Glucose Sensor Using Fe₃O₄ Functionalized MXene Nanosheets as a Promising Sensing Platform: Exploring the Potential of Electrochemical Detection of Glucose

Abstract

Enzymatic electrochemical sensors are promising for real-time glucose monitoring due to their high sensitivity and continuous detection capability. In this work, a magnetic Fe₃O₄@MXene nanocomposite was synthesized hydrothermally. The introduction of Fe₃O₄ not only reduced MXene’s inherent negative surface charge, improving interaction with glucose oxidase (GOD), but also formed a porous structure that enhances enzyme immobilization via physical adsorption. Based on these properties, a Fe₃O₄@MXene/GOD/Nafion/GCE electrode was fabricated. The composite’s high specific surface area, excellent conductivity, and good biocompatibility significantly promoted the direct electron transfer (DET) of GOD. Meanwhile, the apparent electron transfer rate constant (k_s) was calculated to be 9.57 s⁻¹, representing a 1.26-fold enhancement over the MXene-based electrode (7.57 s⁻¹) and confirming faster electron transfer kinetics. The sensor showed a bilinear glucose response in the ranges of 0.05–15 mM, with sensitivity of 120.47 μA·mM⁻¹·cm⁻² and a detection limit of 38 μM. It also exhibited excellent selectivity, reproducibility and stability. Satisfactory recovery rates were achieved in artificial serum samples while demonstrating comparable detection performance to commercial blood glucose meters.

1. Introduction

Diabetes mellitus, a chronic metabolic disorder diagnosed through the measurement of blood glucose concentration in human blood, leads to systemic health deterioration as prolonged hyperglycemia triggers complications, including cardiovascular diseases, nephropathy, and retinopathy [1,2,3]. Therefore, rational monitoring of blood glucose within individualized target ranges is essential to optimize overall health outcomes in people with diabetes. Electrochemical glucose detection methods have attracted significant attention due to their advantages of simplicity, low cost, and high sensitivity, enabling precise and rapid determination of blood glucose levels in clinical samples [4,5]. Third-generation enzymatic electrochemical sensors achieve direct electron transfer (DET) through minimized pathways without requiring electron mediators, rendering them ideal sensing platforms [6,7]. However, the active sites of enzymes are encapsulated within the protein structure, leading to challenges in achieving DET between the enzymes and the electrode interface [8,9,10]. Consequently, identifying suitable electrode materials to enhance the DET between enzymes and electrode surfaces remains a critical challenge in constructing these sensors.

MXenes represent a family of two-dimensional nanomaterials composed of transition metal nitrides, carbides, and carbonitrides [11]. As an emerging class of nanostructured materials, they have attracted significant research interest in fields such as energy storage [12], capacitors [13], and electrochemical sensing [14] due to their large specific surface area and remarkable mechanical stability [15,16]. It is noteworthy that the surface of MXene exhibits a strong negative charge, resulting in significant electrostatic repulsion toward similarly charged enzymes, such as glucose oxidase (GOD). Consequently, this repulsion impedes the effective immobilization of enzymes when MXene is employed as a supporting substrate. Recent studies have indicated that MXene demonstrates exceptional potential as a composite substrate, capable of integrating with diverse nanomaterials to amplify their inherent advantages and realize synergistic interactions within hybrid systems [17]. This demonstrates that effective surface modifications of MXene can modulate its intrinsic surface charge and generate architectures that facilitate enzyme immobilization. These combined characteristics not only establish an enzyme-friendly microenvironment that preserves biocatalytic activity but also significantly enhance interfacial electron transfer efficiency at electrode surfaces.

Fe₃O₄, as a representative member of magnetic nanomaterials, has emerged as a promising candidate in electrochemical catalysis and sensing applications due to its unique combination of magnetic stability, environmental benignity, biocompatibility and exceptional electrical conductivity [18,19,20]. These distinctive physicochemical properties have driven extensive research efforts to exploit Fe₃O₄’s potential in developing advanced sensors and electrocatalytic systems. Research has demonstrated that Fe₃O₄ nanoparticles exhibit favorable electrical conductivity, enabling highly efficient electron conduction pathways in DET systems for redox enzymes and significantly enhancing electron transfer between enzymes and electrode surfaces. Their good biocompatibility also contributes to the stabilization of immobilized enzymes [21]. In recent years, nanostructured Fe₃O₄-based composites, especially those incorporated into two-dimensional nanomaterials, have been developed. These systems retain the intrinsic advantages of Fe₃O₄ while substantially enhancing enzyme loading capacity, thus leading to improved overall performance in sensing applications [22]. Therefore, if Fe₃O₄ nanoparticles are integrated with MXene, the resulting magnetic nanocomposites can synergistically combine the high specific surface area of MXene with the excellent biocompatibility and electrical conductivity inherent to Fe₃O₄ nanoparticles. When coupled with GOD, this composite is anticipated to facilitate enhanced electron transfer efficiency, thereby achieving highly sensitive glucose detection. However, there are no reports yet on direct electron transfer of GOD from Fe₃O₄@MXene nanocomposites.

In this study, we successfully synthesized magnetic MXene (Fe₃O₄@MXene) nanocomposites for the first time through a one-pot hydrothermal synthetic strategy. The Fe₃O₄@MXene/GOD/Nafion/GCE sensing platform was then constructed via electrostatic assembly. Electrochemical research demonstrated that the Fe₃O₄@MXene/GOD/Nafion/GCE exhibited superior sensitivity, lower detection limit, and broader linear range compared to the MXene/GOD/Nafion/GCE. It was successfully applied to the detection of glucose in artificial serum samples.

2. Materials and Methods

2.1. Materials

Glucose oxidase (derived from Aspergillus niger, 50 KU), Ti₃AlC₂ powder (purity ≥ 99%, 300 mesh), lithium fluoride (LiF), hydrochloric acid (HCl, 12 mol/L) and bovine serum albumin (BSA, ≥96%) were obtained from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Dimethyl sulphoxide (DMSO) was purchased from Tianjin Damao Chemical Reagent Factory (Tianjin, China). D-(+)-glucose, Ferric chloride hexahydrate (FeCl₃∙6H₂O), ascorbic acid (AA), uric acid (UA), dopamine (DA), L-cysteine (L-Cys), ammonium hydroxide, and anhydrous ethanol (C₂H₆O) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Deionized double-distilled water was used for preparing all the solutions (18 MΩ·cm⁻¹), and the other chemicals were of analytical reagent grade and were used without further purification.

2.2. Apparatus

The specific morphology of the material was determined using a scanning electron microscope (SEM, Hitachi S-4800, Hitachi High-Tech Corporation, Tokyo, Japan). Morphological features and particle dimensions were characterized by transmission electron microscopy (TEM, JEM-2100, JEOL Ltd., Akishima, Japan). The crystal structure was characterized using an X-ray diffractometer (XRD, Ultima IV, Rigaku Corporation, Beijing, China). The infrared absorption peaks of the material were collected using a Fourier Transform Infrared (FTIR) spectrometer (TENSOR II, Bruker, Bremen, Germany) in transmission mode. X-ray photoelectron spectroscopy (XPS) analysis was performed on an ESCALAB 250 Xi system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a monochromatic Al Kα X-ray source (1486.6 eV, 150 W) and a 500 μm spot size. Zeta potential measurements were conducted using a Malvern Zetasizer (Nano ZS90, Malvern Panalytical, Shanghai, China) at 25 °C. Glucose concentration in artificial serum was measured using a Sinocare blood glucose meter (GA-3, Tianjin Gangyuan Test Instrument Factory, Tianjin, China).

Electrochemical characterization was carried out in a conventional three-electrode configuration using a CHI620B Potentiostat (Shanghai Chenhua Instruments, Shanghai, China).

Glassy carbon electrodes (modified with different MXene nanomaterials), a Pt wire, and an Ag/AgCl (saturated KCl) electrode were used as the working electrode, the counter electrode, and the reference electrode, respectively. Measurements were performed in 0.1 M phosphate-buffered saline (PBS, pH 7.4) under N₂-saturated and air-saturated solution conditions for electrochemical and electrocatalytic evaluations, respectively. Electrochemical impedance spectroscopy (EIS) was conducted using an Autolab PGSTAT302N workstation (Metrohm AG, Beijing, China) with 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] in 0.5 M KCl electrolyte, with scanning frequencies from 10 mHz to 100 kHz with a 5 mV AC signal amplitude.

2.3. Preparation of 2D Layered MXene (Ti₃C₂)

Two-dimensional layered MXene (Ti₃C₂) was synthesized by etching the Al layer from Ti₃AlC₂ MAX phase using an in situ-formed HF solution [23]. Specifically, 1.6 g of LiF was gradually added to 20 mL of 9 M HCl with stirring for 15 min. Then, 1 g of Ti₃AlC₂ powder was slowly introduced and stirred at 35 °C for 24 h. The mixture was diluted with water and centrifuged; the sediment was washed repeatedly until a neutral pH was achieved. The resulting black paste was freeze-dried to obtain multilayer Ti₃C₂ nanosheets. Delamination was carried out via DMSO intercalation: 0.05 g of Ti₃C₂ was dispersed in 10 mL of DMSO and stirred at 35 °C for 24 h. After centrifugation, the sample was redispersed in 50 mL of water and ultrasonicated to yield a 1 mg/mL MXene dispersion.

2.4. Preparation of Fe₃O₄@MXene Nanocomposites

To prepare the Fe₃O₄@MXene nanocomposite, 600 mg of D-glucose and 400 mg of FeCl₃·6H₂O were added to 50 mL of MXene solution (1 mg/mL) under stirring. The mixture was stirred uniformly for 10 min to form a black suspension. Then, 15 mL of ammonia solution was introduced, and stirring was continued for another 15 min under the same conditions. The resulting suspension was transferred into a Teflon-lined autoclave and heated at 180 °C for 48 h. After cooling to room temperature, the black precipitate was collected using a magnet and thoroughly washed with deionized water and ethanol. Finally, it was dried at 60 °C to obtain the Fe₃O₄@MXene nanocomposite and stored in a refrigerator at 4 °C.

2.5. Preparation Fe₃O₄@MXene/GOD/Nafion/GCE

The GCE was sequentially polished using 0.3 μm and 0.05 μm alumina slurry, followed by thorough rinsing and sonication in ethanol and deionized water, respectively. After cleaning, the electrode was dried under a stream of purified nitrogen. The enzyme electrode was fabricated by a simple casting method. Specifically, 1 mL of the Fe₃O₄@MXene suspension (2.5 mg·mL⁻¹) was mixed with 1 mL of GOD (45 mg·mL⁻¹) in the phosphate-buffered saline (PBS). Then, 1 mL of Nafion (1% w/v) was added to the mixture. A volume of 7 μL of the resulting suspension was cast onto the freshly polished GCE surface to obtain the Fe₃O₄@MXene/GOD/Nafion/GCE, which was subsequently stored at 4 °C before use.

For comparison, MXene/GOD/Nafion/GCE, and Fe₃O₄@MXene/Nafion/GCE were prepared following a similar modification procedure. All modified electrodes were thoroughly rinsed with deionized water and stored at 4 °C when not in use.

3. Results and Discussions

3.1. Characterization of Fe₃O₄@MXene Nanocomposites

As for the fabrication of the Fe₃O₄@MXene modified electrode, single-layer MXene was first synthesized through HF etching and DMSO intercalation. Fe₃O₄ nanoparticles were then in situ grown onto the MXene nanosheets via a hydrothermal method. The low isoelectric point of GOD causes it to carry a negative charge at pH 7.4 [24]. Similarly, unmodified MXene nanosheets possess a strongly negative surface charge. Due to the similar charge characteristics of GOD and MXene, the presence of electrostatic repulsion hinders the effective immobilization of GOD on MXene. To address this issue, we introduced Fe₃O₄ nanoparticles to modify the MXene surface. This composite strategy modulates the surface charge characteristics of MXene, reducing its strong innate negative charge and thus improving interactions with GOD. Moreover, the dense packing of Fe₃O₄ nanoparticles on MXene forms a porous architecture, enabling effective immobilization of enzyme molecules through physical adsorption. The Fe₃O₄@MXene nanocomposite exhibits a high specific surface area and excellent biocompatibility, providing a stable platform for GOD immobilization. This facilitates direct electron transfer between the enzyme and electrode, thereby enabling highly specific electrochemical detection of glucose.

Figure 1 shows the morphological images of monolayer MXene (A–C), Fe₃O₄ (D), Fe₃O₄@MXene composites (E and F), and those obtained from TEM and SEM. The intercalated MXene exhibits a monolithic and highly dispersed morphology with lateral dimensions ranging from approximately 38 to 196 nm (Figure 1A,B). Additionally, Figure 1C, as revealed by SEM, shows that the surface of single-layer MXene is smooth, with numerous wrinkles. This structural configuration endows MXene with an enlarged specific surface area, which is advantageous for enhancing interfacial interactions. With a spherical morphology and diameters of approximately 18 to 29 nm, the Fe₃O₄ nanoparticles are densely anchored onto the MXene surface (Figure 1D–F). These results indicate that the hydrothermal treatment effectively facilitates the in situ growth of Fe₃O₄ on the MXene surface. Thus, the negative surface charge state of MXene is altered. The resulting dense architecture forms a porous structure throughout the composite, with pore sizes ranging from approximately 4.16 to 59.20 nm. This porous network offers abundant physical adsorption sites for GOD immobilization.

Figure 2 presents the Energy-Dispersive Spectroscopy (EDS) mapping of the Fe₃O₄@MXene nanocomposite. The EDS analysis reveals homogeneous spatial distributions of C, O, and Ti elements originating from the MXene substrate, along with Fe signals corresponding to the loaded Fe₃O₄ nanoparticles. Notably, the modified MXene nanosheets show a uniform iron distribution (Figure 2), confirming that the in situ hydrothermal treatment achieved homogeneous surface modification of the MXene nanosheets.

Figure 3A displays the X-ray diffraction (XRD) patterns of Fe₃O₄, MXene, and the Fe₃O₄@MXene composite. For Fe₃O₄, distinct diffraction peaks are observed at 2θ = 30.09°, 35.42°, 43.05°, 56.94°, and 62.51°, which correspond to the (220), (311), (400), (511), and (440) crystal planes of magnetite (JCPDS No. 19-0629), respectively [25]. For MXene, a characteristic peak appears at 2θ = 6.9°, indexed to the (002) plane. The absence of the Al (104) peak near 39.1° confirms the complete removal of the Al layer and successful synthesis of MXene [13,16]. In the case of the Fe₃O₄@MXene composite, all characteristic diffraction peaks of Fe₃O₄ are retained. The disappearance of the MXene-associated (002) plane indicates that Fe₃O₄ nanoparticles in the outer layer effectively suppress restacking of MXene nanosheets, endowing the MXene material with a two-dimensional open structure [26]. This two-dimensional open structure provides a favorable environment for enzyme immobilization.

Figure 3B displays the surface charge properties of MXene, GOD, Fe₃O₄@MXene, and Fe₃O₄@MXene-GOD. The exfoliated single-layer MXene nanosheets exhibit a zeta potential of −30.1 mV. However, GOD with an isoelectric point of approximately 4.2 exhibits a negative charge at pH 7.4 (zeta potential = −15.7 mV). Under these conditions, strong electrostatic repulsion exists between MXene nanosheets and GOD, which prevents enzyme immobilization on the material surface efficiently. Following modification with Fe₃O₄, the zeta potential of the composite shifted to +6.1 mV, reflecting a positively charged surface. This shift indicates that the in situ growth of Fe₃O₄ effectively tuned the surface charge characteristics of MXene, thereby mitigating electrostatic repulsion between the electrode and the enzyme. After the interaction between GOD and Fe₃O₄@MXene, it can be seen in Figure 3B that the zeta potential of Fe₃O₄@MXene-GOD shifted to −11.6 mV. The results were similar to those obtained with free GOD, confirming the successful immobilization of GOD on the Fe₃O₄@MXene substrate.

Figure 3C presents the Fourier transform infrared (FTIR) spectra of Fe₃O₄, MXene, and the Fe₃O₄@MXene composite, which reflect their surface functional groups. For bare Fe₃O₄ nanoparticles (blue curve), a broad absorption band in the range of 550–1650 cm⁻¹ is observed, corresponding to Fe-O stretching vibrations characteristic of the metal oxide. After etching, the surface of MXene (green curve) is typically terminated with abundant -OH and =O groups. Furthermore, the infrared absorption bands observed at 3445 cm⁻¹ and 1384 cm⁻¹ in MXene are assigned to the broad O-H stretching vibration and C=O stretching vibration, respectively. The broad peaks located around 617 cm⁻¹ and 877 cm⁻¹ are attributed to combined vibrations of Ti-O and Ti-O-Ti bonds, indicating bond formation within the MXene interlayers [27,28,29,30,31]. In the case of the Fe₃O₄@MXene composite (pink curve), the FTIR spectrum retains all characteristic features of MXene and additionally exhibits the Fe–O vibration band, providing clear evidence for the successful anchoring of Fe₃O₄ nanoparticles onto the MXene substrate.

XPS analysis was performed to investigate the chemical composition and surface modifications of MXene and Fe₃O₄@MXene (Figure 4A,B). The high-resolution XPS spectra of Fe₃O₄@MXene revealed characteristic peaks corresponding to C 1s, O 1s, Ti 2p, and Fe 2p. The high-resolution C 1s spectrum of Fe₃O₄@MXene (Figure 4C) was deconvoluted into several peaks at 281.8 eV, 284.9 eV, 286.1 eV, and 288.3 eV, assigned to C–Ti, C–C, C–OH, and C=O species, respectively. Notably, Fe₃O₄@MXene exhibited a distinct peak at 288.3 eV corresponding to C=O. This suggests chemical binding between Fe₃O₄ and MXene [32,33]. The high-resolution Fe 2p spectrum (Figure 4D) exhibited characteristic doublet peaks corresponding to Fe 2p_3/2 and Fe 2p_1/2 core levels. The deconvoluted peaks at binding energies of 711.2 eV and 715.0 eV are assigned to Fe²⁺ and Fe³⁺ species in the Fe 2p_3/2 region, respectively, while those at 720.1 eV and 725.0 eV are attributed to Fe²⁺ and Fe³⁺ in the Fe 2p_1/2 region. It is noteworthy that the higher binding energy peaks (715.0 eV and 725.0 eV) originate specifically from the Fe³⁺ within the Fe₃O₄ structure, confirming the coexistence of mixed-valence iron states in the composite material [34]. The high-resolution O 1s spectrum (Figure 4E) reveals three deconvoluted peaks at binding energies of 529.9 eV, 531.1 eV, and 532.1 eV, corresponding to Ti–O/Fe–O bonds, Ti–OH/Fe(OH)₂ groups, and C–Ti–(OH)_x bonds, respectively [32]. As shown in Figure 4F, the high-resolution Ti 2p spectrum reveals multiple chemical states. The peaks located at 455.1 eV and 460.7 eV are assigned to Ti–C bonds, while the peak at 456.3 eV corresponds to Ti³⁺ species. Notably, the peaks observed at 458.4 eV and 464.2 eV are attributed to Ti–O–Fe bonding configurations. The enhancement of the characteristic Fe-related peaks attests to the successful in situ generation of Fe₃O₄ on the MXene surface, ruling out mere electrostatic adsorption and pointing to robust interfacial interactions between the nanoparticles and MXene nanosheets [32,35].

3.2. Direct Electrochemistry of Fe₃O₄@MXene/GOD/Nafion/GCE

Figure 5A displays cyclic voltammetry (CV) measurements of different material-modified electrodes in N₂-saturated 0.1 M phosphate-buffered solution (PBS, pH 7.4) at a scan rate of 100 mV/s. As shown in Figure 5, no redox peaks are observed for Fe₃O₄@MXene/Nafion/GCE (curve a), suggesting the absence of intrinsic electroactivity of the Fe₃O₄@MXene nanocomposite. Moreover, when GOD is directly immobilized onto the electrode surface, it lacks efficient electrical communication with the electrode [36]. This leads to negligible electrochemical activity in DET processes. In contrast, well-defined redox peaks appear for both Fe₃O₄@MXene/GOD/Nafion/GCE (curve c) and MXene/GOD/Nafion/GCE (curve b). The formal potentials (E⁰ = Epa/2 + Epc/2) versus Ag/AgCl were calculated to be –0.423 V, respectively. These values correspond closely to the standard electrode potential of the FAD/FADH₂ redox couple at pH 7.0 [37], confirming that both Fe₃O₄@MXene and MXene promote direct electron transfer for GOD. Moreover, notable differences in the electron transfer behavior between the two composite electrodes can be discerned from the comparative analysis. CV measurements revealed a peak current of 4.08 μA for the Fe₃O₄@MXene/GOD/Nafion/GCE electrode, which is approximately three times greater than that recorded for the MXene/GOD/Nafion/GCE electrode (1.34 μA), demonstrating that Fe₃O₄@MXene greatly enhances the electron transfer efficiency of GOD at the electrode interface. The surface coverage (Γ) of GOD on the modified electrodes was calculated using Equation (1), where I_p, A, R, n, T, and F represent peak current, electrode surface area (0.72 cm²), gas constant (8.314 J·K⁻¹·mol⁻¹), electron number (2), temperature (298.15 K), and Faraday constant (96,500 C·mol⁻¹) [38], respectively. The calculated Γ values for MXene/GOD/Nafion/GCE and Fe₃O₄@MXene/GOD/Nafion/GCE were 5.99 × 10⁻¹¹ mol·cm⁻² and 1.70 × 10⁻¹⁰ mol·cm⁻², respectively. These values align closely with the theoretical Γ value of GOD (1.7 × 10⁻¹⁰ mol·cm⁻²) [39], thus verifying the participation of a thin-layer GOD in the electrochemical reaction. It is worth noting that the higher surface coverage observed for the Fe₃O₄@MXene/GOD/Nafion/GCE further validates that Fe₃O₄@MXene, with its large specific surface area and excellent conductivity, serves as a critical factor in improving GOD performance. This enhanced conductivity not only accelerates electron transfer kinetics but also optimizes the electroactive environment for enzyme immobilization, thereby amplifying the redox signal intensity [40].

$$I_p={\displaystyle \frac{n^2{F}^{2}A\Gamma v}{4RT}}$$

(1)

Figure 5B presents the Nyquist plots of four electrodes, and the equivalent circuit model used for EIS fitting is shown by the illustration, which includes the ohmic resistance of the electrolyte (Rs), a double-layer capacitance (CPE₁), the charge-transfer resistance (R₁), and the Warburg impedance (W₁) [41,42]. The red and blue curves correspond to the two GOD-modified electrodes, respectively. The charge-transfer resistance (Rct) of the Fe₃O₄@MXene/Nafion/GCE electrode after GOD immobilization is 812.5 Ω, which is greater than that of the Fe₃O₄@MXene/Nafion/GCE (361.8 Ω), confirming the successful loading of GOD onto the Fe₃O₄@MXene composite. Further comparison with MXene-based electrodes shows that the Rct values for MXene/Nafion/GCE and MXene/GOD/Nafion/GCE are 1431 Ω and 4767 Ω, respectively. In comparison, the Fe₃O₄@MXene-based electrodes exhibit significantly lower Rct values than their MXene-based counterparts, both with and without GOD modification. These trends are consistent with the CV data (Figure 5A), suggesting that the Fe₃O₄@MXene composite possesses a remarkable ability to promote electron transport, thereby leading to a significant enhancement in the electron transfer efficiency of GOD at the electrode surface.

Figure 6 demonstrates the effect of varying scan rates on the electrochemical behavior of the Fe₃O₄@MXene/GOD/Nafion/GCE in N₂-saturated 0.1 M PBS (pH 7.4). As shown in Figure 6B, both the cathodic (Ipc) and anodic (Ipa) peak currents exhibit linear increases with scan rates ranging from 100 to 800 mV·s⁻¹, indicating a surface-controlled electrochemical process [43]. Additionally, the apparent heterogeneous electron transfer rate constant (kₛ) was calculated using Laviron’s equation (Equation (2)) [44]:

$$\ln k_s=\alpha \ln \left(1-\alpha \right)+\left(1-\alpha \right)\ln \alpha -\ln \left({\displaystyle \frac{RT}{nFv}}\right)-\alpha {\displaystyle \frac{\left(1-\alpha \right)nF\Delta E_p}{RT}}$$

(2)

where R is the gas constant, T is the temperature (293 K), α is the electron transfer coefficient, n is the number of electrons transferred, and F is the Faraday constant. The values of n and α were set as 2 and 0.5, respectively [45]. ΔEp is the peak potential separation. The kₛ values of Fe₃O₄@MXene/GOD/Nafion/GCE and MXene/GOD/Nafion/GCE were determined as 9.57 s⁻¹ and 7.57 s⁻¹, respectively (Figure S1). Notably, the k_s value of Fe₃O₄@MXene/GOD/Nafion/GCE demonstrates a 1.26-fold enhancement compared to that of MXene/GOD/Nafion/GCE, confirming superior electron transfer kinetics in the Fe₃O₄@MXene composite. Such remarkably high k_s values signify exceptionally rapid electron transport in the Fe₃O₄@MXene-based system, which primarily originates from the enhanced electrical conductivity of Fe₃O₄@MXene.

Figure 7A shows the cyclic voltammetry (CV) curves of the Fe₃O₄@MXene/GOD/Nafion/GCE in N₂-saturated PBS solutions at varying pH values (4.0–8.0) with a scan rate of 100 mV·s⁻¹, aiming to investigate its electrochemical reaction mechanism [46,47,48]. As observed, both the anodic peak potential (Epa) and cathodic peak potential (Epc) shift negatively as pH increases from 4.0 to 8.0, demonstrating pH-dependent redox behavior. Figure 7B plots the formal potential (E⁰ = Epa/2 + Epc/2) as a function of pH, demonstrating a well-defined linear relationship between E⁰ and pH values. A linear relationship is obtained with the equation E⁰ = −0.061 pH − 0.0026 (R² = 0.996), yielding a slope of −60.6 mV/pH. This value closely matches the theoretical Nernstian slope of −58.6 mV/pH for a two-electron and two-proton transfer process, confirming the involvement of two protons and two electrons in the electrochemical reaction [49].

3.3. Optimization of Experimental Conditions

A systematic optimization of the preparation conditions was carried out in the fabrication process of the electrochemical sensor (Figure 8). As shown in Figure 8A, the maximum current response was observed at a GOD concentration of 45 mg·mL⁻¹. Lower concentrations led to insufficient enzyme loading, reducing catalytic efficiency. A further increase in GOD concentration resulted in a decline in current response, primarily due to its non-conductive nature, which hindered electronic communication. Similarly, the concentration of the Fe₃O₄@MXene suspension significantly affected the current response (Figure 8B). The current increased gradually with higher concentrations of Fe₃O₄@MXene, reaching a maximum at 2.5 mg·mL⁻¹. Beyond this point, the current decreased, mainly owing to the inherent lack of electroactivity of Fe₃O₄@MXene at high concentrations, which impeded electron transfer between GOD and the electrode surface.

The influence of Nafion content is shown in Figure 8C. The optimal peak current occurred at 1% Nafion. Lower Nafion content failed to fully immobilize GOD on the electrode surface, while higher concentrations thickened the Nafion film, obstructing electron transport and reducing peak currents. It is noteworthy that the mixing ratio of the GOD solution and the Fe₃O₄@MXene suspension also significantly influenced the electrochemical performance (Figure 8D). An inadequate amount of Fe₃O₄@MXene resulted in reduced conductivity, which led to a weaker current signal. The strongest response was achieved at a mixing ratio of 1:3. However, a further increase in the proportion of Fe₃O₄@MXene caused a decrease in the current response. This can be attributed to the dilution of the enzyme concentration at higher Fe₃O₄@MXene ratios, thereby reducing the loading of active GOD. Based on these findings, the optimized parameters (45 mg·mL⁻¹ GOD, 2.5 mg·mL⁻¹ Fe₃O₄@MXene, 1% Nafion, and 1:3 GOD/Fe₃O₄@MXene ratio) were selected for subsequent sensor fabrication.

3.4. Electrocatalytic Study of the Fe₃O₄@MXene/GOD/Nafion/GCE

Figure 9 shows that, compared to the cyclic voltammogram (CV) obtained in N₂-saturated PBS, the Fe₃O₄@MXene/GOD/Nafion/GCE in air-saturated PBS shows a markedly increased anodic current and a decreased cathodic current, along with a distinct anodic peak. This is because dissolved oxygen (O₂) effectively re-oxidizes the reduced glucose oxidase (GOD-FADH₂) to its active oxidized form (GOD-FAD), thereby promoting the oxidation process as outlined in Equation (4). When 0.5 mM glucose is introduced into the air-saturated PBS, it undergoes enzymatic oxidation to gluconolactone, consuming GOD-FAD and generating GOD-FADH₂. As a result, the reduction process (Equation (5)) is enhanced. At the same time, the surface concentration of oxidized GOD decreases, and dissolved O₂ is progressively depleted during the reaction, which suppresses the re-oxidation of GOD-FADH₂ (Equations (3) and (4)). Consequently, relative to the glucose-free system, the cathodic current increases while the anodic current decreases. Thus, the anodic current response is directly dependent on the glucose concentration, forming the basis for quantitative detection [6,37,44].

GOD(FAD) + 2H⁺ + 2e⁻ ↔ GOD(FADH₂)

(3)

GOD(FADH₂) + O₂ → GOD(FAD) + H₂O₂

(4)

Glucose + GOD (FAD) ↔ Gluconolactone + GOD (FADH₂)

(5)

3.5. Detection of Glucose by Fe₃O₄@MXene/GOD/Nafion/GCE

To further investigate the relationship between the electrocatalytic current and glucose concentration for the Fe₃O₄@MXene/GOD/Nafion/GCE glucose sensor, differential pulse voltammetry (DPV) measurements were conducted in air-saturated 0.1 M PBS (pH 7.4) over a potential range of −0.3 to −0.6 V (Figure 10). As shown in Figure 10A, the oxidation peak current at −0.450 V progressively decreases with increasing glucose concentration. The resulting calibration curve exhibits two distinct linear ranges: 0.05–1.1 mM and 1.1–15 mM, which are well described by the equations y = −8.674x + 80.854 (R² = 0.989) and y = −1.360x + 71.543 (R² = 0.996), respectively. A limit of detection (LOD) of 38 μM (S/N = 3) was achieved, with sensitivities of 120.47 μA·mM⁻¹·cm⁻² and 18.89 μA·mM⁻¹·cm⁻² corresponding to the low and high-concentration ranges, respectively (Figure 10B). For direct comparison, the glucose sensing performance of the MXene/GOD/Nafion/GCE was also evaluated under identical experimental conditions. As presented in Figure 10C,D, the MXene-based biosensor shows two narrower linear ranges of 0.3–1.6 mM and 1.6–3.8 mM, with a higher LOD of 174 μM (S/N = 3) and lower sensitivities of 52.68 μA·mM⁻¹·cm⁻² and 18.56 μA·mM⁻¹·cm⁻², respectively. In contrast, the Fe₃O₄@MXene/GOD/Nafion/GCE biosensor exhibits a significantly wider linear detection range, lower LOD, and higher sensitivity. Moreover, its extended linear range—compared to the MXene/GOD/Nafion/GCE sensor—fully encompasses the clinically relevant blood glucose concentration range of 3.9–6.1 mmol/L, and concentrations greater than 11.1 mmol/L. This enables the direct detection of blood glucose without dilution, highlighting its enhanced catalytic efficiency and superior analytical performance for glucose sensing [50,51].

When compared with other glucose sensors listed in

Table 1. A comparison of the performance of various GOD based modified electrodes.

Electrode	Linear Range	Sensitivity	LOD	Ks	Reference
	(mM)	(μA·mM−1 cm−2)	(μM)	(s−1)
rGO/Fe3O4/GOD	0.5–10	--	106.5	--	[22]
CHI-GOD/Mo3C2-dPIn	2.5–10.0	3.53	1574	--	[52]
CNT/GOD	0.1–2.0	53.5	--	1.14	[53]
Ti3C2Tx MXene/Graphene/GOD	0.20–5.5	12.10	100		[54]
(air-saturated and O2-saturated)		20.16	130
RFG/rGO/CS/GOD	--	46.71	79.65	--	[2]
β-CD/MWCNTs/GOD	0.05–1.15	32.28	0.42	3.24	[55]
LIG/PB/GOD	0.010–2.0	1.85	57.3	--	[56]
GC/poly(dTT-bT)/GrO/GOD	0.20–10.0	9.40	36	--	[57]
GQD/GOD	0.005–1.27	0.085	1.73	1.12	[58]
FTO-CNTs/PEI/GOD	0.07–0.7	6.38	70	--	[59]
PEDOT:PSS/Ti3C2/GQD-GOD	0–0.5	21.64	65	--	[60]
CF/ZnO/GOD/CS	0.10–1.0	--	100	--	[61]
Fe3O4@Mxene/GOD/Nafion	0.05–1.1	120.47	38	9.57	This work
	1.1–15.0	18.89

, the developed sensor demonstrated a wider detection range, a lower detection limit, and higher sensitivity. In addition, its higher kₛ value relative to those of the listed sensors reflects a faster electron transfer rate, further highlighting the superior analytical performance of the proposed sensor. The enhanced performance is attributed to the synergistic effect between the two components in the composite. MXene offers a large specific surface area, whereas Fe₃O₄ contributes good biocompatibility and high electrical conductivity. The combination of these properties promotes direct electron transfer between GOD and the electrode surface, leading to a significant improvement in the electrocatalytic efficiency of the sensor.

3.6. Study of Analytical Signal Selectivity, Stability, Reproducibility and Repeatability

The selectivity of the sensor was further investigated in the presence of potential interfering substances. As shown in Figure 11A, when 1 mM uric acid (UA), ascorbic acid (AA), dopamine (DA), and L-Cysteine (L-cys) were added to air-saturated 0.1 M PBS (pH 7.4) containing 0.5 mM glucose, the interfering substances exhibited negligible effects on the glucose-derived current signal. This confirms the high selectivity of the developed sensor for glucose detection. The long-term stability was evaluated by testing the sensor after 15 days of storage at 4 °C. The electrochemical response current retained 92.3% of its initial value (Figure 11B), confirming excellent stability.

To assess the reproducibility of the Fe₃O₄@MXene/GOD/Nafion/GCE sensor, five independently prepared electrodes were tested under the same conditions for the detection of 0.5 mM glucose. The results showed a relative standard deviation (RSD) of 2.4% (Figure 11C). Additionally, the repeatability was evaluated by performing ten consecutive measurements of 0.1 mM glucose using the same sensor (Figure 11D), yielding an RSD of 2.04%. These results confirm that the Fe₃O₄@MXene/GOD/Nafion/GCE sensor exhibits excellent reproducibility and repeatability.

3.7. Real Sample Analysis

To validate the practical application of the glucose sensor in clinical analysis, artificial serum was prepared as described in the literature [62,63], and the Fe₃O₄@MXene/GOD/Nafion/GCE was employed to detect glucose in artificial serum samples. As summarized in

Table 2. Analytical detection of glucose in artificial serum samples by Fe₃O₄@MXene/GOD/Nafion/GCE.

		Glucose Concentration Mmol
Sample	Added	Found	Recovery (%)	RSD (%)
	mM	mM
Artificial serum	2.5	2.55	101.80%	2.07%
	3.0	3.10	103.48%	1.93%
	5.0	5.07	101.40%	2.68%
	12.0	12.20	101.67%	2.25%
	15.0	15.43	102.86%	2.34%

, recovery tests were performed by spiking the artificial serum samples with varying concentrations of glucose. The recovery rates ranged from 101.40% to 103.48% with RSD values between 1.93% and 2.68%, demonstrating high reproducibility. Furthermore, the glucose measurements obtained using the fabricated sensor were compared with those from a commercial Sinocare glucometer (Figure S2), showing close agreement. These results confirm the reliability and accuracy of the Fe₃O₄@MXene/GOD/Nafion/GCE electrode, making it suitable for practical clinical analysis of human samples.

4. Conclusions

In this work, we developed a Fe₃O₄@MXene nanocomposite by in situ growth of Fe₃O₄ nanoparticles on monolayer MXene via a hydrothermal method. The Fe₃O₄ modification effectively modulated the strong negative surface charge of MXene and introduced a porous architecture on the nanosheets, which not only mitigated electrostatic repulsion with GOD but also facilitated high enzyme immobilization. Benefiting from the large specific surface area of MXene and the high conductivity and biocompatibility of Fe₃O₄, the resulting sensor exhibited enhanced electron transfer efficiency. The Fe₃O₄@MXene/GOD/Nafion/GCE electrode showed a wide linear range from 0.05 to 15 mM, a low detection limit of 38 μM (S/N = 3), and a high sensitivity of 120.47 μA·mM⁻¹·cm⁻², exhibiting good catalytic performance. Moreover, the sensor achieved satisfactory recovery in artificial serum samples, accuracy comparable to commercial glucometers, excellent selectivity, reproducibility, and storage stability. This Fe₃O₄@MXene-based platform offers a reliable strategy for precise glucose monitoring in clinical diagnostics, demonstrating promising potential for practical application.