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Introduction

Uric acid (UA) is a metabolic product of purine. Due to the antioxidation and radical scavenging of UA, it serves an important role in the metabolic processes of humans. However, abnormal levels of UA additionally serve an important role for the occurrence and development of certain diseases, including gout, hyperuricemia, renal failure, urinary calculi, hypertension, coronary heart disease, leukemia, arthritis and Lesch-Nyhan syndrome (1,2). Therefore, a simple, rapid and accurate detection method of UA may have an important role in the diagnosis, treatment, monitoring and prevention of the aforementioned diseases.

At present, the common detection methods include enzymology, spectrophotometry, high performance liquid chromatography and electrochemical luminescence (3–5). However, these methods have the disadvantages of low sensitivity, complex detection process, a narrow linear range and high cost. In recent years, due to the advantages of high sensitivity, fast detection and low cost, electrochemical sensing methods have been widely investigated. However, the majority of the electrochemical sensors used for UA detection remain unable to avoid the disadvantages of low sensitivity, narrow linear range and poor specificity (7–9). Therefore, identifying an ideal electrochemical sensor has become a popular research focus in recent years.

In 2004, Novoselov et al (6) synthesized graphene for the first time. Graphene is a novel carbon nanomaterial that has a two-dimensional honeycomb crystal structure and is composed of an sp2 hybrid monolayer of carbon atoms connected by covalent bonds. This special structure has the advantages of a high specific surface area, good electrical conductivity, excellent mechanical strength and high electrocatalytic activity (10–12). Graphene has additionally been identified to have a series of other novel unique properties, including good bio-affinity, a perfect quantum tunnel effect, and room-temperature ferromagneticity (13–16). Thus, graphene has been widely used in nano-electronics, energy storage, biochemical sensors and numerous other fields in previous years (17,18). However, due its hydrophobicity, strong van der Waals forces and π-π conjugated bond interactions, the monolayer of graphene tends to aggregate and may even be converted into graphite in aqueous solution (19,20). This greatly limits the applications of graphene in electrochemical biosensors.

Molybdenum disulfide (MoS2) is a member of the transition metal dichacogenide family. MoS2 has a similar crystal structure, physical properties and chemical properties to graphene. The crystal structure of MoS2 is similar to a ‘sandwich’ structure that is composed of ‘S-Mo-S’ units (21,22). The atoms are strongly connected by ‘S-Mo’ covalent bonds, and the triple layers are weakly connected to each other by van der Waals forces, leading to relative sliding between layers (23,24). Due to its specific surface area, strong adsorption capacity, high catalytic activity and weak interlayer-friction, MoS2 is widely used in the manufacturing of battery electrode materials, catalysts, electrochemical devices, hydrogen storage materials and solid lubricants (25–28). Compared with graphene, the MoS2 monolayer is not able to aggregate and has improved chemical stability in aqueous solution. However, the conductivity and structural strength of MoS2 are far inferior to that of graphene, which additionally limits its application in the field of electrochemical biosensors (29,30).

In the present study G-MoS2 composites were synthesized via the hydrothermal method. Due to the synergistic interaction between graphene and MoS2, the G-MoS2 composites have a high catalytic efficiency and electrical conductivity, in addition to avoiding the conversion of graphene into graphite in aqueous solution. The electrochemical behavior of glucose (Glu), ascorbic acid (AA), dopamine (DA), UA and human serum UA at a G-MoS2-Nafion/glassy carbon electrode (GCE) were investigated by cyclic voltammetry (CV), linear sweep voltammetry (LSV) and amperometric i-t curve (i-t).

Materials and methods

Equipment

The following equipment was used: electrochemical workstation (CHI-760E; Shanghai Chenghua Machinery Co., Ltd., Shanghai, China); ultrasonic cleaner (KQ-300DA; Kunshan Ultrasonic Instruments Co., Ltd., Kunshan, China); electronic balance (FA1004; Shanghai Yueping Scientific Instrument Co., Ltd., Shanghai, China); electric heating air blast drying oven (DHG-9023; Shanghai Suopu Instrument Co., Ltd. Shanghai, China; http://suopuyiqi.goepe.com/); freeze drying oven (DZF-6020; Shanghai Xinmiao Medical Equipment Manufacturing Co., Ltd., Hangzhou, China); field-emission scanning electron microscope (SEM; Low Vacuum JSM-6510; JEOL, Ltd., Tokyo, Japan); elemental distribution spectrometer (EDS; Hitachi/S-4800; Hitachi, Ltd., Tokyo, Japan); and X-ray diffractometer (XRD; Dmax/Ultima IV; Rigaku Corporation, Tokyo, Japan).

Electrochemical analysis system

Electrochemical measurements were performed with the three-electrode system (working electrode, reference electrode and auxiliary electrode) electrochemical workstation. The G-MoS2-Nafion/GCE was used as the working electrode. A saturated calomel electrode (Ag/AgCl; CKCl=3 mol/l) and a platinum wire were used as the reference and auxiliary electrodes, respectively.

Reagents

Glu, AA, DA and UA were purchased from Aladdin Biochemical Technology Ltd. (Shanghai, China). Graphite powder, H2SO4 (98%), NaNO3, HCl (5%), H2O2 (30wt%), KMnO4, ammonium molybdatetetrahydrate (H24Mo7N6O24·4H2O) and thioacetamide (C2H5NS) were purchased from the Chengdu Kelong Chemical Reagent Factory (Chengdu, China). Nafion solution (5%) was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). PBS (0.2 M; pH=7.4) was prepared with Na2HPO4 and NaH2PO4. Twice distilled water was used in all of the experiments. All reagents and chemicals were of analytical grade.

Patients

The human serum samples used in the present study were collected from human peripheral blood preserved by the Laboratory Department of The Affiliated Hospital of North Sichuan Medical College (Nanchong, China), between March 2017 and May 2017. A total of 35 human serum samples were collected, including 23 males and 12 females. The age distribution was in the range of 20–60 years old. As the study did not involve direct clinical studies in humans, the interests of the serum donors were not at risk of harm. With the permission of the ethical review committee of the Affiliated Hospital of North Sichuan Medical College, it was necessary to inform the individuals of the primary contents of the present study, and to ensure that the collected serum not be used in other experiments. All patients involved in the present study signed written consent agreements.

Synthesis of graphene oxide (GO)

GO was synthesized from graphite powder using the modified Hummers method (31). Graphite (5 g) was added to concentrated H2SO4 (120 ml) under magnetic stirring at room temperature, to which NaNO3 (2.5 g) was added. The mixture was maintained at 0–5°C. Under vigorous agitation for 30 min, KMnO4 (15 g) was added slowly while the temperature of the mixture solution was maintained at <20°C. The mixed solution was incubated in a 30°C water bath for 12 h to form a thick paste. Subsequently, 150 ml distilled water was slowly added, stirred vigorously, and allowed to react for a further 12 h in a 98°C water bath. A volume of 50 ml H2O2 (30%) was added to the solution, altering the color of the solution from brown to yellow. The mixture was rinsed and centrifuged for 5 min (2,900 × g, 25°C) with HCl (5%) and distilled water numerous times (until pH 7.0 was reached) to remove the SO42−, KMnO4 and other residual substances. The sediment was filtered out and dried in a 60°C vacuum drying oven for 12 h. The GO was obtained as a solid black film.

Synthesis of G-MoS2 composites

G-MoS2 composites were prepared using the hydrothermal process (32,33). GO (60 mg) was dispersed in 20 ml distilled water by ultrasonication (60 kHz, 55°C) for 1 h. H24Mo7N6O24·4H2O (26.5 mg) and C2H5NS (60 mg) were added to the GO aqueous dispersion by ultrasonication for 30 min (60 kHz, 55°C) to form a homogeneous solution. The solution mixture was transferred to a 100-ml Teflon-lined stainless steel autoclave and heated in an electric oven at 200°C for 24 h. The autoclave was naturally cooled to room temperature, and the black solid was obtained by filtration, rinsing and centrifugation (2,900 × g, 5 min, 25°C) three times. The solid was subsequently dried in a −20°C freeze-drying oven, with the obtained black solid samples denoted as G-MoS2.

Element verification of G-MoS2 composites

In order to verify that the G-MoS2 composites were successfully prepared, the morphologies of G-MoS2 composites were characterized by a Low Vacuum JSM-6510 field-emission scanning electron microscope (SEM) with platinum as the sputter coating (20 kV, 1.18×10−3 Pa, 25°C). Briefly, 10 mg G-MoS2 was dispersed in 10 ml absolute ethyl alcohol by ultrasonication (60 kHz, 55°C) for 30 min. Subsequently, the sample was dripped onto a silicon wafer, dried, sprayed with platinum and detected using the SEM. The elemental compositions of G-MoS2 composites were recorded on a Hitachi/S-4800 elemental distribution spectrometer (30 kV, dead-time: 92%) and a Rigaku Dmax/Ultima IV diffractometer with monochromatized Cu Kα radiation (Kα/λ=0.15418 nm). To verify whether the G-MoS2 composites prepared in the present study were consistent with the standard graphene and MoS2, the graphene of the G-MoS2 composites was compared with a previous study (34) and MoS2 of the G-MoS2 composites was compared with the Powder Diffraction File card of MoS2 [Joint Committee on Powder Diffraction Standards (JCPDS) file no. 17-744].

Preparation of the modified electrode

The GCE was sequentially polished with 1.0, 0.3 and 0.05 µm alumina slurry on velvet. The residual alumina slurry was rinsed off with distilled water, and the GCE was placed in anhydrous ethanol and distilled water with ultrasonication (60 kHz, 55°C) for 10 min. The cleaned GCE was dried with an infrared lamp. The G-MoS2 composite (30 mg) and Nafion solution (10 µl) were dispersed in 15 ml anhydrous ethanol with ultrasonication (60 kHz, 55°C) for 30 min to obtain a homogenous suspension. The G-MoS2-Nafion-modified electrode was prepared by casting 5 µl suspension onto the pretreated GCE, followed by drying with an infrared lamp.

Catalytic activity evaluation of the modified electrode

Firstly, the prepared GCE was placed in PBS (20 ml; pH=7.4) for 10 min and the CV curve of PBS was determined. The GCE was subsequently rinsed five times with distilled water and the GCE was modified with G-MoS2-Nafion. The G-MoS2-Nafion/GCE was placed in PBS for 2 h and the CV curve of PBS was determined. Finally, 20, 40, 60, 80 and 100 µl UA (100 mM) were added to the PBS. The resulting concentrations were 100, 200, 300, 400 and 500 µM, and their corresponding CV curves were determined.

Specificity evaluation of the modified electrode

The PBS was detected with the G-MoS2-Nafion/GCE and a control LSV curve was obtained. Subsequently, 10 µl NaOH (1.0 M), Glu (2.0 M), AA (0.1 M), DA (0.1 M) and UA (0.1 M) were added to the PBS and the interference LSV curve was obtained.

Subsequently, an LSV assay was conducted for the human serum samples to confirm whether there may be an effect of other human serum components (including Glu, AA and DA) when detecting the UA in human serum with the G-MoS2-Nafion/GCE. PBS was initially detected with the G-MoS2-Nafion/GCE and a reference LSV curve was obtained. Subsequently, 4 ml PBS was removed and 4 ml of human serum was added to the PBS. The mixed solution of PBS (16 ml) and human serum (4 ml) was subsequently detected with the G-MoS2-Nafion/GCE and the experimental LSV curve was obtained.

Quantitative detection of human serum UA with G-MoS2-Nafion/GCE

Compared with the CV and LSV electrochemical analysis methods, i-t may be used to measure the average current values of different concentrations of UA more easily, stably and accurately. Therefore, i-t was selected to detect the UA in solution and in human serum. The G-MoS2-Nafion/GCE was placed in PBS (20 ml) for 500 sec, and 10 µl different concentrations of UA solution were added into the PBS every 50 sec.

Methodology of the comparison analysis

The UA quantitative detection of the mixed serum was performed using a uricase colorimetric assay. The G-MoS2-Nafion/GCE was placed in PBS (16 ml) for 500 sec prior to detection of the mixed serum samples and 4 ml mixed serum was subsequently added to the PBS. The average current intensity of the mixed serum was measured. The UA concentration values of mixed serum were obtained by the conversion of the calibration curve and the linear correlation was determined. The results from UA determination using uricase colorimetric assay and G-MoS2-Nafion/GCE were further analyzed.

Recovery and repeatability analysis of G-MoS2-Nafion/GCE

Following measurement of the current values of the mixed serum with G-MoS2-Nafion/GCE, a further 10 ml UA solution (100 mM) was added to the PBS immediately. The average current intensity was recorded and the total UA concentration values were obtained using the calibration curve. The MoS2-Nafion/GCE was used to detect the UA solution (200 µM) 11 times.

Statistical analysis

All data are presented as the means ± standard deviation of three experiments. The data were analyzed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) or GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). The relationship between two variables was analyzed using linear regression. Comparison of the results obtained by uricase colorimetric assay and G-MoS2-Nafion/GCE were performed using Student's t-test and one-way analysis of variance with Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The precision test results were verified using Grubbs' test. According to Grubbs' test, when the test is repeated 11 times, P=0.01 or 0.05 as long as the significance level is not greater than the critical value (Ga. n=2.484 or 2.234), indicating that the corresponding result is not an outlier. In addition, according to the Health Industry Standards of the People's Republic of China (http://www.nhfpc.gov.cn/zhuz/s9492/201301/c8dd48222ab14387a6503667be78bec3.shtml), the precision of uric acid detection is considered acceptable when the coefficient of variation <4.5%.

Results

Preparation of a good electrode modified complex

The morphology of G-MoS2 was characterized by SEM (Fig. 1). The MoS2 nanoparticles were densely loaded on the graphene nanosheets, and the flexible graphene nanosheets with wrinkled edges were randomly hybridized with MoS2 nanoparticles. This structure facilitates a good connection and close contact between the graphene and MoS2, thus increasing the contact area with the analytes.

Figure 1. Scanning electron microscopy images of graphene-MoS2 with different magnifications. (A) Magnification, ×10,000; (B) magnification, ×50,000. MoS2, molybdenum disulfide.

The elemental composition of G-MoS2 was analyzed using an EDS (Fig. 2A). The carbon elements of graphene were evenly distributed on the graphene nanosheets. The molybdenum elements and sulfur elements of MoS2 were distributed in the corresponding positions of MoS2 nanoparticles, and the G-MoS2 composite contained no other elements except carbon, molybdenum and sulfur. This indicated that G-MoS2 composites were successfully synthesized.

Figure 2. Verification of the elemental composition in G-MoS2 composites. (A) Elemental distribution spectrometer analysis of G-MoS2. (B) X-ray diffractometer analysis of G-MoS2 (Cu Kα/λ=0.15418 nm). G-MoS2, graphene-molybdenum disulfide; C, carbon; S, sulfur; JCPDS, Joint Committee on Powder Diffraction Standards.

XRD testing was conducted to examine the crystalline characteristics of G-MoS2 (Fig. 2B). The characteristic diffraction peak of graphene may be identified at 25°, which corresponds to C (002), and this is consistent with a previous study (34). The MoS2 demonstrated its characteristic diffraction peaks when compared with the Powder Diffraction File card of MoS2 (JCPDS file no. 17-744) and no other miscellaneous peaks were observed. The diffraction peaks of MoS2 at 14.5, 32.8, 34.2, 38.2, 41.1, 58.2 and 60.4°, were consistent with the (003), (101), (012), (104), (015), (110) and (113) crystal planes of MoS2 (JCPDS file no. 17-744), respectively, indicating that the G-MoS2 composites synthesized in the present study contained a high purity of graphene and MoS2.

G-MoS2-Nafion/GCE has a good electrochemical catalytic activity for UA

The electrochemical catalytic activity of the G-MoS2-Nafion/GCE with the addition of different concentrations of UA was assessed (Fig. 3A). No current intensity alteration or any characteristic peak was observed in the CV curve ‘a’, which was within the potential range of 0–0.6 V, suggesting that no electrochemical reaction occurred on the GCE alone. An increased current intensity was observed in the CV curve ‘b’; however, no oxidation or reduction peak was observed, indicating that although the conductivity of the G-MoS2-Nafion/GCE was enhanced compared with the naked GCE, there was no electrochemical reaction occurring on the G-MoS2-Nafion/GCE. When different concentrations of UA were added into PBS, reduction peaks appeared between 0.28 and 0.32 V, and the reduction current intensity increased gradually as the concentration of UA increased in PBS. As there were no marked oxidation peaks in the CV curves of ‘c-g’, it suggested that electrochemical catalytic activity of UA occurring on the G-MoS2-Nafion/GCE could be monitored.

Figure 3. Evaluation of the catalytic activity of G-MoS2-Nafion/GCE. (A) CV behavior of G-MoS2-Nafion/GCE in different UA solution (scan rate, 0.05 V/sec). (B) The linear fitting of different UA solutions and the corresponding current values. CV, cyclic voltammetry; G-MoS2, graphene-molybdenum disulfide; GCE, glassy carbon electrode; UA, uric acid.

When the UA concentration was fitted with the corresponding current intensity, as demonstrated in Fig. 3B, the oxidation current intensity increased with the enhancement of the UA concentration, exhibiting a strong linear correlation (y=0.256×+29.63; R2=0.9931).

AA, DA and other components did not interfere with the electrochemical catalytic reaction between G-MoS2 and UA

Fig. 4A demonstrated that only electrochemical oxidation reactions of AA, DA and UA occurred at the G-MoS2-Nafion/GCE, and the corresponding potential values of their reduction peaks are −0.04, 0.16 and 0.29 V, which suggests that the reduction peaks of AA, DA and UA were distinct from each other. The reduction peak current intensities of DA and UA were markedly higher compared with AA. Nafion may enhance the adhesion of G-MoS2 composites on the GCE surface, extend the G-MoS2-Nafion/GCE service life and have a shielding effect towards the anion. Therefore, the Nafion may significantly reduce the electrochemical oxidation reaction of AA that occurs on the G-MoS2-Nafion/GCE (35).

Figure 4. Interference test of the electrochemical catalytic reaction between G-MoS2 and UA. (A) LSV electrochemical behavior of G-MoS2-Nafion/GCE in PBS, which contains glucose, NaOH, ascorbic acid, DA and UA (scan rate, 0.1 V/sec). (B) LSV electrochemical behavior of PBS and human serum (scan rate, 0.1 V/sec). LSV, linear sweep voltammetry; G-MoS2, graphene-molybdenum disulfide; GCE, glassy carbon electrode; DA, dopamine; UA, uric acid.

As shown in Fig. 4B, only the UA reduction peak (potential, 0.29 V) was observed, and no other marked reduction peaks were observed at the potential locations of AA (−0.04 V) or DA (0.16 V) compared with the reference LSV curve. This indicates that the G-MoS2-Nafion/GCE had a good selectivity for the determination of UA in human serum.

G-MoS2-Nafion/GCE has a good performance for the determination of UA

The calibration i-t curve of UA is demonstrated in Fig. 5A. With each addition of UA (50 µM) an obvious and uniform current change was observed on the i-t curve (Fig. 5B). The linear correlation between different concentrations of UA and their corresponding current values is demonstrated in Fig. 5C.

Figure 5. Calibration test for the detection of UA by G-MoS2-Nafion/GCE. (A) I-t analysis of different concentrations of UA. (B) The local amplification image of (A). (C) The linear correlation between different concentrations of UA and their corresponding current values (y=0.012×+0.645; R2=0.998). The minimum detectable limit was 13.91 µM (signal-to-noise ratio=3). UA, uric acid.

As indicated in Fig. 5 that the current values increased with an increase in the UA concentration, and there was a linear correlation between them (y=0.012×+0.645; R2=0.998). Furthermore, the minimum detectable value was 13.91 µM (signal-to-noise ratio: S/N=3), and the linear correlation between the concentration of UA and current values was between 25 and 3,000 mM. This suggested that G-MoS2-Nafion/GCE had a high sensitivity and wide linear range for the determination of UA.

Serum UA determined by uricase colorimetric assay and G-MoS2-Nafion/GCE have a high degree of correlation

The UA concentration of the mixed serum was detected by a uricase colorimetric assay and G-MoS2-Nafion/GCE (Table I). The correlation between the two methods is shown in Fig. 6, which demonstrated that there was a good linear correlation between them (y=0.9802×+11.494; R2=0.978).

Figure 6. Linear correlation of serum uric acid concentration, which was measured by uricase colorimetric assay and G-MoS2-Nafion/GCE, respectively (y=0.9802×+11.494; R2=0.978). G-MoS2, graphene-molybdenum disulfide; GCE, glassy carbon electrode.

Table I. Concentration values of serum uric acid detected by uricase colorimetric assay and G-MoS2-Nafion/GCE (n=11).

Table I.

Concentration values of serum uric acid detected by uricase colorimetric assay and G-MoS2-Nafion/GCE (n=11).

Serum sample	Uricase colorimetric assay, µM	G-MoS2-Nafion/GCE, µM
1	154.5	137.6
2	243.1	252.0
3	341.2	340.6
4	167.9	162.1
5	254.8	236.5
6	177.8	180.9
7	264.1	253.0
8	323.1	325.9
9	157.3	148.0
10	242.5	237.5
11	336.8	313.8

[i] G-MoS₂, graphene-molybdenum disulfide; GCE, glassy carbon electrode.

Detection of UA by G-MoS2-Nafion/GCE is accurate and precise

In order to verify the accuracy and precision of using the working electrode prepared in the present study to detect human serum UA, recovery and repeatability analysis was conducted, respectively. As shown in Table II, with an average recovery rate of 95.28%, G-MoS2-Nafion/GCE had a high accuracy for the detection of UA. Therefore, as demonstrated in Table III, no outlier value existed in the present study. Furthermore, the coefficient of variation was 2.04% (<4.5%), which suggested that the G-MoS2-Nafion/GCE used in the present study also had an acceptable precision for UA determination.

Table II. Recovery rate of serum uric acid measured by graphene-molybdenum disulfide-Nafion/glassy carbon electrode (n=11).

Table II.

Recovery rate of serum uric acid measured by graphene-molybdenum disulfide-Nafion/glassy carbon electrode (n=11).

Serum sample	Basic found, µM	Added, µM	Total found, µM	Recovery valuea, µM	Recovery rateb, %
1	137.6	200.0	324.2	186.6	93.3
2	252.0	200.0	448.0	196.0	98.0
3	340.6	200.0	536.6	196.0	98.0
4	162.1	200.0	345.5	183.4	91.7
5	236.5	200.0	427.1	190.6	95.3
6	180.9	200.0	371.1	190.2	95.1
7	253.0	200.0	443.0	190.0	95.0
8	325.9	200.0	518.9	193.0	96.5
9	148.0	200.0	334.8	186.8	93.4
10	237.5	200.0	428.5	191.0	95.5
11	313.8	200.0	506.4	192.6	96.3
Average value	–	–	–	190.56	95.28

a Recovery value = Total found - Basic found.

b Recovery rate = 100 × Recovery value/Added.

Table III. Precision analysis of UA detected by graphene-molybdenum disulfide-Nafion/glassy carbon electrode (n=11).

Table III.

Precision analysis of UA detected by graphene-molybdenum disulfide-Nafion/glassy carbon electrode (n=11).

Test no.	UA value, µM	Significance level
1	186.6	1.02
2	196.0	1.40
3	196.0	1.40
4	183.4	1.84
5	190.6	0.01
6	190.2	0.09
7	190.0	0.14
8	193.0	0.63
9	186.8	0.97
10	191.0	0.11
11	192.6	0.52
Average value	190.56	–
SD	3.89	–
CV (100%)	2.04	–

[i] Significance level = |UA value - Average value|/SD. CV, coefficient of variation; SD, standard deviation; UA, uric acid.

Discussion

At present, the primary methods of preparing GO include the improved Hummers, Brodie and Staudenmaier methods (36–38). The modified Hummers method, used in the present study, is one of the most important methods of preparing GO due to its advantages, including a simple and fast reaction process, and a safer operation compared with other methods. The GO synthesized using the improved Hummers method is rich in oxygen-containing functional groups that contribute to the complex structure of GO. This special structure is beneficial to allow graphene to combine closely with other materials.

The primary methods used for G-MoS2 synthesis include three types of methods: Hydrothermal synthesis; chemical vaporous deposition; and automatic liquid assembly (39–41). The hydrothermal synthesis method, which was used in the present study, is an effective method of synthesizing a more compact structure of G-MoS2. This method additionally has the advantages of mild reaction conditions, simple operation, less environmental pollution and low cost, and the morphology of the synthetic materials may be controlled easily. The morphology, size and structure of the materials may be controlled by altering the conditions of the reaction (42). Under conditions of high temperature and high pressure, due to the effect of electrostatic attraction, the molybdate is adsorbed onto the surface of GO and reacts with sulfur to form MoS2. Simultaneously, the GO is additionally reduced and transformed into graphene by removing its oxygen-containing groups gradually, and the graphene continues to react with MoS2 to form G-MoS2 composites (43,44). In this reaction process, GO acts as a template for the hydrothermal reaction. It provides a good foundation for improving the compactness, avoiding defects and preserving the advantages of MoS2 and graphite.

Based on the above principles, the improved Hummers and hydrothermal synthesis methods were selected in the present study to prepare an electrochemical biosensor, which was derived from the graphite and MoS2 composite, for the determination of UA in human serum. The present study suggested that the G-MoS2-Nafion/GCE had catalytic oxidation activity towards AA, DA and UA; however, the catalytic efficiencies for UA and DA were markedly improved compared with AA. Nafion may not enhance the adhesion of G-MoS2 composites, and may additionally restrain the oxidation or reduction reactions of negatively charged substances, including AA, at the G-MoS2-Nafion/GCE surface.

The present study demonstrated that the reduction peaks of AA, DA and UA may be well-separated, and only the UA reduction peak was detected by the G-MoS2-Nafion/GCE in the human serum samples. This suggested that the method had high specificity for the determination of UA in human serum. Furthermore, the UA concentration had a linear correlation with its corresponding current intensity, a wide linear range and a high sensitivity (the minimum detectable value was 13.91 µM; S/N=3) for the determination of UA. The values of human serum UA, measured using the G-MoS2-Nafion/GCE and an uricase colorimetric assay, had a positive correlation. The average recovery rate of UA detected with G-MoS2-Nafion/GCE was 95.28%, indicating that the electrochemical method used in the present study had a high accuracy for the detection of UA. Additionally, the repeatability tests demonstrated that the electrochemical method had a high precision for the detection of UA.

Although an electrode modifier for serum UA detection was successfully identified, there remain certain limitations to the present research. The feasibility of G-MoS2-Nafion/GCE for the detection of human serum UA was analyzed; however, the specific electrochemical catalytic reaction of UA at the G-MoS2-Nafion/GCE electrode was not discussed. Additionally, the different LSV curves of human serum samples and mixed PBS solutions, which contained AA, DA and UA, in the feasibility analysis may be further examined in future studies. The number of mixed serum samples tested in the comparative analysis of serum UA by uricase colorimetric assay and i-t electrochemical method was low (only 11 cases). Furthermore, the concentration range of serum UA was slightly narrow in the present study. The average recovery rate of UA detected using the G-MoS2-Nafion/GCE was <100%, suggesting that there was a systemic error in the method. The systemic error may be caused by the fact that the G-MoS2-Nafion in the present study was a laboratory-synthesized composite, thus the dampening effect on the electrochemical catalytic ability was unavoidable. In addition, the equivalent amount of UA was added in the recovery analysis, thus, the accuracy of the analysis of the G-MoS2-Nafion/GCE may not be verified in numerous directions. Finally, there was only one concentration of UA standard solution selected in the precision analysis, which does not cover all of the low, medium and high concentrations. Additionally, the number of repeated measurements was <20, thus the precision of the G-MoS2-Nafion/GCE may not be fully verified at present. Therefore, improvements to the experiments to evaluate the performance of the G-MoS2-Nafion/GCE for the detection of human serum UA is required in future studies.

Nafion, which was contained in the G-MoS2-Nafion composites, may inhibit the electrochemical reduction reaction of AA. Therefore, it may be speculated that if another material was identified that had the ability to enhance the adhesion of G-MoS2, and to not affect the catalytic efficiency of G-MoS2 composites towards AA, DA and UA, the simultaneous detection of AA, DA and UA may be possible in the future.

In conclusion, the present study indicated that G-MoS2-Nafion composites may be used as an electrochemical catalytic material for the detection of UA in human serum. Compared with other methods for the determination of UA (including enzymology, points spectrophotometry, high performance liquid phase chromatography and electrochemical chemiluminescence analysis), it has the characteristics of accuracy, high sensitivity, a wide linear range, fast detection, strong anti-interference properties, low cost, a simple preparation process, reusability and being environmentally friendly. Furthermore, human serum samples may be measured immediately without any other treatment following centrifugation. Therefore, the G-MoS2-Nafion composite or its improved analogs as electrochemical catalysts for the determination of human serum UA has good clinical application prospects.

Acknowledgements

The authors thank Professor Jing Jiang from China West Normal University (Nanchong, China) for his assistance with the data analysis.

Funding

The present study was supported by grants from The National Natural Science Foundation of China (grant no. 81641096), the Department of Science and Technology of Sichuan Province (grant no. 2016JY0171) and International S&T Cooperation Program of China (grant no. 2015DFA30420).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

BY and DW designed the research and drafted the manuscript. XL and QD acquired, analyzed and interpreted the data, and performed statistical analysis. QW and QL prepared the figures. XJ and JZ diagnosed the patients and provided treatment information of hyperuricemia, and also helped improve the experimental design. XG and YX performed the element verification of G-MoS2 composites and revised the manuscript for important intellectual content. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of the Affiliated Hospital of North Sichuan Medical College (Nanchong, China). Written informed consent was obtained from all patients.

Patient consent for publication

All patients involved in the present study provided consent for publication.

Competing interests

The authors declare that they have no competing interests.

References