Visual and Fluorescent Detection of Tyrosinase Activity by Using a

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Visual and fluorescent detection of tyrosinase activity by using dual-emission ratiometric fluorescence probe Xu Yan, Hongxia Li, Weishi Zheng, and Xingguang Su Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02037 • Publication Date (Web): 07 Aug 2015 Downloaded from http://pubs.acs.org on August 12, 2015

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Analytical Chemistry

Visual and fluorescent detection of tyrosinase activity by using dual-emission ratiometric fluorescence probe Xu Yana, Hongxia Lib, Weishi Zhengc and Xingguang Sua,* a

Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun, 130012,

P.R. China b

School of Pharmacy, Jilin University, Changchun 130021, P.R. China

c

State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, P.R. China

ABSTRACT In this work, we designed a dual-emission ratiometric fluorescence probe by hybridizing two differently colored quantum dots (QDs), which possess a built-in correction that eliminates the environmental effects and increases sensor accuracy. Red emissive QDs were embedded in the silica nanoparticle as reference while the green emissive QDs were covalently linked to the silica nanoparticle surface to form ratiometric fluorescence probe (RF-QDs). Dopamine (DA) was then conjugated to the surface of RF-QDs via covalent bonding. The ratiometric fluorescence probe functionalized with dopamine (DA) was highly reactive toward tyrosinase (TYR), which can catalyze the oxidization of DA to dopamine quinine, and therefore quenched the fluorescence of the green QDs on the surface of ratiometric fluorescence probe. With the addition of different amounts of TYR, the ratiometric fluorescence intensity of the probe continually varied, leading to color changes from yellow-green to red. So the ratiometric fluorescence probe could be utilized for sensitive and selective detection of TYR activity. There was a good linear relationship between

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the ratiometric fluorescence intensity and TYR concentration in the range of 0.05-5.0 µg mL-1, with the detection limit of 0.02 µg mL-1. Significantly, the ratiometric fluorescence probe has been used to fabricate paper-based test strips for visual detection of TYR activity, which validates the potential on-site application.

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INTRODUCTION Tyrosinase (TYR), a typical polyphenol oxidase, is a copper-containing enzyme widely distributed in all kinds of organisms, such as plants, animal tissues and fungi.1,2 It can catalyze the hydroxylation of phenolic substrates to catechol derivatives, further oxidised to orthoquinone products.3,4 These reactions have been recognized as key processes in the bio-synthetic pathway of some natural pigments.1,5 So, TYR is considered to be a targeting enzyme for establishing treatments to several hypopigmentation-related problems.6,7 Additionally, due to its major effect on dopamine neurotoxicity, TYR imbalance may also cause Parkinson's disease in association with neurodegeneration.8,9 More importantly, the disruption of tyrosinase will lead to severe skin diseases such as oculocutaneous albinism type I,10 and accumulated tyrosinase is regarded as a dependable biomarker of melanoma cancer cells.11 Therefore, sensitive detection of tyrosinase activity is of great urgency to biomedical diagnosis. So far, several methods have been developed for the detection of TYR activity, such as spectrophotometric1 and electrochemical assay.12,13 Although these methods mentioned above have good selectivity and sensitivity, most of them often suffered from time-consume modifying processes or sophisticated instrumentation, which limit their wide applications. Ratiometric fluorescent methods, which detect the analyte by measuring the change of the ratios of photoluminescence (PL) intensities at two wavelengths, have received intense attention in recent years.14-17 Ratiometric fluorescence technique can provide an intrinsic built-in correction to the environment effects and thus possess advantages in terms of improved sensitivity and accuracy.18,19 Most ratiometric sensors employing fluorescent organic dyes are susceptible to photobleaching, low fluorescence quantum yield, and especially the complexity of synthesis and

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purification.20-22

Compared

with

those

organic

dyes

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based

sensors,

ratiometric

fluorescent-quantum dots (RF-QDs) show several advantages, such as better stability with respect to photobleaching, narrower spectral line-width and easier preparation.22,23 Importantly, RF-QDs could be simply immobilized onto filter papers to fabricate test strips for on-site detection.19,23 Thus, RF-QDs could be employed as powerful tools in chemical and biological sensing application. In this paper, we construct a sensitive, selective and reliable ratiometric fluorescence probe (RF-QDs), which shows dual-emission for the visual observation of TYR activity (Scheme 1). The RF-QDs were fabricated by combining two differently colored QDs in one nanoparticle. The red emissive QDs (QDs

651nm)

were embedded in the silica nanoparticle as reference while the green

emissive QDs (QDs

536nm)

were covalently attached to the silica nanoparticle surface as a signal

report unit. Dopamine (DA) was then conjugated to the surface of RF-QDs via a simple covalent bonding. In the presence of TYR and O2, conjugated DA was oxidized into dopamine quinine at RF-QDs interface, and then the electron transfer access (ET) was turn on due to the efficient electron accepting of dopamine quinine. So the PL intensity of the green QDs on the RF-QDs surface can be significantly quenched, while the PL intensity of the entrapped red QDs stayed constant. The fluorescence of green QDs was quenched gradually with the increase of TYR concentration, accompanying continuous fluorescence color changes from yellow-green to red. Furthermore, we prepared portable test strips by transferring the probe on common filter paper for rapid and visual detection of TYR activity. And we also use the proposal sensing system for TYR inhibitor detection.

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Scheme 1 Schematic illustration of the ratiometric probe for the visual detection TYR. EXPERIMENTAL SECTION Chemicals and Materials. All reagents and solvents were at least analytical grade and used directly without further purification. 3-Mercaptopropionic acid (MPA) (99%) was purchased from J&K Chemical Co. and tellurium powder (~200 mesh, 99.8%), CdCl2 (99%) and NaBH4 (99%) were purchased from Aldrich Chemical Co. Hydrogen tetrachloroaurate (III) hydrate (HAuCl4•xH2O) were purchased from Acros Organics Co., Ltd. Tyrosinase (TYR), dopamine (DA), parathion-methyl (PM), tetraethoxysilane (TEOS), 3-aminopropyltriethoxysilane (APTS), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 3-mercaptopropyltrimethoxysilane (MPS) were purchased from Sigma-Aldrich Corporation. Instruments. The fluorescence spectra were obtained by using a RF-5301 PC spectrofluorophotometer (Shimadzu, Japan) equipped with a xenon lamp using right-angle geometry.

Ultraviolet

spectra

were

obtained

on

a

Shimadzu

UV-1700

UV-Visible

spectrophotometer (Shimadzu Co., Kyoto, Japan). All pH measurements were made with a PHS-3C pH meter (Tuopu Co., Hangzhou, China). In both experiments, a 1 cm path-length quartz cuvette was used. Preparation of amino-modified QD@silica nanoparticles. The red emissive QDs (QDs 651nm)

embedded silica nanoparticles were prepared by a modification process based on the Stöber 5



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method.23 Typically, 15 mL ultrapure water, 40 mL ethanol and 5.0 mL red QDs solution were mixed in a 100 mL ﬂask and stirred for 10 min at room temperature. After packing the flask with aluminum foil, 20 µL of MPS was added, and the resultant solution was stirred for 12 h. Then, 0.5 mL of TEOS was introduced to the solution, followed by adding 0.5 mL of ammonium hydroxide, and the mixture was left to react for another 12 h. To modify the silica surface with amino groups, 100 µL APTS was added into the above mixture under vigorous stirring. After reacting 12 h, the products were centrifuged and the precipitate was washed with ethanol and ultrapure water several times. Finally, the amino-modified QD@silica nanoparticles were redispersed in 10 mL of ultrapure water for further use. Preparation of ratiometric fluorescent probe (RF-QDs). 1.0 mL green emissive QDs solution was dispersed in 2.0 mL H2O in the flask. 2.0 mL EDC/NHS (2 mg mL-1) was added, and the solution was stirred for 15min. 3.0 mL of the amino-modified QD@silica nanoparticles was introduced, and the mixture was stirred for 4 h in the dark. The resulting RF-QDs were centrifuged and washed with ultrapure water for three times. The final product RF-QDs was dispersed in 5.0 mL ultrapure water for further use. Preparation of dopamine functionalized RF-QDs (RF-QDs-DA). The RF-QDs were further modified with dopamine (DA). 0.5 mL RF-QDs, 2.0 mL PBS buffer (pH= 5.8, 10 mmol L-1) and 0.5 mL EDC/NHS (2 mg mL-1) were mixed in the flask at 30 0C for 10 min, and then 50 µL DA (0.1 mol L-1) by shaking for 30 min. The obtained RF-QDs-DA were washed with ultrapure water and redispersed in 2 mL of ultrapure water. Detection Assays for TYR Activity. 50 µL RF-QDs-DA solutions were placed in a series of 2.0 mL calibrated test tubes. Subsequently, different amounts of TYR were added, respectively.

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Then, the mixtures were diluted to 2.0 mL with pH 7.0 PBS (10 mmol L-1) and mixed thoroughly. 30 min later, their fluorescence spectra were recorded in the 450-720 nm emission wavelength range with an excitation wavelength of 380 nm. RESULTS AND DISCUSSIONS Characterization of DA Functionalized RF-QDs (RF-QDs-DA). The green emissive QDs (gQDs, curve a) and red emissive QDs (rQDs, curve b) had maximum absorption peak around 490 and 607 nm (Figure S1), and had strong fluorescence emission with the maximal wavelength at 536 and 651 nm, respectively (Figure 1). The fluorescence spectrum of RF-QDs-DA (curve c, Figure 1) showed maximal emission wavelengths around 542 nm and 657 nm under a single wavelength excitation at 380 nm. The photographs of the gQDs, rQDs and RF-QDs-DA under UV light showed green, red and yellow-green fluorescence, respectively (Inset in Figure 1). The stability of the as prepared RF-QDs-DA was systematically investigated within 30 min (Figure 1B). The PL intensity ratio (I542/I657) remains no apparent change, implying that the RF-QDs-DA has good stability and could be applied for TYR detection.

Figure 1 (A) The fluorescence spectra of (a) gQDs, (b) rQDs and (c) RF-QDs-DA. The inset shows the photographs of (a) gQDs, (b) rQDs and (c) RF-QDs-DA under a 365 nm UV lamp. (B) The influence of time on the PL intensity ratio (I542/I657) of RF-QDs-DA. I542/I657 was the PL 7



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intensity of the sensing system at 542 nm versus that at 657 nm.

The Interaction between TYR and RF-QDs-DA. Scheme 1 illustrates the principle of fluorescence sensing system for the detection of TYR based on RF-QDs-DA system. In the presence of TYR, DA could be oxidized into dopamine quinine on the RF-QDs interface. According to previous study, the dopamine quinine could accept the electron from fluorophore and deliver them from the highest occupied molecule orbital (HOMO) to the lowest unoccupied molecule orbital (LUMO).19,24 Thus, as an excellent electron acceptor,24-26 dopamine quinine would effectively quench the fluorescence of green QDs on the surface of RF-QDs-DA due to electron transfer (ET) process. As shown in Figure S2, when 3.5 µg mL-1 TYR was introduced, the PL intensity at 542 nm of the RF-QDs-DA was significantly quenched, whereas the intensity of the embedded rQDs still remained constant. It is because the presence of the coated silica shell could avoid the ET process between the rQDs and the dopamine quinine, thus providing a reliable reference signal for the ratiometric detection of TYR. Additionally, the changes of the PL intensity ratio of RF-QDs-DA led to a noticeable fluorescence color change (Inset of Figure S2), facilitating the visual detection of TYR. Therefore, the RF-QDs-DA could be used as the ratiometric fluorescence probe for TYR detection. The Optimization for TYR Activity Detection. In order to obtain a high sensitivity for the TYR detection, some related factors, such as reaction time, pH of solution and reaction temperature are optimized. From Figure 2A, we can see that the reaction time of TYR and RF-QDs-DA had remarkable influence on the PL intensity ratio (I542/I657)0/ (I542/I657). (I542/I657)0

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and (I542/I657) were the PL intensity ratio of the RF-QDs-DA system in the absence and presence of TYR, respectively. The fluorescence quenching of RF-QDs-DA system occurred immediately after the addition of TYR and completed in 30 min. Thus, the optimal reaction time was chosen 30 min. The PL intensity ratio (I542/I657) of the RF-QDs-DA system was also studied under different pH values. As shown in Figure 2B, when the pH value changed from 5.0 to 8.2, the PL intensity ratio (I542/I657) of RF-QDs-DA and RF-QDs-DA-TYR solution both gradually increased. At pH 7.0, the change of PL intensity ratio of RF-QDs-DA system in the presence and absence of TRY reached maximum. Therefore, pH 7.0 Tris buffer (10 mmol L-1) was chosen as the optimal pH for TYR detection. The influence of the reaction temperature on PL intensity ratio (I542/I657)0/ (I542/I657) was shown in Figure 2C. The results shown that the quenching effect reach the maximum at 37 oC. Thus, we chose 37 oC as the optimal reaction temperature for TYR detection in the further experiments.

Figure 2 (A) The effect of reaction time on the PL intensity ratio (I542/I657)0/ (I542/I657) of RF-QDs-DA system in the presence of 2 µg mL-1 TYR. (I542/I657)0 and (I542/I657) were the PL intensity ratio of the RF-QDs-DA system in the absence and presence of TYR, respectively; (B) The effect of pH on the PL intensity ratio (I542/I657) of RF-QDs-DA probe before and after the addition of 2 µg mL-1 TYR; and (C) The effect of reaction temperature on the PL intensity ratio (I542/I657) of RF-QDs-DA system in the presence of 2 µg mL-1 TYR. I542/I657 was the PL intensity of the sensing system at 542 nm versus that at 657 nm. 9



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Determination of TYR Activity. As shown in Figure 3A, the PL intensity at 542 nm of RF-QDs-DA was continuously decreased with the increasing concentration of TYR. There is a good linear relationship between the PL intensity ratio (I542/I657)0/ (I542/I657) and the TYR concentration in the range of 0.05-5.0 µg mL-1 (Inset in Figure 3A). The regression equation was: (I542/I657)0/ (I542/I657) = 0.983 (±0.018) + 0.526 (±0.009) [TYR], µg mL-1. (I542/I657)0 and (I542/I657) were the PL intensity ratio of the RF-QDs-DA system in the absence and presence of TYR, respectively. The corresponding regression coefficient is 0.996, and the detection limit (LOD) for TYR was 0.02 µg mL-1. Owing to the variation in the PL intensity of RF-QDs-DA, a series of noticeable color changes from yellow-green to red could be observed for the ratiometric fluorescence probe solution under a UV lamp (Figure 3B). Thus, a simple and low-cost fluorescence assay for TYR detection can be realized with the naked eye. Selectivity is a very important parameter to evaluate the performance of fluorescence probe, especially for a sensor with potential applications in biomedical detection. Therefore, the selectivity for TYR activity detection were further investigated by using common interfering substances, including K+, Ca2+, Cl-, threonine, lysine, arginine, histidine, trypsin, pepsin, BSA (bovine serum albumin), lysozyme, urease, OPH (organophosphorus hydrolase) and ALH (albumin human). As shown in Figure S3, after the addition of 10 µg mL-1 above substance, the PL intensity ratio of RF-QDs-DA system in absence and presence of 1.0 µg mL-1 TYR have no obvious change, which indicates that the common inorganic ion, amine acids and proteins do not have evident interference on TYR activity detection. The high selectivity of proposal method was owing to the specifity of TYR. Thus, the established sensor was suitable for selective

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determination of TYR. A comparison between the present method and other reported methods for TYR detection in liner range and sensitivity was summed up in Table S1. It could be observed from Table S1 that the sensitivity of this ratiometric fluorescent probe was comparable or even better than most of the reported methods. Compared to the fluorescent probes based on dyes and gold clusters,27,28 the present method possess a built-in correction, could eliminate environmental effects and improve the accuracy. And the present method also has high selectivity to TYR detection due to the specifity of TYR. Moreover, the fluorescence assay for TYR detection can be realized with the naked eye. The present method for TYR detection could be influenced with some TYR inhibitors, which other dyes and gold clusters-based fluorescent assays also confront with these limitations.

Figure 3 (A) The fluorescence spectra of RF-QDs-DA in the presence of different concentration of TYR. The concentrations of TYR were 0, 0.05, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.125, 3.75 and 5.0

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µg mL-1, respectively. The Inset was the linear plot of PL intensity ratio (I542/I657)0/ (I542/I657) versus the concentration of TYR. (I542/I657)0 and (I542/I657) were the PL intensity ratio of the RF-QDs-DA system in the absence and presence of TYR, respectively. (B) The corresponding fluorescence photographs of the probe solution taken under a 365 nm UV illumination.

Test Strips for TYR Detection. Paper-based sensors provide powerful analytical platforms to revolutionize on-site diagnosis due to low cost, availability, ease of storage, transport, and ease of disposal.29-31 By immobilization of the ratiometric fluorescence probes on the filter paper, test strips were fabricated for TYR activity detection. As shown in Figure 4A, the test strip shows yellow-green fluorescence color under UV illumination, and a bright red color appeared against the yellow-green background after 20 µL TYR solution (5.0 µg mL-1) was dropped on the test strip. The concentration of the TYR could obviously affect the color of filter paper. With the increase of TYR concentration (0, 0.5, 1.0, 2.0 and 5.0 µg mL-1), the color of test strip from yellow-green to red (from a to e) were observed with the naked eye under a UV illumination (Figure 4B). The change of the color was due to the quenching of the gQDs on the RF-QDs surface, which allowing a visual perception of the semi-quantitative for TYR detection.

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Figure 4 (A) Visual detection of TYR by using test strips. The photographs of RF-QDs-DA based test strips were taken (a) under sunlight; (b) under illumination of a 365 nm UV lamp; (c) in the presence of 20 µL TYR solution (5.0 µg mL-1) and under illumination of a 365 nm UV lamp. (B) The photographs of test strips with different concentrations of TYR were taken under a 365 nm UV lamp. The concentrations of TYR (from a to e) were 0, 0.5, 1.0, 2.0 and 5.0 µg mL-1, respectively.

TYR inhibitor study. Considering the TYR inhibitors would reduce the activity of TYR through binding to the enzyme, our method could also be utilized for the determination of TYR inhibitors. According to previous report, organphosphorus pesticides (OPs) are common TYR inhibitor, which could efficiently inhibit the catalytic capacity of TYR.32-34 As a model of OPs, parathion-methyl (PM) is chosen as the inhibitor of TYR. When PM exists in RF-QDs-DA-TYR system, the enzymatic reaction is retarded, and less amount of quinone is generated, showing a decreased quench of PL intensity of the sensing system. For PM detection, different concentrations of PM were first incubated with TYR (1.0 µg mL-1) at 30 0C for 10 min, and then mixed with 13



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RF-QDs-DA. We use the inhibition efficiency (IE %) to TYR as a signal for the detection of PM. IE % was analyzed by the following equation:30, 35

Where Finhibitor and Fno inhibitor refer to the PL intensity ratio of RF-QDs-DA-TYR system in the presence and absence of PM, respectively. F0 refers to the PL intensity ratio of the RF-QDs-DA in the absence of TYR and PM. As shown in Figure 5A, a good linear relationship between the IE and the logarithm of the PM concentration was obtained in the range from 0.001 to 10 µg mL-1. The detection limit (LOD) for PM is 0.45 ng L-1 which was comparable to or even lower than some previous reports of PM detection sensor (Table S2). The maximum residue limit of PM is 100 ng mL-1 in rice set by the Ministry of Agriculture of the People's Republic of China.36 Thus, our proposed methods could satisfy the detection requirements for pesticide residues. Figure 5B showed the interference effect of inorganic ions, amino acid and glucose on the determination of PM. It can be seen that 100-fold of coexistence substances had no influence on the IE value of 0.1 µg mL-1 PM. Thus, the new sensing system displays high selectivity for the determination of PM. The developed fluorescence sensor was also applied for the determination of PM in water and rice samples. The detection results were listed in Table S3. The recovery ranged from 91.6 to 105.5 %, and the relative standard deviation (RSD) was lower than 5.5 %, suggesting that our method can be efficiently applied to detect the inhibition of TYR.

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Figure 5 (A) The fluorescence spectra of RF-QDs-DA-TYR system with different concentrations of PM. The concentrations of PM were 0, 0.001, 0.01, 0.1, 1.0 and 10 µg mL-1, respectively. The Inset was the linear plot of inhibition efficiency versus the logarithm of concentration of PM. (B) The IE value of PM to RF-QDs-DA-TYR system in the presence of the interfering substances. The final concentration of PM and interfering substances were 0.1 and 10 µg mL-1, respectively.

CONCLUSION In conclusion, a dual-emission ratiometric fluorescent probe has been developed for the visual detection of TYR activity. The fluorescent probe which was designed by hybridization of two differently colored QDs, possess a built-in correction that eliminates the environmental effects and increases sensor accuracy. The TYR could catalyze the oxidation of DA at RF-QDs interface into dopamine quinine, which would effectively quench the PL intensity of fluorescent probe. Utilizing the variation of ratiometric fluorescence intensity, a simple and sensitive sensing method for TYR activity were constructed with the detection limits of 0.02 µg mL-1. Moreover, the paper-based test strips based on ratiometric fluorescent probe have been successfully fabricated for TYR activity detection, which validates the potential on-site application. The established

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fluorescence sensing system also could be used for the detection of TYR inhibitor. AUTHOR INFORMATION Corresponding author *Tel.: +86-431-85168352. E-mail address: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21075050, No. 21275063) the Science and Technology Development Project of Jilin Province, China (No. 20110334) and Graduate Innovation Fund of Jilin University (No. 2015022). ASSOCIATED CONTENT Supporting Information Available Additional information mentioned was listed in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. REFERENCES (1) Li, S.; Mao, L. Y.; Tian, Y. P.; Wang, J.; Zhou, N. D., Analyst 2012, 137, 823-825. (2) Kim, T.; Park, J.; Park, S.; Choi, Y.; Kim, Y., Chem. Commun. 2011, 47, 12640-12642. (3) Chang, T. S., Int. J. Mol. Sci. 2009, 10, 2440-2475. (4) Briganti, S.; Camera, E.; Picardo, M., Pigm. Cell Res. 2003, 16, 101-110. (5) Ichikawa, A.; Takagi, H.; Suda, K.; Yao, T., Dermatology 2009, 219, 195-201. (6) Oetting, W. S.; King, R. A., Hum. Mutat. 1999, 13, 99-115. (7) Tomita, Y.; Suzuki, T., Am. J. Med. Genet., Part C 2004 , 131C , 75-81.

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(8) Hastings, T. G., J. Neurochem. 1995, 64, 919-924. (9) Tessari, I.; Bisaglia, M.; Valle, F.; Samorı, B.; Bergantino, E.; Mammi, S.; Bubacco, L., J. Biol. Chem. 2008, 283, 16808-16817. (10) Oetting, W.; King, R., J. Invest. Dermatol. 1994, 103, 131S-136S. (11) Angeletti, C.; Khomitch, V.; Halaban, R.; Rimm, D. L., Diagn. Cytopathol. 2004, 31, 33-37. (12) Yildiz, H. B.; Freeman, R.; Gill, R.; Willner, I., Anal. Chem. 2008, 80, 2811-2816. (13) Freeman, R.; Elbaz, J.; Gill, R.; Zayats, M.; Willner, I., Chem. Eur. J. 2007, 13, 7288-7293. (14) Wu, J. C.; Zou, Y.; Li, C. Y.; Sicking, W.; Piantanida, I.; Yi, T.; Schmuck, C., J. Am.Chem. Soc. 2012, 134, 1958-1961. (15) Chen, L. X.; Fu, X. L.; Li, J. H., Nanoscale 2013, 4, 5905-5911. (16) Yan, X.; Li, H. X.; Li, Y.; Su, X.G., Anal. Chim. Acta 2014, 852, 189-195. (17) Tyrakowski, C. M.; Snee, P. T., Anal. Chem. 2014, 86, 2380-2386. (18) Teng, Y.; Jia, X. F.; Li, J.; Wang, E. K., 2015, 87, 4897-4902. (19) Wang, K.; Qian, J.; Jiang, D.; Yang, Z. T.; Du, X. J.; Wang, K., Biosens. Bioelectron. 2015, 65, 83-90. (20) Wu, C. F.; Chiu, D. T., Angew. Chem. Int. Ed. 2013, 52, 3086-3109. (21) Zong, C. H.; Ai, K.; Zhang, G.; Li, H. W.; Lu, L. H., Anal. Chem. 2011, 83, 3126-3132. (22) Yao, J. L.; Zhang, K.; Zhu, H. J.; Ma, F.; Sun, M. T.; Yu, H.; Sun, J.; Wang, S. H., Anal. Chem. 2013, 85, 6461-6468. (23) Zhang, K.; Zhou, H. B.; Mei, Q. S.; Wang, S. H.; Guan, G. J.; Liu, R. Y.; Zhang, J.; Zhang, Z. P., J. Am. Chem. Soc. 2011, 133, 8424-8427.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(24) Burda, C.; Green, T. C.; Link, S.; El-Sayed, M. A., J. Phys. Chem. B 1999, 103, 1783-1788. (25) Canbay, E.; Akyilmaz, E., Anal. Biochem. 2014, 444, 8-15. (26) Zhu, X. L.; Hu, J.; Zhao, Z. H.; Sun, M. J.; Chi, X. Q.; Wang X. M.; Gao, J. H., small 2015, 11, 862-870. (27) Li, X. H.; Shi, W.; Chen, S. M.; Jia, J.; Ma, H. M.; Wolfbeis, Otto S., Chem. Commun. 2010, 46, 2560-2562. (28) Yang, X. M.; Luo, Y. W.; Zhuo, Y.; Feng, Y. J.; Zhu, S. S, Anal. Chim. Acta 2014, 840, 87-92. (29) Parolo, C.; Merkoci, A., Chem. Soc. Rev. 2013, 42, 450-457. (30) Ge, X. X.; Asiri, A. M.; Du, D.; Wen, W.; Wang, S. F.; Lin, Y. H., Trends in Analytical Chemistry 2014, 58, 31-39. (31) Cate, D. M.; Adkins, J. A.; Mettakoonpitak, J.; Henry, C. S., Analytical chemistry 2014, 87, 19-41. (32) Hou, J. Y.; Dong, J.; Zhu, H. S.; Teng, X.; Ai, S. Y.; Mang, M. L., Biosens. Bioelectron. 2015, 68, 20-26. (33) Shim, J.; Woo, J. J.; Moon, S. H.; Kim, G. Y., J. Membr. Sci. 2009, 330, 341-348. (34) Van Dyk, J. S.; Pletschke, B., Chemosphere 2011, 82, 291-307. (35) Yi, Y. H.; Zhu, G. B.; Liu, C.; Huang, Y.; Zhang, Y. Y.; Li, H. T.; Zhao, J. N.; Yao, S. Z., Anal. Chem. 2013, 85, 11464-11470. (36) http://www.chinapesticide.gov.cn/mprlsv/mprls.html.

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For TOC only

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Visual and Fluorescent Detection of Tyrosinase Activity by Using a

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