In Situ Fluorogenic and Chromogenic Reactions for the Sensitive Dual

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In Situ Fluorogenic and Chromogenic Reactions for the Sensitive Dual-Readout Assay of Tyrosinase Activity Jiahui Zhao, Xingfu Bao, Shuang Wang, Shasha Lu, Jian Sun, and Xiurong Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02739 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 11, 2017

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Analytical Chemistry

In Situ Fluorogenic and Chromogenic Reactions for the Sensitive Dual-Readout Assay of Tyrosinase Activity Jiahui Zhao†,§, Xingfu Bao†, Shuang Wang†,‡, Shasha Lu†,‡, Jian Sun*,†, and Xiurong Yang*,† †State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China §University of Chinese Academy of Sciences, Beijing 100049, China ‡

University of Science and Technology of China, Hefei, Anhui 230026, China

*Fax: +86 431 85689278. E-mail: [email protected] ABSTRACT: As a well-known copper-containing oxidase, tyrosinase has been anticipated to serve as the biomarker of skin diseases. We describe here an exquisite label-free fluorescent and colorimetric dual-readout assay of its activity, inspired by the specific oxidation ability of monophenolamine substrates to catecholamines and a unique fluorogenic reaction between resorcinol and catecholamines. By employing commercially available tyramine as the model substrate (dopamine as the product), it is found that the tyrosinase-incubated tyramine solution exhibits obvious pale yellow with intense blue fluorescence in the presence of resorcinol and O2, where the absorbance and fluorescence intensity are directly related to the concentration of added tyrosinase (i.e., the amount of conversion of tyramine to dopamine). The overall process of sensing tyrosinase activity takes less than 100 minutes at ambient temperature and pressure conditions with exceedingly simple operation procedure, explicit response mechanism, and formation of fluorophore with high quantum yield from scratch. Furthermore, such a convenient, rapid, cost-effective, and highly sensitive dual-readout assay exhibits promising prospect for the tyrosinase activity in extensive bioassays and clinic researches, as well as in screening potential tyrosinase inhibitors.

Tyrosinase is widely distributed in plants and animal tissues, and plays crucial roles in many physiological processes, such as plants browning reactions1, melanogenesis in mammals2, insect cuticle sclerotization3 and so on. As a copper-containing oxidase, tyrosinase possesses two binding sites for aromatic substrates, which has activity for both monophenols and catechols, involving two distinct reactions in melanin synthesis. Its monophenolase activity is catalytic hydroxylation of monophenol to o-diphenol, while the diphenolase activity is processed in the catalytic oxidation of o-diphenol to corresponding o-quinone in the presence of molecular oxygen. In the clinic research, tyrosinase serves as the biomarker of skin diseases such as melanoma cancer and vitiligo, because of its overexpression in melanoma cancer cells4,5 and acting as an autoantigen in vitiligo6,7. Thus, the inhibitors of tyrosinase have been widely exploited as effective constituents for skin-whitening product in cosmetic industry.8 In addition, tyrosinase has implications on dopamine neurotoxicity and neurodegeneration in Parkinson’s disease.9,10 Therefore, the development of highly sensitive tyrosinase activity assays and corresponding inhibitor screening is of great value in providing effective diagnostic approaches or therapeutic targets in biomedical research, as well as in cosmetic industry. Until now, various sensing strategies have been already proposed to determine the tyrosinase activity, including radiometric11, electrochemical12-15, and spectroscopic 16-18 methods . Among them, spectroscopic methods are well-suited for in situ analysis and high-throughput screening

researches.19,20 Particularly, fluorometric assays have attracted considerable attentions due to the simple and rapid implementation, high sensitivity and spatial resolution, as well as real-time monitoring specialties.21-23 As far as we know, serval current fluorescent sensing platforms have typically utilized the fluorescence quenching ability of o-quinone moiety, the tyrosinase-catalyzed oxidation product based on the diphenolase activity.24,25 In virtue of some fluorophores and catechol derivatives26,27 (usually dopamine) as the substrates of tyrosinase, a variety of fluorescence turn-off assays have been developed based on the electron transfer from fluorophore to o-quinone and the resultant fluorescence quenching.25,28 In this regard, various fluorescent materials from simple fluorescent dye or conjugated polymer to functional nanomaterials have been prepared and employed to monitor the tyrosinase activity.29-32 Such diphenolase activity and quinone-based sensing systems have conceivable response mechanism and certain application ability, however, the preparation or modification processes of these fluorescent materials are generally laborious and time-consuming. Meanwhile, the fluorescence turn-off response mechanism is usually accompanied with the high background and relatively low sensitivity. On the other hand, Ma et al. has recently reported several novel tyrosinase-recognition fluorescent small molecules by incorporating substrate moieties into the fluorophore structures.19,33 In presence of tyrosinase, the as-prepared substrate molecules containing monophenol groups can be catalytically oxidized to o-diphenol or corresponding

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o-quinone, which subsequently undergo a spontaneous intramolecular electron rearrangement to eliminate the o-quinone moiety and induce a fluorescence response.34 By adjusting the tyrosinase-response moiety and fluorophore structure, a series of near-infrared fluorescence turn on probes based on the conversion of non-fluorescent substrate molecules to fluorescent ones have been developed and utilized into the tyrosinase detection in complex biosystems.16 However, the preparation and purification processes of such new substrates are still time-consuming and apparently require extensive synthesis experience and formidable background of organic chemistry. Undoubtedly, it is still highly desirable to develop more rational, straightforward and sensitive tyrosinase activity assays, especially taking advantage of the commercially available common molecules with fluorescence turn-on response. As typical catechol derivatives, catecholamines such as dopamine and levodopa have been generally employed as the substrate and oxidized to dopamine-quinone/dopaquinone in several effective tyrosinase activity assays. However, catecholamines should also be considered as the tyrosinase-catalyzed oxidation product of monophenolamines (typically tyramine or tyrosine) based on the monophenolase activity in fact. In 1991, Prota et al. has found that the resorcinol can be used for oxidative coupling of levodopa and significantly inhibit the oxidative conversion of levodopa to dopachrome in an aqueous solution.35 Acuna et al. has recently described in detail the reactions between resorcinol and catecholamines, and identified the rapid generation of fluorescent azocine products at ambient temperature and pressure.36 More significantly, we have further confirmed that similar fluorogenic reactions cannot be implemented and no fluorescent product can be obtained if using the monophenolamines instead of catecholamines. Inspired by such specific fluorogenic reactions between resorcinol and catecholamines, we have proposed to introduce it into the detection of tyrosinase, which is capable of catalyzing the oxidation of monophenolamine substrates to catecholamines. In the subsequent proof of concept of fluorescent tyrosinase activity assay, we have first chosen tyramine as the monophenolamine substrates of tyrosinase, and then demonstrated that the tyrosinase-incubated tyramine solution could emit intense blue fluorescence under ultraviolet light in the presence of resorcinol. The overall process takes less than 100 min with exceedingly facile operation procedure at ambient temperature and pressure conditions, and accompanied with considerable fluorescence quantum yield of 32.5% for the product solution. At the same time, the resultant fluorescent azocine product of the sensing system exhibits obvious pale yellow under visible light and possesses characteristic absorption peak centered at 420 nm. Therefore, we have developed a fluorometric and colorimetric dual read-out assay for tyrosinase activity based on the formation of fluorophore from scratch, and the fluorescence intensities and absorption values of the tyramine-resorcinol system increased gradually with the tyrosinase concentrations. Such innovative in situ fluorogenic reaction-based sensing system can further be extended into the inhibitor screening of tyrosinase. Kojic acid and benzoic acid, the common inhibitors of tyrosinase, are employed to assess the function of inhibitor screening, and IC50 of these two inhibitors are calculated to be approximate 155 µM and 1582 µM, respectively. Furthermore, the proposed system exhibits promising prospect for

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tyrosinase activity assay in extensive bioassays and clinic researches, as well as in screening potential tyrosinase inhibitors. EXPERIMENTAL SECTION

Chemicals and Materials. Sodium carbonate, resorcinol, tyrosine, tyramine, dopamine, levodopa, dibasic dodium phosphate and sodium dihydrogen phosphate were purchased from Aladdin Industrial Corporation (Shanghai, China). Tyrosinase from mushroom (EC 1.14.18.1), EcoR Ⅰ, bovine serum albumin (BSA), human serum albumin (HSA), alkaline phosphatase (ALP), acetylcholinesterase (AchE), glucose oxidase (GOX), lysozyme, and trypsin were purchased from Sigma-Aldrich. The ultrapure water from a Millipore system was used in all aqueous solution. All reagents were analytical grade and used as received without any further purification. Apparatus and Characterization. Fluorescence excitation and emission spectra of all samples were recorded on a Hitachi F-4600 spectrofluorometer (Tokyo, Japan). Absorption spectra were obtained with a CARY 500 UV−Vis−NIR Varian spectrophotometer (CA, USA). The mass spectrum analysis was performed on the electrospray ionization (ESI)-Q-TOF SYNAPT G2 High Definition Mass Spectrometer (Waters, UK) in the negative ion mode. Dopamine Concentration Response. In 2.0 mL microcentrifugals, volumes of 100 µL of phosphate buffer (PB, 200 mM, pH = 7.0) and 1400 µL of water were injected, 200 µL of dopamine aqueous solution with different dopamine concentrations ranging from 0 to 100 µM were added, respectively. 200 µL of 1.0 mM resorcinol solution and 100 µL of 500 mM sodium carbonate solution were successively injected into the above mixture solutions. The fluorescence spectra and measurements were carried out after vibration at room temperature for 5 min. Sensing Tyrosinase Activity. A fluorescent tyrosinase activity assay was performed using the following procedures. Volumes of 100 µL of PB (200 mM, pH = 7.0), 200 µL of 2.0 mM tyramine aqueous solution, 1250 µL of water were injected into a 2.0 mL microcentrifugal tube. Then 200 µL of tyrosinase aqueous solution with different activities ranging from 0 to 8 U mL-1 were added into the mixtures, respectively. After incubation at 37°C for 90 min, 200 µL of 2.0 mM resorcinol solution and 100 µL of 500 mM sodium carbonate solution were successively injected into the above mixture solutions. The fluorescence and absorption spectra measurements were carried out after vibration at room temperature for 5 min. The selectivity of this sensing system was assessed by using other control proteins/enzymes and biological cations in the absence and presence of tyrosinase, respectively. Real Sample Detection. The proposed fluorescence assay was applied to detect the tyrosinase activity in the diluted fetal bovine serum (1%) and human serum samples (1%), respectively. Tyrosinase Inhibition Assay. Kojic acid and benzoic acid, common inhibitors of tyrosinase, were employed to assess the function of inhibitor screening. We initially added various concentrations of kojic acid and benzoic acid solutions into the freshly prepared tyrosinase solution (4 U mL-1), respectively. After vibration at 37°C for 10 min, these inhibitor-treated tyrosinase solutions were injected into tyramine (200 µM) solution in PB (10 mM, pH = 7.0). After incubation at 37°C


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for 90 min, the resorcinol and sodium carbonate solution were successively injected. The fluorescence spectra measurements were carried out after vibration at room temperature for 5 min.

RESULTS AND DISCUSSION

Fluorogenic Reactions between Catecholamines and Resorcinol. As typical catechol derivatives, catecholamines, especially dopamine used to be employed as the substrate in the tyrosinase activity assays, where the chemical or physical properties between dopamine and the corresponding enzymolysis product dopamine-quinone can be differentiated by several sensors.24,25,29 However, dopamine should also be considered as the tyrosinase-catalyzed oxidation product of tyramine based on the monophenolase activity in fact. Recently, it is reported that a category of biocompatible fluorogenic reactions between 1,3-dihydroxybenzene (resorcinol) and 1,2-dihydroxybenzene derivative (catechols or catecholamines) would take place in the aqueous solution, to yield fluorescent oxocines or azocines.36 In the case of dopamine (Scheme 1), the colorless and non-emissive aqueous solution could quickly convert into pale yellow, with bright blue-emitting fluorescence owing to the generation of azamonardine fluorophore in the presence of resorcinol under alkaline condition at room temperature. We have further evaluated the specificity and optimum condition of the reaction by altering experimental parameters. It is suggested that vigorous stirring in the open air can obviously accelerate the fluorogenic reaction due to the ceaseless dissolution of molecular oxygen in aqueous solution (data not shown).

Scheme 1. Chemical structure of monophenolamines substrates (tyramine, tyrosine), catecholamines (dopamine, levodopa), resorcinol, and the corresponding azocine products (azamonardine, carboxyazamonardine).

Particularly, we have carried out several experiments to investigate the influences of pH, resorinol concentrations, reaction time on the fluorogenic reaction between dopamine and resorcinol. The assessment of the pH value of the reaction system was firstly performed at different pH ranging from 5 to 12, and the results were shown in Figure S1B. It could be indicated that the optimal pH value is 11, which happens to be similar to the pH of Na2CO3 solution in fact. Thus, we have chosen to employ Na2CO3 solution as the buffer solution in the subsequent experiments, and a concentration of 25 mM was adopted after the optimization of the concentration of Na2CO3 solution (Figure S1D). At a constant dopamine concentration (100 µM), both the fluorescence intensity and absorption value increased with resorcinol concentrations ranging from 0 to 100 µM (Figure S2), implying that it is equimolar reaction between dopamine and resorcinol. Furthermore, the fluorescence spectra of the reaction solution were discontinuously measured at room temperature to evaluate the effect of reaction time. The results in Figure S3 displayed that the fluorescence

intensities at 460 nm enhanced with the reaction time, and the fluorescence intensities and absorption values reaches the maximum values at 5 min and keep constant after that, demonstrating the reactions could be drastically completed in only 5 min. In this scheme, catecholamine oxidation, first to semiquinone and then to the corresponding quinone, is required for the formation of the fluorescent product, and the nucleophilic attack of resocinol monoanion under alkaline condition to the quinone species is the crucial initial reaction step36. Under alkaline condition, the OH group in azamonardine became anionic, accompanied with intense absorption band and strong fluorescence, and the reaction time shortened considerably as well. Hence, it can be concluded that the fluorogenic and chromogenic reaction between dopamine and resorcinol is rapid and specific with explicit mechanism in an alkaline aqueous solution at ambient temperature and pressure conditions. In contrast, we have speculated that the monophenolamines would not be oxidated under alkaline condition by resorcinol to yield fluorophores owing to the absence of o-diphenol and corresponding o-quinone moiety, which is an essential prerequisite to the aforementioned fluorogenic reactions. In order to verify our assumption, we have investigated the absorption and fluorescence spectra of tyramine, dopamine, resorcinol and the special mixture solutions, respectively (Figure 1). After resorcinol was introduced to dopamine solution under alkaline condition in the presence of O2, a typically intense absorption band centered at 420 nm appeared (line 5), and bright blue-emitting fluorescence was observed when excited at 415 nm, which is analogous to the characteristic peak of the product of the abovementioned fluorogenic reactions as reported in the literature36. In that literature, the NMR and mass spectra analysis of the product solution have further conformed the structural formula of the product (azamonardine) with a molecular weight of 259 Da.36 Under the same reaction condition, we submitted the resultant solution of dopamine reacting with resorcinol to ESI-Mass spectrum in the negative ion mode. As shown in Figure S5A, the ion peak at m/z=258.02 (blue line) was regarded as the quasi-molecular ion peaks of azamonardine and attributed to [azamonardine-H]-, and in our opinion, the product is exactly azamonardine. However, if we choose tyramine instead of dopamine, there is no typical absorption and negligible fluorescence of the mixture solution under the similar experimental conditions (line 4). The above distinction of fluorescence and absorption spectra caused by tyramine and dopamine may lay the foundation for tyrosinase assay based on its monophenol oxidation activity.

Figure 1. (A) Schematic representation of dopamine reacting with resocinol in synthesis of fluorescent azamonardine. (B)



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Absorption and (C) fluorescence spectra of tyramine (1), dopamine (2), resorcinol (3), mixture of tyramine and resorcinol (4) and mixture of dopamine and resorcinol (5) under the same condition, respectively. The insert photographs show the corresponding solution under UV lamp at 365 nm.

Design and Establishment of Tyrosinase Sensing System. Since buffer solution is usually required in clinical and bioassay applications, we carried out the above fluorogenic reactions in 10 mM PB to assess the influence of the buffer on the fluorogenic reactions, and the results displayed no significant change of the fluorescence intensity (Figure S4), indicating the latent ability in bioanalysis and clinic diagnosis values. On the other hand, we have investigated the effect of dopamine concentration on the absorption and fluorescence spectra of the resultant solution at the constant resorcinol level. As shown in Figure 2, the absorbance value at 420 nm and fluorescence intensity at 460 nm of the product solution enhanced gradually with dopamine concentrations ranging from 0 to 100 µM. Besides, there was no change of the shape of the absorbance and fluorescence spectra, reflecting the formation of the same and unitary product with different concentrations of dopamine. Thus, the dopamine concentration-dependent linear relationships further offer favorable conditions for tyrosinase sensing.

Figure 2. (A)Absorption and (C) fluorescence emission spectra at different concentrations of dopamine. (B) Absorption values at 420 nm and (D) fluorescence intensities at 460 nm under different concentrations of dopamine.

Inspired by the different fluorogenic reaction capacity with resorcinol between tyramine and dopamine, we have proposed to utilize tyramine as substrate to assay the activity of tyrosinase. Tyramine is a common and commercial available substrate of tyrosinase, while it has been scarcely employed in tyrosinase activity sensing. Tyrosinase is capable of catalyzing the hydroxylation of tyramine to dopamine based on its monophenol oxidation activity in the presence of O2. After resorcinol was injected into the preincubated tyramine solution in the absence of tyrosinase, the absorption spectra displayed simply the summation of the characteristic peak of tyramine and resorcinol, and there was no new typical absorption peak and negligible fluorescence (black line in Figure 3 B, C). Besides, there was no obvious ion peak at m/z=258.02 in ESI-Mass spectrum of the resultant solution (Figure S5B), which indicates azamonardine could not be generated in the mixture solution of tyramine and resorcinol without tyrosinase preincubation. In contrast, after resorcinol solution was added into the tyramine solution pre-incubated with tyrosinase, the typical absorption band centered at 420 nm (red line in Figure

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3 B, C) attributed to characteristic peak of azamonardine fluorophore (blue line in Figure 3 B, C) with strong blue fluorescence, and the ion peaks at m/z=258.02 attributed to the quasi-molecular ion peaks of azamonardine (blue line in Figure S5C). All of above results demonstrate the pre-introduction of tyrosinase essentially triggers fluorogenic reaction through the hydroxylation of tyramine as we expected (Figure 3A). Thus, the ignorable fluorescence and absorbance signal in the absence of tyrosinase illustrates that tyramine could not participate in the fluorogenic reaction, and fluorescence “turn-on” assay of tyrosinase activity could be accomplished with tyramine as the substrate specifically. Most significantly, the distinction of fluorescence intensities and absorbance values caused by tyrosinase demonstrates tyrosinase-dependent fluorometric and colorimetric signal, which is expected to be exploited for tyrosinase activity assay and its inhibitor screening.

Figure 3. (A) Schematic representation of tyrosinase-enabled in situ synthesis of fluorescent azamonardine. (B) Absorption spectra of product solution of the fluorogenic reactions using tyramine in the absence (black line) and presence (red line) of tyrosinase, respectively, and azamonardine (blue line), fluorescence excitation and emission spectra of the product of tyrosinase-incubated tyramine solution in aqueous solution. The insert photographs show the tyrosinase-incubated tyramine solution under natural light (left) and UV lamp at 365 nm (right), respectively. (C) Fluorescence emission spectra of corresponding product solution excited at 415 nm.

Afterwards several experiments have been implemented to investigate the effect of tyramine concentration, resorinol concentration and incubation time on the fluorescence intensity of the proposed system. The concentrations of both tyramine and resorinol were firstly optimized to 200 µM (Figure S6). Moreover, the incubation time of tyrosinase was evaluated with 200 µM tyramine under different levels of tyrosinase (1, 2, 4 U mL-1) in 10 mM PB (pH=7), and the results in Figure S7 illustrated that the fluorescence intensities enhanced promptly with the incubation time in the first 90 min, and then increased gently or keep constant between 90-150 min. In order to save time, incubation time of 90 min was recommended in the subsequent experiment. More importantly, resorcinol and its derivatives served as the inhibitor of tyrosinase have been widely used to reduce the pigmentation in humans37-39, which means that the addition of resorcinol to the tyrosinase-incubated tyramine solution could terminate the function of tyrosinase in the following fluorogenic reaction. It has been verified by the pre-treatment of resorcinol with tyrosinase, which generates negligible fluorescence under the same condition, as shown in Figure S8.


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In a word, the proposed tyrosinase-enabled fluorogenic and chromogenic reaction could be implemented efficiently with 200 µM tyramine pre-incubated with tyrosinase for 90 min, followed by addition of 200 µM resorcinol with reaction time of 5 min in 25 mM Na2CO3 solution at room temperature. The overall process takes less than 100 minutes with easy operation procedure. Furthermore, the fluorescence quantum yield of the resultant mixture solution have been measured and calculated to be as high as 32.5%, which indicates great potential and high sensitivity in the “turn-on” assay of tyrosinase and its inhibitor screening. Fluorescent and Colorimetric Dual-Readout Assays of Tyrosinase Activity. Under the above optimal conditions, we developed a fluorometric and colorimetric dual read-out sensing assay for tyrosinase activity by using tyramine as the substrate. The assay of tyrosinase activity was carried out by using 200 µM tyramine as substrate in 10 mM PB at pH 7.0 with an incubation time of 90 min, followed by adding of resorcinol and Na2CO3 solutions to ultimate concentrations of 200 µM and 25 mM, respectively. The fluorescence spectra and absorbance spectra measurements were carried out after vibration at room temperature for 5 min. As shown in Figure 4A, the fluorescence intensity at 460 nm of the product solution increased as tyrosinase concentrations ranging from 0 to 8 U mL-1. In particular, there is a linear relationship between fluorescence intensities and tyrosinase activities over the range from 0.005 to 3.5 U mL-1, where the fitted linear equation can be described as I = − 5.06 + 365.4 Ctyrosinase (U mL-1), R2 = 0.993, as depicted in Figure 4B. Moreover, the absorption values at 420 nm increased as tyrosinase concentrations ranging from 0 to 8 U mL-1, and there is a linear relationship between absorption values and tyrosinase activities over the range from 0.01 to 8.0 U mL-1, where the fitted linear equation can be described as I = − 0.0062 + 0.107 Ctyrosinase (U mL-1), R2 = 0.992, as depicted in Figure 4D. In addition, the fluorescent color change of the product solutions induced by 0.1 U mL-1 of tyrosinase activity can be detected with naked-eye readout under the UV lamp, and color change of the product solutions induced by 0.5 U mL-1 of tyrosinase activity can be detected under the natural light (Figure 4E). It is noteworthy that fluorescent readout assay of the proposed system displays higher sensitivity while colorimetric readout assay exhibits wider linear range. More importantly, our label-free fluorescent and colorimetric dual-readout assay reveals a low limit of quantitation and a wide linear range compared to those of spectroscopic tyrosinase assay methods in the previously reported literatures in Table S1.

Figure 4. (A) Fluorescence emission spectra at different tyrosinase concentrations excited at 415 nm. (B) Fluorescence intensities at 460 nm versus different tyrosinase concentrations. (C) Absorbance spectra enhancement with different tyrosinase concentrations. (D) Absorbance values at 420 nm versus different tyrosinase concentrations. (E) Corresponding photographs under 365 nm UV light (1) and natural light (2). (F) Fluorescence responses of the proposed system against the control enzymes (2 U mL-1) or proteins (10 µg mL-1) and biological cation (250 µM) except for Cu2+ (50 µM) in the absence and presence of tyrosinase (2 U mL-1).

Similarly, the assay of tyrosinase activity can also be realized by using tyrosine as substrate, in which tyrosine can be transformed to levodopa by tyrosinase in the presence of O2, followed by generating strong fluorescent carboxyazamonardine36 after the introduce of resorcinol under similar conditions. The fluorescence intensities at 460 nm of the product solution varied as tyrosinase concentrations ranging from 0 to 8 U mL-1 as well, and there is a linear relationship between fluorescence intensities and tyrosinase activities over the range from 0.01 to 3 U mL-1, where the fitted linear equation can be described as I = − 10.7+ 224.4 Ctyrosinase (U mL-1), R2 = 0.98, as depicted in Figure S9. At the same time, colorimetric read-out assay of tyrosinase can also be implemented with tyrosine as substrate under the same condition (data not shown). To further investigate the biospecificity of the sensing system, BSA, HSA, ALP, GOX, AchE, EcoR I, lysozyme, and trypsin, and several kinds of biological cations (K+, Zn2+, Fe3+, Ba2+, Mn2+, Ni2+, Cu2+) were used as controls in the presence and absence of tyrosinase, respectively. The results in Figure 4F have explicitly illuminated that none of these control proteins/enzymes/cations generates distinct fluorescence response in the in situ fluorogenic reaction. Above all, the proposed sensitive and selective probe for tyrosinase activity can be fulfilled in the presence of other proteins and biologically essential metal ions, and it exhibits great potential in extensive applications.



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Determination of Tyrosinase in Serum Sample. In order to further investigate the potential bioassay applications, our developed fluorescent tyrosinase sensing system was carried out in the diluted fetal bovine serum (1%) and human serum samples (1%), respectively. The fluorescence response results in the diluted biological samples were compared with that in the standard colorimetric assay system by using tyrosine as substrate, in which tyrosinase causes an increase of absorbance at A280. The comparative results in Table 1 unambiguously illustrates our proposed label-free system possesses great potential to monitor the tyrosinase activity in biological samples. Table 1. Results of tyrosinase activity in the analysis of serum samples. Sample Fetal bovine serum （ 1%） Human serum

Spiked 0 0.1 0.5 1.0

Found a 0.052 ± 0.003 0.143 ± 0.004 0.578 ± 0.018 1.046 ± 0.039

Found b 0.049 ± 0.002 0.146 ± 0.005 0.565 ± 0.020 1.058 ± 0.050

0 0.075 ± 0.003 0.081 ± 0.004 0.1 0.165 ± 0.003 0.158 ± 0.003 （1%） 0.5 0.597 ± 0.024 0.614 ± 0.033 1.0 1.12 ± 0.035 1.06 ± 0.045 a obtained with proposed fluorescent read-out assay, bobtained with standard colorimetric assay system (U mL-1)

Tyrosinase Inhibition Assay. Considering the importance of tyrosinase in melanin formation and browning process, tyrosinase inhibitors have been widely used in skin hyperpigmentation and food antibrowning treatment. Thus, the development of a convenient and efficient screening method for tyrosinase inhibitors is of great importance to both cosmetic and food industry. Accordingly, the proposed fluorescence assay was further extended to evaluate the efficiency of tyrosinase inhibitor in vitro by using two common inhibitors (kojic acid and benzoic acid) as a model. Tyrosinase was preincubated with each inhibitor before being submitted to the tyramine solution, and the tyrosinase-enabled fluorogenic reaction was monitored by measuring fluorescence intensities at 460 nm. As depicted in Figure 5A, the fluorescence intensities reduced with the kojic acid and benzoic acid concentrations ranging from 20 µM to 155 µM and from 100 µM to 31.62 mM, respectively. Generally, the concentration-dependent slowing down of fluorescence intensities of the proposed system indicates tyrosinase activity has been efficaciously inhibited by each inhibitor. Furthermore, representative sigmoidal curves have been obtained from the plots of fluorescence intensities versus the logarithms of the tyrosinase concentrations. The IC50 values (the inhibitor concentrations where tyrosinase activity is inhibited by 50%) of kojic acid and benzoic acid were calculated to be approximately 155 µM and 1582 µM, respectively, which is of the same order of magnitude compared with those previously reported tyrosinase activity assays19,40.

Figure 5. Fluorescence emission spectra at different concentrations of benzoic acid (A) and kojic acid (C). Kinetic plots of the fluorescence intensity of sensing system in 4 U mL-1 tyrosinase against the logarithm of the kojic acid (B) and benzoic acid (D) concentrations.

CONCLUSION

In summary, it is meticulously demonstrated that the specific reactions between resorcinol and catecholamines can be carried out and generates the typical fluorescent azocine products in aqueous solution at ambient temperature and pressure. Inspired by such fluorogenic/chromogenic reactions and the specific oxidation ability of monophenolamine substrates to catecholamines by tyrosinase, an exquisite label-free fluorescent and colorimetric dual-readout assay of tyrosinase activity have been proposed and evaluated. To the best of our knowledge, it is the first dual-readout assay of tyrosinase activity based on in situ fluorogenic and chromogenic reactions. Under the optimum conditions, this convenient detection system possesses the wide linear detection range and high sensitivity that can effortlessly evaluate 0.1 U mL-1 of tyrosinase activity with naked-eye readout under an ultraviolet lamp. Moreover, the innovative tyrosinase-enabled fluorogenic reaction-based sensing system exhibits promising prospect for tyrosinase activity assay in extensive bioassays and clinic researches, as well as in screening potential tyrosinase inhibitors. We envision that the specific and attractive reactions between resorcinol and catecholamines could become a versatile tool for assaying several other enzymes by rationally designing and utilizing other novel substrate in clinical examination and diagnosis in the near future.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed data of parameter optimization, additional spectra, fluorescent read-out assay data with tyrosine as substrate, and data comparisons (PDF)

AUTHOR INFORMATION Corresponding Author


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*E-mail: [email protected]. Phone: +86 431 85262056. Fax: +86 431 85689278 * E-mail: [email protected]. Phone: +86 431 85262063. Fax:

+86 431 85689278. Author Contributions The manuscript was written through contributions of all authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the financial supports by the National Key Research and Development Program of China (2016YFA0201301), the National Natural Science Foundation of China (No. 21435005, 21627808, 21605139), Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SLH019).

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for TOC only


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In Situ Fluorogenic and Chromogenic Reactions for the Sensitive Dual

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