Electrochemistry-Regulated Recyclable SERS Sensor for Sensitive

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Electrochemistry-Regulated Recyclable SERS Sensor for Sensitive and Selective Detection of Tyrosinase Activity Lu Wang, Zhen-Fei Gan, Dan Guo, Hai-Lun Xia, Fato Tano Patrice, Mahmoud Elsayed Hafez, and Da-Wei Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05341 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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Analytical Chemistry

Electrochemistry-Regulated Recyclable SERS Sensor for Sensitive and Selective Detection of Tyrosinase Activity Lu Wang,‡,a Zheng-Fei Gan,‡,a Dan Guo,a Hai-Lun Xia,a Fato Tano Patrice,a Mahmoud Elsayed Hafez,a,b Da-Wei Li*,a a Key

Laboratory for Advanced Materials, Joint International Laboratory for Precision Chemistry & School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China b Department of Chemistry, Faculty of Science, Beni-Suef University, Beni-Suef 62511, Egypt. ABSTRACT: Tyrosinase (TYR) which can catalyze the oxidation of catechol is recognized as a significant biomarker of melanocytic lesions, thus developing powerful methods for the determination of TYR activity is highly desirable for the early diagnosis of melaninrelated diseases including melanoma. Herein, we develop a novel portable and recyclable surface-enhanced Raman scattering (SERS) sensor, prepared by assembling gold nanoparticles and p-thiol catechol (p-TC) on an ITO electrode, for detecting TYR activity via the SERS spectral variation caused by the conversion of p-TC into its corresponding quinone under TYR catalysis. The developed SERS sensor has a rapid response to TYR within 1 min under the optimized conditions and shows high selectivity for TYR with the detection limit at 0.07 U/mL. Importantly, this SERS sensor can be easily regulated by applying negative voltage to achieve circular utilization, favoring the automation of SERS detection. Furthermore, the presented recyclable SERS sensor can perform well on both the determination of TYR activity in serum and the assessment of TYR inhibitor, demonstrating huge potential in the sensitive, selective, and facile detection of TYR activity for disease diagnosis and drug screening related with TYR.

Tyrosinase (TYR), an important oxidoreductase, plays a critical role in melanin synthesis by catalyzing the hydroxylation of phenols to their respective catechol compounds and the subsequent oxidation of catechols to oquinones 1-3. The abnormal level of TYR has been progressively found to be closely related with a variety of pathological processes such as melanoma cancer 4-7, Parkinson’s disease 8, and vitiligo 9. Therefore, it is of urgency to explore effective approaches for the detection of TYR activity to facilitate the early diagnosis of diseases where TYR is involved in and the deep understanding of physiological functions of TYR. To date, a number of methods have been already developed for TYR activity detection such as colorimetry 10, electrochemistry 11,12, high-performance liquid chromatography 13, and fluorescence with confocal imaging techniques 14-19. Each method has its merits and disadvantages. For instance, although the colorimetry is a good quantitative method, it is usually hampered by low sensitivity or tedious antibody-involved procedures 20; Electrochemical approaches respond rapidly and sensitively, nevertheless they generally have poor capability of preventing from the interference of nonspecific substances in real samples 11; The liquid chromatography can perform well on qualitative and quantitative detections, however it commonly suffers from the sophisticated apparatus 13; Particularly, the fluorescent technique attracts considerable attention for its relatively high sensitivity and spatial resolution 21, whereas it is often hindered by photobleaching, phototoxicity, and background disturbance from autofluorescence of biological samples 22. Surface-enhanced Raman scattering (SERS) which offers amplified spectroscopic molecular fingerprints has increasingly

emerged as a powerful analysis technology due to high sensitivity, excellent selectivity, rapid response, resistance to photobleaching and phototoxity, etc 23-26. These comprehensive superiorities make SERS as an effective detection strategy widely used in biological analysis, for instance protein identification, DNA detection, and cell assay 27-31. However, direct SERS detection of biomolecules, such as proteins, peptides, metabolites, in a biological system is inherently affected by the materials with stronger affinity for metallic SERS substrates, and the complicated structures of the analytes like protein usually render the spectra intricate and analysis difficult 27,32. To address these issues and make the best of SERS advantages to detect biological species, SERS sensors can be prepared through the integration of plasmonic nanostructures and Raman active molecules which have reaction with analytes thus causing the changes of SERS spectra 33-35. On the other hand, handheld and reusable SERS sensors are more attractive because of their advantage in favor of application in remote areas for point-of-care (POC) diagnostics 36,37. However, to the best of our knowledge, portable and recyclable SERS sensors for the selective and sensitive TYR activity assay in serum have not been attempted yet. Inspired by the oxidation of catechols to o-quinones specifically catalyzed by TYR 3, we develop a novel electrochemistry-regulated recyclable SERS sensor to detect TYR activity. As shown in Scheme 1, the sensor is fabricated by assembling plasmonic gold nanoparticles (AuNPs) and the synthesized SERS-active p-thiol catechol (p-TC) in sequence onto an ITO electrode. And the detection of TYR activity is accomplished through the SERS spectral changes of the ITO/AuNPs/p-TC sensor, resulting from the transformation of

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p-TC to its oxidative product p-thiol benzoquinone (p-TB) under the catalysis of TYR. The proposed ITO/AuNPs/p-TC sensor can be facilely reclaimed through applying negative voltage to realize recycling. Additionally, this recyclable SERS sensor allows not only the sensitive, selective and rapid determination of TYR activity in real samples but also the convenient evaluation of TYR inhibitor, thus displaying tremendous potential in the automatable SERS analysis of TYR activity for the related disease diagnosing and drugs screening. Scheme 1. Schematic exhibition of the fabrication of SERS sensors and their electrochemistry-regulated recycling for the detection of TYR activity.

■ EXPERIMENTAL SECTION Reagents and materials All reagents in this work were analytically pure and used without further purifying treatment. Gold chloride hydrate (HAuCl43H2O ≥ 99.9%), trisodium citrate (≥ 99%), potassium chloride (KCl, ≥ 99%), magnesium chloride (MgCl2, ≥ 99%), trypsin, acetycholinesterase (AchE), and TYR from mushroom were purchased from Sigma-Aldrich (St Louis, MO, USA). Glutathione (GSH), glucose, sodium chloride (NaCl, ≥ 99.5%), and 3-mercaptopropyl-trimethoxysilane (MPTS) were obtained from Aladdin Chemical Company (Shanghai, China). Sodium hypochlorite (NaClO, 99.7%) and hydrogen peroxide (H2O2, 30 wt%) were bought from Shanghai LingFeng Chemical Reagent Co., Ltd (Shanghai, China). Urea was supplied by J &K Chemical Ltd (Shanghai, China). Sodium bicarbonate (NaHCO3, ≥ 99.8%) was acquired from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Human TYR enzymelinked immunosorbent assay (ELISA) kits were provided by Tongwei Biotechnology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) was from MoXi Biotech. Co., Ltd (Shanghai, China). Deionized water was collected from MilliQ ultrapure water purification system (˃ 18 MΩ•cm).

Apparatus A JEM-2100 high-resolution transmission electron microscope (TEM, JEOL, Japan) and a S-4800 field emission scanning electron microscope (SEM, Hitachi, Japan) were used to conduct the morphology characterization. USB 2000+ spectrometer (Ocean Optics Inc., USA) was applied to measure UV-Vis absorption spectra. All pH values were recorded on a pH-3c digital pH meter (Shanghai Leici Device Works, Shanghai, China). The temperature was controlled by a thermostatic water bath (Shanghai Boxun Industrial Co., Ltd, China). The transformation of p-TC to p-TB was checked with a time-of-flight secondary ion mass spectrometer (ToF-SIMS V, ION-TOF GmbH, Germany). The Raman spectra were measured by a portable Raman spectrometer (BWS415, B&W Tek Inc, USA) with a 785 nm laser (3 cm-1 resolution). The

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electrochemical tests were performed on a CHI660E electrochemical workstation (Shanghai Chenhua Co., Ltd, China).

Fabrication of ITO/AuNPs/p-TC sensor AuNPs were prepared by a modified citrate reduction process Briefly, deionized water (100 mL) and HAuCl4 solution (4.8 mL, 1.0 wt%) were added into a clean flask at ambient temperature and heated with violent stirring. While the solution began to be boiling, citrate solution (10 mL, 1.0 wt%) was added with the color fast changing from faint yellow to wine red. Next, the mixture was maintained boiling for excess 15 min to further reduce the HAuCl4 and was then cooled to ambient temperature. The obtained AuNPs colloid was treated with centrifugation (5000 rpm, 5 min) to remove redundant trisodium citrate solution. Afterwards, the concentrated AuNPs were stored in refrigerator (4 °C) for the following experiments. In addition, a new ITO glass slide was washed by an ultrasonic cleaner with deionized water and acetone for 30 min successively, and then dried by nitrogen gas. Subsequently, the ITO glass slide was immersed in an acetone solution of 1.0 mM MPTS for 24 h to functionalize its surface and then washed by acetone and dried by nitrogen gas. 2.5 μL AuNPs were dropwise added onto the MPTS-functionalized ITO glass surface, followed by the modification with 10 mM p-TC (2.5 μL) which was synthesized according to the previously described process 39. Thus, the ITO/AuNPs/p-TC SERS sensor was successfully fabricated for the TYR activity detection. 38.

Detection of TYR activity The detection of TYR activity was performed as following procedures. The lyophilized powder of TYR was dissolved and divided into 10 aliquots which were frozen at -20 oC to maintain their activity and were freshly thawed before usage. For SERS tests, the ITO/AuNPs/p-TC sensor was inserted into the testing tube containing TYR reactivated by incubation at 37 °C in PBS solution (10 mM, pH 7.4). The testing tube was then protected from light and put into a constant temperature water bath for a certain time. Finally, the sensor was moved to measure SERS spectra. The determinations of TYR activity for serum samples were conducted in the similar way.

Electrochemistry regulation of the recycling of ITO/AuNPs/p-TC sensor The electrochemistry regulation of the recycling of the ITO/AuNPs/p-TC sensor was conducted by a three-electrode system consisting of a working electrode (ITO/AuNPs/p-TC), a counter electrode of platinum wire, and a reference electrode of Ag/AgCl. The window of potential for the voltammetric tests was from -0.3 to 0.5 V and the scan rate was at 0.05 V/s. The used SERS sensor was regulated by applying constant potential at -0.3 V with 120 s and reused for the SERS detection of TYR activity after rinsing with deionized water. This process was repeated several times to evaluate the recyclability of the ITO/AuNPs/p-TC sensor.

TYR inhibition assay The inhibitory action was carried out by adding different concentrations of inhibitor into the prepared TYR solution (10 U/mL) with 20 min subsequent incubation at 37 oC. Afterwards, the ITO/AuNPs/p-TC sensor was introduced to the inhibitortreated TYR solutions and then moved to perform SERS measurements.


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■ RESULTS AND DISCUSSION Characterization of the ITO/AuNPs/p-TC sensor Characterization by TEM and UV-Vis spectroscopy indicates that the prepared AuNPs displayed spherical shape and good uniformity with particle size approximately 60 nm (Figure S1). Moreover, the synthesized compound p-TC, consisting of a mercapto group for strongly attaching to AuNPs surface and a catechol group for specifically responding to TYR, was 1H-NMR 13C-NMR confirmed with spectroscopy, spectroscopy, and mass spectroscopy (Figure S2-S4). Thus, through assembling AuNPs and p-TC on the ITO surfaces successively, a portable SERS sensor (ITO/AuNPs/p-TC) can be fabricated conveniently (Figure 1A). As shown in Figure 1B and 1C, the SEM images of the sensor indicate that the AuNPs formed a dense gold film on the ITO surface. The highermagnification SEM image (Insert in Figure 1C) further demonstrates that most AuNPs were congregated, which could promote to produce more “hot spots” 40. Consequently, a strong SERS signal of p-TC can be obtained (Figure S5), inferring the possibility of high detection sensitivity of the fabricated sensor. In addition, SERS spectra were collected from 10 randomly selected location of the ITO/AuNPs/p-TC sensor (Figure 1D), and intensities of stochastic peaks at 588 cm-1 and 898 cm-1 (I588 and I898) were used to evaluate the uniformity (Figure 1E). The calculated relative standard deviation (RSD) for I588 and I898 are 4.56% and 5.47%, severally, showing the fabricated sensor was greatly homogeneous. Further SERS investigation revealed that the spectra of ITO/AuNPs/p-TC SERS sensor were generally unchanged after storage for 5 days under air atmosphere and with pH differing in the scope of 5.6-8.0 (Figure S6). Similarly, those spectra of the used sensor almost kept changeless after 24 h storage (Figure S7). This infers that the developed SERS sensor also has outstanding stability and repeatability.

symmetric stretching, respectively, appeared in the SERS spectrum clearly. Meanwhile, some bands disappeared or weakened obviously at 780 cm-1, 898 cm-1, and 1071 cm-1 which could be assigned to the C-O-H symmetric stretching with the mixing of CCC stretching, the C-O-H asymmetric stretching accompanied with the asymmetric stretching of CCC bond, and the C-O-H asymmetric stretching combined with the CCC inplane deformation, respectively (Table S1). The changing in SERS spectrum may imply that the p-TC was transformed into p-TB on the surfaces of the sensor because of the catalysis of TYR. To verify our hypothesis for the SERS results, characterization with UV-Vis spectroscopy and ToF-SIMS were carried out. The UV-Vis test indicated that the absorption peak of the p-TC (100 μM) at 289 nm red-shifted to 293 nm and a new peak at about 450 nm emerged with the addition of 100 U/mL TYR (Figure S8). Furthermore, the negative ion ToFSIMS analysis of the p-TC modified gold film before and after incubation with TYR demonstrated that the intensity ratio between peaks at m/z=138.9 and m/z=141.0 respectively assigned to p-TB (calc. 139.0, [M-H]) and p-TC (calc. 141.0, [M-H]) increased by approximately 2-fold (Figure 2B and 2C). These results further confirm that p-TC on AuNPs surface can be converted to p-TB in the presence of TYR.

Figure 2. (A) SERS spectra of the ITO/AuNPs/p-TC SERS sensors in the (a) absence and (b) presence of TYR (100 U/mL). (B) Negative ion ToF-SIMS spectra of p-TC assembled gold film (a) before and (b) after reaction with TYR. (C) The ratio of I138.9/I141.0 according to the spectra shown in (B).

Optimization of detection conditions

Figure 1. (A) Real image of the ITO/AuNPs/p-TC SERS sensor. (B, C) SEM images of ITO/AuNPs/p-TC SERS sensor. (D) SERS spectra collected at 10 randomly selected locations of the ITO/AuNPs/p-TC SERS sensor (a-j). (E) Plots of the intensities of peaks at 588 cm-1 and 898 cm-1 (I588 and I898) according to SERS spectra shown in (D).

Responsivity of the ITO/AuNPs/p-TC sensor The response ability of the ITO/AuNPs/p-TC sensor to TYR was tested in PBS (10 mM, pH=7.4), which showed that obvious variations of SERS spectrum can be observed for the sensor in the presence of TYR (Figure 2A). In details, new bands at 484 cm-1 and 1017 cm-1, attributed to the C=O rocking and the C=O symmetric stretching with the coupling of CCC

Conditions for TYR activity detection were optimized by investigating the influence of pH value, reaction temperature and detection time on the SERS response of the ITO/AuNPs/pTC sensor toward TYR. As displayed in Figure 3A and 3B, the ratiometric peak intensity of I484/I588 ascended with the pH value varying from 5.6 to 7.4 and then descended with the sequential increase of pH value. From Figure 3C and 3D, it can be observed that I484/I588 values reached the maximum at 37 oC, consisting with the fact that TYR generally possesses the best activity at this temperature 14,15. Moreover, the SERS response of the sensor to TYR in PBS at pH 7.4 and 37 oC can occur instantly and complete within 1 min (Figure 3E and 3F). Taken together, pH 7.4, reaction temperature of 37 oC, and detection time around 1 minute can serve as the optimized conditions for the developed sensor to detect TYR activity.

Sensitivity and selectivity The sensitivity of the ITO/AuNPs/p-TC sensor for TYR activity was evaluated by recording SERS signals under the optimized conditions in the presence of TYR with different concentrations. While the concentration of TYR increased, the peak intensity at 484 cm-1 particular for TYR sensing reaction increased correspondingly (Figure 4A). Furthermore, the ratiometric peak intensities of I484/I588 had an approximately linear dependence on the logarithmic concentration of TYR from 0.1to 100 U/mL (Figure 4B), and the detection limit was



calculated to be 0.07 U/mL on basis of a signal-to-noise ratio of S/N=3. Thus, compared with published approaches (Table S2), the developed SERS sensor has comprehensive superiorities in responding time, sensitivity, and linear range, which renders the SERS sensor capable to detect the TYR activity in biological samples, especially under pathology conditions. Moreover, the selectivity of this SERS sensor was further examined by measuring its response in the presence of different biologically relevant species such as NaClO, KCl, MgCl2, NaCl, NaHCO3, H2O2, glucose, glutathione, trypsin, urea, AchE, and TYR, respectively. The result demonstrated that just TYR triggered a new strong SERS band at 484 cm-1 (Figure 4C and red histogram in Figure 4E). Additionally, when TYR was added into the detection systems in the presence of diverse biologically relevant species severally, such significant SERS bands at 484 cm-1 arose with almost similar I484/I588 values (Figure 4D and blue histogram in Figure 4E). This suggests that the ITO/AuNPs/p-TC sensor has high selectivity towards TYR, probably resulting from the specificity of the TYR-catalyzed reaction and the superiority of the SERS technology on fingerprint 28. Therefore, the presented SERS sensor displays a great promise in the determination of TYR activity in real specimens without significant interference from other biological components.

Figure 3. (A) SERS spectra of the ITO/AuNPs/p-TC SERS sensor after reacting with 100 U/mL TYR at 25 oC under various pH (af): 5.7, 6.2, 6.6, 7.0, 7.4, and 8.0. (B) Ratiometric peak intensities of I484/I588 according to the spectra shown in (A). (C) SERS spectra of the ITO/AuNPs/p-TC SERS sensor after reacting with 100 U/mL TYR under pH 7.4 at various temperature (a-e): 25, 30, 37, 40, and 45 oC. (D) Ratiometric peak intensities of I484/I588 according to the spectra shown in (C). (E) SERS spectra of the ITO/AuNPs/pTC SERS sensor after reacting with 100 U/mL TYR under pH 7.4 at 37 oC for various time (a-g): 0, 1, 3, 5, 15, 30, and 60 min. (F) Ratiometric peak intensities of I484/I588 according to the spectra shown in (E). Error bars represent ± S.D (n = 3).

Electrochemistry-regulated ITO/AuNPs/p-TC sensor

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recycling

of

the

Since p-TC/p-TB is a redox couple and undergoes a reversible change in buffered solution through electrochemistry process 41, one can reasonably speculate that the developed SERS sensor may have the possibility for electrochemistryregulated recycling. In order to validate our speculation, the electrochemical property of the ITO/AuNPs/p-TC sensor was investigated firstly. It was shown that the potential difference (ΔEp) between the anodic and cathodic peaks was 76 mV (Figure 5A), meaning the presented SERS sensor possesses a well reversible voltammetric behavior. Based on this, SERS tests were performed on the sensor with varying potentials. The results showed that the normalized intensity of SERS peak at 484 cm-1 appeared and increased when changing voltage from 0.30 to 0.50 V and then faded after returning voltage to -0.30 V versus Ag/AgCl (Figure 5B). This implies that the reversible transformation between p-TC and p-TB can be accomplished on the ITO/AuNPs/p-TC sensor with application of appropriate potential. Thus, the SERS sensors after usage can be recovered through electrochemical regulation. Further systematical investigation revealed that the SERS spectra of the ITO/AuNPs/p-TC SERS sensor can achieve a graceful recovery by applying -0.3 V potential for 120 s after detecting TYR (Figure S9). Moreover, the recycling capability was tested by measuring SERS response of the sensor to TYR sample with electrochemical regulation. When the electrochemistry-treated sensor was immersed in TYR solution again, the SERS signal at 484 cm-1 reappeared sufficiently (Figure 5C), which means that the recovered sensor maintains good SERS responsiveness to TYR. From Figure 5D, it can be seen that, after repeating the “detection-recovery” procedure for ten times, the SERS sensor still have a good reproducibility. These results suggest that the developed ITO/AuNPs/p-TC sensor can be conveniently regulated with electrochemistry to achieve recycling. This is favor of facilitating the automation of SERS detection and providing a cost-effective and user-friendly approach for pointof-care (POC) diagnosis associated with TYR.

Figure 4. (A) SERS spectra of the ITO/AuNPs/p-TC SERS sensor in the presence of TYR with various concentration (a-m): 0, 0.1, 0.3, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 500 U/mL (pH: 7.4, temperature: 37 oC, time: 1 min). (B) Plot of ratiometric peak intensities of I484/I588 versus the logarithmic concentration of TYR. (C) SERS spectra of the ITO/AuNPs/p-TC SERS sensor in the absence and presence of different biologically relevant species (am): blank, TYR, glucose, GSH, NaClO, KCl, MgCl2, NaCl, NaHCO3, H2O2, trypsin, urea, and AchE. NaClO and H2O2 are 100


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μM, AchE is 1 mg/mL, trypsin is 20 mg/L, others are 500 μM. (D) SERS spectra of the ITO/AuNPs/p-TC SERS sensor in the presence of TYR and diverse relatively biological interferent (a– k): TYR + glucose, TYR + GSH, TYR + NaClO, TYR + KCl, TYR + MgCl2, TYR + NaCl, TYR + NaHCO3, TYR + H2O2, TYR + trypsin, TYR + urea, and TYR + AchE. AchE is 1mg/mL, trypsin is 20 mg/L, and others are 500 μM. TYR is 100 U/mL. (E) Ratiometric peak intensities of I484/I588 with red histogram and blue histogram obtained according to the spectra shown in (C) and (D), respectively. Error bars represent ± S.D (n = 3)

the increase of the inhibitor concentration, illustrating TYR activity has been effectively inhibited by benzoic acid. Furthermore, the inhibitory IC50 value (the inhibitor concentration required to suppress half of the TYR activity under the assayed condition 37) of benzoic acid was calculated to be approximately 1.83 mM agreeing well with the reported results 15,43. Therefore, the ITO/AuNPs/p-TC SERS sensor can also be qualified for screening possible inhibitors besides analyzing TYR activity.

Serum sample test and TYR inhibitor evaluation To assess the feasibility of the presented SERS approach, the detection of TYR in FBS sample was conducted using the developed ITO/AuNPs/p-TC sensor. Figure 6A and 6B depicts the SERS spectra of the ITO/AuNPs/p-TC sensor after incubation with the TYR-contained FBS and the comparison between the founded TYR activity calculated from the standard curves and the spike level of TYR, respectively. It can be observed that the slope value of the correction curve approximately equal to 1 with small error bar, suggesting the presented method has a good recovery percent (detailed data is shown in Table S3). Moreover, the performance of the ITO/AuNPs/p-TC SERS sensor was further evaluated through testing real serum samples provided by Shanghai Changzheng Hospital. It was shown that the TYR activities of real samples determined by the SERS sensor agreed well with those determined by the commercial ELISA Kit (Figure S10 and Table S4). This reveals that the proposed SERS sensor is competent for analyzing the TYR activity in real biological samples.

Figure 6. (A) SERS spectra of the ITO/AuNPs/p-TC SERS sensors after reaction with various activities of TYR in fetal bovine serum (a-e: 0, 3, 15, 30, and 75 U/mL of TYR activity). (B) The correlation between the founded and spiked TYR concentration. (C) SERS spectra of the ITO/AuNPs/p-TC SERS sensors after reaction with TYR (10 U/mL) incubated with different concentration of benzoic acid (a-h: 0, 0.5, 1, 2, 4, 6, 8, and 10 mM). (D) Ratiometric peak intensities of I484/I588 versus benzoic acid concentrations according to the spectra shown in (C). Error bars represent ± S.D (n = 3).

■CONCLUSION

Figure 5. (A) Cyclic voltammetry of ITO/AuNPs/p-TC SERS sensor in 0.1 M PBS (pH 7.4). (B) SERS spectra of ITO/AuNPs/pTC sensor applied different potentials (a-m): -0.3, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.4, 0.3, 0.2, 0.1, 0 and -0.3 V. (C) SERS spectra of the ITO/AuNPs/p-TC sensor with 10 times of electrochemistryregulated recovery and TYR activity detection. TYR: 100 U/mL, voltage at -0.3 V with 120 s. (D) I484/I588 versus recycling times according to the spectra shown in (C). Error bars represent ± S.D (n = 3).

It is well known that TYR inhibitors, such as benzoic acid and cinnamic acid, can reversibly bind to TYR and lower its catalytic ability 42,43, thereby having wide utilization in skin hyperpigmentation and food antibrowning treatment 15. Accordingly, benzoic acid was used as the model to estimate the TYR inhibitors screening property of this SERS strategy. As depicted in Figure 6C and 6D, the ratiometric peak intensities of I484/I588 collected from the sensor decreased with

In summary, a novel electrochemistry-regulated recyclable SERS sensor has been prepared for the assay of TYR activity. Through functionalizing AuNPs and p-TC on ITO electrode, the sensor integrates SERS activity, TYR responsiveness, and electrochemistry-regulated ability. The ITO/AuNPs/p-TC SERS sensor, which is greatly reproducible, highly stable, and rapidly responsive, possesses a wide linear detection range over three orders of magnitude with a LOD of 0.07 U/mL for TYR activity under the optimized conditions. In addition, the specific transformation from p-TC into p-TB caused by TYR combining with the fingerprinting capability of SERS allows the highly selective detection of TYR activity. Notably, the redoxreversible p-TC renders the used sensor to be recovered with electrochemistry reduction and reused in TYR activity detection. To the best of our knowledge, this is the first trial of reproducible SERS sensor regulated by electrochemistry, which could promote the automatable SERS analysis. Furthermore, the proposed SERS strategy demonstrates great potential for diseases diagnosing and inhibitor screening related to TYR activity, and it may also inspire researches of analyzing approaches involved with other oxidoreductases. Even so, there is still space to further improve the sensitivity of the sensor by fabricating it with highly plasmon-active nanostructures to better meet the detection requirement of samples with low TYR activity.



ASSOCIATED CONTENT Supporting Information The representative TEM image and UV-Vis spectra of AuNPs, synthesis details and characterization data, density functional theory calculations, and additional SERS test data of the developed sensors are provided.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡L. W. and Z. F. G. contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The authors greatly appreciate the financial support from National Natural Science Foundation of China (21575041, 21777041), Shanghai Pujiang Program (17PJD010), Shanghai Science and Technology Committee (17520750100).

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Scheme 1. Schematic exhibition of the fabrication of SERS sensors and their electrochemistry-regulated recycling for the detection of TYR activity. 177x99mm (300 x 300 DPI)


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Figure 1. (A) Real image of the ITO/AuNPs/p-TC SERS sensor. (B, C) SEM images of ITO/AuNPs/p-TC SERS sensor. (D) SERS spectra collected at 10 randomly selected locations of the ITO/AuNPs/p-TC SERS sensor (a-j). (E) Plots of the intensities of peaks at 588 cm-1 and 898 cm-1 (I588 and I898) according to SERS spectra shown in (D).



Figure 2. (A) SERS spectra of the ITO/AuNPs/p-TC SERS sensors in the (a) absence and (b) presence of TYR (100 U/mL). (B) Negative ion ToF-SIMS spectra of p-TC assembled gold film (a) before and (b) after reaction with TYR. (C) The ratio of I138.9/I141.0 according to the spectra shown in (B).


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Figure 3. (A) SERS spectra of the ITO/AuNPs/p-TC SERS sensor after reacting with 100 U/mL TYR at 25 oC under various pH (a-f): 5.7, 6.2, 6.6, 7.0, 7.4, and 8.0. (B) Ratiometric peak intensities of I484/I588 according to the spectra shown in (A). (C) SERS spectra of the ITO/AuNPs/p-TC SERS sensor after reacting with 100 U/mL TYR under pH 7.4 at various temperature (a-e): 25, 30, 37, 40, and 45 oC. (D) Ratiometric peak intensities of I484/I588 according to the spectra shown in (C). (E) SERS spectra of the ITO/AuNPs/p-TC SERS sensor after reacting with 100 U/mL TYR under pH 7.4 at 37 oC for various time (a-g): 0, 1, 3, 5, 15, 30, and 60 min. (F) Ratiometric peak intensities of I484/I588 according to the spectra shown in (E). Error bars represent ± S.D (n = 3).



Figure 4. (A) SERS spectra of the ITO/AuNPs/p-TC SERS sensor in the presence of TYR with various concentration (a-m): 0, 0.1, 0.3, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 500 U/mL (pH: 7.4, temperature: 37 oC, time: 1 min). (B) Plot of ratiometric peak intensities of I484/I588 versus the logarithmic concentration of TYR. (C) SERS spectra of the ITO/AuNPs/p-TC SERS sensor in the absence and presence of different biologically relevant species (a-m): blank, TYR, glucose, GSH, NaClO, KCl, MgCl2, NaCl, NaHCO3, H2O2, trypsin, urea, and AchE. NaClO and H2O2 are 100 μM, AchE is 1 mg/mL, trypsin is 20 mg/L, others are 500 μM. (D) SERS spectra of the ITO/AuNPs/p-TC SERS sensor in the presence of TYR and diverse relatively biological interferent (a–k): TYR + glucose, TYR + GSH, TYR + NaClO, TYR + KCl, TYR + MgCl2, TYR + NaCl, TYR + Na-HCO3, TYR + H2O2, TYR + trypsin, TYR + urea, and TYR + AchE. AchE is 1mg/mL, trypsin is 20 mg/L, and others are 500 μM. TYR is 100 U/mL. (E) Ratiometric peak intensities of I484/I588 with red histogram and blue histogram obtained according to the spectra shown in (C) and (D), respectively. Error bars represent ± S.D (n = 3)


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Figure 5. (A) Cyclic voltammetry of ITO/AuNPs/p-TC SERS sensor in 0.1 M PBS (pH 7.4). (B) SERS spectra of ITO/AuNPs/p-TC sensor applied different potentials (a-m): -0.3, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.4, 0.3, 0.2, 0.1, 0 and -0.3 V. (C) SERS spectra of the ITO/AuNPs/p-TC sensor with 10 times of electrochemistryregulated recovery and TYR activity detection. TYR: 100 U/mL, voltage at -0.3 V with 120 s. (D) I484/I588 versus recycling times according to the spectra shown in (C). Error bars represent ± S.D (n = 3).



Figure 6. (A) SERS spectra of the ITO/AuNPs/p-TC SERS sensors after reaction with various activities of TYR in fetal bovine serum (a-e: 0, 3, 15, 30, and 75 U/mL of TYR activity). (B) The correlation between the founded and spiked TYR concentration. (C) SERS spectra of the ITO/AuNPs/p-TC SERS sensors after reaction with TYR (10 U/mL) incubated with different concentration of benzoic acid (a-h: 0, 0.5, 1, 2, 4, 6, 8, and 10 mM). (D) Ratiometric peak intensities of I484/I588 versus benzoic acid concentrations according to the spectra shown in (C). Error bars represent ± S.D (n = 3). 176x139mm (300 x 300 DPI)


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TOC graphic 64x43mm (300 x 300 DPI)


Electrochemistry-Regulated Recyclable SERS Sensor for Sensitive

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