An Electrochemical Sensor Based on Chalcogenide Molybdenum Disulfide-Gold-Silver Nanocomposite for Detection of Hydrogen Peroxid

sensors

Letter An Electrochemical Sensor Based on Chalcogenide Molybdenum Disulﬁde-Gold-Silver Nanocomposite for Detection of Hydrogen Peroxide Released by Cancer Cells

Jinchun Hu †, Congcong Zhang †, Xue Li and Xin Du * Shandong Provincial Key Laboratory of Animal Resistance Biology, Key Laboratory of Food Nutrition and Safety, College of Life Sciences, Shandong Normal University, Jinan 250014, China; [email protected] (J.H.); [email protected] (C.Z.); [email protected] (X.L.) * Correspondence: [email protected]; Tel.: +86-136-5640-1019 These authors contributed equally to this work. † Received: 23 September 2020; Accepted: 23 November 2020; Published: 28 November 2020

Abstract: Hydrogen peroxide (H2O2) as a crucial signal molecule plays a vital part in the growth and development of various cells under normal physiological conditions. The development of H2O2 sensors has received great research interest because of the importance of H2O2 in biological systems and its practical applications in other fields. In this study, a H2O2 electrochemical sensor was constructed based on chalcogenide molybdenum disulfide–gold–silver nanocomposite (MoS2-Au-Ag). Transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and energy dispersive spectroscopy (EDS) were utilized to characterize the nanocomposites, and the electrochemical performances of the obtained sensor were assessed by two electrochemical detection methods: cyclic voltammetry and chronoamperometry. The results showed that the 1 2 MoS2-Au-Ag-modified glassy carbon electrode (GCE) has higher sensitivity (405.24 µA mM− cm− ), wider linear detection range (0.05–20 mM) and satisfactory repeatability and stability. Moreover, the prepared sensor was able to detect the H2O2 discharge from living tumor cells. Therefore, this study offers a platform for the early diagnosis of cancer and other applications in the fields of biology and biomedicine.

Keywords: hydrogen peroxide; tumor cells; molybdenum disulﬁde–gold–silver; electrochemical sensor; detection

1. Introduction

Cells can produce all kinds of reactive oxygen species in metabolic process, and H2O2 is one of the most momentous representatives. H2O2 plays a vital role in numerous processes such as cellular signal transduction, apoptosis and many other physiological processes [1]; however, excess H2O2 is toxic to humans, which leads to various diseases, such as cancer, cardiovascular disease and neurodegenerative diseases [2]. Research has proved that cancer cells will release excessive H2O2 to achieve the abnormal growth of tumors. Therefore, the accurate and fast determination of H2O2 is signiﬁcant in the cellular environment and diagnosing cancer in its early stages. Additionally, H2O2 is widely used as an oxidizer, disinfectant or bleach in the food, environmental analysis, chemical and pharmaceutical industries [3,4]. The traditional methods for the detection of H2O2 include spectrophotometry [5], chemiluminescence [6,7], ﬂuorescence [8,9] and high performance liquid chromatography [10]. However, these methods also have some limitations, such as expensive equipment and cumbersome

Sensors 2020, 20, 6817; doi:10.3390/s20236817 www.mdpi.com/journal/sensors Sensors 2020, 20, 6817 2 of 12 procedures. Electrochemical detection is a detective method which has the advantages of high sensitivity, low cost, high efficiency and good portability [11]. In recent years, the development of nanomaterials has provided many new opportunities for the construction of hydrogen peroxide sensors. Nanomaterials have the unique properties of a strong adsorption capacity, large specific surface area and high catalytic efficiency [12,13]. In the field of electrochemical sensors, the application of nanomaterials not only improves the stability and sensitivity of electrodes, but also improves the detection performance of sensors [13]. MoS2 has attracted extensive attention in the studies of lithium batteries, catalysts and cancer treatment owing to its advantages of good stability, high catalytic efficiency and easy functionalization [14–16]. The structure of single-layer MoS2 is composed of two layers of sulfur sandwiched by a layer of molybdenum. The atoms are covalently bonded to each other, which shows as a sandwich structure [17]. At present, the sensors using molybdenum disulfide as the experimental material are involved in the detection of glucose, hydrogen peroxide, nucleic acid, protein and other substances which showed a good sensitivity and detection performance. Au, Ag and other precious metal nanomaterials not only have the characteristics of general nanometer materials, but also have excellent electrical conductivity, a unique catalytic activity and are non-toxic, which makes them have unique advantages and extensive applications in the field of sensors [18,19]. Herein, we developed a sensitive sensor for the detection of H2O2 discharge from tumor cells by using a MoS2-Au-Ag nanocomposite to accomplish high conductivity and increase the surface area of the electrode, due to the fact that H2O2 is a typical tumor biomarker and the developed MoS2-Au-Ag electrode exhibits an excellent enzyme-mimic electrocatalytic activity in the oxidation of H2O2. We chose cyclic voltammetry (CV) to evaluate the properties of our fabricated sensor for the detection of H2O2, which is a very sensitive electrochemical detection tool used to evaluate the performance of an electrochemical sensor [19]. Our results indicated that this sensor significantly improved the detection signal and consequently enhanced the low detection limit with a wide detection range, and high selectivity and sensitivity. This study demonstrates a functional, non-enzymatic electrochemical sensor based on MoS2-Au-Ag nanocomposite for the detection of H2O2 released by normal and tumor cells.

2. Experimental

2.1. Chemicals and Reagents

MoS2, phthalic diglycol diacrylate (PDDA), K3[Fe(CN)6], phorbol 12-myristate 13-acetate (PMA), chloroauric acid (H[AuCl4]) and AgNO3 were obtained from Sigma (USA). H2O2 was purchased from Shanghai Wokai Biotechnology Co., Ltd. (China). C2H5OH was obtained from Tianjin Chemical Reagent Sixth Factory (China). KCl was purchased from Shanghai Hutan Laboratory Equipment Co., Ltd. (China). Phosphate buﬀer solution (PBS) was obtained from Biological Industries. 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Beijing Solarbio Science & Technology Co., Ltd. (China). The RPE-1 human retinal pigment epithelial cell line and the MCF-7 human breast cancer cell line were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). For the conﬁguration of all aqueous solutions and to clean all materials we used twofold distilled water.

2.2. Electrochemical Measurements The electrochemical detection used a standard three-electrode electrochemical testing device with a glassy carbon electrode modiﬁed by nanomaterials as the working electrode, and a silver/silver chloride (Ag/AgCl) and platinum wire were used as the reference electrode and counter electrode, respectively. The electrochemical test was conducted in PBS or cell samples, which were deoxygenated with N2 and stirred with a magnetic agitator at a speed of 300 rpm to maintain a stable and uniform H2O2 concentration in the solution. Sensors 2020, 20, x FOR PEER REVIEW 3 of 13 Sensors 2020, 20, 6817 3 of 12

2.3. Dispersion of MoS2

2.3. DispersionThe dispersing of MoS 2agent PDDA was used to disperse the MoS2 into a uniform and stable dispersion by taking 3 mg and 7.5 mg MoS2 into a 1.5 mL centrifuge tube and then adding 1% or The dispersing agent PDDA was used to disperse the MoS2 into a uniform and stable dispersion 0.5% PDDA at a constant volume to 1.5 mL. Next, we got a MoS2 solid precipitate via sonication for by taking 3 mg and 7.5 mg MoS2 into a 1.5 mL centrifuge tube and then adding 1% or 0.5% PDDA at a 2 h and centrifuged it at 15,000 rpm for 10 min. Subsequently, four different concentrations of constant volume to 1.5 mL. Next, we got a MoS2 solid precipitate via sonication for 2 h and centrifuged itdispersion at 15,000 rpmwere for obtained 10 min. b Subsequently,y discarding the four supernatant different concentrations and adding ofwater dispersion at a constant were obtained volume byto 1.5 mL, including 2 mg mL–1 and 5 mg mL–1 MoS2 dispersions with 0.5% and 1% PDDA. 1 discarding the supernatant and adding water at a constant volume to 1.5 mL, including 2 mg mL− 1 and 5 mg mL− MoS2 dispersions with 0.5% and 1% PDDA. 2.4. Preparation of MoS2-Au-Ag/GCE 2.4. PreparationThe bare GCE of MoS wa2-Au-Ags pre-polished/GCE with 0.3 µm and 0.05 µm alumina powders to get a smooth mirrorThe surface bare GCE before was fabricating pre-polished with with a 0.3modifiedµm and electrode 0.05 µm alumina[20]. For powders the purpose to get of a w smoothiping off mirror the surfacephysically before adsorbed fabricating substance, with a modified GCE was electrode then sonicated [20]. For with the purposethe mixture of wiping solution off the of physicallyultra-pure adsorbedwater and substance, ethanol for GCE 10 wasmin. then After sonicated that, the with cleaned the mixtureelectrode solution was dried of ultra-pure under N2 water. Briefly, and 10 ethanol µL of –1 forMoS 102 min.suspension After that, (2 mg the mL cleaned) was electrode cautiously was dro driedpped under onto N the2. Briefly, GCE surface, 10 µL of and MoS the2 suspension modified 1 (2electrode mg mL −was) was dried cautiously under air. dropped Then, MoS onto2/GCE the GCE was surface, electrodeposited and the modified in 2.5 mM electrode H[AuCl was4] and dried 2.5 undermM AgNOair. Then,3 solution MoS2/GCEs, respectively was electrodeposited, under in the 2.5 mM condition H[AuCl of4] and−0.4 2.5 V.mM Accordingly, AgNO3 solutions, the respectively,MoS2-Au-Ag/GCE under was the successfully condition of prepared.0.4 V. Accordingly, The whole process the MoS of -Au-Agthe fabrication/GCE was is illustrated successfully in − 2 prepared.Scheme 1. The whole process of the fabrication is illustrated in Scheme1.

Scheme 1. Fabrication and detection of the H O electrochemical sensor. Scheme 1. Fabrication and detection of the H22O2 electrochemical sensor.

2.5. Detection of H2O2 Solutions with Different Concentrations and Released by Cells 2.5. Detection of H2O2 Solutions with Different Concentrations and Released by Cells The 30% pure H2O2 solution was diluted into 100 mM, 400 mM, 2000 mM and 4000 mM H2O2 The 30% pure H2O2 solution was diluted into 100 mM, 400 mM, 2000 mM and 4000 mM H2O2 with PBS, and the corresponding concentration of H2O2 was added to the 20 mL PBS buffer liquid with PBS, and the corresponding concentration of H2O2 was added to the 20 mL PBS buffer liquid system to conduct electrochemical detection of the modified electrodes. RPE-1 cells and MCF-7 cells system to conduct electrochemical detection of the modified electrodes. RPE-1 cells and MCF-7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and were grown in a cell were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and were grown in a culture incubator with 5% CO2 and 37 ◦C. After they were grown to 80–90% confluence, the cells were cell culture incubator with 5% CO2 and 37 °C. After they were grown to 80–90% confluence, the cells washed 2–3 times with PBS, and resuspended into 2 mL oxygen-removing PBS for electrochemical were washed 2–3 times with PBS, and resuspended into 2 mL oxygen-removing PBS for detection of H2O2 generated by the MCF-7 cells. With the cell-free group as the control group and electrochemical detection of H2O2 generated by the MCF-7 cells. With the cell-free group as the three groups of cells with different concentrations as the experimental group, the flux of H O caused control group and three groups of cells with different concentrations as the experimental2 group,2 the by normal cells and living cancer cells upon the addition of PMA at different concentrations and the flux of H2O2 caused by normal cells and living cancer cells upon the addition of PMA at different correlated electrochemical response signals were recorded by amperometric i-t curves. The effect of the concentrations and the correlated electrochemical response signals were recorded by amperometric nanomaterials in the cell culture was assessed via an MTT assay and the detailed experimental steps i-t curves. The effect of the nanomaterials in the cell culture was assessed via an MTT assay and the can be found in Supporting Information. detailed experimental steps can be found in Supporting Information.

Sensors 2020, 20, 6817 4 of 12

3. Results and Discussion Sensors 2020, 20, x FOR PEER REVIEW 4 of 13 3.1. Characterization of MoS2-Au-Ag/GCE Nanocomposite 3. Results and Discussion The morphology and structure of the obtained nanomaterials were investigated by TEM. Figure1A shows3.1. that Characterization the MoS2 ofhas MoS a2- uniformAu-Ag/GCE lamellar Nanocomposite structure. As illustrated in Figure1B,C, the Au and Ag nanoparticles showed clear dendritic and granular morphologies, respectively, and were The morphology and structure of the obtained nanomaterials were investigated by TEM. evenly dispersed over the MoS . As shown in Figure1D, the MoS -Au-Ag mimics uniformly Figure 1A shows that the MoS2 has a uniform lamellar structure. As illustrated2 in Figure 1B,C, the compactAu branchesand Ag nanoparticles in the PDDA showed solution. clear In dendritic summary, and we granular successfully morphologies, synthesized respectively the MoS, 2and-Au-Ag nanocomposite,were evenly dispersed which has over potential the MoS2 application. As shown in value Figure for 1D, improving the MoS2-Au electrochemical-Ag mimics uniformly detection performance.compact branches The chemical in the compositionPDDA solution of. In MoS summary,2-Au-Ag we was successfully verified synthesized by XPS. As the shown MoS2 in-Au Figure-Ag S1, the curvenanocomposite, presented O which 1s peaks, has potential C 1s peaks, application Ag 3d peaks, value Mo for 3d improving peaks, Au electrochemical 4f peaks and detectionS 2p peaks in one spectrum.performance. For The the chemical Mo 3d region, composition the two of characteristicMoS2-Au-Ag was peaks verified at 229.8 by XPS. eV and As shown 233.5 eVin Figure corresponded S1, the curve presented O 1s peaks, C 1s peaks, Ag 3d peaks, Mo 3d peaks, Au 4f peaks and S 2p peaks to Mo 3d5/2 and Mo 3d3/2 of MoS2, respectively, and the single S 2s peak is located at about 227 eV. in one spectrum. For the Mo 3d region, the two characteristic peaks at 229.8 eV and 233.5 eV Meanwhile, the binding energies at 162.1 eV and 163.2 eV can be attributed to the S 2p3/2 and S 2p1/2 corresponded to Mo 3d5/2 and Mo 3d3/2 of MoS2, respectively, and the single S 2s peak is located at species on the edges or surface of the MoS nanosheet, respectively. In addition, the strong peaks about 227 eV. Meanwhile, the binding energies2 at 162.1 eV and 163.2 eV can be attributed to the S of the elements Au and Ag confirmed the successful electrodeposition of two metals on the glassy 2p3/2 and S 2p1/2 species on the edges or surface of the MoS2 nanosheet, respectively. In addition, the carbonstrong electrode peaks modifiedof the elements with Au MoS and2. In Ag addition, confirmed energy the successful dispersive electrodeposition spectroscopy of (EDS) two metals was used to analyzeon the and glassy study carbon the electrode components modified of the with MoS MoS2-Au-Ag.2. In addition The element, energy ofdispersive Mo, S, Au spectroscopy and Ag were clearly(EDS) observed, was used which to analyze originated and study from the the component MoS2, Aus andof the Ag MoS nanomaterials,2-Au-Ag. The element respectively of Mo (Figure, S, Au 1E). The resultsand Ag further were clearlymanifested observed that, MoSwhich2-Au-Ag originated was from constructed the MoS successfully.2, Au and Ag We nanomaterials also studied, the effectsrespectively of different ( materialsFigure 1E on). sensor The results performance further (Figuremanifest2).ed The that resulting MoS2-Au spectra-Ag was manifested constructed that the successfully. We also studied the effects of different materials on sensor performance (Figure 2). oxidation and reduction peaks associated with MoS2-Au/GCE (b) and MoS2-Au-Ag/GCE (c) increased The resulting spectra manifested that the oxidation and reduction peaks associated with substantially compared with MoS2/GCE (a). Moreover, the peak value of redox was the largest for MoS2-Au/GCE (b) and MoS2-Au-Ag/GCE (c) increased substantially compared with MoS2/GCE (a). MoS2-Au-Ag/GCE, demonstrating that the Au-Ag nanocomposite particles have a good effect on Moreover, the peak value of redox was the largest for MoS2-Au-Ag/GCE, demonstrating that the enhancingAu-Ag electrode nanocomposite conductivity. particles The have MoS a2 -Au-Ag good effect composite on enhancing was used electrode to modify conductivity the electrode. The in the subsequentMoS2-Au experiments.-Ag composite was used to modify the electrode in the subsequent experiments.

Figure 1. Characterization of the morphology, structure, and electrochemical performance of the Figure 1. Characterization of the morphology, structure, and electrochemical performance of the nanocomposites. (A–D) Transmission electron microscopy images of MoS (A), MoS -Au (B), MoS -Ag nanocomposites. (A–D) Transmission electron microscopy images of 2 MoS2 (A), 2 MoS2-Au (B), 2 (C) and MoS2-Au-Ag (D). (E) The X-ray energy dispersive spectrometric analysis of MoS2-Au-Ag.

Sensors 2020, 20, x FOR PEER REVIEW 5 of 13

MoS2-Ag (C) and MoS2-Au-Ag (D). (E) The X-ray energy dispersive spectrometric analysis of

SensorsMoS20202-Au, 20-,Ag. 6817 5 of 12

FigureFigure 2. 2. CyclicCyclic voltammetry voltammetry of of nanomaterials. nanomaterials. CV CV profiles proﬁles of of MoS MoS2/GCE2/GCE (a), (a), MoS MoS2-2Au/GCE-Au/GCE (b), (b),

MoSMoS2-2Au-Au-Ag-Ag/GCE/GCE (c) (c) recorded recorded in in K K3[3Fe(CN)[Fe(CN)6]6. ].

3.2. Optimization of Experimental Parameters for the Fabrication of MoS2-Au-Ag/GCE 3.2. Optimization of Experimental Parameters for the Fabrication of MoS2-Au-Ag/GCE Changes in the experimental conditions of the modified electrode will directly affect the Changes in the experimental conditions of the modified electrode will directly affect the electrochemical properties of the electrode, which will lead to alterations of the catalytic properties and electrochemical properties of the electrode, which will lead to alterations of the catalytic properties sensor functions [21]. For the sake of enhancing the sensing performance of the MoS -Au-Ag/GCE, and sensor functions [21]. For the sake of enhancing the sensing performance2 of the the effects of the PDDA concentration of dispersed MoS2, the electrodeposition time, and the modified MoS2-Au-Ag/GCE, the effects of the PDDA concentration of dispersed MoS2, the electrodeposition electrode material were investigated. time, and the modified electrode material were investigated. MoS2 was dispersed with water, PBS buffer and PDDA of different concentrations, and then MoS2 was dispersed with water, PBS buffer and PDDA of different concentrations, and then placed in a 4 C refrigerator for 4 h after ultrasound to observe the dispersion (Figure3A). The results placed in a 4 ◦°C refrigerator for 4 h after ultrasound to observe the dispersion (Figure 3A). The showed that the MoS2 dispersed with water and PBS buffer had obvious precipitation and was not results showed that the MoS2 dispersed with water and PBS buffer had obvious precipitation and dissolved at all. The dispersion of MoS2 with PDDA was better, and a uniform MoS2 dispersion was was not dissolved at all. The dispersion of MoS2 with PDDA was better, and a uniform MoS2 obtained. The effect of PDDA concentration on the electrochemical property of MoS2-Au-Ag/GCE dispersion was obtained. The effect of PDDA concentration on the electrochemical property of 1 was analyzed by CV in K3[Fe(CN)6]. As shown in Figure3B, the peak current values of 2 mg mL − 2 3[ 6] MoS -Au-Ag/GCE was analyzed by CV1 in K Fe(CN) . As shown in Figure 3B, the peak current MoS2 with 1% PDDA–1 (b) and 5 mg mL− MoS2 with 1% PDDA–1 (c) are similar. Based on the principle values of 2 mg mL MoS1 2 with 1% PDDA (b) and 5 mg mL MoS2 with 1% PDDA (c) are similar. of economy, 2 mg mL− MoS2 with 1% PDDA was selected for subsequent experiments. The Au-Ag Based on the principle of economy, 2 mg mL–1 MoS2 with 1% PDDA was selected for subsequent electrodeposited on the surface of MoS2/GCE can provide a large effective surface area and enhance experiments. The Au-Ag electrodeposited on the surface of MoS2/GCE can provide a large effective the electrocatalytic performance of the electrode, which is of great significance for the determination of surface area and enhance the electrocatalytic performance of the electrode, which is of great H2O2 [22]. Thus, the amount of Au-Ag electrodeposited on the surface of MoS2/GCE immediately significance for the determination of H2O2 [22]. Thus, the amount of Au-Ag electrodeposited on the affects the performance of the H2O2 sensor. To enhance the function of Au-Ag on the MoS2/GCE, surface of MoS2/GCE immediately affects the performance of the H2O2 sensor. To enhance the the effect of electrodeposition time from 30 s to 150 s was analyzed by CV in 10 mM H2O2, as shown in function of Au-Ag on the MoS2/GCE, the effect of electrodeposition time from 30 s to 150 s was Figure3D. The analysis results revealed that the potential and the current value of redox peak were analyzed by CV in 10 mM H2O2, as shown in Figure 3D. The analysis results revealed that the different. As we can see from Figure3C, the b and d peak current values are bigger, but the peak potential and the current value of redox peak were different. As we can see from Figure 3C, the b potential value of b is larger. As such, we chose the optimized electrodeposition time in the fabrication and d peak current values are bigger, but the peak potential value of b is larger. As such, we chose of modified electrode to reduce the interference of high voltage. the optimized electrodeposition time in the fabrication of modified electrode to reduce the interference3.3. Electrocatalytic of high Activitiesvoltage. of the Modified Electrodes

We employed CV to research the electrocatalytic activities of MoS2/GCE, MoS2-Au/GCE and MoS2-Au-Ag/GCE in 5 mM H2O2 (Figure4A). The CV experiments revealed that no redox peaks were measured relating to MoS /GCE (a) in the range of 0.80 V–0.00 V, while MoS -Au/GCE (c) 2 − 2 and MoS -Au-Ag/GCE (d) had the presence of a reduction peak of H O at 0.50 V. Therefore, 2 2 2 − MoS2-Au-Ag/GCE had a better reaction performance to H2O2.

Sensors 2020, 20, 6817 6 of 12 Sensors 2020, 20, x FOR PEER REVIEW 6 of 13

Figure 3. Optimization of experimental parameters for the fabrication of MoS2-Au-Ag/GCE. (A) MoS2 Figure 3. Optimization of experimental1 parameters for1 the fabrication of MoS2-1Au-Ag/GCE. (A) dispersion effects of 2 mg mL− water (a), 2 mg mL− PBS buffer (b), 5 mg mL− 0.5% PDDA (c), −1 −1 −1 MoS2 dispersion1 effects of 2 mg mL 1 water (a), 2 mg mL PBS buffer1 (b), 5 mg mL 0.5% PDDA (c), 5 mg mL− 1% PDDA (d), 2 mg mL− 0.5% PDDA (e) and 2 mg mL− 1% PDDA (f). (B) CV curves of 5 mg mL−1 1% PDDA (d), 2 mg mL−1 0.5% PDDA (e) and 2 mg mL−11 1% PDDA (f). (B) CV curves1 of dispersed MoS2 modified electrodes in K3[Fe(CN)6] under 2 mg mL− 0.5% PDDA (a), 2 mg mL− 1% dispersed MoS2 modified1 electrodes in K3[Fe(CN)1 6] under 2 mg mL−1 0.5% PDDA (a), 2 mg mL−1 1% PDDA (b), 5 mg mL− 1% PDDA (c), 5 mg mL− 0.5% PDDA (d). (C) The current line diagram of the PDDA (b), 5 mg mL−1 1% PDDA (c), 5 mg mL−1 0.5% PDDA (d). (C) The current line diagram of the electrode at 10 mM H2O2 with different deposition times. (D) CV curves of electrode modified with electrode at 10 mM H2O2 with different deposition times. (D) CV curves of electrode modified with different depositions time in 10 mM H2O2. Deposition time: (a) Au 30 s Ag 30 s, (b) Au 30 s Ag 90 s, (c)different Au 30 deposition s Ag 150 s,s (d) time Au in 90 10 s AgmM 30 H s,2O (e)2. Deposition Au 90 s Ag time 90 s,: (f)(a) AuAu 9030s sAg150 Ag 30 s,s, (g)(b) AuAu 15030 s s Ag Ag 90 30 s, (h)(c) Au 30 150 s sAg Ag 150 90 s,s, (i)(d) Au Au 150 90 s Ag 15030 s, s. (e) Au 90 s Ag 90 s, (f) Au 90 s Ag150 s, (g) Au 150 s Ag 30 s, (h) Au 150 s Ag 90 s, (i) Au 150 s Ag 150 s.

We then examined the CV results of MoS2-Au-Ag/GCE in PBS and in different concentrations of H3.3.2O Electrocatalytic2 (Figure4B). TheActivities redox of peak the Modified of modified Electrodes electrodes was not measured in PBS (a) while it was shown in 5 mM (b) and 10 mM H O (c). With the increase in concentration of H O , the reductive We employed CV to research2 the2 electrocatalytic activities of MoS2/GCE, MoS2 22-Au/GCE and peak position shifting to the left may be due to the delay of electron transfer caused by the increase MoS2-Au-Ag/GCE in 5 mM H2O2 (Figure 4A). The CV experiments revealed that no redox peaks in H O concentration. In order to prevent interference from other substances, 0.45 V was selected were2 measured2 relating to MoS2/GCE (a) in the range of −0.80 V–0.00 V, while− MoS2-Au/GCE (c) as the catalytical voltage for subsequent current response experiments. Compared with the 5 mM and MoS2-Au-Ag/GCE (d) had the presence of a reduction peak of H2O2 at −0.50 V. Therefore, H O solution, the reduction peak current value measured by the modified electrode in 10 mM H O MoS2 2-Au-Ag/GCE had a better reaction performance to H2O2. 2 2 increased significantly, indicating that MoS2-Au-Ag/GCE had a higher sensitivity to H2O2. The kinetics of the MoS2-Au-Ag/GCE were investigated by analyzing the impacts of the scan rate on the redox current. The electrochemical performance of MoS2-Au-Ag/GCE was examined in 1 10 mM K3[Fe(CN)6] with the increase of scan rate from 10 to 100 mV s− . As displayed in Figure4C, a great linear relationship was shown between the square root of scan rate and the peak current density. The maximum current of the redox peak was increased linearly and the distance of the peak was expanded gradually. According to these results, we made a linear fit of the oxidation peak (Ipa) and 1/2 reduction (Ipc) peak currents with the square root of the scan rate (v ). The relevant linear regression 1/2 1/2 equations were Ipa = 14.89 v 13.74 and Ipc = 15.37 v 15.26 with correlation coefficients of 0.9916 − − −

Sensors 2020, 20, 6817 7 of 12 and 0.9976, respectively (Figure4D). The electrochemical signal of the modiﬁed electrode suggests that theSensors catalysis 2020, 20 process, x FOR PEER is di REVIEWﬀusion controlled. 7 of 13

Figure 4. Electrocatalytic activities of the modified electrodes. (A) CV profiles of electrode modified Figure 4. Electrocatalytic activities of the modified electrodes. (A) CV profiles of electrode modified by MoS2/GCE (a), MoS2-Au/GCE (b) and MoS2-Au-Ag/GCE (c) in 5 mM H2O2.(B) CV curves by MoS2/GCE (a), MoS2-Au/GCE (b) and MoS2-Au-Ag/GCE (c) in 5 mM H2O2. (B) CV curves of of MoS2-Au-Ag/GCE in PBS (a), 5 mM H2O2 (b) and 10 mM H2O2 (c). (C) Kinetic analysis of MoS2-Au-Ag/GCE in PBS (a), 5 mM H2O2 (b) and 10 mM H2O2 (c). (C) Kinetic analysis of MoS -Au-Ag/GCE at scan rates ranging from 10 to 100 mV s 1.(D) Linear fits of the oxidized peak 2 −−1 MoS2-Au-Ag/GCE at scan rates ranging from 10 to 100 mV s . (D) Linear fits of the1/2 oxidized peak current (Ipa) and reduced peak current (Ipc) versus the square root of the scan rate (v ). current (Ipa) and reduced peak current (Ipc) versus the square root of the scan rate (v1/2). 3.4. Amperometric Responses to H2O2 We then examined the CV results of MoS2-Au-Ag/GCE in PBS and in different concentrations To further detect the amperometric current responses of MoS2-Au-Ag/GCE, we added H2O2 at of H2O2 (Figure 4B). The redox peak of modified electrodes was not measured in PBS (a) while it different concentrations continuously at a potential of 0.45 V. To prevent interference, H2O2 was was shown in 5 mM (b) and 10 mM H2O2 (c). With − the increase in concentration of H2O2, the added for the first time after the current was kept at a stable value, and then added every 50 s. reductive peak position shifting to the left may be due to the delay of electron transfer caused by Figure5A shows the amperometric response of MoS 2-Au-Ag/GCE with various concentrations of the increase in H2O2 concentration. In order to prevent interference from other substances, −0.45 V H O . After injecting a certain amount of H O , MoS -Au-Ag/GCE exhibited a fast current response was2 2 selected as the catalytical voltage for2 subsequent2 2 current response experiments. Compared because of the excellent electrocatalytic performance of MoS2-Au-Ag. It can be clearly seen that each with the 5 mM H2O2 solution, the reduction peak current value measured by the modified electrode reduction current response demonstrated an explicit increase as the concentration of H2O2 increased in 10 mM H2O2 increased significantly, indicating that MoS2-Au-Ag/GCE had a higher sensitivity to and the response current attained a steady state within 4 s. The illustration manifests the amperometric H2O2. current response at lower concentrations of H2O2 (0.05 mM–0.75 mM). The kinetics of the MoS2-Au-Ag/GCE were investigated by analyzing the impacts of the scan rate on the redox current. The electrochemical performance of MoS2-Au-Ag/GCE was examined in 10 mM K3[Fe(CN)6] with the increase of scan rate from 10 to 100 mV s−1. As displayed in Figure 4C, a great linear relationship was shown between the square root of scan rate and the peak current density. The maximum current of the redox peak was increased linearly and the distance of the peak was expanded gradually. According to these results, we made a linear fit of the oxidation peak (Ipa) and reduction (Ipc) peak currents with the square root of the scan rate (v 1/2). The relevant linear regression equations were Ipa = 14.89 v1/2 − 13.74 and Ipc = −15.37 v1/2 − 15.26 with correlation coefficients of 0.9916 and 0.9976, respectively (Figure 4D). The electrochemical signal of the modified electrode suggests that the catalysis process is diffusion controlled.

Sensors 2020, 20, x FOR PEER REVIEW 8 of 13

3.4. Amperometric Responses to H2O2

To further detect the amperometric current responses of MoS2-Au-Ag/GCE, we added H2O2 at different concentrations continuously at a potential of −0.45 V. To prevent interference, H2O2 was added for the first time after the current was kept at a stable value, and then added every 50 s. Figure 5A shows the amperometric response of MoS2-Au-Ag/GCE with various concentrations of H2O2. After injecting a certain amount of H2O2, MoS2-Au-Ag/GCE exhibited a fast current response because of the excellent electrocatalytic performance of MoS2-Au-Ag. It can be clearly seen that each reduction current response demonstrated an explicit increase as the concentration of H2O2 increased and the response current attained a steady state within 4 s. The illustration manifests the Sensors 2020, 20, 6817 8 of 12 amperometric current response at lower concentrations of H2O2 (0.05 mM–0.75 mM).

Figure 5. Kinetics of MoS2-Au-Ag/GCE. (A) The i-t curve of the MoS2-Au-Ag/GCE when different Figure 5. Kinetics of MoS2-Au-Ag/GCE. (A) The i-t curve of the MoS2-Au-Ag/GCE when different concentrations of H2O2 were added to the PBS buffer successively. (B) Linear fitting diagram. concentrations of H2O2 were added to the PBS buffer successively. (B) Linear fitting diagram. As shown in Figure5B, the calibration curve was fitted by using multiple sets of repeated experimentalAs shown data. in F Accordingigure 5B, the to the calibration curve fitting, curve we was calculated fitted by the using linear multiple range of sets the of prepared repeated sensorexperimental to be from data. 0.05 According mM to 20 mM,to the with curve a linear fitting, correlation we calculated coefficient the linear of 0.9933, range and of the the sensitivity prepared sensor to be from 10.05 mM2 to 20 mM, 1with a linear correlation coefficient of 0.9933, and the was 405.24 µA mM− cm− (28.63 µA mM− ). It is worth noting that the sensitivity is much higher sensitivity was 405.24 µA mM–1 cm–2 (28.63 µA mM–1). It is worth noting that the sensitivity is much than many reported H2O2 sensors, as shown in Table1. The detection equation of the sensor washigherY = than28.63 many X reported14.88 and H the2O2 minimumsensors, as detectionshown in limitTable was 1. The 7.19 detectionµM. This equation experiment of the further sensor was Y =− −28.63 X− − 14.88 and the minimum detection limit was 7.19 µM. This experiment further demonstrated that the MoS2-Au-Ag composite is an excellent material for H2O2 detection with gooddemonstrated sensitivity. that the MoS2-Au-Ag composite is an excellent material for H2O2 detection with good sensitivity.

Table 1. Comparison of the performances of various H2O2 sensors.

Sensitivity Detection Limit Linear Range Material 1 2 Reference (µA mM− cm− ) (µM) (mM) Pt/OMCs 184.6 1.2 0.5–4.5 [23] Ag-TiO2 65.23 0.83 0.0083–0.0433 [24] ZIF-67/CNFs 323 0.62 0.0025–0.19 [25]

Pt-SnO2@C 241.1 0.1 0.001–0.17 [26] PANI/HRP/SWCNTs 200 900 2.5–50 [27] MoS2-Au-Ag 405.24 7.19 0.05–20 this work

3.5. Repeatability and Stability of MoS2-Au-Ag/GCE The repeatability and stability of the electrochemical sensor are the main parameters affecting the applicability of the H2O2 sensor. The current response values of five modified electrodes when 1 mM H2O2 was successively injected under the same conditions are recorded in Figure6A. The RSD of the relative standard deviation of the five electrodes was calculated to be 3.79%. The stability was evaluated Sensors 2020, 20, x FOR PEER REVIEW 9 of 13

Table 1. Comparison of the performances of various H2O2 sensors.

Sensitivity Detection Limit Linear Range Material Reference (µA mM−1 cm−2) (µM) (mM) Pt/OMCs 184.6 1.2 0.5–4.5 [23] Ag-TiO2 65.23 0.83 0.0083–0.0433 [24] ZIF-67/CNFs 323 0.62 0.0025–0.19 [25] Pt-SnO2@C 241.1 0.1 0.001–0.17 [26] PANI/HRP/SWCNTs 200 900 2.5–50 [27] MoS2-Au-Ag 405.24 7.19 0.05–20 this work

3.5. Repeatability and Stability of MoS2-Au-Ag/GCE The repeatability and stability of the electrochemical sensor are the main parameters affecting the applicability of the H2O2 sensor. The current response values of five modified electrodes when 1 Sensors 2020, 20, 6817 9 of 12 mM H2O2 was successively injected under the same conditions are recorded in Figure 6A. The RSD of the relative standard deviation of the five electrodes was calculated to be 3.79%. The stability was evaluatedby placing by the placing MoS2-Au-Ag the MoS/GCE2-Au in-Ag/GCE PBS bu ffiner PBS and bufferadding and 1 mM adding H2O 2 1 continuously mM H2O2 continuously (Figure6B). (TheFigure modified 6B). The electrode modified was tested electrode every was 4 h tested and then every placed 4 h overnight and then in placed a 4 ◦C refrigerator overnight infor a further 4 °C reftesting.rigerator The for response further current testing. of theThe i-t response curve detected current for of thethe first i-t curve time at detected a large concentration for the first time has some at a largedeviation concentration from other has curves, some but deviation the deviation from other is small, curves, and thebut otherthe deviation curves almost is small coincide,, and the yielding other curvesan RSD almost of 3.09%. coincide Thus,, yielding the MoS a2n-Au-Ag RSD of electrode 3.09%. Th showsus, the a MoS good2-Au repeatability-Ag electrode and shows an acceptable a good repeatabilitystability, indicating and an thatacceptable the MoS stability,2-Au-Ag indicating composite that material the MoS has2-Au a- goodAg composite application material prospect has ina goodH2O2 applicationdetection. prospect in H2O2 detection.

FigureFigure 6. 6. ResultsResults obtained obtained using using the the H 22OO22 sensorsensor are are highly highly sensitive sensitive and and reproducible. reproducible. ( (AA)) The The i i-t-t

curvescurves of of MoS MoS22--Au-AgAu-Ag/GCE/GCE w wereere dripped dripped with with the the same same concentration of H 2OO22 intointo PBS. PBS. ( (BB)) The The i i-t-t

curvescurves of of the the same MoS 2--Au-AgAu-Ag/GCE/GCE when when the the same same concentration of of H 2OO22 waswas dripped dripped into into PBS PBS afterafter the the electrode electrode was was placed placed at at different diﬀerent times.

3.6. Detection of H2O2 Released from Normal Cells and Living Cancer Cells 3.6. Detection of H2O2 Released from Normal Cells and Living Cancer Cells H2O2 is an important representative of the reactive oxygen species related to lots of cellular life H2O2 is an important representative of the reactive oxygen species related to lots of cellular life activities, and is a potential biomarker for cancer cells [28–30]. In order to better explore the physiological activities, and is a potential biomarker for cancer cells [28–30]. In order to better explore the processes of early-stage cancer cells, the monitoring of H2O2 levels in the cell environment is crucial. physiological processes of early-stage cancer cells, the monitoring of H2O2 levels in the cell In this study, we selected human breast cancer cells (MCF-7) for the on-site real-time monitoring of environment is crucial. In this study, we selected human breast cancer cells (MCF-7) for the on-site H2O2 release after stimulation. First, an MTT assay was used to evaluate the effects of the prepared real-time monitoring of H2O2 release after stimulation. First, an MTT assay was used to evaluate the nanomaterials on living cancer cells. The results showed that the survival rate of these living tumor effects of the prepared nanomaterials on living cancer cells. The results showed that the survival cells all reached more than 90% after 24 h of co-incubation, even with 25 µg mL 1 nanomaterials, so the rate of these living tumor cells all reached more than 90% after 24 h of co-incubation− , even with 25 cell growth was almost unchanged and the original activity was maintained (Figure7D). Therefore, μg mL−1 nanomaterials, so the cell growth was almost unchanged and the original activity was it can be proven that the MoS2-Au-Ag nanocomposite was biocompatible with living cells and can be maintained (Figure 7D). Therefore, it can be proven that the MoS2-Au-Ag nanocomposite was used for further electrochemical tests. Moreover, the dynamic release of H O by pure a PBS solution biocompatible with living cells and can be used for further electrochemical2 2 tests. Moreover, the and MCF-7 cells of different concentrations was detected using the MoS2-Au-Ag sensor before and dynamic release of H2O2 by pure a PBS solution and MCF-7 cells of different concentrations was after the stimulation of PMA. As depicted in Figure7A, we observed a significantly reduced current 1 increase upon the addition of 40 µL PMA (0.5 mg mL − ) when the PBS was suspended with MCF-7 cells. The control group with the absence of MCF-7 cells (a) showed no detectable current response to the injection of PMA, indicating that the triggered H2O2 was the result of PMA-stimulated cells. In addition, curves b, c, and d showed the current response curve of the MoS2-Au-Ag sensor at –0.4 V in the presence of the 10%, 30% and 50% concentrations of MCF-7 cells by physiological injection of PMA, respectively. The results revealed that the reduction current increases more significantly as the cell concentration increases. Besides, hydrogen peroxide, released by human retinal pigment epithelial cells (RPE-1), was detected under the same conditions. As shown in Figure7B, we observed that the control group (a) without RPE-1 cells showed no current response after PMA injection, while the experimental group with PMA stimulation showed a slight decrease in current. Compared with the MCF-7 cells, RPE-1 cells showed a very weak reduction in response current, further indicating that the triggered H2O2 was the result of the stimulation of cancer cells by PMA, as exhibited in Figure7C. Sensors 2020, 20, x FOR PEER REVIEW 10 of 13 detected using the MoS2-Au-Ag sensor before and after the stimulation of PMA. As depicted in Figure 7A, we observed a significantly reduced current increase upon the addition of 40 µL PMA (0.5 mg mL −1) when the PBS was suspended with MCF-7 cells. The control group with the absence of MCF-7 cells (a) showed no detectable current response to the injection of PMA, indicating that the triggered H2O2 was the result of PMA-stimulated cells. In addition, curves b, c, and d showed the current response curve of the MoS2-Au-Ag sensor at –0.4 V in the presence of the 10%, 30% and 50% concentrations of MCF-7 cells by physiological injection of PMA, respectively. The results revealed that the reduction current increases more significantly as the cell concentration increases. Besides, hydrogen peroxide, released by human retinal pigment epithelial cells (RPE-1), was detected under the same conditions. As shown in Figure 7B, we observed that the control group (a) without RPE-1 cells showed no current response after PMA injection, while the experimental group with PMA stimulation showed a slight decrease in current. Compared with the MCF-7 cells, RPE-1 Sensors 2020, 20, 6817 10 of 12 cells showed a very weak reduction in response current, further indicating that the triggered H2O2 was the result of the stimulation of cancer cells by PMA, as exhibited in Figure 7C. In conclusion, the proposed MoS2-Au-Ag sensor could be utilized to detect the H2O2 released by living cancer In conclusion, the proposed MoS2-Au-Ag sensor could be utilized to detect the H2O2 released by living cells, and it has the potential to be used as a physiological and pathological H2O2 sensor for the cancer cells, and it has the potential to be used as a physiological and pathological H2O2 sensor for the diagnosisdiagnosis and and treatment treatment of of cancer cancer in in its its early early stages stages..

FigureFigure 7. 7. DetectionDetection of of H H22OO2 2releasedreleased from from normal normal cells cells and and living living cancer cancer cells. cells. ( (AA)) Amperometric Amperometric 1 responseresponse of MoS22--Au-AgAu-Ag/GCE/GCE toto injectioninjection of of 200 200 ng ng mL mL− −1PMA PMA with with MCF-7 MCF- cells7 cells in 0.1in 0.1 M PBSM PBS (pH (pH 7.4). 1 7.4).(B) Amperometric(B) Amperometric response response of MoS of2 MoS-Au-Ag2-Au/GCE-Ag/GCE to injection to injection of 200 of ng 200 mL −ng PMAmL–1 withPMA RPE-1 with cellsRPE- in1 cells0.1 M in PBS 0.1 (pH M PBS 7.4). (pH (C) Comparison7.4). (C) Comparison of response of currentresponse di currentﬀerences differences between MCF-7 between and MCF RPE-1-7 andcells RPEinduced-1 cells by induced H2O2 release. by H2O (D2 )Ereleaseﬀects. of(D prepared) Effects of MoS prepared2-Au-Ag MoS on2 metabolic-Au-Ag on activities metabolic of MCF-7activities cells. of MCF-7 cells. 4. Conclusions

In summary, we constructed an H2O2 sensor based on MoS2-Au-Ag nanocomposites by electrodepositing Au and Ag nanoparticles to modify MoS /GCE. The resulting MoS -Au-Ag/GCE 2 2 1 2 exhibited an excellent electrochemical performance with a sensitivity of 405.24 µA mM− cm− 1 (28.63 µA mM− ) and a linear detection range of 0.05 mM to 20 mM. The H2O2 sensor has good repeatability and stability, and a short response time. The H2O2 sensor constructed in this study has also been successfully applied in the detection of H2O2 generated by living cancer cells, indicating that this work is expected to play a signiﬁcant role in the early diagnosis and treatment of tumors and providing an important basis for the development of eﬀective treatment strategies. Thus, we believe that the prepared MoS2-Au-Ag sensor can be used to analyze H2O2 in all sorts of samples, and we can continue to miniaturize it for better clinical testing in the future. Sensors 2020, 20, 6817 11 of 12

Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/20/23/6817/s1, Figure S1: XPS survey spectrum of MoS2-Au-Ag. Author Contributions: Conceptualization, X.D.; methodology, X.D., J.H. and C.Z.; validation, X.D., J.H., C.Z. and X.L.; investigation, J.H., X.L. and C.Z.; data curation, X.L.; writing—original draft preparation, J.H. and C.Z.; writing—review and editing, X.D.; supervision, X.D.; project administration, X.D.; funding acquisition, X.D.; All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grants from the National Natural Science Foundation of China (Grant nos. 31801200). Conﬂicts of Interest: The authors have declared that no competing interest exists.

References

1. Juang, F.-R.; Chern, W.-C. Octahedral Cu2O nanoparticles decorated by silver catalyst for high sensitivity nonenzymatic H2O2 detection. Mater. Sci. Semicond. Process. 2019, 101, 156–163. [CrossRef] 2. Ohshima, H.; Tatemichi, M.; Sawa, T. Chemical basis of inflammation-induced carcinogenesis. Arch. Biochem. Biophys. 2003, 417, 3–11. [CrossRef] 3. Ye, Y.; Kong, T.; Yu, X.; Wu, Y.; Zhang, K.; Wang, X. Enhanced nonenzymatic hydrogen peroxide sensing with reduced graphene oxide/ferroferric oxide nanocomposites. Talanta 2012, 89, 417–421. [CrossRef] 4. Lu, N.; Zhang, T.; Yan, X.; Gu, Y.; Liu, H.; Xu, Z.; Xu, H.; Li, X.; Zhang, Z.; Yang, M. Facile synthesis of 3D N-doped porous carbon nanosheets as highly active electrocatalysts toward the reduction of hydrogen peroxide. Nanoscale 2018, 10, 14923–14930. [CrossRef] 5. Zou, J.; Cai, H.H.; Wang, D.Y.; Xiao, J.Y.; Zhou, Z.M.; Yuan, B.L. Spectrophotometric determination of trace hydrogen peroxide via the oxidative coloration of DPD using a Fenton system. Chemosphere 2019, 224, 646–652. [CrossRef] 6. Yuan, J.C.; Shiller, A.M. Determination of subnanomolar levels of hydrogen peroxide in seawater by reagent-injection chemiluminescence detection. Anal. Chem. 1999, 71, 1975–1980. [CrossRef] 7. Yang, X.J.; Li, R.S.; Li, C.M.; Li, Y.F.; Huang, C.Z. Cobalt oxyhydroxide nanoflakes with oxidase-mimicking activity induced chemiluminescence of luminol for glutathione detection. Talanta 2020, 215.[CrossRef] 8. Han, H.; He, X.; Wu, M.X.; Huang, Y.B.; Zhao, L.H.; Xu, L.L.; Ma, P.Y.; Sun, Y.; Song, D.Q.; Wang, X.H. A novel colorimetric and near-infrared fluorescence probe for detecting and imaging exogenous and endogenous hydrogen peroxide in living cells. Talanta 2020, 217.[CrossRef] 9. Ren, M.G.; Deng, B.B.; Wang, J.Y.; Kong, X.Q.; Liu, Z.R.; Zhou, K.; He, L.W.; Lin, W.Y. A fast responsive

two-photon fluorescent probe for imaging H2O2 in lysosomes with a large turn-on fluorescence signal. Biosens. Bioelectron. 2016, 79, 237–243. [CrossRef] 10. Wen, Y.; Liu, K.; Yang, H.; Li, Y.; Lan, H.; Liu, Y.; Zhang, X.; Yi, T. A highly sensitive ratiometric fluorescent probe for the detection of cytoplasmic and nuclear hydrogen peroxide. Anal. Chem. 2014, 86, 9970–9976. [CrossRef] 11. Diouf, A.; El Bari, N.; Bouchikhi, B. A novel electrochemical sensor based on ion imprinted polymer and gold nanomaterials for nitrite ion analysis in exhaled breath condensate. Talanta 2020, 209, 120577. [CrossRef] [PubMed] 12. Aragay, G.; Merkoci, A. Nanomaterials application in electrochemical detection of heavy metals. Electrochim. Acta 2012, 84, 49–61. [CrossRef] 13. Liu, M.; Xie, S.B.; Zhou, J. Use of animal models for the imaging and quantification of angiogenesis. Exp. Anim. 2018, 67, 1–6. [CrossRef][PubMed] 14. Akter, R.; Rahman, M.A.; Rhee, C.K. Amplified electrochemical detection of a cancer biomarker by enhanced precipitation using horseradish peroxidase attached on carbon nanotubes. Anal. Chem. 2012, 84, 6407–6415. [CrossRef]

15. Kibsgaard, J.; Chen, Z.; Reinecke, B.N.; Jaramillo, T.F. Engineering the surface structure of MoS2 to preferentially expose active edge sites for electrocatalysis. Nat. Mater. 2012, 11, 963–969. [CrossRef] 16. Hwang, H.; Kim, H.; Cho, J. MoS(2) nanoplates consisting of disordered graphene-like layers for high rate lithium battery anode materials. Nano Lett. 2011, 11, 4826–4830. [CrossRef] Sensors 2020, 20, 6817 12 of 12

17. Zhang, A.; Li, A.; Zhao, W.; Liu, J. Recent Advances in Functional Polymer Decorated Two-Dimensional Transition-Metal Dichalcogenides Nanomaterials for Chemo-Photothermal Therapy. Chemistry 2018, 24, 4215–4227. [CrossRef] 18. Huang, Z.C.; Zhang, A.M.; Zhang, Q.; Pan, S.J.; Cui, D.X. Electrochemical Biosensor Based on Dewdrop-Like

Platinum Nanoparticles-Decorated Silver Nanoﬂowers Nanocomposites for H2O2 and Glucose Detection. J. Electrochem. Soc. 2019, 166, B1138–B1145. [CrossRef] 19. Sangili, A.; Annalakshmi, M.C.; Chen, S.-M.; Balasubramanian, P.; Sundrarajan, M. Synthesis of silver nanoparticles decorated on core-shell structured tannic acid-coated iron oxide nanospheres for excellent electrochemical detection and eﬃcient catalytic reduction of hazardous 4-nitrophenol. Compos. Part B Eng. 2019, 162, 33–42. [CrossRef] 20. Du, X.; Zhou, J. Application of biosensors to detection of epidemic diseases in animals. Res. Vet. Sci. 2018, 118, 444–448. [CrossRef] 21. Fani, M.; Rezayi, M.; Pourianfar, H.R.; Meshkat, Z.; Makvandi, M.; Gholami, M.; Rezaee, S.A. Rapid and label-free electrochemical DNA biosensor based on a facile one-step electrochemical synthesis of rGO-PPy-(L-Cys)-AuNPs nanocomposite for the HTLV-1 oligonucleotide detection. Biotechnol. Appl. Bioc. 2020.[CrossRef][PubMed] 22. Du, X.; Chen, Y.; Dong, W.H.; Han, B.K.; Liu, M.; Chen, Q.; Zhou, J. A nanocomposite-based electrochemical sensor for non-enzymatic detection of hydrogen peroxide. Oncotarget 2017, 8, 13039–13047. [CrossRef] [PubMed] 23. Bo, X.; Ndamanisha, J.C.; Bai, J.; Guo, L. Nonenzymatic amperometric sensor of hydrogen peroxide and glucose based on Pt nanoparticles/ordered mesoporous carbon nanocomposite. Talanta 2010, 82, 85–91. [CrossRef][PubMed]

24. Khan, M.M.; Ansari, S.A.; Lee, J.; Cho, M.H. Novel Ag@TiO2 nanocomposite synthesized by electrochemically active biofilm for nonenzymatic hydrogen peroxide sensor. Mater. Sci. Eng. C Mater. Biol. Appl. 2013, 33, 4692–4699. [CrossRef] 25. Fu, Y.; Dai, J.; Ge, Y.; Zhang, Y.; Ke, H.; Zhang, W. A Novel Non-Enzymatic Electrochemical Hydrogen Peroxide Sensor Based on a Metal-Organic Framework/Carbon Nanofiber Composite. Molecules 2018, 23, 2552. [CrossRef] 26. Lu, H.; Yu, S.; Fan, Y.; Yang, C.; Xu, D. Nonenzymatic hydrogen peroxide electrochemical sensor based on carbon-coated SnO2 supported Pt nanoparticles. Colloids Surf. B Biointerfaces 2013, 101, 106–110. [CrossRef] 27. Tang, N.; Zheng, J.; Sheng, Q.; Zhang, H.; Liu, R. A novel H2O2 sensor based on the enzymatically induced deposition of polyaniline at a horseradish peroxide/aligned single-wall carbon nanotubes modified Au electrode. Analyst 2011, 136, 781–786. [CrossRef] 28. Pei, Y.; Hu, M.; Tang, X.; Huang, W.; Li, Z.; Chen, S.; Xia, Y. Ultrafast one-pot anodic preparation of Co3O4/nanoporous gold composite electrode as an efficient nonenzymatic amperometric sensor for glucose and hydrogen peroxide. Anal. Chim. Acta 2019, 1059, 49–58. [CrossRef] 29. Guler, M.; Turkoglu, V.; Kivrak, A.; Karahan, F. A novel nonenzymatic hydrogen peroxide amperometric

sensor based on Pd@CeO2-NH2 nanocomposites modiﬁed glassy carbon electrode. Mater. Sci. Eng. C Mater. Biol. Appl. 2018, 90, 454–460. [CrossRef]

30. Hussain, S. Comparative eﬃcacy of epigallocatechin-3-gallate against H2O2-induced ROS in cervical cancer biopsies and HeLa cell lines. Wspolczesna Onkol. 2017, 21, 209–212. [CrossRef]

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional aﬃliations.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).