Synthesis of Tumbleweed-like MoSe₂ Nanostructures for Ultrasensitive Electrochemical Detection of Uric Acid

Abstract

Uric acid (UA), the final metabolic product of purines, plays a crucial role in human health monitoring. The UA concentration in biological fluids serves as a diagnostic marker for various disorders, particularly kidney diseases, and represents a potential therapeutic target. Given the growing emphasis on preventive healthcare, developing methods for real-time UA detection has become increasingly significant. Here, we demonstrate the synthesis of novel tumbleweed-like molybdenum diselenide (MoSe₂) nanostructures through a single-step hydrothermal process. The synthesized MoSe₂ was subsequently hybridized with reduced graphene oxide (rGO) to construct electrodes for UA sensing. Differential pulse voltammetry (DPV) measurements revealed that the MoSe₂/rGO-modified glassy carbon electrode (GCE) exhibited excellent UA detection capabilities under optimized conditions. The sensor demonstrated a remarkably low limit of detection (LOD) of 28.4 nM and maintained linearity across a wide concentration range (40 nM to 200 μM). Notably, the sensor showed high selectivity for UA detection even in the presence of common interfering species, including citric acid (CA), dopamine (DA), ascorbic acid (AA), cysteine (Cys), glucose (Glu), oxalic acid (OA), sodium ions (Na⁺), and potassium ions (K⁺). The developed sensor displayed outstanding selectivity, stability, and reproducibility characteristics. This synthetic approach offers promising opportunities for developing MoSe₂-based electrochemical sensing platforms suitable for diverse bioanalytical applications.

1. Introduction

UA, a crucial end product of purine metabolism in humans, requires careful monitoring of its concentrations in serum and urine for effective health management [1]. Contemporary improvements in dietary standards have significantly influenced UA metabolism, leading to an increased prevalence of UA-related disorders [2,3]. In healthy individuals, the normal range of UA concentration in serum is typically 200–420 μM for males and 140–360 μM for females. In human urine, the normal range of UA excretion is generally 1.5–4.5 mmol/24 h (≈0.7–4.4 mM) [4,5]. Insufficient UA levels correlate with elevated risks of various conditions, including diabetes mellitus and multiple sclerosis [6,7]. Conversely, elevated UA concentrations contribute to the development of multiple pathological conditions, including nephrolithiasis, gouty arthritis, obesity, and various cardiovascular and neurological disorders [8,9]. Although numerous analytical techniques have been established for UA detection—including ion chromatography [10], fluorescence spectroscopy [11], spectrophotometry [12], capillary electrophoresis [13], colorimetry [14], and high-performance liquid chromatography [15]—these conventional methods present significant limitations. Their time-intensive nature, requirement for extensive sample preparation, and high operational costs render them impractical for rapid, point-of-care UA monitoring [16,17]. In contrast, electrochemical detection methods have emerged as superior alternatives [18], offering the advantages of cost-effectiveness, operational simplicity [19], enhanced sensitivity [20], and excellent selectivity in UA quantification [21,22].

Graphene and its derivatives have emerged as prominent materials for electrochemical UA detection sensors, owing to their rapid electron transport, high surface area, excellent biocompatibility, and superior mechanical properties [23,24,25,26]. Despite these advantages, electrochemical electrodes fabricated from unmodified carbon-based materials exhibit insufficient sensitivity for practical UA detection applications [27,28]. To enhance performance, researchers have focused on modifying graphene and its derivatives to optimize charge transfer during UA electrocatalytic oxidation, resulting in composite materials with improved catalytic capabilities [29,30]. Notable examples include the work of Aparna et al. [31], who developed Au-Cu₂O/rGO nanocomposites through a one-pot synthesis method, achieving UA detection with a linear range of 100–900 μM and a LOD of 6.5 μM. Similarly, Darabi et al. [32] employed microwave-assisted synthesis to create rGO/polypyrrole–Pt nanoparticle composites, yielding an electrochemical UA sensor with a linear range of 100–350 μM and an impressive LOD of 0.16 μM. While these composite materials demonstrate enhanced catalytic activity for UA oxidation, their reliance on expensive noble metal nanomaterials (Au and Pt) and complex synthesis protocols increases production costs and hinders widespread adoption. Consequently, there remains a critical need for simple, stable, and cost-effective composite materials for highly sensitive UA electrochemical biosensors [33]. Transition metal dichalcogenides (TMDs) have recently emerged as promising alternatives to graphene-based materials for electrochemical sensors, offering remarkable physical, chemical, catalytic, optical, and electronic properties while maintaining cost-effectiveness and straightforward synthesis procedures [34]. TMDs facilitate efficient electron transfer through their large surface area and abundant active sites, while surface defects serve as additional binding sites for small biomolecules, thereby enhancing sensor conductivity and sensitivity [35]. Recent investigations into TMD-based composite materials for UA electrochemical sensing have yielded promising results [36]. For instance, Cogal et al. [37] synthesized two-dimensional WSe₂ nanosheet–carbon black composites via hydrothermal methods, developing a WSe₂@C electrode that demonstrated high sensitivity, selectivity, reproducibility, and stability, with a linear range of 1–185.2 μM and an LOD of 0.42 μM. Similarly, Sha et al. [38] combined electrospinning and hydrothermal synthesis to fabricate MoS₂ nanosheet/nanocarbon fiber composites, producing a UA electrochemical sensor with a linear range of 1–60 μM and an LOD of 0.91 μM, exhibiting excellent reproducibility and stability. These results underscore the exceptional potential of TMD-based composite materials for electrochemical UA detection.

In this study, we developed an efficient hydrothermal method for synthesizing tumbleweed-like MoSe₂ nanostructures, offering the advantages of simplicity, cost-effectiveness, and high yield. The composite electrode, fabricated using MoSe₂/rGO, with its distinctive roll-like morphology, functioned as an electrochemical UA sensor, exhibiting a broad linear detection range from 40 nM to 200 μM and achieving a remarkably low LOD of 28.4 nM under optimized conditions. The electrode demonstrated superior analytical performance in terms of repeatability, anti-interference capability, and long-term stability. Notably, this work represents the first reported application of MoSe₂ in high-sensitivity bioelectronic sensing for nanomolar-level UA detection.

2. Materials and Methods

2.1. Reagents

Phosphate buffer solution (PBS, containing 2.67 mM KCl, 136.89 mM NaCl, 1.76 mM KH₂PO₄, and 8.10 mM Na₂HPO₄) was purchased from Sangon Biotech (Shanghai, China) Co., Ltd. (Shanghai, China). Ammonium molybdate tetrahydrate ((NH₄)₆Mo₇O₂₄·4H₂O), selenium powder (Se), sulfuric acid (H₂SO₄), potassium hexacyanoferrate(Ⅱ) (K₄[Fe(CN)₆]), potassium ferrocyanide (K₃[Fe(CN)₆]), dopamine (C₈H₁₁NO₂), citric acid (C₆H₈O₇), cystine (C₆H₁₂N₂O₄S₂), sodium chloride (NaCl), potassium chloride (KCl), D(+)-glucose anhydrous (C₆H₁₂O₆), ascorbic acid (C₆H₈O₆), dopamine hydrochloride (C₈H₁₁NO₂·HCl), anhydrous oxalic acid (C₂H₂O₄), and UA were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). rGO was obtained from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). All chemicals used in the experiments were of analytical grade and were used without further purification. Deionized water (Milli-Q) was used throughout all experiments.

2.2. Synthesis of Tumbleweed-like MoSe₂/rGO and Electrode Fabrication

First, 0.5 g of (NH₄)₆Mo₇O₂₄·4H₂O and 0.32 g of Se were added to 40 mL of deionized water, and the solution was stirred with a magnetic stirrer for 20 min to achieve homogeneous dispersion. Subsequently, 1.12 g of OA was introduced into the solution and stirred for an additional 15 min. The homogeneous mixture was placed into a 50 mL Teflon-lined stainless-steel autoclave and underwent hydrothermal treatment at 200 °C for 24 h in a muffle furnace. Upon natural cooling to ambient temperature, the reaction product underwent three successive washing cycles with deionized water and ethanol. The resultant black precipitate was isolated via centrifugation and vacuum-dried at 60 °C for 4 h to obtain MoSe₂ powder.

The MoSe₂/rGO composite was synthesized by dispersing MoSe₂ and rGO powders in deionized water at a mass ratio of 3:1, followed by 20 min of sonication to achieve a homogeneous dispersion. The GCE (3 mm diameter) was prepared through sequential surface treatments, beginning with mechanical polishing using 50 nm alumina slurry. Successively, the electrode underwent ultrasonic cleaning in deionized water and ethanol to remove residual particles. Electrochemical activation of the GCE was accomplished through cyclic voltammetry (CV) in 0.5 M H₂SO₄ solution, applying potential sweeps between −1.0 and 1.0 V at 100 mV/s. The modified electrode was fabricated by drop-casting 7 μL of the MoSe₂/rGO aqueous dispersion (1 mg/mL) onto the activated GCE surface, followed by thermal drying at 50 °C for 10 min.

2.3. Electrochemical Sensing of UA

For electrochemical measurements, the electrolyte was prepared using PBS. A three-electrode system was employed, comprising MoSe₂/rGO as the working electrode, platinum (Pt) as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. The electrochemical characterization included DPV, CV, and electrochemical impedance spectroscopy (EIS) to evaluate the voltametric responses of the modified electrodes. DPV analysis was conducted from −0.1 to 0.5 V using optimized parameters (pulse period: 0.5 s; step potential: 0.004 V; amplitude: 0.005 V). CV measurements were performed over five cycles within a potential window of −0.2 to 0.6 V at 0.1 V/s. EIS measurements were conducted at the open circuit potential (OCP), with the equilibrated OCP being 0.193 V. The AC amplitude was set to 10 mV, and the frequency range was 0.1 Hz to 100 kHz. All electrochemical analyses were performed using a CHI660e electrochemical workstation (CH Instruments, Shanghai, China).

2.4. Characterization

The morphology of the modified materials was observed using a transmission electron microscope (TEM, JEM–2100F, JEOL, Tokyo, Japan), energy-dispersive spectroscopy (EDS), and a field-emission scanning electron microscope (FE–SEM, QUANTA 250 FEG, FEI, Hillsboro, OR, USA). Raman spectroscopy (Renishaw inVia Reflex, Renishaw plc, Wotton–under–Edge, London, UK) with a 532 nm laser wavelength was used to analyze the synthesized materials. X-ray diffraction (XRD, D8 Advance, Bruker, Karlsruhe, Germany) with Cu Kα radiation (λ = 1.54 Å) was employed for the elemental and crystallographic analysis of the materials. X-ray photoelectron spectroscopy (XPS, Axis SUPRA+, Shimadzu, Japan) was used to characterize the surface composition and chemical states of the materials.

3. Results and Discussion

3.1. Morphological Characterization of Tumbleweed-like MoSe₂

As shown in Figure 1a, the MoSe₂ exhibited a clustered, tumbleweed-like structure with an average spherical diameter of approximately 300 nm and a nanoflake-rich composition. EDS analysis (Figure 1b,c) demonstrated a uniform distribution of Mo and Se elements throughout the sample. Additionally, Figure 1b and c show that both Mo and Se are evenly distributed across the material. The TEM and HRTEM images (Figure 1d,e) reveal the layered structure of MoSe₂, with an interlayer spacing of approximately 0.649 nm corresponding to the (002) plane. Selected area electron diffraction (SAED) (Figure 1f) indicated that the MoSe₂ material is polycrystalline. These comprehensive morphological and structural characterizations validate the successful synthesis of tumbleweed-like MoSe₂, featuring a well-defined, nanoflake-rich structure suitable for electrochemical applications.

3.2. Chemical Composition and Crystal Structure Analysis of Tumbleweed-like MoSe₂

Raman spectroscopy analysis revealed the characteristic chemical structure of the synthesized MoSe₂, as illustrated in Figure 2a. The peaks at 263.0 cm⁻¹ and 292.2 cm⁻¹ correspond to the out-of-plane (A_1g) and in-plane (E1 2g) vibrational modes of the Se–Mo–Se bond, respectively [39]. The XRD pattern in Figure 2b shows that all the observed diffraction peaks are attributable to MoSe₂ (JCPDS# 29–0914) [40]. These comprehensive characterization results provide compelling evidence for the successful formation of hexagonal MoSe₂ crystals.

XPS analysis was employed to analyze the chemical binding energies and elemental composition of the sample. The XPS spectra of MoSe₂ are presented in Figure 3a–c. The survey spectrum (Figure 3a) confirmed the presence of Mo and Se as the primary constituent elements in MoSe₂. Figure 3b displays the Mo 3d core-level spectrum, with two characteristic peaks at 228.66 eV and 231.77 eV, which are attributed to the Mo 3d_5/2 and Mo 3d_3/2 peaks of Mo⁴⁺ in MoSe₂, respectively. The Se 3d spectrum in Figure 3c shows two characteristic peaks at approximately 54.22 eV and 55.06 eV, corresponding to Se 3d_5/2 and Se 3d_3/2, respectively [41].

3.3. Sensitivity Optimization of MoSe₂/rGO Electrodes for UA Detection

To evaluate the electrochemical performance of various electrode modification materials for UA detection, DPV measurements were performed using bare GCE, rGO, MoSe₂, and MoSe₂/rGO electrodes in PBS electrolyte containing 10 µM UA across a potential window of −0.1 to 0.5 V (Figure 4a). The DPV curves exhibited a characteristic UA oxidation peak at 0.271 V, which served as the analytical signal for UA detection. In comparison to the bare GCE, the MoSe₂/rGO, rGO, and MoSe₂ electrodes demonstrated enhanced current densities of 300.0%, 158.3%, and 32.4%, respectively. The superior electrochemical performance of the MoSe₂/rGO electrode suggested a synergistic interaction between the MoSe₂ and rGO components. This enhancement is attributed to the incorporation of rGO, which provided additional electrochemically active sites and established efficient electron transfer pathways throughout the MoSe₂ nanostructures. The results confirm that the MoSe₂/rGO electrode exhibited remarkable electrochemical sensing capabilities for UA detection.

To optimize the detection performance of the MoSe₂/rGO electrode for UA, several experimental parameters were systematically investigated, including the MoSe₂/rGO loading mass, electrolyte pH, and scan rate. The optimal MoSe₂/rGO loading mass was determined by adjusting the volume of the dispersion drop-cast onto the GCE surface to achieve the deposition of different amounts (1, 3, 5, 7, and 9 µg) of MoSe₂/rGO on the bare GCE, and DPV measurements were taken in PBS electrolyte containing 10 µM of UA. Based on the results shown in Figure 4b, when the loading exceeds 7 μg, the current response shows a downward trend, which may be due to the effect of charge transfer blockage by material stacking. Optimizing the loading amount is critical to balancing active site exposure and mass transport efficiency. Therefore, 7 µg of MoSe₂/rGO loading was selected for subsequent electrochemical studies. Since electrolyte pH significantly influences the electrochemical behavior of UA at the electrode interface, its effect on UA electro-oxidation at the MoSe₂/rGO electrode was examined across pH values ranging from 5.0 to 9.0 (Figure 4c). The electrochemical response reached its maximum at pH 7.0, which was subsequently selected as the optimal condition for UA detection using the MoSe₂/rGO electrode.

To assess the electrochemical behavior of the MoSe₂/rGO electrode surface, CV measurements were performed at scan rates ranging between 20 and 200 mV s⁻¹ in 10 mM of [Fe(CN)₆]^3−/4− electrolyte containing 0.1 M KCl (Figure 4d). The peak current densities for I_ox and I_red showed a linear relationship with the square root of the scan rate (Figure 4e), indicating that the redox reactions occurring on the MoSe₂/rGO electrode are diffusion-controlled [42]. The electroactive surface area (ESA) of various electrodes was calculated using the Randles–Sevcik equation [43,44]:

$$I_{red}=(2.69\times {10}^5)·n^{1.5}·A{·D}^{0.5}·{\nu }^{0.5}·c$$

(1)

In this equation, I_red represents the reduction peak current, n denotes the number of transferred electrons involved the reduction process, A is the ESA of the working electrode, D is the diffusion coefficient of ferricyanide (7.6 × 10⁻⁷ cm² s⁻¹), ν refers to the scan rate, and c is the concentration of ferricyanide in the electrolyte (1 × 10⁻⁵ mol cm⁻³). The results indicated that the ESA of the MoSe₂/rGO electrode was 0.092 cm², while the ESA of the bare GCE was approximated to be its geometric area (0.071 cm²). These findings suggest that the MoSe₂/rGO-modified electrode remarkably enhanced the electroactive surface area of the GCE, attributable to the high conductivity and specific surface area of MoSe₂/rGO.

The electron transfer capability of the MoSe₂/rGO electrode was investigated to assess its electrochemical performance for UA detection. The interfacial charge transfer capabilities of the GCE, rGO, MoSe₂, and MoSe₂/rGO electrodes were evaluated using EIS in 10 mM of [Fe(CN)₆]^3−/4− electrolyte solution (Figure 4f). The obtained Nyquist plots for the MoSe₂ electrode were analyzed using an equivalent circuit [45]:

$$R_s\left(Q_{dl}\right(R_{ct}·Z_w\left)\right)$$

(2)

where $R_s$ is the series resistance. Notably, $R_{ct}$ represents the charge transfer resistance, a critical indicator of the sensor’s charge transfer capability [46]. The $R_{ct}$ values for the GCE, rGO, MoSe₂, and MoSe₂/rGO electrodes were 2514.0, 722.6, 350.7, and 194.1 Ω, respectively. The lower $R_{ct}$ value of the MoSe₂/rGO electrode compared to the other electrodes indicates improved charge transfer capability.

3.4. Detection Performance and Practical Applications of MoSe₂/rGO Sensors

The electrochemical analysis of UA was performed quantitatively using the MoSe₂/rGO electrode through DPV measurements, as demonstrated in Figure 5a. The peak current density exhibited a positive correlation with UA concentration across a range spanning from 40 nM to 200 µM. Figure 5b presents the calibration curve constructed from the averaged peak current responses against UA concentrations. Within the concentration range of 40 nM to 200 µM, the linear regression equation for UA was as follows: $I_{pcd}\left(\mathrm{m}\mathrm{A}{\mathrm{c}\mathrm{m}}^{-2}\right)=0.0012·\mathrm{U}\mathrm{A}+0.253(\mathrm{R}²=0.999)$. Based on the regression results, the LOD was determined to be 28.4 nM (S/N = 3). As presented in

Table 1. Comparison of LOD and linear range of MoSe₂/rGO electrodes with reported TMD nanomodifiers for UA detection.

Modified Electrodes	Measurements	Linear Range (μM)	LOD (nM)	Ref.
WS2	DPV	5.0–1000	1200	[47]
MoS2	DPV	10–400	1169	[38]
FeS2	DPV	10–725	1000	[48]
MoS2@polyaniline@rGO	DPV	1.5–500	840	[27]
WSe2@C	DPV	1.0–185.2	420	[37]
MXene@MoS2/carbon fiber paper (CFP)	DPV	0.50–1000	380	[24]
MnFe2O4@MoS2	DPV	5.0–80	140	[49]
CdTe quantum dot	DPV	1–400	100	[50]
Polypyrrole/α–Fe2O3/MoS2	SWV	0.30–1000	61	[51]
MoSe2/rGO	DPV	0.040–200	28.4	This work

, compared to the other UA sensors based on TMDs, our MoSe₂/rGO sensor demonstrates a very low limit of detection (LOD) and is simple to prepare.

Additionally, to assess the interference resistance of the MoSe₂/rGO sensor, 10 µM of UA was detected in the company of common urine interfering substances, including 1 mM DA, 1 mM AA, 1 mM OA, 1 mM CA, 1 mM Cys, 1 mM Glu, 5 mM Na⁺, and 5 mM K⁺. The i–t technique was applied to the MoSe₂/rGO electrode, as shown in Figure 5c. The MoSe₂/rGO electrode demonstrated remarkable anti-interference capability against various molecular species during electrochemical detection. Reproducibility tests conducted on ten independent samples under identical conditions revealed a minimal relative standard deviation of 1.61% for current density (Figure 5d), confirming the exceptional reliability of the MoSe₂/rGO sensor. The sensor’s reusability was systematically evaluated through consecutive daily measurements, yielding consistently satisfactory performance. The electrode retained 89.4% of its initial current density after eight consecutive days of testing (Figure 5e), while long-term stability assessments revealed the retention of 85.3% of the initial current density following one month of storage (Figure 5f). These comprehensive performance metrics substantiate the practical viability of our sensor for UA detection applications.

4. Conclusions

In this study, tumbleweed-structured MoSe₂ was synthesized via a hydrothermal method and subsequently integrated with rGO to construct a UA sensing platform. The fabricated MoSe₂/rGO electrode demonstrated superior sensitivity toward UA oxidation compared to pristine MoSe₂, rGO, and bare GCE electrodes. The sensor exhibited outstanding analytical performance for UA detection across a broad linear range from 40 nM to 200 μM, achieving a detection limit of 28.4 μM. Comprehensive validation studies confirmed that the MoSe₂/rGO electrode possessed outstanding anti-interference capabilities, excellent reusability, and long-term stability. These findings establish a promising foundation for developing high-performance electrochemical biosensors based on MoSe₂/rGO composite materials.