ ID NO: 32 (5WYA„Mut1), and SEQ ID NO: 33 (5WYA_Mut2) were evaluated by SDS-PAGE and measuring absorbance at 280 nm using calculated extinction coefficients. Fig.3 shows the SDS-PAGE image showing the expression level of SEQ ID NO: 31 (5WYA), SEQ ID NO: 32 (5WYA JVIutl ), and SEQ ID NO: 33 (5WYA_Mut2). The image shown in Fig.3 confirmed that SEQ ID NO: 31 (5WYA), SEQ ID NO: 32 (5WYA„Mut1), and SEQ ID NO: 33 (5WYA_Mut2) all exhibited satisfactory expression level and were suitable for further investigation. Results of the A280 measurement, as demonstrated in Table 9, also confirmed that SEQ ID NOs: 31-33 showed satisfactory expression level.Table 9: A280 results of SEQ ID NOs; 31-33.Racemase A280 mg / mL SEQ ID NO: 31 (Uniport: M1 GRN3) i 1.2(5WYA)SEQ ID NO: 32 (5WYA„Mut1 ) 1.2SEQ ID NO: 33 (5WYA_Mut2) 1.2Example 4: Enzyme Activity (Strategy 2)

[0337] In Strategy 2, the L-amino acids contained in the sample are first converted into D-amino acids by a racemase. Thereafter, the D-amino acids are oxidized into imino acids by a D-amino acid oxidase, generating hydrogen peroxide (H2O2). In the examples, a D-amino acid oxidase (SEQ ID NO: 34) was purchased from Millipore Sigma (Sigma Aldrich). An exemplary reaction equation is illustrated below:Attorney Docket No. 081906-1535050-256510PCIminoD-AAAcid

[0338] The activity of the racemase was evaluated by measuring the production of hydrogen peroxide. Commercial test strips were used for detecting the presence of hydrogen peroxide.

[0339] To set up the substrate, 30 p of each amino acid was dissolved in 50 mM phosphate buffer pH 7.5, 50 mM NaCI. In a 96 well-plate, 100 pL of enzyme and 100 y L of dissolved substrate were combined. The reaction conditions were 30 pL racemase with 30 pL DAAO with 30 pL L-AA. The reaction was allowed to react for 30 minutes at 21°C. After the incubation period, 10 pL of each reaction was placed in an individual test strip for colorimetric analysis using the Bartovation peroxide strips.Detection of hydrogen peroxide:

[0340] Bartovation hydrogen peroxide strips (https ■ / / bartovation. com) were used to detect the presence of hydrogen peroxide. These strips were calibrated at the 0, 1, 3, 10, 50, and 100 ppm. The test strip turns blue If hydrogen peroxide is presented, and bluer color indicates higher concentration of hydrogen peroxide.Specificity of racemase 5WYA and its mutants toward each amino acid

[0341] After 30 minutes of reaction time, the racemases were tested for their activity and specificity against each of the 19 L-amino acids and glycine.

[0342] Figs. 4A-4E show the testing results of the wild type racemase 5WYA (SEQ ID NO: 31); Figs. 5A-5E show the testing results of the mutant 5WYA„Mut1 (SEQ ID NO: 32, 124V); and Figs. 6A-6E shows the testing results of the mutantAttorney Docket No. 081906-1535050-256510PC5WYA_jMut2 (SEQ ID NO: 33, I24A). The strips 1-20 shown in Figs.4A-4E, 5A-5E, 6A-6E represent the testing result of each of the 19 L-amino acids and glycine. The amino acid corresponds to each of strips 1-20 are listed in Table 10 below. The color of each strip indicates the hydrogen peroxide concentration of each sample. Table 11 below lists the hydrogen peroxide concentration of each sample (+++: 70+ ppm; ++: 20-70 ppm; +:1-20 ppm; not active).Table 10: Amino acid corresponds to each of strips 1-20.Strip number Amino acid Strip number Amino acid 1 Alanine (A) 11 Methion ine(M) 2 Cysteine (C) 12 Asparagine (N) 3 Aspartic acid (D) 13 Proline (P) 4 Glutamic acid (E) 14 Glutamine (Q) 5 Phenylalanine (F) 15 Arginine (R) 6 Glycine (G) 16 Serine (S) 7 Histidine (H) 17 Threonine (T) 8 Isoleucine (1) 18 Valine (V) 9 Lysine (K) 19 Tryptophan (W)10 Leucine (L) 20 tyrosine ( Y)Attorney Dodtet No 08190*6 1535050-256510PC Table 11: Specificity of racemases, as indicated by the HjOs concentration.J A C | D E F G H 5W YA [ ' I - +++ + - SWYA. Mut1 J + i - i - +++ + - SWYA Mut2 + - + + - i I i K i is t- M N p Q 5WYA:~ | +++ - - - 5WYA_Mut1 j +-»■<- j -> j +-H- +++5WY A p— _ _ _V w Y5WYA +++ - +5WYA Mut* i - i 0 < - i +++ - -5WYA Mut2 j - 1 - i - + - - 70+ ppm++: 29-70 ppm+:1-2S ppm• not active t aAttorney Docket No. 081906-1535050-256510PC(0343] As shown in Figs. 4A-4E, 5A-5E, 6A-6E and Table 11 above, 5WYA_Mut2 (SEQ ID NO: 33, I24A) exhibited better specificity towards leucine, as demonstrated by the higher hydrogen peroxide production when leucine was used as the substrate. In comparison, 5WYA (SEQ ID NO: 31) and 5WYA„Mut1 (SEQ ID NO: 32, I24V) did not exhibit similar specificity, as evidenced by their high reactivity toward^ phenylalanine, Isoleucine, leucine, methionine* and valine. The experimental data demonstrates that 5WYAJVM2 (SEQ ID NO: 33, I24A) is suitable to be applied in detecting and measuring concentration of specific amino acid (leucine).Specificity of other racemases toward each amino acid

[0344] Other racemases of interest were also expressed, purified, and tested for their activities using the same methods as described above. The reactivity of each racemase against each of the 19 L-amino acids and glycine, as indicated by the hydrogen peroxide concentration, were listed in Tables 12-16 (+++: 70+ ppm; ++: 20-70 ppm; +:1-20 ppm; -: not active).Attorney Dodtet No 08190*6 1535050-256510PC Table 12: Specificity of racemasea, as indicated by the HjOa concentration.A c D E F G H SEC ID NO' 4 - - + - (P 10724)SEQ ID NO: 5 - - - + - (P0A6B4)SEQ IO NO: 6 - - - + - (058403)SEQ ID NO: 7 - - + - (P9WPW9)SEQ ID NO: 8 - - - + - (P84556)SEQ IO NO; 9 - - + - (Q5JGG8)SEQ ED NO: 10 - - - + - (H3ZR39)L M N P Q SEQ IO NO; 4 + ■7- - - - - (P 10724)SEQ ID NO: 5 - - - - (P0A6B4)SEQ ID NO: 6 - + - - - (058403!SEQ IO NO: 7 * - - - - (P9WPW9)SEQ ID NO: 8 4- - - - - (P94556)SEQ ID NO: 9 - - - - - (Q5JGG6)SEQ ID NQ; 10 * + - - -. (H3ZR33);Attorney Dodiet No 081906- 1535050-236510PC § R I S T V w Y S£Q iD HO: 4 - + - (P 10724) SEQ ID NO:: 5 - • - (P0A6R4: | | SEO ID ND: 6 - + - {058403) § § SECHD NO: 7 - - - (P9WPW9) j j SECHD NO:: 8 - + + (P94556) I SEQ {D NO: 9 - ' - (G5JGG6J SECHD NO: 10 J - “ - (H3ZR39) | | +++: 7Q+ ppm +4; 20'70 ppm M-StS ppm■: not activeAttorney Dodtei No 08190*6 1535C50-256510PC Table 13; Specificity of racemasea, as indicated by the HjOa concentration.A C 0 E F G H SEQ ID NO il - - + - (Q4DA80)SEQ ID NO: 12 - - - - - + - (059791)SEQ ID NO: 13 - - + - {M4GGR9)SEO *D NO 14 - - - + - (A0ASX5MRS9)SEQ ID NO. 15 - - - - + - (AOAOFSALZSj} K L M N p Q SEO ID NO; 11 - - - +++ - (Q4OA80)SEQ ID NO; 12 - - - - - (059791)SEQ ID NO 13 -* -N - - - - (M4GGR9)SEQ ID NO' 14 * -v * - - - - (A0A9X5MRS9)SEQ ID NO 15 - - * + - - - (A0A9F6ALZ8;R s T ¥ w YSEQ ID NO: 11 * *(Q4DA80)SEQ ID NO: 12 - - - - *(059791 )SEQ ID NO: 13 - - - - - -(M4GGR9)Attorney Dodtei No 081 06-1535050-256510PC BEQ ID NO: 14 § - § + - - - (A0A9X5MRS9) j j SEQ ID NO: 15 w - - - - - (AOAOFSALZ8) j j [ +++: 70+ ppm ++: 20-70 ppm + 1-20 ppsti■: hot activeAttorney Dodtet No 08190*6 1535C50-256510PC Table 14: Specificity of racemases, as indicated by the HjOs concentration.E F G H SEQ ID NO.’ t? ++ n - + + - (D3L71tjSEQ ID NO: 16 - - ’ - - + - (Q04M87) J i i iaSEQ ID NO: IS ++ - - + - (Q9X1T3) J i iSEQTD N& 'W I - I - - + - (AOAOGCZVai)SEQ ED NO.20 - - + - (A0A7U4DMM4) iuSEO. *D NO 21 - - - - - + - (A0A7W8ISU9') i i iSEO ED NO: 22 " - - + - {AOA023CTQ6) | | |SEQ ID NO: 23 - “ “ - - + - (A0A063YVK8) j j iSEO ID NO: 24 - - " - - + - (A0A167U3S7) | | |SEO ID NO: 25 - - + - (AOAOK2LEBO.: | |SEO ED NO: 26 - - + - (A0A0R1SELS) j i iSEQ ID NO::27 - - - - - + - (AQA0R1YTN2) |SEO ED NO: 28 - - + - (ADA0R2AKA1) i J iSEQ ID NO. 29 - - + -(A0A2P4R6Q6) i i iAttorney Dodtei No 081906- 1535050-236510PC SEO ID NO: 30 • ■> - - - + - i'A0A5P0ZRE3j jj i j k j L M N p Q SEO. ID NO; 17. i - *+ ++ - - - (D3L711) | |SEO ID NO: 18 • + + + - - - (Q04HB7) | | |SEO ID NO: 18 - - - - - - - (Q9X1T3) j jSEQ ID NO: 19 - - - + - - - {AOAOQOZVOn | §SEO ID NO: 20 - " * - - - - (ABA7U4DMM4):SEQ ID NO: 21 i - “ * - - - - (A0A7W8ISL9) | | |SEQ ID NO: 22 - - ++ - - - - (AOA023CTQ6)SEQ ID NO: 23 - - * - - - - (A0A063YVK8)SEQ ID NO.24 - - + - - - - (ADA167U3S7) | j LSEO -D NO, 25 - - - - iAOAOK2LE30;SEO ID NO: 26 - ~ ' - - - - (A0AGR1SEL9)SEQ lD Nd-.27 ' ' ' - - - - (AOAOR1YTN2)SEO ID NO 28 - - - - (A0A0R2AKA1)SEQ ID NO: 29 - ' ' - - - -(ABA2P4R6Q6) J §Attorney Dodtei No 081906- 1535050-236510PCAttorney Oadtei No 081906- 1535050-236510PC SEO iD NO: 30 § - § ■> 1 - - - (A0A5P0ZRE3) § § § +<•+.70+ ppm ++: 2870 ppffl *: 1-30: ppmnot activeAttorney Dodret No 081906- 1535050-236510PCTable 15i Specificrty of racemases, as indicated by the H. j. concentration.A c D E F G H SEQ ID NO 55 + (A0A2N7AUX81)SEQ ID NO 56 - + (A0A1P8Q2648)SEQ ID NO. 57 + (AGA0R2NYU21;SEQ -D NO 58 + (A0A0R1V5L33O)SEQ ID NO: 59 +(A0A2S3U5J9)SEQ ID NO: 60 * +(A0A5P0ZK31)SEQ ID NO; 61 +■(A0A0R1K5T9)SEQ ID NO: 62 + (A0A5R9CV78jSEQ ID NO. 63 -* + 0AOA386PV56)SEQ ID NO 64 +(AQAG9FDC4)SEQ ID NO: 65 + (A0A837R8R14)SEO. ID NO 66 - - + (AOAOR1M960)SEQ ID NO, 67 +(A0A660E419)SEQ ID NO: 68 +CT > cog m I m S ra > g O o O i 57) C S a ® M ri2U2 s CTP o < o Q m s g b> w c o <»t<r 1 | i [IO ss ON QSS - - f 1 I S! 8 i <- -1rIQD SEO0 N: 6- (} § § IAPKZOA5O34 -1 zQEIDO SN::81 i i I - - () j j §KOR15AAOT9SEQ(DNO:62 +++H+ - -- ()A0A5R9C78VQ SED NO: 63 I IA K ) _ 1 (A0386PV58A i - - 1 -0 rQ£? ST5NOs4t I'''''''. ’...| ( | | §)A0GA9FDC4DEQ N: S. IO 65,- () § § |A0A7R8R8314Q SEDNO:66I " " - ()AQA0R1M86Q - - ' o QEID SNO::87 [: - - - |O8>958d0VVi> <9S; ONGD3SI£Q§gl _ I i iAA6E419SEQD: I NO 88 - § UW8XWZNZW:(n IAAORab iOtu [ j SgQN ai O3S: ' - _Attorney Dodtei No 081906- 1535050-236510PC (A0A2N7AUX8i) SEQ iD NO. 56 (A0A1P8Q2648) SEO ID NO 57 - (A0AQR2NYU21'i SEO (O NO: 58 - {AOAOR1V5L330) SEO ID NO: 59 (A0A2S3U5J9) SEQ ID NO: 80 - (A0A5P0ZK31) SEQ ID NO: 61 - (A0A0R1K5T3) SEQ ID NO 62 + {AGA5R9CV78} SEQ ID NO 83 - (A0A386PV56) SEQ ID NO: 64 - (A0AG9FDC4> SEQ ID NO. 55 - (A0A637R8R141 SEQ ID NO 66 - (ACAORW9SO} SEQ ID NO; 67 - (AOA660E419) SEQ ID NO: 58 - (A0AQR1QEU1) ++*■_ 70+ ppm ++: 20- 70 ppm +'i-2O ppm ■; net activeAttorney Dodret No 681906- 1535050-236510PCTable 16: Specificity of racemases, as tndicBted: by the H. j. concentration.A c D E F G H SEO ID NO; 69 - - + - (A0A2R3JU959)SEQ IO NO: 70 - - - + - (A0A1S6QHY01)SEO ID NO: 71 - - - - + - (A0A0R2LA549)SEO ID NO 72 r - - - + - (AOA4O1FJM8)SEO IO NO: 73 - - + - (AQA001A881)SEO ID NO. 74 * * - +++ + - (A0A0R1L1T708)SEO ID NO; 75 - - - + - (AONL? 1 •SEO ID NO: 7g - - ++ + - (A0A0A1GWW4)SEO ID NO. 77 * -* -* - - + - (A0AAU8MP)SEO ID NO; 78 - - - + - (A0A0RMSJ4)SEQ ID NO: 79 - - + - (A0AB37RKA0)SEO. ID NO 80 - - - - - + - (A0A0H4QKL5)SEQ ID NO 8! - - ++ + - (AOAO: Rt£JM2>I K L N pAttorney Dodtei No 081906- 1535050-236510PC SEQ iD NO: 69 § - § ■> 1 - - - - (A0A2R3JU959) j |SEQ ID NO: 70 ++ - - - (A0A1S6QHY01JSEQ ID NO:: 71 - - - - - - - (A0A0R2LA549)SEQ ED NO: 72 - - - - - - - (A0A401FJM8) | | |SEQ sD NO. 73 - - - - - - - $A0A0D^ A881 j L j jS& Q: D NQ, 74 j j - I ++* +++ - - - (A0AGR1LTT70B)SEQ ID NO: 75 - * ' - - - - (A0N1.71)SEQ ID NO: 76 ] ++ j - *+ ++ - - - (A0A0A1GWW4) |SEO ID NO: 77 - - " - - - - (A0AAU8MP)SEQ ID NO: 78 - - - - - - - (A0A0R1WSJ4) ISEO ID NO; 79 - - - - (A0A837RKA0): j jSEQ ID NO: 80 - ' 1 - - - - (AOAOH4QKE5) ISEQ ED NO; 81: +++ - -«-+■ +++ - - - (AOAOR1SJM21 j jR j S j T V w YSEQ ID NO; 6S - - ' - - - (A0A2R3JU959)SEO ED NO: 70 - ' * - - - (A0A1S8QHY01) j ISEQ ID NO: 71 - 1 - f - - -0£tSMpB jWJ:■ UifJfJ £E' p+ Oz-O?: ++ U!<id +Q7:*•;.+ (mt’siuovw) - - +++ ig ON di 035(SDiO^HOVQV) - - - - - 0g: ON 01035(bv>iaz€8wvj - - - - - - 6i ON 01 OSS (t’PSMl-HOVOVJ - - - - - - 8Z: ON d: 035(dfisnwctf) - - -: ON Qi O3S (hWiAOi-VOVOW - - 4-+ - - - 9Z: QN Qi OSS UZTMWi - - - - V - <1A 'ON 0: OSS (8021 Hiaovow - - ++ + - - - W:©N 01 O3S H88VIGOV0V) - - - - - - €2 ON Q; O3S iswrsUwQV) - - - - V. Zl -. ON QI 03S(swTz'Sovw)Od0i-59Sa-DSO5E9i MS! 80 I^WO AW< WAttorney Docket No. 081906-1535050-256510PCExample 5: Enzyme Activity (Strategy 3)

[0345] In Strategy 3, the L-amlno adds contained in the sample are oxidized into imino acids by an alpha-hydroxy acid oxidase, generating hydrogen peroxide (H2O2). An exemplary reaction equation is illustrated below:Native Function:OWaseaHydroxy Add 2-Oxo Acid Cryptic Function:Hydroxy AcirfOxidasesMHIminoL-AAAcid

[0346] The activity of the alpha-hydroxy acid oxidase was evaluated by measuring the production of hydrogen peroxide. Commercial test strips were used for detecting the presence of hydrogen peroxide.

[0347] To set up the substrate, 30 pM of each amino acid was dissolved in 50 mM phosphate buffer pH 7.5, 50 mM NaCI, In a 96 well-plate, 100 pL of enzyme and 100 pL of dissolved substrate were combined. The reaction conditions were 30 pL alpha-hydroxy acid oxidase with 30 pL L~AA. The reaction was allowed to react for 30 minutes at 21 °C. After the incubation period, 10 pL of each reaction was placed in an individual test strip for colorimetric analysis using the Bartovation peroxide strips.Detection of hydrogen peroxide:

[0348] Bartovation hydrogen peroxide strips (https: / / bartovation.com) were used to detect the presence of hydrogen peroxide. These strips were calibrated at the 0:1, 3, 10, 50, and 100 ppm. The test strip turns blue if hydrogen peroxide is presented, and bluer color indicates higher concentration of hydrogen peroxide.of acid oxidasesAttorney Docket No. 081906-1535050-256510PC0349J After 30 minutes of reaction time, the alpha-hydroxy acid oxidases were tested for their activity and specificity against each of the 19 L-amino acids and glycine. Table 17 below lists the testing results of each alpha-hydroxy acid oxidase (+++: 70+ ppm; ++: 20-70 ppm; +11-20 ppm: not active).AHorr.ey Dodtei No 081906- 1535050-236510PC Table 17: Specificity of alpha-hydroxy aci oxidase, as Indicated by the H2O2concentrationE F G H SEQ ID NO: 35 - - - - - - - (A0A0G2K713) nSEQ ID NO: 36 - - ’ - - - - (A0A2KSDG71) | | | jraSEO ID NO 37 - - - - (A0A6P6GWR0) | | |SEQ ED NO. 38 - - ++ - ■A0A73’SXU6)SEQ ED NO.39 - ] - - - - {AOA7J8CQXO5 ioSEQ ID NO: 40 - - - - - - - (A0A7L2A2Y71 | | |SEO ED NO: 41 - - - - - - - (A0A8B8Y3U2) | | |SEQ ID NO.42 - - “ - - - - (A0A8C0MFB5) j | |SEQ ID NO: 43 - - " - - - - (AOA9COUYF9)SEO ID NO: 44 - - ++ - (A0A8C1GCG2) | | |SEQ ED NO: 45 - - ' - - - - (A0A8L2UQG3) j jSEQ ID NO: 46 - 1 - - - - - - (A0A9N7YYJ8) ISEQ ED NO: 47 - " " - - - - ^0AB91TC22) | J |SEQ ID NO. 48 - - - - (A0A8O3K455) J j LSEO ID NO 49 J - | - J - - - -Attorney Dodtei No 081906- 1535050-236510PCAttorney Dodtei No 081906- 1535050-236510PC SEO ID NO: 45 ++ - ++ - - {A0A8L2UQG3)SEO ID NO: 46 - - - - - (A0A9N7YYJ8)SEQ ID NO 47 - - - - - - - (AQA091TC221SEO ID NO: 48 - - - - (A0A803K455)SEO ID NO. 49 - - - - - (A0A851TF6?)SEO ID NO 50 - - - - {G1MH58}SEO ID NO: 51 - - - - - - - (H3DDZ4)SEO ID NO: 52 - - - - - - - (I3M3W0)SEO ID NO 53 - - - - (P05414)SEO ID NO: 54 - — (007523):R: s T V w ¥SEO ID NO 3.5(A0A0G2K713;SEO ID NO: 36 - * * - - - (A0A2K5DG71)SEQ ID NO: 37 * * * - - - (A9A6P6GWR0)SEO ID NO 38 * - * - - - (A0A7J7SXU6)SEO ID NO: 39 - - - - - (A0A7J8CQX0;SEO ID NO: 40 - - -Attorney Docket No 081906- 1535050-236510PC (A0A7L2A2W d 1SEQ ID NO: 41 - (A0A888Y3U2) ( (SEO ID NO 42 - - - (AQA8CGMFB5) j jSEO ED NO: 43 - - - (AOA8GOUYF9) § §SEO ID NO: 44 - - - (A0A8C1GCG2) ( jSEO ID NO 45 - - - (A0A8L2UQG3) ISEO ID NO: 45 - " ++(A0A9N7YY38)SEQ ID NO: 47 I - - - (A0AQ91TC22) ISEQ Ip NO 48 - (A0A803K455) | |SEO ED NO: 49 - - - (A0A851TF67}SEQ ID NO. 50 ~ ' - (G1MH58) | ISEO ID NO 51 - (H3DDZ4jSEO ED NO; 52 - - - (E3M3W0)SEd lb NO: 53 - * - iP05414>SEQ ID NO; 54 - ' -(007623) ( (+++: 70+ ppm28-70 ppmAttorney Dodtei No 081906- 1535050-236510PC +:1 - 0 m-■ not activeAttorney Docket No. 081906-1535050-256510PCExample 6: Investigation of the Transducer for Detecting Hydrogen Peroxide Byproduct

[0350] FIGs. 13A-13B show the results of the investigation of the transducer for detecting hydrogen peroxide byproduct according to an embodiment of the present invention. FIG. 3 A shows the cyclic voltammetric responses of the transducer (Prussian blue-modified screen-printed carbon electrodes) recorded in PBS and PBS spiked with 1 M H2O2, within a potential range of -0.5 to 0.6 V vs Ag / AgCI at a scan rate of 50 mV / s. FIG. 13B shows the chronoamperometric responses measured in PBS spiked with 5 mM and 10 mM H2O2at an applied potential of -0.3 V vs Ag / AgCI for 60 seconds. As shown in FIGs. 13A-13B the detection responses of samples containing no H2O2were distinct from the detection responses of samples containing H2O2. The results indicated that the electrode design of the present invention is sensitive and capable of detecting trace amount of H2O2.Example 7: Preliminary Detecting Results of Leucine and Alanine

[0351] FIGs. 14A-14B show the preliminary detecting results of leucine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. FIGs, 1SA-15B show the preliminary detecting results of alanine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. The tested leucine concentrations ranged from 2 to 10 mM, while alanine concentrations ranged from 0.1 to 1 mM. Both applied a potential of -0.3 V vs Ag / AgCI for 60 seconds. FIG, 14A shows the chronoamperometric response to increasing leucine concentrations (from the lower curve to the upper curve, in sequential order: 2mM, 4mM, 6mM, 8mM, and 10 mM);FIG. 148 shows a data plot of the calibration curve for leucine detection (2mM, 4mMs6mM, 8mM, and 10 mM); FIG. 15A shows the chronoamperometric response to increasing alanine concentrations (from the lower curve to the upper curve, in sequential order: 0.1 mM, 0.2mM, 0,3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, and 1.0mM); and FIG. 15B shows a data plot of the calibration curve for alanine detectton (O. ImM, 0.2mM, 0.3mM, 0.4mMt0.5mM, O. BmM, 0.7mM, O. SmM, 0.9mM, and 1.0mM). As shown in the results, the exemplary electrochemical sensor exhibited a linear correlation of current / leucine concentration and a linear correlation of current / alanine concentration. The results indicated that the concept ofAttorney Docket No. 081906-1535050-256510PCdecentralized electrochemical sensing with the universal slope concept, enabling direct, calibration-free, real-time measurements, is workable with the electrode design of the present invention for detecting leucine and alanine.Example 8: Detecting Results of L-alanine Under Different Incubation Time

[0352] FIGs, 16A-16C show the detecting results of L-alanine in PBS (0.1 M, pH 7.4) under different incubation time according to an embodiment of the present invention. FIG. ISA shows a data plot of the calibration curve for L-alanine detection (0,2mM, 0.4mM, 0.6mM) with an Incubation time of 0.5 minutes; FIG, 168 shows a data plot of the calibration curve for L-alanine detection (0.2mM, 0.4mM, 0,6mM) with an incubation time of 1 minute; and FIG. 16C shows a data plot of the calibration curve for L-alanine detection (0.2mM, 0.4mM, 0.6mM) with an incubation time of 3 minutes. A potential of -0.3 V vs Ag / AgCI was applied for 60 seconds. As shown in the results, the exemplary electrochemical sensor exhibited a linear correlation of current / L-alanine concentration, with alanine racemase (SEQ ID NO: 4, P10724) and D-amino acid oxidase (SEQ ID NO: 34, P00371) being applied to the sensor. The exemplary electrochemical sensor is able to produce satisfactory results with an incubation time of as short as 1 minute or 0.5 minutes. The results indicated that the concept of applying a combination of racemase and D-amino acid oxidase to the working electrode is workable for practical applications, as the incubation time can be minimized.Example 9: Detection of L-alanine in PBS Using Enzymes

[0353] FIGs. 17A-17B show the detecting results of L-alanine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. FIG. 17A shows the chronoamperometric response to increasing L-alanine concentrations (from the lower curve to the upper curve, in sequential order; 0,1 mM, 0.2mM, 0,3mM, 0.4mM. O. SmM, 0.6mM, 0.7mM, 0.8mM, O. SmM, and 1.0mM); and FIG. 17B shows a data plot of the calibration curve for L-alanine detection (0.1 mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, O. SrnM, 0.7mM, 0.8mM, 0.9mM, and I. OmM). The incubation time was 1 minute. A potential of -0.3 V vs Ag / AgCI was applied for 60 seconds. As shown in the results, the exemplary electrochemical sensor exhibited a linear correlation of current / L-alanine concentration, with alanine racemase (SEQ ID NO: 4, P10724) andAttorney Docket No. 081906-1535050-256510PCD-amino add oxidase (SEQ ID NO: 34, P00371 ) being applied to the sensor. The results indicated that the concept of applying a combination of racemase and D- amino acid oxidase to the working electrode is workable for detecting one or more L~ amino acids of interest.Example 10: Detection of L-aianine in PBS in the Presence of Proteins

[0354] FIGs. 18A-18B show the detecting results of L-alanine in PBS (0.1 M, pH 7.4) with proteins (2 mg / mL albumin and 0.5 mg / mL y-gtobulin) according to an embodiment of the present invention. FIG. 18A shows the chronoamperometric response to increasing L-alanine concentrations (from the lower curve to the upper curve, in sequential order: 0.1 mM* 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, O mM, 0.8mM, 0.9mM, and I. OmM); and FIG. 18B shows a data plot of the calibration curve for L-alanine detection (0.1 mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.7mM, 0.8mM, O. SmM, and 1. OmM). A potential of -0.3 V vs Ag / AgCI was applied for 60 seconds. As shown in the results, the exemplary electrochemical sensor exhibited a linear correlation of current / L-alanine concentration, with alanine racemase (SEQ ID NO: 4, P10724) and D-amino acid oxidase (SEQ ID NO: 34, P00371) being applied to the sensor. The results indicated that the concept of applying a combination of racemase and D-amino acid oxidase to the working electrode is workable for detecting one or more L-amino acids of interest, in an environment similar to blood, plasma, or serum (a sample containing serum proteins).Example 11: Detection of L-alanine in PBS Using Enzymes

[0355] FIGs. 19A-19B show the detecting results of L-alanine in PBS (0.05 M, pH 7.4) and 0.05 NaCI according to an embodiment of the present invention. FIG.19A shows the chronoamperometric response to increasing L-alanine concentrations (from the lower curve to the upper curve, In sequential order: 1m, 2mM, 3mM, 4mM, 5m, 6mM, 7mM, 8mM, 9mM, and 10m ); and FIG. 19B shows a data plot of the calibration curve for L-alanine detection (1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8m, 9mM, and 10mM)- The incubation time was 3 minutes. A potential of - 0.3 V vs Ag / AgCI was applied for 60 seconds. As shown in the results, the exemplary electrochemical sensor exhibited a linear correlation of current / L-alanine concentration, with alanine racemase (SEQ ID NO: 4, P10724) and D-amino acidAttorney Docket No. 081906-1535050-256510PCoxidase (SEQ ID NO: 34, P00371) being applied to the sensor. The results Indicated that the concept of applying a combination of racemase and D-amino acid oxidase to the working electrode is workable for detecting one or more L-amino acids of interest at a relatively higher concentration.Example 12: Detection of L-aianine in PBS Using Enzymes

[0356] FIGs. 20A-20B show the detecting results of L-alanine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. FIG, 20A shows the chronoamperometric response to increasing L-alanine concentrations (from the lower curve to the upper curve, in sequential order: 1 M, 2mM, 3mM, 4mM, 5 M, 6mM, 7mM, 8mM, 9mM, and 10mM): and FIG, 20B shows a data plot of the calibration curve for L-alanine detection (1 M, 2m, 3 M, 4mM, 5 M, 6mM, 7mM, 8mM, 9mM, and lOmM). The incubation time was 3 minutes. A potential of -0.3 V vs Ag / AgCI was applied for 60 seconds.

[0357] FIGs. 21 A-21 B show the detecting results of L-alanine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. FIG. 21 shows the chronoamperometric response to increasing L-alanine concentrations (from the lower curve to the upper curve, in sequential order: 0.1 mM, 0.2mM, 0.3mM, 0.4mM„ 0.5mM, O. Sm, 0.7m, 0.8mM, 0.9m, and 1.0mM); and FIG. 21B shows a data plot of the calibration curve for L-alanine detection (0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5m, 0.6mM, 0.7mM, 0.8mM, 0.9mM, and I. OmM). The incubation time was 3 minutes. A potential of -0.3 V vs Ag / AgCI was applied for 60 seconds.

[0353] As shown in the results, the exemplary electrochemical sensor exhibited a linear correlation of current / L-alanine concentration, with alanine racemase (SEQ ID NO: 4, P10724) and D-amino acid oxidase (SEO ID NO: 34, P00371) being applied to the sensor. The linear correlation of current / L-alanine concentration was shown in lower L-alanine concentration range 0.1-1. QmM) and in higher L-alanine concentration range (1-1 OmM). The results indicated that the concept of applying a combination of racemase and D-amino acid oxidase to the working electrode is workable for detecting one or more L-amino acids of interest (such as L-alanine) at either a relatively higher concentration or a relatively lower concentration.Attorney Docket No. 081906-1535050-256510PCExample 13: L-alanine sensors: Selectivity analysis

[0359] FIG. 22 shows the relative current responses upon the addition of various potential interferents according to an embodiment of the present invention. Specifically, 0.4 mM L-alanine (L~Ala), 0.1 mM ascorbic acid (AA), 0.1 mM acetaminophen (ACP), 10 mM glucose (Glu), 3 mM lactate (Lac), 0.2 mM uric acid (UA), and 10 mM L-leucine (L-Leu) were each added to a testing sample to evaluate the selectivity of an exemplary sensor (a combination of racemase (SEQ ID NO: 4, PI 0724) and D-amino acid oxidase (SEQ ID NO: 34, P00371) applied to the working electrode) of the present invention. As shown in FIG. 22, the exemplary sensor exhibited very high sensitivity to the presence of L-alanine (~100% detection ratio). In comparison, the exemplary sensor exhibited very low sensitivity to the presence of other amino acids or small molecules, including ascorbic acid, acetaminophen, glucose, lactate, uric acid, and L-leucine. The results indicate that the sensor of the present invention, by applying a combination of racemase and D-amino acid oxidase to the working electrode, is able to achieve a very high sensitivity and selectivity to the one or more amino acids of interest.Example 14: L-leucine sensors: Detection of L-leucine in PBS using Enzymes

[0360] FIGs. 23A-23B show the detecting results of L-leucine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. Specifically, leucine racemase / lsoleiicine 2-epimerase (SEQ ID NO: 33, 5WYA_Mut2) and D-amino acid oxidase (SEO ID NO: 34, P00371 ) were applied to the working electrode in a 1:1 ratio. FIG. 23A shows the chronoamperometric response to increasing L-leucine concentrations (from the lower curve to the upper curve, in sequential order: 0.1 mM.0.2mM, 0.3mM, 0.4mM, and 0.5mM); and FIG. 238 shows a data plot of the calibration curve for L-leucine detection (0.1 mM, 0.2mM, 0.3mM, 0.4mM, and 0.5m ). The incubation time was 1 minute. A potential of -0.3 V vs Ag / AgCI was applied for 60 seconds. As shown in the results, the exemplary electrochemical sensor exhibited a linear correlation of current / L-leucine concentration. The results indicated that the concept of applying a combination of racemase and D-amino acid oxidase to the Working electrode is workable for detecting one or more L-amino acids ef interest, such as detecting the presence and concentration of L-leucine.Attorney Docket No. 081906-1535050-256510PCExample 15: L-leucine sensors: Different RacemasetOxidase Ratios

[0361] Further experiments were conducted to evaluate the effect of incorporating different racemase: D-amino acid oxidase (OAAO) ratios to the sensor.

[0362] FIGs. 23A 23B show the detecting results of L-leucine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. Specifically, leucine racemase / lsoleucine 2-epimerase (SEQ ID NO: 33, 5WYAJVIut2) and D-amlno acid oxidase (SEQ ID NO: 34, P00371) were applied to the working electrode in a 1:1 ratio. FIG. 23A shows the chronoamperometric response to increasing L-leucine concentrations (from the lower curve to the upper curve, in sequential order: 0.1 mM, 0.2mM, 0.3mM, 0.4mM, and 0.5mM); and FIG. 23B shows a data plot of the calibration curve for L-leucine detection (0.1 mM. 0.2mM,0.4mM, and 0,5m ). The incubation time was 1 minute. A potential of -0.3 V vs Ag / AgCI was applied for 60 seconds.

[0363] FIGs. 24A-24B show the detecting results of L-leucine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. Specifically, leucine racemase / lsoleucine 2-epimerase (SEQ ID NO: 33, 5WYA_Mut2) and D-amino acid oxidase (SEQ ID NO: 34, P00371) were applied to the working electrode in a 1:5 ratio.. FIG.24A shows the chronoamperometric response to increasing L-leucine concentrations (from the lower curve to the upper curve, in sequential order: 0.1mM.0 2mM, 0.3mM, S.4mM, O. SmM, O.e M, 0.7mM, O. SmlVI, 0.9m M, and 1.0mM); and FIG. 24B shows a data plot of the calibration curve for L-leucine detection (0.1 mM.0.2ml, 0.3mM, 0.4ml, 0.5mM, O. SmlVI, 0.7mM, 0.8mM, 0.9mM, and I. OmM). The incubation time was 1 minute. A potential of -0.3 V vs Ag / AgCI was applied for 60 seconds.[G364] FIGs. 25A-25B show the detecting results of L-leucine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. Specifically, leucine racemase / lsoleucine 2-epimerase (SEQ ID NO: 33, 5WYA_Mut2) and D-amino acid oxidase (SEQ ID NO: 34, P00371) were applied to the working electrode in a 1:10 ratio. FIG. 25A shows the chronoamperometric response to increasing L-leucine concentrations (from the lower curve to the upper curve, in sequential order: 0.1mM, 0.2ml, 0,3mM, 0.4mlM, 0.5mM, 0.6mM, 0.7mM, O. SmlVI, 0.9mh / l, and 1.0mM); and FIG. 25B shows a data plot of the calibration curve for L-leucine detection (0.1 M, 0.2mM, 0.3mM, 0.4mM, O. SmlVI, O. SmlVI, 0.7mM, O. SmlVI, 0.9mM, and 1.0mM). TheAttorney Docket No. 081906-1535050-256510PCincubation time was 1 minute. A potential of -0.3 V vs Ag / AgCI was applied far 60 seconds.

[0365] As shown in the results, the exemplary electrochemical sensor exhibited a linear correlation of current / L-leucine concentration when the racemase: DAAO ratio was 1:1, 1:5, and 1:10. In particular, when theracemase: DAAO ratio was 1:10, the exemplary electrochemical sensor exhibited an even better linear correlation of current / L~leucine concentration. The results indicated that the concept of applying a combination of racemase and D-amino acid oxidase to the working electrode is workable throughout the racemase: DAA0 ratio of 1:1 to 1:10; and the performance of the sensor is even better when the racemase: DAAO ratio is about 1:10.Example 16: L-leucme sensors: High RacemaserOxidase Ratio and Different L- leucine Concentration Ranges

[0366] Further experiments were conducted to evaluate the performance of a sensor with high racemase: DAAO ratio under different L-leucine concentration ranges.

[0367] FIGs. 26A-26B show the detecting results of L-leucine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. Specifically, leucine racemase / isoieucine 2-epimerase (SEQ ID NO: 33, 5WYA__Mut2) and D-amino acid oxidase (SEQ ID NO: 34, P00371 ) were applied to the working electrode in a 1:10 ratio. FIG. 26A shows the chronoamperometric response to increasing L-leucine concentrations (from the lower curve to the upper curve, in sequential order: 0.1 m, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, and 1.0mM); and FIG. 26B shows a data plot of the calibration curve for L-leucine detection (0.1 mM, 0.2m, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, Q.8mM, 0.9m, and I. OmM). The incubation time was 1 minute. A potential of -0.3 V vs Ag / AgCI was applied for 60 seconds.

[0368] FIGs. 27A-27B show the detecting results of L-leucine in PBS (0.1 M, pH 7.4) according to an embodiment of the present invention. Specifically, leucine racemase / isoieucine 2-epimerase (SEQ ID NO: 33, 5WYA_Mut2) and D-amino acid oxidase (SEQ ID NO: 34, P00371) were applied to the working electrode in a 1:10 ratio. FIG. 27A shows the chronoamperometric response to increasing L-leucineAttorney Docket No. 081906-1535050-256510PCconcentrations (from the tower curve to the upper curve, in sequential order: 1mM, 2mM, 3m, 4mM, 5mM, Sm, 7mM, 8mM, 9m, and 10mM); and AG.27B shows a data plot of the calibration curve for L-leucine detection (ImM, 2mM, 3mM, 4mM, 5mlM, 6 M, 7mM, 8mM, 9mM, and WmM). The incubation time was 1 minute. A potential of -0.3 V vs Ag / AgCI was applied for 60 seconds.

[0369] As shown in the results, the exemplary electrochemical sensor exhibited a linear correlation of current / L-leucine concentration, with leucine racemase / lsoleucine 2-epimerase (SEQ ID NO: 33, 5WYA_Mut2) and D-amino acid oxidase (SEQ ID NO: 34, P00371) being applied to the sensor in a ratio of 1:10. The linear correlation of current / L-leucine concentration was shown in lower L-leucine concentration range (0,1-1. OmM) and in higher L-leucine concentration range (1- 10mM). The results indicated that the concept of applying a combination of racemase and D-amino acid oxidase to the working electrode is workable for detecting one or more L-amino acids of interest (such as L-leucine) at either a relatively higher concentration or a relatively tower concentration.Example 17: Evaluation of Reproducibility

[0370] The reproducibility of the sensor was also evaluated. Specifically, a sample comprising 0.4 m L-alanine and PBS (0.1 M, pH 7.4) was subjected to 6 L- alanine sensors (alanine racemase (SEQ ID NO: 4, P 10724) and D-amino acid oxidase (SEQ ID NO: 34, P00371) being applied to the sensors), and their respective current responses were recorded. FIG, 28 shows the current responses of these 6 L-alanine sensors. As shown in FIG.28, the relative standard deviation (RSD) among the current responses of these 6 L-alanine sensors was 7.64%. The results indicated that L-alanine sensors of the present invention exhibited good reproducibility.Example 18: Detection of L-alanine in Capillary Blood Using Enzymes

[0371] FIGs. 29A-29B show the detecting results of L-alanine in capillary blood according to an embodiment of the present invention. FIG.29A shows the chronoamperometric responses to a capillary blood sample and a capillary blood sample comprising ImM L-alanine; and FIG.29B shows the current responses of aAttorney Docket No. 081906-1535050-256510PCcapillary blood sample in comparison to a capillary blood sample comprising 1mM L-alanine.

[0372] As shown in the results, the exemplary electrochemical sensor exhibited a noticeable change in the chronoamperometric / current responses when L- alanine was added to the capillary blood sample. The results indicated that the concept of applying a combination of racemase and D-amino acid oxidase to the working electrode is workable for detecting one or more L-amino acids of interest contained in a liquid sample, such as for detecting one or more L-amino acids of interest contained in a capillary blood sample.Example 19: Leucine: Monitoring and Optimizing Leucine Plasma Levels for Muscle Protein Synthesis and Clinical Applications

[0373] Based on the present disclosure, real-time monitoring of leucine could be performed using test strips produced for detecting and measuring plasma leucine concentrations. Leucine in the plasma typically ranges from 100-200 pM, but can increase 10-fold with feeding.

[0374] The technologies provided by the present disclosure enables users to use such test strips to measure leucine levels and where appropriate adjust dietary leucine intake to elevate plasma concentrations, thereby enhancing anabolic signaling and muscle protein synthesis. By providing actionable feedback, such test strips could serve as a tool for optimizing muscle protein synthesis, protein growth and recovery. See, e.g., Katsanos et al., February 28, 2006, A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly, Am J Physiol Endocrinol Metab 291: E381-E387.

[0375] In addition to fitness applications, the test strips could help address clinical conditions linked to altered leucine levels, including prolonged starvation, low- protein diets, liver cirrhosis, urea cycle disorders, skin diseases, and chronic renal failure. By facilitating precise metabolic monitoring, this technology can support both therapeutic intervention and dietary management strategies.Example 20: Alanine: Monitoring and Modulating Alanine Plasma Levels for Muscle Catabolism and Clinical ApplicationsAttorney Docket No. 081906-1535050-256510PC

[0376] Alanine serves as a biomarker for muscle catabolism and gluconeogenesis. Typical plasma concentrations of alanine range from 200 to 500 pM. Elevated alanine ievels exceeding 500 pI may indicate an increase in muscle protein breakdown. The blood meter technology that can be produced based on the present disclosure provides devices which enables users to monitor alanine levels in real time, providing an opportunity for targeted interventions to mitigate muscle catabolism.

[0377] Such interventions could include adjusting exercise intensity, optimizing dietary intake to restore the balance between catabolic and anabolic processes, or administering medical treatments in cases of significant muscle degradation. In addition to its role in muscle preservation, the technologies of the present disclosure could be applied in clinical contexts, such as the management of conditions associated with muscle wasting, including malnutrition, sarcopenia, and muscle degeneration in chronic diseases like cancer cachexia and severe burn injuries. By facilitating timely and precise adjustments to lifestyle or medical strategies, such devices can support enhanced recovery and overall muscle health.Example 21: Developed Biosensor Technology for the Detection of Target Amino Acids

[0378] FIG. 30A is a schematic illustration of the developed biosensor strip featuring a three-electrode system, comprising a reference electrode (RE), counter electrode (GE), and working electrode (WE) integrated onto a single substrate. An insulating layer is applied to define the sensing area and isolate the connection tracks.FIG. 30B is a schematic illustration of the key component structure of working electrode surface in the developed biosensor strips. FIG. 30C is a conceptual representation of the electrochemical reaction principle underlying the biosensing mechanism in strip-based platforms. Amino acid detection is facilitated by a coupled reaction mechanism involving the bioengineered enzyme reagent layer confined to the working electrode transducer along with the Prussian blue (PB) redox mediator. The hydrogen peroxide (H2O2) product of the enzyme biocatalytic reaction is detected amperometrically at a low potential by the PB-based mediated reaction. FIG. 30D is a schematic illustration of the optimized configuration layers of the biosensor. FIG. 30E is a linear response profile of the biosensor for L-alanine within the physiological concentration range of 0.01-1 m. following a 20-second incubation time and a totalAttorney Docket No. 081906-1535050-256510PCanalysis time of 80 seconds, FIG. 30F is the selectivity assessment of the biosensor in the presence of common electroactive species found in human blood. Relative current responses upon the addition of potential interferences 0.4 mM L-alanine, 0.1 mM ascorbic acid (AA), 0.1 mM acetaminophen (ACP), 10 mM glucose (Glu), 3 mM lactate (Lac), 0.2 mM uric acid (UA), and 10 mM L-leucine. FIG. 30G shows the reproducibility of the biosensor. FIGs. 30H-K are corresponding evaluations (layer configuration, linearity, selectivity, and reproducibility) for L-leucine biosensor.[03791 The biosensor technology of the present invention utilizes uniquely engineered enzymes for high selectivity toward each target analyte and optimized biocompatible reagent layers. As shown in the results, the biosensor of the present invention exhibited sensitive detection across the physiological range in human biofluids, high selectivity with insignificant interference from electroactive species presenting in blood, reproducibility suitable for scalable mass production, and rapid analysis enabling real-time, on-site monitoring at low cost per unitExample 22: Investigation of Biosensor Performance for L-alanine Detection in Human Capillary Blood Samples

[0380] FIGs. 31A-31B show the chronoamperometric responses (left) and corresponding current signal profiles (right) for L-alanine level changes in a healthy subject (Subject 1) following protein drink consumption over a 2-hour period. FIGs.3 C-31E show the chronoamperometric responses (left) and corresponding current signal profile (right) for L-alanine level changes in a healthy subject (subject 2) following protein drink consumption over 2-hour (FIG, 31 C), 3-hour (FIG. 31 D), and 4- hour (FIG.31 E) periods, respectively. All measurements were performed using 10 pL of capillary blood diluted with 10 pl of PBS solution (0.1M, pH 7.4) and recorded every 30 minutes using the developed L-alanine biosensors at an applied potential of -0.3 V vs Ag / AgCl. Each detection involved a 20-second incubation and a 60-second measurement, resulting in a total analysis time of 80-second per detection.

[0381] As shown in the results, the exemplary electrochemical sensor was capable of detecting changes of L-alanine level in capillary blood following protein drink consumption. The results indicated that the exemplary electrochemical sensor of the present invention is capable of being applied to detect and / or monitor the blood L-alanine level of a subject.Attorney Docket No. 081906-1535050-256510PCExample 23: Investigation of Biosensor Performance for L-leucine Detection in Human Blood Samples

[0382] FIG. 32A shows the chronoamperometric response of the L-leucine biosensor in capillary blood spiked with different L-leucine concentrations ranging from 0 to 2 mlM, with a 60-second incubation time. FIG.32 B shows the chronoamperometric responses in capillary blood spiked with L-leucine concentrations ranging from 0 to 0.4 mM, using 20-second (dash lines) and 60-second (solid lines) incubation times. All measurements were conducted using 10 pL of capillary blood diluted with 10 pL of PBS solution (0.1 I 1, pH 7.4), recorded at an applied potential of -0.3 V vs Ag / AgCl.

[0383] As shown in the results, the exemplary electrochemical sensor exhibited a noticeable change in the chronoamperometric response when L-leucine was added to the capillary blood sample. The results indicated that the concept of applying a combination of racemase and D-amino acid oxidase to the working electrode is workable for detecting one or more L-amino acids of interest contained in a liquid sample, such as for detecting the L-leucine contained in a capillary blood sample.Example 24: Preireatment and Activation Procedures for the L-leucine Biosensor Strips

[0384] Before modification, the working electrode was electrochemically activated. First, 100 pL of 1M NaOH solution was applied, and chronoamperometry was performed at 0.2 V vs Ag / AgCl for 30 minutes. This was followed by treatment With 100 pL of I HCI solution under chronoamperometry at 0.2 V vs Ag / AgCl for 30 minutes. This activation process was conducted using an external reference and counter electrode. The electrode surface was then rinsed with DI water and air-dried at room temperature. By applying the pretreatment and / or the activation procedures, the sensitivity of the working electrode may be further enhanced.Example 25: Surface Chlorination of the Reference Electrode

[0385] The reference electrode was chlorinated using 30 pL of 10 m AuCIs solution for 30 minutes, followed by rinse with DI water and drying at room temperature. By applying the chlorination process, the silver chloride (AgCI) layer on the screen-printed silver (Ag) or Ag / AgCl reference electrode may be further stabilized.Attorney Docket No. 081906-1535050-256510PCExample 26: Screening of Putative D-Amlno Acid Oxidase Candidates £0386] Screening of several promising D-amino acid oxidase candidates was conducted. The reactivity of each D-amino acid oxidase against each of the 19 D- amino acids and glycine, as indicated by the hydrogen peroxide concentration, were listed in Table 18 (++: > 20 ppm; +:1-2O ppm;not active). A hydrogen peroxide reading was taken using the Bartovation hydrogen peroxide test strips. As shown below, D-amino acid oxidases with SEQ ID NOs: 89, 94, and 97 showed activity against D-leucine but were inactive against D-methionine. This unique property makes these D-amino acid oxidases especialiy suitable for designing selective sensors that are sensitive to leucine but not methionine.Attorney Dodtei No 081906- 1535050-236510PC Table 18: Specificity of O-amina acici oxidase, as indicated by the H2O2concentrationA C E F G H SEQ ID NO: 34 4"fr ++ ++ + ++ (P0Q371)SEQ ID NO: 82 4'+ 4,t - - ++ + ++ < P80324iSEQ ID NO: 83 +4- +4- - ++ ++ + ++ {P 14920}.SEQ ID NO 34 4-4 4-4 ++ ++ + ++ (A0A8C3YS&8)SEQ sD NO. 85 ■4+ - ++ ++ + ++ {A0A4W2F4L01SEQ ID NO: 86 +4- 4-4- - ++ ++ + ++ (A0A6S3DYS?)SEO ED NO: 87 4- + - - ++ ++ + ++ (A0A6P6K364)SEQ ID NO: 88 - - - - - - - (A0ASV0ZP681SEQ ID NO; 89 - ++ - + ++ (A0A8C0URM1}SEQ ID NO: 90 - - - - - - (ApA2i3M6S7)SEQ ED NO: 91 - - - - (A0A6. J0IAK5)SEQ ID NO 92 - - - - (A0A669PGG2)SEQ ED NO: 93 - - - - (APA6P8K3MDSEQ ID NO: 94 X ++ - + ++ (A0A384DFU1)SEQ ID NO: 95.. - - - - -Attorney Dodtei No 081906- 1535050-236510PCAttorney Dodtei No 081906- 1535050-236510PC SEQ ID NO: 94 § • § ++ *+ - ++ ++ ++ (A0A384DFU1) § jSEQ ID NO: 95 - - - - - - (U3K214}SEQ ID NO;: 96 - - - - - - - (A0A2Y9FBA0) JSEQ ID NO. 97 - ++ -F+ - ++ ++ ++ (A0AAA9SXG3) |J R j S T V w YSEQ ID NC>: 34 *+ *+ tH- ++ ++ ++(PO0371) §SEQ ID NO: 82 ++ ++ f-4- ++ ++ ++(P80324)SEQ ID NO: 83 ++ *+ 4.4. ++ ++ ++Ip 14920) | |SEQ ip NO 84 ++ ++ 44* ++ ++ ++ (A0A8C3YS98) §SEQ ID NO: 85 ++ ++ + + ++ ++ ++ {A0A4W2F4L0)SEQ ID NO: 86 J ++ J ++ 4+ ++ - ++ (A0A663DYS7; j |SEQ ID NO: 87 j j ’++ ++ - ++ (A0A6P6K364)SEQ ID NO: 88 * - - - (A0A8V0ZF68}SEQ ID NO 89 ++ ++ * ++ - ++ (A0A8C0URMF> |SEQ ID NO 90 ' ' * - - - (A0A213M6S7)SEQ ID NO: 91 - - - - (A0A630IAK5) J jSEQ ID NO: 32 1 - 1 - - -AftOrney Oodtei No 081906- 1535050-236510PC (A0A669PGG2) 1 1 SEO; D NO: 93 - - - (A0A6P6K3M1) § j | SEO ID NG:: 94 *+ ♦+ + ++ - - (A0A384PFU1) J j § SEO SD NO: 95 - - - - - - (U3K214) § § SEQ SO NO: 96 - - - (A0A2Y9FBA0) j j SEQ! D NO; 97 *+ *+ *+ ++ - - (A0AAA9SXG3) | | § ++: 20* ppm +'1-30 ppm-.: not sctiveAttorney Docket No. 081906-1535050-256510PCExample 27: Blood tests using 2-mm Diameter Working Electrode

[0387] On-body tests using whole blood samples (20 pL for 20 s) following intake of whey protein (60g) were conducted. The biosensing strips were manufactured according to typical commercial strip standards (2 mm diameter working electrode and 20pL sample volume). The configuration of the L-alanine and L-leucine sensors are shown in FIG. 30D and FIG. 30H. Leucine racemase (SEQ ID NO: 33), alanine racemase (SEQ ID NO: 4), and D-amino acid oxidase (SEQ ID NO: 34) were applied in the sensors. FIG. 34A shows the current (I) to time (min) of L-alanine and L-leucine detection; FIG. 34B shows the current change (Al) to time (min) of L-alanine and L-leucine detection. FIGs. 34A-34B show that the sensitivity of the leucine sensor may be improved even without diluting the leucine racemase or treating it with enzyme stabilizer. FIGs. 34C-34D show detecting results of L-alanine and L-leucine. In FIGs.34C-34D, the enzyme layer of the sensors were dried for 24-48 hours before applying the next layer, and that the fully modified sensors were set for at least 1-2 days before use. FIGs. 42A-42B show the detecting results of L-alanine and L-leucine in whole blood samples following intake of whey protein (60g), detected by sensors covered with an additional layer to facilitate filling of the sample {the configuration is further discussed in Example 32).Example 28: Evaluation of. Storage life of L-Alanlne and L-Leucine Sensors when Storing in a Refrigerator (about 1.5~5°C).

[0388] The stability of the sensors under refrigerated conditions was further tested. The biosensing strips were manufactured according to typical commercial strip standards (2 mm diameter working electrode and 20pL sample volume), The configuration of the L-alanine and L-leucine sensors are shown in FIG. 30D and FIG.30H. L-alanine and L-leucine sensors were stored in a refrigerator (about 1.5-5’C) for 0-16 weeks and their sensitivity were evaluated by PBS (0.1 M, pH 7,4) spiked with L- alanine (0.5 m ) or L-leucine (0.5 mM). FIG. 35A shows that the L-alanine sensors maintained an acceptable storage life for up to 2 months (with a variation of approximately ±3%), but the sensitivity decreased to about 65.5% after 4-month storage. FIG. 35B shows that L-leucine sensors maintained a sensitivity of about 78.0% after 4-month storage; the lower sensitivity of L-leucine sensors at 2-month storage indicates that L-leucine sensors were not as stable as L-alanine sensors. FIG.Attorney Docket No. 081906-1535050-256510PC35C shows the stability data of a new batch of L-leucine sensors. The L-leucine sensors were stored in dark, dry conditions using commercial desiccant beads. The results suggest that the L-leucine sensor maintained an acceptable storage life for up to 3 weeks (with a variation of approximately ±1.7%), but the sensitivity decreased to about 66.5% after 4-month storage.Example 29: Evaluation of Storage life of L-Alanine and L-Leucine Sensors Under Room Temperature (about 15-30’0)

[0389] The stability of the sensors under room temperature (about 15-30*0) was further tested. The biosensing strips were manufactured according to typical commercial strip standards (2 mm diameter working electrode and 20yL sample volume). The configuration of the L-alanine and L-leucine sensors are shown in FIG.30D and FIG, 30H. L-alanine and L-leucine sensors were stored under room temperature (about 15-30°C) for 0-16 weeks and their sensitivity were evaluated by PBS (0.1 M, pH 7.4) spiked with L-alanine (0.5 mM) or L-leucine (0.5 mM). The sensors were stored in dark, dry conditions using commercial desiccant beads. FIG.36A shows that the L-alanine sensors maintained an acceptable storage life for up to 2 months, but the sensitivity decreased to about 79.3% after 4-month storage. FIG.368 shows that L-leucine sensors maintained a sensitivity of about 65% after 4-month storage; the tower sensitivity of L-leucine sensors at 2-month storage indicates that L- leucine sensors were not as stable as -alanine sensors.Example 30: Evaluation of Storage life of L-Leucine Sensors (Tests with Trehalose)

[0390] The stability of the sensors under refrigerated conditions was further tested. The biosensing strips were manufactured according to typical commercial strip standards (2 mm diameter working electrode and 20pL sample volume), and coated with stabilizer (10% trehalose). The configuration of the L-leucine sensors is shown In FIG. 30H. L-leucine sensors were stored in a refrigerator (about 1.5-5°C), in dark, dry conditions using commercial desiccant beads, for 0-8 weeks and their sensitivity were evaluated by PBS (0.1 M, pH 7.4) spiked with L-leucine (0.5 mM). FIG. 37A shows the detection signal extremely increased after 3rd and 4th weeks. FIG. 378 shows a recalibrated diagram starting from the 3rd week (as the new week 1 ), and the diagramAttorney Docket No. 081906-1535050-256510PCshows a 30.3% decrease in sensitivity after G-week storage. FIGs, 37-37D are recalibrated diagrams showing the detection sensitivity after 4-week and 6-week storage under room temperature (about 15-30’0), in dark, dry conditions using commercial desiccant beads. FIG. 37D shows a 3.49% change after 6-week storage.Example 31: Custom-Designed Storage Box and Sensor Strips

[0391] FIG. 38A shows a custom-designs storage box holding 51 sensor strips. The custom-designs storage box has a dimension of 2 * 3.6 x 8.5 cm. The container of the custom-designs storage box may be made by conventional molding or 3D- printing.

[0392] FIG. 38B shows that the custom-designs storage box has a channel for storing desiccant beads for moisture control.

[0393] FIGs. 38C-38D are demonstration of handling the custom-designs storage box for delivery.

[0394] FIGs. 38E-38F further show that after placing the sensor strips into the custom-designs storage box, the custom-designs storage box may further be sealed into a sealable plastic bag and purge with nitrogen (N2) to remove oxygen. The nitrogen filling process may be about 15 minutes. After sealing, the custom-designs storage box may then be placed into an ice box containing ice packs and shipped overnight.Example 32: Regulating the Sensing Surface Area by Additional Layers

[0395] The sensor may further comprise one or more coatings and / or spacers made of ion-exchange resins (such as sodium polystyrene sulfonate, polyAPTAC, and polyethylene amine), polyesters, polyethylene terephthalate (PET), PVC, PTFE, and / or polyolefin. The addition of one or more coatings and / or spacers may further protect the sensor as well as facilitate filling of a sample (e g., by capillary-driven filling).

[0396] FIGs. 39A-39B show an exemplary sensor covered with an additional layer. The additional layer (1 * 1.5 cm, with a diameter of 0.72 cm) helps in regulating the filling of a sample liquid such that the sample liquid is contained in the sensing surface area.Attorney Docket No. 081906-1535050-256510PCExample 33: Blood Strip and Touch-Based Sweat Sensors

[0397] FIGs. 41A-41B are schematic illustrations of a blood strip sensor (FIG.41 A) and a touch-based sweat sensor (FIG. 41 B) sensors according to embodiments of the present invention. In the blood strip sensor, the sensing area contacts a blood sample and detects the amino acids (e.g., alanine) and an analyte other than amino acids (e.g., glucose) contained therein. In the touch-based sweat sensor platform, a hydrogel layer may be covering the sensing area to facilitate detection. A user presses his or her finger to the sensing area, such that the sweat secreted from the fingertip is detected by the sensor. This allows a real-time detection of amino acids contained in sweat.

[0398] As shown in the depictions, the sensing area of the detection platform may include multiple sensors targeting different analytes. This allows for the detection platform to be able to detect one or more analytes of interest simultaneously.Example 34: Preliminary Testing of Touch-Based Sweat Sensors

[0399] FIGs. 40A-40B show the detecting results of L-alanine by touch-based sweat sensors following intake of whey protein (60g). The working electrode of the sensors had a diameter of 3 mm. The testing was conducted by touching the sensor for 120 seconds with 60 seconds of detection time. The results show that responses of the L-alanine sensor slightly increased at 60 minute and obviously increased at 75 minute after protein intake.Example 35: Evaluation of Storage life of L-Alanine and L-Leucine Sensors Without Stabilizer

[0400] FIGs. 43A-43B show the evaluation of storage life of L-alanine (FIG.43A) and L-leucine (FIG.43B) sensors when storing in a refrigerator (about 1.5-5°C). The configuration of the L-alanine and L-leucine sensors are shown in FIG, 30D and FIG. 30H. The sensors were not coated with stabilizer. The sensors were stored in dark, dry conditions using commercial desiccant beads. Their sensitivity were evaluated by PBS (0.1 I, pH 7.4) spiked with L-alanine (0.5 mM) or L-leucine (0.5 mM).Attorney Docket No. 081906-1535050-2565 WPCExample 36: Preliminary Evaluation of L-Alanine and L-Leucine Sensors Following Food Consumption

[0401] FIGs. 44A-44D show detecting results of L-alanine and L-leucine in whole blood samples following beef consumption. The biosensing strips were manufactured according to typical commercial strip standards (2 mm diameter working electrode and 20pL sample volume). The configuration of the L-alanine and L-leucine sensors are shown in FIG. 30D and FIG. 30H. Whole blood samples (20 pL for 20 s) were used following food consumption, FIGs. 44A-44B are detecting results of...

Claims

Attorney Docket No. 081906-1535050-256510PCCLAIMS1. A sensor for detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids in a sample, comprising:a substrate;a first electrode formed on the substrate;an entrapment layer formed on the first electrode, wherein the entrapment layer comprises at least one D-amino acid oxidase and at least one racemase, wherein the at least one racemase comprises a racemase that is active against leucine and / or alanine; anda second electrode formed on the substrate, wherein the second electrode is operable as a counter electrode and / or a reference electrode.

2. The sensor of claim 1, wherein the sensor is a wearable or a non wearable sensor.

3. The sensor of claim 1, wherein the sensor is a blood test strip, a sweat sensor, or a microneedle based sensor.

4. A sensor, comprising:a first sensor capable of detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, anda second sensor capable of detecting, measuring, and / or monitoring a concentration of an analyte other than an amino acid,5. The sensor of claim 4, wherein the analyte other than an amino acid is glucose and / or a ketone.

6. The sensor of any one of claims 1 - 5, wherein the entrapment layer comprises at least one additional racemase selected from the group consisting of: an arginine racemase, a histidine racemase, a lysine racemase, an aspartate racemase, a glutamate racemase, a serine racemase, a threonine racemase, an asparagine racemase, a glutamine racemase, a cysteine racemase, a proline racemase, an alanine racemase, a valine racemase, an isoleucine racemase, a leucine racemase,Attorney Docket No. 081906-1535050-256510PCa methionine racemase, a phenylalanine racemase, a tyrosine racemase, and / or a tryptophan racemase, and / or a combination thereof.

7. The sensor of any one of claims 1 - 5, wherein the at least one D-amino acid oxidase is active against one or more of D-arginine, D-histidine, D-lysine, D~ aspartic acid, D-glutamic acid, D-serine, D-threonlne, D-asparagine, D~glutamine D~ cysteine, glycine, D-proline, D-alanine, D-valine, D-isoleucine, D-leudne, D- methionine, D-phenylalanine, D-tyrosine, and / or D-try ptophan.

8. The sensor of any one of claims 1 -5, wherein the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NQs: 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81.

9. The sensor of any one of claims 1 ~ 5, wherein the at least one racemase comprises an amino acid sequence of one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

10. The sensor of any one of claims 1 - 5, wherein the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at ieast 97%, at least 98%, or at least 99% to SEQ ID NO: 33, wherein SEQ ID NO: 33 comprises an alanine at position 24.

11. The sensor of any one of claims 1 ~ 5, wherein the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 4.Attorney Docket No. 081906-1535050-256510PC12. The sensor of any one of claims 1 - 5, wherein the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 31.

13. The sensor of any one of claims 1 - 5, wherein the at least one D-amino acid oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 34.

14. The sensor of any one of claims 1 - 5, wherein the at least one D-amino acid oxidase is active against D-leucine and inactive against D-methionine.

15. The sensor of claim 14, wherein the at least one D-amino acid oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 89, 94, or 97.

16. A sensor, comprising:a substrate;a first electrode formed on the substrate;an entrapment layer formed on the first electrode, wherein the entrapment layer comprises at least one D-teucine oxidase and at least one leucine racemase, or at least one D-alanine oxidase and at least one alanine racemase; anda second electrode formed on the substrate, wherein the second electrode is operable as a counter electrode and / or a reference electrode,wherein the at least one D-leucine oxidase is active against D-leucine, ortho at least one D-alanine oxidase is active against D-alanine,wherein the at least one D-leucine oxidase or the at least one D-alanine oxidase is inactive against one or more other D-amino acids,wherein the sensor exhibits a high degree of specificity against leucine and / or alanine.Attorney Docket No. 081906-1535050-256510PC17. The sensor of any one of claims 1 - 5 and 16, wherein the first electrode is operable as a working electrode, and the second electrode is operable as the counter electrode and the reference electrode,18. The sensor of any one of claims 1 - 5 and 16, wherein the sensor further comprises a third electrode formed on the substrate,wherein the first electrode is operable as a working electrode,wherein the second electrode is operable as the counter electrode, and Wherein the third electrode is operable as the reference electrode.

19. The sensor of any one of claims 1 -5 and 16, further comprising one or more coatings and / or spacers made of ion-exchange resins, polyesters, polyethylene terephthalate (PET), PVC, PTFE, and / or polyolefin,wherein the one or more coatings and / or spacers are formed on the first electrode, the second electrode, and / or the third electrode, and facilitates capillary- driven filling of a sample.

20. The sensor of any one of claims 1 - 5 and 16, wherein the sensor is capable of detecting, measuring, and / or monitoring the amino acid concentration or concentrations of more than one amino acids of a body fluid, wherein the body fluid comprises one or more of whole blood, serum, plasma, a blood fraction other than serum or plasma, lymph, cerebrospinal fluid (CSF)* interstitial fluid (ISF) intracellular fluid, transcellular fluid, saliva, tears, sweat, vaginal discharge, milk, mucus, chyme, pus, bile, semen, urine, amniotic fluid, synovial fluid, peritoneal fluid, pericardial fluid, glandular secretions, exudate, contents of cysts, and / or ascites,21. The sensor of any one of claims 1 -5 and 16, wherein the sensor is used for detecting, measuring, and / or monitoring the amino acid concentration or concentrations of more than one amino adds in urine.

22. A sensor for detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, comprising:a substrate;Attorney Docket No. 081906-1535050-256510PCa first electrode formed on the substrate, wherein the first electrode is operable as a working electrode;an entrapment layer formed on the first electrode, wherein the entrapment layer comprises at least one oxidase and at least one racemase, wherein the at least one racemase comprises a racemase that is active against leucine; anda second electrode formed on the substrate, wherein the second electrode is operable as a counter electrode and / or a reference electrode.

23. The sensor of claim 22, wherein the entrapment layer comprises at least one additional racemase selected from the group consisting of: an arginine racemase, a histidine racemase, a lysine racemase, an aspartate racemase, a glutamate racemase, a serine racemase, a threonine racemase, an asparagine racemase, a glutamine racemase, a cysteine racemase, a proline racemase, an alanine racemase, a valine racemase, an isoleucine racemase, a leucine racemase, a methionine racemase, a phenylalanine racemase, a tyrosine racemase, and / or a tryptophan racemase, and / or a combination thereof.

24. The sensor of claim 23, wherein the at least one additional racemase is selected from the group consisting of: an arginine racemase, a lysine racemase, an alanine racemase, an isoleucine racemase, a leucine racemase, an aspartate racemase, a glutamate racemase, a proline racemase, and / or a serine racemase, and / or a combination thereof.

25. The sensor of any one of claims 23 ~ 24, wherein the at least one additional racemase is active against one or more of alanine, methionine, phenylalanine, serine, proline, arginine, leucine, isoleucine, and / or valine.

26. The sensor of any one of claims 22 - 24, wherein the at least one oxidase comprises an arginine oxidase, a histidine oxidase, a lysine oxidase, an aspartate oxidase, a glutamate oxidase, a serine oxidase, a threonine oxidase, an asparagine oxidase, a glutamine oxidase, a cysteine oxidase, a glycine oxidase, a proline oxidase, an alanine oxidase, a valine oxidase, an isoleucine oxidase, a leucine oxidase, a methionine oxidase, a phenylalanine oxidase, a tyrosine oxidase, and / or a tryptophan oxidase, and / or a combination thereof.Attorney Docket No. 081906-1535050-256510PC27. The sensor of any one of claims 22 - 24, wherein the at least one oxidase is active against one or more of D-arginine, D-histidine, D-lysine, D~aspartic acid, D- glutamic acid, D-serine, D-threonine, D-asparagine, D~glutamine. D-cysteine, glycine, D-proline, D-alanlne, D-valine, D-isoleucine, D-leucine, D-methionine, D-phenylalanine, D-tyrosine, and / or D-tryptophan.

28. The sensor of any one of claims 22 ~ 24, wherein the at least one oxidase is active against one or more of D-alanine, D-Methionine, D-phenylalanine, D-Serine D-proline, D-arginine, D-leucine, D-isoleucine, and / or D-valine.

29. The sensor of any one of claims 22 - 24, wherein the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to one of SEQ ID NOs: 44, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 9, 20, 21, 22. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81.

30. The sensor of any one of claims 22 - 24, wherein the at least one racemase comprises an amino acid sequence of one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 55, 56. 57, 58, 59, 60, 61, 62, 63. 64, 65, 66, 67, 68, 69, 70, 71, 72, 73. 74, 75, 76, 77, 78, 79, 80, or 81, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

31. The sensor of any one of claims 22 - 24, wherein the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%. at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 33.

32. The sensor of any one of claims 22 - 24, wherein the at least one racemase comprises an amino acid sequence at least 80%, at least 85%, at leastAttorney Docket No. 081906-1535050-256510PC90%, at feast 95%, at least 96%, at feast 97%, at least 98%, or at least 99% to SEQ ID NO: 33 comprising, wherein SEQ ID NO: 33 comprises an alanine at position 24.

33. The sensor of any one of claims 22 ~ 24, wherein the at least one racemase comprises an amino acid sequence of SEQ ID NO: 33, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions and wherein SEQ ID NO: 33 comprises an alanine at position 24.

34. The sensor of any one of claims 22 - 24, wherein the at least one oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 34.

35. The sensor of any one of claims 22 - 24, wherein the at least one oxidase comprises an amino acid sequence of SEQ ID NO: 34, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

36. The sensor of any one of claims 22 ~24, wherein the entrapment layer further comprises at least one redox mediator, wherein the at least one redox mediator comprises one or more of Prussian blue, ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complexes, and / or organometallic ruthenium complexes.

37. The sensor of any one of claims 22 ~ 24, wherein the entrapment layer comprises:a transducer layer, wherein the transducer layer comprises the at least one redox mediator; anda receptor layer, wherein the receptor layer comprises the at least one oxidase and the at least one racemase.Attorney Docket No. 081906-1535050-256510PC38. The sensor of any one of claims 22 - 24, further comprises a protective layer formed on the entrapment layer, wherein the protective layer comprises an inner protective layer and / or an outer protective layer.

39. The sensor of claim 38, wherein the inner protective layer and / or the outer protective layer are formed by one or more of polyurethane (PU), polyethylene glycol (PEG) and PEG-related polymers (Macrogol and / or polyethylene glycol¬ polyvinyl alcohol (PEG-PVA)), polyvinyl chloride (PVC), Nafion, silicone and silicone- based polymers (polydimethylsiloxane (POMS), polymethylhydrosiloxane (PMHS). and / or polyvinyl siloxane (PVS)), polyaniline (PANS), poly(methyl methacrylate) (PMMA), polypyrrole (PPy), cellulose, cellulose acetate, polycarbonates (PC), polyacetylene (PAc), Poly(p-phenylene vinylene) (PPV), Rayon, chitosan, and / or Polythiophenes (PTs).

40. The sensor of claim 38, whereinthe inner protective layer is formed by chitosan, andthe outer protective layer is formed by PVC.

41. The sensor of any one of claims 22 - 24, wherein the at least one oxidase and / or the at least one racemase are immobilized to the entrapment layer.

42. The sensor of any one of claims 22 - 24, wherein the entrapment layer comprises a conductive polymer film, and the at least one oxidase and / or the at least one racemase are immobilized to the conductive polymer film.

43. The sensor of claim 42, wherein the conductive polymer film comprises one or more of poly(thionine), polyphenylene vinylene, polypyrrole, polythiophene, polyaniline, and / or polyphenylene sulfide, and / or a combination thereof.

44. The sensor of of claim 42, wherein the at least one oxidase and / or the at least one racemase are cross-linked to the conductive polymer film via a cross-linking agent,Attorney Docket No. 081906-1535050-256510PCwherein the cross-linking agent includes one or more of glutaraldehyde, diisocyanates, carbodiimides, acyl azide, genipin, and / or polyepoxides, and / or a combination thereof,45. The sensor of any one of claims 22 - 24, wherein the substrate is formed of an insulative material,46. The sensor of any one of claims 22 - 24, wherein the first electrode is operable as the working electrode, and the second electrode is operable as the counter electrode and the reference electrode.

47. The sensor of claim 46, whereinthe first electrode is made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, and the second electrode is an Ag / AgCI electrode, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper-copper(ll) sulfate electrode (CSE), a Pd / Hs electrode, or a mercury-mercurous sulfate electrode (MSE).

48. The sensor of any one of claims 22 - 24, wherein the sensor further comprises a third electrode formed on the substrate,wherein the first electrode is operable as the working electrode,wherein the second electrode is operable as the counter electrode, and wherein the third electrode is operable as the reference electrode.

49. The sensor of claim 48, whereinthe first electrode is made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, the second electrode is made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, and the third electrode is an Ag / AgCI electrode, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper-copper(H) sulfate electrode (CSE), a Pd / H2 electrode, or a mercury-mercurous sulfate electrode (MSE).Attorney Docket No. 081906-1535050-256510PC50. A sensor for detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, comprising:a substrate;a first electrode formed on the substrate, wherein the first electrode is operable as a working electrode;an entrapment layer formed on the first electrode, wherein the entrapment layer comprises at least one oxidase; anda second electrode formed on the substrate, wherein the second electrode is operable as a counter electrode and / or a reference electrode.

51. The sensor of claim 50, wherein the at least one oxidase comprises at least one L-amino acid oxidase.

52. The sensor of claim 51, wherein the at least one L-amino acid oxidase is active against one or more of L-arginine, L-histidine, L-lysine, L-aspartic acid, L- glutamic acid, L-serine, L-threonine, L~asparagine, L-glutamine, L-cysteine, glycine, L- proline, L-alanine, L-valine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L- tyrosine, and / or L-tryptophan.

53. The sensor of any one of claims 51 ~ 52, wherein the at least one L- amino acid oxidase is active against one or more of L-alanine, L-proline, L~arginine, L-leucine, L-isoleucine, and / or L-valine.

54. The sensor of claim 50, wherein the at least one oxidase comprises at least one hydroxy acid oxidase.

55. The sensor of claim 54, wherein the at least one hydroxy acid oxidase has cryptic activity against one or more of alanine, proline, arginine, leucine, isoleucine, and / or valine.

56. The sensor of any one of claims 50 - 52, wherein the at least one L- amino acid oxidase comprises an amino acid sequence at least 80%, at least 85%, atAttorney Docket No. 081906-1535050-256510PCfeast 90%, at feast 95%, at feast 96%, at least 97%, at feast 98%, at feast 99%, or at least 99.5% identical to one of SEQ ID NOs: 1, 2, or 3.

57. The sensor of any one of claims 50 - 52, wherein the at least one L~ amino acid oxidase comprises an amino acid sequence of one of SEQ ID NOs: 1, 2, or 3, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

58. The sensor of any one of claims 54 - 55, wherein the at least one hydroxy acid oxidase comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at feast 97%, at least 98%, at least 99%, or at feast 99.5% identical to one of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51. 52, 53, or 54.

59. The sensor of any one of claims 54 - 55, wherein the at least one hydroxy acid oxidase comprises an amino acid sequence of one of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 6, 47, 48, 49, 50, 51, 52, 53, or 54, wherein said amino acid sequence comprises one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions.

60. The sensor of any one of claims 50 - 52, wherein the entrapment layer further comprises at least one redox mediator, wherein the at least one redox me iator comprises one or more of Prussian blue, ferricyanide, phenazine, phenothiazine, thionine, methylene green / blue, tetrathiafulvalene, quinone derivatives, ferrocene, organometallic osmium complexes, and / or organometallic ruthenium comptexes.

61. The sensor of any one of claims 50 - 52, wherein the entrapment layer comprises:a transducer layer, wherein the transducer layer comprises the at least one redox mediator; anda receptor layer, wherein the receptor layer comprises the at least one oxidase.Attorney Docket No. 081906-1535050-256510PC62. The sensor of any one of claims 50 - 52, further comprises a protective layer formed on the entrapment layer, wherein the protective layer comprises an inner protective layer and / or an outer protective layer.

63. The sensor of claim 62, wherein the inner protective layer and / or the outer protective layer are formed by one or more of polyurethane (PU), polyethylene glycol (PEG) and PEG-related polymers (Macrogol and / or polyethylene glycol¬ polyvinyl alcohol (PEG-PVA)), polyvinyl chloride (PVC), Nafion, silicone and silicone- based polymers (poiydimethylsiioxane (PDMS), polymethylhydrosiloxane (PMHS), and / or polyvinyl siloxane (PVS)), polyaniline (PANS), polyfmethyl methacrylate) (PMMA), polypyrrole (PPy), cellulose, cellulose acetate, polycarbonates (PC), polyacetylene (PAc), Poly(p-phenylene vinylene) (PPV), Rayon, chitosan, and / or Polythiophenes (PTs).

64. The sensor of claim 62, whereinthe inner protective layer is formed by chitosan, andthe outer protective layer is be formed by PVC.

65. The sensor of any one of claims 50 - 52, wherein the at least one oxidase is immobilized to the entrapment layer.

66. The sensor of any one of claims 50 ~ 52, wherein the entrapment layer comprises a conductive polymer film, and the at least one oxidase is immobilized to the conductive polymer film.

67. The sensor of claim 66, wherein the conductive polymer film comprises one or more of poly(thionine), polyphenylene vinylene, polypyrrole, polythiophene, polyaniline, and / or polyphenylene sulfide, and / or a combination thereof.

66. The sensor of claim 66, wherein the at least one oxidase is cross-linked to the conductive polymer film via a cross-linking agent,wherein the cross-linking agent includes one or more of glutaraldehyde, diisocyanates, carbodiimides, acyl azide, genipin, and / or polyepoxides, and / or a combination thereof.Attorney Docket No. 081906-1535050-256510PC69. The sensor of any one of claims 50 - 52, wherein the substrate is formed of an insulative material.

70. The sensor of any one of claims 50 ~ 52, wherein the first electrode is operable as the working electrode, and the second electrode is operable as the counter electrode and the reference electrode.

71. The sensor of claim 70, whereinthe first electrode is made of carbon graphite, copper, stiver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, and the second electrode is an Ag / AgCI electrode, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper-copper(ll) sulfate electrode (CSE), a Pd / H electrode, or a mercury-mercurous sulfate electrode (MSE).

72. The sensor of any one of claims 50 - 52, wherein the sensor further comprises a third electrode formed on the substrate,wherein the first electrode is operable as the working electrode,wherein the second electrode is operable as the counter electrode, and wherein the third electrode is operable as the reference electrode.

73. The sensor of claim 72, whereinthe first electrode is made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, the second electrode is made of cartoon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, the third electrode is an Ag / AgCI electrode, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper-copper(ll) sulfate electrode (CSE), a Pd / H₂ electrode, or a mercury-mercurous sulfate electrode (MSE).Attorney Docket No. 081906-1535050-256510PC74. The sensor of claim 72, wherein the first electrode is connected to a first connection, the second electrode is connected to a second connection, and the third electrode is connected to a third connection,wherein the first connection, the second connection, and the third connection are electrically conductive.

75. The sensor of claim 74, wherein a cover layer is formed on the first connection, the second connection, and / or the third connection,wherein the cover layer at least partially covers the first connection, the second connection, and / or the third connection, andwherein the cover layer is made of an electrically insulative material.

76. A system for detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, comprising:the sensor of any one of claims 22 and 50; anda portable device connected to the sensor,wherein the sensor is configured to measure an amino acid concentration or concentrations of more than one amino acids contained within a sample and output a data stream to the portable device,wherein the portable device comprises:a processor configured to process the data stream from the sensor; and an interface configured to display and / or communicate measured amino acid concentration values.

77. The system of claim 76, wherein the sensor is coupled to the portable device via the first connection, the second connection, and / or the third connection.

78. The system of claim 76, wherein the sensor is configured on a disposable test strip.

79. The system of claim 76, wherein the portable device further comprises a signal conditioning circuit to amplify electrical signals detected by the first electrode.Attorney Docket No. 081906-1535050-256510PC80. The system of claim 76, wherein the portable device further comprises a memory,81. The system of claim 76, wherein the portable device further comprises a wireless communications unit to wirelessly transmit the detected electrical signals and / or the data stream to an external computing device.

82. A method of manufacturing a sensor for detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, comprising:providing a substrate;forming a first electrode on the substrate, wherein the first electrode is operable as a working electrode;forming an entrapment layer on the first electrode;forming a second electrode on the substrate, wherein the second electrode is operable as a counter electrode; andforming a third electrode on the substrate, wherein the third electrode is operable as a reference electrode.

83. The method of claim 82, wherein forming the entrapment layer comprises:depositing a conductive polymer material on the first electrode, such that a conductive polymer film is formed on the first electrode; anddepositing at least one oxidase and a cross-linking agent to the conductive polymer film, such that the at least one oxidase is immobilized to the conductive polymer film.

84. The method of claim 83, further comprises depositing at least one racemase to the conductive polymer film, such that the at least one racemase is immobilized to the conductive polymer film.

85. The method of any one of claims 83 - 84, further comprises depositing at least one redox mediator to the conductive polymer film, such that the at least one redox mediator is immobilized to the conductive polymer film.Attorney Docket No. 081906-1535050-256510PC86. The method of any one of claims 82 - 84, further comprises forming a protective layer on the entrapment layer.

87. The method of claim 86, wherein the protective layer is formed by depositing a solution containing Nafion on the entrapment layer, and drying the solution.

88. The method of claim 86,wherein the protective layer comprises an inner protective layer and / or an outer protective layer, andwherein the inner protective layer and / or the outer protective layer are formed by one or more of polyurethane (PU), polyethylene glycol (PEG) and PEG-related polymers (Macrogol and / or polyethylene glycol-polyvinyl alcohol (PEG-PVA)), polyvinyl chloride (PVC), Nafion, silicone and silicone-based polymers (polydimethylsiloxane (PDMS), polymethylhydrosiloxane (PMHS), and / or polyvinyl siloxane (PVS)), polyaniline (PANI), poly(methyl methacrylate) (PMMA), polypyrrole (PPy), cellulose, cellulose acetate, polycarbonates (PC), polyacetylene (PAc), Poly(p- phenylene vinylene) (PPV), Rayon, chitosan, and / or Polythiophenes (PTs).

89. The method of claim 88, whereinthe Inner protective layer is formed by chitosan, andthe outer protective layer is be formed by PVC.

90. The method of any one of claims 82 - 84, whereinthe first electrode is made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, the second electrode is made of carbon graphite, copper, silver, platinum, palladium, rhodium, iridium, nickel, and / or gold, and / or a combination thereof, the third electrode is an Ag / AgCI electrode, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper~copper(ll) sulfate electrode (CSE), a Pd / Ha electrode, or a mercury-mercurous sulfate electrode ( SE).Attorney Docket No. 081906-1535050-256510PC91. The method of any one of claims 82 - 84, further comprises: forming a first connection electronically connected to the first electrode; forming a second connection electronically connected to the second electrode; forming a third connection electronically connected to the third electrode; and forming a cover layer on the first connection, the second connection, and / or the third connection, wherein the cover layer at least partially covers the first connection, the second connection, and / or the third connection, and wherein the cover layer is made of an electrically insulative material.

92. A method of detecting, measuring, and / or monitoring an amino acid concentration or concentrations of more than one amino acids, comprising:subjecting a sample to the sensor of any one of claims 22, 50, and 82; and detecting and / or measuring a presence of hydrogen peroxide.

93. A method of improving health and / or wellness in a subject in need thereof comprising using a sensor, device, er apparatus of any one of claims 22, 50, and 82.

94. The method of claim 93 used in conjunction with a food item or supplement comprising a high amino acid composition.

95. The method of claim 94, wherein the food item or supplement contains more than 1g, more than 2.5g, more than 5g, more than 7.5g, more than 10g, more than 12.5g, more than 15g, more than 17.5g, more than 20g, more than 22.5g, more than 25g, more than 27.5g, more than 30g, more than 32.5g, more than 35g, more than 37.5g, more than 40g, more than 42.5g, more than 45g, more than 47.5g, or more than 50g of branched-chain amino acids (BCAA) per unit.

96. The method of claim 94, wherein the food item or supplement contains more than 1g, more than 2.5g, more than 5g, more than 7.5g, more than 10g, more than 12.5g, more than 15g, more than 17.5g, more than 20g, more than 22.5g, more than 25g, more than 27.5g, more than 30g, more than 32.5g, more than 35g, more than 37.5g, more than 40g, more than 42.5g, more than 45g, more than 47.5g, or more than 50g of leucine per unit.Attorney Docket No. 081906-1535050-256510PC97. The method of claim 94, wherein the food item or supplement contains more than 1g, more than 2.5g, more than 5g, more than 7.5g, more than 10g, more than 12.5g, more than 15g, more than 1.5g, more than 20g, more than 22.5g, more than 25g, more than 27.5g, more than 30g, more than 32.5g, more than 35g, more than 37.5g, more than 40 g, more than 42.5g, more than 45g, more than 47.5g, or more than 50g of alanine per unit.

98. The sensor of any one of claims 22, 50, and 82, wherein the more than one amino acids comprises two, three, four, five, six, seven, eight, nine, or ten amino acids.

99. The sensor of claim 98, wherein the more than one amino acids comprises alanine, leucine, isoleucine, and / or valine.

Images