Integration of redox cycling in a photoelectrochemical sensing platform for tyrosinase activity evaluation

Journal Pre-proofs Short Communication Integration of redox cycling in a photoelectrochemical sensing platform for tyrosinase activity evaluation Kai Yan, Jinnan Wu, Weihao Ji, Junfeng Wu, Jingdong Zhang PII: DOI: Reference:

S1388-2481(19)30218-8 https://doi.org/10.1016/j.elecom.2019.106555 ELECOM 106555

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Electrochemistry Communications

Received Date: Revised Date: Accepted Date:

12 August 2019 13 September 2019 16 September 2019

Please cite this article as: K. Yan, J. Wu, W. Ji, J. Wu, J. Zhang, Integration of redox cycling in a photoelectrochemical sensing platform for tyrosinase activity evaluation, Electrochemistry Communications (2019), doi: https://doi.org/ 10.1016/j.elecom.2019.106555

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Integration of redox cycling in a photoelectrochemical sensing platform for tyrosinase activity evaluation Kai Yan a, Jinnan Wu a, Weihao Ji a, Junfeng Wu b, Jingdong Zhang *a a

Key laboratory of Material Chemistry for Energy Conversion and Storage (Ministry

of Education), School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Luoyu Road 1037, Wuhan 430074, P.R. China b

Henan Province Key Laboratory of Water Pollution Control and Rehabilitation

Technology, Henan University of Urban Construction, Pingdingshan, Henan 467036, P.R. China

*Corresponding

author. Fax: +86-27-87543632. E-mail address:

[email protected] (J. Zhang).

1

Abstract Photoelectrochemical (PEC) sensors have shown great promise in bioanalysis and diagnostic applications while redox cycling has offered a convenient approach for electrochemical signal amplification. Herein, we reported an innovative PEC sensing strategy for sensitive and selective detection of tyrosinase (Tyr) activity by utilizing the redox cycling of enzymatic generated electron donor on a visible light-responsive photoelectrode. In this protocol, phenol was enzymatically oxidized to catechol by dissolved O2 in the presence of Tyr. The subsequent oxidation of catechol by photogenerated-holes of graphene-Bi2S3 nanocomposites under visible light illumination

could

be

cycled

via

the

regeneration

of

catechol

by

tris(2-carboxyethyl)phosphine (TCEP) from the oxidized product (o-benzoquinone). Thus, the PEC platform could generate an amplified photocurrent signal correlated to the activity of Tyr. Our work demonstrated a new perspective for the design and development of highly sensitive and selective PEC sensors by utilizing the high efficiency and diversity of redox cycling strategies.

Keywords: Photoelectrochemical sensor; Redox cycling strategy; Graphene-Bi2S3 nanocomposites; Tyrosinase activity evaluation

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1. Introduction Tyrosinase (Tyr, EC 1.14.18.1) is a binuclear copper-containing single-chain glycoprotein and exists widely in micro-organisms, fungi and animals [1]. It mainly functions as oxidase that catalyzing the hydroxylation of phenolic substrates to catechol derivatives, with subsequent dehydrogenation to orthoquinone products in the presence of molecular oxygen [2]. Since Tyr controls the production of melanin in pigment cells, its activity has been demonstrated as a highly sensitive indicator for the prognostic diagnosis of melanoma [3]. Additionally, it has been reported that Tyr could also act as an autoantigen and serve as a diagnostic marker for vitiligo [4]. Thus, it is of great significance to develop a highly sensitive strategy for Tyr activity evaluation. So far, various methods on the basis of colorimetry [5], electrochemistry [6], surface-enhanced Raman scattering (SERS) [7] have been established for the detection of Tyr activity. As an emerging analytical technique, photoelectrochemical (PEC) sensors have received considerable research interests in recent years owing to their inherent advantages of excellent sensitivity and low background [8]. Among various semiconductors that can be employed as photosensitive material to construct visible light-responsive PEC sensors, Bi2S3 has been considered as one of the most feasible candidates in terms of its low cost, high utilization efficiency of solar light and reasonable photon-electron conversion efficiency [9]. Inspired by the prominent advantages mentioned above, serval PEC sensing platforms have been successfully proposed by utilizing Bi2S3 as photosensitive layer [10,11]. Nevertheless, the 3

application of Bi2S3 intrinsically suffers from low electrical conductivity and a severe recombination of photo-generated electrons and holes [12]. Thus, it is desired to improve the PEC performance of Bi2S3 by coupling with a cocatalyst such as carbonaceous nanomaterials, semiconductors and metal nanoparticles. In particular, as a carbon-based two-dimensional nanomaterial, graphene (G) has been extensively investigated in the last decade for application in PEC field originating from its remarkable physicochemical properties [13]. In this regard, visible light-responsive G-Bi2S3 nanocomposites capable of improved optical and electrochemical properties have been successfully prepared and employed to fabricate high-performance PEC devices [14,15]. In this work, we proposed a novel visible light-driven PEC sensing strategy for the detection of Tyr activity based on G-Bi2S3 photoelectrode. To obtain an amplified electrical signal of the proposed PEC system, a tris(2-carboxyethyl)phosphine (TCEP)-triggered redox cycling was introduced in this platform. As illustrated in Scheme 1, Tyr catalyzed the oxidation of phenol into catechol, which acted as the electron donor in this PEC system. The subsequent oxidation of catechol by the photogenerated-holes of G-Bi2S3 nanocomposites modified photoelectrode would then trigger the chemical redox cycling by utilizing TCEP as reductant. That is, catechol was photoelectrocatalytically oxidized at photoelectrode surface and regenerated via reduction of the oxidized product (o-benzoquinone) by TCEP. Thus, the recovery of electron donor in this PEC system induced an amplified PEC signal [16]. Therefore, a novel PEC method for detection of Tyr activity was developed. 4

2. Material and Methods 2.1. Preparation of G-Bi2S3 nanocomposites The commercial graphene nanosheets (300 mg) was pretreated according to reference [17] and ultrasonically dispersed in 25 mL of glycol, followed by the addition of 3.75 mmol of Bi(NO3)3·5H2O under vigorous stirring. Then, 5.625 mmol of Na2S·9H2O dissolved in 30 mL water was added drop-wisely into the above mixture, leading to the formation of a black solution. After adding 20 mL water containing 32 mmol of urea, the mixture was transferred into a 100 mL Teflon-lined autoclave and stirred at room temperature for 1 h. The autoclave was further sealed in a stainless-steel tank, and heated at 180 °C for 12 h. The product was washed three times with deionized water and ethanol, dried at 60 °C overnight to obtain G-Bi2S3 nanocomposites. G-Bi2S3 suspension was prepared by dispersing suitable amount of G-Bi2S3 nanocomposites in water with the aid of sonication. 2.2 Fabrication of PEC sensor Before modification, indium tin oxide (ITO) substrates were sonicated in acetone, mixed solution of ethanol and 2 M NaOH (v/v, 1:1) and water, respectively for 20 min. After being dried with nitrogen gas, the ITO electrodes with an exposed geometric area of 0.071 cm2 was coated with 7 μL of G-Bi2S3 suspension and dried at 60 °C in an oven, followed by rinsing with water to remove loosely adsorbed materials. The obtained modified electrode was marked as G-Bi2S3/ITO. The photocurrent responses of modified electrodes were recorded in phosphate-buffered saline (PBS, pH7.4, 0.1 M) containing 0.1 mM phenol, 1 mM 5

TCEP and different concentration of Tyr. Electrochemical measurement was carried out after an incubation period of 10 min. 2.3. Apparatus The surface morphology and crystalline phase were characterized by a Quanta 200 field emission scanning electron microscope (SEM, FEI, The Netherlands) and X-ray diffraction (XRD) instrument (Bruker Instruments, Germany), respectively. The UV-Vis absorption spectra were collected on a UV-2550 spectrophotometer (Shimadzu, Japan). Electrochemical measurements were performed on CHI660A electrochemical workstation (Chenhua Instrument Co., Shanghai, China) using a conventional 3-electrode cell. A modified ITO electrode, a saturated calomel electrode (SCE), and a platinum wire were employed as the working, reference and counter electrodes, respectively. A portable violet laser pen with a power of 20 mW at 405 nm and a diameter of ∼3 mm for the illumination area was used as the light source. 3. Results and Discussion 3.1. Characterization of Materials The morphologic structures of the prepared materials were first characterized by SEM. As can be seen, the acid-treated graphene shows sheet-like morphology with a size about 5 μm (Fig. 1A) [17], while the solvothermally synthesized Bi2S3 is composed of a large quantity of nanorods with an average length of 60 to 100 nm (Fig. 1B). For G-Bi2S3 nanocomposites, it can be observed that many Bi2S3 nanorods are attached on the surface of graphene nanosheets with intimate contact (Fig. 1C). XRD 6

analysis (Fig. 1D) further demonstrates that the crystal structures of graphene and Bi2S3 are not changed during the preparation process. The UV-Vis absorption spectra of the prepared Bi2S3 nanorods and G-Bi2S3 nanocomposites were recorded to evaluate their light-harvesting ability. As shown in Fig. 1E, the pure Bi2S3 possesses a certain absorption over the whole UV-visible light region, while the introduction of graphene increases the absorbance of Bi2S3 nanorods. This result demonstrates that the G-Bi2S3 nanocomposites exhibits a superior light absorption property. Moreover, electrochemical impedance spectroscopy (EIS) were performed to investigate the interfacial electron transfer on the electrode surface. The Nyquist plots of ITO electrodes modified with Bi2S3, and G-Bi2S3 nanocomposites (Fig. 1F) reveal that Bi2S3 can accelerate the electron transfer on the electrode interface, and electron transfer resistance further decreases when G-Bi2S3 nanocomposites are employed to modify ITO electrode, attributed to the better electrocatalytic activity of graphene nanosheets. Accordingly, the prepared G-Bi2S3 nanocomposites with improved optical and electrochemical properties can be potentially exploited for high-performance PEC sensing platform. 3.2 Voltammetric and PEC Analysis The feasibility of the proposed Tyr-induced redox cycling strategy was first investigated by recording cyclic voltammogram (CV) of the G-Bi2S3/ITO electrode in PBS containing different species. As shown in Fig. 2A, the CV curve recorded in blank PBS does not show noticeable Faradaic current (curve a in Fig. 2A), and the electrooxidation of phenol is not observed in the given potential range (curve b in Fig. 7

2A). When 20 U/mL of Tyr is added in the electrolyte, the anodic current of the CV curve shows an evident increment over 0.3 V (curve c in Fig. 2A), which could be assigned to the electrochemical oxidation of catechol generated from phenol under the enzymatic catalysis of Tyr. In the presence of TCEP, the anodic current of the enzymatic system increases dramatically over a broad potential range (curve d in Fig. 2A). This result strongly indicates that redox cycling involving catechol and TCEP is occurring. By contrast, the introduction of TCEP into phenol solution in absence of Tyr only induces a slight increase in the anodic current over 0.3 V (curve e in Fig. 2A), demonstrating the crucial role of Tyr in the designed redox cycling system. On the other hand, the photocurrent responses of G-Bi2S3/ITO in presence of various species were recorded to study the performance of chemical redox cycling under light irradiation. As depicted in Fig. 2B, G-Bi2S3/ITO responds sensitively to the visible light irradiation and possesses a good photoelectrical property (curve a in Fig. 2B). Meanwhile, the addition of 0.1 mM phenol has no effect on increasing the photocurrent (curve b in Fig. 2B). While Tyr and phenol co-existe in the electrolyte, both the photocurrent and dark current responses increase, ascribed to the PEC and electrochemical oxidation of catechol (curve c in Fig. 2B). As expected, when TCEP is added in the enzymatic system, an enhanced photocurrent response is observed (curve d in Fig. 2B), further revealing that TCEP could regenerate catechol from the photoelectrochemical oxidized product and promote the photoelectrochemical signal [18]. Additionally, the current in curve d before photoirradiation enhances after the addition of TCEP, owing to the electrochemical-chemical (EC-C) redox cycling of 8

catechol in presence of TCEP in the dark. Nevertheless, the addition of TCEP into phenol solution without Tyr does not induce obvious change in the photocurrent response (curve e in Fig. 2B). These results demonstrate the achievement of the proposed Tyr-induced photoelectrochemical-chemical (PEC-C) redox cycling process, as illustrated in Scheme 1. Obviously, Tyr plays a key role in this system since it catalyzes the oxidation of phenol to catechol, which further participate in the redox cycling process to generate an amplified photocurrent signal. Therefore, the proposed PEC platform can be explored for the evaluation of Tyr activity. Moreover, to obtain the highest Tyr sensing performance, the effect of the amount of G-Bi2S3 nanocomposites on the ΔPI (photocurrent difference before and after incubation with Tyr) response toward 5 U·mL-1 Tyr was optimized, as depicted in Fig. 2C.The electrode modified with 2 g/L G-Bi2S3 shows the optimal response. While the amount of G-Bi2S3 is higher than 2 g/L, the photocurrent decreases, due to enhanced recombination of photogenerated electrons and holes with excessive G-Bi2S3. 3.3 PEC sensing of Tyr. Based on the above evaluation, the proposed PEC sensor was constructed under optimal conditions. The photocurrent responses of G-Bi2S3/ITO toward different concentration of Tyr was recorded in 0.1 M PBS (pH 7.4) containing 0.1 mM phenol and 1 mM TCEP is depicted by Fig. 3A. The ΔPI value is found to be linearly increased with increasing the concentration of Tyr from 0.3 - 20 U·mL-1 (inset of Fig. 3A). The linear regression equation can be expressed as ΔPI/μA= 0.0570C/U·mL-1 + 0.0011 (R2 = 0.998). The limit of detection (LOD) is calculate to be 0.15 U·mL-1 by 9

using the formula LOD=3s/b, where s is the standard deviation of the blank and b is the slope of the calibration curve. Compared with some previously reported methods for Tyr detection (Table 1), this PEC sensor shows lower LOD. Additionally, since the EC-C redox cycling involving catechol and TCEP occurs in the dark, the dark current in Fig. 3A also increases linearly with the increase of Tyr concentration. Nevertheless, for such a EC-C redox cycling scheme for Tyr detection, the LOD is estimated to be 0.90 U/mL, much higher than that obtained using PEC-C redox cycling scheme (0.15 U/mL). This result further demonstrates the superiority of the proposed PEC sensing platform. The selectivity of the fabricated PEC biosensor for Tyr was studied by recording the photocurrent response of the sensor to several representative interfering substances including bovine serum albumin (BSA), laccase (Lac) and glucose oxidase (GOx). No obvious photocurrent signals change can be observed by these potential interfering substances, suggesting that the proposed PEC sensor has a satisfactory selectivity (Fig. 3B). Furthermore, the reproducibility of this PEC sensing platform toward Tyr was investigated by carrying out five independent experiments. The relative standard deviation was calculated to be 3.7%, suggesting good reproducibility of the proposed sensor. The stability of the sensor was also investigated by checking the photocurrent response after the G-Bi2S3/ITO photoanode was stored in dark conditions at 4 °C for 7 days. The results showed that the PFC still maintained at least 97.9% of the initial response, indicating that the sensor has high stability. To further assess the practical application of the proposed PEC sensor, different 10

concentrations (1.0, 5.0, 10.0 U·mL-1) of Tyr in biological samples were evaluated by standard addition methods. The recovery results of spiked 10-fold diluted human serum samples are summarized in Table 2. The RSD was 1.6 ~3.0% and recoveries were 97.0-102.2%, indicating the designed PEC biosensor for Tyr detection is of good accuracy and reliability in practical application in real samples. 4. Conclusions A novel PEC sensing platform coupling with redox cycling strategy for the detection of Tyr activity was proposed based on a visible light-responsive G-Bi2S3 modified photoelectrode. In such a sensor, Tyr catalyse the oxidation of phenol to catechol, which acts as the electron donor of this PEC system. The combination of PEC oxidation of catechol and efficient cycling of catechol by TCEP from the oxidized product, an amplified photocurrent signal correlated to the activity of Tyr is obtained. The proposed PEC sensing platform exhibits a good selectivity, stability and practicability in real sample analysis. This redox cycling strategy-coupled PEC sensing platform offers a new perspective for the development of high-performance PEC bioanalytical protocols. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 61571198), China Postdoctoral Science Foundation (Grant No. 2019M652618) and the opening fund of Henan Province Key Laboratory of Water Pollution Control and Rehabilitation Technology (Grant No. CJSP2018008). We also thank the Analytical and Testing Center of Huazhong University of Science 11

and Technology for the help in materials characterization. References [1] C. Tang, L. Jin, Y. Lin, J. Su, Y. Sun, P. Liu, Q. Li, G. Wang, Z. Zhang, L. Du, M. Li, Organic & Biomolecular Chemistry, 16 (2018) 9197-9203. [2] L. Chai, J. Zhou, H. Feng, C. Tang, Y. Huang, Z. Qian, ACS Appl Mater Inter, 7 (2015) 23564-23574. [3] N.G. Ordóñez, Hum Pathol, 45 (2014) 191-205. [4] C. Angeletti, V. Khomitch, R. Halaban, D.L. Rimm, Diagn Cytopathol, 31 (2004) 33-37. [5] B.W. Liu, P.C. Huang, J.F. Li, F.Y. Wu, Sens Actuators B, 251 (2017) 836-841. [6] B. Shah, A. Chen, Electrochem Commun, 25 (2012) 79-82. [7] L. Wang, Z.F. Gan, D. Guo, H.L. Xia, F.T. Patrice, M.E. Hafez, D.W. Li, Anal Chem, 91 (2019) 6507-6513. [8] K. Yan, Y. Liu, Y. Yang, J. Zhang, Anal Chem, 87 (2015) 12215-12220. [9] Q. Liu, J. Huan, N. Hao, J. Qian, H. Mao, K. Wang, ACS Appl Mater Inter, 9 (2017) 18369-18376. [10] H. Yin, B. Sun, Y. Zhou, M. Wang, Z. Xu, Z. Fu, S. Ai, Biosens Bioelectron, 51 (2014) 103-108. [11] J. Wang, J. Long, Z. Liu, W. Wu, C. Hu, Biosens Bioelectron, 91 (2017) 53-59. [12] L.L. Long, A.Y. Zhang, Y.X. Huang, X. Zhang, H.Q. Yu, J Mater Chem A, 3 (2015) 4301-4306. [13] D. Chen, H. Zhang, Y. Liu, J. Li, Energy Environ Sci, 6 (2013) 1362-1387. [14] O.K. Okoth, K. Yan, Y. Liu, J. Zhang, Biosens Bioelectron, 86 (2016) 636-642. [15] S. Vadivel, A.N. Naveen, V.P. Kamalakannan, P. Cao, N. Balasubramanian, Appl Surf Sci, 351 (2015) 635-645. [16] J.T. Cao, B. Wang, Y.X. Dong, Q. Wang, S.W. Ren, Y.M. Liu, W.W. Zhao, ACS Sensors, 3 (2018) 1087-1092. [17] X. Chen, D. Liu, G. Cao, Y. Tang, C. Wu, ACS Appl Mater Inter, 11 (2019) 9374-9384. 12

[18] S. Noh, H. Yang, Electroanalysis, 26 (2014) 2727-2731. [19] X. Yang, Y. Luo, Y. Zhuo, Y. Feng, S. Zhu, Anal Chim Acta, 840 (2014) 87-92. [20] F. Kong, H. Liu, J. Dong, W. Qian, Biosens Bioelectron, 26 (2011) 1902-1907. [21] H.B. Yildiz, R. Freeman, R. Gill, I. Willner, Anal Chem, 80 (2008) 2811-2816. [22] J.W. Liu, Y.M. Wang, L. Xu, L.Y. Duan, H. Tang, R.Q. Yu, J.H. Jiang, Anal Chem, 88 (2016) 8355-8358.

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Table 1. Comparison of different analytical methods for Tyr activity determination. Method

Linear range (U·mL-1)

LOD (U·mL-1)

Reference

Fluorescent sensor based on Gold nanoclusters

0.5 - 200

0.08

[19]

Colorimetric detection based on SiO2/Gold NPs

1.0 - 15

1

[20]

Electrochemical sensor based on Pt nanoparticles

-

1

[21]

Colorimetric detection based on Ag NPs

0.5 - 4

0.117

[22]

PEC sensor based on G-Bi2S3

0.3 - 20

0.15

this work

14

Table 2. Determination of Tyr activity in spiked diluted serum samples by the proposed PEC method (n = 5). Added

Found

(U·mL-1)

(U·mL-1)

0

RSD

Recovery

0

-

-

1

0.97

2.8%

97.0%

5

5.02

1.6%

100.4%

10

10.22

3.0%

102.2%

15

Scheme and Figure Captions Scheme 1. Schematic illustration of PEC sensing platform coupling with redox cycling for Tyr detection. Fig. 1. SEM images of (A) graphene, (B) Bi2S3, and (C) G-Bi2S3 nanocomposites. (D) XRD patterns of (a) graphene, (b) Bi2S3, and (c) G-Bi2S3 nanocomposites. (E) UV-Vis absorption spectra of (a) Bi2S3 and (b) G-Bi2S3 nanocomposites. (F) EIS plots of (a) ITO, (b) Bi2S3/ITO and (c) G-Bi2S3/ITO electrodes in 0.1 mol L-1 KCl solution containing 5.0 mmol L-1 [Fe(CN)6]3-/4- at bias potential of 0.26 V. The frequency range was 0.1 Hz ~ 100 kHz. The inset represents the corresponding equivalent circuit (Rs = solution resistance, Ret = electron transfer resistance, W = Warburg impedance, CPE = constant phase element). Fig. 2. (A) CV curves and (B) photocurrent responses of G-Bi2S3/ITO recorded in 0.1 M PBS solution (pH 7.4) containing different species: (a) blank PBS solution, (b) 0.1 mM phenol, (c) 0.1 mM phenol and 20 U·mL-1 Tyr, (d) 0.1 mM phenol, 20 U·mL-1 Tyr and 1 mM TCEP, (e) 0.1 mM phenol and 1 mM TCEP. CV curves were recorded in the dark and the corresponding photocurrent responses were recorded at 0 V under chopped light. (C) Influence of amount of G-Bi2S3 nanocomposites on the ΔPI response toward 5 U·mL-1 Tyr. Error bars were derived from the standard deviation of three measurements. Fig. 3. (A) Photocurrent responses of G-Bi2S3/ITO electrode recorded in 0.1 M PBS containing 0.1 mM phenol, 1 mM TCEP and various concentrations of Tyr: (a) 0, (b) 0.3, (c) 1, (d) 5, (e) 10, (f) 20 U·mL-1. Inset: calibration curve for Tyr. (B) Histogram 16

for ratio of ΔPI values of the proposed PEC sensor toward 5 U·mL-1 Tyr without (ΔPI’) and with (ΔPI’’) different potential interferents: BSA (50 μg/mL), Lac (10 U/mL) and GOx (10 U/mL).

17

Scheme 1

18

Fig. 1.

19

Fig. 2.

20

Fig. 3.

21

22

Highlights 1. A redox cycling strategy-coupled photoelectrochemical sensor was developed. 2. Catechol was generated in situ and cycled as electron donor in this platform. 3. Visible light-responsive graphene-Bi2S3 was exploited as photoelectric species. 4. Sensitive and selective detection of tyrosinase activity was achieved.

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