WO2024081853A2

WO2024081853A2 - Monoamine oxidase (mao) electrochemical biosensor and uses thereof

Info

Publication number: WO2024081853A2
Application number: PCT/US2023/076796
Authority: WO
Inventors: James GALAGAN; Karen Allen; Uros KUZMANOVIC; Samantha MUELLERS; Margarita Alexandrovna TARARINA
Original assignee: Trustees Of Boston University
Priority date: 2022-10-14
Filing date: 2023-10-13
Publication date: 2024-04-18

Abstract

Described herein is an amperometric biosensor, e.g., chronoamperometric biosensor for the measurement of the concentration of amine-comprising substates, including dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5-methoxytryptamine, and/or hexylamine. The biosensor disclosed herein comprises a monoamine oxidase (MAO) enzyme, such as Corynebacterium ammoniagenes MAO (caMAO). Also described herein are systems comprising said amperometric biosensor, e.g., chronoamperometric biosensor and methods of using said biosensors.

Description

MONOAMINE OXIDASE (MAO) ELECTROCHEMICAL BIOSENSOR AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/416,342 filed October 14, 2022, the contents of which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

[0002] This invention was made using government support under Contract No. HU00011810022 awarded by the Department of Defense. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on October 12, 2023, is named 701586-000103WOPT_SL.xml and is 23,614 bytes in size.

TECHNICAL FIELD

[0004] The technology described herein relates to monoamine oxidase (MAO) electrochemical biosensor and uses thereof.

BACKGROUND

[0005] There is a need for electrochemical biosensors that can detect a wide range of amine- comprising substrates in samples.

SUMMARY

[0006] The technology described herein is directed to an amperometric biosensor, e.g., chronoamperometric biosensor for the measurement of the concentration of amine-comprising substates, including dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5- methoxytryptamine, and/or hexylamine. The biosensor disclosed herein comprises a monoamine oxidase (MAO) enzyme, such as Corynebacterium ammoniagenes MAO (CαMAO). Also described herein are systems comprising said amperometric biosensor, e.g., chronoamperometric biosensor and methods of using said biosensors.

[0007] Monoamine oxidases (MAOs) play a role in the breakdown of primary and secondary amines. In humans and other eukaryotic organisms, these enzymes are involved in the regulation of monoamine neurotransmitters and the degradation of dietary monoamines. MAOs have also been identified in prokaryotic species, although their role in these organisms is not well understood. [0008] Kinetic and structural characterization were used to gain a better understanding of the specificity, mechanism, and role of the bacterial Corynebacterium ammoniagenes MAO (CαMAO). Described herein is a steady-state kinetic analysis of the promiscuous CαMAO as well as the unliganded, holo structure, as determined by X-ray crystallography. This structure identified an unusual cysteine residue located within the “aromatic cage” - the two residues which flank the isoalloxazine ring of the flavin cofactor. Canonically, these amino acids are aromatic and have been shown to play a steric role in substrate binding as well as a catalytic role- increasing the nucleophilicity of the amino group on the substrate. An “aromatic cage” cysteine has been observed in only one other FAO enzyme structure - a polyamine oxidase (PAO) from .S', cerevisiae - but its role in catalysis was not further investigated. Kinetic studies of the wild-type (WT) CαMAO, as well as several active-site variants, revealed the catalytic importance of this cysteine and the effect on pH- dependence of the enzyme. Furthermore, described herein is the structure-activity relationships of various monoamine and polyamine substrates using seven substrate-bound structures of CαMAO. Computational mapping and site-directed mutagenesis revealed the presence of a secondary binding site, responsible for substrate inhibition. A similar site was identified in the nicotine -degrading enzyme from P. putida, NicA2, allowing the drawing of connections from CαMAO to other members of the FAO superfamily. Analysis of GNDs of those enzymes most closely related to CαMAO indicates roles for bacterial MAOs in amino-acid biosynthesis and polyamine degradation and scavenging of nitrogen.

[0009] Accordingly, in one aspect described here is a biosensor for the measurement of the concentration of at least one amine-comprising substrate comprising: (a) an electrode comprising a surface; (b) an electronically active mediator (Med) deposited on the surface of the electrode; and (c) a plurality of monoamine oxidase (MAO) enzymes deposited on the surface of the electrode, wherein the MAO enzyme catalyzes the at least one amine-comprising substrate to produce hydrogen peroxide (H₂O₂).

[0010] In some embodiments of any of the aspects, the MAO enzyme is Corynebacterium ammoniagenes MAO (CαMAO) or a functional variant or fragment thereof.

[0011] In some embodiments of any of the aspects, the MAO enzyme is encoded by a nucleic acid comprising a sequence that is at least 90% identical to SEQ ID NO: 1.

[0012] In some embodiments of any of the aspects, the MAO enzyme comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 2.

[0013] In some embodiments of any of the aspects, the at least one amine-comprising substrate is a monoamine substrate.

[0014] In some embodiments of any of the aspects, the at least one amine-comprising substrate is a monoamine neurotransmitter or a dietary monoamine. [0015] In some embodiments of any of the aspects, the at least one amine-comprising substrate is a polyamine substrate.

[0016] In some embodiments of any of the aspects, the at least one amine-comprising substrate is selected from the group consisting of dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5- methoxytryptamine, and hexylamine.

[0017] In some embodiments of any of the aspects, the at least one amine-comprising substrate is dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and hexylamine. [0018] In some embodiments of any of the aspects, the MAO enzyme is immobilized on the surface of the electrode with a polymer, and optionally, a top layer above the polymer, wherein the top layer comprises Prussian-Blue (PB).

[0019] In some embodiments of any of the aspects, the polymer comprises low molecular weight (LMW) or medium molecular weight (MMW) chitosan in 0.5% acetic acid, and optionally Prussian- Blue (PB).

[0020] In some embodiments of any of the aspects, in the presence of the at least one amine- comprising substrate, the MAO enzyme produces H₂O₂, wherein breakdown of H₂O₂ to O₂ and H₂O releases electrons to produce an electrochemical signal, wherein the electrochemical signal is detected by current passed to the electrode.

[0021] In some embodiments of any of the aspects, the detectable signal is produced when the at least one amine-comprising substrate is catalyzed by the MAO enzyme and transfers at least one electron from Med_red to hydrogen peroxide (H₂O₂), resulting in its reduction to Med_ox, wherein Med_ox is reduced by the electrode producing a detectable signal.

[0022] In some embodiments of any of the aspects, the Med_ox produces an electrochemical signal, wherein the electrochemical signal is detected by current passed to the electrode.

[0023] In some embodiments of any of the aspects, the biosensor is an amperometric biosensor and the detectable signal is electrochemical.

[0024] In some embodiments of any of the aspects, the electrode is connected to a potentiostat having a current resolution to at least IpA (100 nA).

[0025] In some embodiments of any of the aspects, the biosensor comprises a working electrode and a reference electrode.

[0026] In some embodiments of any of the aspects, the biosensor comprises a counter electrode.

[0027] In some embodiments of any of the aspects, the biosensor does not comprise a counter electrode.

[0028] In some embodiments of any of the aspects, the working electrode is > 10π mm².

[0029] In some embodiments of any of the aspects, the electrode is metallic.

[0030] In some embodiments of any of the aspects, the metallic electrode is gold, silver, platinum, or palladium. [0031] In some embodiments of any of the aspects, the electrode is non-metallic.

[0032] In some embodiments of any of the aspects, the non-metallic electrode comprises carbon.

[0033] In some embodiments of any of the aspects, the amperometric biosensor is a chronoamperometric biosensor.

[0034] In one aspect, described herein is a method of using an amperometric biosensor to measure the concentration of at least one amine-comprising substrate comprising: (a) assembling a biosensor as described herein, wherein the biosensor is an amperometric biosensor; (b) providing a sample; and (c) measuring the current produced by the oxidation of at least one amine-comprising substrate present in the sample.

[0035] In some embodiments of any of the aspects, the sample is selected from the group consisting of: gastric juice, urine, saliva, feces, cerebrospinal fluid, sweat, interstitial fluid, and blood. [0036] In some embodiments of any of the aspects, the method of using the amperometric biosensor is used to measure the physiological concentration of at least one amine-comprising substrate in the range of 0.01μM-80μM range.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A-1D. Structural features of the CαMAO active site. (Fig. 1A) The structure of unliganded, holo CαMAO bound to flavin cofactor (light gray sticks) is depicted as cartoon and is shaded by domain. (Fig. IB) View of the active site with residue sidechains shown as sticks and shaded by domain. (Fig. 1C) Glu207 and Phel75 sit within the active site tunnel (mapped using CAVER3.0 and depicted as gray mesh). (Fig. ID) Schematic of MAO showing specific residues of MAO (e.g., W387, C424) and exemplary analytes detected by MAO.

[0038] Figure 2A-2E. Structures of MAO in complex with substrate. The active site of the following bound MAO structures are shown: (Fig. 2A) tryptamine, (Fig. 2B) dopamine, (Fig. 2C) octopamine, and (Fig. 2D) cadaverine. Interactions are depicted using dashes (dark gray = hydrophobic contacts < 4 A, light gray = hydrogen bonds). Panel (Fig. 2E) summarizes the polar interactions made by each substrate, as determined by structural analysis; asterisks “*” indicates the Thr210 backbone interactions.

[0039] Figure 3A-3D. WT and C424S pH-rate kinetics. Kinetic assays were performed at pH values between 7.0 and 9.0 with the substrate dopamine for (Fig. 3A) WT MAO and (Fig. 3B) C424S variant. Note the difference in scale of y-axis between (Fig. 3A) and (Fig. 3B). Curves were fit to a substrate-inhibition equation using GRAPHPAD PRISM. (Fig. 3C) k_cat/K_m values for the WT and C424S MAO are shown for each pH. (Fig. 3D) Fresh enzyme was spiked into reaction mixtures of MAO and dopamine at pH 7.0 and pH 8.5 after 1200 seconds (indicated by the break in the plot). Solid lines depict the addition of enzyme and dotted lines indicate control samples where buffer was added in place of enzyme. [0040] Figure 4A-4B. 2FoFc maps displaying the rotameric states of residue 424. Electron density maps calculated with coefficients 2FoFc contoured to 1.0 o display the rotamer states of (Fig. 4A) Cys424 in WT CαMAO and (Fig. 4B) Ser424 in the variant. In Fig. 4B, the hydrogen bond formed between the serine hydroxyl and the isoalloxazine ring is depicted with light gray dashes.

[0041] Figure 5A-5C. Substrate inhibition. (Fig. 5A) The binding site identified by FTMap is delineated by the probe cluster in gray lines. These results were aligned to the structure of CαMAO bound to dopamine to show the position of the secondary site with respect to the active site. (Fig. 5B) Overlay of the structure of CαMAO (light gray) and P. putida NicA2 (dark gray, PDB 7KHN) bound to two molecules of nicotine shows the close agreement in the positions of the substrate inhibition sites (CαMAO site is depicted with a circle). (Fig. 5C) Kinetic assays were performed with tryptamine using the WT and E207A MAOs. Substrate inhibition was observed for WT but not for the variant. [0042] Figure 6 is a schematic showing monoamine substrates.

[0043] Figure 7. Structural alignment of CαMAO to homologous FAO structures. The table lists the sequence identities and RMSD values for each homolog in comparison to CαMAO. The plot below, breaks down the alignments of each separate domain of the structure. All alignments were performed using the cealign command in PyMOL.

[0044] Figure 8A-8C. Additional substrate-bound structures. The active site interactions are shown for the following substrates: (Fig. 8A) tyramine, (Fig. 8B) norepinephrine, and (Fig. 8C) 5- aminopentanol. Interactions are depicted using dashes (dark gray = hydrophobic contacts < 4 A, light gray = hydrogen bonds, dark gray with asterisks (“*”) = water-mediated). For (Fig. 8C) both conformations of 5 -aminopentanol are depicted (dark and light grey).

[0045] Figure 9A-9B. Stability and activity of HRP and CαMAO in pH-rate studies. (Fig.

9A) DSF analysis showing the first-derivative of fluorescence/temperature is plotted for the WT CαMAO at pH values between 6.5 and 9.0. (Fig. 9B) Peroxide standard curves were performed with the assay reagent HRP at pH values between 7.0 and 9.0. Similar activity was observed across this pH range.

[0046] Figure 10A-10C. UV-Vis spectroscopy of flavin cofactor at varying pH for WT and C424S CαMAO. Fig. 10A and Fig. 10B show the UV-Vis spectra of the flavin cofactor from 315 nm to 700 nm for the WT and C424S variant, respectively. Fig. 10C depicts the overlay between the spectra taken at pH 8.5 for the WT (solid line) and C424S variant (dashed line).

[0047] Figure 11A-11C. GNN analysis for bacterial MAOs. In each of Fig. 11A-11C, one or two representative operons from Cluster 14 are shown with example chemical reactions depicted beneath. In each operon, the MAO enzyme is located in the center of the view and is shown in a dashed line rectangle. The percent sequence identity between each MAO and CαMAO is listed to the left. Genes encoding other enzymes of interest are labeled. [0048] Fig. 12 is a schematic showing the Amplex® UltraRed mechanism. The amine- comprising substrate is oxidized into its oxidized version and H₂O₂ with presence of MAO and O₂; exemplary amine-comprising substrates of MAO include dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and/or hexylamine. Horseradish peroxidase (HRP) then reduces Amplex® UltraRed in the presence of H₂O₂ into resorufm, which is fluorescent.

[0049] Fig. 13 is a schematic depicting the sequences of events that occur in the MAO biosensor system. Amine-comprising substrate oxidation by MAO results in an oxidized version of the specific amine- comprising substrate; exemplary amine-comprising substrates of MAO include dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and/or hexylamine. H₂O₂ reduction at the Prussian Blue (PB) film is measured by electrons transferred from the working electrode.

[0050] Fig. 14 shows a simplified representation of potentiostat circuitry which allows for the accurate measurement of a current response resulting from analyte addition to the electrochemical biosensor.

[0051] Fig. 15A-15C shows a schematic of an electron flow mechanism for the MAO biosensor. Fig. 15A shows an exemplary embodiment of the MAO biosensor which can be composed of a carbon three-electrode screen printed electrode (SPE) with an Ag/AgCl reference electrode. Before deposition of enzyme onto the SPE, Prussian Blue (PB) mediator is electrodeposited onto the SPE. Once the mediator is electrodeposited, MAO is deposited onto the SPE working electrode (WE), optionally with chitosan, a natural polymer. When analyte is added onto the three electrodes of the SPE the circuit is completed between them, and a current response can be measured when a constant potential is applied by a potentiostat. The presence of the amine- comprising substrate (e.g., dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and/or hexylamine) in the solution causes the creation of a diffusion layer and a gradient between regions of high and low analyte concentrations. This gradient creates flux governed by Fick’s Law and drives the movement of analyte to the working electrode. In another exemplary embodiment, the MAO biosensor comprises a two-electrode SPE. Fig. 15B and 15C shows exemplary representations of a MAO amperometric biosensor, e.g., chronoamperometric biosensor for the detection and/or measurement of amine-comprising substrates (e.g., dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and/or hexylamine).

DETAILED DESCRIPTION

[0001] The technology described herein relates to an amperometric biosensor, e.g., chronoamperometric biosensor for the measurement of at least one amine-comprising substrate. In order to design such a chronoamperometric sensor, a sensing element for amine-comprising substrates was identified from a bacterial source. Bacteria have evolved over 3 billion years to detect and respond to virtually all classes of stimuli or chemicals relevant to their surroundings. For example, the bacteria Corynebacterium ammoniagenes encodes and expresses a promiscuous monoamine oxidase (MAO). In the presence of the substrate, the MAO produces hydrogen peroxide for chronoamperometric measurement. Described herein is a prototype point-of-care (POC) detection device based on the MAO with sensitivity and specificity for clinical use as well as quantification of amine-comprising substrates from samples. Accordingly, in one aspect, described herein is a amperometric biosensor, e.g., chronoamperometric biosensor comprising: (a) an electrode comprising a surface; (b) an electronically active mediator deposited on the surface of the electrode; and (c) a plurality of MAO enzyme deposited on the surface of the electrode, wherein the MAO enzyme is an oxidase that catalyzes at least one amine-comprising substrate to produce hydrogen peroxide (H₂O₂).

I. Elements of a MAO Biosensor device

[0002] Label-free sensing of small molecule analytes such as amine-comprising substrates (e.g., dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and/or hexylamine) is of critical importance to biomedical research, point of care diagnostics, and environmental sensing, among other applications. Connected devices that monitor human biology or the environment in real-time represent the next frontier in biosensors. Monitoring amine-comprising substrates is of significant interest to subjects and medical professionals detecting and/or quantifying monoamine neurotransmitters (e.g., dopamine, norepinephrine). However, the real-time monitoring of analytes such as amine-comprising substrates is challenging from a biology, chemistry, and engineering perspective. Using natural sensing elements from microbial species, e.g., native biomolecules that have evolved sensor and modulator capabilities, provides the opportunity to utilize a detection platform that is distinct from other antibody- or aptamer-based strategies for detecting amine-comprising substrates. Described herein is an amperometric, e.g., a chronoamperometric biosensor for the measurement of at least one amine-comprising substrate in a sample.

[0003] A biosensor is a device comprising a biological sensor element (also referred to as a biorecognition element or biological component) that typically produces electronic signals that are proportional to the concentration of a particular substance to be determined. As used herein the term “amperometric” refers to the measurement of current of an electrode, and “chronoamperometric” refers to the measurement of current of an electrode as a function of time.

[0004] Biosensors, e.g., nanobiosensors are a type of analytical device that use biological molecules to monitor biorecognition events and interactions. Generally, a biosensor comprises a biological component, a redox-mediator alongside nanoelectrodes; the various components can be equated with the electronic elements of a sensor because the components transduce the signal generated at the source (bioelement) to the detector (electrode). In general, a biological component of a biosensor can be a protein (e.g., enzyme or antibody), nucleic acid (DNA or RNA) or even entire cells. [0005] The use of enzymes as bioactive interfaces is well known in the art, and such interfaces are used in analytical methods of detecting electronic transduction of enzyme-substrate reactions. Direct electrical activation of enzymes such as redox enzymes permits stimulation of bioelectrocatalyzed oxidation or reduction of enzyme substrates. Rapid transfer of electrons between an electrode and a given redox enzyme results in current generation corresponding to the rate of turnover of the electron exchange between the substrate and biocatalyst. In other words, the transduced current of the system correlates with enzyme substrate concentration. Electrical contacting of redox proteins in a biosensor and the electrode support contained therein may be mediated by direct electron transfer with electrode surfaces. Redox enzymes lacking direct electrical communication with electrodes may achieve electrical contact by mediated electron transfer via redox mediators that serve as active charge carriers.

[0006] For non-limiting examples of biosensors, see e.g., US Patents 6,241,863, 6,736,777, 7,794,994; US patent publications US 2003/0027239, US 2009/0099434, US 2009/0061451, US 2012/0181189, US 2019/0004005, US 2021/0259585; PCT publications WO 2013/059534, WO 2005/048834, WO 2022/016071; European patent 1194585; Vigneshvar et al. Front Bioeng Biotechnol. 2016, 4: 11; Turner, Chem Soc Rev. 2013 Apr 21, 42(8):3184-96; Hosu et al., Taianta Volume 204, 1 November 2019, Pages 525-532; the contents of each of which, including but not limited to biosensors and methods and systems comprising them, are incorporated herein by reference in their entireties.

[0007] In all aspects of the technology described herein is a MAO biosensor for the measurement of the concentration of at least one amine-comprising substrate, comprising on its surface, a MAO enzyme, where the MAO enzyme is electronically coupled to a redox mediator (referred to herein as a “Med” or an “electronically active mediator”), so that when the MAO enzyme binds to, and catalyzes the at least one amine-comprising substrate, it transfers electrons (i.e., causes a redox reaction) to a redox mediator/electronically active mediator, were redox mediator/electronically active mediator then transfers electrons, either directly (or indirectly, as discussed herein with the use of Readout enzyme) to a suitable electron detection method, for example, to an electrode thereby producing a current, or to an electronically excitable product.

[0008] Without wishing to be limited to theory, in catalyzing at least one amine-comprising substrate to an oxidized product, the MAO enzyme causes a redox event which is coupled to a redox mediator which acts as a conductor of electrons to the detector, typically an electrode, thereby relaying the detection of the at least one amine-comprising substrate. In some embodiments, the amount of electrons produced, and detected by the electron detector is corresponds to the amount of the analyte, in this instance, the amount of the at least one amine-comprising substrate. In some embodiments, the biosensor is set up to allow multiple redox events, e.g., 2, or 3 redox events. In some embodiments, a redox event occurs between the transfer of electrons from the amine-comprising substrate to the redox mediator/electronically active mediator when the amine-comprising substrate is catalyzed to its oxidized form (step 1), and a second redox event occurs the when the reduced redox mediator returns to the oxidized form, thereby transferring electrons to the electron detector, typically an electrode (step 2). In another embodiment, a redox event occurs between the transfer of electrons from the amine-comprising substrate to a redox mediator/electronically active mediator when the amine- comprising substrate is catalyzed to its oxidized form and H₂O₂ (step 1), where the redox mediator/electronically active mediator can only accept the electrons in the presence of an intermediate redox enzyme (IRE) and H₂O₂, and a second redox event occurs the when the reduced redox mediator produces a signal (step 2).

A. MAO Enzymes for use in biosensors

[0009] Herein, the technology relates to a MAO biosensor device detecting at least one amine- comprising substrate, the device comprising a MAO redox-enzyme. Redox-enzymes in general are responsible for the binding and recognition of the special target analyte, whether a small molecule or a large protein partner. The binding of the redox-enzyme to the target analyte is the basis for signal generation and a physical element, such a detectable signal is generated, and an electrode can capture the signal as the output. Thus, coupling the redox-enzyme with a mediator, translates information from the binding of the target analyte and redox-enzyme into a chemical or physical output with a defined sensitivity. The information that is detected can be chemical, energetic, such as detection of light, and/or signal detection and transduction.

[0010] In all aspects of the technology described herein, the redox enzyme in the biosensor is a MAO redox enzyme that catalyzes or detects at least one amine-comprising substrate or a derivative or analogue thereof. In some embodiments, the MAO redox enzyme can detect each optically active amine-comprising substrate variant, for example including the optically active, two enantiomeric forms.

[0011] A “redox enzyme” or “oxidoreductases” are used interchangeably herein and refer to an enzyme that catalyzes a reaction with one or more enzyme substrate(s), resulting in generation or utilization of electrons. Redox enzymes are proteins that catalyze electron transfer by reduction or oxidation of substrates within the redox network. The oxidoreductase (redox) enzyme reaction and the electrode coupling method has become attractive to develop biosensors. In particular, for a range of substrates, i.e., glucose (i.e., glucose oxidase and glucose dehydrogenase reaction, respectively), electrochemical detection of reduced coenzyme nicotinamide adenine dinucleotide (NADH) or hydrogen peroxide has been used in galvanometer biosensors.

[0012] A redox reaction is a chemical reaction in which the oxidation states of atoms are changed. Any such reaction involves both a reduction process and a complementary oxidation process, two key concepts involved with electron transfer processes. Redox reactions include all chemical reactions in which atoms have their oxidation state changed; in general, redox reactions involve the transfer of electrons between chemical species.

[0013] Aspects of the biosensor described herein rely on catalyzing an electrochemical reaction (redox) of the redox-enzyme biosensor in the presence of a target analyte (i.e., substrate; e.g., at least one amine-comprising substrate). In use, an analyte specific to the redox-enzyme biosensor is catalyzed, changing electron flow through the biosensor. In one non-limiting embodiment, the redox- enzyme biosensor (or a functional portion thereof) catalyzes a redox event in the presence of a target analyte (i.e., where the analyte is a substrate of the redox enzyme). The redox event can be coupled to a redox-mediator (referred to herein as a “Med”) which acts as a conductor of electrons to permit detection of the redox event between the target analyte (e.g., at least one amine-comprising substrate) and the redox-enzyme (e.g., MAO enzyme). In some embodiments, the redox event between the target analyte (e.g., at least one amine-comprising substrate) and the redox-enzyme (e.g., MAO enzyme) can be coupled to an intermediate redox enzyme (IRE), that acts as a conductor of electrons between the first redox event (between and the redox-enzyme (e.g., MAO enzyme) and the redox-mediator (Med). In some embodiments, the redox-mediator can be linked to an electrode, nano-electrode, or nanobioneedle, which all act as conductors of electrons to permit detection of any signal changes in the redox-enzyme biosensor.

[0014] In some embodiments, the redox-mediator generates a signal detectable by optical methods, such as, without limitation, fluorescence, surface plasmon resonance, or piezoelectric methods. i. MAO Enzymes

[0015] In some embodiments of any of the aspects, the redox enzyme is a monoamine oxidase (MAO) enzyme. One aspect described herein relates to an amperometric biosensor comprising a MAO enzyme. In all aspects herein, the MAO enzyme is a redox enzyme. In some embodiments, the MAO enzyme is an oxidase enzyme. Accordingly, in one aspect described herein is an amperometric biosensor, e.g., chronoamperometric biosensor comprising a MAO enzyme. In some embodiments of any of the aspects, the biosensor described herein comprises a plurality of MAO enzymes deposited on the surface of the electrode. In some embodiments of any of the aspects, the MAO enzyme is an oxidase that catalyzes at least one amine-comprising substrate to produce hydrogen peroxide (H₂O₂). [0016] In some embodiments of any of the aspects, the MAO enzyme is Corynebacterium ammoniagenes MAO (caMAO). In some embodiments of any of the aspects, the MAO enzyme is from the genus Corynebacterium. In some embodiments of any of the aspects, the MAO enzyme is from the strain Corynebacterium ammoniagenes NCTC 2398 (ATCC).

[0017] In some embodiments of any of the aspects, the biosensor described herein comprises a MAO enzyme encoded by any of SEQ ID NO: 1 or a nucleic acid sequence that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: Ithat maintains the same function when expressed as a polypeptide (e.g., at least one amine-comprising substrate degradation), or a codon-optimized version thereof.

[0018] SEQ ID NO: 1 : Wild Type MAO from Corynebacterium ammoniagenes strain KCCM 40472 chromosome, complete genome GenBank: CP019705.1, complement REGION: 124251- 125597, locus_tag=”CA40472_00570”, 1347 base pairs (bp).

[0019] In some embodiments of any of the aspects, the amino acid sequence of the MAO enzyme comprises SEQ ID NO: 2 or an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 2 that maintains the same function of SEQ ID NO: 2 (e.g., degradation of at least one amine-comprising substrate).

[0020] SEQ ID NO: 2 MAO(WT) [Corynebacterium ammoniagenes], AD(P)/FAD-dependent oxidoreductase, see e.g., NCBI Reference Sequence: WP_040355246.1, 449 amino acids (aa); amino acid region 13-440 corresponds to an amino oxidase domain.

[0021] In some embodiments, the MAO enzyme does not the N-terminal domain. In some embodiments, the MAO enzyme does not the C-terminal domain. In some embodiments, the MAO enzyme does not comprise amino acids 1-12 of SEQ ID NO: 2, which are predicted to be disordered, play a role in localization, and not be critical to the enzyme’s properties. In some embodiments, the MAO enzyme comprises residues 13-440 of SEQ ID NO: 2, or residues 13-449 of SEQ ID NO: 2, or a sequence that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to residues 13-440of SEQ ID NO: 2 or residues 13-449 of SEQ ID NO: 2 that maintains the same function (e.g., degradation of at least one amine-comprising substrate). A MAO variant derived from SEQ ID NO: 2 can comprise a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids from the N-terminus of the peptide. In some embodiments, the variant additionally or alternatively may have a deletion 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids from the C-terminus of the peptide, such as a deletion of the C-terminal residue.

[0022] In some embodiments, the MAO enzyme comprises a substitution in at least one residue involved in the active site (e.g., Phel75, Alal78, Leu211, Phe341, Val342, Cys424, Trp387, Glu207, Thr210); see e.g., Example 1.

[0023] In some embodiments, the MAO enzyme is a mutant MAO enzyme comprising at least one mutation selected from any of: C424V, C424Y, C424S, W387Y, and E207A of SEQ ID NO: 2, or an amino acid comprising at least 85%, or at least 90% or at least 95% sequence identity to SEQ ID NO: 2 and comprising at least one mutation selected from: C424V, C424Y, C424S, W387Y, and E207A. [0024] In some embodiments, the MAO enzyme is a mutant MAO enzyme comprising at least one mutation selected from any of: C424Y or W387Y of SEQ ID NO: 2, or an amino acid comprising at least 85%, or at least 90% or at least 95% sequence identity to SEQ ID NO: 2 and comprising at least one mutation selected from: C424Y or W387Y. [0025] In some embodiments, the MAO enzyme comprises a mutation that enhances catalytic activity of the enzyme.

[0026] In some embodiments, directed evolution of the MAO enzyme can be done to improve selectivity, catalytic rates (k_cat/K_m), and substrate affinity (Km). In alternative embodiments, alternative MAO enzymes can be used that have similar or enzymatic properties to MAO.

[0027] In some embodiments of any of the aspects, the MAO enzyme is from a species selected from Corynebacterium ammoniagenes, Halomoncis alkaliantarica, Amycolatopsis sp. KNN50.9B, and Alteribacillus bidgolensis . In some embodiments of any of the aspects, the MAO enzyme is from a genus selected from Corynebacterium, Halomonas, Amycolatopsis, and Alteribacillus.

[0028] In some embodiments of any of the aspects, the MAO enzyme is from a species selected from Halomonas alkaliantarica, Amycolatopsis sp. KNN50.9B, and Alteribacillus bidgolensis . In some embodiments of any of the aspects, the MAO enzyme is from a genus selected from Halomonas, Amycolatopsis, and Alteribacillus.

[0029] In some embodiments of any of the aspects, the amino acid sequence of the MAO enzyme comprises one of SEQ ID NOs: 3-5 or an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of one of SEQ ID NOs: 3- 5 that maintains the same function of one of SEQ ID NOs: 3-5 (e.g., degradation of at least one amine-comprising substrate).

[0030] In some embodiments of any of the aspects, the MAO enzyme is from Halomonas alkaliantarica (UNIPROT A0A4R7YMS2; see e.g., Fig. 11A; SEQ ID NO: 3). In some embodiments of any of the aspects, the MAO enzyme is from Amycolatopsis sp. KNN50.9B (UNIPROT A0A229TV24; see e.g., Fig. 11A, Fig. 11C; SEQ ID NO: 4). In some embodiments of any of the aspects, the MAO enzyme is from Alteribacillus bidgolensis (UNIPROT A0A1G8QUH9; see e.g., Fig. 11B; SEQ ID NO: 5).

[0031] SEQ ID NO: 3, Halomonas alkaliantarica Monoamine oxidase (UNIPROT A0A4R7YMS2), 495 aa

[0032] SEQ ID NO: 4, Amycolatopsis sp. KNN50.9b Amino acid oxidase (UNIPROT A0A229TV24), 430 aa

[0033] SEQ ID NO: 5, Alteribacillus bidgolensis Monoamine oxidase (UNIPROT A0A1G8QUH9), 435 aa

[0034] In some embodiments of any of the aspects, the MAO enzyme is immobilized on an electrode of the biosensor. In some embodiments of any of the aspects, the MAO enzyme is immobilized on an electrode of the biosensor electrode with a polymer.

[0035] In a preferred embodiment, the MAO enzyme is immobilized on the electrode with chitosan. Chitosan was selected as a matrix for immobilization of the enzyme because of its biocompatibility, non-toxicity, high mechanical strength and excellent membrane forming ability. Chitosan can be divided into three categories, namely low molecular weight (e.g., 50 kDa-190 kDa), medium molecular weight (e.g., 190 kDa-310 kDa), and high molecular weight (e.g., 310 kDa-375 kDa). Chitosan of higher molecular weight possesses longer molecular chains with the availability of more hydroxyl groups. There is also a higher possibility that there are more amino groups, although the number of amino groups is determined by the degree of deacetylation. These amino groups are responsible for crosslinking. In some embodiments of any of the aspects, higher molecular weight chitosan (e.g., medium molecular weight compared to low molecular weight chitosan) can improve enzyme retention activity and loading, and thus function as a suitable matrix for enzyme immobilization. Using this chitosan membrane as a biosensor can provide a better performance in terms of sensitivity and stability. In some embodiments of any of the aspects, an amperometric biosensor, e.g., chronoamperometric biosensor can comprise chitosan of different molecular weights as a matrix for enzyme immobilization using a variety of adsorption and crosslinking techniques. See e.g., Ang et al., Study on Different Molecular Weights of Chitosan as an Immobilization Matrix for a Glucose Biosensor, PLoS One. 2013 Aug 5,8(8):e70597; Teepoo et al., Electrospun Chitosan-Gelatin Biopolymer Composite Nanofibers for Horseradish Peroxidase Immobilization in a Hydrogen Peroxide Biosensor, Biosensors (Basel). 2017 Oct 15 ;7(4). pii: E47. In some embodiments of any of the aspects, the MAO enzyme is immobilized on the working electrode of the biosensor as described further herein.

[0036] In some embodiments of any of the aspects, the MAO enzyme is immobilized on the electrode surface using crosslinking of a redox polymer. Generally, an aqueous mixture containing the enzymes, the redox polymer, and crosslinking agent in an aqueous solution are applied on an electrode and dried or allowed to dry to form a sensing film or coating on the electrode surface. See e.g., US patent publication US 2012/0181189.

[0037] In some embodiments of any of the aspects, the MAO enzyme is immobilized on the electrode surface on a self-assembled monolayer (SAM) comprising chemisorbed alkanethiols. In this embodiment, a gold electrode is preferred as the transducer in the sensor system since thiols chemisorb to gold to give a strong, stably bound layer. Other chemical groups suitable for adsorption to a metal surface include sulfates, sulfonates, phosphates, and selenides. In some embodiments, thiol chemisorption on gold yielding thiolate is preferred, due to the relative stability of the metal-sulfur bond. See e.g., US Patent 6,241,863, the content of which is incorporated by reference herein it its entirety.

[0038] In some embodiments of any of the aspects, the MAO biosensor is at a pH of about 8.0. In some embodiments of any of the aspects, the MAO biosensor is at a pH of about 7.5 to 8.5. In some embodiments of any of the aspects, the MAO biosensor is at a pH of about 7.5 to 9.0. In some embodiments of any of the aspects, the MAO biosensor is at a pH of greater than 7.0. ii. Amine -Comprising Substrates

[0039] In the aspects described herein, the MAO enzyme can specifically bind, react with, and/or degrade at least one amine-comprising substrate. As used herein the term “amine-comprising substrate” is a molecule that comprises at least one amine functional group, such as NH2 or NHU. Amines include functional groups that contain a basic nitrogen atom with a lone electron pair. Amines are derivatives of ammonia (NH₃), wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group.

[0040] In some embodiments, the at least one amine-comprising substrate is a monoamine substrate. A monoamine is a compound having a single amine group in its molecule. In some embodiments, the at least one amine-comprising substrate is a polyamine substrate. A polyamine is an organic compound having two or more (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amine groups. [0041] In some embodiments, the at least one amine-comprising substrate is a primary amine. A primary amine (1° amine) is an amine in which the amino group is directly bonded to one carbon of any hybridization which cannot be a carbonyl group carbon.

[0042] In some embodiments, the at least one amine-comprising substrate is a secondary amine. A secondary amine has two organic substituents (alkyl, aryl or both) bound to the nitrogen together with one hydrogen. In some embodiments, the at least one amine-comprising substrate is not a secondary amine.

[0043] In some embodiments, the at least one amine-comprising substrate is a tertiary amine. A tertiary amine is an amine in which the nitrogen atom is directly bonded to three carbons of any hybridization which cannot be carbonyl group carbons. In some embodiments, the at least one amine- comprising substrate is not a tertiary amine.

[0044] In some embodiments, the at least one amine-comprising substrate is selected from the group consisting of dopamine, octopamine, tyramine, norepinephrine, tryptamine, and hexylamine, or any combinations thereof (see e.g., Table 1).

[0045] In some embodiments, the at least one amine-comprising substrate is selected from the group consisting of dopamine, octopamine, tyramine, tryptamine, and hexylamine, or any combinations thereof (see e.g., Table 1).

[0046] In some embodiments, the at least one amine-comprising substrate is selected from the group consisting of dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and hexylamine, or any combinations thereof (see e.g., Table 6).

[0047] In some embodiments, the at least one amine-comprising substrate is selected from the group consisting of pentylamine, 5 -aminopentanol, 4-aminobutanol, and cadaverine, or any combinations thereof (see e.g., Table 6).

[0048] In some embodiments, the at least one amine-comprising substrate is selected from the group consisting of dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, hexylamine, pentylamine, 5 -aminopentanol, 4-aminobutanol, and cadaverine, or any combinations thereof (see e.g., Table 6).

[0049] In some embodiments, the at least one amine-comprising substrate is 5 -methoxytryptamine and/or hexylamine.

[0050] The MAO biosensors described herein can be used to detected 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and hexylamine, pentylamine, 5 -aminopentanol, 4-aminobutanol, and cadaverine.

[0051] In some embodiments, the at least one amine-comprising substrate is the monoamine dopamine. Dopamine (DA, a contraction of 3,4-dihydroxyphenethylamine) is a neuromodulatory molecule of the catecholamine and phenethylamine families. Dopamine constitutes about 80% of the catecholamine content in the brain. Dopamine is an amine synthesized by removing a carboxyl group from a molecule of its precursor chemical, L-DOPA, which is synthesized in the brain and kidneys. The brain includes several distinct dopamine pathways, one of which plays a major role in the motivational component of reward-motivated behavior. Other brain dopamine pathways are involved in motor control and in controlling the release of various hormones.

[0052] In some embodiments, the at least one amine-comprising substrate is the monoamine octopamine. Octopamine is an organic chemical closely related to norepinephrine, and synthesized biologically by a homologous pathway. Octopamine is often considered the major "fight-or-flight" neurohormone of invertebrates. Its name is derived from the fact that it was first identified in the salivary glands of the octopus. Octopamine is also found naturally in numerous plants, including bitter orange. Octopamine has been sold under trade names such as EPIRENOR, NORDEN, AND NORFEN for use as a sympathomimetic drug.

[0053] In some embodiments, the at least one amine-comprising substrate is the monoamine tyramine. Tyramine is a naturally occurring trace amine derived from the amino acid tyrosine. Tyramine acts as a catecholamine releasing agent. A hypertensive crisis can result from ingestion of tyramine-rich foods in conjunction with the use of monoamine oxidase inhibitors (MAOIs).

[0054] In some embodiments, the at least one amine-comprising substrate is the monoamine norepinephrine. Norepinephrine (NE), also called noradrenaline (NA) or noradrenalin, is an organic chemical in the catecholamine family that functions in the brain and body as both a hormone and neurotransmitter. The general function of norepinephrine is to mobilize the brain and body for action. Norepinephrine release is lowest during sleep, rises during wakefulness, and reaches much higher levels during situations of stress or danger, in the so-called fight-or-flight response. In the brain, norepinephrine increases arousal and alertness, promotes vigilance, enhances formation and retrieval of memory, and focuses attention; it also increases restlessness and anxiety. In the rest of the body, norepinephrine increases heart rate and blood pressure, triggers the release of glucose from energy stores, increases blood flow to skeletal muscle, reduces blood flow to the gastrointestinal system, and inhibits voiding of the bladder and gastrointestinal motility.

[0055] In some embodiments, the at least one amine-comprising substrate is the monoamine tryptamine. Tryptamine is an indolamine metabolite of the essential amino acid, tryptophan. Tryptamine has been shown to activate trace amine-associated receptors expressed in the mammalian brain, and regulates the activity of dopaminergic, serotonergic and glutamatergic systems. In the human gut, symbiotic bacteria convert dietary tryptophan to tryptamine, which activates 5-HT4 receptors and regulates gastrointestinal motility. Multiple tryptamine -derived drugs can be used to treat migraines.

[0056] In some embodiments, the at least one amine-comprising substrate is the monoamine 5- methoxytryptamine. 5 -Methoxytryptamine (5 -MT), also known as mexamine, is a tryptamine derivative closely related to the neurotransmitters serotonin and melatonin. 5-MT has been shown to occur naturally in the body in low levels. 5-MT is biosynthesized via the deacetylation of melatonin in the pineal gland. 5-MT acts as a full agonist at the 5-HT1, 5-HT2, 5-HT4, 5-HT6, and 5-HT7 receptors. 5-MT has no affinity for the 5-HT3 receptor and its affinity for the 5-HT1E receptor is very weak in comparison to the other 5-HT1 receptors.

[0057] In some embodiments, the at least one amine-comprising substrate is the monoamine hexylamine, hexylamine is colorless liquid and is one of the isomeric amines of hexane. At standard temperature and pressure, hexylamine has an ammonia/bleach odor common to amines and is soluble in almost all organic solvents. Hexylamine is primarily of interest in surfactants, pesticides, corrosion inhibitors, dyes, rubber, emulsifiers, and pharmaceuticals.

[0058] In some embodiments, the at least one amine-comprising substrate is the monoamine pentylamine. Pentylamine or 1 -aminopentane is used as a solvent, as a raw material in the manufacture of a variety of other compounds, including dyes, emulsifiers, and pharmaceutical products, and as a flavoring agent.

[0059] In some embodiments, the at least one amine-comprising substrate is the monoamine 5- aminopentanol (e.g., 5 -Amino- 1 -pentanol). 5 -Amino- 1 -pentanol undergoes facile intramolecular cyclocondensation with piperidine and methyl or ethyl piperidine in the presence of methanol or ethanol over zeolite catalyst. As non-limiting examples, 5 -Amino- 1 -pentanol has been used in the synthesis of S-glycosyl amino-acid building blocks, and 5 -Amino- 1 -pentanol has been used as starting reagent in the synthesis of amino-functionalized 4-chloro-2,2':6',2"-terpyridine.

[0060] In some embodiments, the at least one amine-comprising substrate is the monoamine 4- aminobutanol (e.g., 4-Amino-l -butanol). As non-limiting examples, 4 -Amino- 1 -butanol can be used: in the synthesis of cyclic amines; as a linker in the synthesis of highly branched poly([3-amino esters) (HPAEs) for gene delivery; as a side chain to modulate antimicrobial and hemolytic activities of copolymers; and/or in the total synthesis of (+)-fawcettimine,(+)-fawcettidine,[4] and (-)- lycoj apodine A

[0061] In some embodiments, the at least one amine-comprising substrate is the polyamine cadaverine. Cadaverine is an organic compound with the formula (CH₂)₅(NH₂)₂ Classified as diamine, cadaverine is a colorless liquid with an unpleasant odor. It is present in small quantities in living organisms but is often associated with the putrefaction of animal tissue.

[0062] In some embodiments, the at least one amine-comprising substrate is not serotonin, epinephrine, melatonin, putrescine, spermidine, spermine, S-nicotine, tyrosine, tryptophan, arginine, or lysine (see e.g., Table 6).

[0063] In some embodiments, the MAO enzyme of the biosensor has a high catalytic efficiency for the at least one amine-comprising substrate. One method to measure the catalytic efficiency of an enzyme is to determine the k_cat/K_m ratio. k_cat represents the turnover number and describes how many substrate molecules are transformed into products per unit time by a single enzyme. The K_m value gives a description of the affinity of the substrate to the active site of the enzyme. The k_cat/K_m ratio measures how effective the enzyme is on that particular substrate. The greater the k_cat/K_m ratio, the higher the rate of catalysis; conversely, the lower the k_cat/K_m ratio, the slower the catalysis.

[0064] In some embodiments, the MAO enzyme of the biosensor has a k_cat/K_m ratio for the at least one amine-comprising substrate of at least 1 x 10³ s⁴M⁴. In some embodiments, the MAO enzyme of the biosensor has a k_cat/K_m ratio for the at least one amine-comprising substrate of at least 1 x 10⁴ s’ ¹M⁴. In some embodiments, the MAO enzyme of the biosensor has a k_cat/K_m ratio for the at least one amine-comprising substrate of at least 1 x 10³ s⁴M⁴, at least 2 x 10³ s⁴M⁴, at least 3 x 10³ s⁴M⁴, at least 4 x 10³ s⁴M⁴, at least 5 x 10³ s⁴M⁴, at least 6 x 10³ s⁴M⁴, at least 7 x 10³ s⁴M⁴, at least 8 x 10³ S⁴M⁴, at least 9 x 10³ s⁴M⁴, at least 1 x 10⁴ s⁴M⁴, at least 2 x 10⁴ s⁴M⁴, at least 3 x 10⁴ s⁴M⁴, at least 4 x 10⁴ s⁴M⁴, at least 5 x 10⁴ s⁴M⁴, at least 6 x 10⁴ s⁴M⁴, at least 7 x 10⁴ s⁴M⁴, at least 8 x 10⁴ S⁴M⁴, at least 9 x 10⁴ s⁴M⁴, at least 1 x 10⁵ s⁴M⁴, at least 2 x 10⁵ s⁴M⁴, at least 3 x 10⁵ s⁴M⁴, at least 4 x 10⁵ s⁴M⁴, at least 5 x 10⁵ s⁴M⁴, or more (see e.g., Table 1, Table 6). In some embodiments, the MAO enzyme of the biosensor has a k_cat/K_m ratio for the at least one amine-comprising substrate of at least 3.5 x 10² s⁴M⁴. In some embodiments, the MAO enzyme of the biosensor has a K_m for the at least one amine-comprising substrate of at least 1 mM.

B. Detection of a signal from MAO catalysis of at least one amine-comprising substrate.

[0065] In some embodiments, an exemplary biosensor disclosed herein comprises a MAO enzyme as described herein. In one embodiment, the MAO biosensor detecting at least one amine-comprising substrate can operate with the following reaction steps shown in the reaction scheme 1 below:

[0066] where: MAO (ox) is oxidized form of MAO enzyme, MAO (red) - reduced form of the MAO enzyme, Med(ox) - oxidized from of redox mediator, Med(red) - reduced form of redox mediator.

[0067] In step 1, the MAO oxidizes at least one amine-comprising substrate to produce a product and the MAO itself is reduced. In step 2, the reduced form of the MAO oxidase reacts with oxygen in order to produce hydrogen peroxide. In step 3, hydrogen peroxide is oxidized into water while the oxidized form of the redox mediator Med(ox) is reduced to Med(red). In step 4, in the presence of an electric potential, the Med(red) is oxidized to regenerate Med(ox) and a measurable detectable signal is produced in the form of electrons. In some embodiments, the electrons are a detectable signal which can be measured as a current (amperometrically). [0068] A general reaction scheme 1 for detection of at least one amine-comprising substrate using a MAO enzyme through electrochemical methods (2^nd generation) can also be represented as shown in reaction scheme 2 as follows:

[0069] In some embodiments, at least one amine-comprising substrate can be identified through electrochemical methods (1^st generation) via detection with the MAO enzyme as shown in reaction scheme 3 as follows:

[0070] In some embodiments, at least one amine-comprising substrate can be identified through electrochemical methods (3^rd generation) using the MAO enzyme as shown in reaction scheme 4 as follows:

[0071] In some embodiments, at least one amine-comprising substrate can be identified through electrochemical methods using the MAO AD(P)/FAD-dependent oxidoreductase as shown in reaction scheme 5 as follows, where PB refers to “Prussian Blue” and can be substituted with another mediator (Med) (see e.g., Fig. 15A):

i. Redox mediators

[0072] In some embodiments, the MAO enzyme is not capable of transferring electrons to a redox mediator/electrically active mediator directly. That is, a detectable signal is not produced when the MAO enzyme interacts with at least one amine-comprising substrate. Accordingly, in some embodiments, at least one amine-comprising substrate is detected by a MAO enzyme that is capable of interacting directly with at least one amine-comprising substrate, but cannot exchange electrons with a redox mediator. Such an embodiment is useful where some redox-enzymes cannot exchange electrons directly with an electrode because their redox active sites are buried deep within the enzyme protein structure. Therefore, in order to transfer electrons between the redox active site of the enzyme and produce a detectable signal, a redox mediator (Med) also known as an “electron transfer agent” or an “electronically active mediator” is used. In some embodiments, the analyte -specific enzyme is cross-linked to the electron transfer agent. In some embodiments of any of the aspects, the amperometric biosensor, e.g., chronoamperometric biosensor described herein comprises an electronically active mediator deposited on the surface of the electrode.

[0073] In some embodiments, redox mediators are electroreducible and electrooxidizable ions or molecules having redox potentials (voltages) that are a few hundred millivolts above or below the redox potential (voltage) of the standard calomel electrode. In some embodiments, the redox mediators are not more reducing than about -150 mV and not more oxidizing than about +400 mV versus a standard calomel electrode. Examples of suitable redox mediators are disclosed, for example, in Mao et al. (U.S. Pat. No. 6,605,200) the entire content of which is herein incorporated by reference. [0074] Accordingly, the biosensor disclosed herein comprises redox mediators (Med) that serve as electron carriers, or electron signal mediators. That is, they are multi -electron transfer mediators that function as electrochemically detectable signal mediators to produce a detectable signal when transfer of electrons occurs. Preferably a redox-mediator as disclosed herein is linearly or exponentially amplified by magnifying electrochemical signal output via recycling the enzyme substrates.

[0075] In some embodiments of any of the aspects, the electronically active mediator (Med) can be reduced from a reduced form (Med_red) to a oxidized form (Med_ox), wherein the Med_ox produces a detectable signal, n some embodiments of any of the aspects, the detectable signal is produced when at least one amine -comprising substrate is catalyzed by the MAO enzyme and transfers at least one electron from Med_redto hydrogen peroxide (H₂O₂), resulting in its oxidation to Med_ox. In some embodiments of any of the aspects, the electronically active mediator Med_ox is reduced by the electrode, producing a detectable signal due to the flow of electrons. In some embodiments, the redox- mediator is catalyzed to produce a detectable signal that is electrochemical or colorimetric. In some embodiments, the redox-mediator produces an optical readout comprising fluorescence, bioluminescence, or luminescence.

[0076] A redox-mediator can be any of the natural or synthetic mediators commonly used in biosensors known to date, and is preferably selected from the group consisting of cytochromes, quinones, aminophenols, electron-acceptor aromatic compounds (e.g., TTF = tetratiafulvalene and NMP = N-methylphenazine), electron-donor aromatic compounds (e.g., TCNQ = tetrakyano-p- quinodimethane), organic conductive salts (e.g., TTF.TCNQ = tetratiafulvalene-7,7,8,8-tetrakyano-p- quinodimethane and NMP.TCNQ = N-methylphenylene-7 , 7,8,8-tetracyano-p-quinodimethane), organic dyes, metallocenes, organometallic complexes of osmium, ruthenium and duct, inorganic iron complexes. In some embodiments, redox mediators are ferricyanide ferrocene, 1,1 -dimethyl- ferrocene, hexacyanoferrate or hexacyanoferrate.

[0077] In some embodiments, the redox mediator is AUR as disclosed herein. Natural and artificial mediators are shown in Table 7.

[0078] Table 7

[0079] In some embodiments, the redox-mediator is a ferricyanide compound (i.e., Prussian blue). Prussian blue is a dark blue pigment produced by oxidation of ferrous ferrocyanide salts. It has the chemical formula Fe^III4[Fe^II(CN)₆]₃. The IUPAC name of Prussian Blue is iron(II,III) hexacyanoferrate(II,III), but it can also be referred to as Berlin blue, ferric ferrocyanide, ferric hexacyanoferrate, iron(III) ferrocyanide, iron(III) hexacyanoferrate(II), or Parisian blue. Prussian blue nanoparticles (PB NPs) exhibit an intrinsic peroxidase-like catalytic activity towards the hydrogen peroxide (H₂O₂)-mediated oxidation of classical peroxidase substrate 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt to produce a colored product. See e.g., PCT publication WO 1990/012487A2, which is incorporated herein by reference in its entirety. In some embodiments, the electronically active mediator comprises a ferricyanide compound which is reducible in the presence of an electron from hydrogen peroxide (H₂O₂) to produce a ferrocyanide compound. In some embodiments, the electronically active mediator comprises iron(II,III) hexacyanoferrate(II,III) (Prussian blue). ii. Optical or fluorescence detection of at least one amine -comprising substrate using a readout enzyme [0080] In some embodiments, the reaction between the MAO enzyme and at least one amine- comprising substrate is coupled to one or more additional enzymes, herein referred to an intermediate redox enzyme (e.g., IRE) to form a multi-enzyme system for detection of the redox reaction between at least one amine-comprising substrate and the MAO enzyme. In some embodiments of any of the aspects, the oxidase redox-enzyme of the amperometric biosensor, e.g., chronoamperometric biosensor described herein can be coupled to an intermediate redox-enzyme (IRE), for example, a peroxidase enzyme, as disclosed herein.

[0081] In some embodiments, the Med(red) can catalyze a redox reaction with a readout enzyme (ReadE) to convert a readout substrate (ReadS) into a readout product (ReadP), where the ReadP produces a detectable signal which can be measured optically, e.g., by fluorescence or other luminescence methods.

[0082] In some embodiments, a MAO biosensor detecting at least one amine-comprising substrate described herein can also comprise an intermediate redox-enzyme. For example, the Med(ox) is reduced to Med(red) in the presence of hydrogen peroxide and a peroxidase enzyme, thereby avoiding issues of auto-oxidation of the Med(red) and increasing the accuracy of measurement of analyte concentration.

[0083] In some embodiments, the biosensor comprising MAO can comprise use of a readout enzyme (ReadE), also referred to as an intermediate redox- enzyme (IRE), for example, a peroxidase enzyme, where the basic chemical and electrochemical transformation are shown with reference to an oxidase biosensor system in the following reaction scheme 6 below:

[0084] In step 1, the oxidase MAO oxidizes the amine-comprising substrate to produce a product and the MAO enzyme itself becomes reduced. In step 2, the reduced form of MAO reacts with oxygen to produce hydrogen peroxide and the oxidized form of MAO. In step 3, the hydrogen peroxide, in the presence of the readout enzyme (ReadE), oxidizes the readout substrate (ReadS) to form the readout product (ReadP) which is itself a readable signal. In some embodiments, the ReadP is a signal which can be measured optically or by luminescence techniques such as by fluorescence or chemiluminescence, and the measurement can be correlated to the concentration of the amine- comprising substrate.

[0085] A general reaction scheme for detecting an amine-comprising substrate using the MAO biosensor comprising a readout enzyme (ReadE), such as a peroxidase can also be represented as shown in reaction scheme 7 as follows:

[0086] In some embodiments, the conversion of Med(red) back to Med(ox) can produce a signal which can be measured as a current or amperometrically, and the measurement can be correlated to the concentration of the analyte.

[0087] In some embodiments, the readout enzyme (ReadE) (also referred to as “intermediate redox- enzyme (IRE)”) is a peroxidase enzyme, for example, but not limited to, APEX2 which, in the presence of hydrogen peroxidase, catalyzes the conversion of the readout substrate (ReadS) AMPLEX® UltraRed (AUR) to the readout product (ReadP) Resorufm, which produces a detectable signal. An exemplary reaction scheme for identification of an oxidase MRE using APEX2 as a peroxidase as a readout enzyme can be represented as shown in reaction scheme 8 as follows:

[0088] While the MAO biosensor is exemplified using components and electrochemical reactions schemes using caMAO as the monoamine oxidase, other at least one MAO enzymes from other species are encompassed for use herein (see e.g., Fig. 11A-11C, SEQ ID NOs: 3-5). [0089] In some embodiments, the intermediate redox enzyme (IRE) is a peroxidase enzyme, for example, but not limited to, APEX2, and the redox mediator (Med) is Amplex® UltraRed (AUR) that in the presence of hydrogen peroxidase and the peroxidase enzyme APEX2, is converted to Resorufm which produces a detectable signal.

[0090] In some embodiments, the technology encompasses alternative or modified Readout substrates (also referred to herein as intermediate redox enzymes (IREs)), for example, such as modifying the APEX2 enzyme to have improved enzyme kinetics or to use alternative peroxidase enzymes, or HRP derivatives. Additionally, in some embodiments, the redox-mediator, such as AUR, can be replaced with other H₂O₂ responsive probes known in the art, for example, but not limited to PY1, PO1, Amplex® Red, homovanillic acid (HVA), luminol, OPD, DCFH, ABST, K iodide, or ABTS. In some embodiments, other redox enzymes can be used, for example, the peroxidase IRE APEX2 can be substituted for another enzyme, or peroxidase enzyme which is sensitive to other enzyme products. In some embodiments, transcription factors (TFs) can be used to respond to the produced H₂O₂. In some embodiments, OxyR, a TF responsive to H₂O₂, can be used to regulate and induce GFP expression downstream of an OxyR binding site. In some embodiments, the redox- responsive probe (RRP) is catalyzed to produce a detectable signal that is fluorescence, or bioluminescence, luminescence or produce an optical readout or detectable signal.

[0091] By way of an illustrative example only, in some embodiments, the intermediate redox enzyme (IRE) is a peroxidase enzyme, such as, but not limited to, ascorbate peroxidase (APEX2), and the redox-mediator (Med) is, but not limited to, Amplex® UltraRed (AUR), which produces a fluorescent product only in the presence of hydrogen peroxide (H₂O₂) and the peroxidase enzyme as the IRE. In this illustrative example, the biosensor comprising a MAO enzyme which degrades at least one amine-comprising substrate will produce a fluorescent signal, because the oxidase will produce H₂O₂, which is used as a substrate, along with AUR, for the peroxidase enzyme, APEX2 to produce a fluorescent product.

[0092] APEX2 is an engineered ascorbate peroxidase enzyme that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. In some embodiments, APEX2 can be used as an IRE to catalyze the generation of a fluorescent product from Amplex® UltraRed (AUR) only in the presence of hydrogen peroxide (H₂O₂). In some embodiments, the assays, methods, and compositions as disclosed herein can also use modified version of the APEX2 enzyme, e.g., a modified APEX2 enzyme with improved enzyme kinetics or catalyzes a different HRP derivative (e.g., catalyzes a different substrate to AUR).

[0093] In some embodiments, the technology encompasses alternative or modified intermediate redox enzymes (IREs), for example, such as modifying the APEX2 enzyme to have improved enzyme kinetics or to use alternative peroxidase enzymes, or HRP derivatives. Additionally, in some embodiments, the redox-mediator, such as AUR, can be replaced with other H₂O₂ responsive probes known in the art, for example, but not limited to PY1, PO1, Amplex® Red, homovanillic acid (HVA), luminol, OPD, DCFH, AB ST, K iodide, or ABTS. In some embodiments, other redox enzymes can be used, for example, the peroxidase IRE APEX2 can be substituted for another enzyme, or peroxidase enzyme which is sensitive to other enzyme products. In some embodiments, transcription factors (TFs) can be used to respond to the produced H₂O₂. In some embodiments, OxyR, a TF responsive to H₂O₂, can be used to regulate and induce GFP expression downstream of an OxyR binding site. In some embodiments, the redox-responsive probe (RRP) is catalyzed to produce a detectable signal that is fluorescence, or bioluminescence, luminescence or produce an optical readout or detectable signal. [0094] In some embodiments of any of the aspects, the one or more fluorescence probes to detect H₂O₂ is 10-Acetyl-3,7-dihydroxyphenoxazine (N-Acetyl-3,7-dihydroxyphenoxazine or ADHP) (Amplex® Red). 10-Acetyl-3,7-dihydroxyphenoxazine is highly specific and stable. The substrate itself is nearly colorless and nonfluore scent until it is oxidized by H₂O₂ (reacting in a 1 : 1 stoichiometry) in the presence of horseradish peroxidase (HRP) to become the highly red fluorescent resorufin.

[0095] The structure of 10-Acetyl-3,7-dihydroxyphenoxazine (N-Acetyl-3,7-dihydroxyphenoxazine is as follows:

[0096] Additionally, in some embodiments, the redox-mediator, such as AUR, can be replaced with other H₂O₂ responsive probes known in the art, for example, but not limited to PY 1, PO1, Amplex® Red, homovanillic acid (HVA), luminol, OPD, DCFH, ABST, K iodide, or ABTS.

[0097] In some embodiments, other redox enzymes can be used as intermediate redox enzymes, for example, the peroxidase IRE APEX2 can be substituted for another enzyme, or peroxidase enzyme which is sensitive to other enzyme products.

[0098] Other intermediate redox enzymes (IRE) are encompassed for use in this assay system can be selected from a nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase, a flavin adenine dinucleotide (FAD)-dependent oxidase, or a flavin mononucleotide (FMN)-dependent oxidase. For example, in some embodiments, the IRE of this system is selected from 1 ip-hydroxysteroid dehydrogenase type 2 (1 ip-HSD-2), glucose oxidase, NAD-glucose dehydrogenase, FAD-glucose dehydrogenase, lactate oxidase, NAD-lactate dehydrogenase, NAD-alcohol dehydrogenase, pyruvate oxidase, NAD-glutamate dehydrogenase, and xanthine oxidase. [0099] In some embodiments, transcription factors (TFs) can be used as an alternative to an IRE and coupled to the candidate redox-enzyme to respond to the produced H₂O₂. In some embodiments, the transcription factor coupled to detect the interaction of the target analyte and the redox-enzyme is OxyR, which is a transcription factor responsive to H₂O₂, and in some embodiments, be used to regulate and induce gene expression, such as GFP expression, downstream of an OxyR binding site. [00100] Accordingly, in some embodiments, transcription factors (TFs) can be used to respond to the H₂O₂ produced by the MAO enzyme. In some embodiments, OxyR, a TF responsive to H₂O₂, can be used to regulate and induce GFP expression downstream of an OxyR binding site. OxyR encodes a transcription factor that senses H₂O₂ and is activated through the formation of an intramolecular disulfide bond. OxyR activates the expression of a regulon of hydrogen peroxide-inducible genes including but not limited to katG, gor, ahpC, ahpF, oxyS, dps, fur and grxA. OxyR expression is negatively autoregulated by binding to a 43 bp region upstream of its own coding sequence. OxyR is inactivated by reduction of its essential disulfide bond by the product of GrxA, itself positively regulated by OxyR. The following sequence of OxyR is provided: OxyR amino acid-encoding polynucleotide sequence, e.g. P0ACQ4-1 (SEQ ID NO: 6):

C. Electrode(s)

[00101] Optimization of the surface of the electrode can be performed to produce for a functional biosensor comprising MAO enzyme. In particular, multiple parameters of the electrode of the MAO biosensor disclosed herein can be optimized to achieve a biosensor with real-time measurements of at least one amine-comprising substrate, is sensitive to physiological levels of at least one amine- comprising substrate, and has repeated detection of at least one amine-comprising substrate in a variety of different samples. In some embodiments, the surface of the electrode on which MAO enzyme is located can be optimized to comprise low- and medium molecular weight (MW) chitosan in 0.5% acetic acid. In some embodiments, the surface area and size of the electrode can be increased or decreased. In some embodiments, the biosensor device can consist of only two electrodes.

[00102] In some embodiments of any of the aspects, the amperometric biosensor, e.g., chronoamperometric biosensor as described comprises at least one electrode. In some embodiments, the MAO biosensor comprises two electrodes. In some embodiments, the MAO biosensor consists of, or consists essentially of two electrodes, a working electrode and a reference electrode, where the working electrode has a MAO enzyme deposited on it. In some embodiments, the MAO biosensor does not comprise a third electrode. In some embodiments, the MAO biosensor disclosed herein does not comprise a counter electrode. In some embodiments, the MAO biosensor disclosed herein further comprises a counter electrode. In some embodiments, the MAO biosensor comprises three electrodes. In some embodiments, the MAO biosensor consists of, or consists essentially of three electrodes, a working electrode, a reference electrode, and a counter electrode, where the working electrode has a MAO enzyme deposited on it. In some embodiments of any of the aspects, the working electrode comprises a surface, for example to which MAO enzymes and optionally, a Readout enzyme can be immobilized as described herein.

[00103] In some embodiments of any of the aspects, the electrode is metallic. In some embodiments of any of the aspects, the metallic electrode is gold, silver, platinum, or palladium. In some embodiments of any of the aspects, the electrode is non-metallic. In some embodiments of any of the aspects, the non-metallic electrode comprises carbon (e.g., glassy carbon).

[00104] In some embodiments of any of the aspects, the amperometric biosensor, e.g., chronoamperometric biosensor comprises one electrode referred to herein as a “working electrode.” The working electrode is the electrode in an electrochemical system on which the reaction of interest is occurring. Accordingly, herein the MAO biosensor disclosed herein comprises a working electrode comprising MAO enzyme deposited on the working electrode. Typically, the working electrode is often used in conjunction with a counter electrode and a reference electrode in a three-electrode system. However, in specific embodiments of the MAO biosensor disclosed herein, the working electrode is used in conjunction with a reference electrode but not the counter electrode. In some embodiments of any of the aspects, the working electrode is coated with the redox mediator (e.g., Prussian blue). In some embodiments of any of the aspects, the MAO enzyme is immobilized to the surface of the working electrode with a polymer.

[00105] In some embodiments of any of the aspects, the amperometric MAO biosensor, e.g., chronoamperometric biosensor disclosed herein further comprises an auxiliary electrode. As used herein, the term “counter electrode” (also referred to as the “auxiliary electrode”) is an electrode used in an electrode electrochemical cell for voltammetric analysis or other reactions in which an electric current is expected to flow.

[00106] In some embodiments of any of the aspects, the amperometric MAO biosensor, e.g., chronoamperometric MAO biosensor can optionally comprise a reference electrode. As used herein, the term “reference electrode” is an electrode which has a stable and well-known electrode potential. The high stability of the electrode potential is usually reached by employing a redox system with constant (buffered or saturated) concentrations of each participant of the redox reaction. The counter (or auxiliary) electrode is distinct from the reference electrode, which establishes the electrical potential against which other potentials may be measured, and the working electrode, at which the analyte detection (e.g., at least one amine-comprising substrate detection) by MAO takes place. [00107] In some embodiments, the MAO biosensor disclosed herein is a two-electrode system (e.g., working and reference electrodes), where either a known current or potential is applied between the working and reference electrodes and the other variable may be measured. The counter electrode functions as a cathode whenever the working electrode is operating as an anode and vice versa. In some embodiments, the counter electrode often has a surface area much larger than that of the working electrode to ensure that the half-reaction occurring at the counter electrode can occur fast enough so as not to limit the process at the working electrode.

[00108] In some embodiments, the size of the working electrode can influence the current produced from the MAO biosensor. Accordingly, in some embodiments, the working electrode has an area of >4π mm². In some embodiments, the size of the working electrode is in the range of 5-8 π mm², or 8- 10π mm², or 10-12π mm², or 12-15π mm², or greater than 15π mm². In some embodiments, the shape of the working electrode is circular, and in some embodiments the shape is oval. In some embodiments, the reference electrode is a geometric shape which wraps or circles around the working electrode.

[00109] When a three-electrode cell (e.g., working, counter, and reference electrodes) is used to perform electroanalytical chemistry, the auxiliary electrode, along with the working electrode, provides a circuit over which current is either applied or measured. Here, the potential of the counter electrode is usually not measured and is adjusted so as to balance the reaction occurring at the working electrode. This configuration allows the potential of the working electrode to be measured against a known reference electrode without compromising the stability of that reference electrode by passing current over it.

[00110] In some embodiments of any of the aspects, the amperometric MAO biosensor, e.g., chronoamperometric biosensor comprises, or consist essentially of, a working electrode and a reference electrode. In some embodiments of any of the aspects, the biosensor can comprise a multi- electrode configuration including a working electrode, a counter electrode, and a reference electrode. In one example, the MAO enzyme and optionally, readout enzyme or redox mediator are immobilized on the working electrode. The working electrode can be, for example, carbon, glassy carbon, metal, metal oxides or a mixture of carbon and metal or metal oxides. In one example, the working electrode is a glassy carbon electrode. The reference electrode can be, for example, a saturated calomel reference electrode (SCE), Ag/AgCl, or saturated Hg₂Cl₂. In some embodiments, the counter or reference electrode can be, for example, a metal such as gold, silver, platinum, or stainless steel, such as a metal wire counter or reference electrode. In some embodiments, the working and/or the reference electrodes, and optionally the counter electrode are screen printed electrodes (SPE). [00111] The biosensor electrodes, such as active electrodes, can be formed by coating a fine metal wire with a formulation (e.g., comprising the MAO enzyme, redox mediator, and/or polymer such as chitosan) and drying the coating in place on the wire. For example, a fine platinum wire can be coated with the enzyme-formulation and the coating dried in place. The coated wire can be arranged in a syringe or other suitable flow cell or channel device that can be placed, for example, in-line or into the flow of an analyte stream to be monitored for the concentration of at least one amine-comprising substrate.

[00112] Platinum, silver, carbon, and Ag/AgCl ink also can be used in screen-printing methods, or photolithographically patterned metal vapor deposition methods, to form film sensors for the fabrication of miniaturized, planar, solid-state electrodes. These electrodes can be used in electrode strips, biochips, and other miniaturized sensor configurations. The biosensor can be, for example, a screen-printed (e.g., a screen-printed electrode (or SPE)) or photolithographically patterned three- electrode transducer with a carbon or platinum working electrode. In some embodiments, the biosensor can be screen-printed or inkjet printed as disclosed in WO2019/224628, which is incorporated herein in its entirety by reference. As a non-limiting example, in some embodiments of any of the aspects, the working electrode comprises a Prussian blue screen-printed electrode. See e.g., US patent publication 2008/0160625, PCT publications WO2 017210465, WO2018202793 A2, Chinese patent publications CN110573868, CN 102053161, CN105136885; the contents of each of which are incorporated herein by reference in their entireties. Other transducer configurations also can be used.

[00113] Referring to FIG. 15B, in one example, an amperometric MAO biosensor for use herein has the configuration of an electrode test strip 1 having an electrode support layer 6, a redox-enzyme and redox-mediator coated-working electrode 2 a disposed on the support layer 6, and a counter electrode 2 b and reference electrode 2 c spaced from the working electrode 2 a and disposed on the support. A covering layer 7 defines an aperture 4 that opens into a recessed space or well 8 having walls defined by layer 7 and a bottom defined by layer 6. As shown, the electrodes 2 a, 2 b, and 2 c are situated in well 8. The electrodes are left exposed in well 8, such that sample fluid can be received in well 8 to contact the electrodes. The working electrode 2 a comprises a coating of the redox-enzyme and redox-mediator composition immobilized to a conductive electrode material, such as referenced herein. The counter electrode 2 b is a conductive electrode material without the coating of the redox-enzyme and redox-mediator composition. The electrode support 6, typically an elongated strip of electrical insulating polymeric material, e.g., PVC, polycarbonate, or polyester, supports two or more printed tracks of electrically conducting carbon ink 5. The conducting inks 5 are hidden in the view of FIG. 15B, and are represented by hatched lines. These printed tracks define the positions of the working, counter, and reference electrodes, and of the electrical contacts 3 that are operable to be inserted into an appropriate measurement device (not shown; e.g., a potentiostat). The covering layer 7 also can be an electrical insulating polymeric material. The insulating layers 6 and 7 can be, for example, hydrophobic insulating polymeric material. FIG. 15C further shows the electrodes as positioned in the well 8, where they can be contacted and covered by fluid sample during measurements. In addition to the arrangement shown, the working, counter, and reference electrodes can be arranged in other configurations relative to each other within recess well 8. The working, reference and counter electrodes can be spaced, for example, from about 0.25 mm to about 0.5 mm, and the working, counter, and reference electrodes can have a width, for example, of about 0.5 mm to 1.5 mm, and a length, for example, of from about 1.5 mm to about 2.5 mm, or other dimensions.

[00114] In some embodiments of any of the aspects, the amperometric MAO biosensor, e.g., chronoamperometric biosensor comprises an electrochemical sensor strip. In some embodiments of any of the aspects, the electrochemical sensor strip comprises: an insulation substrate; a first conduction film mounted on the insulation substrate and having a first and a second ends; and an insulation layer mounted on the first conduction film to cover the first end, wherein the second end serves as a signal output terminal, the first end has a first conduction section exposed between the insulation layer and the insulation substrate, and the first conduction section serves as a working surface of an electrode (i.e., working electrode) of the electrochemical sensor strip. See e.g., European patent publication 1845371, the content of which is incorporated herein by reference in its entirety. [00115] In some embodiments, the biosensor electrode is contained within a miniaturized device to further facilitate sample quantification. This amperometric microbiosensor comprises several components. First, a metal wire with a working end to be further electroplated with a noble metal serves as the working electrode. This biosensing electrode forms the working electrode about which an encasement is then drawn. The working electrode within the encasement is further drawn to a tip of about 1-20 μm in diameter. A Ag/AgCl wire is then inserted into the encasement wherein the Ag/AgCl wire serves both as a reference and counter electrode. Finally, an electrolyte filler is inserted into the encasement to complete the microbiosensor.

D. Detection of signals from Redox Mediators

[00116] The technology described herein relates to a biosensor useful for the accurate, reliable and sensitive measurement of at least one amine-comprising substrate in at least the environmental, industrial, or clinical setting.

[00117] The redox-enzyme biosensor generates an electrochemically or non-electrochemically detectable product or by-product directly, or alternatively, the enzyme system can also include at least one further component, such as an intermediate redox enzyme (IRE) as disclosed herein. In some embodiments, the further component may be: one or more additional enzyme(s) forming an enzymatic pathway utilizing the product or by-product of the initial redox-enzyme reaction to thereby generate a photometrically or electrochemically detectable product or by-product; or at least one signal mediator; or both the additional enzyme(s) and the signal mediator(s). The signal mediator(s) may be selected from, for example: indicators, such as a pH-change indicators; electron transfer mediators; photometric mediators, and other components.

[00118] In some embodiments in an electrochemical embodiment of the assay, the redox-enzyme system utilizes an electrochemically detectable cofactor, such as FADH₂ or NADH, or generates a by- product, such as H₂O₂, during the course of the enzymatic reaction with at least one amine-comprising substrate.

[00119] For illustrative purposes only, the oxidase enzyme can be conjugated to an electroactive molecule and the analyte probe is attached or on the surface of a conducting surface of a semiconductor device, such that when the oxidase enzyme is bound to an amine-comprising substrate, the electroconductive molecule conjugated to the oxidase enzyme and the amine-comprising substrate are in close proximity to allow electron transfer, and the flow of electrons to the semiconductor device which is detected by an increase in current on the surface.

[00120] In another embodiment, electrochemical impedance spectroscopy can be used to measure the resistance of the system by using redox markers.

[00121] Other methods to modify the MAO enzyme and analyte probes for electrochemical detection of the analyte and generating electroconductive biosensors for use herein are described in Electrochemical methods - Fundamentals and applications, 2Ed., Allen Bard and Larry Faulkner, and Electrochemistry for biomedical researchers, Richie L C Chen, World Scientific Press, each of which are incorporated herein in their entirety by reference.

[00122] In some embodiments of any of the aspects, detection of at least one amine-comprising substrate is not limited to electrochemical means, and the redox-enzyme system disclosed herein may employ different detection methods, e.g., UV, fluorescence, or other suitable methods of detecting the target analyte and redox-enzyme interactions.

[00123] Non-electrochemical detection of redox-mediators (Med) involves, for example, any calorimetric or photometric detection mode known in the art (for example, any colorimetric, spectrometric, spectrophotometric, luminescence-based, chemiluminescence-based, or fluorescence- based detection method.)

[00124] A fluorescence detection device can comprise the following conditions or components: it can be light-tight to eliminate stray light from its surroundings, its fluorophores can be stored in the dark to prevent photobleaching (that is increase shelf life), and its optics can be at a 90° angle. A diode emitting the desired excitation wavelength can function as the light source, and a PMT can function as the detector. These need not be elaborate since both the excitation and emission λmax of the fluor are known, and these are the only wavelengths required. The same redox-enzyme and redox-mediator used in an enzyme electrochemical device can be used in a fluorescence device. A portable fluorescence detector for aflatoxin has been described in the literature (M A Carlson et al., An automated handheld biosensor for aflatoxin, Biosens. Bioelectr. 14:841 (2000)).

[00125] Both direct and indirect fluorescence allows the detection of at least one amine-comprising substrate and redox-enzyme interactions, via the redox-mediator. The tCCf-gcncrating systems can use H₂O₂ and an additional fluor. In these systems, H₂O₂ production causes an increase in fluorescence intensity that is proportional to the at least one amine-comprising substrate concentration.

[00126] Indirect fluorescence of FADH₂ or NADH can be detected using the dye, such as rhodamine 123. In some embodiments, non-radiative energy transfer (also called fluorescence resonance energy transfer, FRET) occurs between the excited states of FADH₂ or NADH and rhodamine 123. FRET is a well-known technique for determining the proximity of two species, i.e., FRET is utilized as a “molecular yardstick” both in vitro and in vivo. In this context of the target analyte and redox-enzyme interactions, a donor fluorophore, e.g., FADH₂ orNADH, transfers its excited state energies to the acceptor fluorophore, e.g., rhodamine 123. (R P Haugland, Handbook of Fluorescent Probes and Research Products, 2002 (9th ed.; Molecular Probes, Inc.; Eugene, Oreg.); K Van Dyke et al., eds. Luminescence Biotechnology. Instruments and Applications, 2002 (CRC Press; Boca Raton, Fla.) and references contained therein). The NADH-rhodamine 123 FRET method has been successfully employed in other enzymatic assays (M H Gschwend et al., Optical detection of mitochondria) NADH content in intact human myotubes, Cell. Mol. Biol. 47:OL95 (2001); H. Schneckenberger et al., Time-gated microscopic imaging and spectroscopy in medical diagnosis and photobiology, Opt. Eng. 33:2600 (1994)). Bioluminescence resonance energy transfer, or BRET, may also be used in conjunction with a MAO enzyme system. In BRET, the donor fluorophore is replaced by a luciferase. Bioluminescence from luciferase in the presence of a substrate excites the acceptor fluorophore. BRET has also been applied in vitro and in vivo (K Van Dyke et al., 2002).

[00127] ATP can be derivatized with a fluorophore for indirect fluorescence. Several commercially available dyes include BODIPY ATP and trinitrophenyl ATP (Haugland, 2002). These analogs change their fluorescence intensity or become fluorescent when bound to an enzyme's ATP binding site.

[00128] Indirect fluorescence detection of H₂O₂ has also been reported (Carr & Bowers, 1980). These methods utilize dyes that reduce the peroxide to H₂O and are themselves oxidized. Homovanillic acid (4-hydroxy-3 -phenylacetic acid) and p-hydroxyphenylacetic acid are among the most commonly used in clinical chemistry (Can and Bowers, 1980). A commercially available kit uses the dye Amplex® Red for fluorescence detection of H₂O₂ (Haugland, 2002).

[00129] Any fluorescent dyes and fluorescence -detectable enzyme substrate or cofactor analogs can be used in a fluorescence device to detect at least one amine-comprising substrate and MAO redox- enzyme interactions. [00130] In one embodiment, fluorescent molecule detection can be achieved using a number of detection systems. The choice of a proper detection system for a particular application is well within the abilities of one skilled in the art. Exemplary optical detection system capable of detecting the fluorescence means include, but are not limited to, detection by unaided eye, Fluorescence activated cell sorting (FACS), light microscopy using the eye or an optical sensor as the detector, confocal microscopy, laser scanning confocal microscopy, imaging using quantum dot color, fluorescence spectrum or other quantum dot property and wide-field imaging with a 2 D CCD camera and a high numerical aperture microscope objective. An exemplary laser-based microscope system capable of detecting and spectrally resolving the fluorescence from single semiconductor nanocrystals is known in the art.

[00131] In some embodiments, the optical detection system may or may not comprise at least one source of excitatory light, such as at least one laser. A source of excitatory light is not needed to detect objects which luminesce independently of light absorption, such as can be generated via bioluminescence or chemiluminescence, for example. In some embodiments, an optical detection system useful herein to measure fluorescence may comprise a light detector detecting light emitted from the object. The light detector is capable of at least partially absorbing light incident thereon and generating output signals in response to the light. The light detector can comprise a control circuit for controlling the operation of the light detector. The control circuit can comprise a circuit of signal amplifier, A/D convertor, integrator, comparator, logic circuit, readout circuit, memory, microprocessor, clock, and/or address.

[00132] In some embodiments, the detecting apparatus may comprise a computer for processing output signals from the light detector and generating a determination result. The detecting apparatus may further comprise a blind sheet with a pinhole. The apparatus may further comprise an excitation light source. The object may absorb light emitted from the excitation light source and then emit another light to be detected by the detecting apparatus. The light emitted from the object may have different wavelength than the light emitted from the excitation light source.

[00133] In some embodiments, the detection device, e.g., optical sensor or semiconductive device allows point of care testing (POCT), that is, the subject can perform all the relevant step in analyte (e.g., at least one amine-comprising substrate) detection, including obtaining the sample, applying the sample to the biosensor, placement in the reader device (e.g., optical sensor or semiconductive device), which can transmit the results to a mobile device (e.g., a mobile phone or smartphone, IPAD, tablet, smartwatch), or other interface, e.g., cloud to be accessed by the subjects clinical practitioner. [00134] Chemiluminescence (CL) and electrogenerated chemiluminescence (ECL) (collectively referred to herein as “(E)CL”) are widely used in medical diagnostics and analytical chemistry (C Dodeigne et al., Chemiluminescence as a diagnostic tool: A review, Taianta 2000, 51:415; K A Fahnrich et al., Recent applications of electrogenerated chemiluminescence in chemical analysis, Taianta 2001, 54:531). Enzyme-based (E)CL systems are sensitive and specific, and many CL systems are used with enzyme cycling to detect H₂O₂ (Dodeigne et al., 2000). (E)CL can detect picomolar (pM; 10-12M) concentrations of analyte over a wide linear range (Dodeigne et al., 2000; Fahnrich et al., 2001). An (E)CL device can be constructed in accordance with the following principles. Since the reaction itself emits light, an (E)CL device does not need a light source. A photomultiplier tube (PMT) can function as the detector; (E)CL is visible to the unaided, dark-adapted eye. A battery can be the power source for ECL. ECL requires electrodes and a source of applied potential. Like a fluorescence detection device, (E)CL devices can be light-tight and their reagents can be protected from light until use. Also, like fluorescence, (E)CL can use derivatized reagents or additional enzymes and reagents to detect the at least one amine-comprising substrate. (E)CL devices can be used with disposable strips (B D Leca et al., Screen-printed electrodes as disposable or reusable optical devices for luminol electrochemiluminescence, Sens. Actuat. B. 2001, 74: 190) and can be miniaturized (Y Lv et al., Chemiluminescence biosensor chip based on a microreactor using carrier airflow for determination of uric acid in human serum, Analyst 2002, 127: 1176).

[00135] An optical electrode (or optrode) can be fabricated using for detection of the target analyte and MAO redox-enzyme interactions. For example, an optrode such as that used in a glucose optrode that uses ECL, can be employed (see C H Wang et al., Co-immobilization of polymeric luminol, iron(II) tris(5 -aminophenanthroline) and glucose oxidase at an electrode surface, and its application as a glucose optrode, Analyst 2002, 127: 1507)).

[00136] CL systems can involve the detection of H₂O₂ or another reactive oxygen species (Carr & Bowers, 1980; Haugland, 2002; Dodeigne et al., 2000; K Van Dyke et al., 2002) and references contained therein). One exemplary system is luminol-peroxidase. In basic solution, H₂O₂ oxidizes luminol to an excited amino-phthalate ion; the excited amino-phthalate ion emits a 425 -nm photon to return to its ground state. When used in medical diagnostics, this reaction is catalyzed with horseradish peroxidase (HRP) (Carr & Bowers, 1980; Dodeigne et al., 2000). Thus, any enzyme system, such as MAO, that produces H₂O₂ or requires a cofactor that can react with additional reagents to form H₂O₂ can be used in a CL device. The H₂O₂-generating systems described herein can use luminol -HRP directly for detection of at least one amine-comprising substrate. These enzyme cycling schemes increase the light emission over time because the substrates are continuously recycled (Dodeigne et al., 2000). While luminol itself is frequently used in CL, its improved analogs can also be used in a CL-based detector, in place of luminol, in order to increase the sensitivity. Examples of such analogs are those described in Carr & Bowers, 1980; and Dodeigne et al., 2000. [00137] FADH₂ or NADH detection using CL can be used (Dodeigne et al., 2000). For example, in the presence of l-methoxy-5-methylphenazinium methylsulfate, NADH (or FADH₂) reduces O₂ to H₂O₂ which generates light using the luminol-peroxidase system (Dodeigne et al., 2000). For a MAO biosensor, the O₂ in ambient air can be sufficient to detect at least one amine-comprising substrate using this system. NADH also reacts with oxidized methylene blue to form H₂O₂ that reacts with luminol (Carr and Bowers, 1980). FADH₂ or NADH can also act as a CL quencher. The fluorescence intensity of the substrate ALPDO is decreased in the presence of NADH and HRP (Van Dyke et al., 2002). FADH₂ or NADH also can be used with Ru(bpy)3 2+ for ECL (E S Jin et al., An electrogenerated chemiluminescence imaging fiber electrode chemical, sensor for NADH, Electroanal. 2001, 13(15): 1287). Rhodamine B isothiocyanate can also be used for ECL detection of H₂O₂ (Fahnrich et al., 2001). ECL also offers another advantage in that, by use of a properly poised electrode, the electroactive species can be regenerated at the electrode surface. Regeneration both conserves reagents and allows durable and/or “reagentless” sensors. All these systems can be used in a (E)CL device interfaced to a MAO enzyme system as described herein.

[00138] MAO reactions using co-enzymes FAD or NAD can use ATP. CL is widely used to quantitate ATP simply and sensitively (Carr & Bowers, 1980). The enzyme luciferase catalyzes the reaction of ATP and luciferin to produce excited-state oxyluciferin, which returns to its ground state with the emission of a 562-nm photon (Carr & Bowers, 1980; Haugland, 2002). The quantum yield for this reaction is very high; 10-14 mol ATP can be detected. A kit for this reaction is commercially available (Haugland, 2002). Because luciferase is the enzyme that causes fireflies to “glow,” this reaction is referred to as bioluminescence. Both native and recombinant luciferase are commercially available, and several groups have reported using bioluminescence ATP assays to quantify biological analytes (P Willemsen et al., Use of specific bioluminescence cell lines for the detection of steroid hormone [ant]agonists in meat producing animals, Anal. Chim. Acta 2002, 473: 119; S J Dexter et al., Development of a biolumine scent ATP assay to quantify mammalian and bacterial cell number from a mixed population, Biomat. 2003, 24:nb27).

[00139] In addition to the luminol-HRP system, H₂O₂ can also be detected using peroxyoxalic acid derivatives (Dodeigne et al., 2000). H₂O₂ can also be detected with CL non-enzymatically with ferricyanide as the catalyst (Dodeigne et al., 2000). In these (E)CL systems, detection of the target analyte and redox-enzyme interactions as described herein either produce H₂O₂ or require cofactors that can be utilized to form H₂O₂.

[00140] Optical biosensors can use photometric detection (that is, absorbance, fluorescence) of substrates consumed or products formed by the reaction catalyzed by the enzyme system incorporated into the sensor. The target analyte and redox-enzyme reactions as described can be monitored by several photometric methods-namely by measuring NAD(P)H or FAD absorbance at about 340 nm for the pyridine nucleotide-dependent enzymes or absorbance of the quinoneimine dye for the H₂O₂ forming enzyme systems. For the later, addition of a peroxidase allows detection of H₂O₂ by catalyzing the reduction of H₂O₂ with concomitant oxidation of a dye compound that upon oxidation absorbs at a specified wavelength. Peroxidase enzymes (for example, commercially available horseradish peroxidase) can have broad substrate specificities so several different electron donor compounds can be used. NAD(P)H or FAD consumption may also be measured by fluorescence detection (excitation at 350 nm and emission at 450 nm).

[00141] Calorimetry may be employed as a detection means to detect the target analyte and redox- enzyme interactions. Chemical reactions can be either exo- or endothermic; that is, they release or absorb heat as they occur. Calorimeters detect and measure this heat by measuring a change in the temperature of the reaction medium (K Ramanathan & B Danielsson, Principles and applications of thermal biosensors, Biosens Bioelectr. 16:417 (2001); B Danielsson, Enzyme Thermistor Devices. In Biosensor Principles and Applications. Vol. 15, pp. 83-105 (L J Blum & P R Coulet, eds.; Bioprocess Technology Series, volume 15; Marcel Dekker, Inc: New York, 1991, pp. 83-105, and references contained therein). Thus, the action of the target analyte (e.g., at least one amine-comprising substrate) and MAO redox-enzyme interactions can be monitored calorimetrically. Calorimeters have been designed that are sensitive enough to detect protein conformational changes, and calorimetry has been used to study many enzymatic reactions in detail (M. J. Todd & J Gomez, Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal. Biochem. 2001, 296: 179 (2001)).

[00142] The major advantage of calorimetry is the lack of derivatization required for analysis (Danielsson, 1991). Since most reactions involve heat exchange, and this heat is detected, no chromophores, fluorophores, luminophores, “mediators,” or other modifications of the analyte are required. Reagents and analytes can be used “as is.” This allows the analysis of both reactions that lack a chromophore or fluorophore and/or would be difficult or impossible to derivatize or couple to the generation of an electroactive species.

[00143] Miniaturized or chip-based thermosensors can be used (Ramanathan & Danielsson, 200: 1; B Xie & B Danielsson, Development of a thermal micro-biosensor fabricated on a silicon chip. Sens. Actuat. B 6: 127 (1992); P Bataillard et al., An integrated silicon thermopile as biosensor for the thermal monitoring of glucose, urea, and penicillin. Biosen. Bioelect. 8:89 (1993)). These devices range from radically arranged thermopiles on freestanding membranes to groups of thermopiles constructed on silicon/glass microchannels. These devices have been used to detect specific, single enzymatic reactions (Danielsson, 1991; Xie & Danielsson, 1992; Bataillard et al., 1993). Moreover, two groups have reported thermosensors for glucose (B Xie et al., Fast determination of whole blood glucose with a calorimetric micro-biosensor, Sens. Actuat. B 15-16: 141 (1993); M J Muehlbauer et al., Model for a thermoelectric enzyme glucose sensor, Anal. Chem. 61:77 (1989); B C Towe & E J Guilbeau, Designing Medical Devices, 1998.

IL MAO biosensor devices

[00144] In one aspect, described herein is a system comprising a MAO amperometric biosensor, e.g., in some instances, a chronoamperometric MAO biosensor as described herein and a potentiostat. A potentiostat is the electronic hardware required to control a three -electrode cell or a two-electrode cell and run most electroanalytical experiments. The system functions by maintaining the potential of the working electrode at a constant level with respect to the reference electrode by adjusting the current at an auxiliary electrode. A potentiostat is a control and measuring device; as such it can be referred to as a power supply unit, a processing unit, and/or a display unit. A potentiostat comprises an electric circuit which controls the potential across the cell by sensing changes in its resistance, varying accordingly the current supplied to the system: a higher resistance will result in a decreased current, while a lower resistance will result in an increased current.

[00145] In some embodiments of any of the aspects, the potentiostat allows for chronoamperometry, an electrochemical technique in which the potential of the working electrode is stepped and the resulting current from faradaic processes occurring at the electrode (caused by the potential step) is monitored as a function of time. In some embodiments of any of the aspects, the potentiostat measures the current of the chronoamperometric biosensor. In some embodiments of any of the aspects, the current readings are output onto a display (e.g., a display unit of the potentiostat or a separate display module).

[00146] Fig. 14 shows an exemplary system comprising amperometric biosensor, e.g., a chronoamperometric biosensor that is electrically coupled to a potentiostat, for example through electrical leads to at least one electrode of the biosensor. In some embodiments of any of the aspects, the potentiostat is electrically coupled to the working electrode of the biosensor. In some embodiments of any of the aspects, the potentiostat is electrically coupled to the reference electrode of the biosensor. In some embodiments of any of the aspects, the potentiostat is electrically coupled to the counter electrode of the biosensor. In some embodiments of any of the aspects, the potentiostat is electrically coupled to the working electrode and the reference electrode of the biosensor. In some embodiments of any of the aspects, the potentiostat is electrically coupled to the working electrode, reference electrode, and counter electrode of the biosensor.

[00147] In some embodiments of any of the aspects, the amperometric biosensor, e.g., chronoamperometric biosensor is contained in a Faraday cage. A Faraday case is a grounded metal screen surrounding a piece of equipment to exclude electrostatic and electromagnetic influences. [00148] In some embodiments of any of the aspects, the system comprises a portable device; as a non- limiting example, the biosensor and/or the potentiostat can be portable. In some embodiments of any of the aspects, the system comprises a wearable device; as a non-limiting example, the biosensor and/or the potentiostat can be wearable. See e.g., US patent publication 2015/0260674, Shiwaku et al., Scientific Report (2018) 8:6368; Steinberg et al., Taianta. 2015 Oct 1 ; 143 : 178-183; the contents of each of which are incorporated by reference herein in their entireties.

[00149] In some embodiments, a MAO biosensor as disclosed herein can be fabricated by using screen-printing technology, or inkjet- or other 3D printing technology to print components of the biosensor, where the biosensor comprises a substrate, e.g., a backing layer, and at least one set of three electrodes. In some embodiments, the electrodes are printed from a conducting polymer onto the backing layer. An exemplary MAO biosensor device useful herein includes a three-electrode geometry which include a reference electrode, a working electrode, which preferably includes a biofunctional polymeric coating, and optionally a counter electrode. Typically, each electrode includes an active area, an electrical interconnect, and a contact area.

[00150] The electrodes can have a length between about 2 mm and about 20 mm, a width between about 0. 1 mm and about 2 mm, and a height between about 0. 1 mm and about 2 mm. The sensors can include an array of sets of three electrodes. The sensor can be connected to a data acquisition system, a display system, or both an acquisition and a display system, forming a sensor system.

[00151] In some embodiments, the working electrode includes a coating positioned over its active, and MAO enzyme in or on the coating. The sensor typically includes a sensing area. The sensing area is usually formed of at least a portion of the active areas of the reference electrode, the working electrode, and/or the counter electrode. In some embodiments, the sensing area is formed of all of the active area of the reference electrode, all of the active areas of the working electrode and/or all of the active areas of the counter electrode. In some embodiments, the sensing area can include a protective coating. The contact areas of the reference electrode, the working electrode, and/or the counter electrode connect the sensor to a data acquisition system, a display system, or both an acquisition and a display system, forming a sensor system. The electrical interconnects that connect the sensing area and the contact areas of the electrodes can include an insulation coating.

[00152] The electrodes of the biosensor can be printed from a conducting polymer. Suitable conducting polymers include poly(4,4- dioctylcyclopentadithiophene), poly(isothianapthene), poly (3 ,4- ethylenedioxythiophene), polyacetylene (PAC), polyaniline (PANI), polypyrrole (PPY) or polythiophenes (PT), poly(p-phenylene sulfide) (PPS), and poly(3,4 ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). The sensing area can include a protective coating. Typically, the protective coating is a polymer that reduces or prevents the non-specific interaction or interference of different molecules in the biological sample with the biofunctional coating of the sensor. The protective coating can be a cation exchange membrane containing a polymer that prevent negatively charged interferences from reaching the sensor surface. Exemplary polymers that can be used as or in a protective coating include polystyrene sulfonate, perfluorinated sulfonated ionomer such as Nafion® (E. I. Du Pont De Nemours And Company Corporation, Wilmington, DE), AQUIVION® (Solvay SA Corporation, Brussels, Belgium), or a combination thereof.

[00153] Typically, the coating includes a mediator, such as a multivalent metal ion or an organometallic compound, and/or a polymer matrix formed of a positively charged polymer such as alginate amine, chitosan, dextran amine, heparin amine, and/or any combination thereof. The coating also includes a MAO enzyme as described herein, which is capable of oxidizing at least one amine- comprising substrate in a test sample. The coating can also comprise other redox mediators (Med) as described herein, as well as intermediate redox enzymes as described herein having the capability of acting as both electron donors and electron acceptors. Additionally, the coating can also comprise multivalent metal ions such as copper, iron, magnesium, manganese, molybdenum, nickel and zinc, and co-factors such as nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+), ascorbic acid, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), coenzyme F420, coenzyme B, Coenzyme Q, glutathione, heme, lipoamide, and pyrroloquinoline quinone. In some embodiments, the sensors may include either electrodes for amperometric tests or cyclovoltammetry.

[00154] Generally, the sensor is small enough to be applied onto a medical device or onto a subject. The surface of the biosensor, e.g., the substrate (e.g., backing layer) can be a planar surface, such as a paper, a tattoo, a tape, a textile, a wound dressing or bandage, a medical implant, a contact lens, or a pad. The sensor can be part of a catheter, a contact lens, or a medical implant. The sensor can be worn by a subject on a patch or a bandage, or can be provided in a kit, ready to be used as needed. In some embodiments, the sensor can be inserted in whole or in part into a biological sample such as, but not limited to, blood, plasma, serum, urine, saliva, gastric juice, fecal matter, cerebrospinal fluid, or cervicovaginal mucosa. In some embodiments, the sensor can be connected to a data or signal acquisition system, such as a potentiostat, and, optionally, to a display system. The display system can be a portable display system with a screen to display sensor reading. Portable display systems include smartphones, tablets, laptops, desktop, pagers, watches, and glasses.

[00155] Typically, the sensor permits non-invasive detection of a presence, absence, or a concentration of, at least one amine-comprising substrate in a biological sample. Exemplary biological samples include, but are not limited to, bodily fluids or mucus, such as saliva, sputum, tear, sweat, urine, exudate, blood, plasma, cerebrospinal fluid, gastric juice, or vaginal discharge.

[00156] A MAO biosensor comprising a MAO enzyme can be configured according to a biosensor described in WO₂019/224628, which is incorporated herein in its entirety by reference.

[00157] Printed MAO enzymatic sensors and sensor systems as disclosed herein are capable of detecting concentrations of at least one amine-comprising substrate in the relevant range from biological samples obtained non-invasively. The MAO biosensors can show long term stability use with accurate and reproducible measurement of levels of at least one amine-comprising substrate. [00158] The MAO biosensor device system typically includes a sensor (also referred to as a biosensor), which may be attached to a reader containing an acquisition and/or a display component. In some embodiments, the biosensor system is portable, and the acquisition and/or one or more display components can be attached or disconnected from the sensor as needed.

A. MAO biosensor [00159] The MAO biosensors typically include at least one backing layer, and at least one set of three electrodes (or two electrodes) printed from a conducting material onto the backing layer. Typically, the electrodes include an active area, an electrical interconnect, and a contact area. The electrodes can be formed from the same conducting material or different conducting materials. Typically, all electrodes are formed from the same conducting material, e.g., a conducting polymer. In some instances, all electrodes can be printed from the same conducting polymer on the backing layer in one step. The combination of the active areas of the reference electrode, the working electrode, and the counter electrode forms the sensing area. Each printed electrode can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 layers of a conducting polymer, for example, but not limited to, poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) anions (PEDOT PSS), which is widely used in various organic optoelectronic devices. PEDOT: PSS is a blend of cationic polythiophene derivative, doped with a poly anion. In some embodiments, the working electrode with all layers (electrode, dielectric, MAO enzyme and redox mediator, and protective coating) can be printed successively.

[00160] An exemplary set includes a three electrode geometry with a reference electrode, a working electrode with a biofunctional coating, and a counter electrode. Typically, the electrodes have a length between about 2 mm and about 20 mm, a width between about 0. 1 mm and about 2 mm, and a height between about 0. 1 mm and about 2 mm.

[00161] The sensors can include an array of sets of three electrodes. The sensor can be connected to an acquisition system, a display system, or both an acquisition and a display system. The contact areas of the reference electrode, the working electrode, and the counter electrode can connect the sensor to a data acquisition system, a display system, or both an acquisition and a display system, forming a sensor system.

[00162] Generally, the sensors include a coating positioned over a surface of the working electrode, e.g., the active area of the working electrode, and an electron-generating MAO enzyme in the biofunctional coating. The coating can further include a redox mediator and/or a polymer matrix. The sensor can include a sensing area, which is formed of active areas of the reference electrode, the working electrode, and/or the counter electrode. The sensing area typically includes a protective coating. Typically, the protective coating is a polymer that reduces or prevents the non-specific interaction or interference of different molecules in the biological sample with the biofunctional coating of the sensor. The protective coating can also stabilize the MAO enzyme and/or the redox mediators in the coating. The protective coating can be a cation exchange membrane containing a polymer that prevent negatively charged interferences from reaching the sensor surface. The electrical interconnects that connect the sensing area and the contact areas of the electrodes can include an insulation coating, such as a dielectric coating. The dielectric coating can separate or insulate the sensing area from the contact areas. The sensor system can include a printed metabolite sensor on a backing layer, which can be as simple as a commercial disposable paper. The fabrication of a MAO biosensor as described herein using ink-jet technology or other 3D printing technology permits the production of a highly sensitive, selective, portable, inexpensive, stable, and user-friendly MAO biosensor device. The printed MAO biosensor can be tested over a period of one month and its long- term stability confirmed. The MAO biosensor can be used in real world applications with bodily fluids such as sweat, blood and saliva, permitting non-invasive monitoring. In some embodiments, the MAO biosensor as described herein can be configured as an all-polymer “smart e-paper MAO biosensor” providing the next generation of disposable low cost and eco-friendly high-performance MAO biosensor devices.

[00163] Such a MAO biosensor can have high sensitivity and can repeatedly measure at least one amine-comprising substrate in numerous samples. The MAO biosensor can have a sensitivity to be able to detect at least one amine-comprising substrate within a 0.01 μM - 80 μM. and/or 2μM -50μM, and/or 50μM -lOOOμM range. As a non-limiting examples, the MAO biosensor has a sensitivity for at least one amine-comprising substrate of 0.01 μM - 80 μM, 0.01 μM - 10 μM, 0.01 μM - 1 μM, 0.01 μM - 0.1 μM, or of at least 0.01 μM, at least 0.1 μM, at least 1.0 μM, at least 5.0 μM, at least 10 μM, at least 20 μM, at least 30 μM, at least 40 μM, at least 50 μM, at least 60 μM, at least 70 μM, or at least 80 μM. i. Reference Electrode

[00164] The reference electrode is an electrode having a maintained potential, used as a reference for measurement of other electrodes. Exemplary reference electrodes are, but not limited to, silver, silver chloride, silver/silver chloride, gold, copper, carbon, and conducting polymer. The reference electrode can be screen-printed or inkjet-printed from the above-mentioned materials. Typically, the reference electrode is inkjet-printed from a conducting polymer. In some embodiments, the MAO biosensor disclosed herein does not comprise a reference electrode. ii. Working Electrode

[00165] The working electrode typically includes a biofunctional coating. The biofunctional coating can contain the MAO enzyme. The biofunctional coating can further include a redox mediator, and optionally an intermediate redox enzyme (IRE) and/or a polymer matrix.

[00166] The mechanism of the detection of at least one amine-comprising substrate is based on the specific MAO enzyme and the cycle of electrochemical reactions, which alternatively oxidize/reduce the compounds immobilized at the surface of the sensor, e.g., at the surface of the working electrode. Typically, the electrons are transferred from at least one amine-comprising substrate (the analyte) to the conducting polymer through the cycle of electrochemical reactions, generating a current between the working and counter electrodes detected by the acquisition system. An exemplary cycle of reactions is depicted herein, where upon reacting with at least one amine-comprising substrate, the MAO enzyme, i.e. MAO gets reduced, and the reduced MAO enzyme cycles back via the redox mediator, which mediates electron transfer from the MAO enzyme to the conducting polymer, e.g., PEDOTPSS.

[00167] In some embodiments, the size of the working electrode can influence the current produced from the MAO biosensor. Accordingly, in some embodiments, the working electrode has an area of >4π mm². In some embodiments, the size of the working electrode is in the range of 5-8 π mm², or 8- 10π mm², or 10-12π mm², or 12-15π mm², or greater than 15π mm². In some embodiments, the shape of the working electrode is circular, and in some embodiments the shape is oval. In some embodiments, the counter electrode is a geometric shape which wraps or circles around the working electrode.

[00168] In some embodiments, the working electrode comprises MAO enzyme in a low- or medium- molecular weight (MW) chitosan in 0.5% acetic acid layer. In some embodiments, the working electrode comprises NAFION. In some embodiments, the working electrode does not comprise NAFION.

Hi. Redox Mediators

[00169] Typically, a mediator is a small molecule compound participating in an electron donor/acceptance. Exemplary mediators include compounds containing multivalent metal ions such as copper, iron, magnesium, manganese, molybdenum, nickel and zinc, organometallic compounds, phenazine methosulfate, dichlorophenol indophenol, short chain ubiquinones, ferrocene complex, and co-factors such as nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+), ascorbic acid, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), coenzyme F420, coenzyme B, Coenzyme Q, glutathione, heme, lipoamide, and pyrroloquinoline quinone. In some instances, the mediator is a ferrocene complex. In some embodiments, a FAD redox mediator is not used. iv. Counter Electrode

[00170] The counter electrode, often also called the auxiliary electrode, is an electrode used in a three- electrode electrochemical cell for voltammetric analysis or other reactions in which an electric current is expected to flow. Exemplary counter electrodes are, but not limited to, gold, copper, carbon, and conducting polymer. The counter electrode may be screen-printed or inkjet-printed from the above- mentioned materials. Typically, the counter electrode is inkjet-printed from a conducting polymer. In some embodiments, the MAO biosensor disclosed herein does not comprise a counter electrode. v. Materials Forming the Electrodes

[00171] Generally, the sensor includes at least one set of three electrodes. Each electrode in the sensor can include one or more coatings. The electrode and the coatings can be inkjet-printed, in sequential manner, to obtain the sensor arrangement described herein.

[00172] Materials forming the electrodes and its coatings include conductive polymers, dielectric inks, charged biocompatible polymers, and synthetic ionic polymers. Typically, the reference electrode, the working electrode, and/or the counter electrode are formed of conductive polymers. The reference electrode, the working electrode, and/or the counter electrode can also include a dielectric coating formed of dielectric ink. The working electrode typically includes a biofunctional coating containing a biofunctional molecule, a mediator, and polymer matrix formed of a charged biocompatible polymer. At least a portion, e.g., the active area of the reference electrode, the working electrode, and/or the counter electrode can be coated with a protective coating containing a synthetic ionic polymer. a. Conducting Polymer

[00173] Conducting polymers can be used to form the reference electrode, the working electrode, and/or the counter electrode. Exemplary conducting polymers include poly(3,4- ethylenedioxythiphene) (PEDOT), poly(hydrooxymethyl 3,4-ethylenedioxythiphene) (PEDOT-OH), polystyrenesulfonate (PSS), F8BT, F8T2, J51, MDMO-PPV, MEH-PPV, PBDB-T, PBDTBO-TPD, PBDT(EH)-TPD, PBDTTT-C-T, PBDTTT-CF, PBTTPD, PBTTT-C14, PCDTBT, PCPDTBT, PDTSTPD, PffBT4T-20D, PffBT4T-C9C13, PFO-DBT, Poly([2,6'-4,8-di(5- ethylhexylthienyl)benzo [1, 2 -b ; 3 ,3 -b] dithiophene] { 3 -fluoro-2 [(2- ethylhexyl)carbonyl]thieno [3 ,4- b]thiophenediyl}), Poly(3-dodecylthiophene- 2, 5 -diyl), Poly (3-hexyl thiophene-2, 5 -diyl), Poly(3 - octylthiophene-2, 5 -diyl), PSiF-DBT, poly(triaryl amine) (PTAA), PTB7, TQ1, N2300, P(NDI-T2), poly(diketopyrrolopyrrole) (DPP), poly(benzimidazobenzophenanthroline), poly(2, 5 -di(3,7- dimethyloctyloxy)cyanoterephthalylidene), poly(2,5- di(hexyloxy)cyanoterephthalylidene), poly(5- (3 ,7-dimethyloctyloxy)-2- methoxy-cyanoterephthalylidene), poly(2,5 - di(octyloxy)cyanoterephthalylidene), poly(5-(2-ethylhexyloxy)-2 -methoxy- cyanoterephthalylidene), poly(4,4-dioctylcyclopentadithiophene), poly(isothianapthene), poly(3,4-ethylenedioxy thiophene), poly acetylene (PAC), polyaniline (PANI), polypyrrole (PPY) or polythiophenes (PT), and poly(p- phenylene sulfide) (PPS). In some instances, the conductive polymer is a combination of two or more conductive polymers described above. For example, the conductive polymer can be poly(3,4 ethylenedioxythiophene) :polystyrene sulfonate (PEDOT: PSS). b. Dielectric Coating

[00174] The dielectric coating can be a dielectric/insulator ink layer. The dielectric ink layer can be a dielectric polymer, copolymer, block polymer, or polymer-inorganic composite. The dielectric polymer can be polyimide, polyurethane, polysiloxane, polyacrylate, polyethylene, polystyrene, polyepoxide, polytetrafluoroethylene, polyarylene ether, methylsilsesquioxone, fluorinated polyimide, or a combination thereof. Dielectric polymer-inorganic composite can include a polymer and an inorganic compound such as barium titanate(IV) (BaTiCF). Titanium carbide (TiC or TiCb), aluminum oxide (AI₂O₃), zirconium dioxide (ZrO₂). An exemplary dielectric polymer-inorganic composite can be polyimidc-BaTiCF.

[00175] Commercially available dielectric/insulator inks or pastes can be EMD 6200 (SUN CHEMICAL CORPORATION, Parsippany, NJ), KA 701 (DUPONT), 125-17, 116-20, 113-48, 111- 27, 118-02, 122-01, 119-07, 118-08, 118-12 (CREATIVE MATERIALS®), D2070423P5, D2071120P1, D2140114D5, D2020823P2, D50706P3, D2030210D1, D2070412P3, D2081009D6, D50706D2, D2130510D2 (SUN CHEMICAL CORPORATION, Parsippany, NJ), LOCTITE® EDAG 1020A E&C, LOCTITE® EDAG 452SS E&C, LOCTITE® EDAG PD 038 E&C, LOCTITE® EDAG PF 021 E&C, LOCTITE® EDAG PF 455B E&C, or LOCTITE® M 7000 A BLU E&C (HENKEL CORPORATION). c. Polymer Matrices Immobilizing MAO enzyme

[00176] In some instances, the biofunctional coating of the working electrode includes a redox mediator, a MAO enzyme, and a polymer matrix for immobilizing the redox mediator and the MAO enzyme. The polymer matrix can entrap the redox mediator and the MAO enzyme within its matrix to prevent leaking and to improve the processability of the MAO enzyme. The polymer matrix can be biocompatible.

[00177] The polymer matrices for immobilizing the mediator and the MAO enzyme can be formed of positively charged polymers, such as alginate amine, chitosan, dextran amine, heparin amine, and any combination thereof. d. Protective Coating

[00178] The protective coating is typically placed on top, e.g., inkjet-printed over, the electrodes, over a portion of the electrodes, and can be the outermost-layer of on the electrodes. The protective coating can be formed of synthetic ionic polymer, such as polystyrene sulfonate and perfluorinated sulfonated ionomers, such as NAFION®, AQUIVION® (Solvay Sa Corporation, Brussels Belgium), or a combination thereof. In some embodiments, NAFION® is not present. vi. Sensing Area

[00179] The sensing area (6) of the MAO biosensor as disclosed herein typically includes a portion of the working, counter and/or reference electrodes, e.g., the active areas of the working, counter, and reference electrodes. Typically, the active area of the working electrode containing at least a portion of the biofunctional coating. The sensing area may include a polymer coating. The polymer coating typically reduces or prevents the non-specific interaction or interference of different molecules in the biological sample with the MAO enzyme on the sensor. The polymer coating reduces or prevents any interaction or interference with the electron transport in the sensor from the different molecules in the biological sample. vii. Backing Layer or Substrate

[00180] In some embodiments, the surface of the biosensor is a substrate. In some embodiments, the surface of the sensor is a backing layer, and can be a planar surface such as paper, a tattoo, a tape, a textile, a wound dressing or bandage, a medical implant such as catheter, a contact lens, a patch, a pad, glass, or plastics. Typically, the backing layer is a paper. The paper can be disposable after one use or multiple uses, e.g., four times. viii. Wearable MAO biosensor

[00181] Another aspect of the technology described herein is a wearable MAO biosensor device. In some embodiments, a wearable MAO device comprises two parts, an electroconductive part comprising a housing with a removable lid, and within the housing an electric control circuit to control the screen-printed electrode (SPE), a battery, and PDMS layer housing a paper channel to wick a subject’s sweat from their skin into the SPE. Magnets or other means can be used to attach the removable lid to the housing. The housing is positioned above or adjacent to a two electrode-screen printed electrode (SPE), where at least one electrode has a MAO enzyme deposited on, where the MAO serves as the biorecognition element of at least one amine-comprising substrate, and where the SPE is in fluid communication via the paper channel to a wicking apparatus, where the wicking apparatus contacts the skin of the wearer (i.e., the subject) and wicks sweat from the surface of a subject’s skin. Accordingly, an exemplary wearable at least one amine-comprising substrate electrochemical biosensor device can comprise a housing portion of the biosensor and underneath or adjacent to the housing is the placement of a 2-electrode SPE where one electrode is deposited with MAO, and where the SPE is in fluid communication with a sweat sample. In particular, the SPE is in fluid communication via a paper channel with a wicking paper, where the wicking paper wicks sweat from the skin surface of a subject, and 1) the sweat is wicked onto the SPE by the paper channel, and then, 2) sweat is drawn towards the sink by capillary action and passed onto of the 2-electrode sensor comprising the MAO biorecognition element, and 3) sweat is collected at the sink after measurement. Such a wearable MAO biosensor can have high sensitivity and can repeatedly measure at least one amine-comprising substrate in numerous samples, and can have a sensitivity to be able to detect at least one amine-comprising substrate within a 0.01 μM - 80 μM. and/or 2μM -50μM, and/or 50μM - 1000μM range.

[00182] In some embodiments, the wearable MAO biosensor can be adapted by one of ordinary skill in the art, including using an adhesive sheet to attach the wicking apparatus to the surface of the wearer’s skin, as disclosed in US Patents 9,820,692, or use of sweat collection pads as disclosed in US patents 10,182,795 and 10,646,142, each of which are incorporated herein their entirety by reference. [00183] In some embodiment, a wearable MAO biosensor disclosed herein does not necessarily include all features needed for operation, examples being a battery or power source which is required to power electronics, or for example, a wax paper backing that is removed prior to applying an adhesive patch, or for example, a particular antenna design, that allows wireless communication with a particular external computing and information display device. Several specific, but non-limiting, examples can be provided as follows. In a particular embodiment, a wearable MAO biosensor as disclosed herein is a type of sweat sensor device. In some embodiments, the wearable MAO biosensor can take on forms including patches, bands, straps, portions of clothing, wearables, or any mechanism suitable to affordably, conveniently, effectively, intelligently, or reliably bring sweat stimulating, sweat collecting, and/or sweat sensing technology into intimate proximity with sweat as it is generated. In some embodiments of the wearable MAO biosensor disclosed herein can require adhesives to the skin, but devices can also be held by other mechanisms that hold the device secure against the skin such as strap or embedding in a helmet. The wearable MAO biosensor disclosed herein can benefit from chemicals, materials, sensors, electronics, microfluidics, algorithms, computing, software, systems, and other features or designs, as commonly known to those skilled in the art of electronics, biosensors, patches, diagnostics, clinical tools, wearable sensors, computing, and product design.

[00184] The wearable MAO biosensor disclosed herein can include all known variations of the MAO biosensors disclosed herein, and the description herein shows sensors as simple individual elements. It is understood that many sensors require two or more electrodes, reference electrodes, or additional supporting technology or features which are not captured in the description herein. The wearable MAO biosensor disclosed herein are preferably electrical in nature such as ion-selective, potentiometric, amperometric, and impedance (faradaic and non-faradaic), but can also include optical, chemical, mechanical, or other known biosensing mechanisms.

[00185] Sensors can allow for continuous monitoring of at least one amine-comprising substrate in the sweat of a subject. In some embodiments, the at least one amine-comprising substrate that is detectable in sweat is selected from the group consisting of dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and hexylamine. As a non-limiting example, dopamine can be detected in sweat samples; see e.g., Sun et al., Analytica 2023, 4(2), 170-181 (2023), the contents of which are incorporated herein by reference in their entirety.

[00186] In some embodiments, a wearable MAO biosensor disclosed herein can be in duplicate, triplicate, or more, to provide improved data and readings. Many of these auxiliary features of the device can also require additional aspects as described herein or known in the art.

B. Reader

[00187] The biosensors as disclosed herein can be connected to a system, optionally including a display. i. Acquisition System

[00188] In some embodiments, an acquisition system can be a potentiostat, a biosensor, or a galvanostat. In some embodiments, a potentiostat that has a current resolution of as low as IpA (lOOnA range) is used, for example, a DROPSENS potentiostat. Typically, the acquisition system is connected to software that converts data into a graph, chart, or table, for the at least one amine- comprising substrate. ii. Display System [00189] The display system can be a portable display system with a screen to display sensor reading. Portable display systems include smartphones, tablets, laptops, and monitors.

C. Packaging

[00190] The MAO biosensor as disclosed herein can be packaged to protect the electrodes prior to use. Examples of packaging are known in the art and include molded or sealed pouches with temperature and/or humidity control. The pouches can be foil pouches, paper pouches, cardboard boxes, polymeric pouches, or a combination thereof. In some embodiments, the MAO biosensor as disclosed herein, and sensor systems can be packaged as one unit.

[00191] Alternatively, in some embodiments, the MAO biosensors can be packaged separately, and used as needed with an acquisition and/or display system provided by the end user.

D. Computer, Hardware, and Software Systems

[00192] In some embodiments of any of the aspects, the system further comprises a computing device, a server, a network, and/or a database. As a non-limiting example, data output from the biosensor and potentiostat as described herein can be displayed on a computing device and/or input into a program that can be stored in a database. In some embodiments, the data is wirelessly transmitted from the biosensor and potentiostat to a computing device, such as but not limited to a mobile phone; see e.g., Ainla et al., April 2018 Analytical Chemistry 90(10). The computing device and server can be connected by a network and the network can be connected to various other devices, servers, or network equipment for implementing the present disclosure. A computing device can be connected to a display. Computing device can be any suitable computing device, including a desktop computer, server (including remote servers), mobile device, or other suitable computing device. In some examples, algorithm(s) as described herein and other software can be stored in database and run on server. Additionally, data and data processed or produced by said algorithms or programs can be stored in a database.

[00193] It should be understood that the disclosure herein can be implemented with any type of hardware and/or software and can be a pre-programmed general purpose computing device. For example, the system can be implemented using a server, a personal computer, a portable computer, a thin client, or any suitable device or devices. The disclosure and/or components thereof can be a single device at a single location, or multiple devices at a single, or multiple, locations that are connected together using any appropriate communication protocols over any communication medium such as electric cable, fiber optic cable, or in a wireless manner.

[00194] It should also be noted that the disclosure can be illustrated and discussed herein as having a plurality of modules which perform particular functions. It should be understood that these modules are merely schematically illustrated based on their function for clarity purposes only, and do not necessary represent specific hardware or software. In this regard, these modules can be hardware and/or software implemented to substantially perform the particular functions discussed. Moreover, the modules can be combined together within the disclosure or divided into additional modules based on the particular function desired. Thus, the disclosure should not be construed to limit the present technology as disclosed herein, but merely be understood to illustrate one exemplary implementation thereof.

[00195] The computing system can include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other. In some implementations, a server transmits data (e.g., an HTML page) to a client device (e.g., for purposes of displaying data to and receiving user input from a user interacting with the client device). Data generated at the client device (e.g., a result of the user interaction) can be received from the client device at the server.

[00196] Implementations of the subject matter described herein can be implemented in a computing system that includes a back end component, e.g., as a data server, or that includes a middleware component, e.g., an application server, and/or that includes a front end component, e.g., a client computer having a graphical user interface or a Web browser through which a user can interact with an implementation of the subject matter described in this specification, or any combination of one or more such back end, middleware, or front end components. The components of the system can be interconnected by any form or medium of digital data communication, e.g., a communication network. Examples of communication networks include a local area network (“LAN”) and a wide area network (“WAN”), an inter-network (e.g., the Internet), and peer-to-peer networks (e.g., ad hoc peer to-peer networks).

[00197] Implementations of the subject matter and the operations described herein can be implemented in digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed herein and their structural equivalents, or in combinations of one or more of them. Implementations of the subject matter described herein can be implemented as one or more computer programs, e.g., one or more modules of computer program instructions, encoded on computer storage medium for execution by, or to control the operation of, data processing apparatus. Alternatively or in addition, the program instructions can be encoded on an artificially generated propagated signal, e.g., a machine-generated electrical, optical, or electromagnetic signal that is generated to encode information for transmission to suitable receiver apparatus for execution by a data processing apparatus. A computer storage medium can be, or be included in, a computer-readable storage device, a computer-readable storage substrate, a random or serial access memory array or device, or a combination of one or more of them. Moreover, while a computer storage medium is not a propagated signal, a computer storage medium can be a source or destination of computer program instructions encoded in an artificially generated propagated signal. The computer storage medium can also be, or be included in, one or more separate physical components or media (e.g., multiple CDs, disks, or other storage devices).

[00198] The operations described in this specification can be implemented as operations performed by a “data processing apparatus” on data stored on one or more computer-readable storage devices or received from other sources.

[00199] The term “data processing apparatus” encompasses all kinds of apparatus, devices, and machines for processing data, including by way of example a programmable processor, a computer, a system on a chip, or multiple ones, or combinations, of the foregoing. The apparatus can include special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application specific integrated circuit). The apparatus can also include, in addition to hardware, code that creates an execution environment for the computer program in question, e.g., code that constitutes processor firmware, a protocol stack, a database management system, an operating system, a cross- platform runtime environment, a virtual machine, or a combination of one or more of them. The apparatus and execution environment can realize various different computing model infrastructures, such as web services, distributed computing, and grid computing infrastructures.

[00200] A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages, and it can be deployed in any form, including as a standalone program or as a module, component, subroutine, object, or other unit suitable for use in a computing environment. A computer program can, but need not, correspond to a file in a file system. A program can be stored in a portion of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub programs, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a communication network.

[00201] The processes and logic flows described in this specification can be performed by one or more programmable processors executing one or more computer programs to perform actions by operating on input data and generating output. The processes and logic flows can also be performed by, and apparatus can also be implemented as, special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application specific integrated circuit).

[00202] Processors suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor can receive instructions and data from a read only memory or a random access memory or both. The essential elements of a computer are a processor for performing actions in accordance with instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto optical disks, or optical disks. However, a computer need not have such devices. Moreover, a computer can be embedded in another device, e.g., a mobile telephone, a personal digital assistant (PDA), a mobile audio or video player, a game console, a Global Positioning System (GPS) receiver, or a portable storage device (e.g., a universal serial bus (USB) flash drive), to name just a few. Devices suitable for storing computer program instructions and data include all forms of nonvolatile memory, media and memory devices, including by way of example semiconductor memory devices, e.g., EPROM, EEPROM, and flash memory devices; magnetic disks, e.g., internal hard disks or removable disks; magneto optical disks; and CD ROM and DVD-ROM disks. The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.

III. Methods of Making the Sensors and the Sensor Systems

[00203] The biosensor can be assembled as described herein. As a non-limiting example, described herein is a method of preparing amperometric biosensor, e.g., a chronoamperometric biosensor capable of measuring the concentration of at least one amine-comprising substrate comprising: (a) depositing an electronically active mediator on the electrode surface; (b) depositing a polymer on the electrode surface; and (c) depositing a MAO enzyme on the electrode surface. These steps can occur in any order. In some embodiments of any of the aspects, an electrode is used that already comprises an electronically active mediator on the electrode surface. Accordingly, in another aspect the method of assembling or preparing the biosensor can comprise (a) depositing a polymer (e.g., chitosan) on the electrode surface and (b) depositing a MAO enzyme on the electrode surface. In another aspect the method of assembling or preparing the biosensor can comprise (a) depositing an electronically active mediator on the electrode surface and (b) depositing a MAO enzyme on the electrode surface. In some embodiments of any of the aspects, the step of assembling the system can comprise electrically coupling the biosensor to a potentiostat and/or other devices such as a portable cellular device.

[00204] In some embodiments, inkjet technology may be used in all the steps for the fabrication of a MAO biosensor as disclosed herein, according to the methods disclosed in international patent application WO₂019/224628, which is incorporated herein in its entirety by reference. Other additive printing technologies, such as screen printing or inkjet printing can also be used, e.g., to yield high performance MAO biosensor devices.

[00205] For the deposition of the electronic components as well as the biological layers (such as MAO enzyme for the detection of at least one amine-comprising substrate), inkjet technology not only allows for the controlled deposition of a variety of different materials but also constitutes a low temperature process which is a critical factor when it comes to the integration of biological molecules such as enzymes. Ink jetting enables the patterning of customizable geometries and can easily be integrated in roll-to-roll processes.

[00206] A general method of making the sensors include using a conducting polymer ink dedicated for ink-jetting and adjusting the ink formulation to meet the substrate requirements for the formation of a uniform and conducting layer. For example, a cross linker, e.g., 3- glycidoxypropyltrimethoxysilane (GOPS) and/or a surfactant, e.g., dodecyl benzene sulfonic acid (DBSA) can be added to the conducting polymer ink to prevent delamination of the conducting pattern from the backing layer and to improve the wettability of the ink and fdm formation during printing, respectively. The cross linker can be added at a concentration between about 0.01 percentage by weight (wt%) and about 5 wt%, between about 0. 1 wt% and about 5 wt%, between about 0.5 wt% and about 5 wt%, between about 0.5 wt% and about 4 wt%, between about 0.5 wt% and about 2 wt%, between about 1 wt% and about 5 wt%, and between about 0.1 wt% and about 1 wt%. In some instances, the cross linker can be added at a concentration of about 1 wt%. In some instances, the cross linker is absent. The surfactant can be added at a concentration of between about 0.01% (v/v) and about 1% (v/v), between about 0.05% (v/v) and about 1% (v/v), between about 0.1% (v/v) and about 1% (v/v), between about 0.1% (v/v) and about 0.5% (v/v), between about 0. 1% (v/v) and about 0.4% (v/v), and between about 0.2% (v/v) and about 0.5% (v/v). In some instances, the surfactant can be added at a concentration of about 0.4% (v/v). The ink can be printed on most planar surfaces, including paper, such as a commercial glossy paper. The ink is printed on the planar surface to form all electrodes (e.g., reference, working, and/or counter electrodes) in the set. All electrodes in the set can be formed of the same material or different materials. In some embodiments, all the electrodes in the set are formed of the same conducting polymer. All electrodes can be printed in a single step.

[00207] To insulate and/or separate the sensing area from the contact areas, one, two, three, or more layers of dielectric ink can be printed on top of the electrodes. In some instances, the dielectric ink is printed over a surface of at least one of the electrodes in a set of electrodes. In some instance, the dielectric ink is printed over a surface of all three electrodes in a set of electrodes. In some instances, the dielectric ink is printed over the electrical interconnects of the working, reference, and/or counter electrodes. Typically, the dielectric ink is UV-curable.

[00208] For the biofunctionalization of the sensor, a biological ink containing a mediator (e.g. ferrocene), a polymer matrix, (e.g., chitosan, a polymer for forming a biocompatible matrix and entrapping the mediator in a polymeric biocompatible matrix) and the MAO enzyme, is printed on top of the working electrode to form a biofunctional coating. The MAO enzyme can be immobilized on or in the polymer matrix via non-covalent or covalent bonding, such as via chemical conjugation, e.g., EDC-NHS coupling reaction where carboxyl groups of the enzyme can be conjugated to the amine groups of the polymeric matrix. In some instances, both the redox mediator and the MAO enzyme are physically entrapped in the polymer matrix. In some instances, the MAO enzyme is covalently immobilized on or in the polymer matrix, and the mediator is physically entrapped in the polymer matrix. This can form the biofunctional coating of the working electrode.

[00209] A protective coating can be applied onto the electrodes, including onto the biofunctional coating, by printing a coating polymer on top of the electrodes. The protective coating can be printed on the entire surface of the electrodes, including on the biofunctional coating of the working electrode, or on a portion of the electrodes and on a portion of the biofunctional coating of the working electrode. In some instances, the protective coating is printed on the active areas of the working, reference, and/or counter electrode. In some instances, the protective coating is printed on the active area of the working electrode. In some instances, the protective coating is printed on the biofunctional coating of the working electrode.

[00210] For example, the coating polymer or a polymer mixture, such as a mixture containing Nafion® can be printed on top of the sensing area (comprising the active areas of the working, counter, and/or reference electrodes) to block the interferents present in biologic milieu/media such as, but not limited to, saliva or sweat.

[00211] An acquisition system, such as a potentiostat, is commercially available. It can be attached to the sensor by connecting each electrode to a lead in the acquisition system. The acquisition system can then be connected to a display system, such as a device with a display screen. Exemplary display systems include smartphones, tablets, laptops, desktops, and smartwatches, and are commercially available. The display systems can include electronic conversion means, such as software, to convert the signals received from the acquisition system to a concentration value or a graph, which is then displayed on the screen. Such conversion means are known in the art.

[00212] Variations to the MAO biosensor described herein can include changes to the screen-printed electrodes (SPEs). Changes in size, shape, and material of the SPEs can be made to yield a similar result or improve the biosensor. Also, changes to the matrix which holds the MAO enzyme can be made. Using a two or three electrode circuit with the potentiostat can affect the current response. Furthermore, the choice of a potentiostat for sensitivity to current can improve the biosensor. Mediators on the SPE can be changed or optimized by changing the ions concentrations that constitutive it to yield a higher current response and thus increase sensitivity of the biosensor.

IV Uses and Applications of the MAO biosensor devices

[00213] In some embodiments of any of the aspects, the sample analyzed by the MAO biosensor as described herein is preferably a fluid, which is contacted with the electrode surface. That is, a fluid sample contacts the MAO present on the substrate, and if at least one amine-comprising substrate is present in the fluid sample, it relays a redox event that is transmitted to the electrodes as describe above. As a non-limiting example, a liquid sample can be applied to the area of the biosensor comprising at least one electrode. In some embodiments of any of the aspects, a non-liquid sample can be transformed into a liquid sample; as a non-limiting example a solid or gaseous sample can be dissolved in a liquid, such as an aqueous solvent that does not interfere with the redox reactions. [00214] The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a blood or plasma sample from a subject. In some embodiments of any of the aspects, the present invention encompasses several examples of a biological sample. In some embodiments of any of the aspects, the biological sample is cells, or tissue, or peripheral blood, or bodily fluid. Exemplary biological samples include, but are not limited to, a biopsy, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; gastric fluid; feces; and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any of the aspects, a test sample can comprise cells from a subject. In some embodiments of any of the aspects, the test sample can be sweat, gastric juice, urine, saliva, feces, cerebrospinal fluid, interstitial fluid (ISF), or blood (e.g., whole blood, plasma, or serum). [00215] The test sample can be obtained by removing a sample from a subject, but can also be accomplished by using a previously isolated sample (e.g., isolated at a prior time point by the same or another person).

[00216] In some embodiments of any of the aspects, the test sample can be an untreated test sample. As used herein, the phrase “untreated test sample” refers to a test sample that has not had any prior sample pre -treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing, and thawing, and combinations thereof. In some embodiments of any of the aspects, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays, and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays, and systems described herein. In some embodiments of any of the aspects, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments of any of the aspects, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, homogenization, sonication, filtration, thawing, purification, and any combinations thereof. In some embodiments of any of the aspects, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed, for example, to protect and/or maintain the stability of the sample during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an analyte as described herein (e.g., at least one amine-comprising substrate). [00217] In some embodiments of any of the aspects, the pH of the sample is or is adjusted to about 8.0. In some embodiments of any of the aspects, the pH of the sample is or is adjusted to about 7.5 to

8.5. In some embodiments of any of the aspects, the pH of the sample is or is adjusted to about 7.5 to

9.0. In some embodiments of any of the aspects, the pH of the sample is or is adjusted to greater than

7.0.

[00218] In some embodiments of any of the aspects, the methods described herein can further comprise a step of obtaining or having obtained a test sample from a subject. In some embodiments of any of the aspects, the subject can be a human subject. In some embodiments of any of the aspects, the subject can be a subject in need of monitoring of at least one amine-comprising substrate.

[00219] In some embodiments of any of the aspects, measuring the current produced by the oxidation of an amine-comprising substrate present in the sample allows for the determination of the concentration of the amine-comprising substrate. In some embodiments of any of the aspects, a known concentration of the amine-comprising substrate results in a predictable current; thus, an unknown amine-comprising substrate concentration can be determined using a known current and a standard curve of currents at known concentrations of the amine-comprising substrate.

[00220] In some embodiments of any of the aspects, the concentration of at least one amine- comprising substrate is measured in the range of 0.4 uM-100 uM. As a non-limiting example, the amperometric MAO biosensor, e.g., chronoamperometric MAO biosensor as described herein can detect at least one amine-comprising substrate in a sample at a concentration of at least 0.1 uM, at least 0.2 uM, at least 0.3 uM, at least 0.4 uM, at least 0.5 uM, at least 0.6 uM, at least 0.7 uM, at least 0.8 uM, at least 0.9 uM, at least 1.0 uM, at least 2.0 uM, at least 3.0 uM, at least 4.0 uM, at least 5.0 uM, at least 6.0 uM, at least 7.0 uM, at least 8.0 uM, at least 9.0 uM, at least 10 uM, at least 15 uM, at least 20 uM, at least 25 uM, at least 30 uM, at least 35 uM, at least 40 uM, at least 45 uM, at least 50 uM, at least 55 uM, at least 60 uM, at least 65 uM, at least 70 uM, at least 75 uM, at least 80 uM, at least 85 uM, at least 90 uM, at least 95 uM, at least 100 uM, at least 150 uM, at least 200 uM, at least 250 uM, at least 300 uM, at least 350 uM, at least 400 uM, at least 450 uM, at least 500 uM, at least 550 uM, at least 600 uM, at least 650 uM, at least 700 uM, at least 750 uM, at least 800 uM, at least 850 uM, at least 900 uM, at least 950 uM, or at least 100 uM. In some embodiments of any of the aspects, the amperometric MAO biosensor, e.g., chronoamperometric MAO biosensor as described herein can detect at least one amine-comprising substrate in a sample at a concentration between 0.4 uM-100 uM, 0.1 uM-100 uM, 0.1 uM-500 uM, or 0.1 uM-1000 uM.

[00221] As described herein, the current of the amperometric MAO biosensor, e.g., chronoamperometric MAO biosensor is not altered in the presence of an interferent. Such interferents can include but are not limited to L-(+)-lactic acid, ascorbic acid , uric acid, dopamine, (-)- epinephrine, creatinine, S-(+)-glucose, sodium, calcium, magnesium, potassium, phosphate, albumin, and/or amino acids. [00222] In some embodiments of any the aspects, the amperometric MAO biosensor, e.g., chronoamperometric MAO biosensor as described herein and the systems and methods comprising it can be used for monitoring levels of at least one amine-comprising substrate of a subject for a physician.

[00223] In some embodiments of any the aspects, the MAO biosensor can be used to monitor levels of at least one amine-comprising substrate in a subject. A person of skill in the art can recognize specific applications of the MAO biosensor described herein, depending on the health and condition of the specific subject. As a non-limiting example, the MAO biosensor can be used to monitor levels of at least one monoamine neurotransmitter (e.g., dopamine, norepinephrine) in a subject, e.g., a sample from a subject.

[00224] In some embodiments, the subject is taking a neurotransmitter-effecting drug. In some embodiments, the subject is taking a drug that modulates (e.g., increases and/or decreases) monoamine neurotransmitters (e.g., norepinephrine and/or dopamine) levels in the subject (e.g., compared to the subject not taking the drug). In some embodiments, the subject is taking a monoamine reuptake inhibitor (MRI) including but not limited to a Norepinephrine reuptake inhibitor (NRI), Selective norepinephrine reuptake inhibitor (sNRI), Dopamine reuptake inhibitor (DRI), Serotonin-norepinephrine reuptake inhibitor (SNRI), Serotonin-dopamine reuptake inhibitor (SDRI), Norepinephrine-dopamine reuptake inhibitor (NDRI), and/or Serotonin-norepinephrine-dopamine reuptake inhibitor (SNDRI), as known in the art.

[00225] In some embodiments, the subject is taking a drug that modulates (e.g., increases and/or decreases) dopamine levels in the subject (e.g., compared to the subject not taking the drug) including but not limited to opiates; alcohol; nicotine; amphetamines; cocaine; marijuana; heroin; chocolate; methylphenidate (RITALIN); a dopaminergic drug such as L-dopa (levodopa); carbidopa (LODOSYN), which protects levodopa from early conversion to dopamine outside the brain; monoamine oxidase B (MAO B) inhibitors such as selegiline (ZELAPAR), rasagiline (AZILECT) and/or safmamide (XADAGO) that help prevent the breakdown of brain dopamine; catechol O- methyltransferase (COMT) inhibitors such as Entacapone (COMTAN) and/or opicapone (ONGENTYS) that blocking a dopamine -degrading enzyme; adenosine receptor antagonists (A2A receptor antagonists) that allow more dopamine to be released, such as istradefylline (NOURIANZ); dopamine agonists including pramipexole (MIRAPEX ER), rotigotine (NEUPRO), and/or apomorphine (APOKYN); a dopamine reuptake inhibitor (DRI) including but not limited to amineptine, dexmethylphenidate, difemetorex, fencamfamine, lefetamine, levophacetoperane, medifoxamine, mesocarb, methylphenidate, nomifensine, pipradrol, prolintane, and/or pyrovalerone; and/or a dopamine antagonist drug include but not limited to chlorpromazine (THORAZINE), metoclopramide (REGLAN), promethazine (PHENERGAN), paliperidone (INVENGA), risperidone (RISPERDAL), quetiapine (SEROQUEL), and/or clozapine (CLOZARIL). In some embodiments, the subject is undertaking an activity that modulates (e.g., increases and/or decreases) dopamine levels in the subject (e.g., compared to the subject not undertaking the activity) including but not limited to exercise or sex.

[00226] In some embodiments, the subject is being monitored by a medical professional for high levels of dopamine, which can indicate dopamine dysregulation syndrome (DDS), which is characterized by compulsive dopaminergic medication use markedly exceeding the amount required for adequate control of Parkinson's disease (PD) symptoms.

[00227] In some embodiments, the subject is being monitored for dopamine levels in relation to treatment for Parkinson's disease, restless legs syndrome, depression, schizophrenia, bipolar disorder, attention deficit hyperactivity disorder (ADHD), addiction, nausea, and/or vomiting.

[00228] In some embodiments, the subject is taking a drug that modulates (e.g., increases and/or decreases) norepinephrine levels in the subject (e.g., compared to the subject not taking the drug) including but not limited to a norepinephrine reuptake inhibitor (NRI, NERI) or noradrenaline reuptake inhibitor or adrenergic reuptake inhibitor (ARI), including but not limited to Atomoxetine (STRATTERA), Reboxetine (EDRONAX, VESTRA), Viloxazine (QELBREE, VIVALAN), Amedalin (UK-3540-1), Daledalin (UK-3557-15), Edivoxetine (LY-2216684), Esreboxetine (AXS- 14; PNU-165442G), Lortalamine (LM-1404), Nisoxetine (LY-94,939), Talopram (tasulopram) (Lu 3- 010), Talsupram (Lu 5-005), Tandamine (AY-23,946), Amedalin (UK-3540-1), Daledalin (UK-3557- 15), Edivoxetine (LY-2216684), Esreboxetine (AXS-14; PNU-165442G), Lortalamine (LM-1404), Nisoxetine (LY-94,939), Talopram (tasulopram) (Lu 3-010), Talsupram (Lu 5-005), Tandamine (AY-23,946), Amedalin (UK-3540-1), Daledalin (UK-3557-15), Edivoxetine (LY-2216684), Esreboxetine (AXS-14; PNU-165442G), Lortalamine (LM-1404), Nisoxetine (LY-94,939), Talopram (tasulopram) (Lu 3-010), Talsupram (Lu 5-005), and/or Tandamine (AY-23,946); and/or a beta- blocker.

[00229] In some embodiments, the subject is being monitored by a medical professional for high levels of norepinephrine, which can indicate chronic stress or anxiety.

[00230] In some embodiments, the subject is being monitored for norepinephrine levels in relation to treatment for pheochromocytoma, pituitary tumors, adrenal gland disorders such as adrenocortical carcinoma, high blood pressure (hypertension), rapid or irregular heartbeat, excessive sweating, anxiety, depression, post-traumatic stress disorder, and substance abuse, panic attacks, hyperactivity. [00231] In some embodiments, the subject is being monitored for norepinephrine levels in relation to treatment for human norepinephrine (NE) deficiency, severe allergic reactions (anaphylaxis), sudden asthma attacks, shock, low blood pressure, and/or slow heart rate.

[00232] As such, the data obtained from the biosensor can be communicated to social applications, support groups, physicians, and/or insurance companies for the purpose of monitoring and/or reducing and/or increasing at least one amine-comprising substrate in a subject. In some embodiments of any the aspects, the MAO biosensor can be used to detect at least one amine-comprising substrate in the environment. In some embodiments of any the aspects, the MAO biosensor can be used to monitor levels of at least one amine-comprising substrate in the blood of a subject for a physician. One of ordinary skill in the art can use the MAO biosensor as described herein for various other applications requiring monitoring.

[00233] Aspects of the assays described herein rely on catalyzing an electrochemical reaction (redox) of the redox-enzyme biosensor in the presence of at least one amine-comprising substrate. In use, at least one amine-comprising substrate is catalyzed, changing electron flow through the biosensor. In one non-limiting embodiment, the redox-enzyme (or a functional portion thereof) catalyzes a redox event in the presence of at least one amine-comprising substrate. In some embodiments, the redox- enzyme is a MAO enzyme. The redox event can be coupled to a redox-mediator (referred to herein as a “Med”) which acts as a conductor of electrons to permit detection of the redox event between at least one amine-comprising substrate and the redox-enzyme biosensor, thereby detecting the presence of the redox-enzyme biosensor. In some embodiments, the redox event between at least one amine- comprising substrate and redox-enzyme can be coupled to an intermediate redox enzyme (IRE), that acts as a conductor of electrons between the first redox event (between at least one amine-comprising substrate and the MAO redox-enzyme biosensor) and the redox-mediator (Med) to permit detection of the activity of the redox-enzyme biosensor reacting with at least one amine-comprising substrate. [00234] In some embodiments, the redox-mediator generates a signal detectable by optical methods, such as, without limitation, fluorescence, surface plasmon resonance, or piezoelectric methods.

[00235] Accordingly, described herein is a method of using an amperometric biosensor, e.g., chronoamperometric biosensor to measure the concentration of at least one amine-comprising substrate comprising: (a) assembling the amperometric biosensor, e.g., chronoamperometric biosensor (or system comprising said biosensor) as described herein; (b) providing a sample; and (c) measuring the current produced by the oxidation of at least one amine-comprising substrate present in the sample.

[00236] The MAO biosensor system described herein may be portable, wearable, or attachable to a subject. In some aspects, the sensor is small enough to be applied onto a medical device or onto a subject. In some embodiments, the MAO biosensor has a backing layer which may be a planar surface, such as a paper, a tape, a bandage, a catheter, a lens, a patch, an implant, or a pad. The MAO biosensor, therefore, may be part of a contact lens, or a medical implant or patch. In some embodiments, the MAO biosensor as disclosed herein may be worn by a subject as a patch or on a bandage, or may be provided in a kit, ready to be used as needed.

[00237] The sensor can be connected to an acquisition system, such as a potentiostat, and, optionally, to a display system. The display system can be a portable display system with a screen to display sensor reading. Portable display systems include smartphones, tablets, laptops, desktop, pagers, watches, and glasses.

[00238] An exemplary method of use includes applying a test sample, e.g., a fluid biological sample onto the sensing area of the sensor, and obtaining a reading indicating that at least one amine- comprising substrate is detected. Optionally, a polymeric well is used on top of the sensing area of the MAO biosensor to confine the test sample. Alternatively, if an acquisition system and/or a portable system is used, the method can include also obtaining a concentration of at least one amine- comprising substrate in the sample.

[00239] The information obtained from the sensors or sensor systems may be used to determine levels of at least one amine-comprising substrate in a subject, or metabolism of at least one amine- comprising substrate in a subject, or provide guidance on intake of at least one amine-comprising substrate, or appropriate treatment of the subject is needed.

[00240] There are numerous additional applications for a MAO sensor. The MAO biosensor as disclosed herein can also be used as a personal health monitor which lets a wearer know the levels of at least one amine-comprising substrate.

[00241] In some embodiments, the MAO biosensor described herein is an implantable device. More particularly, the MAO biosensor described herein is designed to provide, and in conjunction with a suitable signal processing unit, a current which is proportional to the concentration of the analyte of interest, e.g., at least one amine-comprising substrate. In some embodiments, the MAO biosensor described herein may be implanted in vivo, including intra-cerebral, sub-cutaneous, intra-muscular, inter-peritoneal oral, serum, and vascular implantation, for systemic monitoring and used to monitor at least one amine-comprising substrate levels in the subject in real-time. In some embodiments, the MAO biosensor described herein can be joined, or electronically coupled with one or more other biosensors, to allow for the simultaneous recording of at least one amine-comprising substrate and one or more multiple analytes of interest. In addition to the in vivo applications, the MAO biosensors described herein may also find use in medical monitoring, industrial processes, environmental monitoring, and waste water stream monitoring

V Definitions

[00242] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Lrederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. [00243] As used herein, the term “biological sample” refers to a sample obtained from a subject. The sample may be from a subject who has been treated with a drug, or may be from an untreated or drug naive subject. Exemplary samples include, but are not limited to serum, plasma, cell lysate, milk, saliva, vitreous fluid, and other secretions, synovial fluid, peritoneal cavity fluid, lacrimal fluid, and tissue homogenate. In some embodiments, the sample is a bodily fluid, including sweat, blood, cerebrospinal fluid (CSF), plasma, whole blood, serum, semen, synovial fluid, saliva, vaginal lubrication, breast milk, amniotic fluid, urine, human feces, phlegm tears, saliva, lymph, peritoneal intracellular fluid, feces, gastric fluid, or an original tissue from fetuses, newborn babies, children, teenagers, adults or animals. Moreover, the sample can be in various forms including but not limited to a liquid, frozen, chilled, lyophilized sample. The sample may be subjected to additional purification or treatment steps prior to and/or following the affinity purification step herein.

[00244] As used herein, the term “substrate” or “analyte” refers to a substance which is catalyzed by the MAO redox-enzyme biosensor. In some embodiments, an analyte can be a “biological marker” or “biomarker”, which is an analyte in a biological system and may be used as an indicator of the risk or progression of disease.

[00245] The term “redox” or “oxidation-reduction” or “oxidoreduction” reaction describes any reaction in which electrons are transferred from one molecule, compound, molecular group, etc. to another. The process of oxidation occurs in conjunction with (is coupled with) a reduction reaction, thus resulting in the transfer of electrons.

[00246] As used herein, a “redox enzyme” or “oxidoreductase” are used interchangeably herein and refer to an enzyme that catalyzes a reaction with one or more enzyme substrate(s), resulting in generation or utilization of electrons. The oxidoreductases (redox) enzyme reaction and the electrode coupling method can be used to develop biosensors. In some instances, redox enzymes employ no prosthetic group, such as those that use reversible formation of a disulfide bond between two cysteine residues, as in the case of thioredoxin. Other redox enzymes use prosthetic groups, such as flavins, NAD, transition metal ions or clusters of such metal ions, etc. The use of the transition metal ions in these enzymes is due to their ability to attain multiple oxidation and spin states.

[00247] As used herein, the term “oxidase” refers to an enzyme that catalyzes an oxidation-reduction reaction, especially one involving dioxygen (O₂) as the electron acceptor. In reactions involving donation of a hydrogen atom, oxygen is reduced to water (H₂O) or hydrogen peroxide (H₂O₂).

[00248] As used herein, the term “biosensor device” refers to an analytical device which integrates a biorecognition element (e.g., the MAO enzyme) with a physical transducer to generate a measurable signal proportional to the concentration of an analyte (e.g., at least one amine-comprising substrate) recognized by the biorecognition element (e.g., the MAO enzyme). In some embodiments, the biosensor is also referred to as a “sensor” and can be described as device containing elements required for generating an electrical current when a biological sample is applied to the sensor. The sensor can include additional elements, such as an acquisition system and/or a display system, forming a sensor system.

[00249] As used herein, the terms “redox molecule”, “redox mediator” and “electroactive molecule” are used interchangeably herein and relate to any molecule that is able to undergo an electrochemical reaction. Upon which one or more electrons are either added to or removed from the molecule, converting it into a different oxidative state. For example, 1,4-Benzoquinone is an electroactive molecule that can be converted to hydroquione upon the reduction of the molecule with an addition of two electrons and two protons according to a specific embodiment.

[00250] As used herein, the term “redox mediator” as refers to a molecule capable of participating in an electron exchange between at least one amine-comprising substrate, a MAO enzyme, and/or the conducting polymer.

[00251] As used herein, the term “biofunctional” in the context of a molecule or a coating refers to a property of the molecule or the coating capable of electron exchange.

[00252] As used herein, the term “metabolite” refers to a small molecule formed during or after a metabolic reaction, or a metabolic pathway.

[00253] As used herein, the term “detection” or “detecting” in the context of detecting at least one amine-comprising substrate using a MAO biosensor, refers to an act of obtaining a value or a reading indicating the presence or absence of the at least one amine-comprising substrate in a sample. The detection can require a comparison of the obtained value or reading for at least one amine-comprising substrate from a test sample to a value or reading obtained from a control sample for at least one amine-comprising substrate and tested in the same way as the test sample.

[00254] As used herein, the term “planar surface” refers to a surface with a region that is sufficiently planar, e.g., sufficiently flat, over a surface area sufficient to accommodate an electrode. For example, if a planar surface is a contact lens, the contact lens has a sufficiently planar region to accommodate an electrode having a length of about 2 mm, and a width of about 2 mm.

[00255] As used herein, the term “ink” refers to a solution or suspension of a material to be deposited using inkjet printing onto a surface, such as a conducting polymer or metal, or a polymeric coating [00256] As used herein, the term “open reading frame” refers to a reading frame that has the ability to be translated. An ORF is a continuous stretch of codons that begins with a start codon (usually AUG) and ends at a stop codon (usually UAA, UAG or UGA). An ATG codon (AUG in terms of RNA) within the ORF (not necessarily the first) may indicate where translation starts. The transcription termination site is located after the ORF, beyond the translation stop codon. If transcription were to cease before the stop codon, an incomplete protein would be made during translation. In eukaryotic genes with multiple exons, introns are removed, and exons are then joined together after transcription to yield the final mRNA for protein translation. In the context of gene finding, the start-stop definition of an ORF therefore only applies to spliced mRNAs, not genomic DNA, since introns may contain stop codons and/or cause shifts between reading frames. An alternative definition says that an ORF is a sequence that has a length divisible by three and is bounded by stop codons.

[00257] In some embodiments, a polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to an assay known in the art or described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[00258] In some embodiments, a polypeptide described herein can be a variant of a polypeptide or molecule as described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions, or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity of the non-variant polypeptide. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[00259] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into lie or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Vai, into lie or into Leu.

[00260] As used herein, the term “affinity” refers to the strength of the binding interaction between a single biomolecule (e.g., a MAO redox-enzyme) to it substrate or analyte.

[00261] As used herein, a “nucleoside” is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the intemucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

[00262] As used herein, the term “small molecule” refers to low molecular weight molecules (< 900 Daltons) that include lipids, monosaccharides, second messengers, other natural products and metabolites, as well as drugs and other xenobiotics. They are distinct from macromolecules such as proteins. A small molecule is able to enter cells easily because it has a low molecular weight. Once inside the cells, it can affect other molecules, such as proteins. This is different from drugs that have a large molecular weight, such as monoclonal antibodies, which are not able to get inside cells very easily. [00263] As used herein, the term “conjugate” or “conjugation” refers to the attachment of two or more entities to form one entity. The attachment can be by means of linkers, chemical modification, peptide linkers, chemical linkers, covalent or non-covalent bonds, or protein fusion or by any means known to one skilled in the art. The joining can be permanent or reversible. In some embodiments, several linkers can be included in order to take advantage of desired properties of each linker and each protein in the conjugate. Flexible linkers and linkers that increase the solubility of the conjugates are contemplated for use alone or with other linkers as disclosed herein. Peptide linkers can be linked by expressing DNA encoding the linker to one or more proteins in the conjugate. Linkers can be acid cleavable, photocleavable and heat sensitive linkers. Methods for conjugation are well known by persons skilled in the art.

[00264] As used herein, the term “ligand” refers to a substance that forms a complex with a biomolecule to serve a biological purpose. In protein-ligand binding, the ligand is usually a molecule, which produces a signal by binding to a site on a target protein. The binding typically results in a change of conformational isomerism(conformation) of the target protein. In DNA-ligand binding studies, the ligand can be a small molecule, ion, or protein, which binds to the DNA double helix. The relationship between ligand and binding partner is a function of charge, hydrophobicity, and molecular structure. The instance of binding occurs over an infinitesimal range of time and space, so the rate constant is usually a very small number.

[00265] As used herein, the term “binding” refers to an association between proteins or nucleotides that occurs through intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces. The association of docking can be reversible through dissociation. Measurably irreversible covalent bonding between a ligand and target molecule is atypical in biological systems. Ligand binding to a receptor protein or to an allosteric transcription factor can alter the conformation by affecting the three-dimensional shape orientation. The conformation of a receptor protein or allosteric transcription factor composes the functional state. Ligands include small molecules, hormones, inhibitors, activators, and neurotransmitters.

[00266] As used herein, the term “fluorescent molecule” refers to a fluorescent chemical compound that can reemit light upon light excitation. Fluorescent molecules typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds. Current fluorescence imaging probes typically consist of single conventional fluorophore (e.g., organic dyes, fluorescent proteins), fluorescent proteins (e.g., GFP) and semiconductor quantum dots (Q-dots). Single fluorophores are usually not stable and have limited brightness for imaging. Similar to dyes, the fluorescent proteins tend to exhibit excited state interactions which can lead to stochastic blinking, quenching and photobleaching. Fluorescent molecules are known in the art and include florescent proteins (e.g., CAP, WFP, BFP, and other GFP derivatives). Other suitable fluorescent molecules are known in the art and commercially available from, for example, MOLECULAR PROBES (Eugene, Oreg.). These include, e.g., donor/acceptor (i.e., first and second signaling moieties) molecules such as: fluorescein isothiocyanate (FITC) /tetramethylrhodamine isothiocyanate (TRITC), FITC/Texas Red TM Molecular Probes), FITC/N-hydroxysuccinimidyl 1 -pyrenebutyrate (PYB), FITC/eosin isothiocyanate (EITC), N-hydroxysuccinimidyl 1 -pyrenesulfonate (PYS)ZFITC, FITC/Rhodamine X (ROX), FITC/tetramethylrhodamine (TAMRA), and others. In addition to the organic fluorophores already mentioned, various types of nonorganic fluorescent labels are known in the art and are commercially available from, for example, QUANTUM DOT CORPORATION, Inc. Hayward Calif.). These include, e.g., donor/acceptor (i.e., first and second signaling moieties) semiconductor nanocrystals (e.g., “quantum dots”) whose absorption and emission spectra can be precisely controlled through the selection of nanoparticle material, size, and composition.

[00267] As used herein, the term “device” refers to an electrically addressable unit that performs some task, such as switching, storing a single bit of information, or sensing a particular molecule or class of molecules according to an embodiment as described herein. Depending upon the embodiment, other examples of definitions also exist.

[00268] As used herein, the term “circuit” refers to a group of devices, each of which are designed to carry out similar tasks according to a specific embodiment. For example, a transistor is a switching device. A multiplier is a logic circuit constructed from many transistors, which is a circuit. As another example, a nanowire is a chemical sensing device. An array of nanowires each coated with a different molecular probe, constitutes a sensor circuit designed to sense many different molecular targets according to a specific embodiment. Depending upon the embodiment, other examples of definitions also exist.

[00269] As used herein, the term “integrated circuit” refers to a group of circuits, each design to carry out different specific tasks, but operating together to perform some larger function. For example, a multiplier circuit can retrieve two numbers from a memory circuit, multiply them together, and store them back into the memory circuit. Depending upon the embodiment, other examples of definitions also exist.

[00270] The term “percent (%) amino acid sequence identity” or “% sequence identity to amino acids” with respect to a particular SEQ ID NO is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the comparative sequence identified by the SEQ ID NO, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et al., Methods in Enzymology 266, 460-480 (1996); available on the world wide web at blast.wustUedu/blast/README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values; overlap span=l, overlap fraction=0.125, world threshold (T)=l 1. HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above. [00271] A variant amino acid or DNA sequence can be at least 70%, at least 75%, at least 80%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g., BLASTp or BLASTn with default settings). [00272] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[00273] The terms “a,” “an,” “the” and similar references used in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Further, ordinal indicators - such as “first,” “second,” “third,” etc. - for identified elements are used to distinguish between the elements, and do not indicate or imply a required or limited number of such elements, and do not indicate a particular position or order of such elements unless otherwise specifically stated. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[00274] Furthermore, the term "about," as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, is meant to encompass variations of± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even± 0.1% of the specified amount.

[00275] Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[00276] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference.

[00277] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[00278] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00279] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[00280] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00281] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely fortheir disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00282] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

1. A biosensor for the measurement of the concentration of at least one amine-comprising substrate comprising:

(a) an electrode comprising a surface;

(b) an electronically active mediator (Med) deposited on the surface of the electrode; and

(c) a plurality of monoamine oxidase (MAO) enzymes deposited on the surface of the electrode, wherein the MAO enzyme catalyzes the at least one amine-comprising substrate to produce hydrogen peroxide (H₂O₂).

2. The biosensor of paragraph 1, wherein the MAO enzyme is Corynebacterium ammoniagenes MAO (CαMAO) or a functional variant or fragment thereof.

3. The biosensor of paragraph 2, wherein the MAO enzyme is encoded by a nucleic acid comprising a sequence that is at least 90% identical to SEQ ID NO: 1.

4. The biosensor of paragraph 2, wherein the MAO enzyme comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 2.

5. The biosensor of paragraph 1, wherein the at least one amine-comprising substrate is a monoamine substrate.

6. The biosensor of paragraph 1, wherein the at least one amine-comprising substrate is a monoamine neurotransmitter or a dietary monoamine.

7. The biosensor of paragraph 1, wherein the at least one amine-comprising substrate is a polyamine substrate.

8. The biosensor of paragraph 1, wherein the at least one amine-comprising substrate is selected from the group consisting of dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5- methoxytryptamine, and hexylamine.

9. The biosensor of paragraph 1, wherein the at least one amine-comprising substrate is dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and hexylamine.

10. The biosensor of paragraph 1, wherein the MAO enzyme is immobilized on the surface of the electrode with a polymer, and optionally, a top layer above the polymer, wherein the top layer comprises Prussian-Blue (PB). 11. The biosensor of paragraph 10, wherein the polymer comprises low molecular weight (LMW) or medium molecular weight (MMW) chitosan in 0.5% acetic acid, and optionally Prussian- Blue (PB). 12. The biosensor of paragraph 1, wherein in the presence of the at least one amine-comprising substrate, the MAO enzyme produces H₂O₂, wherein breakdown of H₂O₂ to O₂ and H₂O releases electrons to produce an electrochemical signal, wherein the electrochemical signal is detected by current passed to the electrode. 13. The biosensor of paragraph 1, wherein the detectable signal is produced when the at least one amine-comprising substrate is catalyzed by the MAO enzyme and transfers at least one electron from Med_red to hydrogen peroxide (H₂O₂), resulting in its reduction to Med_ox, wherein Med_ox is reduced by the electrode producing a detectable signal. 14. The biosensor of paragraph 13, wherein the Med_ox produces an electrochemical signal, wherein the electrochemical signal is detected by current passed to the electrode. 15. The biosensor of paragraph 1, wherein the biosensor is an amperometric biosensor and the detectable signal is electrochemical. 16. The biosensor of paragraph 1, wherein the electrode is connected to a potentiostat having a current resolution to at least 1pA (100 nA). 17. The biosensor of paragraph 1, wherein the biosensor comprises a working electrode and a reference electrode. 18. The biosensor of paragraph 1, wherein the biosensor comprises a counter electrode. 19. The biosensor of paragraph 1, wherein the biosensor does not comprise a counter electrode. 20. The biosensor of paragraph 1, wherein the working electrode is > 10 ^ mm². 21. The biosensor of paragraph 1, wherein the electrode is metallic. 22. The biosensor of paragraph 21, wherein the metallic electrode is gold, silver, platinum, or palladium. 23. The biosensor of paragraph 1, wherein the electrode is non-metallic. 24. The biosensor of paragraph 23, wherein the non-metallic electrode comprises carbon. 25. The biosensor of paragraph 1, wherein the amperometric biosensor is a chronoamperometric biosensor. 26. A method of using an amperometric biosensor to measure the concentration of at least one amine-comprising substrate comprising: (a) assembling the biosensor of paragraph 1, wherein the biosensor is an amperometric biosensor; (b) providing a sample; and (c) measuring the current produced by the oxidation of at least one amine-comprising substrate present in the sample. 27. The method of paragraph 26, wherein the sample is selected from the group consisting of: gastric juice, urine, saliva, feces, cerebrospinal fluid, sweat, interstitial fluid, and blood.

28. The method of paragraph 22, wherein the method of using the amperometric biosensor is used to measure the physiological concentration of at least one amine-comprising substrate in the range of 0.01μM-80 μM range.

[00283] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[00284] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[00285] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.

EXAMPLES

Example 1: Structural insights into the substrate range of a bacterial monoamine oxidase [0052] Monoamine oxidase (MAO) enzymes, belonging to the flavoenzyme amine oxidase (FAO) superfamily, catalyze the oxidation of primary and secondary amines. In humans, MAO-A and MAO-B play a vital role in the breakdown of dietary monoamines as well as in the regulation of monoamine neurotransmitters such as dopamine and norepinephrine. The presence of MAO activity has been reported in prokaryotic species, yet it is unclear what role these enzymes play in prokaryotes. Bioinformatics analysis of the FAO superfamily have identified numerous bacterial mono- and polyamine oxidase enzymes. It has been proposed that bacterial MAOs are involved in degradation pathways which are necessary for bacteria grown with amines as a nitrogen source. These pathways are not well explored, however, so it is unclear what other enzymes may be operating in concert with these bacterial MAOs to obtain nitrogen and carbon from the environment. To gain a better understanding of the role that MAO enzymes play, a genome-neighborhood network (GNN) has been constructed for the FAO superfamily, and bioinformatic analysis identified degradation pathways in which MAO enzymes can play a role.

[0053] Oxidase activity toward monoamine substrates was first identified in Corynebacterium ammoniagenes (formerly Brevibacterium) by Murooka, et al. in 1979. They posited that bacteria grown with amines as nitrogen sources require MAOs within related amine degradation pathways. These MAOs catalyze the oxidation of primary and secondary amines present in a wide range of substrates releasing ammonia as a by-product and an aldehyde product. A potentiometric assay was used to detect ammonia within cell extracts treated with various monoamine substrates. Of the fifteen bacterial strains screened, C. ammoniagenes had the most promiscuous monoamine oxidase activity, displaying activity with tyramine, octopamine, dopamine, norepinephrine, and tryptamine. No activity was observed for the other two compounds tested, histamine and benzylamine. To further study this bacterial monoamine oxidase activity through structural and biophysical methods, the flavin- dependent enzyme, NCBI WP_040355246.1 from Corynebacterium ammoniagenes (CαMAO), was cloned and expressed. The inclusion of this enzyme in a sequence similarity network (SSN) of the flavin amine oxidase (FAO) superfamily (Pfam: PF01593) identified that CαMAO falls within the nicotine oxidase (NicOx) cluster. Other notable enzymes within this cluster include P. putida NicA2 (PDB 6C71), Shinella sp. (S)-6-hydroxynicotine oxidase, and A. niger MAO. In addition to understanding the specificity and metabolic role of the enzyme, the sequence similarity of CαMAO to enzymes involved in nicotine degradation makes its structure/fiinction analysis applicable to the engineering of related enzymes.

[0054] Described herein are the biophysical and structural properties of a promiscuous, bacterial MAO from Corynebacterium ammoniagenes (CαMAO). CαMAO catalyzes the oxidation of a number of monoamine substrates including dopamine and norepinephrine, as well as exhibiting some activity with polyamine substrates such as cadaverine. The X-ray crystal structures of Michaelis complexes with seven different substrates shows that conserved hydrophobic interactions and hydrogen-bonding pattern (for polar substrates) allows the broad specificity range. The structure of CαMAO identifies an unusual cysteine (Cys424) residue in the so-called “aromatic cage” which flanks the flavin isoalloxazine ring in the active site. Site-directed mutagenesis, steady-state kinetics and UV-vis spectroscopy revealed that Cys424 plays a role in the pH-dependence and modulation of electrostatics within the CαMAO active site. Notably, bioinformatic analysis shows a propensity for variation at this site within the “aromatic cage” of the flavin amine oxidase (FAO) superfamily. Structural analysis also identified the conservation of a secondary substrate inhibition site, present in a homologous member of the superfamily. Finally, genome neighborhood diagram analysis of CαMAO in the context of the FAO superfamily allows identification of roles for these bacterial MAOs in amino-acid biosynthesis, polyamine degradation, and catabolic pathways related to scavenging of nitrogen.

Materials and Methods

[0055] Protein expression and purification

[0056] Corynehacterium ammoniagenes NCTC 2398 (ATCC) was propagated in 5 mb M3 media at 30 °C with shaking at 200 rpm. CαMAO (WP_040355246.1) was amplified with primers using the Q5® High-Fidelity kit (NEW ENGLAND BIOLABS). The linearized backbone was then digested with Dpnl (NEW ENGLAND BIOLABS) for 1 hour at 37 °C. Amplified CαMAO was purified using the QIAQUICK PCR Purification Kit (QIAGEN) and inserted into a pET-52b(+) vector containing a cleavable C-terminal lOx-histidine tag by Gibson Assembly (NEW ENGLAND BIOLABS). Plasmids were transformed into One Shot™ TOP10 chemically competent A. coli cells (THERMOFISHER SCIENTIFIC) and plated on agar plates supplemented with carbenicillin. Gene incorporation was verified by both colony PCR and Sanger Sequencing (GENEWIZ). The resulting plasmid was transformed into E. coli BL21(DE3) competent cells (NEW ENGLAND BIOLABS). These cells were plated on a Miller’s Luria Bertani (LB) agar plate containing 100 pg/mL carbenicillin and incubated at 37 °C overnight to allow colony growth. Colonies were then selected and grown overnight in small cultures of LB broth. Starter culture (5 mL) of was used to inoculate 1 L of LB broth supplemented with carbenicillin. Cells were grown at 37 °C with shaking until an OD600 of 0.6-0.8 was reached. Protein expression was induced with the addition of 1 mM isopropyl-[3-D- thiogalactopyranoside (IPTG, GOLDBIO) and the temperature was lowered to 16 °C. After 20 hours, the cells were pelleted by centrifugation at 5,000 rpm for 20 minutes.

[0057] The cell pellet was resuspended in lysis buffer containing 50 mM Tris (pH 8.2), 500 mM NaCl, 1 mM DTT, 10 mM imidazole, 600 μL of 10 mg/mL DNase I (MILLIPORE SIGMA), and 1 tablet of protease inhibitor cocktail (THERMO FISHER SCIENTIFIC). Cells were lysed using a microfluidizer (Microfluidics Model #MI 10P) and the lysate was clarified by ultracentrifugation (38,000 x g for 35 minutes). The supernatant was applied to a 5 mL Ni-NTA column (CYTIVA LIFE SCIENCES) equilibrated with lysis buffer and was purified using an AKTA PURE FPLC system (CYTIVA LIFE SCIENCES). The column was washed five times with wash buffer (50 mM Tris, 500 mM NaCl, 1 mM DTT, pH 8.2) and the protein was eluted using a linear gradient of elution buffer (50 mM Tris, 500 mM NaCl, 1 mM DTT, 300 mM imidazole, pH 8.2). The fractions were assessed by UV absorbance using the characteristic peaks of 350 and 475 nm and those containing CαMAO were pooled and dialyzed overnight in the presence of tobacco etch virus (TEV) protease to facilitate cleavage of the His-tag. The dialysate was applied to the Ni-NTA resin to separate the His-tagged from the cleaved CαMAO protein. Purified protein was concentrated and quantified using the absorbance at 280 nm using a NANODROP instrument (THERMOFISHER SCIENTIFIC) and purity was assessed by SDS-PAGE. Aliquots of protein were flash frozen using liquid nitrogen in the presence of 15% glycerol. The final yield of protein was 12.5 mg/g cell pellet.

[0058] Generation of caMAO variants using site-directed mutagenesis

[0059] The CαMAO variants (C424V, C424Y, C424S, W387Y, and E207A) were generated using the Q5 site-directed mutagenesis kit (NEW ENGLAND BIOLABS). Primers (listed in Table 4) were designed using the NEBase Changer (NEW ENGLAND BIOLABS). Plasmids encoding the mutations were purified using the MONARCH Plasmid Miniprep Kit (NEW ENGLAND BIOLABS) and incorporation of the base changes was confirmed by Sanger sequencing (GENEWIZ). Expression and purification of these variants was carried out in the same manner as for wild-type enzyme.

[0060] Steady-state kinetic analysis and pH-rate profile

[0061] Enzyme activity for both wild-type enzyme and each of the variants was determined using the AMPLEX ULTRARED Assay kit (THERMOFISHER SCIENTIFIC). Substrate concentration was varied from 1000 μM to 1 μM through serial dilution in water to a final volume of 25 μL in a black, polypropylene 96-well plate (CORNING). A master mix containing 2 U mL- Ihorseradish peroxidase (HRP, THERMOFISHER SCIENTIFIC) and 125 μM AMPLEX ULTRARED (THERMOFISHER SCIENTIFIC) diluted in 50 mM sodium phosphate assay buffer at pH 7.4 was added to each well at 50 μL. Lastly, 25 μL of CαMAO (final concentration 2 nM) was added to each well to initiate the reaction. Fluorescence at Xex = 490 nm, λcm = 585 nm was monitored every 20 seconds using a SPECTRAMAX M5 microplate reader (MOLECULAR DEVICES). The resulting data were fit using Michaelis-Menten kinetic equations by GRAPHPAD PRISM (GRAPHPAD SOFTWARE) to obtain apparent K_m and V_max values. A standard curve was generated with varying hydrogen peroxide concentrations (2500, 1250, 625, 312, 156, 78, 39 nM), 2 U mL-IHRP and 125 μM AMPLEX ULTRARED to a final volume of 100 μL. The plate was covered and incubated at 25 °C for 30 minutes and endpoint fluorescence was read at the same excitation and emission wavelengths as described above.

[0062] Activity of the wild-type CαMAO and the C424S variant was tested using dopamine as a substrate across a pH range of 7.0 - 9.0. Five buffer solutions were prepared containing 50 mM Tris, 50 mM sodium phosphate adjusted to pH values of 7.0, 7.5, 8.0, 8.5, and 9.0. Before kinetic analysis, the stability of the enzyme was confirmed at each of these buffers using differential scanning fluorimetry (DSF). At a final volume of 20 μL, 10 μM CαMAO and SYPRO orange dye (5X, THERMOFISHER SCIENTIFIC) were mixed in a 96-well PCR plate format with 3 replicate wells for each pH. A REALPLEX MASTERCYCLER RT PCR instrument (EPPENDORF) was used to monitor fluorescence at 520 nm with increasing temperature at a rate of 1 °C min"¹ from 4 °C to 95 °C. The T_m values were calculated as the peak of the first derivative of the melting curve. After confirming that CαMAO and the coupling enzyme, HRP, are both stable within this pH range, a separate hydrogen peroxide standard curve was generated for each pH using the same AMPLEX ULTRARED assay method as described above. Steady-state kinetics with dopamine concentrations ranging from 2 mM to 8 μM were performed using the same assay setup. Fluorescence values were converted to micromoles of product using the respective standard curve for each pH. Data were fit to Michaelis-Menten kinetics using GRAPHPAD PRISM (GRAPHPAD SOFTWARE) to obtain apparent K_m and V_max values for dopamine at each pH.

[0063] Using a discontinuous assay, activity at the crystallization condition pH was assessed by comparison of the activity of the MAO in 50 mM MES/Tris buffer at pH 7.5 and pH 5.5. Two reactions with 12.5 nM CαMAO and 1 mM dopamine were performed in buffers of pH 5.5 and pH 7.5 and incubated at room temperature for 15 minutes. HRP and AMPLEX ULTRARED were added to the reaction (at final concentrations of 2 U/mL and 125 μM, respectively) and fluorescence was immediately read. Positive controls containing 2.5 μM H₂O₂ were performed at both pH 5.5 and pH 7.5 to confirm HRP activity. To confirm that HRP remained in solution at pH 5.5, each solution was transferred to a clear-bottom 96-well assay plate (CORNING) and absorbance was read at 600 nm. [0064] A test for enzyme inactivation was performed for the wild-type CαMAO by testing activity with 250 μM of dopamine at pH 7.0 and pH 8.5. After monitoring the reaction for 20 minutes, additional enzyme was spiked into each reaction well (or the equivalent volume of assay buffer in the control wells) and monitoring of fluorescence was resumed.

[0065] Crystallization and data collection

[0066] Wild-type CαMAO at 10 mg/mL in 50 mM Tris, 100 mM NaCl, 1 mM DTT, pH 8.2 was crystallized using the hanging-drop vapor diffusion method by combining protein and well solution in a 1: 1 ratio using the following condition: 10 - 15% w/v PEG-3350, 0.1 M sodium malonate, pH 5.0 (HAMPTON RESEARCH). Crystals usually grew within 5 days and were harvested and transferred to a solution containing mother liquor and 15% glycerol before flash-freezing in liquid nitrogen. Data for the wild-type CαMAO (PDB 8EEI) structure and both the unbound (PDB 8EEN) and dopamine- bound (PDB 8EEI) structures of the C424S variant were collected at the National Synchrotron Light Source-II (NSLS-II) at Brookhaven National Laboratory on beamline AMX (17-ID-l) which is equipped with an EIGER 9 M detector. Data for the CαMAO structures bound to the following substrates were also collected at NSLS-II on the FMX beamline (17-ID-2) equipped with an EIGER 16 M detector: dopamine (PDB 8EEG), cadaverine (PDB 8EEO), octopamine (PDB 8EEF). Lastly, datasets for CαMAO structures bound to the following substrates were collected at Stanford Synchrotron Radiation Lightsource on beamline 12-2 equipped with a DECTRIS PILATUS 6 M detector: tryptamine (PDB 8EEH), norepinephrine (PDB 8EEM), tyramine (PDB 8EEK), and 5- aminopentanol (PDB 8EEL). The structures bound to cadaverine and dopamine were obtained by co- crystallization with substrates included at a final concentration of 2 mM in the drop. All other substrate-bound structures were obtained by soaking CαMAO crystals in the well solution mixed with 15% glycerol and 1-2 mM substrate for 5-20 min before cryo-protection.

[0067] Phase determination and structure refinement

[0068] A truncated molecular replacement search model of the nearest structural homolog, (S)-6- hydroxynicotine oxidase (PDB 6CR0, 28.6 % sequence identity to CαMAO), was generated with Phenix. Sculptor using the default parameters (mainchain deletion = gap, sidechain pruning = schwarzenbacher) and a sequence alignment of CαMAO obtained using the BLASTp suite (NCBI). These truncated coordinates were used as the input for Phenix.Phaser using the default parameters except for the following: allowed clashes cutoff = 20, allow all solutions (no packing test), and high- resolution limit for refinement = 3 A. The resulting model and map files were provided to Phenix. Autobuild (the option to morph input model into density was selected) which solved a partial solution of this structure (comprising 397 of the 443 residues). Iterative rounds of refinement were performed on this model using Phenix.Refme with the following refinement parameters: individual B- factors, translation-liberation-screw (TLS) parameters, and simulated annealing. Remaining missing residues and sidechains were built manually in Coot during this process. Phases were calculated for each of the structures of CαMAO bound to substrate using the unliganded structure and were refined in the same manner. The flavin adenine dinucleotide (FAD) cofactor and ligands were placed using Phenix.LigandFit and validated using both omit and Polder electron density maps. Crystallographic data and refinement statistics are provided in Table 5. Solvent accessible tunnels and cavity volumes were calculated using CaverWeb vl.l (LOSCHMIDT LABORATORIES). Ligand interactions were confirmed using both PyMOL 2.5 (Schrodinger) and LigPlot+ v2.2 (EMBL-EBI) with default parameters.

[0069] Genome neighborhood diagram analysis

[0070] A colored SSN was generated using the EFI -EST SSN tool for the FAO superfamily using the Pfam PF01593 with the sequence for the CαMAO added. An alignment score of 55 was used and sequence length cutoffs of no shorter than 200 and no longer than 1000 amino acids. This SSN was visualized and analyzed using CYTOSCAPE. The EFI-EST genome neighborhood tool (GNT) was then applied to this SSN using standard parameters to calculate the GND. Operons within the cluster containing the CαMAO (Cluster 14) were visually inspected in the GND explorer (EFI-EST). [0071] UV-Vis flavin spectroscopy

[0072] Fresh stocks of WT CαMAO and C424S variant were concentrated to 20 mg/mL and diluted to 1 mg/mL in buffers containing 50 mM Tris and 50 mM sodium phosphate adjusted to pH values of 7.0, 7.5, 8.0, 8.5, and 9.0. Each sample was filtered using a 0.22 pm PVDF filter (CELLTREAT) and centrifuged on a benchtop centrifuge at 14,000 rpm for 10 minutes. Each sample was transferred to a 1 mb quartz cuvette. UV-Vis spectra were collected from 200 nm to 800 nm in triplicate using a BECKMANN COULTER DU-800 spectrophotometer. After collecting initial spectra, each sample was treated with 10% sodium dodecyl sulfate (SDS) to unfold the MAO. After 20 minutes of incubation at room temperature, each sample was once again filtered and centrifuged. Triplicate spectra were collected for the post-SDS samples in the same manner as above and flavin concentration was calculated for each sample using E450 = 11,300 M-ls-1. The pre-treatment spectra were then normalized by flavin concentration before further analysis.

Results and Discussion

[0073] caMAO kinetics

[0074] Kinetic characterization of the purified CαMAO enzyme using a fluorescence-based peroxide detection assay confirmed a broad substrate range toward both monoamine and polyamine substrates. Unless otherwise stated, all kinetic experiments were performed at room temperature in a 50 mM sodium phosphate buffer at pH 7.4. Similar to the observations with cell extracts, the monoamine substrates identified include tryptamine, tyramine, octopamine, and dopamine (see e.g., Table 1). The k_cat and K_m values reported in Table 1 are apparent values, as all measurements were performed at a single concentration of O₂. Lower activity was observed for norepinephrine (Table 1) despite sharing structural features with the other monoamine substrates (see e.g., Fig. 6). Using dopamine as a substrate, inhibition of CαMAO by the poor substrate norepinephrine was observed (Ki was not measured). Furthermore, no activity was observed with epinephrine, indicating that CαMAO preferentially oxidizes primary amines as opposed to secondary amines. Surprisingly, CαMAO also exhibited some activity (detection above background at 1 mM substrate) with the polyamine substrate cadaverine, but not with the shorter polyamine, putrescine, or longer polyamines: spermine and spermidine (see e.g., Table 6). Replacement of the second amino group on cadaverine with a hydroxyl group (resulting in the monoamine substrate 5 -aminopentanol) allowed detection above background (with 1 mM substrate) whereas replacement with a methyl group (in the nonpolar, monoamine substrate hexylamine) resulted in a substantial improvement in catalytic efficiency to 1.9 x 10⁴M^-1S^-1 (see e.g., Table 6). The same replacements on the scaffold of putrescine (4-aminobutanol and pentylamine, respectively) had similar effects wherein the detection of activity above background could not be measured for either polar substrate at 1 mM. For cadaverine, 5 -aminopentanol, 4- aminobutanol, and pentylamine, accurate K_m and k_cat values could not be measured due to solubility limitations, but activity was compared at 1 mM substrate. Lastly, CαMAO displayed no activity with nicotine analogs (S-nicotine and S-6-hydroxynicotine) or amino acids (tryptophan, tyrosine, arginine, lysine) indicating that this enzyme does not exhibit nicotine oxidase or L-amino acid oxidase (LAAO) activity (see e.g., Table 6).

[0075] Unliganded structure of caMAO

[0076] The structure of the unliganded, holo enzyme was determined using X-ray crystallography. Molecular replacement was performed using a truncated model of the homologous enzyme, S-6 hydroxynicotine oxidase (PDB 6CR0) from Shinella sp. HZN7. The overall fold of this enzyme includes the canonical Rossmann-core fold (flavin-binding domain; FBD), a hotdog-like fold (substrate-binding domain I; SBD-I), and a helical bundle (substrate-binding domain II; SBD-II) (see e.g., Figure 1A). The active site, a cavity adjacent to the face of the flavin isoalloxazine ring with a volume of 457 A3, is comprised predominantly of aromatic and aliphatic residues including Phel75, Alal78, Leu211, Phe341, and Val342 which come together at the interface of the three domains to form the active site (see e.g., Figure IB). Only one polar sidechain extends into the active site, Glu207, positioned within the tunnel connecting the active site to bulk solvent (see e.g., Figure 1C). The two so-called “aromatic cage” residues flanking the isoalloxazine ring are Trp387 and a noncanonical cysteine residue, Cys424 (see e.g., Figure IB). A cysteine residue within the aromatic cage has only been observed in one other structurally-characterized FAO, a polyamine oxidase (PAO) from .S', cerevisiae (PDB 4ECH). The role of an aromatic cage cysteine has not been previously investigated.

[0077] The DALI server identified A. niger MAO (29% ID, PDB 2VVL) as the closest structural homolog to CαMAO, with a backbone RMSD of 2. 1 A (see e.g., Figure 7). The second nearest structural homolog identified by DALI (RMSD of 3.2 A) was P. nicotinovorans y-N- methylaminobutyrate oxidase (21% ID, PDB 7RT0), a bacterial MAO with preferential activity toward secondary amines (see e.g., Figure 7). Inspection of the P. nicotinovorans MAO structure revealed that parts of the active site are not resolved, presumably due to disorder. Other close structural homologs identified by DALI included various LAAO and PAO enzymes. Two other enzymes located in the NicOx cluster of the FAO SSN, P. putida NicA2 (PDB 6C71) and Shinella sp. S-6-hydroxynicotine oxidase (PDB 6CR0) aligned to CαMAO with RMSD values of 3.89 and 3.91 A, respectively (see e.g., Figure 7). Separate alignment of domains identified that the FBD had high structural similarity (RMSD 1.4 - 2.8 A), whereas the SBD-II had lower structural similarity, as reflected by higher RMSD values (2 - 6.5 A) (see e.g., Figure 7). This is expected, as the flavin cofactor is held in common between all FAO members whereas the substrate-binding domain differs to provide specificity.

[0078] Structure-activity relationships of MAO substrates

[0079] Structures of the MAO in complex with the polyamine substrate cadaverine as well as various monoamine substrates including: dopamine, octopamine, tryptamine, tyramine, norepinephrine, and 5 -aminopentanol were resolved via co-crystallization or soaking. In the crystalline state no reduction was observed with addition of substrate (crystals remained yellow), consistent with the assignment of the molecule observed in the active site of each structure as the substrate and not product. Although it is impossible to discern substrate from the aldehyde product from electron density alone, the excess substrate and lack of color change is consistent with very slow turnover in the crystalline state, possibly due to the low pH (5.5) of the solution in which the crystals are grown or the constraints of the crystal lattice. The effect of pH on activity was assessed using a discontinuous form of the assay (see e.g., Materials and Methods), showing that the rate of the reaction with 1 mM dopamine was ca. 10-fold lower at pH 5.5 compared to pH 7.5. Thus, even in the co-crystallization experiments, the low pH and substrate inhibition (vide infra) allow visualization of bound substrate.

[0080] Each substrate bound in the expected orientation with the amino group positioned within 3 Å of the N5 atom of the flavin isoalloxazine ring. Tryptamine binding is achieved by hydrophobic contacts alone with no polar interactions (see e.g., Figure 2A) whereas hydroxyl groups of other monoamine substrates form hydrogen bonds with active-site residues. Octopamine, tyramine, dopamine, and norepinephrine make similar interactions through hydroxyl groups that decorate the benzene ring (see e.g., Figure 2B, 2C, Figure 8A, 8B). In dopamine and norepinephrine, the hydroxyl group in the position meta to the aminoethyl moiety forms the sole hydrogen bond with an amino-acid sidechain- Glu207 (see e.g., Figure 2B, Figure 8B) and, in the case of norepinephrine, through a water-mediated interaction. In octopamine and norepinephrine, hydroxyl groups in the para position and on the aminoethyl group form hydrogen bonds with the backbone carbonyl group of Phel75 and Thr210, respectively (see e.g., Figure 2C, Figure 8B). These same interactions are present in the structures of CαMAO bound to tyramine, dopamine, norepinephrine, and octopamine (see e.g., Figure 2B, 2C, 2E; Figure 8A, 8B). The structure of cadaverine shows that the N5 amino group forms a similar interaction to that of a substrate hydroxyl with the backbone of Phel75 (see e.g., Figure 2D). The monoamine substrate 5 -aminopentanol binds to the active site in two distinct conformations, as confirmed using Polder maps. In one conformation, the same interaction is formed between the substrate hydroxyl group and Phel75 (see e.g., Figure 8C). In the other conformation, the hydroxyl group of 5 -aminopentanol instead forms a hydrogen bond with the sidechain of Glu207. None of the substrates interacted directly with Cys424, indicating that the cysteine does not directly play a role in substrate coordination or positioning.

[0081] These substrate -complex structures also provide insight into the structure -activity relationships for substrates screened for activity with CαMAO (see e.g., Table 6). For example, there is a lack of activity observed with putrescine, with a carbon chain one atom shorter than cadaverine. The structure of the MAO bound to cadaverine indicates that a shorter carbon chain would place the C4 amino group too distant from Phel75 to form the same interaction (see e.g., Figure 2D). Hexylamine and pentylamine exhibit greater activity than their polar and polyamine analogs owing to the hydrophobic nature of the active site. 5 -Aminopentanol (see e.g., Figure 8C) and cadaverine (see e.g., Figure 2D) exhibit some activity over background as their polar moieties can form interactions with Phel75 (T see e.g., able 6). The charges of the amino and carboxyl groups present in the L-amino acid substrates screened were not tolerated by the hydrophobic active site, leading to no activity measurable above background (see e.g., Table 6). [0082] Kinetic analysis of active-site variants

[0083] To better understand the role of residues in the active site and aromatic cage, variants were generated using site-directed mutagenesis. The kinetics of each variant were compared to the wild-type CαMAO with respect to the monoamine substrates of highest activity: dopamine, norepinephrine, tyramine, tryptamine, and octopamine. Elimination of the two backbone interactions formed between CαMAO and the substrates was attempted by altering backbone conformation with the following substitutions: F175P and L21 IP. However, both substitutions resulted in inactivity (see e.g., Table 2), as a significant portion of enzyme unfolded (as determined by DSF experiments and visible formation of aggregates). Glu207, which contributes the only polar sidechain involved in substrate binding, was substituted with alanine. This variant displayed a significant 15 -fold decrease in activity with dopamine corresponding to a decrease in k_cat and an increase in K_m values (see e.g., Table 2). Without wishing to be bound by theory, it is posited that this change in activity results from the loss of the hydrogen bond between Glu207 and the hydroxyl group on the substrate (see e.g., Figure 2B). Although this sidechain makes a similar interaction with norepinephrine, activity with this substrate remained similar to that of the WT enzyme, as the two additional hydrogen bonds that norepinephrine forms are retained (see e.g., Figure 2E; Figure 8B).

[0084] Additionally, site-directed mutagenesis was used to determine the contributions of the aromatic cage residues. Trp387 was substituted for tyrosine - the amino acid present in the analogous position of the closest structurally-characterized homolog, Shinella sp. S-6-hydroxynicotine oxidase (PDB 6CR0; (note P. putida NicA2, with lowest RMSD also has a Trp in this position)). This substitution resulted in no significant change in k_cat or K_m (see e.g., Table 2), indicating that preservation of an aromatic group at that position retains activity. Mutation of Cys424 to an aromatic group, specifically tyrosine (also present in the analogous position in PDB 6CR0) resulted in inactivity (see e.g., Table 2). Melting temperature analysis using differential scanning fluorimetry (DSF) confirmed destabilization of the enzyme with a shift from 55.5 °C for WT to 48.9 °C for Cys424Y and a more significant portion of the enzyme was unfolded during purification, likely due to steric issues caused by introduction of a bulky sidechain at that position. Sequence alignment of CαMAO with the other proteins in the NicOx cluster of the SSN identified a gap present in almost all sequences aligned to the Cys424 position, making primary structure analysis difficult. One homolog, G. terrae PAO (UNIPROT ID: A0A4U9ZY38) has a valine in the analogous sequence position. The substitution C424V resulted in decreased activity in comparison to the WT CαMAO enzyme (see e.g., Table 2), most notably, a 30- and 10-fold decrease in k_cat/K_m values for tryptamine and norepinephrine, respectively. In both cases, the decreased activity is attributed to an increase in the K_m of the substrates and a decrease in k_cat. This residue was also substituted with a serine to maintain steric bulk and polarity of the sidechain without the propensity to ionize. The C424S variant displayed significantly lower k_cat/K_m values for dopamine, octopamine, and tryptamine, ranging from 7.71 x 10² to 1.85 x 10³(s^-1 M ¹) (see e.g., Table 2). The enzyme exhibited no activity with the poorest substrate, norepinephrine, but, notably, no change in activity with tyramine as compared to WT (see e.g., Table 2).

[0085] pH-rate profile

[0086] To further study the contribution of Cys424 to activity, pH-rate profiles were compared between the WT CαMAO and C424S variant. After confirming the stability of AMPLEX assay reagents and CαMAO within the pH range tested (see e.g., Figure 9), the activity of WT and C424S with the substrate dopamine was tested at the following pH values: 7.0, 7.5, 8.0, 8.5, and 9.0. The WT CαMAO exhibited increased activity with increasing pH with a total 10-fold increase in k_cat/K_m between pH 7.0 and pH 9.0 (see e.g., Table 3, Figure 3A, Figure 3C). Dopamine had no significant change in K_m across this pH range, highlighting that the differences in catalytic efficiency can be attributed to changes in k_cat alone. A decrease in rate at high substrate concentrations indicated substrate inhibition. Fitting to a substrate inhibition curve was consistent with a decrease in Ki of dopamine with increasing pH (see e.g., Table 3). To confirm that this trend was due to substrate inhibition and not enzyme inactivation, additional fresh enzyme was spiked into the reaction mixture after 20 minutes (see e.g., Figure 3D). Activity did not increase with the addition of fresh enzyme (see e.g., Figure 3D), indicating that the decreasing rates observed at high substrate concentrations is due to substrate inhibition rather than enzyme inactivation. All monoamine substrates tested exhibited substrate inhibition. It should also be noted that substrate inhibition has been observed with other members of the FAO family, including P. putida NicA2.9. As can be seen in Figure 3D, a decrease was also observed in the rate of the reaction at extended time points; an effect which occurs more rapidly in the reaction tested at pH 8.5 than the reaction at pH 7.0. This can be due to the buildup of peroxide in the AMPLEX ULTRARED assay. It was tested whether the curvature was due to product inhibition using one of the MAO products, hexanal (the oxidized product of the reaction with hexylamine). As hexanal does not inhibit strongly (50% loss of activity at 1 mM), and significant product is not produced during the assay period, product inhibition is not the likely cause of the curvature.

[0087] Unlike the WT enzyme, the C424S variant did not display changes in k_cat/K_m with varying pH (see e.g., Figure 3B-3C, Table 3). Therefore, ionization of Cys424 can be responsible for the pH- rate profile observed for WT enzyme. To test whether the deprotonated form of cysteine is responsible for the pH-rate profiles, the C424D variant was generated to observe the effect of an enforced negative charge in the aromatic cage. This variant displayed very little activity with all substrates at a concentration of 1 mM, likely due to the steric bulk of the aspartate sidechain (as observed with the C424Y variant). Because substrate inhibition was also observed with the C424S variant, the ionization of Cys424 is not responsible. However, the effect of pH on substrate inhibition was slightly offset (by approximately half of a pH unit) between WT and the C424S variant. For example, at pH 8.0, dopamine had a Ki of 491 μM with WT whereas dopamine had a Ki of 1.2 mM for the C424S variant at that same pH (see e.g., Table 3).

[0088] UV-Vis spectroscopy of flavin cofactor

[0089] The pH-dependence observed for the WT CαMAO can be a result of changes to the protein active-site residues or changes to the flavin cofactor. To probe the latter possibility, UV-Vis spectra were recorded on both the WT and C424S proteins at varying pH to observe any changes to the flavin spectra. Spectra were collected for both proteins in buffers adjusted to pH values of 7.0, 7.5, 8.0, 8.5, and 9.0 (see e.g., Figure 10). For both proteins, two distinct peaks were observed at wavelengths near 375 nm and 460 nm which are characteristic of the spectrum of oxidized flavin bound to enzyme. Comparison of the WT CαMAO spectra from pH 7.0 to pH 9.0 shows no large or systematic changes with increasing pH (see e.g., Figure 10A). Therefore, there are no significant changes in the electronic character of the chromophore or its environment within the pH range being tested. Taken together with the kinetic analysis, this indicates that the variance in k_cat/K_m between pH 7.0 and 9.0 is likely not caused by drastic changes in the state of the cofactor. Instead, the changes in pH can affect the protonation state of Cys424 and/or the substrate amine. There was also observed little to no difference in the spectra with pH for the C424S variant, which can be expected due to the elevated pKa of the serine sidechain in comparison to that of cysteine (see e.g., Figure 10B).

[0090] Comparison of the WT and C424S variant spectra at each pH reveals additional features associated with the presence or absence of cysteine at position 424. First, there is a large increase in the absorbance for the peak centered at -375 nm for the variant as compared with WT CαMAO (see e.g., Figure 10C). A red shift was also observed in the wavelength of the second peak from - 464 nm for the WT protein to -469 nm for the variant (see e.g., Figure 10C), and the shoulder near 490 nm is more resolved in the spectrum of the variant (see e.g., Figure 10C). Similar changes to the longer- wavelength peak have been reported resulting from changes in hydrogen bonding with the isoalloxazine moiety. Although Cys424 does not engage in direct interaction with the flavin in any of our X-ray crystal structures (see e.g., Figure 4A) and without wishing to be bound by theory, it was hypothesized that the less-bulky hydroxyl of Ser424 can permit the side chain to assume a different rotamer conformation and form a hydrogen bond with the flavin. This type of direct interaction can produce the changes observed in the peak observed at 469 nm. To probe this hypothesis, the structure of the CαMAO C424S variant was obtained in an unliganded form as well as in complex with the substrate dopamine. In both structures, Ser424 assumes two rotamer conformations (each with approximately 50% occupancy, see e.g., Figure 4B). In the structure of the variant bound to dopamine, two conformations of the substrate were also observed, one of which engages in direct interaction between the amino moiety and the hydroxyl of Ser424, contributing to the lower activity of the C424S variant by binding the substrate in a conformation which is not catalytically competent. In the other conformation of the serine, the hydroxyl moiety of Ser424 forms a hydrogen bond with the carbonyl at C2 of the flavin isoalloxazine ring (see e.g., Figure 4B). Taken together, these results indicate that the chromophore is environmentally sensitive to the amino acid present at that position of the aromatic cage. Some of the aromatic cage substitutions tested - C424Y and C424D - likely perturb the active site as a result of steric changes whereas others - C424S and C424V - can be responsible for subtle electronic effects on the flavin isoalloxazine, ultimately effecting the chemistry of the enzyme. The electronic properties of the site will also be altered by the presence of the substrate.

[0091] Substrate inhibition

[0092] Further structural and kinetic studies aided in the identification of the mechanism of substrate inhibition. The computational binding-hotspot program FTMap was used to identify secondary binding sites in CαMAO. FTMap identified a secondary hotspot within the tunnel that leads to the active site (see e.g., Figure 5A). A similar substrate inhibition site was identified in P. putida NicA2 (see e.g., Figure 5B) using both computational and crystallographic methods. In the FTMap results for CαMAO, the hotspot probes cluster together in close proximity to the sidechain of Glu207, which spans both the active site and this secondary site (see e.g., Figure 5A). Activity of the E207A variant (discussed above) was assessed with tryptamine at pH 8.5 to determine if the glutamate sidechain plays a role in substrate inhibition. Unlike the WT enzyme, no substrate inhibition was observed with this variant at pH 8.5. Glu207 therefore plays a role in both substrate recognition in the active site, as well as binding a substrate molecule within this secondary binding site.

[0093] Genome neighborhood diagram analysis

[0094] A GND was calculated from the SSN of the FAO superfamily to better understand the genomic context of these bacterial MAO enzymes. CαMAO is located in the SSN cluster previously designated as the NicOx cluster. Operons in this cluster containing bacterial MAO enzymes were analyzed using the GND explorer to identify potential pathways in which these enzymes operate (see e.g., Figure 11). Many of the operons within this cluster were observed to contain an aldehyde dehydrogenase (ALDH) enzyme downstream of the MAO (see e.g., Figure 11A). For monoamine substrates such as tyramine or tryptamine, the conversion of the MAO product (an aldehyde) by ALDH would result in the formation of amino acid products tyrosine and tryptophan, respectively. Other operons were found to contain polyamine aminotransferase enzymes upstream of the MAO (see e.g., Figure 1 IB). The product of a polyamine aminotransferase would be a monoamine which could then be further altered by these bacterial MAOs. Lastly, in a few operons, the enzyme agmatinase, was observed (see e.g., Figure 11C). This enzyme is responsible for the breakdown of agmatine - a by-product of arginine degradation - into putrescine and urea. Putrescine can then be further processed through several routes. In some cases, this molecule may be used as a substrate for a PAO enzyme. Alternatively, a polyamine aminotransferase can remove one of the amino groups, making it a suitable substrate for an MAO enzyme. Taken together, this analysis indicates roles for bacterial MAOs including: (1) amino acid biosynthesis, (2) polyamine degradation, and (3) part of degradation pathways related to scavenging of nitrogen.

Discussion

[0095] It is unclear why C. ammoniagenes utilizes these pathways, but the promiscuity of CαMAO permits oxidation of a wide variety of monoamine substrates. This broad specificity observed with CαMAO is in stark juxtaposition to other enzymes within the same cluster of the FAO SSN, such as P. putida NicA2 which exhibits strong preference for a single substrate, S-nicotine. The difference in substrate range between NicA2 and CαMAO, with high structural similarity (rmsd 1.89 A) but low sequence identity (26.8 %) demonstrates that this scaffold can be tuned for specificity through changes to the active-site residues and/or more extensive substitutions within the protein sequence. The retention of shared structural/functional features is also reflected in the fact that CαMAO demonstrates the secondary substrate binding site and substrate inhibition observed in NicA2. Moreover, NicA2 and CαMAO both position a non-canonical amino acid within the aromatic cage. Our spectroscopic analysis of CαMAO shows that the C424S substitution in the aromatic cage may alter active-site electrostatics and/or the redox potential of the isoalloxazine ring of the flavin cofactor. To assess the propensity of non-canonical amino acids in the aromatic cage, a sequence alignment of all proteins in the nicotine oxidase (NicOx) cluster of the FAO SSN was used (and alignment verified by AlphaFold-predicted models of 29 of the homologs). Approximately 70% of the proteins within this cluster have canonical aromatic amino acids in both positions of the cage. The sequence/structure alignment also identified that the most common aromatic residue near the methylated C7 and C8 of the isoalloxazine ring is tryptophan (87%) whereas the most common aromatic residue near C2 and N3 of the isoalloxazine ring is phenylalanine (55%). In 16% of sequences, one position is an aromatic residue and the other position is not and in the remaining 4% of the sequences analyzed, both residues are non-aromatic. Of these occurrences of non-aromatic residues, approximately half maintain hydrophobic amino acids in one or both of the “aromatic cage” positions. Unexpectedly, in a small subset of sequences, the non-canonical amino acid is either proline or glycine. The remaining non- canonical residues identified include the polar or charged amino acids cysteine, serine, threonine, asparagine, or arginine. As observed with CαMAO, these non-canonical residues can alter the electrostatics of the active site and/or may alter the redox potential of the isoalloxazine ring, suggesting this position as an evolutionary handle to tune the reactivity and specificity of these FAO enzymes.

[0096] ACCESSION CODE: CαMAO Uniprot ID A0A807MR40

[0097] ABBREVIATIONS: MAO, monoamine oxidase; FAO, flavin amine oxidase; CαMAO, Corynebacterium ammoniagenes monoamine oxidase; SSN, sequence similarity network; GND, genome neighborhood diagram; PAO, polyamine oxidase; FBD, flavin-binding domain; SBD, substrate-binding domain

Tables

[0098] Table 1. MAO kinetics with monoamine substrates.

[0099] Table 2. CαMAO variant kinetics

[00100] Table 3. pH-rate kinetics for WT and C424S MAO.

[00101] Table 4. Primers used for site-directed mutagenesis

[00102] Tables 5A-5E. X-ray crystallography data collection and refinement statistics

[00103] Table 6. All compounds screened for activity with C. ammoniagenes MAO (CαMAO).

[00104] The following references cited herein are each incorporated herein by reference in their entireties: Gaweska et al. Biomol. Concepts 2011, 2 (5), 365-377; Edmondson et al. Arch. Biochem. Biophys. 2007, 464(2), 269-276; Binda et al. ACS Med. Chem. Lett. 2012, 3 (1), 39-42; Weyler et al. Pharmacol. Ther. 1990, 47 (3), 391-417; Murooka et al. Appl. Environ. Microbiol. 1979, 38 (4); Tararina et al. J.Mol. Biol. 2020, 432 (10), 3269-3288; Zeng et al. J. Bacteriol. 2013, 195 (22), 5141; Hacisalihoglu et al. Microbiology 1997, 143 (2), 505-512; Tararina et al. Biochemistry 2021, 60 (4), 259-273; Tararina et al. Biochemistry 2018, 57 (26), 3741-3751; Li et al. Biochemistry 2006, 45, 15, 4775-4784; Adachi et al. Biochemistry 2012, 51 (24), 4888-4897; Stourac et al. Nucleic Acids Res. 2019, 47 (Wl), W414-W422; Laskowski et al. J. Chem. Inf. Model. 2011, 51 (10), 2778-2786; Zallot et al. Biochemistry 2019, 58 (41), 4169-4182; Shannon et al. Genome Res. 2003, 13 (11), 2498- 2504; Gelin et al. ACS Infect. Dis. 2020, 6 (3), 422-435; Holm et al. Trends Biochem. Sci. 1995, 20 (11), 478-480; Atkin et al. J. Mol. Biol. 2008, 384 (5), 1218-1231; Chiribau et al. FEBS J. 2006, 273 (7), 1528-1536; Tararina et al. Biochemistry 2018, 57 (26), 3741; Deay et al. Arch. Biochem.

Biophys. 2022, 718, 109122; Massey et al. Biochemistry 1965, 4 (6), 1161-1173; Harbury et al. Proc. Natl. Acad. Sci. U. S. A. 1958, 44 (7), 662; Kozakov et al. Nat. Protoc. 2015, 10 (5), 733-755.

Example 2: MAO Electrochemical Biosensors

[00105] Described herein in an enzyme-based electrochemical monoamine biosensor that can specifically and continuously detect a wide spectrum of monoamines in real-time and at concentrations appropriate for a range of commercial and health applications. The underlying sensing part is a bacterial monoamine degrading enzyme which functions similarly to glucose oxidase found in commercially available continuously monitoring glucose biosensors. Commercially available parts can thus be used to similarly develop a small scale, portable biosensor that measures monoamine levels in fluids. No commercial real-time enzyme-based biosensor which can measure multiple monoamines continuously currently exists. The sensor can be used in any application and/or sample where the accurate, specific, and rapid sensing of monoamines is useful.

[00106] Described herein is a MAO electrochemical biosensor comprising the MAO from Corynebacterium ammoniagenes . Such a MAO electrochemical biosensor reacts to a wide range of compounds (see e.g., Example 1). [00107] Corynebacterium ammoniagenes (Cooke and Keith) Collins (ATCC® 6871™) was used from ATCC. The gene locus tag CAMM_RS00195 was amplified from bacterial genomic DNA, isolated, and cloned into the pET-52b(+) E. coli bacterial expression vector. The MAO, WP 040355246.1, was purified from cells and tested with the AMPLEX ULTRARED Assay (THERMO FISHER SCIENTIFIC) for hydrogen peroxide production in the presence of tryptamine, tyramine, octopamine, dopamine, norepinephrine, histamine, benzylamine, and serotonin by fluorescent detection (see e.g., Fig. 6). The MAO was deposited on a DROPSENS 710 screen-printed electrode and exposed to the same group of eight amines under amperometric detection with a potential of -0.2V and the resulting current measured. See e.g., Fig. 3A-3D, 5C, 9A-9B, 10A-10C. [00108] It is contemplated herein that the MAO can include any mutations to the enzyme which allow it to be more specific to a particular monoamine or provide it sensitivity to a new monoamine the enzyme was previously not reactive to. Other mutations can allow for improved kinetics which can improve the specificity, reading time, or dynamic range of the electrochemical sensor.

[00109] The following references cited herein are each incorporated herein by reference in their entireties: Sun et al., Biosensors and Bioelectronics 26, 3450-3455 (2011); Wang et al., Sensors and Actuators B: Chemical 204, 302-309 (2014); Yang et al., Biosensors and Bioelectronics 56, 300-306 (2014); Feng et al., Nanoscale 7, 2427-2432 (2015); de Jesus et al., Journal of pharmaceutical and biomedical analysis 33, 983-990 (2003); Minamide et al., Sensors and Actuators B: Chemical 108, 639-645 (2005); Xu et al., Biosensors and Bioelectronics 107, 184-191 (2018); Joshi et al., Microchimica Acta 169, 383-388 (2010); Medyantseva et al., Journal of Analytical Chemistry 63, 275 (2008); Miyazaki et al., Materials Science and Engineering: C 58, 310-315 (2016); Henao-Escobar et al., Microchimica Acta 180, 687-693 (2013); Vela et al., Electroanalysis: 15, 133-138 (2003); Baranwal et al., Biosensors and Bioelectronics 121, 137-152 (2018); Kacar et al., Analytical and Bioanalytical Chemistry, 1-14 (2020); Ishida et al., Journal of neuroscience research 96, 817-827 (2018); Medyantseva et al., Pharmaceutical Chemistry Journal 48, 478-482 (2014); Yagodina et al., Sensors and Actuators B: Chemical 44, 566-570 (1997); Medyantseva et al., Journal of Analytical Chemistry 70, 535-539 (2015); Aigner et al., Microchimica acta 182, 925-931 (2015); Budantsev et al., Analytica chimica acta 249, 71-76 (1991); Si et al., Chemosensors 6, 1 (2018).

Claims

CLAIMS What is claimed herein is:

(a) an electrode comprising a surface;

2. The biosensor of claim 1, wherein the MAO enzyme is Corynebacterium ammoniagenes MAO (CαMAO) or a functional variant or fragment thereof.

3. The biosensor of claim 2, wherein the MAO enzyme is encoded by a nucleic acid comprising a sequence that is at least 90% identical to SEQ ID NO: 1.

4. The biosensor of claim 2, wherein the MAO enzyme comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 2.

5. The biosensor of claim 1, wherein the at least one amine-comprising substrate is a monoamine substrate.

6. The biosensor of claim 1, wherein the at least one amine-comprising substrate is a monoamine neurotransmitter or a dietary monoamine.

7. The biosensor of claim 1, wherein the at least one amine-comprising substrate is a polyamine substrate.

8. The biosensor of claim 1, wherein the at least one amine-comprising substrate is selected from the group consisting of dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5- methoxytryptamine, and hexylamine.

9. The biosensor of claim 1, wherein the at least one amine-comprising substrate is dopamine, octopamine, tyramine, norepinephrine, tryptamine, 5 -methoxytryptamine, and hexylamine.

10. The biosensor of claim 1, wherein the MAO enzyme is immobilized on the surface of the electrode with a polymer, and optionally, a top layer above the polymer, wherein the top layer comprises Prussian-Blue (PB).

11. The biosensor of claim 10, wherein the polymer comprises low molecular weight (LMW) or medium molecular weight (MMW) chitosan in 0.5% acetic acid, and optionally Prussian-Blue (PB).

12. The biosensor of claim 1, wherein in the presence of the at least one amine-comprising substrate, the MAO enzyme produces H₂O₂, wherein breakdown of H₂O₂ to O₂ and H₂O releases electrons to produce an electrochemical signal, wherein the electrochemical signal is detected by current passed to the electrode.

13. The biosensor of claim 1, wherein the detectable signal is produced when the at least one amine-comprising substrate is catalyzed by the MAO enzyme and transfers at least one electron from Med_red to hydrogen peroxide (H₂O₂), resulting in its reduction to Med_ox, wherein Med_ox is reduced by the electrode producing a detectable signal.

14. The biosensor of claim 13, wherein the Med_ox produces an electrochemical signal, wherein the electrochemical signal is detected by current passed to the electrode.

15. The biosensor of claim 1, wherein the biosensor is an amperometric biosensor and the detectable signal is electrochemical.

16. The biosensor of claim 1, wherein the electrode is connected to a potentiostat having a current resolution to at least 1pA (100 nA).

17. The biosensor of claim 1, wherein the biosensor comprises a working electrode and a reference electrode.

18. The biosensor of claim 1, wherein the biosensor comprises a counter electrode.

19. The biosensor of claim 1, wherein the biosensor does not comprise a counter electrode.

20. The biosensor of claim 1, wherein the working electrode is > 10 π mm².

21. The biosensor of claim 1, wherein the electrode is metallic.

22. The biosensor of claim 21, wherein the metallic electrode is gold, silver, platinum, or palladium.

23. The biosensor of claim 1, wherein the electrode is non-metallic.

24. The biosensor of claim 23, wherein the non-metallic electrode comprises carbon.

25. The biosensor of claim 1, wherein the amperometric biosensor is a chronoamperometric biosensor.

26. A method of using an amperometric biosensor to measure the concentration of at least one amine-comprising substrate comprising: (a) assembling the biosensor of claim 1, wherein the biosensor is an amperometric biosensor; (b) providing a sample; and (c) measuring the current produced by the oxidation of at least one amine-comprising substrate present in the sample.

27. The method of claim 26, wherein the sample is selected from the group consisting of: gastric juice, urine, saliva, feces, cerebrospinal fluid, sweat, interstitial fluid, and blood.

28. The method of claim 22, wherein the method of using the amperometric biosensor is used to measure the physiological concentration of at least one amine-comprising substrate in the range of 0.01 μM-80 μM range.