silver nanoparticles under illumination

Accepted Manuscript Title: Colorimetric detection of tyrosinase during the synthesis of kojic acid/silver nanoparticles under illumination Authors: Bo-Wen Liu, Peng-Cheng Huang, Jian-Fang Li, Fang-Ying Wu PII: DOI: Reference:

S0925-4005(17)30954-1 http://dx.doi.org/doi:10.1016/j.snb.2017.05.129 SNB 22413

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

5-1-2017 20-5-2017 23-5-2017

Please cite this article as: Bo-Wen Liu, Peng-Cheng Huang, Jian-Fang Li, Fang-Ying Wu, Colorimetric detection of tyrosinase during the synthesis of kojic acid/silver nanoparticles under illumination, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.05.129 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Colorimetric detection of tyrosinase during the synthesis of kojic acid/silver nanoparticles under illumination

Bo-Wen Liu, Peng-Cheng Huang*, Jian-Fang Li, Fang-Ying Wu

College of Chemistry, Nanchang University, Nanchang 330031, China

*Corresponding Author: Peng-Cheng Huang, [email protected]. Tel: + 86 79183969882, Fax: + 86 7918396951.

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Highlights 1. A green, reliable and one-step colorimetric assay for tyrosinase (Tyr) is developed during the synthesis of silver nanoparticles (AgNPs). 2. The assay was based on specific binding between Tyr and kojic acid which prevented the formation of AgNPs. 3. The use of light makes the assay rapid, economical, and environmentally friendly. 4. The assay was applied to detect Tyr in human serum samples.

Abstract: We report a green, reliable and single-step colorimetric assay for tyrosinase (Tyr) using the silver nanoparticles (AgNPs) as the signal readout. With the aid of light as the catalyst, kojic acid can rapidly reduce Ag+ ions and stablize the produced AgNPs. While uopn addition of Tyr during the synthesis of AgNPs, Tyr can effectively interrupt the formation of AgNPs due to its preferential combination with kojic acid, eventually leading to color fading and signal decrease of AgNPs solution. The presented method shows a good linear response toward Tyr over the range 0.5~4 u/mL with low detection limit of 0.117 u/mL. Common coexisting substances including metal ions, amino acids and biomacromolecules exert no effect on the determination of Tyr. This colorimetric sensor was successfully employed to quantify Tyr in human serum samples.

Keywords: Tyrosinase, AgNPs, kojic acid, light, green synthesis

1. Introduction

Tyrosinase (Tyr), a typical polyphenol oxidase, is a copper-containing enzyme that controls the production of melanin by catalyzing the oxidation of phenolic substrates into respective quinones. This enzyme is widely distributed in all kinds of organisms, such as fungi, plants and animal tissues[1,2] and has been considered as an important biomarker of melanoma cancer because of its overexpression in melanoma cancer cells[3,4]. In addition, an abnormal level of tyrosinase may cause serious skin diseases such as vitiligo and neurological syndromes like Parkinson’s disease[5,6]. It is also an influencing factor in the appearance of human beings and the nutritional 2

value of fruits and vegetables[7,8]. Therefore, tyrosinase assay possesses vast importance for both fundamental research and practical applications in clinical diagnosis, cosmetic, and food industry. Despite being quantitative, the traditional colorimetric assay is limited by its low sensitivity[9,10]. Recently, several other methodologies have been developed for the detection of Tyr, including electrochemical[11-13], spectrophotometric[14-17] and fluorescent[18-24] methods. Among these methods, noble metal nanoparticles, like gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs), have been used as promising probes due to their simplicity, high sensitivity and biocompatibility[14-16]. To date, noble metal nanoparticles based colorimetric assays mainly rely on apparent color changes of the monodispersed or aggregated state of Au or AgNPs owing to their high extinction coefficients and localized surface plasmon resonance (LSPR) effect[25,26]. Although this feature enables the detection of target-induced molecular events, external factors in complicated application environments (e.g., the impurities, extreme temperature, or high ionic strength) may induce undesirable aggregation of Au or AgNPs, leading to unreliable detection possibility[27]. Furthermore, these colorimetric assays generally require three steps: synthesis of nanoparticles, modification of nanoparticles, and detection of targets. The first two steps usually involve toxic reagents and/or complex chemical procedures, which limits their applicability[14,28,29]. It is thus highly desirable to establish a more facile and rapid assay for reliable detection of Tyr[30-32]. Herein, we developed a simple, rapid, and label-free colorimetric assay for the determination of Tyr based on the specific binding between Tyr and kojic acid which prevented the formation of AgNPs. As illustrated in Scheme 1, under illumination, the AgNPs were readily prepared by using kojic acid (KA) as both reducing and capping agents in aqueous solution. While in the presence of Tyr, KA would strongly bind with it by chelating the copper ions normally present in the active site of tyrosinase[33], consequently resulting in an obvious inhibition effect on the formation of AgNPs. According to our method, the synthesis, modification of AgNPs and the detection of Tyr can be accomplished in a single step, which is more facile and rapid than traditional three-step methods. This kind of signal-generated method via the involvement of the target during the generation of AgNPs is also much more reliable and specific than the aggregated colorimetric signal method. In our assay, the solution color gradually faded from bright yellow to light yellow and even to colorless, which was easily recognized by the naked eye. Moreover, light-driven green 3

synthesis of AgNPs with KA as the reducing agent makes this method economical and environmentally benign[34-37]. Thanks to light irradiation, AgNPs can be produced much faster than under normal conditions. Besides, compared with previous reports about synthesis of AgNPs through light irradiation strategy[34-36], the preparation of synthetic reducing agents or natural extracts by tedious pretreatment is not included in our method. Both of them would further save some time and simplify operation procedures.

2. Experimental

2.1. Materials

All reagents and solvents were at least analytical grade and used directly without further purification. AgNO3, NaOH, kojic acid, NaNO3, KNO3, Mg(NO3)2, Ca(NO3)2, glucose, Bovine Serum Albumin (BSA), glycine (Gly), alanine (Ala), glucose oxidase (GOD), phenylalanine (Phe), leucine(Leu) and isoleucine(Ile) were purchased from Shanghai Qingxi Technology Co., Ltd. (Shanghai, China). Mushroom tyrosinase (EC 1.14.18.1) was purchased from Sigma (St. Louis, MO, USA). Milli-Q-purified distilled water was used to prepare all the solution in this study. All glassware was cleaned thoroughly with freshly prepared aqua regia (3:1 (v/v) HCl/HNO3) and rinsed thoroughly with doubly distilled water prior to use. All experiments were operated at room temperature.

2.2. Instruments

UV–vis absorption spectra were examined on a UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan) using a 1.0-cm quartz cell at room temperature. Fourier transform infrared (FT-IR) spectra were measured with KBr pellets on a Nicolet 5700 FTIR Spectrometer (Nicolet, USA). Transmission electron microscopy (TEM) was recorded by a JEM-2100 transmission electron microscope (JEOL Ltd. Japan). The data of dynamic light scattering (DLS) were obtained on NPA152 Nanoparticle size analyzer (Microtrac Inc., USA). The Simulated light source is a LED

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folding lamp (AS-CT104, Aisan-led Inc., China). Its power and luminous flux are 3 W and 240 LM, respectively. The light wavelength range is between 400 nm and 800 nm.

2.3. Colorimetric detection of tyrosinase

First, 20 μL of kojic acid (4 mM) and corresponding concentrations of tyrosinase were mixed in a 5 mL centrifugal tube. Then, 30 μL of NaOH (0.1 M) and 40 μL of AgNO3 (8 mM) were added to the tube in sequences. Finally, the solution was added to 3910 μL ultrapure water. The mixture was incubated at room temperature for 40 minutes under the light before UV-vis measurents and photograph-taken. Here a LED folding lamp mentioned above was used as the light source. Experimental conditions including the concentration of NaOH (0-1 mM), kojic acid (0-60 μM), AgNO3 (0-120 μM), and reaction time were studied with UV-vis absorption spectra. The spiked-recovery detection of tyrosinase in the human serum sample was manipulated according to the same procedure. Human serum was obtained from Nanchang University Hospital from healthy donors. Human serum (0.5 mL) was placed in a centrifuge tube and acetonitrile (2.0 mL) was added to precipitate proteins. After vortex-mixing, the sample was centrifuged at 12, 000 rpm for 15 minutes, and the supernatant was transferred into a 25 mL volumetric flask and diluted to the mark with deionized water.

3. Results and discussion

3.1. Establishment of colorimetric assay for Tyr

The hydroxyl groups of KA were reported to show moderate reducing ability[38]. In our experiment, KA was used to reduce Ag+ ions under illumination. The formed KA/AgNPs in bright yellow color (Fig. 1A-c) displayed a strong SPR absorption band at 410 nm (Fig. 1B-c), indicative of the characteristics of monodispersed AgNPs[30]. This is mainly because KA serves as the capping agent on the surface of AgNPs to protect them from aggregation. During the reaction 5

process, the hydroxyl groups lost electrons to form quinone structure, which would be attached onto the AgNPs surface and stabilize the monodispersed AgNPs. FTIR measurement further testified the modification of AgNPs with KA (Fig. S1). The intense absorption peaks at ca. 3450, 2917, and 1652 cm-1 were ascribed to the –OH, -CH-, and C=O groups of KA, respectively[39]. These characteristic peaks could also be discerned in the pure KA sample. It should be noted that compared with KA, the band of the –OH group of KA/AgNPs blueshifted to some extent, probably due to the combination between the –OH group of KA and the Ag atoms. It was also demonstrated that illumination treatment was an important factor which had significant influence on the synthesis of AgNPs. As shown in Fig. 1A-a and 1B-a, a similar control solution kept in the dark for the same time did not show any color or spectral change. And even after 5 hours, the solution exhibited very slight yellow color together with subtle increase of the absorbance at 410 nm (Fig. 1A-b and 1B-b). These results indicated that light plays an indispensable role in accelerating the reaction rate during the synthesis of KA/AgNPs. As reported previously, light may facilitate efficient and fast electron transfer for the reduction of Ag+ ions to Ag(0) state during the formation of AgNPs[34-37]. While upon addition of Tyr during the synthesis of KA/AgNPs, and the solution color finally faded away (Fig. 1A-d), accompanied by sharp decrease of the corresponding absorption peak (Fig. 1B-d). Such spectral and color changes of KA/AgNPs upon addition of Tyr could be explained well by the coordination interaction between copper ions in the active site of Tyr and the ketone group and hydroxyl group of KA[33]. The formation of the adduct composed of Tyr and KA decreased the amount of free KA to reduce Ag+ ions, eventually producing a small quantity of pale-colored AgNPs. We investigated UV-vis absorption spectra of kojic acid after the addition of Tyr (Fig. S2). As shown in Fig. S2, two main peaks at 216 and 270 nm corresponded to the characteristic absorption bands of KA. After addng Tyr, the absorbance of these two peaks both decreased, and simultaneously, a new shoulder absorption band near ca. 320 nm appeared, which indicated the formation of stable adduct consisting of KA and Tyr due to their high affinity. To better understand this binding, Cu2+ was introduced as the alternative of Tyr to this detection system. This is because two Cu2+ ions, present in the active site of Tyr coordinated by six histidine residues, play a main role in catalyzing the oxidation of phenolic substrates into respective quinones[33]. The similar phenomenon was observed (1A-e and 1B-e), substantially confirming 6

high affinity between Tyr and KA to prevent the generation of AgNPs. The TEM images revealed the monodispersed KA/AgNPs in the absence and presence of Tyr. It can be seen that from Fig. 1C the size of generated AgNPs in the former case (Fig. 1C-a) was larger than AgNPs in the latter (Fig. 1C-b). The size distributions (Fig. S3) also clearly showed that the mean particle sizes of KA-AgNPs in the absence and presence of Tyr were around 31 and 13 nm, respectively. The decrease in size also suggested the absence of aggregation of AgNPs after the addition of Tyr, which was consistent with the fact that there was no increase at about 550 nm for related plasmonic peak which otherwise was present in other methods of synthesis followed by sensing[29]. We also tried to use AuNPs as the colorimetric reporter for Tyr detection. It was found that although AuNPs could be produced, however, the introduction of Tyr failed to prevent the formation of AuNPs, which rendered it difficult to determine Tyr similar to that using AgNPs. For this phenomenon, we speculate that weaker binding between HAuCl4 and KA led to less AuNPs to be produced. When Tyr was added, the change in the absorption spectra could almost be neglected due to similar absorption to the background (Fig. S4). Finally, we verify whether other tyrosinase inhibitors like benzoic acid, benzaldehyde, and anisic acid could also be the alternatives of kojic acid for Tyr detection. Unfortunately, it was found that in Fig. S5, these tyrosinase inhibitors could not play a similar role as KA. For benzoic acid and anisic acid, they could not reduce AgNO3 into AgNPs. For benzaldehyde, it could easily reduce AgNO3 into AgNPs, but in the presence of Tyr, the inhibition effect on the formation of AgNPs was not observed. Therefore, owing to the remarkable inhibition effect of Tyr during the synthesis of KA/AgNPs under illumination, a simple and one-step KA/AgNPs-based colorimetric nanosensor for Tyr is well established, in which the color fading of KA/AgNPs induced by Tyr can be visualized by the naked eye and also be recorded by UV-vis absorption spectra.

3.2. Optimization of detection conditions

3.2.1. Effect of kojic acid concentration

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Since kojic acid acts as not only the stabilizer but the reductant, which makes in situ detection of Tyr during the synthesis of KA/AgNPs possible, the concentration of KA was firstly investigated. As depicted in Fig. 2, when the concentration of KA was 20 μM, the highest absorbance of KA/AgNPs was obtained. Such a strong signal would greatly imporve detection sensitivity because our assay was based on the decrease of the characteristic absorption peak at 410 nm. In addition, under this condition, the absorbance changed gradually with the increase of Tyr. Compared with those at other concentrations of KA (e.g., 30 and 40 μM), it showed a wider response concentration range for Tyr. Thus, 20 μM KA was selected for the following experiments.

3.2.2. Effect of NaOH Tyr detection can be influenced by the system pH as the deprotonation of the hydroxyl groups in KA is necessary by strongly chelating with Ag+ ions before Ag+ ions was reduced. In this experiment, pH value was adjusted with NaOH. As shown in Fig. 3, the AgNPs could not be synthesized without NaOH since under this condition the binding between protonated KA and Ag+ was too weak. However, the largest change in the absorbance at 410 nm emerged when the concentration of NaOH was 0.75 mM (the pH of the solution was 10.8), in which KA was fully deprotonated benefiting for robust coordination with Ag+ and thus sequential reduction of Ag+. Furthermore, excess amounts of NaOH were also not suitable mainly due to the generation of Ag2O. So the concentration of NaOH was fixed at 0.75 mM for Tyr detection.

3.2.3. Effect of incubation time

To determine the incubation time required for Tyr detection during the synthesis of KA/AgNPs, the change in the absorbance at 410 nm after the addition of Tyr was monitored and plotted against time. As shown in Fig. 4, the absorbance of AgNPs without Tyr increased very rapidly within 40 minutes and then almost with faint changing happened, indicating that the synthesis of AgNPs almost completed within 40 min under this condition. But the absorbance of AgNPs had a tiny change before 40 min and started increasing slowly after 40 minutes with the

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addition of Tyr. Therefore, to maximize the sensitivity toward Tyr, all the measurements were performed after incubation for 40 minutes.

3.3. Sensitivity of the colorimetric assay for Tyr

In order to evaluate the detectability of the developed colorimetric method, under the above optimized conditions, the absorption spectra of KA-AgNPs of different concentrations of Tyr were recorded for sensitivity investigation. Fig. 5 showed the photographs and the corresponding UV–vis absorption spectral changes of KA/AgNPs in the presence of 20 μM KA and different concentrations of Tyr under 40-min illumination. It was observed that the color of the KA-AgNPs gradually changed from yellow to faint yellow and finally to colorless with the increase of Tyr concentration, and meanwhile, the absorbance at 410 nm in the absorption spectra decreased accordingly. It is because Tyr effectively inhibited the formation of the AgNPs by binding with the reductant KA. As shown in the inset of Fig. 5B, a good linear correlation existed between the absorbance at 410 nm and Tyr concentration in the range from 0.5 to 4.0 u/mL, and the calibration equation obtained was: A410 = -0.01509 [C]/(u/mL) + 0.7862 with a correlation coefficient of R2 = 0.998. The limit of detection (LOD) was calculated to be 0.117 u/mL (defined as 3S0/K, where S0 is the standard deviation of blank measurements (n=10) and K is the slope of calibration curve).

3.4. Selectivity of the colorimetric assay for Tyr

In order to evaluate the selectivity of this assay, metal ions, amino acids, and biomacromolecules including Na+, K+, Mg2+, Ca2+, glucose, Gly, Ala, Ile, Phe, Leu, BSA, and GOD (the concentration of metal ions are all 1.0 μM, and those of other substances are all 1.0 mg/L) were examined under the same experimental conditions. With the addition of the mentioned-above substances, the spectra were pretty much the same as that of pure KA/AgNPs and all colorimetric tubes demonstrated bright yellow color (Fig. S6), suggesting that these substances alone cannot inhibit the formation of KA/AgNPs. The effect of coexisting components was further studied by adding them with Tyr to the detection system. As shown in Fig. 6, the coexistence of following amounts of foreign species compared with the concentration of Tyr (3.0 9

u/mL) resulted in less than ±10% error: the concentration of Phe is 100.0 mg/L, concentration of Na+ and K+ is 70.0 mg/L, the concentration of Leu, Ile, and Gly is 50.0 mg/L, the concentration of Ala is 30.0 mg/L, the concentration of GOD is 10.0 mg/L, the concentrations of Mg2+ and Ca2+ is 1.0 μM, and the concentrations of glucose and BSA is 1.0 mg/L. Thus, the developed simple strategy was highly selective towards Tyr and had strong anti-interference ability to other common substances.

3.5. Analysis in human serum samples

In order to validate the feasibility of this assay, we applied it to determine Tyr in human serum samples spiked with different concentrations of Tyr. As listed in Table 1, good recoveries in the range of 96.0% to 108.6% suggested that other chemical species present in human serum samples did not interfere with the quantification of Tyr. Compared with existing colorimetric assays for Tyr, our assay bears some advantages as summarized in Table S1. Because of the integration of the synthesis, modification of AgNPs and the detection of Tyr, along with the promotion of light in the reaction rate, in our assay synthesis and detection time is faster than other methods. Also, the strong combination of Tyr and KA endows the detection with high sensitivity. Finally, this method is very eco-friendly and involves no toxic reagents or organic solvents. Therefore, the present colorimetric assay may be extended to probe Tyr in real samples with high speed and accuracy.

4. Conclusion

In summary, a green, low-cost and one-step strategy for rapid colorimetric detection of tyrosinase (Tyr) was established on the basis of the inhibition effect of Tyr on the formation of AgNPs via the specific binding with the reducing agent kojic acid. In this assay, the detection could be completed quickly and visualized from the solution color during the synthesis of AgNPs by using light irradiation to accelerate the reaction rate. This colorimetric nanosensor exhibited good selectivity and sensitivity and was capable of quantitatively monitoring Tyr in human serum samples with satisfactory recoveries. Compared with conventional three-step and 10

aggregation-based metal nanoparticle colorimetric assays, our assay is much simpler, less time-consuming and more reliable and holds great promise in medical diagnosis and food quality control associated with Tyr.

Acknowledgements

This work is financially supported by National Natural Science Foundation of China (No. 21505067).

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Fang-Ying Wu is currently a professor of College of Chemistry, Nanchang University, China. She received her BSc and MSc degrees from Jiujiang Normal College (1987) and Nanchang University (1996), respectively. She obtained her PhD in Xiamen University in 2003 and pursued her postdoctoral studies at Department of Chemistry, Seoul National University (2005-2006). Her work mainly focuses on design, synthesis, and related applications of novel organic dyes- and nanomaterials-based fluorescent and colorimetric sensors for metal ions, anions, and biomolecules.

Captions Fig. 1 (A) The photograph of and (B) UV-visible absorption spectra under different experiment conditions: (a) AgNO3 + kojic acid, in the dark for 40 minutes; (b) AgNO3 + kojic acid, in the dark for 5 hours; (c) AgNO3 + kojic acid, under illumination for 40 minutes; (d) AgNO3 + kojic acid + Tyr, under illumination for 40 minutes; (e) AgNO3 + kojic acid + Cu2+, under illumination for 40 minutes. (C) TEM images of KA/AgNPs (a) in the absence and (b) presence of 3 u/mL Tyr. The concentrations of AgNO3, KA, Tyr, Cu(NO3)2 and NaOH are 80 μM, 20 μM, 3 u/mL, 40 mM, and 0.75 mM, respectively. Fig. 2 The effect of the concentration of KA on the absorbance at 410 nm in the presence of 0, 3 and 6 u/mL Tyr during the synthesis of KA/AgNPs under 40-min illumination. ΔA0-3 and ΔA3-6 represent difference values of the absorbance at 410 nm between 0 and 3 u/mL and between 3 and 6 u/mL Tyr in the presence of various concentrations of KA, respectively. Fig. 3 The effect of NaOH concentration on the absorbance at 410 nm in the absence and presence of 6 u/mL Tyr during the synthesis of KA/AgNPs under 40-min illumination. Fig. 4 Temporal profiles of the absorbance at 410 nm in the absence and presence of 6 u/mL Tyr during the synthesis of KA/AgNPs under illumination. Fig. 5 (A) Photographs and (B) UV-visible spectra of KA/AgNPs solutions in the presence of 20 μM KA and various concentrations of Tyr (0, 0.5, 1, 2, 3, 4, and 5 u/mL) under 40-min illumination. Inset is the linear calibration curve between the absorbance A410 and Tyr concentration. Fig. 6 The absorbance at 410 nm of KA/AuNPs when foreign species were added alone ( along with Tyr 3.0 u/mL ( ) to the detection system.

) and

Scheme 1 Schematic representation for colorimetric detection of Tyr during the synthesis of kojic acid/Ag nanoparticles under illumination. Table 1 Determination of human serum samples with different amounts of Tyr. 16

Fig. 1

Fig. 2

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Fig. 3

Fig. 4

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Fig. 5

Fig. 6

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Scheme 1

Table 1 Sample

Added (u/mL)

Measured (u/mL)

Recovery (%)

RSD (%, n=3)

1 2 3 4

0 1.25 2.50 3.50

Not found 1.20 ± 0.12 2.41 ± 0.10 3.82 ± 0.30

96.0 96.4 108.6

9.61 3.37 7.96

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