A fluorometric assay for tyrosinase activity and its inhibitor screening based on graphene quantum dots

Abstract

In this work, a sensitive and selective fluorescence sensing platform was developed for the detection of tyrosinase (TYR) activity and its inhibitor screening using graphene quantum dots (GQDs) as probes. Upon excitation at 383 nm, GQDs displayed an intense emission at 445 nm. TYR, a typical polyphenol oxidase, can catalyze the oxidation of dopamine (DA) to dopaquinone and then a fluorescence resonance energy transfer (FRET) process between GQDs and dopaquinone took place. Therefore, the fluorescence of GQDs could be quenched. Thus, quantitative evaluation of TYR activity was established in terms of the relationship between fluorescence quenching efficiency and TYR activity. A linear range was obtained from 0.005 to 0.5 U mL⁻¹ and from 0.5 to 5.0 U mL⁻¹, respectively. A detection limit of 0.0015 U mL⁻¹ was obtained, which was lower than those of reported papers. In addition, it constructed a useful platform for TYR inhibitor screening.

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1. Introduction

Tyrosinase (EC 1.14.18.1, TYR), which contains a binuclear copper center as the active site, is a ubiquitous enzyme found in bacteria, fungi, animals, and humans.¹ It can catalyze the oxidation of diphenol into o-quinones in the presence of molecular oxygen.² These specific activities of TYR can initiate the formation of melanin which plays a vital protective role against skin photocarcinogenesis. Thus, TYR acts as an autoantigen and serves as a marker for vitiligo.³ More importantly, TYR activity has been recognized as an important biomarker of melanoma cancer because of its evaluated amounts in melanoma cancer cells.^4,5 Therefore, it is urgent to develop a highly sensitive assay for detecting TYR activity and screening its inhibitors for diagnostic purposes.

So far, several methods for the detection of tyrosinase activity have been developed on the basis of colorimetry,⁶ electrochemistry,⁷ and fluorometry.^8–10 Among them, fluorometry still attracts much attention due to its accessibility and high sensitivity. However, very few fluorescent probes for tyrosinase have been reported for the detection of TYR activity, with the exception of quantum dots,¹¹ noble metal nanoclusters,¹² conjugated polymers,¹³ organic dyes¹⁴ and dopamine functionalized carbon quantum dots (Dopa-CQDs).¹⁵ However, these fluorophores might suffer from some drawbacks, such as high toxicity for CdSe QDs, high cost for metal nanoclusters, poor photostability and solubility in water for dyes, laborious synthesis procedure for conjugated polymers, and complex functionalization process for Dopa-CQDs. Therefore, it is highly necessary to develop new fluorescent probes for the determination of TYR activity.

Graphene quantum dots (GQDs) are a new class of zero-dimensional graphic nanomaterials with lateral dimensions of less than 100 nm in a single layer, double layers, and multi-layers (3 to <10).¹⁶ As a novel fluorescent nanomaterial, GQDs have sparked significant excitement due to their ease of production, low cost, processability, chemical stability, low cytotoxicity, and photostability.^17,18 Therefore, GQDs have been considered as a low-toxicity, eco-friendly candidate of fluorescent sensor materials. Up to now, various analytes, such as metal ions, pH, glucose, monosaccharides, trinitrotoluene, free chlorine, and biomolecules have been detected based on GQDs sensors.^19–23 However, the development and application of GQDs in fluorometric assay is still in its initial stage, and very little work has been done for the evaluation of enzyme activity. Tian et al. demonstrated a novel fluorescent biosensor for trypsin on the basis of cytochrome c-induced self-assembled GQDs.²⁴ Zhang's group constructed a highly sensitive fluorescent nanosensor to determine acetylcholinesterase activity and to screen its inhibitors based on the GQDs.²⁵ To the best of our knowledge, there is no report about the determination of TYR activity and the screening of its inhibitors based GQDs up to date.

Herein, we developed a sensitive fluorescent detection assay for probing the TYR activity and its inhibitors employing GQDs as the fluorescent probe. The pristine GQDs displayed an emission peak at 445 nm upon excitation at 383 nm. In the presence of TYR, dopamine (DA) could be oxidized to dopaquinone, which would effectively quench the fluorescence of GQDs.^15,26,27 Thus, quantitative evaluation of TYR activity can be established in terms of the correlation between fluorescence quenching efficiency and present TYR activity. However, when an inhibitor of TYR was present, TYR activity could be heavily inhibited to lead to the increase in fluorescence as the rise in amount of the inhibitor. Especially, compared with the fluorescent platform based on the functionalized carbon quantum dots,¹⁵ this developed sensing platform was simple and time-saving without any functionalization towards GQDs.

2. Experimental

2.1 Reagents and apparatus

The GQDs were obtained from XFNANO. DA hydrochloride, kojic acid (KA), benzaldehyde (BD), benzoic acid (BA), cinnamic acid (CA), Na₂HPO₄·12H₂O, and NaH₂PO₄·2H₂O were purchased from Aladdin Company (Shanghai, China). The TYR from mushroom, bovine serum albumin (BSA), trypsin (Try), ExoIII, glucose oxidase (GOx), horseradish peroxidase (HRP), alkaline phosphatase (ALP), and immunoglobulin G (IgG) were purchased from Sigma Aldrich (Shanghai, China). Phosphate buffer of various pH were prepared with different ratios of Na₂HPO₄ and NaH₂PO₄. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazoliumbromide (MTT) assay kit was acquired from KeyGEN Biotech Co. (Nanjing, China). DEME high glucose medium, fetal bovine serum, penicillin, and streptomycin were achieved from Hyclone (Thermo Scientific, USA). All reagents were of analytical reagent grade, and used as received. Doubly deionized water was used throughout.

Transmission electron microscope (TEM) images were obtained using a Philips CM200 FEG microscope. Atomic force microscopy (AFM) images were captured using a MultiMode Nanoscope IIIa controller atomic force microscope (Veeco, USA). All fluorescence measurements were carried out on an F-7000 spectrometer (Hi-tachi, Japan) operated at an excitation wavelength at 383 nm, with both excitation and emission slit widths of 5.0 nm. The fluorescence intensities were collected at 445 nm. UV-vis spectra were recorded using a Varian cary-300 UV-vis spectrophotometer. MTT assay was accomplished by using RS-232C elisa (Japan, BIO-RAD Company). The desired pH buffer solutions were adjusted with pHS-3C pH meter (Shanghai, China).

2.2 Fluorescent TYR assay

For the evaluation of TYR activity, 50 μL GQDs, 10 μL DA (50 mM), and 5 μL TYR (1 kU mL⁻¹) were added into 435 μL phosphate buffer (10 mM, pH 6.0). After reaction for 20 min at room temperature, the fluorescence spectra of the mixtures were monitored using fluorescence spectrometer. To evaluate the interference of biological metal ions, since TYR was well-known as a metal-containing enzyme, a variety of metal ions (e.g., Ca²⁺, Fe³⁺, K⁺, Zn²⁺, Mg²⁺, and Mn²⁺) were introduced with the identical concentration of 500 μM. Furthermore, the selectivity of this assay has been evaluated in the presence of other biological protein (BSA, 1 mg mL⁻¹) and enzymes (e.g. Try, GOx, HRP, ExoIII, ALP, and IgG, 10 U mL⁻¹).

2.3 Inhibitor screening

Kojic acid (KA), benzaldehyde (BD), benzoic acid (BA) and cinnamic acid (CA) as models were used for inhibitor screening of TYR. To detect the inhibition effect, the mixtures containing TYR (5 μL, 1 kU mL⁻¹) and different concentrations of inhibitor ranging from 0.001 mM to 1.0 mM were incubated for 10 min, and then were separately added into the GQDs solution containing 1.0 mM DA. After 20 min incubation, the fluorescence of the resulting mixture was recorded.

2.4 Cytotoxicity test

The cytotoxicity of both GQDs and inhibitor (KA as a model) on human HeLa cells was evaluated by the standard MTT assay. Briefly, HeLa cells were seeded in 96-well U-bottom plates at a density of 7000 cells per well, and incubated with GQDs or KA at varying concentrations at 37 °C for 24 h. Then, the culture media were discarded, and 0.1 mL of the MTT solution (0.5 mg mL⁻¹ in DMEM) was added to each well, followed by incubation at 37 °C for 4 h. The supernatant was abandoned, and 150 μL of DMSO was added to each well to dissolve the formed fromazan. After shaking the plates for 10 min, absorbance values of the wells were recorded with a microplate reader at 490 nm. The cell viability rate (VR) was calculated according to the following equation: VR = A/A₀ × 100%, where A is the absorbance of the experimental group (i.e., the cells were treated by GQDs or KA) and A₀ is the absorbance of the control group (i.e., the cells were untreated). The cell survival rate from the control group was considered to be 100%.

3. Results and discussion

3.1 Structural characterization of GQDs

The TEM image of GQDs and their size distribution were shown in Fig. 1A and B. The average size of GQDs is about 2.9 nm in diameter. The atomic force microscopy (AFM) image was shown in Fig. 1C. As shown, the heights were between 1.2 and 3.9 nm, corresponding to 1–3 graphene layers, similar to those previously reported works.^28,29

Fig. 1 (A) The TEM image, (B) the size distribution histogram, and (C) the AFM image of GQDs.

3.2 Optical properties of GQDs

To explore the properties of GQDs, UV-vis absorption and fluorescence spectra characterizations were carried out. As shown in Fig. 2A, the GQDs suspension showed the UV-vis absorption peak around 300 nm, which was consistent with the previous report.³⁰ The fluorescence emissions of GQDs with different excitation wavelengths were shown in Fig. 2B. Like most fluorescent carbon dots, the GQDs also exhibit an excitation-dependent fluorescence behaviour. With increasing excitation wavelengths, the emission peak position shifts to longer wavelengths and the intensity decreases rapidly. Such phenomena have also been observed from other carbon-based quantum dots, which may arise from the different particle size and different emission sites of the formed GQDs.^31,32 The maximum emission intensity for GQDs was achieved at 445 nm when excited at 383 nm.

Fig. 2 The UV-vis absorption (A) of GQDs and fluorescence spectra of GQDs at different excitation wavelengths (B).

3.3 Assessment of detection strategy for TYR activity based on GQDs

Firstly, the feasibility of GQDs for the qualitative evaluation of TYR activity was assessed with the results shown in Fig. 3. The fluorescent emission spectra of GQDs under different conditions were recorded. As shown in Fig. 3, GQDs had a 445 nm emission peak, which could not be influenced by TYR or DA separately. When both TYR and DA existed, the fluorescence at 445 nm was quenched efficiently. The effective quenching of the fluorescence of GQDs by TYR and DA can be used for the quantitative determination of TYR activity.

Fig. 3 Fluorescence emission spectra of GQDs under different conditions. [GQDs] = 0.1 mg mL⁻¹; [DA] = 1.0 mM; [TYR] = 10 U mL⁻¹; pH = 6.0.

DA, as one of the most important catecholamine neurotransmitters, shows a characteristic redox property and can be specifically oxidized by TYR in the presence of molecular oxygen to dopaquinone.^25–27 Herein, we explored the underlying quenching mechanism between GQDs and the generated dopaquinone. As shown in Fig. S1,† DA or TYR has no obvious UV-vis absorption in the range of 350–700 nm.

However, after DA and TYR incubated for 20 min, the mixed solution has an obvious UV-vis absorption in the range of 350–700 nm with a maximum absorption peak at 474 nm, which indicates the generation of dopaquinone oxidized from DA by TYR in the presence of molecular oxygen. Since there is spectral overlap between the emission spectrum of GQDs and the absorption spectrum of dopaquinone, a fluorescence resonance energy transfer (FRET) quenching mechanism is supported (Scheme 1). In order to further confirm the quenching effect of dopaquinone on GQDs, ascorbic acid (AA) was introduced into the GQDs–TYR–DA system because AA is a well-known efficient reducing agent for dopaquinone,³³ generating DA again. As shown in Fig. S2,† when AA was added into the GQDs–TYR–DA system, the fluorescence of quenched GQDs was recovered. These results demonstrated that dopaquinone was a real quencher for GQDs, providing an efficient energy transfer process from GQDs to oxidized DA.

Scheme 1 Schematic illustration of detection strategy for TYR activity based on GQDs fluorescent probe.

3.4 Optimization of detection conditions

Reaction conditions including the concentration of DA, incubation time, and media pH were optimized to gain the best results.

Firstly, we investigated the effect of DA concentration on the TYR detection. As shown in Fig. S3,† the fluorescence intensity decreased quickly with the increasing of DA concentration, indicating the increasingly concentration of dopaquinone. When the concentration of DA is up to 1.0 mM, the fluorescence intensity changed slightly with the increasing of DA concentration, indicating the generating dopaquinone reached the maximum value. Thus, 1.0 mM DA was chosen in the following experiments.

Considering the TYR activity and the easy oxidation of DA in alkaline conditions,³⁴ weakly acidic buffer was chosen for the reaction. The effects of pH ranging from 4.0 to 7.0 on the detection of TYR activity were investigated. As shown in Fig. S4,† the fluorescence intensity of GQDs alone changed slightly in the pH range of 4.0–7.0, indicating GQDs was very stable in the weakly acidic solution. While the quenching efficiency F₀/F, where F₀ and F are the fluorescence intensity of GQDs in the absence and presence of both DA and TYR, respectively, increased with the increasing pH from 4.0 to 6.0 and then decreased with the increasing pH. Obviously, the optimal pH was 6.0. This result might be from the effect of pH on 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. Therefore, the time optimization was investigated in the buffer with pH 6.0 at 25 °C. As shown in Fig. S5,† the fluorescence of GQDs almost did not change with the increasing time, indicating that GQDs alone was stable in the pH 6.0 buffer. While the fluorescence quenching of GQDs occurred immediately in the presence of both DA and TYR and completed in 20 min. Therefore, the incubation time of 20 min was chosen in the following experiments.

3.5 Detection of TYR activity

Under the optimal conditions, for the detection of TYR activity, GQDs were mixed with 1.0 mM DA and different concentrations of TYR for 20 min at 25 °C. As shown in Fig. 4A, as the concentration of TYR rising, the fluorescence intensity at 445 nm decreased gradually while the quenching efficiency (F₀/F, where F₀ and F are the fluorescence intensity of GQDs–DA system in the absence and presence of TYR, respectively) increased systematically. The quenching degree exhibited a good linear relationship with TYR in the concentration ranges of 0.005 to 0.5 U mL⁻¹ and 0.5 to 5.0 U mL⁻¹ (Fig. 4B). The regression equation for the calibration curve can be expressed as (F₀/F) = 1.079 + 3.254C_TYR (U mL⁻¹) and (F₀/F) = 1.253 + 0.515C_TYR (U mL⁻¹) with a correlation coefficient of 0.998 and 0.990, respectively. The detection limit can reach as low as 0.0015 U mL⁻¹ (S/N = 3), which is better than those based on Dopa-CQDs (0.007 U mL⁻¹),²⁷ glutathione (GSH) protected Au NCs (0.006 U mL⁻¹),²⁵ organic molecules (0.01–0.02 U mL⁻¹),^14,35 and gold nanoclusters templated with L-tyrosine (0.08 U mL⁻¹).⁹ Overall, these results indicated that the GQDs are suitable for the measurement of TYR activity.

Fig. 4 (A) Fluorescence emission spectra of GQDs in the presence of various concentrations of TYR; (B) plot of the quenching efficiency (F₀/F) versus the concentrations of TYR introduced. The inset was the linear range of TYR concentrations from 0.005 to 5.0 U mL⁻¹. The other conditions are the same as Fig. 2.

3.6 Interference and selectivity studies

As been well known, TYR played a role as a metal-containing enzyme. Thus, we investigated the effect of foreign coexisting substance on the activity of TYR. The interference experiments were performed in the presence of TYR (10 U mL⁻¹) together with other biologically significant metal ions (Zn²⁺, Ca²⁺, K⁺, Mg²⁺, Fe³⁺, and Mn²⁺). As shown in Fig. 5A, there was no obvious variation for the fluorescence intensity, indicating that these foreign coexisting metal ions showed scarce effect on assaying TYR activity.

Fig. 5 (A) Interference of biological meal ions (500 μM) on the TYR activity (10 U mL⁻¹), F₀ and F represent the fluorescence intensity of GQDs–DA–TYR system in the absence of presence of different composition; (B) selectivity test of assay toward TYR. F₀ and F represent the fluorescence intensity of GQDs–DA system in the absence of presence of different composition. The concentration of each composition is 10 U mL⁻¹, except BSA is 1 mg mL⁻¹.

To evaluate the selectivity of this proposed method, experiments were carried out separately in the presence of a series of other enzymes including GOx, HRP, ExoIII, Try, ALP, IgG, and a typically biological protein of BSA. As shown in Fig. 5B, about 80% decrease of the fluorescence intensity was observed upon addition of TYR. In contrast, no obvious decrease was found by introducing other enzymes or the biological protein into GQDs–DA system, revealing that the satisfactory selectivity of GQDs for TYR detection.

3.7 Inhibitor screening for TYR

As described in the preceding part, the presence of inhibitor could block the FRET process and then further reduce or stop the quenching of the fluorescence, which could be utilized to screen TYR inhibitors. KA, BD, BA and CA, as well-known TYR inhibitors, could inhibit its catalytic capacity.^1,36–38 Firstly, KA was taken the example to test the screening function of this assay. For KA detection, different concentrations of KA were first incubated with TYR at 25 °C for 10 min and then mixed with DA and GQDs. As shown in Fig. 6, the fluorescence of GQDs was quenched by 77% in the presence of TYR and DA within 20 min, but the addition of 0.25 mM KA caused only 40% of fluorescence quenching under the same conditions. Further progressive increase of the concentration of KA leads to stepwise decrease of fluorescence quenching from 40% to almost zero, and no apparent fluorescence quenching can be observed when the concentration of added KA is over 1.0 mM, which provides solid evidence that KA is an effective inhibitor for TYR activity.

Fig. 6 Fluorescence emission spectra of GQDs–DA–TYR system with different concentrations of KA. The concentrations of KA were 0.01, 0.03, 0.07, 0.1, 0.16, 0.25, 0.5, 0.75 and 1.0 mM, respectively. The inset was the linear plot of inhibition efficiency versus the concentration of KA.

We use the inhibition efficiency (IE%) to TYR as a signal for the detection of KA. IE% was analyzed by the following equation:^39,40

where F_inhibitor and F_{no inhibitor} refer to the fluorescence intensity of GQDs–DA–TYR system in the presence and absence of KA, respectively. F₀ refers to the fluorescence intensity of GQDs–DA. As shown in Fig. 6 (inset), a good linear relationship between IE% and the concentration of KA was obtained in the range of 0.01–0.25 mM. The detection limit for KA is 5 μM. Similarly, BD, BA, and CA can also obviously inhibit the activity of TYR. As shown in Fig. S6–S8,† the detection range for BD, BA, and CA is 0.05–1.0 mM, 0.001–0.25 mM, and 0.01–1.0 mM, respectively. Thus, it can be concluded from the preceding results that GQDs can be used as new fluorescent probe for TYR activity analysis and the screening of potential inhibitors.

3.8 Cytotoxicity test

Cellular cytotoxicity of both GQDs and KA is evaluated by using human HeLa cells in a MTT assay and the results were given in Fig. S9 and S10.† It is seen that GQDs are of low toxicity to HeLa cells because total cell viability of tested cells remains at the high level even when the concentration of GQDs is up to 200 μg mL⁻¹. Similarly, KA is also of low toxicity to HeLa cells, giving rise to a cell viability of >95% at a concentrations of 1.0 mM. These results described herein well demonstrated that GQDs with low cytotoxicity are promising for the applications in bio-imaging.

4. Conclusions

In summary, empolying GQDs as the probes, a fluorometric assay for the TYR activity and its inhibitor has been developed. The TYR could catalyze the oxidation of DA into dopaquinone, which could effectively quench the fluorescence intensity of the GQDs. Utilizing the decrease of fluorescence intensity, a simple and sensitive sensing method for TYR activity were constructed with the detection limit of 0.0015 U mL⁻¹. Moreover, the system also provides a powerful platform for TYR inhibitor study. More importantly, the GQDs were used directly without any modification or functionalization. Considering the high sensitivity, selectivity, cost effective, the high stability as well as low cytotoxicity for GQDs, we expect the developed assay to be a promising candidate for clinical diagnosis and medicine discovery.

Acknowledgements

This work is kindly supported by the National Natural Science Foundation of China (No. 21405094, 81303179, and 21405093), the Natural Science Foundation of Shandong Province (No. ZR2013BQ018), the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (No. SKLEAC201506), the Student Research Training Program of Qufu Normal University (No. 2015A071), and the Taishan Scholar Foundation of Shandong Province, China.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra13325h

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