L-DOPA sensor employing multi-copper oxidase

Abstract

Sensors, kits, and methods for assaying L-DOPA using engineered multi-copper oxidase are disclosed. Engineered multi-copper oxidase has improved catalytic activity at room temperature and can selectively dehydrogenate L-DOPA as a substrate when immobilized on an electrode for electrochemical detection. Embodiments include a sensor for assaying L-DOPA, the sensor comprising engineered multi-copper oxidase, which has increased catalytic activity toward the substrate compared to the catalytic activity of wild-type multi-copper oxidase.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 505,278, filed May 31, 2023, which is incorporated herein by reference in its entirety. Reference to Sequence Listing

[0002] The sequence listing described in the file named 614156SEQLIST.xml is 7.4 kilobytes, created on May 23, 2024, and is incorporated herein by reference.

Background Art

[0003] Parkinson's disease (PD) affects more than 10 million people worldwide and is a chronic disorder that clinically presents as speech difficulties, swallowing, tremors, slowness, mood disorders, and psychosis. PD biologically manifests through the degeneration of dopaminergic neurons in the substantia nigra of the brain, which leads to low dopamine production. The most effective currently used treatment for PD is levodopa (l - 3,4 - dihydroxyphenylalanine; L - DOPA), a dopamine precursor that can cross the blood - brain barrier and is later converted to dopamine. However, due to the lack of a standardized L - DOPA measurement platform, most L - DOPA administrations have to rely on patient feedback and other subjective measures. This administration system presents numerous challenges further complicated by the fact that L - DOPA has a short therapeutic window and is cytotoxic at high concentrations.

[0004] Accurate detection of L-DOPA would enable improved administration, better management of mid- and late-stage Parkinson's disease symptoms, and dramatically improve the quality of life for millions of people worldwide. There is a great need for a selective and continuous L-DOPA sensing system to better treat patients with PD. As described herein, engineered multi-copper oxidases with enhanced catalytic activity have been developed with improved properties for biosensing applications. The bioelectrochemical devices disclosed herein address this need through the invention of engineered multi-copper oxidases for highly sensitive detection of L-DOPA. [Overview of the project]

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

[0006] The embodiments are sensors for assaying L-DOPA, the sensors comprising engineered multicopper oxidase, the engineered multicopper oxidase having increased catalytic activity toward the substrate compared to the catalytic activity of wild-type multicopper oxidase. In the embodiments, the engineered multicopper oxidase comprises a sequence having one or more amino acid residue modifications of wild-type multicopper oxidase, the modification resulting in enhanced electron transfer between the enzyme and the substrate at the type 1 (T1) copper center, and optionally, the modification resulting 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 the catalytic activity of 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 manipulated multi-copper 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 located at the position corresponding to position 262 of SEQ ID NO: 1. In some embodiments, the modification is a conserved amino acid substitution. In some embodiments, the modification is the substitution of a phenylalanine amino acid residue by a different nonpolar amino acid residue. In one embodiment, the modification is the substitution of a phenylalanine amino acid residue by an isoleucine residue. In another embodiment, the modification is the substitution of a phenylalanine amino acid residue by a leucine residue. In one embodiment, the modification is the substitution of a phenylalanine amino acid residue by a valine residue. In further embodiments, the modification is the substitution of a phenylalanine amino acid residue by an alanine residue.

[0008] In some embodiments, the manipulated multi-copper oxidase contains 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 with any one of SEQ ID NOs: 2-5. In some embodiments, the manipulated multi-copper oxidase contains any one of the amino acid sequences shown in SEQ ID NOs: 2-5.

[0009] In some embodiments, the manipulated multi-copper oxidase is immobilized on an electrode, which comprises a conductive material. In some embodiments, the conductive material is selected from the group consisting of carbon paper, glassy carbon, carbon nanotubes, gold, and palladium.

[0010] In some embodiments, the L-DOPA assay involves measuring electrons transferred to an electrode in the sample, which are generated from the dehydrogenation of L-DOPA. In some embodiments, the measurement is a current 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 manipulated multi-copper oxidase exhibits improved catalytic activity for L-DOPA as a substrate compared to substrates other than L-DOPA. In embodiments, the manipulated multi-copper oxidase is selective for the detection of L-DOPA in the presence of one or more physiological substrates other than L-DOPA, and a reduced signal bias is observed compared to the signal bias observed for wild-type multi-copper 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 includes a three-electrode configuration comprising (I) 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 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, the 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, comprising a sensor and instructions for use.

[0015] Another embodiment is a method for assaying L-DOPA in a sample, comprising the steps of bringing the sample into contact with a sensor and measuring the amount of L-DOPA.

[0016] In some embodiments, the measurement is current. In some embodiments, a voltage potential is applied between an electrode containing manipulated multi-copper oxidase and another electrode, and the voltage is between -0.1V and -0.5V. In some embodiments, the measurement is performed over a period ranging from 5 seconds to 1 month.

[0017] In other embodiments, the measurement is potential. In some embodiments, the potential is open-circuit potential. In some embodiments, the chronoamperometry signal is pulsed with a voltage over a certain length of time. In some embodiments, the pulsed voltage is 0.2V to 0.4V, and the length of time is 30 seconds to 300 seconds. In some embodiments, the measurement is taken after the chronoamperometry signal has been pulsed. In some embodiments, the measurement is taken 5 seconds to 600 seconds after the signal has been pulsed.

[0018] In some embodiments, the limit of detection (LOD) is less than 0.01 μM, less than 0.1 μM, less than 0.2 μM, less than 0.3 μM, less than 0.4 μM, less than 0.5 μM, less than 0.6 μM, less than 0.7 μM, less than 0.8 μM, less than 0.9 μM for DOPA, less than 1.0 μM, less than 2.0 μM, less than 5 μM, less than 10 μM, less than 20 μM, or less than 55 μM for L-DOPA. [Brief explanation of the drawing]

[0019] [Figure 1] This provides a general overview of multi-copper oxidases. [Figure 2] This outlines its use for manipulated multi-copper oxidase and levodopa (L-DOPA) sensing (created on BioRender.com). [Figure 3A]Chronoamperometric evaluation of three different gold disk electrodes with engineered PaMCO Phe290Ile enzyme immobilized on the surface for measuring dopaquinone reduction current is shown. Figure 3A shows raw data, Figure 3B shows a calibration curve for L-DOPA from 0 to 1.2 μM, and Figure 3C shows a calibration curve for L-DOPA from 0 to 55 μM. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 4A] Chronoamperometric evaluation of three different gold disk electrodes with mushroom-derived tyrosinase immobilized on the surface for measuring dopaquinone reduction current is shown. Figure 4A shows raw data, Figure 4B shows a calibration curve for L-DOPA from 85 to 185 μM. Figure 4C shows raw data, and Figure 4D shows a calibration curve for L-DOPA from 0 to 55 μM. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 5A] Chronoamperometric evaluation of three different gold disk electrodes with Aspergillus sp.-derived laccase immobilized on the surface for measuring dopaquinone reduction current is shown. Figure 5A shows raw data, and Figure 5B shows a calibration curve between 0 and 55 μM of L-DOPA. [Figure 5B] Same as above. [Figure 6] Chronoamperometric signal bias from the addition of various physiological interferents at the upper limit of physiological concentration in the presence of 180 μM of L-DOPA is shown for electrodes with engineered PaMCO Phe290Ile enzyme (fifth from the left) or mushroom-derived tyrosinase (fifth from the right) immobilized on the surface.

Mode for Carrying Out the Invention

[0020] Currently, Parkinson's disease (PD) treatment is hampered by the lack of technology for detecting L-DOPA. Providing appropriate therapeutic doses is difficult without data, and both under- and over-dosing of L-DOPA have serious side effects that affect patients' quality of life. There are no commercially available L-DOPA sensors, and no practical L-DOPA sensors have been reported in the scientific literature. There have been several attempts to develop either a clinical-grade L-DOPA sensor or a continuous platform. 6、7、12 Challenges related to enzyme substrate specificity, stability, and biosensor performance have hindered the development of commercially available L-DOPA sensors. Specifically, the development of commercial products is limited by the fact that the majority of reported L-DOPA sensors utilize enzymes such as tyrosinase, which are not specific to L-DOPA. This specification discloses a biosensor that utilizes engineered multi-copper oxidase to specifically detect L-DOPA levels and address this long-standing need in the field of Parkinson's disease treatment. The serial L-DOPA biosensor, point-of-care (POCT), and personal-use L-DOPA monitoring system disclosed herein have the potential to significantly enhance the management of stage II PD and minimize “off-time” PD symptoms.

[0021] The subject matter of this disclosure is described more fully below. However, many modifications and other embodiments of the subject matter of this disclosure described herein will be recalled by those skilled in the art to which the subject matter of this disclosure pertains, who are of interest in the teachings presented in the preceding description. Therefore, it should be understood that the subject matter of this disclosure is not 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 encompasses all substitutes, modifications, and equivalents. If one or more incorporated documents, patents, and similar materials, including but not limited to defined terms, use of terms, and described techniques, differ from or conflict with this application, this application shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All publications, patent applications, patents, and other references described herein are incorporated in their entirety by reference.

[0022] I. Overview Multi-copper oxidases (MCOs) belong to a protein superfamily of enzymes that catalyze the four-electron reduction of oxygen to water by oxidation of various substrates. They catalyze the one-electron oxidation of a wide range of compounds, including substituted phenols, arylamines, and aromatic thiols, as shown in Figure 1. MCOs are classified as type 1 (T1), type 2 (T2), or type 3 (T3) and contain at least four copper atoms. 1、2、3Substrate oxidation begins at the T1 mononuclear copper center, followed by the internal transfer of electrons to the trinuclear copper center T2 / T3, where oxygen (O2) is reduced by four electrons to produce two water molecules. The family of multi-copper oxidase enzymes includes laccase (EC 1.10.3.2), bilirubin oxidase (EC 1.3.3.5), and ascorbate oxidase (EC 1.10.3.3). Various types of organisms, including bacteria, archaea, and fungi, possess multi-copper oxidase enzymes. One example is Pyrobaculum aerophilum, an aerobic hyperthermophilic archaeon with an optimal temperature of 100°C. Other studies have recently elucidated the structure of a novel hyperthermally stable Pyrobaculum aerophilum multi-copper oxidase (PaMCO). 4 Wild-type multi-copper oxidase exhibits high structural stability at an optimal reaction temperature of 85°C and maintains its activity over a wide pH range (e.g., 4.5–11), but shows low catalytic activity. Subsequently, the same research group used directed evolution to identify mutants of Pyrobaculum aerophilum multi-copper oxidase with enhanced catalytic activity. 5

[0023] Multi-copper oxidases, when immobilized on electrodes, enable direct electron transfer to the electrodes, making them suitable for bioelectrochemical applications. Biosensors capable of accurately and sensitively detecting L-DOPA would be extremely beneficial for the treatment of Parkinson's disease patients; however, issues with enzymatic stability, sensitivity, and specificity for L-DOPA have hindered the development of commercially available L-DOPA sensors. The Pyrobaculum aerophilum multi-copper oxidases, engineered for increased catalytic activity and disclosed herein, meet this need and can be used in biosensors for L-DOPA detection.

[0024] In embodiments, amino acid sequences of multicopper oxidases, such as those derived from Pyrobaculum aerophilum, are provided herein, which have been engineered to have increased catalytic activity compared to wild-type multicopper oxidase. In embodiments, methods for producing engineered multicopper oxidases with increased catalytic activity and their applications as biosensors, including but not limited to electrochemical L-DOPA sensors, are provided herein. Engineered multicopper oxidases shown herein can selectively dehydrogenate L-DOPA at room temperature and / or body temperature while maintaining thermal stability.

[0025] In some embodiments, the catalytic activity of the manipulated multicopper oxidase is measured as specific activity. For example, manipulated multicopper oxidases having specific activities about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 times higher than the specific activity of wild-type multicopper oxidase are provided herein.

[0026] It should be noted that the multi-copper oxidase enzymes disclosed herein may or may not contain an initial signal peptide sequence, which is found at the N-terminus of the wild-type multi-copper oxidase sequence and typically consists of a cytoplasmic domain and a transmembrane helix, typically a 28-amino acid residue. Mutations are described herein by their numbered positions within the entire enzyme sequence, including the signal peptide sequence (e.g., Phe290Ile). However, in certain embodiments provided herein, the enzymes were manipulated and produced without the initial signal peptide sequence. Therefore, amino acid residue mutations in the multi-copper oxidases disclosed herein may be referred to using numbered positions that differ from the actual locations of the mutations in the disclosed sequences, due to the absence of the inclusion of the signal peptide. For example, the mutation referred to as Phe290Ile is located at position 262 of SEQ ID NO: 1, because the first 28 amino acid residues containing the initial signal peptide are not included in SEQ ID NO: 1.

[0027] In the embodiments provided herein, the manipulated multicopper oxidase is derived from Pyrobaculum aerophilum multicopper oxidase, and the wild-type enzyme has the amino acid sequence shown in SEQ ID NO: 1.

[0028] Wild-type multi-copper oxidase from Pyrobaculum aerophilum MTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKNKLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHGLTAKQ FYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPMEMIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVPMRLIAVDQGFLA RPIEVRALFLAPAERAEVVVELGEGVYLLKNTPFDPMHLEMGHGMQEALPEGSEYTIATFLVEGKGEAVPVEALSDPPPEPPKPTRTRRFALSLSGMQWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERKDSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHCHNLEHEDGGMMINIAVK(Sequence ID 1)

[0029] In embodiments disclosed herein, the engineered multi-copper oxidase derived from Pyrobaculum aerophilum possesses one or more mutations to improve catalytic activity, such as a mutation in Phe290 corresponding to position 262 of the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the mutation to improve catalytic activity is Phe290Ile, which includes the amino acid sequence shown in SEQ ID NO: 2.

[0030] Multicopper oxidase from Pyrobaculum aerophilum containing Phe290Ile MTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKNKLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHGLTAKQ FYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPMEMIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVPMRLIAVDQGFLA RPIEVRALFLAPAERAEVVVELGEGVYLLKNTPIDPMHLEMGHGMQEALPEGSEYTIATFLVEGKGEAVPVEALSDPPPEPPKPTRTRRFALSLSGMQWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERKDSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHCHNLEHEDGGMMINIAVK(Sequence ID 2)

[0031] In another embodiment, the mutation for improving catalytic activity is Phe290Leu, which includes the amino acid sequence shown in SEQ ID NO: 3.

[0032] Multicopper oxidase from Pyrobaculum aerophilum containing Phe290Leu MTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKNKLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHGLTAKQ FYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPMEMIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVPMRLIAVDQGFLA RPIEVRALFLAPAERAEVVVELGEGVYLLKNTPLDPMHLEMGHGMQEALPEGSEYTIATFLVEGKGEAVPVEALSDPPPEPPKPTRTRRFALSLSGMQWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERKDSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHCHNLEHEDGGMMINIAVK(Sequence ID 3)

[0033] In one embodiment, the mutation for improving catalytic activity is Phe290Val, which includes the amino acid sequence shown in SEQ ID NO: 4.

[0034] Multi-copper oxidase from Pyrobaculum aerophilum containing Phe290Val MTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKNKLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHGLTAKQ FYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPMEMIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVPMRLIAVDQGFLA RPIEVRALFLAPAERAEVVVELGEGVYLLKNTPVDPMHLEMGHGMQEALPEGSEYTIATFLVEGKGEAVPVEALSDPPPEPPKPTRTRRFALSLSGMQWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERKDSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHCHNLEHEDGGMMINIAVK(Sequence ID 4)

[0035] In a further embodiment, the mutation for improving catalytic activity is Phe290Ala, which includes the amino acid sequence shown in SEQ ID NO: 5.

[0036] Multicopper oxidase from Pyrobaculum aerophilum containing Phe290Ala MTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKNKLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHGLTAKQ FYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPMEMIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVPMRLIAVDQGFLA RPIEVRALFLAPAERAEVVVELGEGVYLLKNTPADPMHLEMGHGMQEALPEGSEYTIATFLVEGKGEAVPVEALSDPPPEPPKPTRTRRFALSLSGMQWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERKDSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHCHNLEHEDGGMMINIAVK(Sequence ID 5)

[0037] In one embodiment, a sensor is provided for assaying L-DOPA in a sample, and the sensor comprises an engineered multi-copper oxidase having increased catalytic activity as described herein. In another embodiment, a kit is provided for assaying L-DOPA in a sample, and the kit comprises an engineered multi-copper oxidase as described herein. In yet another embodiment, a method is described for the detection of L-DOPA using an engineered multi-copper oxidase as described herein.

[0038] This specification provides engineered multi-copper oxidases having increased catalytic activity capable of selectively dehydrogenating L-DOPA as a substrate. As described herein, the engineered multi-copper oxidases are highly specific and selective to L-DOPA as a substrate and do not react with other physiologically relevant compounds. These engineered multi-copper oxidases are useful as enzymes in biomolecular recognition elements of electrochemical biosensors such as L-DOPA monitoring kits and for implantable or wearable sensors for continuous L-DOPA monitoring. This specification provides engineered multi-copper oxidases suitable for L-DOPA biosensing.

[0039] II. Definition Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having the meaning consistent with their meaning in relation to this application and the relevant art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. The terms used in the description of the invention herein are for the purpose of describing specific aspects and are not intended to be limitations of the invention. In case of any terminological conflict, this specification shall prevail.

[0040] The term "subject" refers to mammals (e.g., humans) requiring L-DOPA concentration analysis. Subjects may include dogs, cats, pigs, cattle, sheep, goats, horses, rats, mice, non-human mammals, and humans. The term "subject" does not necessarily exclude individuals that are healthy in all respects and do not have or show signs of Parkinson's disease.

[0041] As used herein, the term “physiological conditions” refers to the range of temperature, pH, and tonic (or osmotic) conditions that are typically encountered within the tissues of a living human body.

[0042] As used herein, the term "body temperature" refers to the physiological temperature of a human or animal. The average human body temperature is approximately 37°C.

[0043] As used herein, “room temperature” refers to a range of temperatures commonly chosen for indoor settings, such as 16–27°C.

[0044] The term "in vitro" refers to artificial environments, and processes or reactions that occur within artificial environments (e.g., test tubes).

[0045] The term "in vivo" refers to the natural environment (e.g., cells, organisms, or bodies), and processes or reactions that occur within the natural environment.

[0046] As used herein, the term "and / or" encompasses all possible combinations of one or more of the related enumerated items, as well as the absence of any combination, as interpreted by the alternative ("or").

[0047] The term "or" refers to any one of the components of a particular list.

[0048] Unless otherwise made clear from the context, the term “approximately” includes the standard error of measurement of the described value (e.g., SEM) or values ​​within ±0.5%, 1%, 5%, or 10% of the specified value.

[0049] The singular articles "a," "an," and "the" include plural references unless the context explicitly indicates otherwise. For example, the terms "a protein" or "at least one protein" can include multiple proteins, including mixtures of them.

[0050] A composition or method that "comprises" (or includes) one or more of the listed elements may include other elements not specifically listed. For example, a composition that "comprises" (or includes) protein may contain protein alone or in combination with other components.

[0051] Other definitions are provided below.

[0052] III. Composition Manipulated multi-copper oxidase In one embodiment, an isolated, engineered multicopper oxidase is provided that exhibits increased catalytic activity compared to wild-type multicopper oxidase. In this embodiment, the engineered multicopper oxidase having increased catalytic activity is capable of selectively utilizing L-DOPA as a substrate. In this embodiment, the engineered multicopper oxidase having increased catalytic activity also has increased specific activity. In this embodiment, the engineered multicopper oxidase exhibits increased catalytic activity at room temperature and / or body temperature (e.g., about 37°C). In this embodiment, the engineered multicopper oxidase maintains thermal stability at temperatures higher than room temperature (e.g., 27–100°C).

[0053] As used herein, “isolated” with respect to polypeptides (and polynucleotides) means molecules (e.g., polypeptides, proteins, or polynucleotides) that are isolated from their natural environment or prepared using synthetic methods known to those skilled in the art. Complete purification is not required in any case. The molecules described herein can be isolated and purified from materials typically associated by conventional methods such that the molecule is the dominant species in the purified preparation. At a minimum, the degree of purification is such that foreign substances in the preparation do not interfere with the use of the molecule in the manner disclosed herein. The molecules are at least about 85% pure, alternatively at least about 90% pure, alternatively at least about 95% pure, alternatively at least about 99% pure.

[0054] As used herein, “about” means within a statistically significant range of one or more values, such as concentration, length, molecular weight, pH, sequence identity, time frame, temperature, or volume. Such values ​​or ranges may be within one order of magnitude of a given value or range, typically within 20%, more typically within 10%, and even more typically within 5%. The acceptable variation encompassed by “about” depends on the particular system under study and will be readily apparent to those skilled in the art.

[0055] The term "wild-type" refers to an entity that possesses the structure and / or activity found in a normal state or context (as opposed to mutants, pathogenic or modified organisms). Wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles). A "wild-type amino acid residue" at a given position refers to the amino acid present at a given position in a wild-type polypeptide.

[0056] As used herein, “variant” means, when used in relation to polypeptides or proteins such as enzymes, a variant comprising an insertion, deletion, or substitution of one or more amino acid residues on the polypeptide or protein at the indicated position. “Variant” is also used to refer to polynucleotides encoding such a mutant polypeptide or protein.

[0057] As used herein, “corresponding position” means the position of an amino acid residue in a query amino acid sequence that is aligned with an amino acid residue in a reference amino acid sequence using software such as Vector NTI’s AlignX with default parameters (available from Invitrogen; see Lu & Moriyama (2004) Brief Bioinform. 5:378-88). Therefore, “amino acid (AA) residue at the position corresponding to position Y in the amino acid sequence shown in SEQ ID NO: X” means an AA residue in the query amino acid sequence that is aligned with AA Y in SEQ ID NO: X using Vector NTI’s AlignX with default parameters. Note that AA Y in SEQ ID NO: X itself is also encompassed by this term.

[0058] 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.

[0059] As used herein, “dehydrogenate” or “Dh” refers to an enzymatic process by an enzyme that catalyzes the oxidation of a substrate through the removal of hydrogen atoms and electrons. Multicopper oxidase can dehydrogenate L-DOPA as a substrate, producing L-dopaquinone as an oxidation product. The term “dehydrogenation” of a substrate (e.g., L-DOPA) may be used interchangeably with the term “oxidation” of a substrate.

[0060] As used herein, “oxidase activity” means the enzymatic activity of an engineered multi-copper oxidase that utilizes oxygen as an electron acceptor. Oxidase activity can be assayed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate by monitoring the increase in absorbance at 420 nm based on the formation of reduced ABTS and / or by any method known in the art.

[0061] 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, engineered multicopper oxidase is X

number

number

[0062] As used herein, “thermal stability” refers to the ability of an enzyme to carry out its catalytic reaction at high temperatures (e.g., above room temperature). A multi-copper oxidase is described as “thermally stable” if it retains the ability to catalyze the dehydrogenation of its substrate at temperatures above room temperature.

[0063] As used herein, “high temperature” refers to a temperature higher than room temperature, such as a temperature higher than 27°C. Wild-type multi-copper oxidase from Pyrobaculum aerophilum is efficient at high temperatures, with an optimal reaction temperature of 85°C.

[0064] Therefore, with respect to electrochemical biosensors, it is desirable to manipulate enzymes to have high catalytic activity at room temperature and / or body temperature with high specificity for L-DOPA as a substrate. In embodiments, manipulated multi-copper oxidases having increased catalytic activity at room temperature are disclosed herein.

[0065] As used herein, “biosensor” means a device containing a biorecognition element (e.g., enzyme, aptamer, antibody, etc.) that reacts with a target molecule and subsequently converts the biological signal into an analyzable electrical signal. In this specification, the term “biosensor” may be used interchangeably with “enzyme sensor” or “sensor.”

[0066] In embodiments provided herein, the engineered multicopper oxidase comprises a sequence having one or more amino acid residue modifications of wild-type multicopper oxidase, the modifications resulting in increased catalytic activity for a substrate (e.g., L-DOPA). In some embodiments, the engineered multicopper oxidase exhibits approximately 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, and 12-fold increased catalytic activity compared to wild-type multicopper oxidase. In some embodiments, catalytic activity is measured as specific activity. In some embodiments, catalytic activity is determined by an assay using ABTS as a substrate and monitoring the increase in absorbance at 420 nm based on the formation of reduced ABTS, and / or by any method known in the art.

[0067] In embodiments, the manipulated multi-copper 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., sugar, carbidopa, dopamine, etc.), and a reduced signal bias is observed compared to the signal bias observed for wild-type multi-copper oxidases. The signal bias can be measured by detecting L-DOPA alone in the presence of physiological substrates at physiologically relevant concentrations to determine interference 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%.

[0068] In the embodiments disclosed herein, the manipulated multi-copper oxidase may be derived from a hyperthermophilic organism (e.g., archaea or bacteria). As used herein, "hyperthermophilic" refers to an organism that survives in high-temperature environments at temperatures above 60°C. In some embodiments, the manipulated multi-copper oxidase is derived from Pyrobaculum aerophilum.

[0069] In embodiments, the engineered multicopper oxidase includes one or more mutations that improve electron transfer between the enzyme and substrate at the T1 copper center. In embodiments, one or more mutations provide increased loop flexibility near the T1 copper center, enhancing the catalytic activity of the engineered multicopper oxidase compared to the catalytic activity of wild-type multicopper oxidase. In embodiments, one or more mutations may increase the current density of an electrode with immobilized engineered multicopper oxidase compared to an electrode with immobilized wild-type multicopper oxidase.

[0070] In some embodiments, the mutations for increasing catalytic activity are amino acid insertions, deletions, and / or substitutions in the sequence of wild-type multicopper oxidase. In some embodiments, the mutations for increasing oxidase activity are amino acid substitutions in the sequence of wild-type multicopper oxidase. In some embodiments, the substitutions are conservative amino acid substitutions.

[0071] In some embodiments, the mutation is the substitution of a phenylalanine amino acid residue with another nonpolar amino acid residue and / or a non-aromatic amino acid residue. In embodiments, the nonpolar amino acid residue is selected from the group consisting of isoleucine, leucine, valine, and alanine.

[0072] In some embodiments, the manipulated multicopper oxidase includes a substitution at the corresponding 262 position of the amino acid residue at the corresponding position in SEQ ID NO: 1 (e.g., Phe290 in wild-type Pyrobaculum aerophilum multicopper oxidase). In some embodiments, the substitution is the Phe290 substitution of wild-type Pyrobaculum aerophilum multicopper oxidase. In some embodiments, the amino acid sequence of the manipulated multicopper oxidase includes the Phe290Ile, Phe290Leu, Phe290Val, or Phe290Ala substitution of wild-type Pyrobaculum aerophilum multicopper oxidase (SEQ ID NO: 1).

[0073] In embodiments, manipulated multi-copper oxidases 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 with any one of SEQ ID NOs: 2 to 5 are provided herein, provided that the amino acid residue at the position corresponding to position 268 in SEQ ID NO: 1 is different from the amino acid residue occupying the corresponding position in SEQ ID NO: 1.

[0074] In some embodiments, the manipulated multi-copper oxidase contains the amino acid sequence shown in any one of SEQ ID NOs: 2-5.

[0075] The numbering of amino acid sequences of the manipulated multicopper oxidases disclosed herein begins with start Met, and it should be understood that the multicopper oxidase may or may not have an initial signal peptide sequence.

[0076] In relation to two polynucleotide or polypeptide sequences, “sequence identity” or “identity” refers to residues in the two sequences that are identical when aligned for maximum correspondence across a specified comparison window. When the percentage of sequence identity is used in relation to proteins, it is recognized that non-identical residue positions are often differed by conserved amino acid substitutions, where the amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. If sequences differ in a conserved substitution, the percentage of sequence identity may be adjusted upward to compensate for the conserved nature of the substitution. Sequences differing by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those skilled in the art. Typically, this involves scoring the conservative substitution as a partial mismatch rather than a complete mismatch, thereby increasing the percentage of sequence identity. Thus, for example, if identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, then conservative substitutions are given a score of 0 to 1. The scoring of conservative substitutions is calculated, for example, as performed by the program PC / GENE (Intelligenetics, Mountain View, California).

[0077] The "sequence identity ratio" refers to a value determined by comparing two optimally aligned sequences (the maximum number of perfectly matched residues) across a comparison window, where some polynucleotide sequences within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The ratio is calculated by determining the number of positions in which identical nucleic acid bases or amino acid residues occur in both sequences, finding the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity ratio. Unless otherwise specified (e.g., including heterogeneous sequences with concatenated shorter sequences), the comparison window is the full length of the shorter of the two sequences being compared.

[0078] Unless otherwise stated, sequence identity / similarity values ​​refer to values ​​obtained using GAP version 10, using the following parameters: nucleotide sequence identity % and similarity % using 50 GAP weights and 3 length weights, and the nwsgapdna.cmp scoring matrix; amino acid sequence identity % and similarity % using 8 GAP weights and 2 length weights, and the BLOSUM62 scoring matrix, or any equivalent program thereof. “Equivalent program” includes any sequence comparison program that, for any two sequences in question, produces alignments having identical nucleotide or amino acid residue matches and identical sequence identity percentages compared to corresponding alignments produced by GAP version 10.

[0079] The term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a sequence with an amino acid of similar size, charge, or polarity but different in nature. Examples of conservative substitutions include the substitution of a nonpolar (hydrophobic) residue such as isoleucine, valine, or leucine for another nonpolar residue. Similarly, examples of conservative substitutions include the substitution of a single polar (hydrophilic) residue for another residue, such as between arginine and lysine, glutamine and asparagine, or glycine and serine. In addition, substitutions of a basic residue such as lysine, arginine, or histidine for another residue, or substitutions of a single acidic residue such as aspartic acid or glutamic acid for another acidic residue are examples of additional conservative substitutions. Examples of non-conservative substitutions include the substitution of a nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue for a nonpolar residue. A typical amino acid classification is summarized below. [Table 1]

[0080] In embodiments, the manipulated multi-copper oxidase thus obtained can 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, isoelectrophoresis, and dialysis.

[0081] IV. Devices L-DOPA biosensor In embodiments, a sensor for assaying L-DOPA in a sample is disclosed herein, the sensor comprising an engineered multi-copper oxidase having increased catalytic activity as described herein. As shown in Figure 2, an L-DOPA biosensor comprising the engineered multi-copper oxidase is used to measure physiologically relevant L-DOPA concentrations.

[0082] In some embodiments, manipulated multi-copper oxidases exhibit improved substrate specificity for L-DOPA compared to biologically relevant phenols, dopamine analogs, and sugars. L-DOPA as described herein is measured using electrochemical techniques. In embodiments, L-DOPA is quantified by measuring electrons transferred to an electrode in a sample, which are generated from the dehydrogenation of L-DOPA. In embodiments, L-DOPA is detected by current or potentiometric measurements. In some embodiments, L-DOPA biosensors measure L-DOPA through open-circuit potential, chronoamperometry, square wave voltammetry, and / or extended-gate field-effect transistor (EG-FET) detection. In embodiments provided herein, L-DOPA biosensors provide highly sensitive and specific detection of L-DOPA.

[0083] In some embodiments, the manipulated multi-copper oxidase is immobilized on 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 electrode. In some embodiments, the L-DOPA biosensor has a three-electrode configuration including 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 including a WE and a shared Ref / CE. See Figure 2. The concentration of L-DOPA in the sample can be determined by measuring the amount of electrons generated or lost due to the enzymatic reaction, the reaction between the product and the electrode, or the change in charge resulting from the interaction between L-DOPA and the enzyme relative to a reference value. In some embodiments, the 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.

[0084] In one embodiment, a non-biological or biological sample, such as blood, serum, saliva, tears, urine, sweat, or interstitial fluid sample, is added to a sample receiver, and the L-DOPA contained in the sample reacts with a manipulated multi-copper oxidase, transferring electrons to an electrode to generate a current that indicates the amount of L-DOPA in the sample.

[0085] In some embodiments, the manipulated multi-copper oxidase is immobilized on the electrode by any method known in the art. Examples of means for immobilizing molecules such as the manipulated multi-copper oxidase include, but are not limited to, crosslinking, encapsulation in a polymer matrix, coating with a dialysis membrane, optically crosslinked polymers, conductive polymers, redox polymers, and any combination thereof. In some embodiments, the manipulated multi-copper oxidase is immobilized on the electrode with a dithiobis(succinimidyl hexanoate) self-assembled monolayer (DSH-SAM) solution.

[0086] The term "immobilized" refers to a compound that adheres to a surface (e.g., an electrode). Immobilization can be achieved through adsorption, crosslinking, covalent bonding, and / or affinity tagging.

[0087] The term "crosslinking" refers to the polymerization reaction of a crosslinking reagent. A "crosslinking reagent" refers to a molecule that contains two or more reactive ends capable of forming a covalent bond.

[0088] The electrodes of the L-DOPA biosensors disclosed herein include a conductive material. In some embodiments, the conductive material includes carbon paper, glassy carbon, carbon nanotubes, gold, and / or palladium. In some embodiments, the cathode and / or anode include a conductive ink.

[0089] The term "conductive material" refers to a substance that is capable of transmitting electricity.

[0090] In the embodiment, the electrodes are screen-printed carbon electrodes, planar gold electrodes, or inter-mating electrode arrays. When the measurement is performed in a current measurement system using carbon (C) electrodes, a gold (Au) or platinum (Pt) electrode provided with immobilized enzymes is used as the 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 solution containing a mediator and maintained at a predetermined temperature.

[0091] A predetermined voltage can be applied to the working electrode, then the sample is added, and the increased current value is measured. Generally, it is also possible to use a so-called two-electrode system having one working electrode and one counter electrode or pseudo-reference electrode.

[0092] In one embodiment, the working electrode can be inserted into a buffer solution together with a counter electrode (such as a Pt electrode) and a reference electrode (such as an Ag / AgCl electrode) and maintained at a predetermined temperature. As shown above, a predetermined voltage can be applied to the working electrode, then the sample is added, and the increased value of the current is measured.

[0093] In some embodiments, the L-DOPA biosensor can continuously assay for L-DOPA in a sample.

[0094] In some embodiments, the manipulated multi-copper 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 strip may be single-use and / or disposable. The L-DOPA sensor strip can be used for point-of-care testing (POCT) and for personal use L-DOPA monitoring.

[0095] Furthermore, this specification also provides a biosensor for the detection of phospholevodopa ((2S)-2-amino-3-(3-hydroxy-4-phosphonooxyphenyl)propanoic acid, dopa 4-phosphate, or levodopa-4'-monophosphate), which is a drug for the treatment of PD patients. The sensor comprises the previously described MCO and further comprises an enzyme that catalyzes the dephosphorylation of phospholevodopa to L-DOPA. In some embodiments, the enzyme is a phosphatase. In some embodiments, the enzyme that catalyzes the phosphorylation of phospholevodopa is immobilized as a layer on the working electrode. In some embodiments, the enzyme that catalyzes the phosphorylation of phospholevodopa is provided in a solution within the device. Once phospholevodopa is dephosphorylated by the enzyme (e.g., phosphatase), the MCO sensor is used to detect the resulting L-DOPA product, as previously described. Thus, phospholevodopa can be detected using a device containing both enzymes.

[0096] Therefore, these devices can be used as continuous phospholevodopa biosensors for point-of-care trials (POCT) in PD patients treated with phospholevodopa. The phospholevodopa monitoring systems disclosed herein have the potential to significantly enhance the management of stage II PD and minimize "off-time" PD symptoms.

[0097] V. Kit In another embodiment, a kit for assaying L-DOPA in a sample, the kit comprising at least the manipulated multi-copper oxidases described herein.

[0098] In addition, the kit may include the buffer solution required for the measurement, a standard solution of L-DOPA for creating a calibration curve, and / or instructions for use. The manipulated multi-copper oxidase may be supplied in various forms, such as a lyophilized reagent or a solution in a suitable storage solution.

[0099] In some embodiments, the kit includes a sensor strip that may be screen-printed and / or disposable.

[0100] Some or all of the kit reagents can be provided in containers that protect them from the external environment, such as in a sealed container. Positive and / or negative controls can be included in the kit to verify the activity and correct use of the reagents employed in accordance with the concept of the present invention. The controls may include samples known to be either positive or negative for the presence of a given concentration of L-DOPA.

[0101] VI. Method The manipulated multi-copper oxidases disclosed herein can be used in a variety of ways. For example, they can be used in a method for assaying L-DOPA in a sample derived from a subject. In embodiments, the sample includes a substance selected from the group consisting of blood, serum, saliva, tears (i.e., lacrimal gland secretions), urine, sweat, and interstitial fluid.

[0102] The method may include at least the steps of contacting a sample with a manipulated multi-copper oxidase and measuring the amount of L-DOPA oxidized by the manipulated multi-copper oxidase, as described above and further below. In embodiments, the method includes continuous measurement of the amount of L-DOPA oxidized by the manipulated multi-copper oxidase. In embodiments, the measurement may be an amperometric or potentiometric measurement.

[0103] In some embodiments, the measurement performed to determine the concentration of L-DOPA is current. In some embodiments, a voltage potential is applied between electrodes containing the operated multi-copper oxidase disclosed herein. In some embodiments, the applied voltage potential is -0.1V to -0.5V. In embodiments, the measurement is performed over a period of time. In some embodiments, the measurement is performed over a period ranging from 5 seconds to 10 minutes. In some embodiments, the measurement is performed continuously. In some embodiments, the sensor is operationally stable and capable of providing measurements over a period ranging from 30 seconds to 1 month.

[0104] In some embodiments, the measurement performed to determine the concentration of L-DOPA is potential. In some embodiments, the potential is the open-circuit potential. In some embodiments, a chronoamperometry signal is pulsed with a voltage over a certain period of time. In some embodiments, the pulsed voltage is 0.2V to 0.4V, and the duration is 30 seconds to 300 seconds. In some embodiments, the measurement is performed after the chronoamperometry signal has been pulsed. In some embodiments, the measurement is performed 5 seconds to 600 seconds after the signal has been pulsed.

[0105] In the embodiments, the detection limit (LOD) of the L-DOPA sensor described herein is less than 0.01 μM, less than 0.2 μM, less than 0.3 μM, less than 0.4 μM, less than 0.5 μM, less than 0.6 μM, less than 0.7 μM, less than 0.8 μM, less than 0.9 μM of DOPA, less than 1.0 μM, less than 2.0 μM, less than 5 μM, less than 10 μM, less than 20 μM, or less than 55 μM of L-DOPA.

[0106] In some embodiments, the MCO provided herein is used to detect L-DOPA derived from phospholevodopa (((2S)-2-amino-3-(3-hydroxy-4-phosphonooxyphenyl)propanoic acid, or dopa 4-phosphate, levodopa-4'-monophoric acid). Phosphorus levodopa in a sample derived from the subject is first dephosphorylated to produce L-DOPA. In some embodiments, the L-DOPA assayed by the method is derived from phospholevodopa, and the method further comprises contacting the sample with an enzyme capable of dephosphorylating phospholevodopa to L-DOPA before contacting the sample with the MCO. A phosphatase enzyme can be used for dephosphorylation. The resulting L-DOPA is then quantified by a device containing the MCO as described.

[0107] These methods may be adapted with necessary modifications for assays of other substrates modified by the manipulated enzymes disclosed herein.

[0108] The disclosed subject matter is further described in the following non-limiting embodiments. It should be understood that these embodiments are given for illustrative purposes only, while illustrating preferred embodiments of the subject matter.

[0109] References [1]Multicopper Oxidases and Oxygenases,EISolomon 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 multicopper 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 7;12(2):102.doi:10.3390 / bios12020102.PMID:35200363;PMCID:PMC8869619.

[12] Barbara Brunetti, Gabriela Valdes-Ramirez, Irene Litvan, Joseph Wang, A disposable electrochemical biosensor for l-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. [Examples]

[0110] Example 1: L-DOPA sensing platform using multi-copper oxidase derived from Pyrobaculum aerophilum In previous studies, random and site-directed mutagenesis was used to identify the F290I mutant of Pyrobaculum aerophilum multicopper oxidase (PaMCO) exhibiting increased catalytic activity. The F290I mutant maintained pH and thermal stability while showing increased catalytic activity with a specific activity of 24.2 units / mg (a 12.4-fold increase) compared to 1.95 units / mg of wild-type PaMCO. In addition, mutants F290Y, F290L, F290V, and F290A were produced, found to have specific activities of 3.54, 10.5, 27.1, and 27.7 units / mg, respectively. The results suggest that the F290I mutant increases the flexibility of the loop adjacent to the T1 Cu center, improving electron transfer between the enzyme and substrate. Due to this observed improvement in catalytic activity, the F290I PaMCO mutant was tested as an enzyme sensor for L-DOPA detection.

[0111] Electrodes were prepared using immobilized PaMCO. Gold disk electrodes were polished with 0.3 and 0.05 μM alumina powder and then immersed for 1 hour in a piranha solution consisting of a 3:1 sulfuric acid to hydrogen peroxide solution. Subsequently, the electrodes were electrochemically washed using cyclic voltammetry sweeping at 0V to 1.2V in a 50 mM KOH solution. The electrodes were then washed with acetone and incubated overnight at 25°C and 300 rpm in a 100 μM dithiobis(succinimidyl hexanoate) self-assembled monolayer (DSH-SAM) solution. Next, the electrodes were incubated overnight at 25°C and stirred at 300 rpm in a 0.014 mg / ml enzyme solution. Finally, the electrodes were washed with 100 mM potassium phosphate buffer (PPB) and then stored in 100 mM PPB until use. The electrodes were prepared using manipulated PaMCO Phe290Ile enzyme (SEQ ID NO: 2), mushroom-derived tyrosinase (Sigma Aldrich T3824, CAS 9002-10-2, EC 1.14.18.1), and Aspergillus sp.-derived laccase (Novozym 51003, CAS 80498-15-3, EC 1.10.3.2).

[0112] As shown in Figures 3A–3C, three different electrodes with immobilized manipulated PaMCO Phe290Ile enzyme (SEQ ID NO: 2) were subjected to chronoamperometry evaluation. After waiting approximately 10–15 minutes to allow the three electrodes to stabilize, L-DOPA was added in the range of 0.0–55.0 μM (Figure 3C). During measurement, -0.3 V was continuously applied, and the manipulated multi-copper oxidase enzyme oxidized L-DOPA to dopaquinone, which was then reduced on the electrode surface, leading to an increased reduction current. The chronoamperometry responses were then averaged to generate a calibration curve. As shown in Figure 3B, the L-DOPA sensor showed a linear increase in reduction current from 0–1.2 μM, accompanied by a calculated LOD of 0.619 μM.

[0113] Next, a similar test was performed using electrodes containing mushroom-derived tyrosine enzymes. Three different electrodes containing immobilized mushroom-derived tyrosinase were evaluated by chronoamperometry, as shown in Figures 4A-4C. After waiting approximately 10-15 minutes to allow the three electrodes to stabilize, L-DOPA was added in the range of 85-185 μM, as shown in the raw data in Figure 4A and the calibration curve in Figure 4B. This L-DOPA concentration range indicates that L-DOPA is not an ideal substrate for tyrosinase, as visualized by the raw data (Figure 4C) and the poor L-DOPA calibration curve (Figure 4D), because the tyrosinase L-DOPA sensor showed no signal response to L-DOPA in the range of 0.0-55.0 μM, unlike the PaMCO Phe290Ile L-DOPA sensor. During the measurement, the dopaquinone reduction current at -0.3V was indistinguishable from noise, so -0.4V was continuously applied instead of -0.3V to further demonstrate the near-optimal performance of tyrosinase as a biorecognition element adopted for the L-DOPA sensor.

[0114] Next, the test was repeated using electrodes containing laccase derived from Aspergillus sp. As shown in Figures 5A-5B, three different electrodes containing immobilized laccase derived from Aspergillus sp. underwent chronoamperometry evaluation. After waiting approximately 10-15 minutes to allow the three electrodes to stabilize, L-DOPA was added in the range of 0.0-55.0 μM, and a potential of -0.3 V was continuously applied. Unlike the engineered PaMCO Phe290Ile enzyme (SEQ ID NO: 2), the laccase derived from Aspergillus sp. failed to measure dopaquinone reduction, indicating that L-DOPA is not an ideal substrate for Aspergillus sp.-derived laccase and further highlighting the value of utilizing engineered multi-copper oxidases as biorecognition elements for sensing L-DOPA.

[0115] Finally, electrodes with immobilized and manipulated PaMCO Phe290Ile enzyme (SEQ ID NO: 2), mushroom-derived tyrosinase, and Aspergillus sp.-derived laccase were compared in the presence of physiological interfering substances at the upper limit of physiological concentrations in the presence of 180 μM L-DOPA. Figure 6 clearly shows that the manipulated PaMCO Phe290Ile enzyme (SEQ ID NO: 2) sensor signal was less affected in the presence of interfering substances compared to the mushroom-derived tyrosinase and Aspergillus sp.-derived laccase, which showed minimal signals for L-DOPA. It is important to note that the interference measurements were performed in the presence of 180 μM L-DOPA because the mushroom-derived tyrosinase L-DOPA sensor did not show a significant L-DOPA current response in the same reduced L-DOPA range (0.0~55.0 μM) as the PaMCO Phe290Ile enzyme (SEQ ID NO: 2) L-DOPA sensor. The PaMCO Phe290Ile enzyme (SEQ ID NO: 2) L-DOPA sensor exhibits reduced interference effects and highlights improved substrate specificity and preference for L-DOPA compared to other biorecognition elements employed for L-DOPA sensing.

[0116] Many modifications and other embodiments of the invention described herein will be recalled by those skilled in the art to which the invention pertains, who benefit from the teachings presented in the foregoing description and the accompanying drawings. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Specific terms are used herein, but they are used only in a general and descriptive sense, and not for limiting purposes.

Claims

1. A sensor for assaying L-DOPA, wherein the sensor comprises manipulated multi-copper oxidase, The sensor wherein the manipulated multicopper oxidase has increased catalytic activity toward a substrate compared to the catalytic activity of wild-type multicopper oxidase.

2. The sensor according to claim 1, wherein the manipulated multicopper oxidase comprises a sequence having one or more amino acid residue modifications of wild-type multicopper oxidase, the modifications resulting in enhanced electron transfer between the enzyme and the substrate at type I (T1) copper centers, and optionally the modifications resulting in increased loop flexibility.

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

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

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

6. The sensor according to claim 5, wherein the hyperthermophilic organism is Pyrobaculum aerophyllum.

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

8. The sensor according to any one of claims 1 to 7, wherein the modification is located at the position corresponding to position 262 of sequence number 1.

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

10. The sensor according to claim 9, wherein the modification is the substitution of a phenylalanine amino acid residue with a different nonpolar amino acid residue.

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

12. The sensor according to claim 10, wherein the modification is the substitution of a phenylalanine amino acid residue with a leucine residue.

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

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

15. The sensor according to any one of claims 1 to 14, wherein the manipulated multi-copper 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 with any one of sequence numbers 2 to 5.

16. The sensor according to any one of claims 1 to 15, wherein the manipulated multi-copper oxidase comprises one of the amino acid sequences shown in Sequence ID No. 2 to 5.

17. The sensor according to any one of claims 1 to 16, wherein the manipulated multi-copper oxidase is immobilized on an electrode, and the electrode comprises a conductive material.

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

19. The sensor according to any one of claims 1 to 18, wherein the L-DOPA assay comprises measuring electrons transmitted to the electrode in a sample, the electrons being generated from the dehydrogenation of L-DOPA.

20. The sensor according to claim 19, wherein the measurement is current measurement or potential difference measurement.

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

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

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

24. The sensor according to any one of the prior claims, wherein the manipulated multi-copper oxidase has improved catalytic activity for L-DOPA as a substrate compared to substrates other than L-DOPA.

25. The sensor according to any one of the prior claims, wherein the manipulated multi-copper oxidase is selective for detecting L-DOPA in the presence of one or more physiological substrates other than L-DOPA, and a reduced signal bias is observed compared to the signal bias observed for wild-type multi-copper oxidase.

26. The sensor according to any one of the prior 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. (I) A three-electrode configuration including a working electrode, a reference electrode, and a counter electrode, or (II) The sensor according to any one prior claim, further comprising a two-electrode configuration including a working electrode and a shared reference and counter electrode.

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

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

30. The sensor according to any one of the prior claims, wherein the sensor is a sensor strip.

31. The sensor according to claim 30, wherein the sensor strip is screen printed.

32. The sensor according to claim 30 or 31, wherein the sensor strip is for single use and / or disposable.

33. The sensor according to any one of the prior claims, further comprising an enzyme capable of dephosphorylating phosphorylated

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

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

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

37. The method according to claim 36, wherein the measurement is electric current.

38. The method according to claim 36 or 37, wherein the measurement is a current over time.

39. The method according to claim 37 or 38, wherein a voltage potential is applied between an electrode containing manipulated multi-copper oxidase and another electrode, and the voltage is between -0.1V and -0.5V.

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

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

42. The method according to claim 41, wherein the potential is the open-circuit potential.

43. The method according to claim 42, wherein the potential is the transient open circuit potential.

44. The method according to claim 42 or 43, wherein the chronoamperometry signal is pulsed with a voltage over a certain period of time.

45. The method according to claim 44, wherein the pulsed voltage is 0.2V to 0.4V, and the duration of the pulse is 0.1 seconds to 300 seconds.

46. The method according to claim 45, wherein the pulsed voltage is 0.2V to 0.4V, and the duration of the pulse is 30 seconds to 300 seconds.

47. The method according to claim 45 or 46, wherein the measurement is performed after the chronoamperometry signal has been pulsed.

48. The method according to claim 47, wherein the measurement is performed between 5 seconds and 600 seconds after the signal is pulsed.

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

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

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