Ratiometric Fluorescence Detection of Tyrosinase Activity and

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Ratiometric fluorescence detection of tyrosinase activity and dopamine using thiolate-protected gold nanoclusters Ye Teng, Xiaofang Jia, Jing Li, and Erkang Wang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 07 Apr 2015 Downloaded from http://pubs.acs.org on April 7, 2015

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Analytical Chemistry

Ratiometric fluorescence detection of tyrosinase activity and dopamine using thiolate-protected gold nanoclusters Ye Tengab, Xiaofang Jiaab, Jing Li*a, Erkang Wang*a a

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, China. b University of the Chinese Academy of Sciences, Beijing, 100049, China. Corresponding author: Prof. Erkang Wang, Associate Prof. Jing Li, Tel: +86-431-85262003, Fax: +86-431-85689711, Email: [email protected] and [email protected]

ABSTRACT: In this work, a sensitive and selective ratiometric fluorescence sensing platform was built for the detection of tyrosinase (TYR) activity and dopamine (DA) using glutathione (GSH) protected gold nanoclusters (Au NCs) as probes. Upon excitation at 350 nm, Au NCs displayed an intense red emission, which could be effectively quenched by quinones. TYR, a typical polyphenol oxidase, can catalyze the oxidization of DA to o-quinone, and therefore quenched the fluorescence of Au NCs. Moreover, the reaction of TYR and DA gave rise to an emission band at 400 nm, which increased in a TYR/DA-concentration-dependent manner. The ratiometric signal variations were utilized for facile, sensitive and selective detection of TYR activity and DA. A linear range was obtained from 0.006~3.6 unit mL-1 of TYR activity, while the linear range for detection of DA was 1.0 nM to 1.0 mM. Additionally, it constructed a useful platform for TYR inhibitor screening in biomedical research.

INTRODUCTION Tyrosinase (EC 1.14.18.1, TYR) is a typical polyphenol oxidase, containing a binuclear copper center as the active site. It can catalyze the oxidation of phenolic substrates into oquinones, which is considered to be a key reaction in the biosynthetic pathway of melanin1-2. TYR activity is a very important index which is over-expressed in melanoma cells3-5. It also an influencing factor in the nutritional value of fruits and vegetables. Therefore the detection of tyrosinase activity is valuable for both clinical diagnosis and food industry6-7. Dopamine (3,4-dihydroxyphenyl ethylamine, DA), which can be catalyzed by TYR, is a neurotransmitter in the mammalian central nervous system. It regulates many biological processes, which is critical to the communication between neurons. The abnormity of DA concentration is considered to be the indicator of some neurological syndromes, such as schizophrenia and Parkinson’s disease. Therefore DA detection is very important for the disease diagnosis and monitoring8-12. The development of highly sensitive TYR activity and DA assays is urgently needed for both fundamental research and clinical application. So far, lots of technologies have been applied for the detection of TYR activity and DA, such as electrochemistry3, 10, 13-18 and fluorescence method12, 19-20. Though electrochemical methods have improved sensitivity, they often suffer from poor repeatability and the interference of environment. Ratiometric fluorescence measurement, which could provide two significant emission signals at different wavelengths under one excitation wavelength, has been widely used in the detection of ions and imaging of live cells21-25. It brings out an intrinsic correction to the effect of environment, and expands the detection range.

Recently, noble metal nanoclusters26 have attracted considerable attention as a new generation of fluorescent probes because of their strong luminescence, high photostability and ultrasmall size. Particularly, the interest in thiolate-protected Au NCs has grown tremendously owing to their chemical stability, good biocompatible and tailorable surface properties. They show great promise as a platform for biomedical imaging and biosensing. Nowadays, great progress has been achieved in the development of biosensing platform based on fluorescent thiolate-protected Au NCs27-30. Herein, we developed a facile, sensitive, selective and reliable ratiometric fluorescence method to detect TYR activity and DA employing glutathione (GSH) protected Au NCs as the fluorescence probe (Scheme 1). The prepared GSH protected Au NCs displayed an emission peak at 610 nm upon excitation at 350 nm. In the presence of TYR, DA could be oxidized to o-quinone, which would effectively quench the fluorescence of Au NCs. On the other side, the reaction of TYR and DA generated a new emission at 400 nm, which enhanced with the concentration of TYR/DA. The two signal changes were utilized for the ratiometric fluorescence method to detect TYR activity and DA. This developed sensing platform was facile and cost-effective, as well as highly sensitive, selective and reliable, making it promising as a candidate for TYR activity and DA analysis. In addition, it constructed a useful platform for TYR inhibitor screening in biomedical research. EXPERIMENTAL SECTION Chemicals and Materials. GSH, DA and kojic acid (KA) were purchased from Acros Organics, Alfa Aesar and Aladdin respectively. Tyrosinase (TYR) from mushroom was obtained from Sigma-Aldrich. Other reagents were bought from Si-

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nopharm Chemical Reagent Beijing Co., Ltd. All the chemicals were of analytical reagent grade and without any further purification. All the solutions were prepared in doubledistilled water purified by a Milli-Q system (Millipore, Bedford, MA, USA). Preparation of GSH protected Au NCs. GSH protected Au NCs were prepared according to the previous literature31. Briefly, 0.50 mL HAuCl4 (20 mM) were added to 0.15 mL GSH (100 nM), mixing with 4.35 mL double-distilled water. The mixture was stirring constantly for 24 h at 70°C. A light yellow aqueous solution was obtained as the fluorescent GSH protected Au NCs, and stored at 4°C for further research. Detection Assays for TYR Activity and DA. Au NCs diluted with 0.05 M PB (pH 6.6) to a final concentration of 80 µM were used in fluorescence measurement. For the detection of TYR activity, 1.0 mM DA were mixed with 80 µM Au NCs at 25°C by adding different concentrations of TYR, and the fluorescence spectra were recorded after incubating for 1.5 h. For the detection of DA, different concentrations of DA were mixed with 80 µM Au NCs at 25°C by adding 1.5 unit mL-1 TYR and the fluorescence spectra were recorded after incubating for 1.5 h. For the detection of TYR inhibitor, different concentrations of KA were mixed with 1.5 unit mL-1 TYR at 25 °C for 2 min, and then incubated with 80 µM Au NCs and 1.0 mM DA for 1.5 h. Characterizations. UV-visible absorbance spectra were obtained with an Agilent Cary 60 UV-Vis spectrophotometer at room temperature. The fluorescence spectra were measured on an Agilent Cary Eclipse fluorescence spectrophotometer, and the slit widths of excitation and emission were both 20 nm. Transmission electron microscopy (TEM) images were recorded on a JEM-2100F high-resolution transmission electron microscope operating at 200 kV. The luminescence decay curves were performed by a FLS920 spectrofluorometer. RESULTS AND DISCUSSIONS Characterization of Au NCs. GSH protected Au NCs were synthetized following the previous report31. The obtained Au NCs were light yellow under day light and exhibited red emission under the UV light. The fluorescent Au NCs were stable for more than 4 months sealed stored in a dark place at 4°C. TEM was employed for the characterizations of the asprepared Au NCs. As shown in Figure 1A, Au NCs were well dispersed and had an ultrasmall size with the average diameter of 1.76 nm, close to the previous report31. The optical properties were shown in Figure 1B. There was a shoulder peak at 400 nm in the UV-Vis spectra, and Au NCs could be excited in a wide range from 300 nm to 400 nm. In our experiments, upon exciting at 350 nm, the fluorescence emission peak was at 610 nm. The strong emission was caused by a mechanism of aggregation-induced emission31. The interaction between TYR, DA and Au NCs. Au NCs had a 610 nm emission peak, which would not be influenced by TYR or DA separately. When TYR and DA both existed, the fluorescence at 610 nm was quenched and a strong emission at 400 nm was appeared. To better understand the fluorescence at 400 nm, a series of control experiments were performed. As shown in Figure 2, TYR itself exhibited no fluorescence emission from 380 nm to 660 nm, and DA had a very weak emission peak at 620 nm. Both DA and TYR had almost no influence on the fluorescence of Au NCs. However, the reaction mixture of DA and TYR showed a strong emission at 400 nm, indicating the emission peak at 400 nm originated

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from DA and TYR. In the detecting condition, with the reaction of DA and TYR, the fluorescence emission intensity at 400 nm was gradually enhanced, while the emission of Au NCs at 610 nm was quenched, which provided the basis for ratiometric fluorescence detection of TYR activity and DA. The fluorescence quenching of Au NCs at 610 nm was considered to be related to the formation of o-quinone. Quinones were reported as an efficient fluorescence quencher for fluorescence probes such as quantum dots and noble metal NCs13, 32 . For further explanation of the mechanism, fluorescence lifetimes of Au NCs were investigated. Fluorescence decay curves were illustrated in Figure 3, and the fitting results of decay time were listed in Table S1. Au NCs had three components including 36.26 ns (98.20%), 0.31 µs (1.21%) and 1.75 µs (0.59%), and the average lifetime was 49.72 ns. After reacting with DA and TYR, the average lifetime of Au NCs was decreased to 12.03 ns, containing 10.88ns (99.86%) and 0.85 µs (0.14%). The lifetimes of Au NCs were obviously changed after adding DA and TYR, indicating a dynamic quenching process. The proposed quenching mechanism was that quinones would accept the electron and deliver them from the highest occupied molecule orbital (HOMO) to the lowest unoccupied molecule orbital (LUMO) 32. The emission peak at 400 nm, as mentioned above, was considered to be related to the reaction of DA and TYR. According to the results of control experiments (Figure 2), the emission peak at 400 nm would only appear when TYR and DA were both existed in the system, suggesting it was related to the products of TYR and DA, including the oxidation products of DA, such as o-quinone and its further polymerization products, and intermediate products of TYR and oxidation products of DA. To further study of the fluorescence origin, different paralled tests were investigated. Figure S2 showed the results of Au NCs reacted with p-benzoquinone. The fluorescence of Au NCs was quenched by p-benzoquinone, which was coincident with the reaction of Au NCs, TYR and DA, demonstrating the fluorescence of Au NCs could be efficiently quenched by quinones. There was no fluorescence peak formed at 400 nm in neither p-benzoquinone nor the mixture with Au NCs, therefore the fluorescence didn’t originate in quinones or the interaction between quinones and Au NCs. Furthermore the oxidization of DA in alkaline buffer (PB buffer, pH 8.0) was also tested for comparison. DA could be easily oxidized by oxygen in alkaline solution, and then DA oxide polymerized to polydopamine33. As shown in Figure S3, there was only a weak emission peak at 500 nm, and no fluorescence appeared at 400 nm. It demonstrated the fluorescence peak at 400 nm was not derived from the oxidation products of DA or its polymerization. Another possible fluorescence origin was the intermediate complex of TYR and polymerized oxidized DA. Tyrosinamide, which had an analogous structure to DA, was examined to react with TYR. The oxidation of tyrosinamide could also be catalyzed by TYR. The products of tyrosinamide and TYR formed a new fluorescence peak at 460 nm (Figure S4). This phenomenon was similar to DA and TYR, except the emission wavelength showed a 60 nm red shift, which might be attributed to the different structures of tyrosinamide and DA. In view of the above, we conjectured that the emission peak at 400 nm might relate to the intermediate product of TYR and polymerized DA oxide, and the exactly mechanism still need further research.


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The detection of TYR activity and DA. Reaction conditions were optimized to gain best results. Considering the TYR activity and the easy oxidation of DA in alkaline condition, faintly acid buffer were chosen for the reaction. To optimize the ratiometric fluorescence intensity, PB buffers with pH ranging from 6.0 to 7.0 were investigated (Figure 4). The black line in Figure 4A showed that the fluorescence intensity of Au NCs decreased with the reduction of pH. However, the emission at 400 nm showed a different trend, which had better intensity at pH 6.6 and 6.8. Figure 4B presented fluorescence intensity ratio at the wavelength of 400 nm and 610 nm I400/I610 in different pH buffers, indicating that pH 6.6 was the optimal pH. This result might be from the effect of pH on both the activity of TYR and the stability of DA aqueous solution. Reaction time was also an important factor which had significant influence on the fluorescence intensity ratio I400/I610, therefore the time optimization was investigated in the PB buffer (pH 6.6) at 25°C. As shown in Figure 4C, the fluorescence quenching of Au NCs occurred immediately and completed in 30 min. The emission at 400 nm was gradually formed in 30 min and reached the maximum in 90 min. So the ratiometric fluorescence intensity was recorded at 90 min after adding reagents in our experiments. Under the optimal condition, for the detection of TYR activity, 80 µM Au NCs were mixed with 1.0 mM DA and different concentrations of TYR (0, 0.006, 0.012, 0.06, 0.12, 0.24, 0.6, 1.2, 2.4, 3.6, 6.0, 8.4 unit mL-1) for 1.5 h at 25°C. As the concentration of TYR rising, the fluorescence at 610 nm was decreased while the fluorescence at 400 nm was increased (Figure 5A). As shown in Figure 5B, in the range from 0.006 unit mL-1 to 3.6 unit mL-1, the ratiometric fluorescence intensity I400/I610 exhibited an excellent linear relationship to the concentration of TYR (R2=0.998), and the detection limit of TYR was 0.006 unit mL-1. It presented a high sensitivity comparing with other reported detection methods for TYR activity4, 34. This method can also be applied to phenols detection. Taking DA as a template, 80 µM Au NCs were mixed with 1.5 unit mL-1 of TYR and different concentrations of DA for 1.5 h incubating at 25°C. The fluorescence at 610 nm was gradually quenched with the addition of DA, and the fluorescence peak at 400 nm was formed. The results of I400/I610 were collected and two linear ranges were found (Figure 6). In low concentrations, I400/I610 had a fine linear relationship to the logarithm of DA concentrations. The linear range was from 1.0 nM to 10 µM (R2=0.993). Besides, at high concentration, I400/I610 exhibited a linear relationship with DA, and the linear range was 10 µM to 1.0 mM (R2=0.988). The detection limit of DA in this sensing method was 1.0 nM which was comparable to or even lower than the other reports of fluorescence DA detection12, 35. The selectivity of TYR activity and DA detection were further investigated using ordinary interfering chemicals, including ascorbic acid (AA), glucose (Glu), lactose (Lac), K+, Zn2+ and some protein, such as immunoglobulin G (IgG), bovine serum albumin (BSA) and histone from calf thymus (His) (Figure 7). It was observed that other proteins had no effective influence to our detection of TYR activity, and the biocompetitors of DA exhibited no remarkable interference to DA detection. Among them, Zn2+ had increased the fluorescence of Au NCs due to the interaction between charged Au NCs and the counter cations36. However the ratiometric fluorescence measurement resulted in almost no change comparing with blank sample and made samples free from the influence of environ-

ment. The high selectivity of our method was owing to the specifity of tyrosinase and the intrinsic correction of ratiometric measurement. Our method was further applied on DA determination in real samples. The hydrochloride dopamine injection with specified DA concentration of 10 mg mL-1 was diluted to 10 µM with PB buffer. Then different concentrations of standard DA solution were added for the detection. The detection results were listed in Table 1. The recovery ranged from 93.5% to 108.6%, suggesting that our method can be applied to DA determination in real samples. TYR inhibitor study. As an important part in melanin synthesis, the inhibition of TYR could efficiently delay the procedure of enzymatic browning. Now TYR inhibitors were widely used in food antibrowning and skin hyperpigmentation treatment. In our method, considering the inhibitor of TYR would reduce the activity of TYR through binding to the enzyme, this system could also be utilized for the detection of TYR inhibitors. Taking KA as a model, the detection of TYR inhibitor was examined. KA is a common inhibitor of TYR, which could chelate the copper active site in TYR and inhibit its catalytic capacity2, 37. To detect the content of KA, different concentrations were incubated with TYR (1.5 unit mL-1) at 25 °C for 2 min, and then mixed with 80 µM Au NCs and 1.0 mM DA. After 1.5 hours, the ratiometric fluorescence were recorded and results were shown in Figure 7. When the concentration of KA ran up to 100 µM, the activity of TYR was almost completely inhibited, exhibiting an efficient inhibiting ability. KA showed a linear relationship to the ratiometric fluorescence signal from 0.2 to 100 µM (R2=0.970), with the detection limit of 0.2 µM. Thus it could be seen that our method could efficiently detect the inhibition of TYR, which was prospected for inhibitor screening and medicine discovery. CONCLUSION In conclusion, employing Au NCs as the probes, a ratiometric fluorescence method was developed for the detection of TYR activity and DA. The reaction of TYR and DA could form a 400 nm emission increasing with concentrations, and the oxidation products o-quinone could effectively quench the fluorescence of Au NCs at 610 nm. Utilizing the variation of ratiometric fluorescence intensity, a simple, sensitive and selective sensing method for TYR activity and DA were constructed with the detection limits of 0.006 unit mL-1 and 1.0 nM for TYR activity and DA, respectively. Moreover, the system we built provides a powerful platform for TYR inhibitor study. Considering the high sensitivity, selectivity and reliability, as well as the ease of use and cost effectiveness, we expect the developed assay to be a promising candidate for clinical diagnosis and medicine discovery.



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SCHEMES

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Scheme 1. Schematic demonstration of the fluorescence method with TYR, DA and Au NCs. FIGURES

Figure 1. (A) The TEM image of Au NCs. The inset is the size distribution of Au NCs. (B) The UV-Vis (blue), fluorescence emission and excitation spectra (black) of Au NCs. The insets are the photograms of Au NCs under day light and UV light.

Figure 2. The fluorescence emission spectra of (a) Au NCs, (b) DA, (c) TYR, (d) Au NCs + DA, (e) Au NCs + TYR, (f) DA + TYR, (g) Au NCs + DA + TYR. The concentrations of Au NCs, TYR and DA were 80 µM, 1.5 unit mL-1 and 1.0 mM respectively. Mixtures were incubated at 25°C for 90 min, and then the fluorescence spectra were recorded upon a 350 nm excitation.


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Figure 3. The lifetime decay of Au NCs (black) and Au NCs + TYR + DA (grey). The inset is the proposed schematic demonstration of quinone’s quenching mechanism.

Figure 4. (A) Fluorescence emission at 400 nm and 610nm, and (B) the ratiometric fluorescence intensity of Au NCs (80 µM) with DA (1.0 mM) and TYR (1.5 unit mL-1) in different pH PB buffer; (C) The fluorescence at the emission of 400 nm, 610nm, and (D) ratiometric fluorescence intensity variation with reaction time, including Au NCs (80 µM), DA (1.0 mM) and TYR (1.5 unit mL-1).



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Figure 5. (A) The fluorescence spectra of Au NCs with DA and different concentrations of TYR. The concentrations of TYR were 0, 0.006, 0.012, 0.06, 0.12, 0.24, 0.6, 1.2, 2.4, 3.6, 6.0, 8.4 unit mL-1 respectively. (B) The linear relationship of TYR concentration and the ratiometric fluorescence intensity. The sensing linear range was from 0.006 to 3.6 unit mL-1.

Figure 6. (A) The relationship of the logarithm of DA concentration with I400/I610 (the ratio of the fluorescence intensity at 400 nm to 610 nm under the excitation of 350 nm) in the range from 1.0 nM to 10 µM. The concentrations of DA were 1.0, 5.0, 10, 100, 1000, 2000, 5000, 10000 nM. (B) The relationship of DA concentration with I400/I610 in the range from 10 µM to 1.0 mM. The concentrations of DA were 10, 50, 100, 200, 500, 1000 µM.

Figure 7. The relationship between KA concentrations and the ratiometric fluorescence signals. The concentrations of KA were 0.2, 5.0, 10, 50, 100, 500, 1000 µM. The inset was the linear range of KA from 0.2 µM to 100 µM.


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Figure 8. The selectivity investigations of our assay. The concentrations of DA and TYR were 1.0 mM and 1.5 unit mL-1 respectively. AA, Glu, Lac, K+ and Zn2+ had the same concentration to DA. The concentrations of proteins, including IgG, BSA and His were 0.1 mg mL-1 and 0.01 mg mL-1 for the selectivity of DA and TYR activity respectively.

Table 1. The determination of DA in hydrochloride dopamine injection samples. Original (µM)

Added (µM)

Measured (µM)

Recovery (%)

R.S.D. (%) (n=3)

1

10

0

10.86

108.6

6.08

2

10

10

19.93

99.3

0.25

3

10

15

24.03

93.5

2.74

4

10

20

29.48

97.4

1.23



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ASSOCIATED CONTENT Supporting Information Additional information mentioned was listed in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Prof. Erkang Wang, Associate Prof. Jing Li, Tel: +86-43185262003, Email: [email protected] and [email protected].

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (NSFC) with the Grant No. 21190040 and 21427811.

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Ratiometric Fluorescence Detection of Tyrosinase Activity and

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