L-dopa sensors employing multicopper oxidases

Abstract

Sensors, kits, and methods are disclosed for assaying L-DOPA with an engineered multicopper oxidase. The engineered multicopper oxidase has improved catalytic activity at room temperature and can selectivity dehydrogenate L-DOPA as a substrate when immobilized on an electrode for electrochemical detection.

Description

L-DOPA SENSORS EMPLOYING MULTICOPPER OXIDASESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 505,278, filed May 31, 2023, which is hereby incorporated by reference in its entirety.REFERENCE TO A SEQUENCE LISTING

[0002] The Sequence Listing written in file name 614156SEQLIST.xml is 7.4 kilobytes, was created on May 23, 2024, and is hereby incorporated by reference.BACKGROUND

[0003] Parkinson’s disease (PD) is a chronic illness that affects more than 10 million people worldwide and clinically presents as the difficulty to speak, swallow, tremors, slowness, mood disorders, and schizophrenia. PD biologically presents through the degeneration of dopaminergic neurons in the substantia nigra of the brain which leads to low production of dopamine. The most effective currently used treatment for PD is levodopa (1-3,4- dihydroxyphenylalanine; L-DOPA), a dopamine precursor capable of crossing the blood-brain barrier, which is later converted into dopamine. However, there is no standardized L-DOPA measurement platform, so most L-DOPA dosing must rely on patient feedback and other subjective measures. This dosing system presents many challenges further complicated by the fact that L-DOPA has a short therapeutic window and is cytotoxic in high concentrations.

[0004] Accurate detection of L-DOPA would allow for improved dosing and enable better management of middle and late-stage Parkinson’s symptoms, which would dramatically improve quality of life for millions worldwide. There is great need for a selective and continuous L-DOPA sensing system to better treat patients with PD. As described herein, engineered multicopper oxidases with enhanced catalytic activity were developed with improved properties for biosensing applications. The bioelectrochemical device disclosed herein addresses this need through the invention of engineered multicopper oxidases for the sensitive detection of L-DOPA.BRIEF SUMMARY

[0005] Compositions, devices, kits, and methods are provided for assaying L-DOPA in a sample from a subject.

[0006] An embodiment is a sensor for assaying L-DOPA, wherein the sensor comprises an engineered multicopper oxidase, wherein the engineered multicopper oxidase has an increased catalytic activity for a substrate compared to the catalytic activity of a wild-type multicopper oxidase. In embodiments, the engineered multicopper oxidase comprises a sequence having a modification of one or more amino acid residues of a wild-type multicopper oxidase, wherein the modification results in enhanced electron transfer between the enzyme and the substrate at a type 1 (Tl) copper center, optionally wherein the modification results in increased loop flexibility. In some embodiments, the catalytic activity of the engineered multicopper oxidase is increased by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, or about 12-fold compared to a catalytic activity of the wild-type multicopper oxidase. In some embodiments, the substrate is 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid (ABTS) or levodopa (L-DOPA).

[0007] In some embodiments, the engineered multicopper oxidase is derived from a hyperthermophilic organism. In some embodiments, the hyperthermophilic organism is Pyrobaculum aerophilum. In some embodiments, the modification is a substitution. In some embodiments, the modification is at position corresponding to position 262 of SEQ ID NO: 1. In some embodiments, the modification is a conservative amino acid substitution. In some embodiments, the modification is a substitution of a phenylalanine amino acid residue with a different non-polar amino acid residue. In one embodiment, a substitution of a phenylalanine amino acid residue with an isoleucine residue. In another embodiment, the modification is a substitution of a phenylalanine amino acid residue with a leucine residue. In one embodiment, the modification is a substitution of a phenylalanine amino acid residue with a valine residue. In a further embodiment, the modification is a substitution of a phenylalanine amino acid residue with an alanine residue.

[0008] In some embodiments, the engineered multicopper oxidase comprises a sequence having 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%, at least 99.5%, or at least 99.9% sequence identityto any one of SEQ ID NOs: 2-5. In some embodiments, said engineered multicopper oxidase comprises any one of the amino acid sequences set forth in SEQ ID NO: 2-5.

[0009] In embodiments, the engineered multicopper oxidase is immobilized on an electrode, wherein the electrode comprises an electroconductive material. In some embodiments, the electroconductive material is selected from the group consisting of carbon paper, glassy carbon, carbon nanotubes, gold, and palladium.

[0010] In embodiments, the assay for L-DOPA comprises a measurement of electrons transferred to the electrode in a sample, wherein the electrons are generated from the dehydrogenation of L-DOPA. In some embodiments, the measurement is amperometric measurement or a potentiometric measurement. In some embodiments, the measurement is an open-circuit potential, chronoamperometry, square-wave voltammetry, or extended gate field effect transistor (EGFET) measurement. In some embodiments, the measurement is a continuous measurement.

[0011] In some embodiments, the engineered multicopper oxidase has an improved catalytic activity for L-DOPA as the substrate as compared to a substrate other than L-DOPA. In embodiments, the engineered multicopper oxidase is selective for the detection of L-DOPA in the presence of one or more physiological substrates other than L-DOPA, wherein reduced signal biased is observed as compared to signal bias observed for a wild-type multicopper oxidase. In embodiments, the measurement of L-DOPA in the presence of one or more physiological substrates other than L-DOPA has a signal bias of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0012] In some embodiments, the sensor further comprises (I) a three-electrode configuration comprising a working electrode, a reference electrode, and a counter electrode; or (II) a two-electrode configuration comprising a working electrode and a shared reference and a counter electrode. In some embodiments, one or more electrodes are disk electrodes or needle electrodes. In one embodiment, one or more electrodes are gold electrodes.

[0013] In some embodiments, said sensor is a sensor strip. In some embodiments, the sensor strip is screen-printed. In some embodiments, the sensor strip is single-use and / or disposable.

[0014] One embodiment is a kit for assaying L-DOPA in a sample, the kit comprising said sensor, and instructions for use.

[0015] Another embodiment is a method of assaying L-DOPA in a sample, the method comprising the steps of: contacting the sample with said sensor; and measuring an amount of L- DOPA.

[0016] In some embodiments, the measurement is an electrical current. In some embodiments, a voltage potential is applied between the electrode comprising the engineered multicopper oxidase and another electrode, wherein the voltage is between -0.1 V and -0.5V. In some embodiments, the measurement is performed for a period of time ranging from 5 seconds to 1 month.

[0017] In other embodiments, the measurement is an is an electric potential. In some embodiments, the electric potential is an open circuit potential. In some embodiments, a chronoamperometry signal is pulsed at a voltage for a length of time. In some embodiment, the voltage pulsed is between 0.2 V and 0.4 V, and wherein the length of time is between 30 seconds and 300 seconds. In some embodiments, the measurement is taken at a time after the chronoamperometry signal is pulsed. In some embodiments, the measurement is taken at a time between 5 seconds and 600 seconds after the signal is pulsed.

[0018] In some embodiments, the limit of detection (LOD) is less than 0.01 pM, less than 0.1 pM, less than 0.2 pM, less than 0.3 pM, less than 0.4 pM, less than 0.5 pM, less than 0.6 pM, less than 0.7 pM, less than 0.8 pM, less than 0.9 pM DOPA, less than 1.0 pM, less than 2.0 pM, less than 5 pM, less than 10 pM, less than 20 pM, or less than 55 pM of L-DOPA.BRIEF DESCRIPTION OF FIGURES

[0019] Figure 1 shows a schematic overview of a multicopper oxidase.

[0020] Figure 2 shows a summary overview of the engineered multicopper oxidase and its use for levodopa (L-DOPA) sensing (created with BioRender.com).

[0021] Figures 3A-3C show chronoamperometric evaluation of three different gold disk electrodes with engineered PaMCO Phe290Ile enzyme immobilized on the surface measuring dopaquinone reduction current. Figure 3A shows the raw data, Figure 3B shows a calibration curve between 0-1.2 pM of L-DOPA, and Figure 3C shows a calibration curve between 0-55 pM of L-DOPA.

[0022] Figures 4A-4D show chronoamperometric evaluation of three different gold disk electrodes with tyrosinase from mushroom immobilized on the surface measuring dopaquinonereduction current. Figure 4A shows the raw data and Figure 4B shows a calibration curve between 85-185 pM of L-DOPA. Figure 4C shows the raw data and Figure 4D shows a calibration curve between 0-55 pM of L-DOPA.

[0023] Figures 5A-5B show chronoamperometric evaluation of three different gold disk electrodes with laccase from Aspergillus sp. immobilized on the surface measuring dopaquinone reduction current. Figure 5A shows the raw data and Figure 5B shows a calibration curve between 0-55 pM of L-DOPA.

[0024] Figure 6 shows chronoamperometry signal bias from the addition of various physiological interferents at high end of physiological concentrations in the presence of 180 pM L-DOPA for electrodes with engineered PaMCO Phe290Ile enzyme (5 from left) or tyrosinase from mushroom (5 from right) immobilized on the surface.DETAILED DESCRIPTION

[0025] Currently, Parkinson’s Disease (PD) treatment is hindered by a lack of technology for the detection of L-DOPA. Providing a proper therapeutic dose is challenging without data, and both under-administering and over-administering L-DOPA has serious side effects which effect patients’ quality of life. There are no L-DOPA sensors available on the market, and no practical L-DOPA sensors have been reported in scientific literature. While there have been several attempts to develop either a L-DOPA point of care sensor, or continuous platform,6,7 12challenges with enzyme substrate specificity, stability, and biosensor performance have prevented the development of commercially available L-DOPA sensors. Specifically, the development of commercial products has been limited by the fact that majority of reported L-DOPA sensors utilize enzymes such as tyrosinase which are not specific towards L-DOPA. Disclosed herein is a biosensor which utilizes an engineered multicopper oxidase to specifically detect L-DOPA levels, addressing this longstanding need in the field of Parkinson’s treatment. Continuous L-DOPA biosensors, point- of-care testing (POCT), and personal use L-DOPA monitoring systems disclosed herein have the potential to significantly enhance the management of stage II PD and minimize “off-time” PD symptoms.

[0026] The presently disclosed subj ect matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subjectmatter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents and other references mentioned herein are incorporated by reference into their entirety.I. Overview

[0027] Multicopper oxidases (MCOs) belong to a protein superfamily of enzymes which catalyze the four-electron reduction of oxygen to water by the oxidation of various substrates. They catalyze the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals as shown in Figure 1. MCOs contain at least four copper atoms, which are classified as type 1 (T 1 ), type 2 (T2) or type 3 (T3)1’2-3. The oxidation of a substrate starts at a T1 mononuclear copper center, then electrons are transferred internally to the trinuclear copper center T2 / T3 where oxygen (O2) is reduced by four electrons, yielding two water molecules. The family of multicopper oxidase enzymes includes laccases (EC 1.10.3.2), bilirubin oxidases (EC 1.3.3.5), and ascorbic acid oxidases (EC 1.10.3.3). Various types of organisms including bacteria, archaeon and fungi have multicopper oxidase enzymes. One example is Pyrobaculum aerophilum, an aerobic hyperthermophilic archaeon with an optimal temperature of 100 °C. Other work recently elucidated the structure of a novel hyper-thermostable Pyrobaculum aerophilum multicopper oxidase (PaMCO).4The wild-type multicopper oxidase has high structural stability with an optimum reaction temperature of 85 °C and retains its activity across a broad pH range (e.g., 4.5 - 11), but exhibits low catalytic activity. The same research group then used directed evolution to identify mutants of Pyrobaculum aerophilum multicopper oxidase with enhanced catalytic activity.5

[0028] Multicopper oxidases are capable of direct electron transfer to an electrode when they are immobilized on the electrode, which make them suitable for bioelectrochemical applications. A biosensor capable of accurately and sensitively detecting L-DOPA would be extremely beneficial for the treatment of Parkinson’ s disease patients, however issues with enzyme stability, sensitivity, and specificity for L-DOPA have prevented the development of a commercially available L-DOPA sensor. Disclosed herein, Pyrobaculum aerophilum multicopper oxidases engineered for increased catalytic activity meet this need and can be used in biosensors for the detection of L-DOPA.

[0029] In embodiments, provided herein are amino acid sequences of multicopper oxidases, such those derived from Pyrobaculum aerophilum engineered to have increased catalytic activity compared to wild-type multicopper oxidases. In embodiments, provided herein are methods of making the engineered multi copper oxidases with increased catalytic activity and their application as biosensors, including but not limited to electrochemical L-DOPA sensors. Shown herein, engineered multicopper oxidases can maintain thermostability while being able to dehydrogenate L-DOPA selectively at room temperature and / or body temperature.

[0030] In some embodiments, catalytic activity of an engineered multicopper oxidase is measured as a specific activity. For example, provided herein are engineered multicopper oxidases with a specific activity of about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11 -fold, and about 12-fold greater than the specific activity of a wild-type multicopper oxidase.

[0031] It should be known that the multicopper oxidase enzymes disclosed herein may or may not include an initial signal peptide sequence comprising a cytoplasmic domain and transmembrane helix, which is found at the N-terminus of wild-type multicopper oxidase sequences and is typically 28 amino acid residues in length. Mutations are herein described by their numbered position in the entire enzyme sequence, which includes the signal peptide sequence (e.g., Phe290Ile). However, in certain embodiments provided herein, enzymes were engineered and produced without the initial signal peptide sequence. Therefore, mutations of amino acid residues of multicopper oxidases disclosed herein may be referred to using numbered positions different from the actual location of the mutation in the disclosed sequence, due to the lack of inclusion of the signal peptide. For example, the mutation referred to as Phe290Ile is at position262 of SEQ ID NO: 1, as the first 28 amino acid residues comprising the initial signal peptide are not included in SEQ ID NO: 1.

[0032] In embodiments, provided herein, engineered multicopper oxidases are derived from Pyrobaculum aerophilum multicopper oxidase, wherein the wild-type enzyme has an amino acid sequence set forth in SEQ ID NO: 1.

[0033] Wild-type multicopper oxidase from Pyrobaculum aerophilumMTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKN KLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHG LTAKQFYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPME MIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVP MRLIAVDQGFLARPIEVRALFLAPAERAEVVVELGEGVYLLKNTPFDPMHLEMG HGMQEALPEGSEYTIATFL VEGKGEAVP VEAL SDPPPEPPKPTRTRRF AL SL SGM QWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERK DSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHC HNLEHEDGGMMINIAVK (SEQ ID NO: 1)

[0034] In embodiments, disclosed herein, engineered multicopper oxidases derived from Pyrobaculum aerophilum harbor one or more mutations to improve catalytic activity, such as mutations of Phe290, which corresponds to position 262 of the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the mutation to improve catalytic activity is Phe290Ile and comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0035] Multicopper oxidase from Pyrobaculum aerophilum with Phe290IleMTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKN KLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHG LTAKQFYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPME MIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVP MRLIAVDQGFLARPIEVRALFLAPAERAEVVVELGEGVYLLKNTPIDPMHLEMG HGMQEALPEGSEYTIATFL VEGKGEAVP VEAL SDPPPEPPKPTRTRRF AL SL SGM QWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERK DSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHC HNLEHEDGGMMINIAVK (SEQ ID NO: 2)

[0036] In another embodiment, the mutation to improve catalytic activity is Phe290Leu and comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0037] Multicopper oxidase from Pyrobaculum aerophilum with Phe290LeuMTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKN KLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHG LTAKQFYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPME MIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVP MRLIAVDQGFLARPIEVRALFLAPAERAEVVVELGEGVYLLKNTPLDPMHLEMG HGMQEALPEGSEYTIATFLVEGKGEAVPVEALSDPPPEPPKPTRTRRFALSLSGM QWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERK DSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHCHNLEHEDGGMMINIAVK (SEQ ID NO: 3)

[0038] In one embodiment, the mutation to improve catalytic activity is Phe290Val and comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0039] Multicopper oxidase from Pyrobaculum aerophilum with Phe290ValMTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKN KLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHG LTAKQFYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPME MIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVP MRLIAVDQGFLARPIEVRALFLAPAERAEVVVELGEGVYLLKNTPVDPMHLEMG HGMQE ALPEGSE YTIATFL VEGKGEAVP VEAL SDPPPEPPKPTRTRRF AL SL SGM QWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERK DSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHCHNLEHEDGGMMINIAVK (SEQ ID NO: 4)

[0040] In a further embodiment, the mutation to improve catalytic activity is Phe290Ala and comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0041] Multicopper oxidase from Pyrobaculum aerophilum with Phe290AlaMTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKN KLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHG LTAKQFYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPME MIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVPMRLIAVDQGFLARPIEVRALFLAPAERAEVVVELGEGVYLLKNTPADPMHLEMG HGMQE ALPEGSE YTIATFL VEGKGEAVP VEAL SDPPPEPPKPTRTRRF AL SL SGM QWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERK DSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHC HNLEHEDGGMMINIAVK (SEQ ID NO: 5)

[0042] In embodiments, a sensor is provided for assaying L-DOPA in a sample, where the sensor includes an engineered multicopper oxidase with increased catalytic activity as described herein. In another embodiment, a kit is provided for assaying L-DOPA in a sample, where the kit includes an engineered multicopper oxidase as described herein. In other embodiments, a method is described for the detection of L-DOPA with the engineered multicopper oxidase described herein.

[0043] Provided herein are engineered multicopper oxidases with increased catalytic activity capable of selectively dehydrogenating L-DOPA as a substrate. As described herein, engineered multicopper oxidases are highly specific and selective for L-DOPA as a substrate and do not react with other physiologically relevant compounds. These engineered multicopper oxidases are useful as an enzyme in a L-DOPA monitoring kit, biomolecular recognition element of electrochemical biosensors such as for L-DOPA monitoring, and for implantable or wearable sensors for continuous L-DOPA monitoring. Provided herein are engineered multicopper oxidases suitable for L-DOPA biosensing.II. Definitions

[0044] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular aspects only and is not intended to be limiting of the invention. In case of a conflict in terminology, the present specification is controlling.

[0045] The term “subject” refers to a mammal (e.g., a human) in need of a L-DOPA concentration analysis. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, non-human mammals, and humans. The term “subject” does not necessarily exclude an individual that is healthy in all respects and does not have or show signs of Parkinson’s Disease.

[0046] As used herein, the term “physiological conditions” refers to the range of conditions of temperature, pH, and tonicity (or osmolality) normally encountered within tissues in the body of a living human.

[0047] As used herein, the term “body temperature” refers to the physiological temperature of a human or an animal subject. The average human body temperature is approximately 37 °C.

[0048] As used herein, “room temperature” refers to a range of air temperatures generally chosen for indoor settings, such as temperatures between 16-27 °C.

[0049] The term “zzz vitro" refers to artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube).

[0050] The term “z ? vzvo” refers to natural environments (e.g., a cell or organism or body) and to processes or reactions that occur within a natural environment.

[0051] As used herein, the term “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”).

[0052] The term “or” refers to any one member of a particular list.

[0053] Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations ± 0.5%, 1%, 5%, or 10% from a specified value.

[0054] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0055] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients.

[0056] Other definitions are provided below.III. CompositionsEngineered Multicopper Oxidases

[0057] In one embodiment, an isolated, engineered multicopper oxidase that exhibits increased catalytic activity compared to a wild-type multicopper oxidase is provided. In embodiments, an engineered multicopper oxidase with increased catalytic activity is capable of selectively utilizing L-DOPA as a substrate. In embodiments, the engineered multicopper oxidase with increased catalytic activity also has increased specific activity. In embodiments, the engineered multicopper oxidase exhibits increased catalytic activity at room temperature and / or body temperature (e.g., approximately 37 °C). In embodiments, the engineered multicopper oxidase maintains thermal stability at temperatures greater than room temperature (e.g., 27-100 °C).

[0058] As used herein, “isolated,” with respect to a polypeptide (and also a polynucleotide), means a molecule (e.g., polypeptide, protein or polynucleotide) isolated from its natural environment or prepared using synthetic methods such as those known to one of skill in the art. Complete purification is not required in either case. The molecules described herein can be isolated and purified from normally associated material in conventional ways, such that in the purified preparation the molecule is the predominant species in the preparation. At the very least, the degree of purification is such that extraneous material in the preparation does not interfere with use of the molecule in the manner disclosed herein. The molecule is at least about 85% pure; alternatively, at least about 90% pure, alternatively, at least about 95% pure; and alternatively, at least about 99% pure.

[0059] As used herein, “about” means within a statistically meaningful range of a value or values such as a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.

[0060] The term “wild-type” refers to entities having a structure and / or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles). A “wild-type aminoacid residue” at a given position refers to the amino acid present at a given position in a wild-type polypeptide.

[0061] As used herein, “mutant,” when used in connection with a polypeptide or protein such as an enzyme, means a variant containing an insertion, deletion, or substitution in one or more of the amino acid residues on the polypeptide or protein at the indicated position(s). Mutant also is used for a polynucleotide encoding such a mutant polypeptide or protein.

[0062] As used herein, “a position corresponding to” means the position of an amino acid residue in a query amino acid sequence that is aligned with the amino acid residue in a reference amino acid sequence using software such as AlignX of Vector NTI with default parameters (available from Invitrogen; see, Lu & Moriyama (2004) Brief Bioinform. 5:378-88). Thus, “amino acid (AA) residue at a position corresponding to the position Y of the amino acid sequence set forth in SEQ ID NO: X” means the AA residue in a query amino acid sequence that is aligned with AA Y of SEQ ID NO: X when the query amino acid sequence is aligned with SEQ ID NO: X using AlignX of Vector NTI with default parameters. It should be noted that the AA Y of SEQ ID NO: X itself is also encompassed by this term.

[0063] As used herein, “catalytic activity” refers to the ability of an engineered multicopper oxidase to dehydrogenate a substrate (e.g., L-DOPA) compared to a wild-type multicopper oxidase.

[0064] As used herein, “dehydrogenate” or “Dh” refers to an enzymatic process of an enzyme to catalyze the oxidation of a substrate through the removal of hydrogen atoms and electrons. A multicopper oxidase can dehydrogenate L-DOPA as a substrate, producing L- dopaquinone as the oxidized product. The “dehydrogenation” of a substrate (e.g., L-DOPA) may be used interchangeably with “oxidation” of a substrate.

[0065] As used herein, “oxidase activity” means an enzymatic activity of the engineered multicopper oxidase to utilize oxygen as an electron acceptor. The oxidase activity may be assayed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate by monitoring the increase in absorbance at 420nm based on the formation of reduced ABTS, and / or by any method known in the art.

[0066] As used herein, “specific activity” refers to the catalytic activity of an enzyme per unit weight of protein (e.g., moles of substrate converted per unit time per gram of enzyme). For example, an engineered multicopper oxidase may be capable of dehydrogenating L-DOPA at Xfimol and a wild-type multicopper oxidase may be capable of dehydrogenating L-DOPA at Y m i n mg fj.rn.ol min jng

[0067] As used herein, “thermal stability” refers to the ability of an enzyme to perform its catalytic reaction at an elevated temperature (e.g., higher than room temperature). A multicopper oxidase is referred to as “thermally stable” if it retains its ability to catalyze the dehydrogenation of its substrate at temperatures higher than room temperature.

[0068] As used herein, “elevated temperature” refers to a temperature greater than room temperature, such as temperatures greater than 27 °C. Wild-type multicopper oxidase from Pyrobaculum aerophilum is efficient at elevated temperatures with an optimum reaction temperature of 85°C.

[0069] It is therefore desired with respect to electrochemical biosensors to engineer an enzyme to have high catalytic activity at room temperature and / or body temperature with high specificity towards L-DOPA as a substrate. In embodiments disclosed herein are engineered multicopper oxidases with increased catalytic activity at room temperature.

[0070] As used herein, “biosensor” means a device containing bio-recognition elements (e g., enzymes, aptamers, antibodies, etc.) that react with a target molecule and then transduce the biological signal into an electrical signal that can be analyzed. Herein, the terms “biosensor” may be used interchangeably with “enzyme sensor” or “sensor.”

[0071] In embodiments provided herein, an engineered multicopper oxidase comprises a sequence having a modification of one or more amino acid residues of a wild-type multicopper oxidase, wherein the modification results in increased catalytic activity for a substrate (e.g., L- DOPA). In some embodiments, the engineered multicopper oxidase is increased by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11 -fold, and about 12-fold greater than a catalytic activity of the wild-type multicopper oxidase. In some embodiments, the catalytic activity is measured as specific activity. In some embodiments, the catalytic activity is determined by an assay using ABTS as a substrate and monitoring the increase in absorbance at 420nm based on the formation of reduced ABTS and / or by any method known in the art.

[0072] In embodiments, engineered multicopper oxidases disclosed herein are selective for the detection of L-DOPA in the presence of one or more physiological substrates other than L-DOPA (e.g., sugars, carbidopa, dopamine, etc), wherein reduced signal biased is observed as compared to signal bias observed for a wild-type multicopper oxidase. Signal bias may be measured by detecting L-DOPA alone and in the presence of a physiological substrate at a physiologically relevant concentration to determine interferent effects. In embodiments, the measurement of L-DOPA in the presence of one or more physiological substrates other than L- DOPA has a signal bias of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0073] In embodiments, disclosed herein, engineered multicopper oxidases may be derived from hyperthermophilic organisms (e.g., archaea or bacteria). As used herein, “hyperthermophilic” refers to an organisms that survives in hot environments in temperatures greater than 60 °C. In some embodiments, the engineered multicopper oxidase is derived from Pyrobacuhtm aerophilum.

[0074] In embodiments, an engineered multicopper oxidase comprises one or more mutations that improve electron transfer between the enzyme and a substrate at a T1 copper center. In embodiments, one or more mutations provide increased loop flexibility near the T1 copper center and to enhance the catalytic activity of the engineered multicopper oxidase as compared to the catalytic activity of a wild-type multicopper oxidase. In embodiments, the one or more mutations may increase an electric current density of an electrode with an immobilized engineered multicopper oxidase as compared to an electric current density of an electrode with an immobilized wild-type multicopper oxidase.

[0075] In embodiments, the mutation to increase catalytic activity is an insertion, a deletion, and / or a substitution of an amino acid in a sequence of a wild-type multicopper oxidase. In some embodiment, the mutation to increase oxidase activity is a substitution of an amino acid in a sequence of a wild-type multicopper oxidase. In some embodiments, the substitution is a conservative amino acid substitution.

[0076] In some embodiment, the mutation is a substitution of a phenylalanine amino acid residue with another non-polar amino acid residue and / or a non-aromatic amino acid residue. In an embodiment, the non-polar amino acid residue is selected from the group consisting of isoleucine, leucine, valine, and alanine.”

[0077] In some embodiments, an engineered multicopper oxidase comprises a substitution at an amino acid residue corresponding position 262 of the corresponding position in SEQ ID NO: 1 (e.g., Phe290 of the wild-type Pyrobaculum aerophilum multicopper oxidase). In someembodiments, the substitution is a Phe290 substitution of the wild-type Pyrobaculum aerophilum multicopper oxidase. In some embodiments, the amino acid sequence of the engineered multicopper oxidase comprises a Phe290Ile, a Phe290Leu, a Phe290Val, or a Phe290Ala substitution of the wild-type Pyrobaculum aerophilum multicopper oxidase (SEQ ID NO: 1).

[0078] In embodiments, provided herein is an engineered multicopper oxidase having 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% sequence identity to any one of SEQ ID NOs:2- 5, provided an amino acid residue at a position corresponding to position 268 of SEQ ID NO: 1 is different from the amino acid residue occupying the corresponding position in SEQ ID NO: 1.

[0079] In some embodiments, the engineered multicopper oxidase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-5.

[0080] It should be understood that the numbering of the position of the amino acid sequence for engineered multicopper oxidases disclosed herein begins at an initial Met and that the multicopper oxidase may or may not have the initial signal peptide sequence.

[0081] “Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0082] “Percentage of sequence identity” refers to the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0083] Unless otherwise stated, sequence identity / similarity values refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalent program” includes any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0084] The term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of nonconservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for apolar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue. Typical amino acid categorizations are summarized below.Alanine Ala A Nonpolar Neutral 1.8Arginine Arg R Polar Positive -4.5Asparagine Asn N Polar Neutral -3.5Aspartic acid Asp D Polar Negative -3.5Cysteine Cys C Nonpolar Neutral 2.5Glutamic acid Glu E Polar Negative -3.5Glutamine Gin Q Polar Neutral -3.5Glycine Gly G Nonpolar Neutral -0.4Histidine His H Polar Positive -3.2Isoleucine He I Nonpolar Neutral 4.5Leucine Leu L Nonpolar Neutral 3.8Lysine Lys K Polar Positive -3.9Methionine Met M Nonpolar Neutral 1.9Phenylalanine Phe F Nonpolar Neutral 2.8Proline Pro P Nonpolar Neutral -1.6Serine Ser S Polar Neutral -0.8Threonine Thr T Polar Neutral -0.7Tryptophan Trp W Nonpolar Neutral -0.9Tyrosine Tyr Y Polar Neutral -1.3Valine Vai V Nonpolar Neutral 4.2

[0085] In embodiments, the engineered multicopper oxidase thus obtained may be purified by any of the known purification techniques including, but not limited to, ion exchange column chromatography, affinity chromatography, liquid chromatography, filtration, ultrafiltration, salt precipitation, solvent precipitation, immunoprecipitation, gel electrophoresis, isoelectric electrophoresis and dialysis.IV. DevicesL-DOPA Biosensors

[0086] In embodiments disclosed herein is a sensor for assaying L-DOPA in a sample, where the sensor includes an engineered multicopper oxidase with increased catalytic activity as described herein. As shown in Figure 2, the L-DOPA biosensor comprising the engineered multicopper oxidase is used to measure physiologically relevant L-DOPA concentrations.

[0087] In some embodiments, the engineered multicopper oxidase has improved substrate specificity for L-DOPA over biologically relevant phenols, dopamine analogues, and sugars. Described herein, L-DOPA is measured utilizing electrochemical techniques. In embodiments, L- DOPA is quantified by the measurement of electrons transferred to the electrode in a sample, wherein the electrons are generated from the dehydrogenation of L-DOPA. In embodiments, L- DOPA is detected by amperometric or potentiometric measurements. In some embodiments, the L-DOPA biosensor measures L-DOPA through open-circuit potential, chronoamperometry, square-wave voltammetry, and / or extended gate field-effect transistor (EG-FET) detection. In embodiments provided herein, the L-DOPA biosensor provides for sensitive and specific detection of L-DOPA.

[0088] In embodiments, the engineered multicopper oxidase is immobilized onto an electrode. In some embodiments, the electrode is a disk electrode or a needle electrode. In one embodiment the electrode is a gold disk rod. In another embodiment the electrode is a gold wire needle type electrode. In some embodiments the L-DOPA biosensor has a three-electrode configuration comprising a working electrode (WE), a reference electrode (Ref), and a counter electrode (CE). In some embodiments the L-DOPA biosensor has a two-electrode configuration comprising a WE and a shared Ref / CE. See Figure 2. The concentration of L-DOPA in a sample may be determined by measuring the amount of electrons generated or lost by the enzyme reaction, the reaction between the product with an electrode, or the change in charge due to the interaction between L-DOPA and the enzyme relative to a reference value. In embodiments, a sensor system may comprise a gold (Au) electrode, a silver chloride (Ag / AgCl) electrode, a platinum (Pt) electrode, a palladium electrode, a carbon (C) electrode, and / or another metal electrode.

[0089] In one embodiment, a sample of non-biological derived or biological derived, such as a blood, a serum, a saliva, a tear, a urine, a sweat or interstitial fluid sample, is added to the sample receiver, L-DOPA contained in the sample will react with the engineered multicopper oxidase, transferring electrons to the electrode to generate a current, which is indicative of the amount of L-DOPA in the sample.

[0090] In embodiments, the engineered multicopper oxidase is immobilized on an electrode by any method known in the art. Examples of means for immobilizing molecules such as the engineered multicopper oxidase include, but are not limited to, cross-linking, encapsulating into a macromolecular matrix, coating with a dialysis membrane, optical cross-linking polymer,electrocon du ctive polymer, oxidation-reduction polymer, and any combination thereof. In some embodiments, the engineered multicopper oxidase is immobilized on the electrode with a dithiobis(succinimidyl hexanoate) self-assembled monolayer (DSH-SAM) solution.

[0091] The term “immobilized” refers to a compound which is attached to a surface (e.g., electrode). Immobilization can be performed through adsorption, cross-linking, covalent bonding and / or affinity -tag binding.

[0092] The term “cross-linking” refers to a polymerization reaction of a cross-linking reagent. A “cross-linking reagent” refers to a molecule that contains two or more reactive ends capable of forming covalent bonds.

[0093] The electrode of the L-DOPA biosensor disclosed herein comprises an electroconductive material. In some embodiments, the electroconductive material comprises carbon paper, glassy carbon, carbon nanotubes, gold, and / or palladium. In some embodiments, the cathode and / or anode comprises conductive ink.

[0094] The term “electroconductive material” refers to a substance capable of transmitting electricity.

[0095] In embodiments, the electrode is a screen-printed carbon electrode, a planar gold electrode, or an interdigitated electrode array. When the measurement is conducted in an amperometric system using a carbon (C) electrode, gold (Au) electrode or platinum (Pt) electrode provided with an immobilized enzyme is used as a working electrode, together with a counter electrode (such as a Pt electrode) and a reference electrode (such as an Ag / AgCl electrode). The electrodes can be inserted into a buffer containing a mediator and kept at predetermined temperature.

[0096] A predetermined voltage can be applied to the working electrode, and then a sample is added and an increased value in electric current is measured. It is generally also possible to use so-called two-electrode systems with one working electrode and one counter or pseudo-reference electrode.

[0097] In one embodiment, the working electrode can be inserted into buffer together with a counter electrode (such as a Pt electrode) and a reference electrode (such as an Ag / AgCl electrode) and kept at a predetermined temperature. As indicated above, a predetermined voltage can be applied to the working electrode, and then the sample is added and increased value in electric current is measured.

[0098] In some embodiments, the L-DOPA biosensor can continuously assay L-DOPA in a sample.

[0099] In some embodiment, the engineered multicopper oxidase is immobilized on a sensor strip, which may be screen-printed or fabricated in any other manner known in the art. In embodiments, the sensor strips may be single-use and / or disposable. L-DOPA sensor strips can be used for point-of-care testing (POCT) and for personal use L-DOPA monitoring.

[0100] Also provided herein are biosensors for the detection of foslevodopa ((2S)-2- amino-3-(3-hydroxy-4-phosphonooxyphenyl)propanoic acid, dopa 4-phosphate, or levodopa-4'- monophosphate), a medication for the treatment of patients with PD. The sensors comprise a MCO as previously described and further comprise an enzyme to catalyze the dephosphorylation of foslevodopa to L-DOPA. In some embodiments, the enzyme is a phosphatase. In some embodiments, the enzyme that catalyzes the phosphorylation of foslevodopa is immobilized as a layer on the working electrode. In some embodiments, the enzyme that catalyzes the phosphorylation of foslevodopa is provided in solution in the device. Once the foslevodopa has been dephosphorylated by the enzyme (e.g., a phosphatase), the MCO sensor is used to detect the resulting L-DOPA product as previously described. Thus, a device comprising both enzymes can be used to detect foslevodopa.

[0101] Accordingly, these devices can be used as continuous foslevodopa biosensors for point of care testing (POCT) for PD patients treated with foslevodopa. The foslevodopa monitoring systems disclosed herein have the potential to significantly enhance the management of stage II PD and minimize “off-time” PD symptoms.V. Kits

[0102] In another embodiment, a kit for assaying L-DOPA in a sample, where the kit include at least an engineered multicopper oxidase as described herein.

[0103] Additionally, the kit can include a buffer necessary for the measurement, a standard solution of L-DOPA for preparing a calibration curve and / or an instruction for use. The engineered multicopper oxidase may be provided in various forms such as, for example, a freeze-dried reagent or a solution in an appropriate storage solution.

[0104] In some embodiments, the kit includes sensor strips, which may be screen-printed and / or disposable.

[0105] Any or all of the kit reagents can be provided within containers that protect them from the external environment, such as in sealed containers. Positive and / or negative controls can be included in the kit to validate the activity and correct usage of reagents employed in accordance with the inventive concept. Controls can include samples known to be either positive or negative for the presence of a predetermined concentration of L-DOPA.VI. Methods

[0106] The engineered multicopper oxidases disclosed herein can be used in various methods. For example, they can be used in methods of assaying L-DOPA in a sample from a subject. In embodiments, the sample comprises material selected from the group consisting of blood, serum, saliva, tears (i.e., lacrimal gland secretions), urine, sweat, and interstitial fluid.

[0107] The method can include at least a step of contacting the sample with the engineered multicopper oxidase and a step of measuring the amount of the L-DOPA oxidized by the engineered multicopper oxidase as described above and further below. In embodiments, the method includes continuous measurement of the amount of L-DOPA oxidized by the engineered multicopper oxidase. In embodiments, the measurement can be an amperometric measurement or a potentiometric measurement.

[0108] In some embodiments, the measurement taken to determine the concentration of L- DOPA is an electrical current. In some embodiments, a voltage potential is applied between the electrode comprising the engineered multi copper oxidase disclosed herein. In some embodiments, the voltage potential applied is between -0.1V and -0.5V. In embodiments, the measurement is performed for a period of time. In some embodiments the measurement is taken for a period of time ranging from 5 seconds to 10 minutes. In some embodiments, the measurement is taken continuously. In some embodiments, the sensor is operationally stable and capable of providing measurements for a period of time ranging from 30 seconds to 1 month.

[0109] In some embodiments, the measurement taken to determine the concentration of L- DOPA is an electric potential. In some embodiments, the electric potential is an open circuit potential. In some embodiments a chronoamperometry signal is pulsed at a voltage for a length of time. In some embodiments, the voltage pulsed is between 0.2 V and 0.4 V, and the length of time is between 30 seconds and 300 seconds. In some embodiments, the measurement is taken at a timeafter the chronoamperometry signal is pulsed. Tn some embodiments the measurement is taken at a time between 5 seconds and 600 seconds after the signal is pulsed.

[0110] In embodiments, the limit of detection (LOD) of the L-DOPA sensor described herein is less than 0.01 pM, 0.2 pM, less than 0.3 pM, less than 0.4 pM, less than 0.5 pM, less than 0.6 pM, less than 0.7 pM, less than 0.8 pM, less than 0.9 pM DOPA, less than 1.0 pM, less than 2.0 pM, less than 5 pM, less than 10 pM, less than 20 pM, or less than 55 pM of L-DOPA.[0U1] In some embodiments, the MCOs provided herein are used to detect L-DOPA derived from foslevodopa (((2S)-2-amino-3-(3-hydroxy-4-phosphonooxyphenyl)propanoic acid, or dopa 4-phosphate, levodopa-4'-monophosphate). The foslevodopa in the sample from the subject is first dephosphorylated to produce L-DOPA. In some embodiments, the L-DOPA assayed by the method is derived from foslevodopa, and the method further comprises contacting the sample with an enzyme capable of dephosphorylating foslevodopa to L-DOPA before contacting the sample with the MCO. Phosphatase enzymes can be used for the dephosphorylation. The resulting L-DOPA is then quantified by the device comprising the MCO as described.

[0112] These methods may be adapted, mutatis mutandis, for the assay of other substrates modified by the engineered enzymes disclosed herein.

[0113] The disclosed subject matter is further described in the following non-limiting Examples. It should be understood that these Examples, while indicating preferred embodiments of the subject matter, are given by way of illustration only.REFERENCES[1] Multicopper Oxidases and Oxygenases, E.I. Solomon et al., Chem. Rev. 1996, 96, 2563-2605[2] Laccase versus Laccase-Like Multi-Copper Oxidase: A Comparative Study of Similar Enzymes with Diverse Substrate Spectra, R. Reiss et al., PLOS ONE, June 2013 | Volume 8 | Issue 6 | e65633[3] Laccases: structure, function, and potential application in water bioremediation, L.Arregui et al., Microb Cell Fact (2019) 18:200[4] Structure of a multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum, H.Sakuraba et al., Acta Cryst. (2011). F67, 753-757, doi: 10.1107 / S1744309111018173[5] Activity enhancement of multi copper oxidase from a hyperthermophile via directed evolution, and its application as the element of a high performance biocathode, T.Satomura et al., Journal of Biotechnology 325 (2021) 226-232 https: / / doi.Org / 10.1016 / j.jbiotec.2020.10.019[6] Wearable Electrochemical Microneedle Sensor for Continuous Monitoring of Levodopa: Toward Parkinson Management. K. Yugender Goud, Chochanon Moonla, Rupesh K. Mishra, Chunmei Yu, Roger Narayan, Irene Litvan, and Joseph Wang. ACS Sensors 2019 4 (8), 2196- 2204. DOI: 10.1021 / acssensors.9b0112[7] Fang L, Ren H, Mao X, Zhang S, Cai Y, Xu S, Zhang Y, Li L, Ye X, Liang B. Differential Amperometric Microneedle Biosensor for Wearable Levodopa Monitoring of Parkinson's Disease. Biosensors (Basel). 2022 Feb 7; 12(2): 102. doi: 10.3390 / biosl2020102. PMID: 35200363; PMCID: PMC8869619.

[0012] Barbara Brunetti, Gabriela Valdes-Ramirez, Irene Litvan, Joseph Wang, A disposable electrochemical biosensor for 1-DOPA determination in undiluted human serum, Electrochemistry Communications, Volume 48, 2014, Pages 28-31, ISSN 1388-2481, https: / / doi.Org / 10.1016 / j.elecom.2014.08.007.EXAMPLESExample 1: An L-DOPA sensing platform using a multicopper oxidase derived from Pyrobaculum aerophilum

[0114] In previous studies, random and site directed mutagenesis was used to identify a F290I mutant of Pyrobaculum aerophilum multicopper oxidase (PaMCO) which showed increased catalytic activity. The F290I mutant retained pH stability and thermostability, while exhibiting an increased catalytic activity with a specific activity of 24.2 units / mg as compared to 1.95 units / mg for the wild type PaMCO (a 12.4-fold increase). Additionally, mutants F290Y, F290L, F290V, and F290A were produced and found to have specific activities of 3.54, 10.5, 27.1, and 27.7 units / mg, respectively. The results suggested that the F290I mutation increased the flexibility of the loop adjacent to the T1 Cu center and improved electron transfer between the enzyme and the substrate. With this observed improvement in catalytic activity, the F290I PaMCO mutant was tested as an enzyme sensor for the detection of L-DOPA.

[0115] Electrodes were prepared with immobilized PaMCOs. Gold disk electrodes were polished with 0.3 and 0.05 pM alumina powder and then soaked in a piranha solution consisting of a 3: 1sulfuric acid to hydrogen peroxide solution for one hour. Afterwards, the electrodes were electrochemically cleaned using cyclic voltammetry in a solution of 50 mM KOH, sweeping between 0V-1.2 V. Afterwards, electrodes were washed with acetone and then incubated overnight in 100 pM dithiobis(succinimidyl hexanoate) self-assembled monolayer (DSH-SAM) solution at 25°C, 300 rpm. Next, electrodes were incubated overnight in 0.014 mg / ml solution of enzyme at 25°C, stirring at 300 rpm. Finally, electrodes were washed by 100 mM potassium phosphate buffer (PPB) and then stored in 100 mM PPB until use. Electrodes were prepared with the engineered PaMCO Phe290Ile enzyme (SEQ ID NO: 2), tyrosinase derived from mushroom (Sigma Aldrich T3824; CAS 9002-10-2; EC 1.14.18.1), and laccase derived from Aspergillus sp (Novozym 51003; CAS 80498-15-3; EC 1.10.3.2).

[0116] As shown in Figures 3A-3C, three different electrodes with immobilized engineered PaMCO Phe290Ile enzyme (SEQ ID NO: 2) underwent chronoamperometric evaluation. After waiting approximately 10-15 minutes to allow for the three electrodes to stabilize, L-DOPA was added between the range of 0.0-55.0 pM L-DOPA (Figure 3C). During measurement, -0.3 V was applied continuously, where the engineered multicopper oxidase enzyme oxidizes L-DOPA to dopaquinone, which is then reduced on the electrode surface leading to increased reduction current. The chronoamperometric response was then averaged and a calibration curve was generated. As shown in Figure 3B, the L-DOPA sensor showed linear increase in reduction current between 0-1.2 pM, with a calculated LOD of 0.619 pM.

[0117] Next, similar testing was performed with an electrode with tyrosinase derived from mushroom. As shown in Figures 4A-4C, three different electrodes with immobilized tyrosinase from mushroom underwent chronoamperometric evaluation. After waiting approximately 10-15 minutes to allow for the three electrodes to stabilize, L-DOPA was added between the range of 85- 185 pM L-DOPA, as shown in the raw data in Figure 4A and the calibration curve of Figure 4B. This L-DOPA concentration range is different than the PaMCO Phe290Ile L-DOPA sensor, as the tyrosinase L-DOPA sensor did not show any signal response towards L-DOPA between the range of 0.0-55.0 pM L-DOPA, indicating that L-DOPA is not an ideal substrate of tyrosinase, which is visualized by the raw data (Figure 4C) and poor L-DOPA calibration curve (Figure 4D). During the measurement, -0.4 V was applied continuously, instead of -0.3 V, as the dopaquinone reduction current at -0.3 V was indistinguishable from noise, further indicating the suboptimal performance of tyrosinase as the employed biorecognition element for a L-DOPA sensor.

[0118] Then, testing was repeated using an electrode with laccase derived from Aspergillus sp. As shown in Figures 5A-5B, three different electrodes with immobilized laccase from Aspergillus sp. underwent chronoamperometric evaluation. After waiting approximately 10-15 minutes to allow for the three electrodes to stabilize, L-DOPA is added between the range of 0.0- 55.0 pM L-DOPA with a potential of -0.3V being applied continuously. Unlike the engineered PaMCO Phe290Ile enzyme (SEQ ID NO: 2), laccase from Aspergillus sp. was not able to successfully measure dopaquinone reduction, indicating that L-DOPA is not an ideal substrate of laccase from Aspergillus sp., further emphasizing the value of utilizing engineered multicopper oxidases as the biorecognition element to sense L-DOPA.

[0119] Finally, electrodes with immobilized engineered PaMCO Phe290Ile enzyme (SEQ ID NO: 2), tyrosinase from mushroom, and laccase from Aspergillus sp. were compared in the presence of physiological interferents at the high end of physiological concentrations in the presence of 180 pM L-DOPA. Figure 6 clearly shows that the engineered PaMCO Phe290Ile enzyme (SEQ ID NO: 2) sensor signal is less impacted in the presence of interferents when compared to tyrosinase from mushroom and laccase from Aspergillus sp, which showed minimal signal towards L-DOPA. It is important to note that the interferent measurements were taken in the presence of 180 pM L-DOPA as the tyrosinase from mushroom L-DOPA sensor was not able to show significant L-DOPA current response in the same lowered L-DOPA range as PaMCO Phe290Ile enzyme (SEQ ID NO: 2) L-DOPA sensor (0.0-55.0 pM). The PaMCO Phe290Ile enzyme (SEQ ID NO: 2) L-DOPA sensor shows lowered interference impact, highlighting the improved substrate specificity and preference towards L-DOPA when compared to other biorecognition elements employed for L-DOPA sensing.

[0120] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which the inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

CLAIMSWhat is claimed is:

1. A sensor for assaying L-DOPA, wherein the sensor comprises an engineered multicopper oxidase, wherein the engineered multicopper oxidase has an increased catalytic activity for a substrate compared to the catalytic activity of a wild-type multicopper oxidase.

2. The sensor of claim 1, wherein the engineered multicopper oxidase comprises a sequence having a modification of one or more amino acid residues of a wild-type multicopper oxidase, wherein the modification results in enhanced electron transfer between the enzyme and the substrate at a type 1 (Tl) coppercenter, optionally wherein the modification results in increased loop flexibility.

3. The sensor of claim 1 or 2, wherein the catalytic activity of the engineered multicopper oxidase is increased by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, or about 12-fold compared to a catalytic activity of the wild-type multicopper oxidase.

4. The sensor of claim 3, wherein the substrate is 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid (ABTS) or levodopa (L-DOPA).

5. The sensor of any one of claims 1-4, wherein the engineered multi copper oxidase is derived from a hyperthermophilic organism.

6. The sensor of claim 5, wherein the hyperthermophilic organism is Pyrobaculum aer ophilum.

7. The sensor of any one of claims 1-6, wherein the modification is a substitution.

8. The sensor of any one of claims 1-7, wherein the modification is at position corresponding to position 262 of SEQ ID NO: 1.

9. The sensor of claim 7 or 8, wherein the modification is a conservative amino acid substitution.

10. The sensor of claim 9, wherein the modification is a substitution of a phenylalanine amino acid residue with a different non-polar amino acid residue.

11. The sensor of claim 10, wherein the modification is a substitution of a phenylalanine amino acid residue with an isoleucine residue.The sensor of claim 10, wherein the modification is a substitution of a phenylalanine amino acid residue with a leucine residue.

13. The sensor of claim 10, wherein the modification is a substitution of a phenylalanine amino acid residue with a valine residue.The sensor of claim 10, wherein the modification is a substitution of a phenylalanine amino acid residue with an alanine residue.

15. The sensor of any one of claims 1-14, wherein the engineered multicopper oxidase comprises a sequence having 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%, at least 99.5%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 2-5.

16. The sensor of any one of claims 1-15, wherein the engineered multicopper oxidase comprises any one of the amino acid sequences set forth in SEQ ID NO: 2-5.

17. The sensor of any one of claims 1-16, wherein the engineered multicopper oxidase is immobilized on an electrode, wherein the electrode comprises an electroconductive material.

18. The sensor of claim 17, wherein the electroconductive material is selected from the group consisting of carbon paper, glassy carbon, carbon nanotubes, gold, platinum and palladium.

19. The sensor of any one of claims 1-18, wherein the assay for L-DOPA comprises a measurement of electrons transferred to the electrode in a sample, wherein the electrons are generated from the dehydrogenation of L-DOPA.

20. The sensor of claim 19, wherein the measurement is amperometric measurement or a potentiometric measurement.21 . The sensor of claim 19 or 20, wherein the measurement is an open-circuit potential, chronoamperometry, square-wave voltammetry, or extended gate field effect transistor (EGFET) measurement.

22. The sensor of claim 21, wherein the measurement is a transient open-circuit potential.

23. The sensor of any one of claims 19-22, wherein the measurement is a continuous measurement.

24. The sensor of any of the previous claims, wherein the engineered multicopper oxidase has an improved catalytic activity for L-DOPA as the substrate as compared to a substrate other than L-DOPA.

25. The sensor of any of the previous claims, wherein the engineered multicopper oxidase is selective for the detection of L-DOPA in the presence of one or more physiological substrates other than L-DOPA, wherein reduced signal biased is observed as compared to signal bias observed for a wild-type multicopper oxidase.

26. The sensor of any of the previous claims, wherein the measurement of L-DOPA in the presence of one or more physiological substrates other than L-DOPA has a signal bias of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

27. The sensor of any of the previous claims, further comprising:(I) a three-electrode configuration comprising a working electrode, a reference electrode, and a counter electrode; or(II) a two-electrode configuration comprising a working electrode and a shared reference and a counter electrode.

28. The sensor of any of the previous claims, wherein one or more electrodes are disk electrodes or needle electrodes.

29. The sensor of any of the previous claims, wherein one or more electrodes are gold electrodes.

30. The sensor of any of the previous claims, wherein the sensor is a sensor strip.

31. The sensor of claim 30, wherein the sensor strip is screen-printed.

32. The sensor of claim 30 or 31, wherein the sensor strip is single-use and / or disposable.

33. The sensor of any of the previous claims, wherein the sensor further comprise an enzyme capable of dephosphorylating foslevodopa to L-DOPA.

34. The sensor of claim 33, wherein the enzyme capable of dephosphorylating foslevodopa is a phosphatase.

35. A kit for assaying L-DOPA in a sample, the kit comprising the sensor of any of the previous claims, and instructions for use.

36. A method of assaying L-DOPA in a sample, the method comprising the steps of: contacting the sample with the sensor of any of claims 1 -32; and measuring an amount of L-DOPA.

37. The method of claim 36, wherein the measurement is an electrical current.

38. The method of claim 36 or 37, wherein the measurement is an electrical current over time.

39. The method of claim 37 or 38, wherein a voltage potential is applied between the electrode comprising the engineered multicopper oxidase and another electrode, wherein the voltage is between -0.1 V and -0.5V.

40. The method of any one of claims 37-39, wherein the measurement is performed for a period of time ranging from 5 seconds to 1 month.

41. The method of claim 36, wherein the measurement is an is an electric potential.

42. The method of claim 41, wherein the electric potential is an open circuit potential.

43. The method of claim 42, wherein the electric potential is a transient open circuit potential.

44. The method of claim 42 or 43, wherein a chronoamperometry signal is pulsed at a voltage for a length of time.

45. The method of claim 44, wherein the voltage pulsed is between 0.2 V and 0.4 V, and wherein the length of time is between 0.1 seconds and 300 seconds.

46. The method of claim 45, wherein the voltage pulsed is between 0.2 V and 0.4 V, and wherein the length of time is between 30 seconds and 300 seconds.

47. The method of claim 45 or 46, wherein the measurement is taken at a time after the chronoamperometry signal is pulsed.

48. The method of claim 47, wherein the measurement is taken at a time between 5 seconds and 600 seconds after the signal is pulsed.

49. The method of any one of claims 36-48, wherein the limit of detection (LOD) is less than 0.01 pM , less than 0.1 pM less than 0.2 pM, less than 0.3 pM, less than 0.4 pM, less than 0.5 pM, less than 0.6 pM, less than 0.7 pM, less than 0.8 pM, less than 0.9 pM DOPA, less than 1.0 pM, less than 2.0 pM, less than 5.0 pM, less than 10.0 pM, less than 20.0 pM, or less than 55.0 pM of L-DOPA.

50. The method of any one of claims 36-49, wherein the L-DOPA is derived from foslevodopa, and the method further comprises contacting the sample with an enzyme capable of dephosphorylating foslevodopa to L-DOPA before contacting the sample with the engineered multicopper oxidase.

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