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

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 MoS₂-Au-Ag-modified glassy carbon electrode (GCE) has higher sensitivity (405.24 µA mM⁻¹ cm⁻²), 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.

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 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 [14,15,16]. 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.

2.3. Dispersion of MoS₂

The dispersing agent 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 0.5% PDDA at a constant volume to 1.5 mL. Next, we got a MoS₂ solid precipitate via sonication for 2 h and centrifuged it at 15,000 rpm for 10 min. Subsequently, four different concentrations of dispersion were obtained by discarding the supernatant and adding water at a constant volume to 1.5 mL, including 2 mg mL⁻¹ and 5 mg mL⁻¹ MoS₂ dispersions with 0.5% and 1% PDDA.

2.4. Preparation of MoS₂-Au-Ag/GCE

The bare GCE was pre-polished with 0.3 µm and 0.05 µm alumina powders to get a smooth mirror surface before fabricating with a modified electrode [20]. For the purpose of wiping off the physically adsorbed substance, GCE was then sonicated with the mixture solution of ultra-pure water and ethanol for 10 min. After that, the cleaned electrode was dried under N₂. Briefly, 10 µL of MoS₂ suspension (2 mg mL⁻¹) was cautiously dropped onto the GCE surface, and the modified electrode was dried under air. Then, MoS₂/GCE was electrodeposited in 2.5 mM H[AuCl₄] and 2.5 mM AgNO₃ solutions, respectively, under the condition of −0.4 V. Accordingly, the MoS₂-Au-Ag/GCE was successfully prepared. The whole process of the fabrication is illustrated in Scheme 1.

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 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 were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and were grown in a cell culture incubator with 5% CO₂ and 37 °C. After they were grown to 80–90% confluence, the cells were washed 2–3 times with PBS, and resuspended into 2 mL oxygen-removing PBS for electrochemical detection of H₂O₂ generated by the MCF-7 cells. With the cell-free group as the control group and three groups of cells with different concentrations as the experimental group, the flux of H₂O₂ caused by normal cells and living cancer cells upon the addition of PMA at different concentrations and the correlated electrochemical response signals were recorded by amperometric i-t curves. The effect of the nanomaterials in the cell culture was assessed via an MTT assay and the detailed experimental steps can be found in Supporting Information.

3. Results and Discussion

3.1. Characterization of MoS₂-Au-Ag/GCE Nanocomposite

The morphology and structure of the obtained nanomaterials were investigated by TEM. Figure 1A shows that the MoS₂ has a uniform lamellar structure. As illustrated in Figure 1B,C, the Au and Ag nanoparticles showed clear dendritic and granular morphologies, respectively, and were evenly dispersed over the MoS₂. As shown in Figure 1D, the MoS₂-Au-Ag mimics uniformly compact branches in the PDDA solution. In summary, we successfully synthesized the MoS₂-Au-Ag nanocomposite, which has potential application value for improving electrochemical detection performance. The chemical composition of MoS₂-Au-Ag was verified by XPS. As shown in Figure S1, the curve presented O 1s peaks, C 1s peaks, Ag 3d peaks, Mo 3d peaks, Au 4f peaks and S 2p peaks in one spectrum. For the Mo 3d region, the two characteristic peaks at 229.8 eV and 233.5 eV corresponded to Mo 3d_5/2 and Mo 3d_3/2 of MoS₂, respectively, and the single S 2s peak is located at about 227 eV. Meanwhile, the binding energies at 162.1 eV and 163.2 eV can be attributed to the S 2p_3/2 and S 2p_1/2 species on the edges or surface of the MoS₂ nanosheet, respectively. In addition, the strong peaks of the elements Au and Ag confirmed the successful electrodeposition of two metals on the glassy carbon electrode modified with MoS_2. In addition, energy dispersive spectroscopy (EDS) was used to analyze and study the components of the MoS₂-Au-Ag. The element of Mo, S, Au and Ag were clearly observed, which originated from the MoS₂, Au and Ag nanomaterials, respectively (Figure 1E). The results further manifested that MoS₂-Au-Ag was constructed successfully. We also studied the effects of different materials on sensor performance (Figure 2). The resulting spectra manifested that the oxidation and reduction peaks associated with MoS₂-Au/GCE (b) and MoS₂-Au-Ag/GCE (c) increased substantially compared with MoS₂/GCE (a). Moreover, the peak value of redox was the largest for MoS₂-Au-Ag/GCE, demonstrating that the Au-Ag nanocomposite particles have a good effect on enhancing electrode conductivity. The MoS₂-Au-Ag composite was used to modify the electrode in the subsequent experiments.

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 electrochemical properties of the electrode, which will lead to alterations of the catalytic properties and sensor functions [21]. For the sake of enhancing the sensing performance of the MoS₂-Au-Ag/GCE, the effects of the PDDA concentration of dispersed MoS₂, the electrodeposition time, and the modified electrode material were investigated.

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 (Figure 3A). The results showed that the MoS₂ dispersed with water and PBS buffer had obvious precipitation and was not dissolved at all. The dispersion of MoS₂ with PDDA was better, and a uniform MoS₂ dispersion was obtained. The effect of PDDA concentration on the electrochemical property of MoS₂-Au-Ag/GCE was analyzed by CV in K₃[Fe(CN)₆]. As shown in Figure 3B, the peak current values of 2 mg mL⁻¹ MoS₂ with 1% PDDA (b) and 5 mg mL⁻¹ MoS₂ with 1% PDDA (c) are similar. Based on the principle of economy, 2 mg mL⁻¹ MoS₂ with 1% PDDA was selected for subsequent experiments. The Au-Ag electrodeposited on the surface of MoS₂/GCE can provide a large effective surface area and enhance the electrocatalytic performance of the electrode, which is of great significance for the determination of H₂O₂ [22]. Thus, the amount of Au-Ag electrodeposited on the surface of MoS₂/GCE immediately affects the performance of the H₂O₂ sensor. To enhance the function of Au-Ag on the MoS₂/GCE, the effect of electrodeposition time from 30 s to 150 s was analyzed by CV in 10 mM H₂O₂, as shown in Figure 3D. The analysis results revealed that the potential and the current value of redox peak were different. As we can see from Figure 3C, the b and d peak current values are bigger, but the peak potential value of b is larger. As such, we chose the optimized electrodeposition time in the fabrication of modified electrode to reduce the interference of high voltage.

3.3. Electrocatalytic Activities 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 (Figure 4A). The CV experiments revealed that no redox peaks were measured relating to MoS₂/GCE (a) in the range of −0.80 V–0.00 V, while MoS₂-Au/GCE (c) and MoS₂-Au-Ag/GCE (d) had the presence of a reduction peak of H₂O₂ at −0.50 V. Therefore, MoS₂-Au-Ag/GCE had a better reaction performance to H₂O₂.

We then examined the CV results of MoS₂-Au-Ag/GCE in PBS and in different concentrations of H₂O₂ (Figure 4B). The redox peak of modified electrodes was not measured in PBS (a) while it was shown in 5 mM (b) and 10 mM H₂O₂ (c). With the increase in concentration of H₂O₂, the reductive peak position shifting to the left may be due to the delay of electron transfer caused by the increase in H₂O₂ concentration. In order to prevent interference from other substances, −0.45 V was selected as the catalytical voltage for subsequent current response experiments. Compared with the 5 mM H₂O₂ solution, the reduction peak current value measured by the modified electrode in 10 mM H₂O₂ 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 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 (I_pa) and reduction (I_pc) peak currents with the square root of the scan rate (v ^1/2). The relevant linear regression equations were I_pa = 14.89 v^1/2 − 13.74 and I_pc = −15.37 v^1/2 − 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.

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 amperometric current response at lower concentrations of H₂O₂ (0.05 mM–0.75 mM).

As shown in Figure 5B, the calibration curve was fitted by using multiple sets of repeated experimental data. According to the curve fitting, we calculated the linear range of the prepared sensor to be from 0.05 mM to 20 mM, with a linear correlation coefficient of 0.9933, and the sensitivity was 405.24 µA mM⁻¹ cm⁻² (28.63 µA mM⁻¹). It is worth noting that the sensitivity is much higher than many reported H₂O₂ sensors, as shown in

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

Material	Sensitivity (µA mM−1 cm−2)	Detection Limit (µM)	Linear Range (mM)	Reference
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

. The detection equation of the 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 good sensitivity.

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 Figure 6A. The RSD of the relative standard deviation of the five electrodes was calculated to be 3.79%. The stability was evaluated by placing the MoS₂-Au-Ag/GCE in PBS buffer and adding 1 mM H₂O₂ continuously (Figure 6B). The modified electrode was tested every 4 h and then placed overnight in a 4 °C refrigerator for further testing. The response current of the i-t curve detected for the first time at a large concentration has some deviation from other curves, but the deviation is small, and the other curves almost coincide, yielding an RSD of 3.09%. Thus, the MoS₂-Au-Ag electrode shows a good repeatability and an acceptable stability, indicating that the MoS₂-Au-Ag composite material has a good application prospect in H₂O₂ detection.

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 activities, and is a potential biomarker for cancer cells [28,29,30]. In order to better explore the physiological processes of early-stage cancer cells, the monitoring of H₂O₂ levels in the cell environment is crucial. In this study, we selected human breast cancer cells (MCF-7) for the on-site real-time monitoring of H₂O₂ release after stimulation. First, an MTT assay was used to evaluate the effects of the prepared nanomaterials on living cancer cells. The results showed that the survival rate of these living tumor cells all reached more than 90% after 24 h of co-incubation, even with 25 μg mL⁻¹ nanomaterials, so the cell growth was almost unchanged and the original activity was maintained (Figure 7D). Therefore, it can be proven that the MoS₂-Au-Ag nanocomposite was biocompatible with living cells and can be used for further electrochemical tests. Moreover, the dynamic release of H₂O₂ by pure a PBS solution and MCF-7 cells of different concentrations was 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 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 cells, and it has the potential to be used as a physiological and pathological H₂O₂ sensor for the diagnosis and treatment of cancer in its early stages.

4. Conclusions

In summary, we constructed an H₂O₂ sensor based on MoS₂-Au-Ag nanocomposites by electrodepositing Au and Ag nanoparticles to modify MoS₂/GCE. The resulting MoS₂-Au-Ag/GCE exhibited an excellent electrochemical performance with a sensitivity of 405.24 µA mM⁻¹ cm⁻² (28.63 µA mM⁻¹) 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.