Spectrophotometric Assays for Sensing Tyrosinase Activity and Their Applications

biosensors

Review Spectrophotometric Assays for Sensing Tyrosinase Activity and Their Applications

Yu-Fan Fan 1,†, Si-Xing Zhu 2,†, Fan-Bin Hou 1, Dong-Fang Zhao 1, Qiu-Sha Pan 1, Yan-Wei Xiang 3, Xing-Kai Qian 1, Guang-Bo Ge 1 and Ping Wang 1,*

1 Shanghai Frontiers Science Center for Chinese Medicine Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shangha 201203, China; [email protected] (Y.-F.F.); [email protected] (F.-B.H.); [email protected] (D.-F.Z.); [email protected] (Q.-S.P.); [email protected] (X.-K.Q.); [email protected] (G.-B.G.) 2 Institute of Science, Technology and Humanities, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; [email protected] 3 School of Rehabilitation Science, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; [email protected] * Correspondence: [email protected] † These authors contributed equally to this work.

Abstract: Tyrosinase (TYR, E.C. 1.14.18.1), a critical enzyme participating in melanogenesis, catalyzes the ﬁrst two steps in melanin biosynthesis including the ortho-hydroxylation of L-tyrosine and the oxidation of L-DOPA. Previous pharmacological investigations have revealed that an abnormal level of TYR is tightly associated with various dermatoses, including albinism, age spots, and malignant melanoma. TYR inhibitors can partially block the formation of pigment, which are always used for improving skin tone and treating dermatoses. The practical and reliable assays for monitoring

TYR activity levels are very useful for both disease diagnosis and drug discovery. This review comprehensively summarizes structural and enzymatic characteristics, catalytic mechanism and

Citation: Fan, Y.-F.; Zhu, S.-X.; Hou, substrate preference of TYR, as well as the recent advances in biochemical assays for sensing TYR F.-B.; Zhao, D.-F.; Pan, Q.-S.; Xiang, activity and their biomedical applications. The design strategies of various TYR substrates, alongside Y.-W.; Qian, X.-K.; Ge, G.-B.; Wang, P. with several lists of all reported biochemical assays for sensing TYR including analytical conditions Spectrophotometric Assays for Sensing and kinetic parameters, are presented for the ﬁrst time. Additionally, the biomedical applications Tyrosinase Activity and Their and future perspectives of these optical assays are also highlighted. The information and knowledge Applications. Biosensors 2021, 11, 290. presented in this review offer a group of practical and reliable assays and imaging tools for sensing https://doi.org/10.3390/bios11080290 TYR activities in complex biological systems, which strongly facilitates high-throughput screening TYR inhibitors and further investigations on the relevance of TYR to human diseases. Received: 9 July 2021 Accepted: 19 August 2021 Keywords: tyrosinase (TYR); enzymatic activity; optical substrates; TYR inhibitors; high-throughput Published: 23 August 2021 screening

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Tyrosinase (TYR, E.C. 1.14.18.1), a type-3 binuclear copper-containing oxidoreductase, efficiently catalyzes o-hydroxylation of monophenols to diphenols (monophenolase activity) and the oxidation of diphenols to quinones (diphenolase activity), without any additional cofactors (Figure1)[ 1,2]. It is ubiquitously distributed in organisms ranging from bacteria Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. to eukaryotes and plays a pivotal role in the enzymatic browning of fruit or fungi, as well This article is an open access article as mammalian melanin synthesis [3,4]. In mammals, melanin is exclusively synthesized in distributed under the terms and melanosomes via complex biochemical reactions (Figure2), and this endogenous substance conditions of the Creative Commons is primarily responsible for the pigmentation of retina and skin [5,6]. TYR catalyzes the Attribution (CC BY) license (https:// first two steps in melanin biosynthesis: the o-hydroxylation of L-tyrosine and the oxidation creativecommons.org/licenses/by/ of L-DOPA. Since the remainder of the reaction sequence can proceed spontaneously at 4.0/). physiological pH, the conversion of L-tyrosine to dopaquinone (DQ) has been implicated as

Biosensors 2021, 11, 290. https://doi.org/10.3390/bios11080290 https://www.mdpi.com/journal/biosensors Biosensors 2021, 11, x FOR PEER REVIEW 2 of 22 Biosensors 2021, 11 , x FOR PEER REVIEW 2 of 22

Biosensors 2021 11 L-tyrosine and the oxidation of L-DOPA. Since the remainder of the reaction sequence , , 290L-tyrosine and the oxidation of L-DOPA. Since the remainder of the reaction sequence 2 of 22 can proceed spontaneously at physiological pH, the conversion of L-tyrosine to do- can proceed spontaneously at physiological pH, the conversion of L-tyrosine to dopaquinone (DQ) has been implicated as a crucial rate-limiting procedure in melanogene- paquinone (DQ) has been implicated as a crucial rate-limiting procedure in melanogenesis [7,8]. DQ could spontaneously convert into dopachrome, which gradually decom- sis [7,8]. DQ acould crucial spontaneously rate-limiting procedureconvert into in melanogenesisdopachrome, which [7,8]. DQ gradually could spontaneously decom- convert poses into 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid poses into 5,6-dihydroxyindoleinto dopachrome, which (DHI) gradually and decomposes5,6-dihydroxyindole-2-carboxylic into 5,6-dihydroxyindole acid (DHI) and 5,6- (DHICA) through a succession of redox reactions [5,9]. Ultimately, these dihydroxyin- (DHICA) throughdihydroxyindole-2-carboxylic a succession of redox reacti acidons (DHICA) [5,9]. Ultimately, through a succession these dihydroxyin- of redox reactions [5,9]. doles are oxidized to eumelanin. Alongside, in the presence of cysteine or glutathione, doles are oxidizedUltimately, to eumelanin. these dihydroxyindoles Alongside, in the are oxidizedpresence toof eumelanin.cysteine or Alongside,glutathione, in the presence DQ is converted to 5-S-cysteinyl dopa or glutothionyl dopa, finally yielding pheomelanin DQ is convertedof cysteineto 5-S-cysteinyl or glutathione, dopa or glutothionyl DQ is converted dopa, to finally 5-S-cysteinyl yielding dopa pheomelanin or glutothionyl dopa, [10,11]. The types and relative amounts of these melanin constitute color-based ethnic [10,11]. The typesﬁnally and yielding relative pheomelanin amounts of [these10,11 ].melanin The types constitute and relative color-based amounts ethnic of these melanin diversification. Three tyrosinase-like enzymes co-regulate melanogenesis, including TYR diversification.constitute Three tyrosinase-like color-based ethnicenzymes diversiﬁcation. co-regulate melanogenesis, Three tyrosinase-like including enzymes TYR co-regulate and TYR-related proteins 1 (TRP-1) and 2 (TRP-2). TRP-1 shows DHICA oxidase and low and TYR-relatedmelanogenesis, proteins 1 (TRP-1) including and TYR2 (TRP-2). and TYR-related TRP-1 shows proteins DHICA 1 oxidase (TRP-1) and and low 2 (TRP-2). TRP-1 tyrosine hydroxylase activity when zinc is replaced by copper. TRP-2 contains two zinc tyrosine hydroxylaseshows DHICA activity oxidase when zinc and lowis replaced tyrosine by hydroxylase copper. TRP-2 activity contains when zinctwo iszinc replaced by cop- ions at the active site and isomerizes dopachrome to DHICA. They are metal-containing ions at the activeper. site TRP-2 and contains isomerizes two dopa zinc ionschrome at the to activeDHICA. site They and isomerizesare metal-containing dopachrome to DHICA. glycoproteins and share ~40% amino acid sequence identity and ~70% similarity [1]. glycoproteinsThey and areshare metal-containing ~40% amino acid glycoproteins sequence and identity share ~40%and ~70% amino similarity acid sequence [1]. identity and Despite TYR and TYR-related proteins 1 (TRP-1) and 2 (TRP-2) being necessary for mel- Despite TYR ~70%and TYR-related similarity [ 1proteins]. Despite 1 (TRP-1) TYR and and TYR-related 2 (TRP-2) proteinsbeing necessary 1 (TRP-1) for and mel- 2 (TRP-2) being anogenesis, TYR is the most critical rate-limiting enzyme [10]. anogenesis, TYRnecessary is the most for melanogenesis, critical rate-limiting TYR is enzyme the most [10]. critical rate-limiting enzyme [10].

Figure 1. The slow ortho-hydroxylation of monophenol and the fast oxidation of catechol catalyzed FigureFigure 1. The 1. slow The orthoslow-hydroxylation ortho-hydroxylation of monophenol of monophenol and the and fast the oxidation fast oxidation of catechol of catechol catalyzed catalyzed by TYR. by TYR. by TYR.

Figure 2. Biosynthetic pathway of melanin [7,8,10]. DQ: dopaquinone; L-Dopa: Figure 2. BiosyntheticFigure 2. Biosyntheticpathway of pathway melanin of melanin[7,8,10]. [7DQ:,8,10 ].dopaquinone; DQ: dopaquinone; L-Dopa: L-Dopa: L-3,4- L-3,4-dihydroxyphenylalanine; DHICA: 5,6-dihydroxyindole-2 carboxylic acid; DHI: L-3,4-dihydroxyphenylalanine;dihydroxyphenylalanine; DHICA: DHICA: 5,6- 5,6-dihydroxyindole-2dihydroxyindole-2 carboxylic acid;acid; DHI: 5,6-dihydroxyindole;DHI: 5,6-dihydroxyindole; ICAQ: indole-2-carboxylic acid-5,6-quinone; IQ: indole-5,6-quinone; HBTA: 5,6-dihydroxyindole;ICAQ: ICAQ: indole-2-carboxylic indole-2-carboxylic acid-5,6-quinone; acid-5,6-quinone; IQ: IQ: indole-5,6-quinone; indole-5,6-quinone; HBTA: HBTA: 5-hydroxy-1,4- 5-hydroxy-1,4-benzothiazinylalanine. 5-hydroxy-1,4-benzothiazinylalanine.benzothiazinylalanine. Recent pharmacological investigations have revealed that the abnormal metabolism Recent pharmacologicalRecent pharmacological investigations investigations have revealed have that revealedthe abnormal that themetabolism abnormal metabolism of melanocytes and imbalance in TYR activity are indirectly or directly responsible for of melanocytesof and melanocytes imbalance and in imbalanceTYR activity in are TYR indirectly activity areor directly indirectly responsible or directly for responsible for various dermatoses [1,12,13]. For example, functional mutations in the gene encoding TYR (TYR, 11q14-21, MIM 606933) would inactivate TYR and cause a deﬁciency in melanin, thereby resulting in oculocutaneous albinism type 1 (OCA1, MIM 203100), an autosomal Biosensors 2021, 11, 290 3 of 22

recessive disorder characterized by the absence of pigment in hair, skin, and eyes [14]. On the contrary, excess melanin accumulation or abnormal distribution would give rise to hyperpigmentation disorders, including age spots, post-inflammatory hyperpigmentation, and even malignant melanoma [13,15]. In particular, the overexpression of TYR and TRP 1 is significantly associated with the risk of melanoma, a fatal skin carcinoma [16–18]. To this end, it has been viewed as a relatively specific biomarker and therapeutic target for melanoma lesions. Additionally, the abnormal level of TYR induces dopamine neurotoxicity and neurodegeneration, which is related to Parkinson’s disease (PD) [19–21]. Monitoring TYR activity in complex biosystems undoubtedly remains critical and challenging for biomedical research and drug high-throughput screening (HTS). In recent decades, various analytical techniques, including immunochemical analysis, mass spectrometry-based proteomics, and substrate-based biochemical assays, have flourished for the quantification of TYR. However, only the substrate-based biochemical assay could rapidly and sensitively determine the real activity of TYR in complex biological systems, leading to its common use in drug discovery and clinical studies [22]. Herein, we will review the role of biochemical detection based on optical substrates in TYR detection in the past 40 years. This review covers breakthroughs in the development of probe substrates and corresponding analytical methodologies for sensing TYR activity. By listing the substrate information and kinetic parameters of several optical methods for the first time, the advantages and defects of these various approaches are sufficiently compared and analyzed. Ultimately, the challenges and future perspectives in this field are highlighted. Collectively, this review provides a practical reference for developing new TYR substrates and methods, which is of great significance for related diseases studies and medical screening.

2. Biochemical Characteristics of TYR 2.1. Structural Feature and Catalytic Mechanism of TYR Human TYR is a glycoprotein (13% carbohydrate) predominantly located in the melanosome membrane of melanocytes [23,24]. The presence of the transmembrane do- main and glycans renders it difficult to isolate homogeneous TYR from melanocytes, which impedes crystallographic studies. Fortunately, the most characteristic TYR can be acquired from Streptomyces glausescens, the fungi Neurospore crassa, or Agaricus bisporus [7,25,26]. To compare the conservation of catalytic cavity, the crystal structures of TYR from bacteria (Streptomyces castaneoglobisporus [25] and Bacillus megaterium [27]), fungi Agaricus bisporus [28], and walnut leaves [29] were retrieved from Protein Date Bank. A salient feature of TYR from various sources (Figure3) is the presence of strictly conserved binuclear copper atoms at the active site, each of copper atoms is coordinated with three conserved histidines, respectively [3]. Moreover, the normal redox state of copper atoms is exceedingly significant for enzyme activity. Due to the relatively high similarity and homology with mammalian TYR, mushroom TYR from Agaricus bisporus acts as a model for enzyme kinetics and inhibitor screening [28,30,31]. Although extensive studies are devoted to TYR, its catalytic mechanism remains controversial. According to the presence/absence of oxygen and the oxidation state of copper ions [Cu (II)/Cu (I)], three enzymatic forms (Eoxy,Emet, and Edeoxy) participate in the catalytic cycle (Figure4)[ 3,32]. The resting form of TYR is found to be a mixture of 85% met and 15% oxy forms, while only the latter could act on the monophenol [7,33,34]. During the monophenolase cycle, to form Eoxy-monophenol complex (EoxyM), the oxygen atom on the deprotonated monophenol is coordinated with the coppers of Eoxy. Then, the phenol is o-hydroxylated to generate Emet-diphenol (D) complex (EmetD) [26,35]. Reducing agents could well draw the Emet into the Edeoxy, with concomitant oxidation to the corresponding o-quinone [36]. Since the deoxy form is the only one capable of reacting with oxygen to regenerate Eoxy and continue the catalytic action, monophenolase activity usually manifests as a characteristic lag time until a sufficient amount of catechol helps Emet to become Edeoxy [36]. Remarkably, this period depends on several factors, including enzyme concentration, monophenol concentration and the Biosensors 2021, 11, x FOR PEER REVIEW 4 of 22

Biosensors 2021, 11, 290 the deoxy form is the only one capable of reacting with oxygen to regenerate Eoxy4 of and 22 continue the catalytic action, monophenolase activity usually manifests as a characteristic lag time until a sufficient amount of catechol helps Emet to become Edeoxy [36]. Remarkably, this period depends on several factors, including enzyme concentration, monophenol presenceconcentration of reducing and agents,the presence especially of reducingo-diphenol agents, derivatives especially (such aso-diphenol L-DOPA) thatderivatives could shorten(such as and L-DOPA) even abolish that could the lag shorten time [ 30and,33 even,37]. Inabolish the diphenolase the lag time cycle, [30,33,37]. Eoxy continues In the di- to bind o-diphenol to originate the EoxyD complex, while both Eoxy and Emet are capable phenolase cycle, Eoxy continues to bind o-diphenol to originate the EoxyD complex, while of oxidizing the diphenol to the o-quinone. After this, Emet is regenerated to complete the both Eoxy and Emet are capable of oxidizing the diphenol to the o-quinone. After this, Emet is catalytic cycle continuously [30,34]. regenerated to complete the catalytic cycle continuously [30,34].

FigureFigure 3. 3.The The conserved conserved cavity cavity of TYRof TYR from from different different sources. sources. (a) The (a) crystalThe crystal of TYR of fromTYR Streptomycesfrom Strepto- myces castaneoglobisporus (PDB ID: 2ZMX). (b) The crystal of TYR from Bacillus megaterium (PDB ID: castaneoglobisporus (PDB ID: 2ZMX). (b) The crystal of TYR from Bacillus megaterium (PDB ID: 3NQ1). 3NQ1). (c) The Crystal of TYR from fungus (PDB ID: 2Y9W, Agaricus bisporus). (d) The Crystal of (c) The Crystal of TYR from fungus (PDB ID: 2Y9W, Agaricus bisporus). (d) The Crystal of TYR from TYR from plant (PDB ID: 5CE9, Juglans regia). Two copper ions (orange) are coordinated with three planthistidine (PDB residues, ID: 5CE9, respectively.Juglans regia ). Two copper ions (orange) are coordinated with three histidine residues, respectively.

2.2. Substrate Specificity of TYR Based on the broad substrate spectrum, in principle, any simple monophenol or corresponding catechol appears to be its substrate [38]. Besides, TYR also oxidizes various aromatic amines, o-aminophenols, and aromatic o-diamines (Figure5), despite the reaction rates being orders of magnitude smaller than the corresponding phenols or catechol [39,40]. In terms of phenols, mammalian TYR tends to be relatively specific for its physiological substrate (L-tyrosine and L-DOPA) and has a higher affinity for the L-isomers [41]. A prevalent characteristic in monophenol substrates is without substituents in the ortho-position of the phenolic hydroxyl group. Understandably, large side-chain substituents increase the difficulty of substrate interaction with the key catalytic residues; this is unpropitious for the recognition and catalytic process between the enzyme and ligand [36]. A kinetic study [42] Biosensors 2021, 11, 290 5 of 22

quantitatively discussed the effects of substituents in the 1-position of the aromatic ring on the rate of hydroxylation catalyzed by TYR. The results revealed that monophenols with a high electron donor tend to be oxidized faster [42]. In sharp contrast, the oxidation Biosensors 2021, 11, x FOR PEER REVIEW rate of catechol is positively correlated with the electron-withdrawing capacity of5 theof 22para -

substituents [36]. As such, the steric hindrance, stereochemical characteristics, and electronic effects of substituents have a distinct influence on the rate of TYR-mediated catalysis.

Biosensors 2021, 11, x FOR PEER REVIEW 6 of 22

Figure 4. Catalytic cycle of TYR. Figure 4. Catalytic cycle of TYR.

2.2. Substrate Specificity of TYR Based on the broad substrate spectrum, in principle, any simple monophenol or corresponding catechol appears to be its substrate [38]. Besides, TYR also oxidizes various aromatic amines, o-aminophenols, and aromatic o-diamines (Figure 5), despite the reaction rates being orders of magnitude smaller than the corresponding phenols or catechol [39,40]. In terms of phenols, mammalian TYR tends to be relatively specific for its physiological substrate (L-tyrosine and L-DOPA) and has a higher affinity for the L-isomers [41]. A prevalent characteristic in monophenol substrates is without substituents in the ortho-position of the phenolic hydroxyl group. Understandably, large side-chain substituents increase the difficulty of substrate interaction with the key cata- Figurelytic 5. Figure Theresidues; catalytic 5. The reactionthis catalytic is ofunpropitious reaction TYR-mediated of TYR- aromaticformediated the aminerecognition aromatic and o-aminophenol. amine and and catalytic o-aminophenol Adapted process with [43]. permission between from the ref. [enzyme43]. 1987, American and ligand Chemical [36]. Society. A kinetic study [42] quantitatively discussed the effects of sub- According to the chemical stability of the corresponding o-benzoquinone, phenolic stituents in the 1-position of the aromatic ring on the rate of hydroxylation catalyzed by substrates (Table 1) can be roughly divided into the following three categories [42,44]. (1) TYR. The results revealed that monophenols with a high electron donor tend to be oxi- The first sort of substrates catalyzed by TYR could yield stable o-quinones. For example, dized4- tertfaster-butylcatechol [42]. In sharp (TBC) contrast, is detectable the oxidation for the diphenolase rate of catechol activity, is whose positively o-quinone correlated is withexceedingly the electron-withdrawing stable. (2) The second capacity substrates of the producepara-substituents a highly unstable [36]. As o -quinonesuch, the but steric hindrance,evolve intostereochemical a stable product char viaacteristics, a first-order and reaction. electronic Targeted effects at ofdiphenolase substituents activity, have a distinctthe common influence determination on the rate ofis basedTYR-mediated on the formation catalysis. of dopachrome using L-DOPA as a substrate. Moreover, there is 3,4-dihydroxymandelic acid (DOMA) [45], dopamine (DA) [46], and isoproterenol (ISO) [47]. (3) The third kind of substrates are oxidized to an unstable o-quinone that is vulnerable to potent nucleophiles (N) and yields chromatic adducts (NQ) with a clear stoichiometry. Commonly used nucleophiles include L-proline (Pro) [48], L-cysteine (Cys) [49], and especially 3-methyl-2-benzothiazolinone hydrazone (MBTH) [42]. Related substrates mainly include 4-hydroxyphenylacetic acid, 4-hydroxyphenylpropionic acid, L-DOPA, DA, etc.

Table 1. Properties of the detectable species from several substrates.

Metabolite Coupled Detectable λ ε Substrates Chemical Structures Ref. Stability Reagent Species (nm) (M−1·cm−1)

4-Tert-butylcatechol (TBC) Stable - o-Quinone 400 1200 [44]

L-Tyrosine Unstable - Dopachrome 475 3600 [45]

- Dopachrome 475 3600 [45] L-DOPA Unstable MBTH MBTH-adduct 484 22,300 [42]

Dopamine (DA) Unstable MBTH MBTH-adduct 503 42,500 [42]

Isoproterenol (ISO) Unstable MBTH MBTH-adduct 497 31,500 [42]

3. Optical Assays for Sensing TYR Activity The development of analytical techniques mainly focuses on improving and monitoring bioactive species, especially real-time analysis in vivo [50–53]. The current optical methods, including spectrophotometry and fluorometric detection, exhibit distinct performance. In this paper, we review the research advances of various methods by empha- Biosensors 20212021,, 1111,, xx FORFOR PEERPEER REVIEWREVIEW 66 ofof 2222

BiosensorsBiosensors 2021 2021, ,11 11, ,x x FOR FOR PEER PEER REVIEW REVIEW 66 of of 22 22

Figure 5. The catalytic reaction of TYR-mediated aromatic amine and oo-aminophenol-aminophenol [43].[43]. Biosensors 2021, 11, 290 6 of 22 FigureFigureAccording 5. 5. The The catalytic catalytic to the reaction reaction chemical of of TYR- TYR- stabilitymediatedmediated of thearomatic aromatic corresponding amine amine and and o o-aminophenol-benzoquinone,-aminophenol-benzoquinone, [43]. [43]. phenolic phenolic substrates (Table 1) can be roughly divided intoto thethe followingfollowing threethree categoriescategories [42,44].[42,44]. (1)(1) The firstAccording sort of substratesto the chemical catalyzedlyzed stability byby TYRTYR of the couldcould corresponding yieldyield stablestable oo-benzoquinone,-quinones.-quinones. ForFor example, example,phenolic AccordingAccording to the chemical to the chemical stability stabilityof the corresponding of the corresponding o-benzoquinone,o-benzoquinone, phenolic phenolic 4-substratesterttert-butylcatechol-butylcatechol (Table 1) (TBC)(TBC)can be is isroughly detectabledetectable divided forfor the theinto diphenolasediphenolase the following activity,activity, three categories whosewhose o-quinone-quinone [42,44]. (1)isis substratessubstratessubstrates (Table (Table 1) 1) (Tablecan can be be1 )roughly roughly can be divided roughlydivided in in dividedtoto the the following following into the followingthree three categories categories three [42,44]. categories[42,44]. (1) (1) [ 42,44]. exceedinglyThe first sort stable. of substrates (2) The cata secondlyzed su bybstrates TYR could produce yield a stablehighly o -quinones.unstable o -quinoneFor example, but TheexceedinglyThe first first(1) sort sort The stable.of of ﬁrstsubstrates substrates sort(2) The of cata substrates catasecondlyzedlyzed su by catalyzedbybstrates TYR TYR could could produce by TYR yield yield could a stable stablehighly yield o o-quinones. -quinones.unstable stable o-quinones. o For -quinoneFor example, example, Forbut example, evolve4-tert-butylcatechol into a stable (TBC)product is viadetectable a first-order for the reaction. diphenolase Targeted activity, at diphenolase whose o-quinone activity, is 4-evolve4-terttert-butylcatechol-butylcatechol into4-tert a -butylcatecholstable (TBC)(TBC)product isis (TBC) detectableviadetectable a first-order is detectable forfor thethe reaction. diphenolasediphenolase for the Targeted diphenolase activity,activity, at diphenolase activity, whosewhose oo-quinone whose-quinone activity,o-quinone isis is theexceedingly common stable.determination (2) The issecond based suonbstrates the formation produce of adopachrome highly unstable using o L-DOPA-quinone asbut a exceedinglytheexceedingly commonexceedingly stable.determinationstable. (2)(2) stable. TheThe issecondsecond (2) based The su suon secondbstratesbstrates the formation substrates produceproduce of produce adopachromea highlyhighly a unstableunstable highly using unstable o o-quinoneL-DOPA-quinoneo-quinone as butbut a but substrate.evolve into Moreover, a stable productthere is 3,4-dihydroxymandelicvia a first-order reaction. acid Targeted (DOMA) at [45],diphenolase dopamine activity, (DA) evolvesubstrate.evolve intointoevolve Moreover, aa stablestable into a product productthere stable is product via3,4-dihydroxymandelicvia aa first-orderfirst-order via a ﬁrst-order reaction.reaction. reaction. acid TargetedTargeted (DOMA) Targeted atat diphenolase[45],diphenolase at diphenolasedopamine activity,activity, (DA) activity, the [46],the common and isoproterenol determination (ISO) is [47]. based (3) on The the third formation kind of of substrates dopachrome are usingoxidized L-DOPA to an asun- a the[46],the common common andcommon isoproterenol determination determination determination (ISO) is is [47].based based is based(3) on on The the the on third formation formation the formationkind ofof of substratesdopachrome dopachrome of dopachrome are using usingoxidized using L-DOPA L-DOPA L-DOPAto an as un-as a a as a sub- stablesubstrate. o-quinone Moreover, that thereis vulnerable is 3,4-dihydroxymandelic to potent nucleophiles acid (DOMA)(N) and yields[45], dopamine chromatic (DA) ad- substrate.stablesubstrate. o-quinonestrate. Moreover, Moreover, Moreover, that there thereis vulnerable there is is 3,4-dihydroxymandelic 3,4-dihydroxymandelic is 3,4-dihydroxymandelic to potent nucleophiles acid acid acid(DOMA) (DOMA)(N) (DOMA) and [45], yields[45], [ 45dopamine dopamine ],chromatic dopamine (DA) (DA) ad- (DA) [46], [46], and isoproterenol (ISO) [47]. (3) The third kind of substrates are oxidized to an un- [46],ducts[46], and and(NQ)and isoproterenol isoproterenol with isoproterenol a clear (ISO) (ISO) stoichiometry. (ISO) [47]. [47]. [47 (3) (3)]. The (3)The Commonly The third third third kind kind kind usedof of substrates ofsubstrates nucleophiles substrates are are areoxidized oxidizedinclude oxidized to L-prolineto an toan un- anun- unstable stable(Pro)(Pro)stable [48],[48], oo-quinone-quinoneo-quinone L-cysteineL-cysteine thatthat that (Cys)(Cys) isis is vulnerablevulnerable vulnerable [49],[49], andand to to especiallyespecially potent potentpotent nucleophilesnucleophiles 3-methyl-2-benzothiazolinone3-methyl-2-benzothiazolinone (N) (N)(N) and andand yields yieldsyields chromatic chromaticchromatic hydrazonehydrazone adducts ad-ad- (NQ) ducts(MBTH)(MBTH)ducts (NQ)(NQ)with [42].[42]. withwith a clear RelatedaRelateda clearclear stoichiometry. stoichiometry. stoichiometry.substratessubstrates Commonly mainlymainly CommonlyCommonly include usedinclude usedused nucleophiles nucleophilesnucleophiles4-hydroxyphenylacetic4-hydroxyphenylacetic include includeinclude L-proline L-prolineL-proline acid,acid, (Pro) [48], (Pro)4-hydroxyphenylpropionic(Pro) [48], [48],L-cysteine L-cysteine L-cysteine (Cys) (Cys) (Cys) [49 [49], ],acid,[49], and and andL-DOPA, especially especially especially DA, 3-methyl-2-benzothiazolinone 3-methyl-2-benzothiazolinone etc.3-methyl-2-benzothiazolinone hydrazone hydrazone hydrazone (MBTH) [42]. (MBTH)(MBTH) Related [42].[42]. substratesRelatedRelated mainlysubstratessubstrates include mainlymainly 4-hydroxyphenylacetic includeinclude 4-hydroxyphenylacetic4-hydroxyphenylacetic acid, 4-hydroxyphenylpropionic acid,acid, Table 1. Properties of the detectable species from several substrates. Table4-hydroxyphenylpropionic4-hydroxyphenylpropionic 1. Propertiesacid, L-DOPA, of the detectable DA, acid, etc.acid, species L-DOPA, L-DOPA, from DA, DA,several etc. etc. substrates. Metabolite Coupled Detectable λ εε Substrates ChemicalTable 1. Structures TableProperties 1. Properties of the detectable of the detectable species from species several from substrates. several substrates. −1 −1 Ref. Table 1. Properties of theStability detectable speciesReagent from several substrates.Species (nm)(nm) (M(M−1·cm·cm−1)) Metabolite Coupled Detectable λ ε Substrates Chemical Structures MetaboliteMetabolite CoupledCoupledCoupled DetectableDetectableDetectable λ λλ εε ε Ref. SubstratesSubstratesSubstrates ChemicalChemical Chemical Structures Structures −1− −1− Ref.Ref. Ref. 4-Tert-butylcatechol (TBC) StabilityStabilityStabilityStable ReagentReagentReagent- Speciesoo-QuinoneSpecies-QuinoneSpecies (nm)(nm)(nm)400400 (M(M(M−120012001−·cm1·cm1·cm −1−)1 ) 1)[44][44]

4-Tert- 4-Tert-butylcatechol4-Tert-butylcatecholbutylcatechol (TBC) (TBC) StableStableStable -- - o-Quinoneoo-Quinone-Quinone 400400400 12001200 1200 [44][44] [44] (TBC) L-Tyrosine Unstable - Dopachrome 475 3600 [45]

L-TyrosineL-TyrosineL-Tyrosine UnstableUnstableUnstable ---- DopachromeDopachromeDopachromeDopachrome 475475475475 360036003600 3600 [45][45][45] [45]

L-DOPA Unstable MBTH-- DopachromeMBTH-adductDopachrome 475484475 22,3003600 3600 [42][45] [45] L-DOPA - - DopachromeDopachrome 475475 36003600 [45][45] Unstable MBTH MBTH-adduct 484 22,300 [42] L-DOPA Unstable L-DOPA Unstable MBTH MBTH-adduct 484 22,300 [42] Dopamine (DA) Unstable MBTHMBTH MBTH-adductMBTH-adduct 484503 22,30042,500 [42][42] DopamineDopamine (DA) (DA) UnstableUnstable MBTHMBTH MBTH-adductMBTH-adduct 503503 42,500 42,500 [42] [42]

DopamineDopamine (DA) (DA) UnstableUnstable MBTHMBTH MBTH-adductMBTH-adduct 503503 42,50042,500 [42][42] IsoproterenolIsoproterenolIsoproterenol (ISO)(ISO) (ISO) UnstableUnstable MBTHMBTH MBTH-adductMBTH-adduct 497497 31,500 31,500 [42] [42]

IsoproterenolIsoproterenol (ISO) (ISO) UnstableUnstable MBTHMBTH MBTH-adductMBTH-adduct 497497 31,50031,500 [42][42] 3. Optical Assays for Sensing TYR Activity 3. Optical Assays for Sensing TYR Activity The development of analytical techniques mainly focuses on improving and moni- The development of analytical techniques mainly focuses on improving and moni- 3.toring3. Optical Optical bioactive Assays Assays species, for for Sensing Sensing especially TYR TYR real-timeActivity Activity analysis in vivo [50–53]. The current optical toring bioactivetoring bioactive species, species,especially especially real-time real-time analysis analysisin vivo [50–53].in vivo [The50– 53current]. The optical current optical methods,The developmentincluding spectrophotometry of analytical techniques and fluo mainlyrometric focuses detection, on improving exhibit distinct and moni- per- methods,TheThemethods, developmentdevelopmentincluding including spectrophotometry ofof analyticalanalytical spectrophotometry techniquestechniques and fluo andmainlymainlyrometric fluorometric focusesfocuses detection, onon detection, improvingimproving exhibit exhibit distinct andand distinctmoni-moni- per- perfor- formance.toring bioactive In this species, paper, weespecially review real-timethe research analysis advances in vivo of various [50–53]. methods The current by empha- optical toringformance.toring bioactive bioactivemance. In this Inspecies, species, paper, this paper, weespecially especially review we review real-timethereal-time research the research analysis analysis advances advances in in vivo vivo of various[50–53]. [50–53]. of various methods The The methods current current by empha-byoptical optical emphasizing methods, including spectrophotometry and fluorometric detection, exhibit distinct per- methods,methods,on includingincluding both their spectrophotometryspectrophotometry pros and cons, and andand we fluo alsofluorometricrometric summarize detection,detection, the non-fluorescent exhibitexhibit distinctdistinct substrates per-per- and formance. In this paper, we review the research advances of various methods by empha- formance.formance.fluorescent In In this this paper, paper, substrates. we we review review the the research research advances advances of of various various methods methods by by empha- empha-

sizing on both their pros and cons, and we also summarize the non-fluorescent substrates and fluorescent substrates.

3.1. Spectrophotometric Assays Due to its intrinsic sensitivity, low cost, and continuous study of the reaction process, the spectrophotometric technique has become the most widely used method [44,54]. Hitherto, in vitro assays of the oxidase activity of TYR are predominantly comprised of the dopachrome formation methods that use L-tyrosine or L-DOPA as substrate [55–58]. However, this mainstream approach also has inherentinherent flaws.flaws. ItIt reliesrelies onon thethe hypothesishypothesis that DQ (an oxidation product of L-DOPA) is completely converted to dopachrome, instead of directly measuring DQ [57]. The instability and relatively low absorption coefficient of dopachrome in an aqueous system mean that the test must be performed quickly and at a low sensitivity. Considering the high reactivity and instability of intermediates Biosensors 2021, 11, 290 and the interference of external factors (such as temperature and oxygen), many other 7 of 22 assays are subsequently improved. Many nucleophiles start appearing on the stage by capturing highly reactive DQ as stable-colored products [59]. Under acidic conditions, MBTH generate a pink adduct with high molar absorptivity and solubility, whose clear MBTHhighly generate reactive a pink DQ adduct as stable-colored with high mo productslar absorptivity [59]. Under and acidic solubility, conditions, whose MBTH clear gener- stoichiometry and high stability endow great measurability [42,44,60]. stoichiometryate a pink and adduct high stability with high endow molar great absorptivity measurability and solubility, [42,44,60]. whose clear stoichiometry Distinct from the above, the quinonization product of 4-tert-butylcatechol (TBC) is Distinctand high from stability the above, endow the great quinonization measurability product [42,44 ,of60 ].4-tert-butylcatechol (TBC) is remarkably stable, simple to accumulate in the reaction mixture, and facile to detect. remarkablyDistinct stable, fromsimple the to above, accumulate the quinonization in the reaction product mixture, of 4-tert-butylcatechol and facile to detect. (TBC) is Lamentably, its affinity (Km = 990 μM) toward TYR is exceedingly poor, which restricts its Lamentably,remarkably its affinity stable, (K simplem = 990 μ toM) accumulate toward TYR in is the exceedingly reaction mixture,poor, which and restricts facile to its detect. application [37]. More typical substrates and their optical parameters are documented in applicationLamentably, [37]. More its afﬁnity typical ( Ksubstratesm = 990 µ M)and toward their optical TYR is parameters exceedingly are poor, documented which restricts in its Table 2. Table application2. [37]. More typical substrates and their optical parameters are documented in Table2. Table 2. Non-fluorescent substrates of TYR. Table 2. Non-ﬂuorescent substrates of TYR. Vmax Vmax Enzyme Km Substrates Chemical Structures Enzyme Sources Metabolites Enzyme Km (nmol/min Ref. Substrates Chemical Structures EnzymeEnzymeEnzyme SourcesSources Metabolites Metabolites Enzyme K (nmol/min(nmol/minV Ref. Substrates Chemical Structures Metabolites Activity m((μM) max Ref. Sources Activity (µM) (nmol/min/mg)/mg)/mg)

Monophenolase MonophenolaseMonophenolase L-TyrosineL-Tyrosine MushroomMushroom L-DOPAL-DOPA 270270 - -[41] [41] activityactivity

DiphenolaseDiphenolase L-DOPAL-DOPA MushroomMushroom DopachromeDopachrome 800800 - -[41] [41] activityactivity

3- Cinnabarinic DiphenolaseDiphenolase 3-HydroxyanthranilicHydroxyanthranilic acid MushroomMushroom Cinnabarinic acid Diphenolase 780780 12 12 [61] [61] 3-Hydroxyanthranilic acid Mushroom Cinnabarinicacid acid activity 780 12 [61] acid activity

4-Tert-butyl- 4-Tertbutyl- 4-Tert-butyl-1,2-bDiphenolaseDiphenolase 4-Tertbutyl-catechol Mushroom 4-Tert-butyl-1,2-b1,2- Diphenolase 990990 - -[37] [37] 4-Tertbutyl-catecholcatechol Mushroom activity 990 - [37] benzoquinoneenzoquinone activity

In recentIn recent years, years,some breakthroughs some breakthroughs have b haveeen made been madein substrates in substrates identificationidentification identification andand and detectiondetection means. means. Several Several sensing sensing platforms platforms utilized utilized the reducibility the reducibility of catechol of catechol to capture to capture + some chromogenicsome chromogenic reagents reagents for quantitative for quantitative analysis analysis [62,63]. [For62,63 instance,]. For instance, Ag+ couldcould Ag ox-ox-+ could idizeidize 3,33,3oxidize′′,5,5,5,5′′-tetramethylbenzidine 3,30,5,50-tetramethylbenzidine (TMB) to (TMB) the oxidized to the oxidized 3,3′′,5,5,5,5 3,3′′-tetramethylbenzidine0,5,50-tetramethylbenzidine (oxTMB),(oxTMB), accompanied accompanied by remarkable by remarkable changes changes inin colorcolor in color andand andabsorbanceabsorbance absorbance [64,65].[64,65]. [64, 65TheThe]. The in-in- intro- troductionduction of reducing of reducing substances substances (such (such as as DA) DA) directly directly decreases decreases oxTMB, oxTMB, resulting inin faded faded blueblue andand aadecrease decrease in in absorbance absorbance (Figure (Figureigure6 ). 6).6). Accordingly, Accordingly,Accordingly, using usingusing TMB TMBTMB as asas achromogenic aa chro-chro- mogenicprobe, probe, a facile a facile colorimetric colorimetric assay assay was wa proposeds proposed to to sense sense TYR TYR activity activity in in human human serum serumsamples samples and and to to screenscreen inhibitorsinhibitors [[66].66]. In a similar vein, Deng Deng et et al. al. demonstrated demonstrated that that catecholcatechol could could suppress suppress the the activity activity of of oxidase-mimickingoxidase-mimicking oxidase-mimicking chitosan-stabilizedchitosan-stabilized chitosan-stabilized plati-plati- platinum num nanoparticlesnanoparticles (ChPtNPs), (ChPtNPs), thereby thereby significantly significantly decreasing decreasing acidified acidified TMB TMB products products [67]. With the oxidation of catechol, a linear relationship between the amount of restored color and the TYR activity was evaluated. In terms of detection means, inspired by specific chromogenic and fluorogenic reactions between resorcinol and catecholamines [68], an absorbance–fluorescence dual-readout assay was established. With tyrosine as a substrate, this assay achieved the determination of TYR in serum samples and inhibitor screening [69]. Notably, these innovative assays are consistent with the L-DOPA oxidation-based method. As a classical analytical method, the spectrophotometry assay still exhibits promising prospects for in situ quantitative analysis. Furthermore, when establishing an experimental methodology, the enzyme activity in the presence of substrates, absorption coefficient or stability of products, anti-interference and sensitivity, incubation time, and even reagent consumption, ought to be taken into account [54,67]. Biosensors 2021, 11, x FOR PEER REVIEW 8 of 22

[67]. With the oxidation of catechol, a linear relationship between the amount of restored color and the TYR activity was evaluated. In terms of detection means, inspired by specific chromogenic and fluorogenic reactions between resorcinol and catecholamines [68], an absorbance–fluorescence dual-readout assay was established. With tyrosine as a sub- Biosensors 2021, 11, 290 strate, this assay achieved the determination of TYR in serum samples and inhibitor8 of 22 screening [69]. Notably, these innovative assays are consistent with the L-DOPA oxidation-based method.

Figure 6. Detection system of of Ag Ag++-TMB-TMB with with dopamine dopamine as as substrate substrate.. (a (a) )Mechanism Mechanism of of the the colori- colori- + metric assay based on Ag +-TMB-TMB system system for for TYR TYR activity. activity. (b (b) )The The metabolic metabolic reaction reaction of of dopamine dopamine catalyzed byby TYR.TYR.

3.2. FluorometricAs a classical Assays analytical method, the spectrophotometry assay still exhibits promising prospectsDespite spectrophotometry for in situ quantitative being commonlyanalysis. Furthermore, used for the detectionwhen establishing of TYR activities an ex- inperimental vitro, this methodology, method is insufficient the enzyme when activity it comes in the to presence high-throughput of substrates, screening absorption (HTS) orcoefficient dynamic or tracking stability [ 70of]. products, Owing to anti-interference their superior sensitivity,and sensitivity, ultrahigh incubation spatiotemporal time, and resolution,even reagent and consumption, without isolation ought to and be derivative,taken into account the fluorescent [54,67]. substrate-based techniques have shown unprecedented developments in real-time visualizing and detecting biomolecules3.2. Fluorometricin vitroAssaysor in vivo [71–73]. To date, a great number of TYR-activated fluorescent substratesDespite spectrophotometry have flourished, being primarily commonly including used organic for the fluorescentdetection of molecules TYR activities and nanometerin vitro, this material-based method is insufficient probes [ 74when]. it comes to high-throughput screening (HTS) or dynamic tracking [70]. Owing to their superior sensitivity, ultrahigh spatiotemporal res- 3.2.1.olution, Small and Molecule-Based without isolation Fluorescent and derivative Substrates, the fluorescent substrate-based techniques haveThe shown current unprecedented probes are mainly developments designed by in the real-time specific phenolicvisualizing substrates and detecting (recognition bio- moiety)molecules in in conjugation vitro or in with vivo fluorophore [71–73]. To scaffoldsdate, a great through number an appropriateof TYR-activated linker fluores- [75,76]. Givencent substrates the metabolic have characteristicsflourished, primarily of TYR, including the recognized organic fragment fluorescent should molecules contain and a phenolicnanometer hydroxyl, material-based without probes substituents [74]. in the ortho position; this facilitates the formation of catechol [77]. To the best of our knowledge, 4-hydroxyphenyl and 3-hydroxyphenyl are the3.2.1. two Small main Molecule-Based types of responsive Fluorescent unit (Figure Substrates7a). However, 4-hydroxyphenyl, as a classical responsive moiety for TYR, could react with both reactive oxygen species (ROS) and TYR The current probes are mainly designed by the specific phenolic substrates (recog- in most cases [70]. Owing to H O , HOCI and some free radicals are usually at a relatively nition moiety) in conjugation 2with2 fluorophore scaffolds through an appropriate linker high concentration (about µM levels) especially in tumor cells; this cross-interference may [75,76]. Given the metabolic characteristics of TYR, the recognized fragment should con- result in false-positive signals and inaccurate results [78,79]. Rejoicingly, the replacement of tain a phenolic hydroxyl, without substituents in the ortho position; this facilitates the 4-hydroxyphenyl with 3-hydroxyphenyl not only preserves binding affinity towards TYR formation of catechol [77]. To the best of our knowledge, 4-hydroxyphenyl and but also avoids the influence of cross-interference from ROS. Mainstream designs primarily 3-hydroxyphenyl are the two main types of responsive unit (Figure 7a). However, include the oxidization-cleavage mechanism and the inhibited photo-induced electron 4-hydroxyphenyl, as a classical responsive moiety for TYR, could react with both reactive transfer (PET) process (Figure7b) [ 80]. The former is oxidized to an unstable o-quinone oxygen species (ROS) and TYR in most cases [70]. Owing to H2O2, HOCI and some free in presence of oxygen and TYR, which undergoes an intramolecular 1,6-rearrangement- elimination,radicals are furtherusually releasingat a relatively free fluorophore high concentration and triggering (about a fluorescenceμM levels) especially response. Inin thetumor latter, cells; the this initial cross-interference hydroxyphenyl group may exertsresult ain PET false-positive effect on the signals parent. and Accompanied inaccurate byresults the formation[78,79]. Rejoicingly, of o-quinone the and replacemen the blockedt of PET 4-hydroxyphenyl effect, the probe with is lit 3-hydroxyphenyl up. By rationally adjusting the TYR-recognition unit and fluorophore structure, a variety of probes could be acquired. The newly developed TYR fluorescent substrates and their biological parameters are presented in Table3. Biosensors 2021, 11, x FOR PEER REVIEW 9 of 22

Biosensors 2021, 11, x FOR PEER REVIEWnot only preserves binding affinity towards TYR but also avoids the influence9 of 22 of

cross-interference from ROS. Mainstream designs primarily include the oxidization-cleavage mechanism and the inhibited photo-induced electron transfer (PET) pro- cessnot (Figureonly preserves 7b) [80]. binding The former affinity is oxidizedtowards toTYR an butunstable also avoids o-quinone the influencein presence of of ox- ygencross-interference and TYR, which from undergoes ROS. Mainstream an intramolecular designs primarily 1,6-rearrangement-elimination, include the oxidiza- fur- thertion-cleavage releasing mechanismfree fluorophore and the and inhibited triggering photo-induced a fluorescence electron response. transfer (PET)In the pro- latter, the initialcess (Figurehydroxyphenyl 7b) [80]. The group former exerts is oxidized a PET toeffect an unstable on the parent.o-quinone Accompanied in presence of by ox- the for- mationygen and of oTYR,-quinone which and undergoes the blocked an intramolecular PET effect, the1,6-rearrangement-elimination, probe is lit up. By rationally fur- adjust- ther releasing free fluorophore and triggering a fluorescence response. In the latter, the ing the TYR-recognition unit and fluorophore structure, a variety of probes could be ac- initial hydroxyphenyl group exerts a PET effect on the parent. Accompanied by the for- quired. The newly developed TYR fluorescent substrates and their biological parameters quired.mation The of o -quinonenewly developed and the blocked TYR fluorescentPET effect, the su bstratesprobe is litand up. their By rationally biological adjust- parameters Biosensors 2021, 11, 290 areing presented the TYR-recognition in Table 3. unit and fluorophore structure, a variety of probes could be ac- 9 of 22 quired. The newly developed TYR fluorescent substrates and their biological parameters are presented in Table 3.

FigureFigureFigure 7. 7. Several SeveralSeveral strategies strategies reported reported reported for for for designin designin designingg theg the fluorescent ﬂuorescentfluorescent substrates substratessubstrates of TYR. ofof ( TYR. aTYR.) Some ( a()a typ-) Some Some typical typical warheads of fluorescent probe for TYR detection. (b) Two sensing mechanisms of OFF-ON icalwarheads warheads of ﬂuorescentof fluorescent probe probe for TYRfor TYR detection. detection. (b) Two (b) sensingTwo sensing mechanisms mechanisms of OFF-ON of OFF-ON probes probes towards TYR. probestowards towards TYR. TYR. Table 3. Fluorescent substrates of TYR. TableTable 3. 3. FluorescentFluorescent substrates substrates of of TYR. TYR. λex/em Km Vmax LOD Biological Names Chemical Structures Folds −1 −1 Ref. λλ(nm)ex/em (μM)Km K(μM·minVmax) V (U·mLLOD) LOD ApplicationsBiologicalBiological NamesNames Chemical Chemical Structures ex/em FoldsFolds m max Ref.Ref. (nm) (μM)(µ M)(μM·min(µM·min−1) −1)(U·mL(U·−mL1) −1) ApplicationsApplications

425/467 L3 - 43.5 1.87 0.2 A375 cells [81] 425/535 425/467 L3L3 - -43.5 43.5 1.87 1.87 0.2 0.2 A375 A375 cells [81 [81]] 425/535

B16 and HeLa cells, Probe 1 670/708 - 156 4.58 2.76 [82] zebrafish BiosensorsBiosensors 2021 2021, 11, 11, x, xFOR FOR PEER PEER REVIEW REVIEW B16 andB16 and HeLa HeLa cells, 1010 of of 22 22 ProbeProbe 1 1 670/708 - - 156 156 4.58 4.58 2.76 2.76 [82[82]] cells,zebrafish zebraﬁsh

B16, HepG2, B16,B16, A549,HepG2, HepG2, HeLa, A549, A549, HeLa,HeLa,CCC-HPF-1 CCC-HPF-1 CCC-HPF-1 HB-NPHB-NPHB-NP 480/675480/675 4848 48 87.35 87.35 87.35 1.07 1.07 1.07 0.5 0.5 0.5 and [80[80][80]] andand CCC-HSF-1 CCC-HSF-1CCC-HSF-1 cells; cells; inhibitorinhibitorcells; inhibitor screening. screening. screening.

Imaging of ImagingImagingTYR inof of B16 TYR TYR in in B16B16cells cells cells and and and ProbeProbeProbe 1 1 1 670/708670/708 ------0.11 0.11 0.11 zebraﬁsh, [83[83][83]] zebrafish,zebrafish,melanoma melanoma melanoma diagnosisdiagnosis in ina amouse. diagnosismouse. in a mouse.

B16,B16, HepG2 HepG2 and and ProbeProbe 1 1 550/586550/586 - - 30 30 0.72 0.72 0.04 0.04 [84][84] MCF-7MCF-7 cells cells

ProbeProbe 1 1 550/583550/583 - - 6.5 6.5 0.0009 0.0009 0.5 0.5 B16 B16 and and HepG2 HepG2 cells cells [85] [85]

NHUNHU 382/503382/503 12 12 ------B16-F1 B16-F1 and and HeLa HeLa cells cells [86] [86]

ProbeProbe 1 1 460/540460/540 1212 ------Screening Screening inhibitors inhibitors [87][87]

ImagingImaging of of TYR TYR in in B16F10B16F10 and and HeLa HeLa NBR-APNBR-AP 580/660580/660 7 7 229.5 229.5 5.75 5.75 - - [88][88] cells,cells, in in vivo vivo imaging imaging ofof zebrafish zebrafish and and mice mice

B16B16 and and HeLa HeLa cells, cells, Mela-TYRMela-TYR 425/547425/547 5050 - - - - 0.07 0.07 thethe subcellular subcellular local- local- [89][89] izationization

HO N N HO 630/760630/760 B16,B16, HeLa, HeLa, MCF-7 MCF-7 Cy-tyrCy-tyr NHNH ------0.02 0.02 [90][90] 516/556516/556 andand HUVEC HUVEC cells cells

N N I- I-

ProbeProbe 1 1 650/720650/720 ------0.01 0.01 ScreeningScreening inhibitors inhibitors [91][91]

BiosensorsBiosensorsBiosensorsBiosensors 20212021 2021 2021,,, ,1111 11, 11,,, ,xxx x, FORFORFOR xFOR FOR PEERPEERPEER PEER PEER REVIEWREVIEWREVIEW REVIEW REVIEW 10101010 ofof of of 2222 22 22 Biosensors 20212021,, 1111,, xx FORFOR PEERPEER REVIEWREVIEW 1010 ofof 2222

B16,B16, HepG2, HepG2, A549, A549, B16,B16, HepG2, HepG2, A549, A549, HeLa,HeLa,HeLa,HeLa, CCC-HPF-1CCC-HPF-1 CCC-HPF-1 CCC-HPF-1 HB-NPHB-NPHB-NP 480/675480/675480/675 484848 87.35 87.35 87.35 1.07 1.07 1.07 0.5 0.5 0.5 HeLa, CCC-HPF-1 [80][80][80] HB-NP 480/675480/675 4848 87.35 87.35 1.07 1.07 0.5 0.5 andand CCC-HSF-1 CCC-HSF-1 cells; cells; [80][80] andandand CCC-HSF-1CCC-HSF-1 CCC-HSF-1 cells;cells; cells; inhibitorinhibitor screening. screening. inhibitorinhibitorinhibitor screening.screening. screening.

Biosensors 2021, 11, 290 10 of 22 ImagingImaging of of TYR TYR in in ImagingImagingImaging ofof of TYRTYR TYR inin in B16B16B16B16 cellscells cells cells andand and and ProbeProbeProbe 1 1 1 670/708670/708670/708 ------0.11 0.11 0.11 B16 cells and [83][83][83] Probe 1 670/708670/708 ------0.11 0.11 zebrafish,zebrafish, melanoma melanoma [83][83] Table 3. Cont. zebrafish,zebrafish,zebrafish, melanomamelanoma melanoma diagnosisdiagnosis in in a a mouse. mouse. diagnosisdiagnosis in in a mouse.a mouse. λex/em Km Vmax LOD Biological Names Chemical Structures Folds −1 −1 Ref. (nm) (µM) (µM·min ) (U·mL ) Applications

B16,B16,B16,B16,B16, HepG2HepG2 HepG2 HepG2 HepG2 andand and and ProbeProbeProbeProbe 11 1 1 550/586550/586550/586550/586 -- - - 30 30 30 30 0.72 0.72 0.72 0.72 0.04 0.04 0.04 0.04 B16, HepG2 and [84][84][84][84] ProbeProbe 1 1 550/586 -- - 30 30 30 0.72 0.72 0.72 0.04 0.04 0.04 MCF-7MCF-7MCF-7andMCF-7 MCF-7 cellscells cells cells [84[84][84]] MCF-7cells cells

Probe 1 550/583 - 6.5 0.0009 0.5 B16 andB16 HepG2 and cells [85] ProbeProbeProbeProbe 11 1 1 550/583550/583550/583 - - - - 6.5 6.5 6.5 6.5 0.0009 0.0009 0.0009 0.0009 0.5 0.5 0.5 0.5 B16 B16 B16 andand and HepG2HepG2 HepG2 cellscells cells [85 [85] [85]] [85] Probe 1 550/583550/583 - - 6.5 6.5 0.0009 0.0009 0.5 0.5 B16 B16 andandHepG2 HepG2HepG2 cells cellscells [85] [85]

B16-F1 and NHUNHUNHUNHU 382/503382/503382/503382/503 12 12 12 12 ------B16-F1 B16-F1 B16-F1 and and and HeLa HeLa HeLa cells cells cells [ 86 [86] [86]] [86] NHU 382/503382/503 12 12 ------B16-F1 B16-F1 HeLaandand HeLa cellsHeLa cellscells [86] [86]

Screening ProbeProbeProbeProbe 1 1 1 460/540460/540460/540460/540 121212 12 ------Screening Screening Screening inhibitors inhibitors inhibitors [ 87[87][87]][87] Probe 1 460/540460/540 1212 ------Screening Screeninginhibitors inhibitorsinhibitors [87][87]

Imaging of ImagingImaging of of TYR TYR in in ImagingImagingImagingTYR in ofof B16F10 of TYRTYR TYR inin in B16F10B16F10B16F10B16F10and andand HeLaand and HeLaHeLa HeLa HeLa NBR-APNBR-APNBR-APNBR-AP 580/660580/660580/660580/660 77 7 7 229.5 229.5 229.5 229.5 5.75 5.75 5.75 5.75 - - - - B16F10cells, and in vivo HeLa [88[88][88]][88] NBR-AP 580/660580/660 77 229.5 229.5 5.75 5.75 - - cells,cells, in in vivo vivo imaging imaging [88][88] cells,cells,cells, ininimaging in vivovivo vivo imagingimaging of imaging ofofofof zebrafishzebrafish zebrafish zebrafishzebraﬁsh andand and and micemice mice mice of zebrafishmice and mice

B16B16 and and HeLa HeLa cells, cells, B16B16 andB16 and and HeLa HeLa HeLa cells, cells, B16 andcells, HeLa the cells, Mela-TYRMela-TYRMela-TYR 425/547425/547425/547 5050 50 ------0.07 0.07 0.07 thethe subcellular subcellular local- local- [89[89][89]] Mela-TYRMela-TYR 425/547425/547425/547 505050 ------0.07 0.07 0.07 thethethe subcellularsubcellular subcellularsubcellular local-local- local-[89][89][89] localizationizationization izationizationization

HO NN HOHOHO N N B16, HeLa, HOHO NH NN 630/760630/760630/760 B16,B16, HeLa, HeLa, MCF-7 MCF-7 Cy-tyr NHNHNH 630/760630/760 - - - 0.02 B16,B16, HeLa,MCF-7 HeLa, and MCF-7 MCF-7 [90] Cy-tyrCy-tyrCy-tyrCy-tyr NHNH 516/556630/760 ------0.02 0.02 0.02 0.02 B16, HeLa, MCF-7 [90][90][90][90] Cy-tyr 516/556516/556 ------0.02 0.02 andand HUVECHUVEC HUVEC cells cells cells [90][90] 516/556516/556516/556 andandand HUVECHUVEC HUVEC cellscells cells - NN I--I - N NII--- I NN II-

Biosensors 2021, 11, x FOR PEER REVIEW Screening 11 of 22 ProbeProbeProbe 1 1 650/720650/720650/720 ------0.01 0.01 0.01 ScreeningScreening inhibitors inhibitors [91[91][91]] ProbeProbe 1 1 650/720650/720650/720 ------0.01 0.01 0.01 ScreeningScreeningScreeninginhibitors inhibitorsinhibitors inhibitors [91][91][91]

B16F10B16F10 and and HeLa HeLa cells, MB1MB1 640/706 >100 >100 4.6 4.6 0.45 0.45 - - cells, photodynamic [92[92]] photodynamic therapytherapy

B16F10 and HeLa Tyro-1 400/452 12.5 - - 0.025 [93] cells

Probe 1 460/515 - 5.3 6.36 - B16F10 cells [94]

CHMC-DO HepG2 cells, 538/629 - - - 0.003 [95] PA zebrafish

4-Hydroxyphenyl Recognition Units The classic structure of 4-hydroxyphenyl is covalently coupled with fluorophore via a carbamate linkage. A novel probe Mela-TYR with melanosome-targeting ability first imaged the distribution of TYR in organelles (Figure 8) [89]. This probe utilized morpholine as a melanosome-targeting group and 4-aminophenol as a responsive warhead. Since the acidic environment (about pH 4.2–4.6) of melanosomes, the protonated form of morpholine enhanced the hydrophilicity of morpholine and facilitated its accumulation in melanosomes. Subsequently, the colocalization experiments with mCherry-tagged melanosomes and DND-99 (a commercial dye) validated this targeting ability. Through Mela-TYR imaging, it was found that TYR was significantly up-regulated in live B16 cells stimulated by psoralen/ultraviolet light A, which was further verified by standard colorimetric methods. To observe another representative example, the near-infrared (NIR) probe NBR-AP was activated through an oxidization-cleavage reaction and displayed a linear relationship over the range of 1~200 U/L [88]. Moreover, NBR-AP accomplished the sensitive and selective detection of endogenous TYR activity in B16F10 cells and zebrafish (Figure 9). Real-time in vivo imaging of melanoma and metastasis in xenogeneic mouse models suggested that NBR-AP may be a reliable approach for the early diagnosis of metastatic melanoma during cancer surgery. To the best of our knowledge, this was the first investigation to utilize a fluorescent substrate for the diagnosis of early melanoma in a rodent model. Follow-up studies demonstrated that a longer linker between the recognition moiety and the dye skeleton might prominently decrease the steric hindrance of the probe entering the catalytic site. By combining thermodynamic computation with molecular docking simulation, Li et al. analyzed the Gibbs free energy change of different urea bonds during spontaneous hydrolysis, as well as the distance between the phenolic hydroxyl group (metabolic site) and the catalytic site [92]. Thus, a rapidly responsive and ultra-sensitive NIR probe MB1 was rationally designed. As a specific substrate of TYR (Km = 4.6 μM; Vmax = 0.45 μM/min), the fluorescence intensity of MB1 could increase >100-fold within 20 min, providing immense convenience for drug Biosensors 2021, 11, x FOR PEER REVIEW 11 of 22

Biosensors 2021, 11, 290 11 of 22

B16F10 and HeLa MB1 640/706 >100 4.6 0.45 - cells, photodynamic [92] Table 3. Cont. therapy

λ K V LOD Biological Names Chemical Structures ex/em Folds m max Ref. (nm) (µM) (µM·min−1) (U·mL−1) Applications

B16F10B16F10 and and HeLa Tyro-1Tyro-1 400/452 12.5 12.5 - - - - 0.025 0.025 [93[93]] HeLacells cells

ProbeProbe 1 1 460/515 - - 5.3 5.3 6.36 6.36 - - B16F10 B16F10 cellscells [94[94]]

CHMC-DOCHMC- HepG2HepG2 cells, 538/629 ------0.003 0.003 [95[95]] PADOPA zebrafishzebraﬁsh

4-Hydroxyphenyl4-Hydroxyphenyl Recognition Recognition Units Units TheThe classic classic structure structure of of4-hydroxyphenyl 4-hydroxyphenyl is covalently is covalently coupled coupled with with fluorophore fluorophore via a viacarbamate a carbamate linkage. linkage. A novel A novelprobe probe Mela-TYR Mela-TYR with melanosome-targeting with melanosome-targeting ability ability first imagedfirst imaged the distribution the distribution of TYR ofin TYRorganelles in organelles (Figure 8) (Figure [89]. This8)[ 89probe]. This utilized probe morpho- utilized linemorpholine as a melanosome-targeting as a melanosome-targeting group and group 4-aminophenol and 4-aminophenol as a responsive as a responsive warhead. warhead. Since theSince acidic the acidicenvironment environment (about (about pH 4.2–4.6) pH 4.2–4.6) of melanosomes, of melanosomes, the the protonated protonated form form of of morpholinemorpholine enhanced enhanced the the hydrophilicity hydrophilicity of of mo morpholinerpholine and and facilitated facilitated its its accumulation accumulation inin melanosomes. melanosomes. Subsequently, Subsequently, the the colocalization colocalization experiments experiments with with mCherry-tagged mCherry-tagged melanosomesmelanosomes and and DND-99 DND-99 (a (a commercial commercial dye) dye) validated validated this this targeting targeting ability. ability. Through Through Mela-TYRMela-TYR imaging, imaging, it was it was found found that that TYR TYR was wassignificantly significantly up-regulated up-regulated in live in B16 live cells B16 stimulatedcells stimulated by psoralen/ultravi by psoralen/ultravioletolet light A, light which A, whichwas further was further verified verified by standard by standard col- orimetriccolorimetric methods. methods. To observe To observe another another repres representativeentative example, example, the the near-infrared near-infrared (NIR) (NIR) probeprobe NBR-AP NBR-AP was was activated activated through through an an oxidization-cleavage oxidization-cleavage reaction reaction and and displayed displayed a a linearlinear relationship relationship over over the rangerange ofof 1~200 1~200 U/L U/L [ 88[88].]. Moreover, Moreover, NBR-AP NBR-AP accomplished accomplished the thesensitive sensitive and and selective selective detection detection of endogenous of endogenous TYRactivity TYR activity in B16F10 in B16F10 cells and cells zebrafish and zebrafish(Figure9 ).(Figure Real-time 9). Real-timein vivo imaging in vivo ofimag melanomaing of melanoma and metastasis and metastasis in xenogeneic in xenoge- mouse neicmodels mouse suggested models suggested that NBR-AP that mayNBR-AP be a ma reliabley be a approach reliable approach for the early for the diagnosis early di- of agnosismetastatic of metastatic melanoma melanoma during cancer during surgery. cancer To surgery. the best To of the our best knowledge, of our knowledge, this was the thisfirst was investigation the first investigation to utilize a to fluorescent utilize a fl substrateuorescent for substrate the diagnosis for the ofdiagnosis early melanoma of early melanomain a rodent in model.a rodent Follow-up model. Follow-up studies demonstrated studies demonstrated that a longer that a linker longer between linker be- the tweenrecognition the recognition moiety and moiety the dye and skeleton the dye mightskeleton prominently might prominently decrease thedecrease steric the hindrance steric hindranceof the probe of the entering probe the entering catalytic the site. catalytic By combining site. By thermodynamiccombining thermodynamic computation com- with putationmolecular with docking molecular simulation, docking Li etsimulation al. analyzed, Li theet al. Gibbs analyzed free energy the Gibbs change free of differentenergy changeurea bonds of different during urea spontaneous bonds during hydrolysis, sponta asneous well ashydrolysis, the distance as betweenwell as the the distance phenolic betweenhydroxyl the group phenolic (metabolic hydroxyl site) group and (metabolic the catalytic site) site and [92 the]. Thus, catalytic a rapidly site [92]. responsive Thus, a rapidlyand ultra-sensitive responsive and NIR ultra-sensitive probe MB1 was NIR rationally probe MB1 designed. was rationally As a specific designed. substrate As a of µ µ specificTYR (K substratem = 4.6 M; of VTYRmax =(K 0.45m = 4.6M/min), μM; Vmax the = 0.45 fluorescence μM/min), intensity the fluorescence of MB1 could intensity increase of MB1>100-fold could withinincrease 20 >100-fold min, providing within immense 20 min, convenience providing immense for drug screening.convenience Notably, for drug this sensor could effectively kill melanoma cells by photodynamic therapy (PDT). As such, this sensor held great potential in melanoma-specific imaging and treatment. Biosensors 2021, 11, x FOR PEER REVIEW 12 of 22

Biosensors 2021, 11, 290screening. Notably, this sensor could effectively kill melanoma cells by photodynamic 12 of 22 therapy (PDT). As such, this sensor held great potential in melanoma-specific imaging and treatment.

Figure 8. (a) TheFigure structure 8. (a) ofThe Mela-TYR structure and of Mela-TYR its reaction and mechanism its reaction with mechanism TYR. (b) Colocalization with TYR. (b) of Colocalization Mela-TYR and of lysosome Mela-TYR and lysosome tracker DND-99 in B16 cells. The cells were pretreated with inulavosin (10 tracker DND-99 in B16 cells. The cells were pretreated with inulavosin (10 µM) for (1) 0, (2) 6, and (3) 12 h, respectively, and μM) for (1) 0, (2) 6, and (3) 12 h, respectively, and then incubated with Mela-TYR and DND-99 [89]. then incubated with Mela-TYR and DND-99 [89]. Copyright permission is granted by American Chemical Society. Copyright permission is granted by American Chemical Society. 3-Hydroxyphenyl Recognition Units When ROS reacts with the hydroxyl recognition unit, it tends to form quinone derivatives rather than hydroxylated product. On this premise, to eliminate the interference from ROS, 3-hydroxyphenyl is proposed. Wu et al. developed NIR probe 1. This sensor displayed a specific response to TYR, even when the concentration of ROS was much higher than that found at physiological levels. The high specificity of probe 1 facilitated the accurate detection of TYR activity in live cells and zebrafish, which was further verified by ELISA [82]. Inspired by the excellent work of predecessors, Peng et al. constructed a NIR melanosome-targeting probe (HB-NP) for the highly selective detection of TYR at the subcellular level (Figure 10) by incorporating 3-hydroxyphenyl moiety and the morpholine unit (melanosome-targeting group) into the salicyladazine skeleton [80]. Compared to Mela-TYR, the probe exhibited a large Stokes shift (195 nm) after PET effect inhibition. HB-NP successfully visualized and quantified intracellular TYR activity in various living cells. Moreover, HB-NP distinguished two human uveal melanoma cells with different in- Biosensors 2021, 11, 290 13 of 22

vasive behaviors and evaluated the effects of the inhibitor (kojic acid) and the up-regulating treatment (psoralen/ultraviolet A). Zhang et al. designed a novel, water-soluble probe that detected the endogenous TYR in living cells and zebraﬁsh [83]. The recognition fragment, Biosensors 2021, 11, x FOR PEER REVIEW3-hydroxybenzyloxy, could speciﬁcally identify TYR instead of ROS. In particular,13 of the 22

probe successfully realized the diagnosis of melanoma in a xenogeneic mouse model.

Figure 9. (a) The schematic diagram of NBR-AP for TYR detection.detection. ( (b)) Images for 3-day-old zebrafish zebrafish incubated with various concentrations of the probe and kojic acid. ( cc)) Fluorescent and and bright field field images of 4-week-old mice injected with the probe upon injection with B16F10 cells. ( d)) Images of of dissected organs organs of the mice injected with the probe upon injection with B16F10 cells for 14 days. (e) Relative intensity values (n = 3) obtained from (d) and calculated using Image injection with B16F10 cells for 14 days. (e) Relative intensity values (n = 3) obtained from (d) and calculated using Image J2x software, while the relative intensity from tumor is defined as 1.0 [88]. Copyright permission is granted by American J2x software, while the relative intensity from tumor is defined as 1.0 [88]. Copyright permission is granted by American Chemical Society. Chemical Society.

3-HydroxyphenylIn brief, each Recognition of the above Units probes exhibits unique performance. For high-throughput screeningWhen at ROS the reacts target levelwith inthe vitro hydroxyl, the sensitivity recognition and unit, rapid it tends response to form of tool quinone molecular de- rivativesdemonstrate rather more than importance hydroxylated [96 product.]. On the On other this premise, hand, the to tracingeliminate and the visualization interference fromanalysis ROS, of 3-hydroxyphenyl TYR activity in vivo is requiresproposed. long Wu emission et al. developed wavelengths NIR and probe speciﬁcity 1. This [sensor97,98]. displayedIn the near a future,specific combiningresponse to computational TYR, even when means the suchconcentration as molecular of ROS docking, was much more highersuperior than sensors that forfound various at physiological purposes are levels expected. The to high be rationally specificity designed; of probe these 1 facilitated sensors thehave accurate broad applicationdetection of potential TYR activity in the in ﬁeld live ofcells bioanalysis. and zebrafish, which was further verified by ELISA [82]. Inspired by the excellent work of predecessors, Peng et al. constructed a NIR melanosome-targeting probe (HB-NP) for the highly selective detection of TYR at the subcellular level (Figure 10) by incorporating 3-hydroxyphenyl moiety and the morpholine unit (melanosome-targeting group) into the salicyladazine skeleton [80]. Compared to Mela-TYR, the probe exhibited a large Stokes shift (195 nm) after PET effect inhibition. HB-NP successfully visualized and quantified intracellular TYR activity in various living cells. Moreover, HB-NP distinguished two human uveal melanoma cells Biosensors 2021, 11, x FOR PEER REVIEW 14 of 22

with different invasive behaviors and evaluated the effects of the inhibitor (kojic acid) and the up-regulating treatment (psoralen/ultraviolet A). Zhang et al. designed a novel, water-soluble probe that detected the endogenous TYR in living cells and zebrafish [83]. Biosensors 2021, 11, 290The recognition fragment, 3-hydroxybenzyloxy, could specifically identify TYR instead 14 of 22 of ROS. In particular, the probe successfully realized the diagnosis of melanoma in a xenogeneic mouse model.

Figure 10. (a)Figure The mechanism 10. (a) The mechanism of TYR activity of TYR detected activity by detected HB-NP. by (b) HB-NP. Colocalization (b) Colocalization of HB-NP of and HB-NP Alexa and Fluor 488 (a commercial dye)Alexa in B16Fluor cells 488 [80 (a]. commercial (1) Bright-field dye) image in B16 of cells the B16 [80] cells;. (1) Bright-field (2) fluorescence image image of the of theB16 red cells; channel (2) flu- for HB-NP; orescence image of the red channel for HB-NP; (3) fluorescence image of the green channel for (3) fluorescence image of the green channel for Alexa Fluor 488 (λex = 488 nm, λem = 500–555 nm); (4) the merged image of Alexa Fluor 488 (λex = 488 nm, λem = 500–555 nm); (4) the merged image of (1)–(3). (4) Intensity (1)–(3). (4) Intensity correlation plot of HB-NP and Alexa Fluor 488. (6) Intensity profile of the linear ROI across the cell correlation plot of HB-NP and Alexa Fluor 488. (6) Intensity profile of the linear ROI across the cell (yellow line in images (2)–(4)). Copyright permission is granted by American Chemical Society. (yellow line in images (2)–(4)). Copyright permission is granted by American Chemical Society. 3.2.2. Nanoparticle-Based Fluorescent Probes In brief, each of the above probes exhibits unique performance. For high-throughput screening at the targetOther level than in small vitro, molecular the sensitivity probes, and nanometer rapid response material-based of tool molecular biosensors have also demonstrate arousedmore importance considerable [96]. interest On the in other the detectionhand, the of tracing biological and analytes.visualization Some emerging analysis of TYRnanomaterials activity in utilizevivo requires the characteristics long emission of TYR-mediated wavelengths metabolism and specificity to trigger a linear fluorescence response with intermediates. For example, under aerobic and alkaline con- [97,98]. In the near future, combining computational means such as molecular docking, ditions, dopamine is converted to polydopamine via oxidation and self-polymerization. more superior sensors for various purposes are expected to be rationally designed; these Enlightened by the intrinsic fluorescence properties of polydopamine, Liu et al. prepared sensors have broad application potential in the field of bioanalysis. the fluorescent polymethyldopa nanoparticles (PMNPs) [99]. Metyrosine acted as a substrate of TYR to yield methyldopa (a dopamine analog). The latter further reacted with 3.2.2. Nanoparticle-Based Fluorescent Probes ethanolamine to produce PMNPs (Figure 11a). Ultimately, the strategy of in situ formation Other thanof fluorescentsmall molecular PMNPs probes, performed nanomete wellr inmaterial-based screening inhibitors. biosensors Using have a similaralso principle, aroused considerableDing et al. interest introduced in the tyraminedetection as of a biological model substrate, analytes. which Some could emerging be converted na- into DA nomaterials utilizeby TYR. the Based characteristics on the specific of TYR-mediated sensing between metabolism silicon nanoparticles to trigger a (Si linear NPs) and DA, the solution color and fluorescence emission changed significantly (Figure 11b). Subsequently, a novel ratiometric fluorescence analysis was established for screening TYR activators and inhibitors [100]. Wang et al. developed a fluorescence-sensing platform utilizing rare-earth-doped upconversion nanoparticles (UCNPs) [101]. Tyramine was oxidized to DA and further yielded melanin-like polymers, leading to the effective quenching of UC- NPs (Figure 11c). Collectively, most nanoparticle-based fluorescent probes have fulfilled Biosensors 2021, 11, x FOR PEER REVIEW 15 of 22

fluorescence response with intermediates. For example, under aerobic and alkaline conditions, dopamine is converted to polydopamine via oxidation and self-polymerization. Enlightened by the intrinsic fluorescence properties of polydopamine, Liu et al. prepared the fluorescent polymethyldopa nanoparticles (PMNPs) [99]. Metyrosine acted as a substrate of TYR to yield methyldopa (a dopamine analog). The latter further reacted with ethanolamine to produce PMNPs (Figure 11a). Ultimately, the strategy of in situ formation of fluorescent PMNPs performed well in screening inhibitors. Using a similar principle, Ding et al. introduced tyramine as a model substrate, which could be converted into DA by TYR. Based on the specific sensing between silicon nanoparticles (Si NPs) and DA, the solution color and fluorescence emission changed significantly (Figure 11b). Subsequently, a novel ratiometric fluorescence analysis was established for screening Biosensors 2021, 11, 290TYR activators and inhibitors [100]. Wang et al. developed a fluorescence-sensing plat- 15 of 22 form utilizing rare-earth-doped upconversion nanoparticles (UCNPs) [101]. Tyramine was oxidized to DA and further yielded melanin-like polymers, leading to the effective quenching of UCNPs (Figure 11c). Collectively, most nanoparticle-based fluorescent probes have fulfilledrelevant relevant drug screening drug screening protocols. protocols. Some potential Some potential factors, including factors, including complicated synthesis, complicated synthesis,time-consuming time-consuming sample pretreatment, sample pretreatment, homogeneity, homoge and stabilityneity, and of nanoparticles, stability deserve of nanoparticles,adequate deserve consideration. adequate consideration. Furthermore, Furthermore, compared to traditionalcompared colorimetry,to traditional both methods colorimetry, bothseem methods to be suitable seem onlyto be forsuitable TYRactivity only for evaluation TYR activity in vitro.evaluation in vitro.

FigureFigure 11. Schematic 11. Schematic illustration illustration of the PMNPsof the PMNPs (a), Si NPs (a), Si (b ),NPs and (b UCNPs), and UCNPs (c) for the (c) TYRfor the activity TYR activity detection. detection. 4. Biomedical Applications of TYR Activity Assays 4. Biomedical4.1. Applications Sensing and of Imaging TYR Activity TYR Activities Assays in Biological Systems 4.1. Sensing and ImagingMelanoma TYR Activities is the most in Biological aggressive Systems malignancy in skin cancer. It is characterized by Melanomahigh is metastaticthe most aggressive potential, malignancy poor prognosis, in skin and cancer. the up-regulation It is characterized of melanocytes by [102]. high metastaticReintgen potential, et al. poor determined prognosis, the and order the ofup-regulation melanomanodal of melanocytes metastases [102]. and showed that Reintgen et al.cutaneous determined melanoma the order usually of me firstlanoma metastasized nodal metastases to the regional and nodal showed basin that via the regional cutaneous melanomalymphatics usually [103,104 first]. About metastasized one-third to of the melanoma regional patientsnodal basin have avia local the recurrence, re- while gional lymphaticsdistant [103,104]. metastasis About at the one-third initial site of ofmelanoma relapse is patients also relatively have a commonlocal recur- [105]. The most rence, while distantcharacteristic metastasis metastatic at the initial sites aresite lymphof relapse nodes, is also lungs, relatively liver, brain,common and [105]. bones [106]. The The most characteristicprecise detection metastatic of subclinical sites are metastaseslymph nodes, for earlylungs, diagnosis liver, brain, and and treatment bones of melanoma [106]. The preciseis a matter detection of urgency. of subclinical Human metastases TYR (11q14-21, for early MIM diagnosis 606933) and is primarily treatment expressed of in epi- melanoma is dermal,a matter follicular, of urgency. and Human ocular melanocytes. TYR (11q14-21, It is MIM essential 606933) for pigmentis primarily formation ex- [107]. The overexpression of TYR and TRP 1 is significantly associated with the risk of melanoma [1]. B16F10 cells are often selected as cell models due to the high TYR expression. TYR has acted as a valuable tumor marker and therapeutic target for the early diagnosis and treatment of melanoma lesions. As a critical melanoma-associated antigen, TYR can be recognized by autologous T lymphocytes, thus inducing effective tumor-specific responses. Currently, a serum assay remains the mainstream approach for biomarker detection [88]. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was developed to detect circulating melanoma cells (CMCs) in the peripheral blood. This method was based on the amplification of the messenger RNA (mRNA) for TYR, while normal melanocytes are not thought to circulate in the peripheral blood [108–110]. However, test results are often controversial and can show false positives; the latter may be due to sample processing and the transient presence of metastasizing tumor cells [111]. By adopting in vivo fluorescent imaging strategy, the elevation of the TYR level (or activity) at the melanoma focus can be localized spatially, thereby lessening the risk of false-positive signals. The NIR probe NBR-AP successfully realized the early diagnosis of melanoma Biosensors 2021, 11, x FOR PEER REVIEW 16 of 22 Biosensors 2021, 11, x FOR PEER REVIEW 16 of 22

Biosensors 2021, 11, x FOR PEER REVIEW 16 of 22

pressed in epidermal, follicular, and ocular melanocytes. It is essential for pigment for- pressed in epidermal, follicular, and ocular melanocytes. It is essential for pigment formation [107]. The overexpression of TYR and TRP 1 is significantly associated with the pressedmation in [107]. epidermal, The overexpression follicular, and ofocular TYR melanocytes.and TRP 1 is Itsignificantly is essential associatedfor pigment with for- the risk of melanoma [1]. B16F10 cells are often selected as cell models due to the high TYR mationrisk of [107]. melanoma The overexpression [1]. B16F10 cells of TYRare often and TRPselected 1 is assignificantly cell models associated due to the with high the TYR expression. TYR has acted as a valuable tumor marker and therapeutic target for the early riskexpression. of melanoma TYR [1]. has B16F10 acted as cells a valuable are often tumor selected marker as cell and models therapeutic due totarget the highfor the TYR early diagnosis and treatment of melanoma lesions. As a critical melanoma-associated antigen, expression.diagnosis TYR and hastreatment acted as of a melanoma valuable tumor lesions. marker As a criticaland therapeutic melanoma-associated target for the antigen,early TYR can be recognized by autologous T lymphocytes, thus inducing effective tu- diagnosisTYR can and be treatment recognized of melanoma by autologous lesions. T As lymphocytes, a critical melanoma-associated thus inducing effective antigen, tumor-specific responses. TYRmor-specific can be recognized responses. by autologous T lymphocytes, thus inducing effective tu- Currently, a serum assay remains the mainstream approach for biomarker detection mor-specificCurrently, responses. a serum assay remains the mainstream approach for biomarker detection [88]. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was developed [88].Currently, Reverse atranscription-polymerase serum assay remains the chainmainstream reaction approach (RT-PCR) for analysis biomarker was detection developed to detect circulating melanoma cells (CMCs) in the peripheral blood. This method was [88].to Reversedetect circulating transcription-polymerase melanoma cells chain(CMCs) reaction in the (RT-PCR) peripheral analysis blood. wasThis developedmethod was based on the amplification of the messenger RNA (mRNA) for TYR, while normal mel- to baseddetect oncirculating the amplification melanoma of cellsthe messenger(CMCs) in RNAthe peripheral (mRNA) forblood. TYR, This while method normal was melanocytes are not thought to circulate in the peripheral blood [108–110]. However, test basedanocytes on the are amplification not thought of to the circulate messenger in th RNAe peripheral (mRNA) blood for TYR, [108–110]. while normalHowever, mel- test results are often controversial and can show false positives; the latter may be due to anocytesresults areare notoften thought controversial to circulate and incan th showe peripheral false positives; blood [108–110]. the latter However, may be duetest to sample processing and the transient presence of metastasizing tumor cells [111]. By resultssample are processingoften controversial and the andtransient can showpresen falsece of positives; metastasizing the latter tumor may cells be due[111]. to By Biosensors 2021, 11, 290 adopting in vivo fluorescent imaging strategy, the elevation of the TYR level (or activity)16 of 22 sampleadopting processing in vivo fluorescentand the transient imaging presen strategy,ce ofthe metastasizing elevation of the tumor TYR cellslevel [111].(or activity) By at the melanoma focus can be localized spatially, thereby lessening the risk of adoptingat the inmelanoma vivo fluorescent focus imagingcan be strategy,localized the spatially, elevation thereby of the TYR lessening level (or the activity) risk of false-positive signals. The NIR probe NBR-AP successfully realized the early diagnosis of at false-positivethe melanoma signals. focus The can NIR be probe localized NBR-AP spatially, successfully thereby realized lessening the early the diagnosis risk of of melanoma and metastasis in a mouse model by imaging TYR activity [88]. The levels of false-positivemelanomaand metastasis andsignals. metastasis in The a mouse NIR in probe modela mouse NBR-AP by model imaging successfully by TYRimaging activity realized TYR [ 88activity ].the The early [88]. levels diagnosis The of TYRlevels of in of the TYR in the tumor and metastatic organs analyzed by Western blot were consistent with melanomaTYRtumor in the and and tumor metastasis metastatic and metastatic in organs a mouse analyzed organs model analyzed by by Western imaging by blotWestern TYR were activity blot consistent were[88]. Theconsistent with levels the fluores-withof the fluorescence results (Figure 9e), in which TYR in the tumor, lung, and spleen was TYRthe cencein fluorescence the resultstumor (Figureand results metastatic9 e),(Figure in which organs 9e), TYRin analyzed which in the TYR by tumor, Westernin the lung, tumor, blot and were spleenlung, consistent and was spleen found with was to be found to be over-expressed. These results demonstrate that small-molecule fluorescent thefound fluorescenceover-expressed. to be over-expressed. results These (Figure results These 9e), demonstrate in results which de TYRmonstrate that in small-molecule the tumor,that small-molecule lung, fluorescent and spleen fluorescent probes was have probes have great promise in the early diagnosis of melanoma and the analysis of bio- foundprobesgreat to behave promise over-expressed. great in promise the early These in diagnosis the results early of diag melanomademonstratenosis of and melanoma that the small-molecule analysis and ofthe biological analysis fluorescent samples.of biological samples. probeslogical have samples. great promise in the early diagnosis of melanoma and the analysis of bio- logical4.2. samples. Screening and Characterization of TYR Inhibitors 4.2. Screening and Characterization of TYR Inhibitors 4.2. ScreeningTYR is and the Charact initiatingerization and rate-limitingof TYR Inhibitors factor in melanin biosynthesis, serving as a TYR is the initiating and rate-limiting factor in melanin biosynthesis, serving as a 4.2. ScreeningprominentTYR is and the target Charact initiating forerization pigmentation and ofrate-limiting TYR disordersInhibitors factor [9, 10in, 112melanin,113]. Accordingly,biosynthesis, TYRserving inhibitors as a prominent target for pigmentation disorders [9,10,112,113]. Accordingly, TYR inhibitors prominentTYRcan block is the target the initiating formation for pigmentation and of rate-limiting pigment disorders and exhibitfactor [9,10,112,113]. in broad melanin application biosynthesis,Accordingly, prospects servingTYR in inhibitors agricultural, as a can block the formation of pigment and exhibit broad application prospects in agricul- prominentcanmedicinal, block target the and formation for cosmetic pigmentation of industries pigment disorders and [114 ].exhi [9,10,112,113]. Mostbit compoundsbroad application Accordingly, are reported prospects TYR as TYRinhibitors in agricul- inhibitors tural, medicinal, and cosmetic industries [114]. Most compounds are reported as TYR cantural, blockdue medicinal, tothe their formation function and cosmeticof inpigment copper industries and chelation exhi [114].bit or broad competition Most application compounds with prospects substrates, are reported in while agricul- as theTYR for- inhibitors due to their function in copper chelation or competition with substrates, while tural,inhibitorsmer medicinal, could due give toand their rise cosmetic tofunction the irreversibleindustries in copper [114]. inactivation chelation Most or compounds of competition TYR [26,36 are ,with115 reported].Popular substrates, as whitening TYR while the former could give rise to the βirreversible inactivation of TYR [26,36,115].Popular inhibitorstheagents, former due such couldto their as hydroquinone,give function rise toin copperthe-arbutin irreversib chelation (ale hydroquinone inactivationor competition derivative),of withTYR substrates,[26,36,115].Popular or kojic while acid, have whitening agents, such as hydroquinone, β-arbutin (a hydroquinone derivative), or kojic thewhitening formeralways could been agents, regardedgive such rise as as tohydroquinone, positive the irreversib controls, βle-arbutin butinactivation they (a alsohydroquinone haveof TYR certain [26,36,115].Popular derivative), drawbacks or (Table kojic 4 ). acid, Efficientlyhave always discovering been regarded potent as TYR positive inhibitors controls, with superiorbut they safety also have profiles certain remains draw- a main- whiteningacid, have agents, always such been as hydroquinone,regarded as positive β-arbutin controls, (a hydroquinone but they also derivative), have certain or kojic draw- backsstream (Table concern 4). Efficiently in hyperpigmentation discovering potent therapy TYR [ 116inhibitors]. with superior safety pro- acid,backs have (Table always 4). beenEfficiently regarded discovering as positive potent controls, TYR inhibitorsbut they also with have superior certain safety draw- pro- files remains a mainstream concern in hyperpigmentation therapy [116]. backsfiles (Table remains 4). a Efficiently mainstream discovering concern in potent hyperpigmentation TYR inhibitors therapy with superior [116]. safety pro- Table 4. The commonly used positive inhibitors and their adverse effects. Tablefiles 4. Theremains commonly a mainstream used positive concern inhibi intors hyperpigmentation and their adverse effects. therapy [116]. Inhibitors ChemicalTable Structures 4. The commonly Sources used positive Adverse inhibitors Effects and their adverse Dosage effects. Group Ref. Inhibitors ChemicalTable Structures 4. The commonly Sources used positive inhibiAdversetors and Effects their adverse effects.Dosage Group Ref. Inhibitors Chemical Structures Sources Adverse Effects Dosage Group Ref. (1) (1)IrritantIrritant contact contact dermatitis. HydroquinoneInhibitorsHydroquinone Chemical Structures Sources (1) Irritantdermatitis.Adverse contact Effects dermatitis. Dosage Group Ref. Hydroquinone PlantPlant (2) Exogenous ochronosis. <4%<4% Forbidden Forbidden [117,118] [117,118 ] (HQ)(HQ) Plant (1) (2)(2)Irritant ExogenousExogenous contact ochronosis. ochronosis.dermatitis. <4% Forbidden [117,118] Hydroquinone(HQ) (3) (3)TransientTransient erythema. erythema. Plant (2) (3)Exogenous Transient ochronosis. erythema. <4% Forbidden [117,118] (HQ) (3) Transient erythema. β-Arbutin Plant Facial tingling <3% Approved [119,120] β-Arbutinβ-Arbutin PlantPlant FacialFacial tingling tingling <3%<3% ApprovedApproved [119,120] [119,120]

β-Arbutin Plant Facial tingling <3% Approved [119,120] (1) Contact dermatitis (especially in (1) Contact dermatitis (especially in (1)sensitiveContact skins). dermatitis (1) Contactsensitive(especially dermatitis skins). in sensitive (especially in Kojic acid (2) Long-term use may make the skin Kojic acid Fungus (2)sensitive Long-termskins). skins). use may make the skin <1% Approved [121,122] (KA) Fungus (2)moreLong-term prone to sunburn. use may <1% Approved [121,122] KojicKojic (KA)acid acid (2) Long-termmore prone use tomay sunburn. make the skin FungusFungus (3) Usingmake KA theon damaged skin more or broken <1%<1% Approved Approved [121,122] [121,122 ] (KA)(KA) (3)more Usingprone prone KA to to sunburn.on sunburn. damaged or broken skins can lead to cancer. (3) (3)UsingskinsUsing KA can on KA lead damaged on damagedto cancer. or broken skinsor can broken lead skinsto cancer. can lead Over the past few decades,to increasing cancer. attempts have been devoted to identifying Over the past few decades, increasing attempts have been devoted to identifying effective TYR inhibitors from natural products and synthetic compounds through in vitro effectiveOver the TYR past inhibitors few decades, from natural increasing produc attemptsts and synthetic have been compounds devoted to through identifying in vitro effective TYR inhibitors from natural products and synthetic compounds through in vitro Over the past few decades, increasing attempts have been devoted to identifying effective TYR inhibitors from natural products and synthetic compounds through in vitro and in silico procedures. Generally speaking, spectrophotometry is the most commonly used in the determination of TYR activity [30,112,123]. Most inhibitors are assessed by dopachrome formation using L-tyrosine or L-DOPA as substrates. Tajima et al. synthesized a series of bibenzyl derivatives and found bibenzyl xyloside 2 to be a potent inhibitor (IC50 = 0.43 µM) that was 17 times more effective than kojic acid [124]. Ishioka et al. developed some novel TYR inhibitors based on the structure of rhododendron, with IC50 values ranging from 0.39 µM to 35.9 µM[125]. Using L-tyrosine and L-DOPA as substrates, Jung et al. designed thirteen (E)-benzylidene-1-indanone derivatives, in which BID3 was the most potent inhibitor of mushroom tyrosinase (IC50 = 0.034 µM, monophenolase activity; IC50 = 1.39 µM, diphenolase activity) [126]. Durai et al. applied evolutionary chemical binding similarity (ECBS) to screen a virtual chemical database for human TYR, which effectively identified seven potential TYR inhibitors [127]. In summary, candidate drugs with high affinity and great druggability can be rapidly identified through virtual screening in combination with the HTS methods presented in this review. All these compounds could Biosensors 2021, 11, 290 17 of 22

be used as lead compounds to design novel potent TYR inhibitors for the treatment of diseases associated with TYR-overexpression [128].

5. Conclusions and Perspectives Mammalian TYR catalyzes the initial and rate-limiting reactions of the melanin biosynthetic pathway, which is a relatively specific biomarker for malignant cutaneous melanoma [2,129]. Monitoring TYR activity remains significant and challenging for the discovery of novel therapeutics. In recent decades, the specific substrate-based optical method has been used for detecting TYR activity in real samples and high-throughput screening of TYR inhibitors. Herein, we reviewed the research advances of various assays, with an emphasis on their respective pros and cons. More substrate preferences and kinetic parameters were also outlined. Among them, the spectrophotometric technique is the most widely used method. Traditional assays for TYR activity mainly depend on the characteristic absorbance of colored products from the substrate L-tyrosine or L-DOPA. Recently, other means have also become more widely used, such as the introduction of nucleophiles to capture DQ to generate stable-colored adducts. To achieve better performance in cell imaging applications, several TYR fluorescent substrates with high specificity and excellent optical properties have gradually emerged, including organic small molecules and nanocomposites. Owing to multiple advantages, such as superior selectivity, high sensitivity, and the potential for dynamic tracking, fluorescent probes could serve as versatile tools for analytical sensing and optical imaging analysis [130]. This not only facilitates the realization of high-throughput screening (HTS) of inhibitors but also evaluates the inhibitory potential of enzyme inhibitors in living cells, living tissues, and even in vivo; these findings significantly improve the efficiency and accuracy of drug discovery [131,132]. Notably, fluorescent probes-based molecular imaging can spatially localize the elevation of the TYR level (or activity) at the melanoma focus, thereby greatly reducing the risk of false-positive signals [133]. The safety of biosensors is a significant prerequisite for the biological studies of TYR in vivo. NIR probes or two-photo probes are capable of deepening photon penetration, reducing photo damage, and producing low background fluorescence, which hold great promise in biomedical imaging [134]. Furthermore, the conjugation of NIR dyes with anticancer agents assists in the synergistic management of cancer, thus integrating the merits of imaging and therapeutic effects to realize the ultimate objective of simultaneous diagnosis and treatment [135,136].

Author Contributions: P.W. and G.-B.G. conceived and designed the paper. Y.-F.F., S.-X.Z., X.-K.Q. and G.-B.G. wrote the manuscript. F.-B.H., Q.-S.P. and D.-F.Z. collected the information. Y.-W.X., X.-K.Q. and P.W. revised the article. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Key Research and Development Program of China (2021YFE0200900, 2017YFC1700200, 2017YFC1702000), the NSF of China (81922070, 81973286), the Three- year Action Plan of Shanghai TCM Development [ZY-(2018-2020)-CCCX-5001], Program of Shanghai Academic/Technology Research Leader (18XD1403600), Shuguang Program (18SG40) supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. Lai, X.; Wichers, H.J.; Soler-Lopez, M.; Dijkstra, B.W. Structure and Function of Human Tyrosinase and Tyrosinase-Related Proteins. Chemistry 2018, 24, 47–55. [CrossRef] 2. Min, K.; Park, G.W.; Yoo, Y.J.; Lee, J.S. A perspective on the biotechnological applications of the versatile tyrosinase. Bioresour. Technol. 2019, 289, 121730. [CrossRef] [PubMed] Biosensors 2021, 11, 290 18 of 22

3. Olivares, C.; Solano, F. New insights into the active site structure and catalytic mechanism of tyrosinase and its related proteins. Pigment. Cell Melanoma Res. 2009, 22, 750–760. [CrossRef][PubMed] 4. Lee, S.H.; Baek, K.; Lee, J.E.; Kim, B.G. Using tyrosinase as a monophenol monooxygenase: A combined strategy for effective inhibition of melanin formation. Biotechnol. Bioeng. 2016, 113, 735–743. [CrossRef][PubMed] 5. Pillaiyar, T.; Manickam, M.; Jung, S.H. Downregulation of melanogenesis: Drug discovery and therapeutic options. Drug Discov. Today 2017, 22, 282–298. [CrossRef] 6. Brenner, M.; Hearing, V.J. The protective role of melanin against UV damage in human skin. Photochem. Photobiol. 2008, 84, 539–549. [CrossRef] 7. Chang, T.S. An updated review of tyrosinase inhibitors. Int. J. Mol. Sci. 2009, 10, 2440–2475. [CrossRef] 8. Pillaiyar, T.; Namasivayam, V.; Manickam, M.; Jung, S.H. Inhibitors of Melanogenesis: An Updated Review. J. Med. Chem. 2018, 61, 7395–7418. [CrossRef] 9. Lee, S.Y.; Baek, N.; Nam, T.G. Natural, semisynthetic and synthetic tyrosinase inhibitors. J. Enzyme Inhib. Med. Chem. 2016, 31, 1–13. [CrossRef] 10. Pillaiyar, T.; Manickam, M.; Namasivayam, V. Skin whitening agents: Medicinal chemistry perspective of tyrosinase inhibitors. J. Enzyme Inhib. Med. Chem. 2017, 32, 403–425. [CrossRef] 11. Hearing, V.J.; Tsukamoto, K. Enzymatic control of pigmentation in mammals. FASEB J. 1991, 5, 2902–2909. [CrossRef][PubMed] 12. Ando, H.; Ichihashi, M.; Hearing, V.J. Role of the ubiquitin proteasome system in regulating skin pigmentation. Int. J. Mol. Sci. 2009, 10, 4428–4434. [CrossRef] 13. Costin, G.E.; Hearing, V.J. Human skin pigmentation: Melanocytes modulate skin color in response to stress. FASEB J. 2007, 21, 976–994. [CrossRef] 14. Ray, K.; Chaki, M.; Sengupta, M. Tyrosinase and ocular diseases: Some novel thoughts on the molecular basis of oculocutaneous albinism type 1. Prog. Retin. Eye Res. 2007, 26, 323–358. [CrossRef][PubMed] 15. dos Santos, V.I.F.; Lima, M.D.F.; Sofia, M. Mechanisms regulating melanogenesis. An. Bras. Dermatol. 2013, 88, 76–83. 16. Ghanem, G.; Fabrice, J. Tyrosinase related protein 1 (TYRP1/gp75) in human cutaneous melanoma. Mol. Oncol. 2011, 5, 150–155. [CrossRef] 17. Gradilone, A.; Cigna, E.; Agliano, A.M.; Frati, L. Tyrosinase Expression as a Molecular Marker for Investigating the Presence of Circulating Tumor Cells in Melanoma Patients. Curr. Cancer Drug Tar. 2010, 10, 529–538. [CrossRef] 18. Jawaid, S.; Khan, T.H.; Osborn, H.M.; Williams, N.A. Tyrosinase activated melanoma prodrugs. Anticancer. Agents Med. Chem. 2009, 9, 717–727. [CrossRef][PubMed] 19. Carballo-Carbajal, I.; Laguna, A.; Romero-Gimenez, J.; Cuadros, T.; Bove, J.; Martinez-Vicente, M.; Parent, A.; Gonzalez-Sepulveda, M.; Penuelas, N.; Torra, A.; et al. Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson’s disease pathogenesis. Nat. Commun. 2019, 10, 973. [CrossRef] [PubMed] 20. da Silva, G.F.; Ming, L.J. Alzheimer’s disease related copper(II)- beta-amyloid peptide exhibits phenol monooxygenase and catechol oxidase activities. Angew. Chem. Int. Ed. Engl. 2005, 44, 5501–5504. [CrossRef][PubMed] 21. Hasegawa, T. Tyrosinase-expressing neuronal cell line as in vitro model of Parkinson’s disease. Int. J. Mol. Sci. 2010, 11, 1082–1089. [CrossRef] 22. Wang, F.Y.; Wang, P.; Zhao, D.F.; Gonzalez, F.J.; Fan, Y.F.; Xia, Y.L.; Ge, G.B.; Yang, L. Analytical methodologies for sensing catechol-O-methyltransferase activity and their applications. J. Pharm. Anal. 2021, 11, 15–27. [CrossRef] 23. Cordes, P.; Sun, W.; Wolber, R.; Kolbe, L.; Klebe, G.; Rohm, K.H. Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase. Biol. Chem. 2013, 394, 685–693. [CrossRef][PubMed] 24. Dolinska, M.B.; Wingfield, P.T.; Sergeev, Y.V. Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector. Curr. Protoc. Protein. Sci. 2017, 89, 6.15.1–6.15.12. 25. Matoba, Y.; Kumagai, T.; Yamamoto, A.; Yoshitsu, H.; Sugiyama, M. Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis. J. Biol. Chem. 2006, 281, 8981–8990. [CrossRef] 26. Kim, Y.J.; Uyama, H. Tyrosinase inhibitors from natural and synthetic sources: Structure, inhibition mechanism and perspective for the future. Cell Mol. Life Sci. 2005, 62, 1707–1723. [CrossRef] 27. Zolghadri, S.; Bahrami, A.; Hassan Khan, M.T.; Munoz-Munoz, J.; Garcia-Molina, F.; Garcia-Canovas, F.; Saboury, A.A. A comprehensive review on tyrosinase inhibitors. J. Enzyme Inhib. Med. Chem. 2019, 34, 279–309. [CrossRef][PubMed] 28. Ismaya, W.T.; Rozeboom, H.J.; Weijn, A.; Mes, J.J.; Fusetti, F.; Wichers, H.J.; Dijkstra, B.W. Crystal structure of Agaricus bisporus mushroom tyrosinase: Identity of the tetramer subunits and interaction with tropolone. Biochemistry 2011, 50, 5477–5486. [CrossRef][PubMed] 29. Ionita, E.; Aprodu, I.; Stanciuc, N.; Rapeanu, G.; Bahrim, G. Advances in structure-function relationships of tyrosinase from Agaricus bisporus-investigation on heat-induced conformational changes. Food Chem. 2014, 156, 129–136. [CrossRef][PubMed] 30. Sendovski, M.; Kanteev, M.; Ben-Yosef, V.S.; Adir, N.; Fishman, A. First structures of an active bacterial tyrosinase reveal copper plasticity. J. Mol. Biol. 2011, 405, 227–237. [CrossRef] 31. Bijelic, A.; Pretzler, M.; Molitor, C.; Zekiri, F.; Rompel, A. The Structure of a Plant Tyrosinase from Walnut Leaves Reveals the Importance of “Substrate-Guiding Residues” for Enzymatic Specificity. Angew. Chem. Int. Ed. Engl. 2015, 54, 14677–14680. [CrossRef][PubMed] Biosensors 2021, 11, 290 19 of 22

32. Fenoll, L.G.; Peñalver, M.J.; Rodríguez-López, J.N.; Varón, R.; García-Cánovas, F.; Tudela, J. Tyrosinase kinetics: Discrimination between two models to explain the oxidation mechanism of monophenol and diphenol substrates. Int. J. Biochem. Cell Biol. 2004, 36, 235–246. [CrossRef] 33. Fenoll, L.G.; Rodriguez-Lopez, J.N.; Garcia-Sevilla, F.; Garcia-Ruiz, P.A.; Varon, R.; Garcia-Canovas, F.; Tudela, J. Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones. Biochim. Biophys. Acta 2001, 1548, 1–22. [CrossRef] 34. Sanchez-Ferrer, A.; Rodriguez-Lopez, J.N.; Garcia-Canovas, F.; Garcia-Carmona, F. Tyrosinase: A comprehensive review of its mechanism. Biochim. Biophys. Acta 1995, 1247, 1–11. [CrossRef] 35. Qu, Y.; Zhan, Q.; Du, S.; Ding, Y.; Fang, B.; Du, W.; Wu, Q.; Yu, H.; Li, L.; Huang, W. Catalysis-based specific detection and inhibition of tyrosinase and their application. J. Pharm. Anal. 2020, 10, 414–425. [CrossRef] 36. Rescigno, A.; Sollai, F.; Pisu, B.; Rinaldi, A.; Sanjust, E. Tyrosinase inhibition: General and applied aspects. J. Enzyme Inhib. Med. Chem. 2002, 17, 207–218. [CrossRef] [PubMed] 37. Sanjust, E.; Cecchini, G.; Sollai, F.; Curreli, N.; Rescigno, A. 3-Hydroxykynurenine as a substrate/activator for mushroom tyrosinase. Arch. Biochem. Biophys 2003, 412, 272–278. [CrossRef] 38. McLarin, M.A.; Leung, I.K.H. Substrate specificity of polyphenol oxidase. Crit. Rev. Biochem. Mol. Biol. 2020, 55, 274–308. [CrossRef] 39. Gasowska, B.; Kafarski, P.; Wojtasek, H. Interaction of mushroom tyrosinase with aromatic amines, o-diamines and o-aminophenols. Biochim. Biophys. Acta 2004, 1673, 170–177. [CrossRef] 40. Rescigno, A.; Bruyneel, F.; Padiglia, A.; Sollai, F.; Salis, A.; Marchand-Brynaert, J.; Sanjust, E. Structure-activity relationships of various amino-hydroxy-benzenesulfonic acids and sulfonamides as tyrosinase substrates. Biochim. Biophys. Acta 2011, 1810, 799–807. [CrossRef] 41. Espin, J.C.; Garcia-Ruiz, P.A.; Tudela, J.; Garcia-Canovas, F. Study of stereospecificity in mushroom tyrosinase. Biochem. J. 1998, 331, 547–551. [CrossRef] 42. Espin, J.C.; Varon, R.; Fenoll, L.G.; Gilabert, M.A.; Garcia-Ruiz, P.A.; Tudela, J.; Garcia-Canovas, F. Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase. Eur. J. Biochem. 2000, 267, 1270–1279. [CrossRef] 43. Toussaint, O.; Lerch, K.J.B. Catalytic oxidation of 2-aminophenols and ortho hydroxylation of aromatic amines by tyrosinase. Biochemistry 1987, 26, 8567–8571. [CrossRef] 44. Garcia-Molina, F.; Munoz, J.L.; Varon, R.; Rodriguez-Lopez, J.N.; Garcia-Canovas, F.; Tudela, J. A review on spectrophotometric methods for measuring the monophenolase and diphenolase activities of tyrosinase. J. Agric. Food Chem. 2007, 55, 9739–9749. [CrossRef] 45. Rodríguez-López, J.N.; Serna-Rodríguez, P.; Tudela, J.; Varón, R.; Garcia-Cánovas, F. A continuous spectrophotometric method for the determination of diphenolase activity of tyrosinase using 3,4-dihydroxymandelic acid. Anal. Biochem. 1991, 195, 369–374. [CrossRef] 46. Jimenez, M.; Garcia-Carmona, F.; Garcia-Canovas, F.; Iborra, J.L.; Lozano, J.A.; Martinez, F. Chemical intermediates in dopamine oxidation by tyrosinase, and kinetic studies of the process. Arch. Biochem. Biophys. 1984, 235, 438–448. [CrossRef] 47. Jiménez, M.; García-Cánovas, F.; García-Carmona, F.; Iborra, J.L.; Lozano, J.A. Isoproterenol oxidation by tyrosinase: Intermediates characterization and kinetic study. Biochem. Int. 1985, 11, 51–59. 48. Rzepecki, L.M.; Waite, J.H. A chromogenic assay for catecholoxidases based on the addition of L-proline to quinones. Anal. Biochem. 1989, 179, 375–381. [CrossRef] 49. Gauillard, F.; Richard-Forget, F.; Nicolas, J. New spectrophotometric assay for polyphenol oxidase activity. Anal. Biochem. 1993, 215, 59–65. [CrossRef][PubMed] 50. Ge, G.B.; Feng, L.; Jin, Q.; Wang, Y.R.; Liu, Z.M.; Zhu, X.Y.; Wang, P.; Hou, J.; Cui, J.N.; Yang, L. A novel substrate-inspired fluorescent probe to monitor native albumin in human plasma and living cells. Anal. Chim. Acta 2017, 989, 71–79. [CrossRef] [PubMed] 51. Wang, Y.-R.; Feng, L.; Xu, L.; Hou, J.; Jin, Q.; Zhou, N.; Lin, Y.; Cui, J.-N.; Ge, G.-B. An ultrasensitive and conformation sensitive fluorescent probe for sensing human albumin in complex biological samples. Sens. Actuators B Chem. 2017, 245, 923–931. [CrossRef] 52. Kamel, S.; Khattab, T.A.J.B. Recent Advances in Cellulose-Based Biosensors for Medical Diagnosis. Biosensors 2020, 10, 67. [CrossRef][PubMed] 53. Zhu, Y.D.; Pang, H.L.; Zhou, Q.H.; Qin, Z.F.; Jin, Q.; Finel, M.; Wang, Y.N.; Qin, W.W.; Lu, Y.; Wang, D.D.; et al. An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems. J. Pharm. Anal. 2020, 10, 263–270. [CrossRef] 54. Haghbeen, K.; Tan, E.W. Direct spectrophotometric assay of monooxygenase and oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates. Anal. Biochem. 2003, 312, 23–32. [CrossRef] 55. Munoz, J.L.; Garcia-Molina, F.; Varon, R.; Rodriguez-Lopez, J.N.; Garcia-Canovas, F.; Tudela, J. Calculating molar absorptivities for quinones: Application to the measurement of tyrosinase activity. Anal. Biochem. 2006, 351, 128–138. [CrossRef] 56. Lin, Y.F.; Hu, Y.H.; Lin, H.T.; Liu, X.; Chen, Y.H.; Zhang, S.; Chen, Q.X. Inhibitory effects of propyl gallate on tyrosinase and its application in controlling pericarp browning of harvested longan fruits. J. Agric. Food Chem. 2013, 61, 2889–2895. [CrossRef] [PubMed] Biosensors 2021, 11, 290 20 of 22

57. Winder, A.J.; Harris, H. New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase. Eur. J. Biochem. 1991, 198, 317–326. [CrossRef] 58. Moonrungsee, N.; Shimamura, T.; Kashiwagi, T.; Jakmunee, J.; Higuchi, K.; Ukeda, H. Sequential injection spectrophotometric system for evaluation of mushroom tyrosinase-inhibitory activity. Talanta 2012, 101, 233–239. [CrossRef] 59. Nolan, L.C.; O’Connor, K.E. A spectrophotometric method for the quantification of an enzyme activity producing 4-substituted phenols: Determination of toluene-4-monooxygenase activity. Anal. Biochem. 2005, 344, 224–231. [CrossRef] 60. Mann, T.; Scherner, C.; Röhm, K.H.; Kolbe, L. Structure-Activity Relationships of Thiazolyl Resorcinols, Potent and Selective Inhibitors of Human Tyrosinase. Int. J. Mol. Sci. 2018, 19, 690. [CrossRef] 61. Rescigno, A.; Sanjust, E.; Soddu, G.; Rinaldi, A.C.; Sollai, F.; Curreli, N.; Rinaldi, A. Effect of 3-hydroxyanthranilic acid on mushroom tyrosinase activity. Biochim. Biophys. Acta 1998, 1384, 268–276. [CrossRef] 62. Teng, Y.; Jia, X.; Li, J.; Wang, E. Ratiometric fluorescence detection of tyrosinase activity and dopamine using thiolate-protected gold nanoclusters. Anal. Chem. 2015, 87, 4897–4902. [CrossRef] 63. Zhang, W.H.; Ma, W.; Long, Y.T. Redox-Mediated Indirect Fluorescence Immunoassay for the Detection of Disease Biomarkers Using Dopamine-Functionalized Quantum Dots. Anal. Chem. 2016, 88, 5131–5136. [CrossRef] 64. Liu, S.; Tian, J.; Wang, L.; Sun, X. Highly sensitive and selective colorimetric detection of Ag(I) ion using 3,30,5,50,- tetramethylbenzidine (TMB) as an indicator. Sens. Actuators B Chem. 2012, 165, 44–47. [CrossRef] 65. Ni, P.; Sun, Y.; Dai, H.; Hu, J.; Jiang, S.; Wang, Y.; Li, Z. Highly sensitive and selective colorimetric detection of glutathione based on Ag [I] ion-3,30,5,50-tetramethylbenzidine (TMB). Biosens. Bioelectron. 2015, 63, 47–52. [CrossRef] 66. Lei, C.; Zhao, X.E.; Sun, J.; Yan, X.; Gao, Y.; Gao, H.; Zhu, S.; Wang, H. A simple and novel colorimetric assay for tyrosinase and inhibitor screening using 3,30,5,50-tetramethylbenzidine as a chromogenic probe. Talanta 2017, 175, 457–462. [CrossRef] 67. Deng, H.H.; Lin, X.L.; He, S.B.; Wu, G.W.; Wu, W.H.; Yang, Y.; Lin, Z.; Peng, H.P.; Xia, X.H.; Chen, W. Colorimetric tyrosinase assay based on catechol inhibition of the oxidase-mimicking activity of chitosan-stabilized platinum nanoparticles. Mikrochim Acta 2019, 186, 301. [CrossRef][PubMed] 68. Acuna, A.U.; Alvarez-Perez, M.; Liras, M.; Coto, P.B.; Amat-Guerri, F. Synthesis and photophysics of novel biocompatible fluorescent oxocines and azocines in aqueous solution. Phys. Chem. Chem. Phys. 2013, 15, 16704–16712. [CrossRef] 69. Zhao, J.; Bao, X.; Wang, S.; Lu, S.; Sun, J.; Yang, X. In Situ Fluorogenic and Chromogenic Reactions for the Sensitive Dual-Readout Assay of Tyrosinase Activity. Anal. Chem. 2017, 89, 10529–10536. [CrossRef] 70. Sun, W.; Li, M.; Fan, J.; Peng, X.J.A. Activity-Based Sensing and Theranostic Probes Based on Photoinduced Electron Transfer. ACC Chem Res. 2019, 52, 2818–2831. [CrossRef] 71. Zou, L.; Wang, P.; Qian, X.; Feng, L.; Yu, Y.; Wang, D.; Jin, Q.; Hou, J.; Liu, Z.; Ge, G.; et al. Bioelectronics, a highly specific ratiometric two-photon fluorescent probe to detect dipeptidyl peptidase IV in plasma and living systems. Biosens. Bioelectron. 2017, 90, 283–289. [CrossRef] 72. Zhang, B.; Zhang, H.; Zhong, M.; Wang, S.; Xu, Q.; Cho, D.-H.; Qiu, H. A novel off-on fluorescent probe for specific detection and imaging of cysteine in live cells and in vivo. Chin. Chem. Lett. 2020, 31, 153–155. [CrossRef] 73. Li, X.; Wang, H.; Zhang, Y.; Cao, Q.; Chen, Y.J.C.C.L. A GSH-responsive PET-based fluorescent probe for cancer cells imaging. Chin. Chem. Lett. 2020, 32, 1541–1544. [CrossRef] 74. Yang, S.; Jiang, J.; Zhou, A.; Zhou, Y.; Ye, W.; Cao, D.S.; Yang, R. Substrate-Photocaged Enzymatic Fluorogenic Probe Enabling Sequential Activation for Light-Controllable Monitoring of Intracellular Tyrosinase Activity. Anal. Chem. 2020, 92, 7194–7199. [CrossRef][PubMed] 75. Zhang, S.; Li, H.; Yao, Q.; Ghazali, S.; Fan, J.; Wang, J.; Gao, F.; Li, M.; Wang, H.; Dong, C.; et al. A unique two-photon fluorescent probe based on ICT mechanism for imaging palladium in living cells and mice. Chin. Chem. Lett. 2020, 31, 2913–2916. [CrossRef] 76. Jin, Q.; Feng, L.; Zhang, S.J.; Wang, D.D.; Wang, F.; Zhang, Y.; Cui, J.N.; Guo, W.Z.; Ge, G.B.; Yang, L.J.A.C. Real-Time Tracking the Synthesis and Degradation of Albumin in Complex Biological Systems with a near-Infrared Fluorescent Probe. Anal. Chem. 2017, 89, 9884–9891. [CrossRef][PubMed] 77. Singh, H.; Tiwari, K.; Tiwari, R.; Pramanik, S.K.; Das, A. Small Molecule as Fluorescent Probes for Monitoring Intracellular Enzymatic Transformations. Chem. Rev. 2019, 119, 11718–11760. [CrossRef] 78. Zhao, C.; An, J.; Zhou, L.; Fei, Q.; Wang, F.; Tan, J.; Shi, B.; Wang, R.; Guo, Z.; Zhu, W.H. Transforming the recognition site of 4-hydroxyaniline into 4-methoxyaniline grafted onto a BODIPY core switches the selective detection of peroxynitrite to hypochlorous acid. Chem. Commun. 2016, 52, 2075–2078. [CrossRef] 79. Lu, P.; Zhang, X.; Ren, T.; Yuan, L.J.C.C.L. Molecular engineering of ultra-sensitive fluorescent probe with large Stokes shift for imaging of basal HOCl in tumor cells and tissues. Chin. Chem. Lett. 2020, 31, 2980–2984. [CrossRef] 80. Peng, M.; Wang, Y.; Fu, Q.; Sun, F.; Na, N.; Ouyang, J. Melanosome-Targeting Near-Infrared Fluorescent Probe with Large Stokes Shift for in Situ Quantification of Tyrosinase Activity and Assessing Drug Effects on Differently Invasive Melanoma Cells. Anal. Chem. 2018, 90, 6206–6213. [CrossRef] 81. Sidhu, J.S.; Singh, A.; Garg, N.; Kaur, N.; Singh, N. A highly selective naphthalimide-based ratiometric fluorescent probe for the recognition of tyrosinase and cellular imaging. Analyst 2018, 143, 4476–4483. [CrossRef] 82. Wu, X.; Li, L.; Shi, W.; Gong, Q.; Ma, H. Near-Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design, Synthesis, and Application in Living Cells and Zebrafish. Angew. Chem. Int. Ed. Engl. 2016, 55, 14728–14732. [CrossRef][PubMed] Biosensors 2021, 11, 290 21 of 22

83. Zhang, J.; Li, Z.; Tian, X.; Ding, N. A novel hydrosoluble near-infrared fluorescent probe for specifically monitoring tyrosinase and application in a mouse model. Chem. Commun. 2019, 55, 9463–9466. [CrossRef][PubMed] 84. Wu, X.; Li, X.; Li, H.; Shi, W.; Ma, H. A highly sensitive and selective fluorescence off-on probe for the detection of intracellular endogenous tyrosinase activity. Chem. Commun. 2017, 53, 2443–2446. [CrossRef] 85. Li, H.; Liu, W.; Zhang, F.; Zhu, X.; Huang, L.; Zhang, H. Highly Selective Fluorescent Probe Based on Hydroxylation of Phenylboronic Acid Pinacol Ester for Detection of Tyrosinase in Cells. Anal. Chem. 2018, 90, 855–858. [CrossRef][PubMed] 86. Yan, S.; Huang, R.; Wang, C.; Zhou, Y.; Wang, J.; Fu, B.; Weng, X.; Zhou, X. A two-photon fluorescent probe for intracellular detection of tyrosinase activity. Chem. Asian. J. 2012, 7, 2782–2785. [CrossRef] 87. Wang, C.; Yan, S.; Huang, R.; Feng, S.; Fu, B.; Weng, X.; Zhou, X. A turn-on fluorescent probe for detection of tyrosinase activity. Analyst 2013, 138, 2825–2828. [CrossRef] 88. Zhan, C.; Cheng, J.; Li, B.; Huang, S.; Zeng, F.; Wu, S. A Fluorescent Probe for Early Detection of Melanoma and Its Metastasis by Specifically Imaging Tyrosinase Activity in a Mouse Model. Anal. Chem. 2018, 90, 8807–8815. [CrossRef] 89. Zhou, J.; Shi, W.; Li, L.; Gong, Q.; Wu, X.; Li, X.; Ma, H. Detection of Misdistribution of Tyrosinase from Melanosomes to Lysosomes and Its Upregulation under Psoralen/Ultraviolet A with a Melanosome-Targeting Tyrosinase Fluorescent Probe. Anal. Chem. 2016, 88, 4557–4564. [CrossRef] 90. Zhang, P.; Li, S.; Fu, C.; Zhang, Q.; Xiao, Y.; Ding, C. A colorimetric and near -infrared ratiometric fluorescent probe for the determination of endogenous tyrosinase activity based on cyanine aggregation. Analyst 2019, 144, 5472–5478. [CrossRef] 91. Li, X.; Shi, W.; Chen, S.; Jia, J.; Ma, H.; Wolfbeis, O.S. A near-infrared fluorescent probe for monitoring tyrosinase activity. Chem. Commun. 2010, 46, 2560–2562. [CrossRef] 92. Li, Z.; Wang, Y.F.; Zeng, C.; Hu, L.; Liang, X.J. Ultrasensitive Tyrosinase-Activated Turn-On Near-Infrared Fluorescent Probe with a Rationally Designed Urea Bond for Selective Imaging and Photodamage to Melanoma Cells. Anal. Chem. 2018, 90, 3666–3669. [CrossRef] 93. Naidu Bobba, K.; Won, M.; Shim, I.; Velusamy, N.; Yang, Z.; Qu, J.; Kim, J.S.; Bhuniya, S. A BODIPY-based two-photon fluorescent probe validates tyrosinase activity in live cells. Chem. Commun. 2017, 53, 11213–11216. [CrossRef][PubMed] 94. Kim, T.I.; Park, J.; Park, S.; Choi, Y.; Kim, Y. Visualization of tyrosinase activity in melanoma cells by a BODIPY-based fluorescent probe. Chem. Commun. 2011, 47, 12640–12642. [CrossRef] 95. Li, Z.; Xia, X.; You, Y.; Lu, C.; Yang, G.; Ma, C.; Nie, J.; Sun, Q.; Wu, S.; Ren, J.; et al. Fast-response fluorescent probe with favorable water solubility for highly sensitive imaging of endogenous tyrosinase in living cells and zebrafish model. Chin. Chem. Lett. 2021, 32, 1785–1789. [CrossRef] 96. Zhou, Q.H.; Qin, W.W.; Finel, M.; He, Q.Q.; Ge, G.B. A broad-spectrum substrate for the human UDP-glucuronosyltransferases and its use for investigating glucuronidation inhibitors. Int. J. Biol. Macromol. 2021, 180, 252–261. [CrossRef] 97. Qiang, J.A.; Hm, A.; Lei, F.B.; Ping, W.A.; Rh, A.; Jing, N.B.; Ling, Y.A.; Gg, A.J.C.C.L. Sensing cytochrome P450 1A1 activity by a resorufin-based isoform-specific fluorescent probe. Chin. Chem. Lett. 2020, 31, 2945–2949. 98. Dl, A.; Wc, A.; Sheng, H.; Xc, B.; Jya, B.J.C.C.L. The regulation of biothiol-responsive performance and bioimaging application of benzo[c][1,2,5]oxadiazole dyes. Chin. Chem. Lett. 2020, 31, 2891–2896. 99. Liu, G.; Zhao, J.; Lu, S.; Wang, S.; Sun, J.; Yang, X. Polymethyldopa Nanoparticles-Based Fluorescent Sensor for Detection of Tyrosinase Activity. ACS Sens. 2018, 3, 1855–1862. [CrossRef][PubMed] 100. Ding, Y.Z.; Wang, W.F.; Chai, T.; Qiang, Y.; Shi, Y.P.; Yang, J.L. Ratiometric target-triggered fluorescent silicon nanoparticles probe for quantitative visualization of tyrosinase activity. Talanta 2019, 197, 113–121. [CrossRef] 101. Wang, H.; Lu, Y.; Wang, L.; Chen, H. Detection of tyramine and tyrosinase activity using red region emission NaGdF4:Yb, Er@NaYF4 upconversion nanoparticles. Talanta 2019, 197, 558–566. [CrossRef] 102. Pinho, J.O.; Matias, M.; Gaspar, M.M. Emergent Nanotechnological Strategies for Systemic Chemotherapy against Melanoma. Nanomaterials 2019, 9, 1455. [CrossRef][PubMed] 103. Kounalakis, N.; Goydos, J.S. Tumor cell and circulating markers in melanoma: Diagnosis, prognosis, and management. Curr. Oncol. Rep. 2005, 7, 377–382. [CrossRef] 104. Reintgen, D.; Cruse, C.; Wells, K.; Berman, C.; Fenske, N.; Glass, F.; Schroer, K.; Heller, R.; Ross, M.; Lyman, G.; et al. The orderly progression of melanoma nodal metastases. Ann. Surg. 1994, 220, 759–767. [CrossRef] 105. Parkin, D.M.; Pisani, P.; Ferlay, J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int. J. Cancer 1999, 80, 827–841. [CrossRef] 106. Heo, J.R.; Kim, N.H.; Cho, J.; Choi, K.C. Current treatments for advanced melanoma and introduction of a promising novel gene therapy for melanoma (Review). Oncol. Rep. 2016, 36, 1779–1786. [CrossRef][PubMed] 107. King, R.A.; Pietsch, J.; Fryer, J.P.; Savage, S.; Brott, M.J.; Russell-Eggitt, I.; Summers, C.G.; Oetting, W.S.J.H.G. Tyrosinase gene mutations in oculocutaneous albinism1 (OCA1): Definition of the phenotype. Hum Genet. 2003, 113, 502–513. [CrossRef] [PubMed] 108. Smith, B.; Selby, P.; Southgate, J.; Pittman, K.; Bradley, C.; Blair, G.E. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 1991, 338, 1227–1229. [CrossRef] 109. Palmieri, G.; Ascierto, P.A.; Perrone, F.; Satriano, S.M.; Ottaiano, A.; Daponte, A.; Napolitano, M.; Caraco, C.; Mozzillo, N.; Melucci, M.T.; et al. Prognostic value of circulating melanoma cells detected by reverse transcriptase-polymerase chain reaction. J. Clin. Oncol. 2003, 21, 767–773. [CrossRef][PubMed] Biosensors 2021, 11, 290 22 of 22

110. Quaglino, P.; Savoia, P.; Osella-Abate, S.; Bernengo, M.G. RT-PCR tyrosinase expression in the peripheral blood of melanoma patients. Expert Rev. Mol. Diagn. 2004, 4, 727–741. [CrossRef][PubMed] 111. Visús, C.; Andres, R.; Mayordomo, J.I.; Martinez-Lorenzo, M.J.; Murillo, L.; Sáez-Gutiérrez, B.; Diestre, C.; Marcos, I.; Astier, P.; Godino, J.J.M.R. Prognostic role of circulating melanoma cells detected by reverse transcriptase-polymerase chain reaction for tyrosinase mRNA in patients with melanoma. Melanoma Res. 2007, 17, 83–89. [CrossRef][PubMed] 112. Mendes, E.; Perry, M.D.J.; Francisco, A.P. Design and discovery of mushroom tyrosinase inhibitors and their therapeutic applications. Expert Opin. Drug Dis. 2014, 9, 533–554. [CrossRef] 113. Ullah, S.; Son, S.; Yun, H.Y.; Kim, D.H.; Chun, P.; Moon, H.R. Tyrosinase inhibitors: A patent review (2011–2015). Expert Opin. Ther. Pat. 2016, 26, 347–362. [CrossRef] [PubMed] 114. Leyden, J.J.; Shergill, B.; Micali, G.; Downie, J.; Wallo, W. Natural options for the management of hyperpigmentation. J. Eur. Acad Derm. Venereol 2011, 25, 1140–1145. [CrossRef] 115. Zhu, T.H.; Cao, S.W.; Yu, Y.Y. Synthesis, characterization and biological evaluation of paeonol thiosemicarbazone analogues as mushroom tyrosinase inhibitors. Int. J. Biol. Macromol. 2013, 62, 589–595. [CrossRef][PubMed] 116. Kubo, I.; Kinst-Hori, I.; Chaudhuri, S.K.; Kubo, Y.; Sanchez, Y.; Ogura, T. Flavonols from Heterotheca inuloides: Tyrosinase inhibitory activity and structural criteria. Bioorg. Med. Chem 2000, 8, 1749–1755. [CrossRef] 117. Jow, T.; Hantash, B.M. Hydroquinone-induced depigmentation: Case report and review of the literature. Dermatitis 2014, 25, e1–e5. [CrossRef] 118. Draelos, Z.D. Skin lightening preparations and the hydroquinone controversy. Dermatol. Ther. 2007, 20, 308–313. [CrossRef] 119. Searle, T.; Al-Niaimi, F.; Ali, F.R. The top 10 cosmeceuticals for facial hyperpigmentation. Dermatol. Ther. 2020, 33, e14095. [CrossRef] 120. Zhou, H.; Zhao, J.; Li, A.; Reetz, M.T. Chemical and Biocatalytic Routes to Arbutin (dagger). Molecules 2019, 24, 3303. [CrossRef] 121. Burnett, C.L.; Bergfeld, W.F.; Belsito, D.V.; Hill, R.A.; Klaassen, C.D.; Liebler, D.C.; Marks, J.G., Jr.; Shank, R.C.; Slaga, T.J.; Snyder, P.W.; et al. Final report of the safety assessment of Kojic acid as used in cosmetics. Int. J. Toxicol. 2010, 29, 244S–273S. [CrossRef] 122. Saeedi, M.; Eslamifar, M.; Khezri, K. Kojic acid applications in cosmetic and pharmaceutical preparations. Biomed. Pharm. 2019, 110, 582–593. [CrossRef][PubMed] 123. Ashooriha, M.; Khoshneviszadeh, M.; Khoshneviszadeh, M.; Rafiei, A.; Kardan, M.; Yazdian-Robati, R.; Emami, S. Kojic acid- natural product conjugates as mushroom tyrosinase inhibitors. Eur. J. Med. Chem. 2020, 201, 112480. [CrossRef] 124. Tajima, R.; Oozeki, H.; Muraoka, S.; Tanaka, S.; Motegi, Y.; Nihei, H.; Yamada, Y.; Masuoka, N.; Nihei, K. Synthesis and evaluation of bibenzyl glycosides as potent tyrosinase inhibitors. Eur. J. Med. Chem. 2011, 46, 1374–1381. [CrossRef][PubMed] 125. Ishioka, W.; Oonuki, S.; Iwadate, T.; Nihei, K.I. Resorcinol alkyl glucosides as potent tyrosinase inhibitors. Bioorg. Med. Chem. Lett. 2019, 29, 313–316. [CrossRef][PubMed] 126. Jung, H.J.; Noh, S.G.; Park, Y.; Kang, D.; Chun, P.; Chung, H.Y.; Moon, H.R. In vitro and in silico insights into tyrosinase inhibitors with (E)-benzylidene-1-indanone derivatives. Comput. Struct Biotechnol. J. 2019, 17, 1255–1264. [CrossRef] 127. Durai, P.; Ko, Y.J.; Kim, J.C.; Pan, C.H.; Park, K. Identification of Tyrosinase Inhibitors and Their Structure-Activity Relationships via Evolutionary Chemical Binding Similarity and Structure-Based Methods. Molecules 2021, 26, 566. [CrossRef] 128. Wang, D.; Zou, L.; Jin, Q.; Hou, J.; Ge, G.; Yang, L. Human carboxylesterases: A comprehensive review. Acta Pharm. Sinica. B 2018, 8, 699–712. [CrossRef][PubMed] 129. Ando, H.; Kondoh, H.; Ichihashi, M.; Hearing, V.J. Approaches to identify inhibitors of melanin biosynthesis via the quality control of tyrosinase. J. Investig. Derm. 2007, 127, 751–761. [CrossRef][PubMed] 130. Park, S.J.; Kim, Y.J.; Kang, J.S.; Kim, I.Y.; Choi, K.S.; Kim, H.M. Carboxylesterase-2-Selective Two-Photon Ratiometric Probe Reveals Decreased Carboxylesterase-2 Activity in Breast Cancer Cells. Anal. Chem. 2018, 90, 9465–9471. [CrossRef][PubMed] 131. Wu, X.; Shi, W.; Li, X.; Ma, H. Recognition Moieties of Small Molecular Fluorescent Probes for Bioimaging of Enzymes. Acc. Chem. Res. 2019, 52, 1892–1904. [CrossRef] 132. Li, S.; Hu, R.; Wang, S.; Guo, X.; Zeng, Y.; Li, Y.; Yang, G. Specific Imaging of Tyrosinase in Vivo with 3-Hydroxybenzyl Caged D-Luciferins. Anal. Chem. 2018, 90, 9296–9300. [CrossRef] 133. Yi, X.; Wang, F.; Qin, W.; Yang, X.; Yuan, J. Near-infrared fluorescent probes in cancer imaging and therapy: An emerging field. Int. J. Nanomed. 2014, 9, 1347–1365. [CrossRef][PubMed] 134. Vendrell, M.; Zhai, D.; Er, J.C.; Chang, Y.T. Combinatorial strategies in fluorescent probe development. Chem. Rev. 2012, 112, 4391–4420. [CrossRef][PubMed] 135. Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P.L.; Urano, Y. New strategies for fluorescent probe design in medical diagnostic imaging. Chem. Rev. 2010, 110, 2620–2640. [CrossRef][PubMed] 136. Pode, Z.; Peri-Naor, R.; Georgeson, J.M.; Ilani, T.; Kiss, V.; Unger, T.; Markus, B.; Barr, H.M.; Motiei, L.; Margulies, D. Protein recognition by a pattern-generating fluorescent molecular probe. Nat. Nanotechnol. 2017, 12, 1161–1168. [CrossRef][PubMed]