Electrochemical sensor based on a three dimensional nanostructured MoS2 nanosphere-PANI/reduced graphene oxide composite for simultaneous detection of ascorbic acid, dopamine, and uric acid

A three dimensional (3D) nanostructured composite based on the self-assembly of MoS2 nanospheres and polyaniline (PANI) loaded on reduced graphene oxide (denoted by 3D MoS2-PANI/rGO) was prepared via a feasible one-pot hydrothermal process. The 3D MoS2-PANI/rGO nanocomposite not only exhibits good functionality and bioaffinity but also displays high electrochemical catalytic activity. As such, the developed 3D MoS2-PANI/rGO nanocomposite can be employed as the sensing platform for simultaneously detecting small biomolecules, i.e., ascorbic acid (AA), dopamine (DA), and uric acid (UA). The peak currents obtained from the differential pulse voltammetry (DPV) measurements depended linearly on the concentrations in the wide range from 50 μM to 8.0 mM, 5.0 to 500 μM, and 1.0 to 500 μM, giving low detection limits of 22.20, 0.70, and 0.36 μM for AA, DA, and UA, respectively. Furthermore, the 3D MoS2-PANI/rGO-based electrochemical sensor also exhibited high selectivity, good reproducibility and stability toward small molecule detection. The present sensing strategy based on 3D MoS2-PANI/rGO suggests a good reliability in the trace determination of electroactive biomolecules.


Introduction
Ascorbic acid (AA), dopamine (DA), and uric acid (UA) are crucial biomolecules that usually coexist in human physiological uids. [1][2][3] Abnormal DA level in the central nervous system may lead to several neurological diseases, e.g., schizophrenia and Parkinson's disease. 4,5 Similarly, AA associates to atherosclerosis, 6 and abnormality of UA level in the human metabolism causes diseases like hyperuricemia and gout. 7 Therefore, sensitive and rapid detection of AA, DA, and UA in biological systems is vital for healthcare, routine analysis and clinical diagnostics. Several analytical techniques including chromatography, 8 surface plasmon resonance spectroscopy, 9 chemiluminescence, 10 uorescence, 11 and electrochemical analysis, 12 have been conducted for detecting biomolecules. Among them, electrochemical techniques have been receiving considerable interest because of the low cost, fast response, easy operation, and inexpensive instrumentation required. 13,14 Notably, as electroactive molecules, AA, DA, and UA can be detected simultaneously by using electrochemical technique. 15 However, the electro-catalytic oxidation of these biomolecules at bare electrodes occur at very similar potentials, leading to poor selectivity. 16 Many efforts have been made to develop effective electrochemically active and large surface area sensors with highly selective and sensitive electrochemical current responses. To address these issues, various sensing materials, e.g., metal nanoparticles, semiconductors, polymers and carbon materials, have been employed. [17][18][19] With high electron transfer rate, electrochemical active surface area, and chemical functionality, reduce graphene oxide (rGO) has been investigated widely in electrochemical sensing. 20,21 Chen et al. reported that cobalt manganese oxides increased the amount of graphene captured in the composites and improved sensing activities. 22,23 Moreover, as a two-dimensional (2D) layered nanomaterial, molybdenum disulde (MoS 2 ) has been focused on the elds like energy conversion and storage, electronic devices, catalysts, and sensors, 24-28 due to its highly optical transmittance, unique electrochemical properties, and large electrochemical active surface area. MoS 2 -based nanocomposites have been used as matrix for simultaneously detecting of AA, DA, and UA. For instance, the gold nanoparticle-decorated MoS 2 nanocomposite modied electrode exhibited good electro-catalytic oxidation of AA, DA, and UA, giving detection limits of 100, 0.05, and 10 mM, respectively. 29 Interestingly, MoS 2 /graphene hierarchical frameworks have been reported where Mo 6+ cations were found to induce the self-assembly of graphene oxide, and the aerogel structures depend strongly on different MoS 2 loadings. 30 What's more, most of the reported MoS 2 used as the sensing materials for biomolecules detection were still based on 2D planar structures. Considering that the effective electrochemically active surface areas of MoS 2 for adsorption and electro-catalytic oxidation of biomolecules may be closely associated with their morphology, the structure modulation needs to be further studied. 31,32 Diverse shapes of nanoparticles distributed and anchored on graphene sheets can be achieved by varying synthesis conditions. 33,34 Additionally, conductive polymers such as polyaniline (PANI) have also been used to functionalize MoS 2 to improve the electro conductivity of the materials for the detection of dopamine. 35 MoS 2 /PANI/rGO aerogel nanocomposite exhibited excellent long-term cycling stability and a high capacity. 36 RGO/MoS 2 /PANI@AuNPs nanocompositebased electrochemical aptasensor was also reported for detection of aatoxin B1. 37 However, the nanocomposites were synthesized via multiple steps.
This work aims to enhance the electrochemical activity of the MoS 2 /graphene and improve the bioaffinity between the small biomolecules and the sensing layer. 3D nanostructured composite based on the self-assembly of MoS 2 nanospheres and PANI loaded on rGO (3D MoS 2 -PANI/rGO) was synthesized by a one-pot hydrothermal reaction, following by the application as electrochemical catalyst toward the trace determination of biomolecules in blood and urine samples (Scheme 1). As expected, the synergistic effects of the effective electro-catalytic performance of MoS 2 , electrochemically activity of PANI, and the excellent charge transfer of graphene impart superior electro-catalytic activity of 3D nanostructured MoS 2 -PANI/rGO composite for the oxidation reactions of AA, DA, and UA.

Preparation of 3D MoS 2 -PANI/rGO nanocomposite
The preparation of GO and PANI are described in detail in the ESI (S1). † MoS 2 -PANI/rGO nanocomposite was synthesized by a one-pot hydrothermal approach. Briey, 0.40 g Na 2 MoO 4 -$2H 2 O, 0.66 g thiourea, 0.20 g PANI and 0.25 g PVP were added in 40 mL of GO aqueous dispersion (1.25 mg mL À1 ) to form a homogeneous solution under stirring. Then the solution was transferred into a 50 mL Teon-lined stainless steel autoclave and heated at 180 C for 24 h. Aer cooling to ambient temperature, the precipitate was collected by centrifugation and washed thoroughly using ultrapure water and ethanol. Finally, the solid was dried in a vacuum oven at 60 C for 12 h and the 3D MoS 2 -PANI/rGO nanocomposite was then obtained. MoS 2 / rGO composite was prepared in a similar manner in the absence of PANI.
Apparatus X-ray diffraction (XRD) spectra were performed using a D8 advance X-ray diffraction instrument (Germany) in the 2q range of 5-80 . Fourier-transform infrared (FT-IR) spectra were recorded on a Bruker TENSOR27 instrument from 400 to 4000 cm À1 . The X-ray photoelectron spectroscopy (XPS) analysis was measured by a Thermo Fisher ESCALAB 250Xi spectrometer equipped with an Al anode (Al-Ka 1486.6 eV). Surface morphologies of the samples were investigated by eldemission scanning electron microscopy (FE-SEM, JSM-6490LV, Japan) and transmission electron microscopy (TEM, JEM-2100F, Japan), respectively.

Fabrication of biosensor and electrochemical measurements
The glassy carbon electrodes (GCE, 3.0 mm in diameter) were polished using 0.3 mm and 0.05 mm alumina slurry, followed by sonicating in nitric acid, ethanol, and ultrapure water sequentially, and drying at room temperature. Aerward, 10 mL of the MoS 2 -PANI/rGO suspension (1.0 mg mL À1 ) was dropped onto GCE electrode surface, then dried in the ambient air to develop the sensor (MoS 2 -PANI/rGO/GCE). For comparison, the MoS 2 / rGO/GCE and PANI/GCE were made in the same way.
Electrochemical measurements were performed on a CHI660D electrochemical workstation (Chenhua, Shanghai, China) with a conventional three-electrode system. The GCE or modied GCEs, platinum slide, and Ag/AgCl (saturated KCl) Scheme 1 Schematic diagram of MoS 2 -PANI/rGO-based electrochemical biosensor for simultaneous detection of AA, DA, and UA. electrode were used as working electrode, counter electrode, and reference electrode, respectively. Cyclic voltammetry (CV) was recorded in 0.1 M PBS (pH 7.0; containing 0.1 M KCl). The potential range was from À0.2 to 0.8 V and the scan rate was 50 mV s À1 . Differential pulse voltammetry (DPV) was collected at from À0.2 V to 0.8 V with pulse amplitude of 50 mV.

Characterization
The chemical structure and components of nanomaterials were determined by FT-IR (S2 †) and XPS. The FT-IR spectra (Fig. S1 †) suggested that PANI was present in MOS 2 -PANI/rGO nanocomposite. The chemical composition and valence states of C, N, Mo and S containing in MoS 2 /rGO and MoS 2 -PANI/rGO (Fig. 1, S2 and Table S1 †) nanocomposites were investigated by XPS in detail. From the XPS survey spectrum (Fig. S2a †), C, N, O, Mo and S can be clearly identied in MoS 2 -PANI/rGO with atomic% of 74.5, 11.8, 10.8, 0.9, and 2.0, respectively. Fig. 1a shows the C 1s characteristic peak which is caused by the component of rGO and PANI. Three main peaks are centered at 284.6, 285.6, and 287.7 eV, which are assigned with the groups of C-C/C]C/C-H, C-OH/C-N, and C]O, respectively. 38 The graphene possess numerous edge planes and defects, which are conductive to the fast electron transfer as well as electrocatalytic activity. 39,40 Apparently, the C-N was originated from PANI. The N 1s core-level XPS spectrum (Fig. 1b) was tted into three components, 398.8, 399.8 and 400.8 eV, which are corresponded to imine (]N-), amine (-NH), and protonated amine units (-NH + /]N + -) containing in PANI, respectively. 41 The amine and imine groups can form hydrogen bonds with hydroxyl groups present in the small biomolecules and eventually results strong bioaffinity. 42 Fig. 1c and d depict the highresolution scan XPS spectra of the Mo 3d, S 2s and S 2p electrons. The binding energies of Mo 3d 3/2 , Mo 3d 5/2 and S 2s peaks are located at 232.4, 229.2 and 226.5 eV, whereas S 2p 1/2 and S 2p 3/2 peaks are observed at 163.3, and 162.1 eV, respectively, 30,43 suggesting that Mo 4+ chemical state existed and the formation of crystalline MoS 2 . The combination of the MoS 2 , rGO and PANI may effectively increase the conductivity, bioaffinity and catalytic activity of the nanocomposite.

Morphological studies
The surface morphology of as-prepared MoS 2 /rGO, PANI, and MoS 2 -PANI/rGO were investigated by FE-SEM, as shown in Fig. 2 and S3. † The results show that the MoS 2 nanospheres were formed with a diameter of 300-400 nm (Fig. S3a †), approximately, whereas the PANI exhibits a rod-like shape (Fig. S3b †). The FE-SEM images of MoS 2 /rGO reveal spherical-shaped MoS 2 nanoparticles wrapped by crumpled structure of graphene sheets ( Fig. S3c and d †). As for the MoS 2 -PANI/rGO, the honeycomb-like structure was formed with MoS 2 nanospheres interpenetrating in 3D graphene sheets ( Fig. 2a and b), which could increase the electrochemical active surface area of the nanocomposite. Further structural insights were obtained in TEM analysis. The graphene appears a curved and crinkled texture (Fig. 2c, d and S4a-c †), which is well anchored by MoS 2 nanospheres. For MoS 2 -PANI/rGO, dendritic hierarchical structure with several layers can be found in high-resolution TEM (HR-TEM) image. In Fig. 2e, the layered structures with an interlayer distance of $0.62 nm corresponds to the (002) crystal planes of MoS 2 . The corresponding selected area electron diffraction (SAED) patterns of MoS 2 /rGO (Fig. S4d †) and MoS 2 -PANI/rGO (inset of Fig. 2e) conrm the hexagonal  This journal is © The Royal Society of Chemistry 2019 structure of MoS 2 with different crystallographic orientations and present separated diffraction rings that can be indexed to the XRD patterns (Fig. 2f). The peaks that appeared in the XRD patterns of all the three samples are indexed based on MoS 2 .

Electrochemical performance of the as-synthesized nanomaterials
To evaluate the electrochemical performance of the different materials, CV curves were tested on the bare GCE, MoS 2 /rGO, PANI, and 3D MoS 2 -PANI/rGO-modied glassy carbon electrodes. The MoS 2 -PANI/rGO/GCE (0.125 cm 2 ) exhibited larger electrochemical active surface area than MoS 2 /rGO/GCE (0.101 cm 2 ) and PANI/GCE (0.102 cm 2 ), suggesting it can provide quick mass transport of molecules to the electrocatalyst (Fig. S5 †). When the AA, DA and UA biomolecules were added in 0.1 M PBS solution (Fig. 3a), the GCE and PANI/GCE showed broad peak from À0.2 to 0.8 V, while MoS 2 /rGO/GCE had two obvious peak responses to DA and UA. In comparison, the MoS 2 -PANI/rGO/ GCE exhibited the largest anodic peaks in the CV measurements. Three peaks were observed on MoS 2 -PANI/rGO/GCE in DPV measurements, indicating a simultaneous electrochemical detection of the three bio-molecules (Fig. 3b). The MoS 2 -PANI/ rGO/GCE exhibits the highest peak current intensity and three well-resolved peaks at 0.020, 0.196, and 0.320 V (DE AA-DA ¼ 176 mV, DE DA-UA ¼ 124 mV, DE AA-UA ¼ 300 mV), implying that synergistic effect existed among the MoS 2 , rGO and PANI toward the electrochemical catalysis of three small biomolecules. The observed anodic peaks are ascribed to the oxidation of hydroxyl groups to carbonyl groups in AA, catechol to o-quinone in DA, and bridging double bond to hydroxyl groups in UA. 16 Moreover, the effect of pH on electrochemical oxidation of AA, DA, and UA with MoS 2 -PANI/rGO/GCE was investigated (Fig. 4). The results show that the anodic peak potential of AA, DA, and UA shied negatively with increasing of their pH value (pH 4.0-10.0) (Fig. 4a), and oxidation peak potential of AA, DA, and UA were linearly proportional to the pH value (Fig. 4b), demonstrating that the proton took part in the electrochemical oxidation reaction process. 6,29 The peak current of AA, DA, and UA was also changed with the pH value. The AA, DA, and UA achieved the maximum value at pH 6.0, 7.0, and 7.0, respectively   ( Fig. 4c). Considering sensitivity and selectivity, the electrolyte solution with pH 7.0 was selected for further measurements of the analytes.

Simultaneous detection of AA, DA and UA
Individually or simultaneously analyze AA, DA, and UA on MoS 2 -PANI/rGO/GCE was studied by DPV. It is found that the response current corresponding to the analyte increases with increasing concentration of the ternary mixture. In Fig. 5a-b, the peak currents increased linear with the AA concentrations in the range of 50 mM to 8.0 mM. The regression equation of I p,AA (mA) ¼ 11.596 + 0.0034C AA (mM) (R 2 ¼ 0.996) was obtained. The limit of detection (LOD) was determined to be 22.20 mM for AA at a signal-to-noise ratio of 3. Similarly, the peak currents of DA ( Fig. 5c and d) and UA ( Fig. 5e and f)  In the simultaneous detection of AA, DA, and UA experiments, three well-resolved anodic peaks at potentials of 0.052, 0.196, and 0.304 mV were presented at MoS 2 -PANI/rGO/GCE from DPV plots (Fig. 6). The three peak currents were linearly dependent on the concentrations ranging from 30 mM to 3.0 mM for AA, 3.0 to 300 mM for DA, and 2.0 to 200mM for UA while the LOD of AA, DA, and UA were 27.49, 0.65, and 0.40 mM, respectively. The results demonstrate that the 3D MoS 2 -PANI/ rGO-based sensor exhibited wide linear ranges and low detection limits for detection of biomolecules comparative to those of other sensors (Table 1).

Selectivity, reproducibility, and stability of the fabricated biosensor
Several potential interferential species were used to examine the selectivity at MoS 2 -PANI/rGO/GCE for simultaneously detecting of AA (1.0 mM), DA (75 mM) and UA (75 mM) (Fig. 7a).
There were negligible interferences detected in the presence of NaNO 3 , ZnSO 4 , glucose, glycine, and citric acid, whose concentrations are 1.0 mM. The peak response changes were less than AE3%, revealing a good selectivity of the developed electrochemical sensor.
Five MoS 2 -PANI/rGO modied GCEs were prepared independently and their peak currents for AA, DA and UA mixture were investigated (Fig. S6 †). The relative standard deviation (RSD) were 4.0% for AA, 4.56% for DA, and 4.24% for UA,   indicating the good reproducibility of the developed sensor. In successive 15 times DPV measurements, the RSD values of 1.13%, 1.70%, and 2.49% for AA, DA and UA were observed. Furthermore, the stability of the electrode was also determined by DPV measurements for 7 days. The oxidation currents decreased by less than 5.0% of the initial currents (Fig. 7b), conrming a good stability of the biosensor.

Real sample analysis
To evaluate the applicability of the MoS 2 -PANI/rGO-based sensor, the simultaneous detection of AA, DA, and UA in human serum and urine samples was investigated by using standard addition method. Human serum (obtained from Beijing Solarbio Science & Technology Co., Ltd.) and human urine (supplied by an adult male student in the laboratory) were ltered through 0.22 mm lter, and diluted 100 times with 0.1 M PBS (pH 7.0) to prepare the real samples. Subsequently, DPV curves were collected before and aer spiking with different concentrations of UA, DA and AA in the real samples (Fig. S7 †). As shown in Table 2, the recoveries of the three kinds of small biomolecules varied from 99.0% to 103.5% in human serum, whereas they changed from 96.2 to 103.6% in human urine samples. The RSD values were also accurate and precise, indicating the applicability of the developed sensor for detection of these electroactive molecules in real biological system.

Conclusion
A 3D nanostructured MoS 2 -PANI/rGO nanocomposite based on the self-assembly of MoS 2 nanospheres and PANI loaded on reduced graphene oxide was synthesized by a one-pot hydrothermal reaction, and employed as the platform for electrochemical oxidation of biomolecules. The result showed that synergistic effect existed among MoS 2 , rGO and PANI. The ternary composite exhibited good electrochemical activity, high bioaffinity and strong catalytic effect on the oxidation of biomolecules. The 3D MoS 2 -PANI/rGO-based sensing platform exhibited high sensitivity for simultaneous detection of AA, DA, and UA with three distinguished oxidation peaks (DE AA-DA ¼ 176 mV, DE DA-UA ¼ 124 mV, DE AA-UA ¼ 300 mV) in DPV measurements. The MoS 2 -PANI/rGO/GCE showed excellent responses toward AA, DA, and UA in the linear range of 50 mM to 8.0 mM, 5.0-500 mM, and 1.0-500 mM with low detection limit of 22.20, 0.70, and 0.36 mM (S/N ¼ 3), respectively. The sensor also exhibited good selectivity, reproducibility and stability for simultaneous detection of these biomolecules in human serum and urine samples.

Ethical conduct of research
The authors state that for investigations involving human subjects, informed consent was obtained from all human subjects.

Conflicts of interest
There are no conicts to declare.