Bi2Fe4O9/reduced graphene oxide nanocomposites with enhanced peroxidase activity for sensitive glutathione detection

Bi2Fe4O9/reduced graphene oxide nanocomposites with enhanced peroxidase activity for sensitive glutathione detection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bi2Fe4O9/reduced graphene oxide nanocomposites with enhanced peroxidase activity for sensitive glutathione detection Jinxiu Guo, Haozhe Jia, Runtian Ma, Zuoxiang Ma, Yanjun Cui, Bing Hu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9180770/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Bi 2 Fe 4 O 9 nanoparticles (Bi 2 Fe 4 O 9 NPs) and Bi 2 Fe 4 O 9 /reduced graphene oxide nanocomposites (Bi 2 Fe 4 O 9 /rGO NCs) were successfully synthesized via a hydrothermal method. Morphological and compositional characterizations revealed that the introduction of graphene oxide (GO) not only modifies the lamellar morphology of Bi 2 Fe 4 O 9 NPs but also enhances their dispersion, thereby increasing the specific surface area of the composites. Bi 2 Fe 4 O 9 /rGO NCs exhibited excellent peroxidase (POD)-like activity, efficiently catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H 2 O 2 to form a blue oxidation product (oxTMB) with a characteristic absorption peak at 652 nm. Steady-state kinetic analysis confirms Bi 2 Fe 4 O 9 /rGO NCs have superior catalytic activity and substrate affinity to pure Bi 2 Fe 4 O 9 NPs, and density functional theory calculations reveal the atomic-level mechanism for the enhanced POD-like activity, as the nanocomposites show much lower adsorption energies for TMB (-5.67 eV) and H 2 O 2 (-3.44 eV) than Bi 2 Fe 4 O 9 NPs (TMB: -2.18 eV, H 2 O 2 : -1.49 eV). Leveraging the competitive inhibitory effect of glutathione (GSH) on the Bi 2 Fe 4 O 9 /rGO-TMB-H 2 O 2 catalytic system, a sensitive and selective colorimetric platform for GSH detection was established, achieving a linear range of 10–250 nM and a limit of detection of 3.1 nM. Furthermore, the platform exhibited satisfactory recovery rates (94.4%-99.3%) in fetal bovine serum samples, demonstrating its great potential for practical application in complex biological matrices and analytical sensing fields. Bi2Fe4O9 reduced graphene oxide peroxidase-like activity glutathione colorimetric assay Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Glutathione (GSH) is a tripeptide consisting of glutamic acid, cysteine, and glycine condensed by peptide bonds [1]. GSH has become an important metabolic regulators and biomarkers due to its vital physiological functions, such as detoxication [2], protection against oxidative and free radical damage [3] and defense against liver injury [4]. Therefore, it is indispensable to establish an efficient and sensitive method to determine GSH. Spectroscopy, chromatography and electrochemical methods have been applied in the detection of GSH [5–7]. These traditional analytical methods each have their inherent advantages, but they are plagued by some limitations involving expensive equipment, cumbersome sample pretreatment, time-consuming procedures, excessive solvent usage, laborious electrode modification and sensitivity to matrix interference [8]. In contrast, ultraviolet-visible (UV-vis) spectrophotometry based on the inhibition of nanozyme-catalyzed 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by GSH has emerged as a promising alternative. This method combines the advantages of low cost, simple operation, rapid readout, and visual color change, making it suitable for on-site and large-scale sample analysis [9]. Natural enzymes, represented by horseradish peroxidase (HRP), have long been the gold standard for catalytic reactions in biosensing. However, their practical application is severely hindered by inherent limitations: complex and costly preparation/purification processes, poor stability under extreme conditions, and difficulty in recycling and reuse. For instance, HRP loses over 50% of its activity below pH 3.0 or at temperatures above 50 ℃ [10]. Iron-based nanozymes, as one of the earliest developed synthetic enzyme mimics, have successfully addressed these drawbacks [11]. They feature facile synthesis, low toxicity, excellent stability, and tunable catalytic performance, making them ideal candidates for replacing natural enzymes in various fields [12]. Among iron-based nanozymes, metal ferrite nanomaterials have garnered extensive attention due to their versatile enzyme-mimetic activities and structural adjustability. Han et al. synthesized MnFe 2 O 4 nanozymes modified with different functional groups (-COOH, -NH 2 , -PEG) and found that the surface functionalization not only enhanced their peroxidase activity but also endowed them with tailored antibacterial properties. MnFe 2 O 4 -COOH showed preferential inhibition of Gram-positive bacteria, while MnFe 2 O 4 -NH 2 exhibited the most significant and broad-spectrum antibacterial activity [13]. Lu and colleagues constructed MoS 2 -bPEI-CeFe 2 O 4 nanoflowers with a core-shell structure, where MoS 2 served as the photothermal core and branched polyethylenimine (bPEI)-modified CeFe 2 O 4 nanoparticles (NPs) acted as the catalytic shell. The CeFe 2 O 4 NPs exhibited superior Fenton-like activity, efficiently generating intracellular reactive oxygen species (ROS), while the MoS 2 core enhanced photothermal therapy efficacy, achieving a synergistic effect in tumor treatment [14]. Feng et al. further reported that SnFe 2 O 4 NPs possess dual catalase-like and peroxidase-like activities, showcasing promising potential for tumor therapy [15]. Bi 2 Fe 4 O 9 NPs, a functional metal oxide with excellent gas sensing, photocatalytic, and photoelectric conversion properties [16–18], have been identified as promising peroxidase mimics and successfully used for the colorimetric detection of dopamine [19]. Fe active sites and the d-orbitals of Fe 3+ in Bi 2 Fe 4 O 9 NPs was considered to operate in the catalytic oxidation of TMB in presence of H 2 O 2 [19]. However, pristine Bi 2 Fe 4 O 9 NPs tend to form irregular sheet-like aggregates due to van der Waals forces and hydrogen bonding interactions. This aggregation not only reduces their specific surface area but also shields the active Fe 3+ sites, limiting the exposure of catalytic centers and thus compromising their enzyme-mimetic activity. Therefore, optimizing the morphology and dispersion of Bi 2 Fe 4 O 9 NPs is essential to enhance their catalytic performance for practical applications. Modulation of properties of nanozymes can be performed by controlling the structure, morphology, surface properties, and combining with other materials [20, 21]. Among various modifiers, two-dimensional (2D) nanomaterials stand out as ideal carriers or synergistic components for nanozyme optimization. Their unique planar structure provides a large specific surface area, which promotes nanozyme dispersion and increases the exposure of active sites; meanwhile, the abundant functional groups on their surface enhance the affinity between the nanozyme and substrates, and the high electron conductivity accelerates charge transfer during catalytic reactions [22, 23]. Graphene, graphene oxide (GO), and reduced graphene oxide (rGO) are typical 2D materials that have been widely used in the construction of TMB/H 2 O 2 -based biosensors [24–26]. To address the aggregation issue of pristine Bi 2 Fe 4 O 9 NPs and further enhance their peroxidase (POD)-like activity, we fabricated Bi 2 Fe 4 O 9 /rGO nanocomposites by integrating Bi 2 Fe 4 O 9 with GO via a hydrothermal method. The nanocomposites can efficiently catalyze the oxidation of TMB to form deep-blue oxTMB in the presence of H 2 O 2 , enabling quantitative evaluation of peroxidase activity through absorbance measurement. Density functional theory (DFT) calculations were employed to gain mechanistic insights into the rGO-mediated enhancement of enzyme-mimetic activity [27]. Based on the competitive inhibition of GSH on the Bi 2 Fe 4 O 9 /rGO-TMB-H 2 O 2 system, a highly sensitive and selective colorimetric method for GSH detection was constructed. Its applicability was validated in complex biological samples such as fetal bovine serum (FBS), providing a reliable analytical tool for GSH-related biological research and clinical diagnosis. 2 Experimental 2.1 Chemicals and reagents Bi(NO 3 ) 3 ·5H 2 O (≥ 99%), Fe(NO 3 ) 3 ·9H 2 O (98%), GO solution (≥ 98%, 1–5 µm, 1–2 layers, 1–3 wt% dispersion in H 2 O, pH = 1–2), TMB (≥ 98%), p-Benzoquinone (p-BQ, 99%), terephthalic acid (TA, 99%), L-glutathione (GSH, 99%), D-(+)-glucose (Glu, 99%), lactose (Lac, 98%), fructose (Fru, 99%), uric acid (UA, ≥ 98.0%), D-alanine (Ala, 98%), D-cysteine (Cys, 97%), L-arginine (Arg, ≥ 99%), L(+)-ascorbic acid (AA, 99%), KCl (≥ 99.8%), NaCl (99.99%), MgCl 2 ·6H 2 O (≥ 98%), CaCl 2 ·6H 2 O (98%), FBS were purchased from Shanghai Titan Scientific Co., Ltd. (Shanghai, China). HNO 3 (65–68% w/w), acetone and dopamine hydrochloride (DA, 98.0%) were bought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). NaAc (99%) and ethanol were gotten from Guangdong Guanghua Sci-Tech Co., Ltd. (Shantou, China). H 2 O 2 (30 wt%) was purchased from Fuchen Chemical Reagent Factory (Tianjin, China). HAc (≥ 99.8%) was obtained from Chengdu Kelong Chemical Co., Ltd (Chengdu, China). NaOH was bought from Damao Chemical Reagent Co., Ltd. (Tianjin, China). Isopropanol (IPA) was bought by Shanghai Jianxin Chemical Co., Ltd. (Shanghai, China). NaN 3 (≥ 99.5%) was gotten from Bomei Biotechnology Co., Ltd. (Hefei, China). Unless stated otherwise, all other reagents were of analytical grade, deionized water (18.2 MΩ·cm at 25°C) was used throughout the experiments, and all reagents were used as received without further purification. 2.2 Preparation of Bi 2 Fe 4 O 9 /rGO NCs As shown in Fig. 1 a, Bi 2 Fe 4 O 9 /rGO NCs were synthesized via a slightly modified hydrothermal method reported previously [17]. Briefly, 0.485 g of Bi(NO 3 ) 3 ·5H 2 O and 0.404 g of Fe(NO 3 ) 3 ·9H 2 O were first dissolved in a mixture of 13 mL of deionized water and 2 mL of HNO₃, followed by magnetic stirring at room temperature for 30 min. An 8 M NaOH solution was added dropwise to the above mixture until the pH reached 10–11, resulting in the formation of a red-brown suspension. Subsequently, 100 µL of GO dispersion was introduced under continuous stirring. The resulting suspension was filtered and rinsed with deionized water until the filtrate reached neutral pH. The obtained brown precipitate was mixed with 40 mL of 12 M NaOH solution, and the mixture was vigorously stirred for 1 h. The suspension was then transferred to a Teflon-lined stainless-steel autoclave, which was heated at 200 ℃ for 24 h. After cooling to room temperature naturally, the precipitate was collected via centrifugation, rinsed three times alternately with deionized water and ethanol, and dried at 60 ℃ to obtain the final product. For comparison, pure Bi 2 Fe 4 O 9 NPs were synthesized via the identical procedure but without the addition of the GO. 2.3 Characterization of Bi 2 Fe 4 O 9 /rGO NCs The morphologies and elemental compositions of the samples were characterized using a field-emission scanning electron microscope (FESEM, Regulus 8100, Hitachi, Japan), a transmission electron microscope (TEM, Talos F200x, FEI, USA), and energy dispersive X-ray spectroscopy (EDS) mapping. The chemical states of elements were analyzed via X-ray photoelectron spectroscopy (XPS, K-Alpha, Thermo Scientific, USA). Fourier transform infrared spectroscopy (FT-IR) was used to identify the functional groups in the materials by a FT-IR-650 spectrometer (Gangdong Co. Ltd. Tianjin, China). The rGO was characterized by Raman spectroscopy (LabRAM HR Evolution, Horiba, Japan) using a laser with an excitation wavelength of 633 nm. Thermogravimetric (TG) and derivative thermogravimetric (DTG) analyses were performed on a thermal analyzer (STA 449 F5 Jupiter, NETZSCH, Germany) at a heating rate of 10°C/min. The crystal structures were determined via X-ray diffraction (XRD, XD-3, Purkinje General Instrument Co., Ltd., Beijing, China) with Cu Kα radiation, at a scanning rate of 2°/min from 10° to 80°. 2.4 Catalytic activity of Bi 2 Fe 4 O 9 /rGO NCs and steady-state kinetic study In order to evaluate the catalytic activity of Bi 2 Fe 4 O 9 /rGO, we performed the catalytic experiment of the composites for the oxidation of TMB in the presence of H 2 O 2 . The typical assay was carried out by adding 96 µL of 1 mM TMB solution (in acetone), 60 µL of 10 mM H 2 O 2 solution (in H 2 O) and 25 µL of 30 µg/mL catalyst into 819 µL of 0.1 M acetic acid-sodium acetate (HAc-NaAc) buffer solution (pH 4.5) with reacted at 40 ℃ for 20 min. All chemical reagents were freshly prepared and all reactions were monitored at 652 nm by a Ultraviolet-visible (UV-vis) spectrophotometer (T2602S, YOKE, Shanghai, China). The steady-state kinetic values were determined by changing the concentration of TMB (0-0.128 mM) or H 2 O 2 (0-1.8 mM) as substrate in 0.1 M acetic acid buffer (pH = 4.5) with 0.75 µg/mL catalyst. The Michaelis-Menten parameters were calculated by using the Lineweaver-Burk double reciprocal plot according to the Michaelis-Menten model [28]. 2.5 Detection of ROS To clarify the POD-like catalytic mechanism, radical trapping experiments were conducted in 0.1 M HAc-NaAc buffer (pH 4.5) at 40°C for 20 min. The reaction system contained the catalyst at 0.75 µg/mL, TMB at 0.096 mM, and H 2 O 2 at 0.9 mM. Specific scavengers were introduced at optimized concentrations to selectively quench distinct reactive species: IPA for hydroxyl radicals (•OH), NaN 3 for singlet oxygen ( 1 O 2 ), and p-BQ for superoxide anion (O 2 •− ) [29]. Furthermore, the generation of •OH was verified using TA as a fluorescent probe [30]. 0.5 mM TA, 0.9 mM H 2 O 2 , and 0.75 µg/mL catalyst were mixed in the above buffer, followed by incubation at 40℃ for 20 min. Fluorescence emission spectra were recorded in the range of 400–550 nm at the maximum excitation wavelength (λ ex = 315 nm) using a Shimadzu RF-600 fluorometer. 2.6 DFT Calculations All theoretical calculations were carried out with the Vienna Ab Initio Simulation Package (VASP) [31] integrated with the projector-augmented-wave (PAW) method [32]. The simulations were grounded in spin-polarized DFT within the generalized gradient approximation (GGA) framework using the Perdew-Burke-Ernzerhof (PBE) functional [33]. The on-site Coulomb interaction of Fe 3d electrons was described using the DFT + U method (Dudarev scheme) with an effective U value (Ueff) of 3.0 eV. Long-range van der Waals interactions were incorporated via the DFT-D3 method [27], the plane-wave cutoff energy was set to 500 eV, and the energy convergence criterion was fixed at 1 × 10 − 5 eV. The first Brillouin zone was sampled with a 1×1×1 k-point Monkhorst-Pack grid for all calculations. 2.7 GSH detection assay For colorimetric detection, GSH at varied concentrations were added to the solution containing 0.096 mM TMB, 0.9 mM H 2 O 2 and 0.75 µg/mL Bi 2 Fe 4 O 9 /rGO NCs. The final volume of the solution was adjusted to 1.0 mL with 0.1 M acetic acidic buffer (pH 4.5). The reaction mixture was incubated at 40 ℃ for 20 min, then analyzed at 652 nm to obtain the calibration curve and the limit of detection (LOD = 3σ/K) of GSH, where σ is the standard deviation of the blank sample measurements (n = 8) and K is the slope of the calibration curve. 2.8 Preparation of FBS FBS Samples were stored at -18°C until analysis. In order to eliminate the influence of proteins in the serum on the test results, a protein precipitant needs to be used. Noticing that using other reagents might affect the detection conditions, protein precipitation was conducted by adding acetone to serum in a ratio of 1:10 (v/v) [34]. Then the mixture was vortexed for 30 s, followed by incubation in an ice bath for 10 min, and then centrifuged at 4000 rpm for 5 min. The content and recovery rate of GSH in the FBS samples were determined using the standard addition method. Instead of GSH, the pure FBS and spiked samples were added to the above reaction solution in section 2.7 . 3 Results and discussion 3.1 Preparation and characterization of Bi 2 Fe 4 O 9 /rGO NCs Dilute nitric acid effectively addresses the poor solubility of bismuth nitrate, but markedly lowers the pH of the precursor solution. Since the formation of Bi 2 Fe 4 O 9 requires a relatively high pH environment, the use of high-concentration NaOH as a mineralizer is necessitated for the successful synthesis of Bi 2 Fe 4 O 9 crystals [14]. To prepare Bi 2 Fe 4 O 9 /rGO NCs with varying GO mass fractions (0.375%, 0.75%, 1.5%, and 3%), different volumes of GO dispersion (50, 100, 200, and 400 µL) were added to the mixed solution of Bi(NO 3 ) 3 ·5H 2 O and Fe(NO 3 ) 3 ·9H 2 O, which were designated as Bi 2 Fe 4 O 9 /rGO-50, Bi 2 Fe 4 O 9 /rGO-100, Bi 2 Fe 4 O 9 /rGO-200, and Bi 2 Fe 4 O 9 /rGO-400, respectively. Photographs of the as-prepared samples are displayed in Fig. S1 . As the GO content increases, the color of the final product deepens gradually. Comparison of FESEM images in Figs. 1 b-f reveals that pure Bi 2 Fe 4 O 9 NPs exhibit aggregated block-like morphologies, whereas Bi 2 Fe 4 O 9 /rGO NCs transform into nanosheet structures with the introduction of GO. When the GO addition volume reaches 100 µL (corresponding to a mass fraction of 0.75%), the sheet-like Bi 2 Fe 4 O 9 /rGO NCs exhibit excellent dispersibility and uniformity, with a length of 500–1000 nm with a thickness of 100–200 nm. Further increasing the GO addition leads to a reduction in the material’s uniformity. Thus, Bi 2 Fe 4 O 9 /rGO specifically refers to Bi 2 Fe 4 O 9 /rGO-100 in the following text. As displayed in Fig. 1 g, the TEM image of Bi 2 Fe 4 O 9 /rGO-50 clearly reveals thin and transparent lamellar structures assigned to GO nanosheets, which act as a supporting matrix to facilitate the uniform dispersion of Bi 2 Fe 4 O 9 sheets on its surface. The results of EDS elemental distribution mapping and analysis are presented in Fig. S2 clearly illustrate of Bi, Fe, C and O elements, which display the presence and homogeneous distribution of mentioned elements in the entire of the nanohybrids. The phase purity of the as-prepared Bi 2 Fe 4 O 9 /rGO NCs was examined using XRD (Fig. 1 h). All characteristic diffraction peaks could be indexed to the pure mullite phase Bi 2 Fe 4 O 9 of the orthorhombic structure (JCPDS No. 00-025-0090). Among them, the diffraction peaks at 61.8°, 56.6°, 47.0°, 39.0°, 37.5°, 33.7°, 29.8°, 28.9°, 28.2° and 14.8°correspond to the crystal planes (004), (332), (141), (212), (202), (130), (002), (211), (121) and (001), respectively [19]. No impurity peaks were detected, confirming that GO addition did not alter the crystal structure or phase purity of Bi 2 Fe 4 O 9 . Additionally, the characteristic GO (001) diffraction peak near 11.1° was not observed, likely due to its low content in the composite [17]. XPS was employed to analyze the chemical states of elements in the composite. The XPS survey spectrum (Fig. 1 i) confirms the presence of Bi, Fe, O, and C, with a higher carbon content than pure Bi 2 Fe 4 O 9 , verifying GO incorporation. The trace carbon in pure Bi 2 Fe 4 O 9 likely arises from residual impurities. Bismuth elements are split into Bi 4f 7/2 and Bi4f 5/2 peaks at 164.8 eV and 159.5 eV, and these binding energy positions indicate that bismuth elements in the composite material exist in the form of Bi 3 ⁺. High-resolution XPS spectra of Fe 2p, O 1s and C 1s are shown in Fig. 1 j-l. The Fe 2p spectrum displays peaks at 710.9 eV (Fe 2p 3/2 ) and 724.8 eV (Fe 2p 1/2 ), which deconvolve into Fe 2+ and Fe 3+ respectively. In addition, the two satellite peaks at 717.9 eV (Sat. I) and 732.2 eV (Sat. II) further confirm Fe 3+ species. The O 1s spectrum deconvolves two peaks with binding energies of 530.3 eV (lattice oxygen) and 532.0 eV (adsorbed oxygen) [19]. The C 1s spectrum shows peaks at 284.5 eV (C-C = C groups) and 288.6 eV (O-C = O groups) [17]. Raman spectroscopy was used to investigate structural changes in GO before and after composite formation. GO exhibits two characteristic Raman peaks: the D peak (1332 cm − 1 , defect-induced) and G peak (1590 cm − 1 , indicative of graphitic order). GO typically has a high I D /I G ratio due to abundant oxygenated groups and defects. However, the I D /I G ratio decreases significantly in Bi 2 Fe 4 O 9 /rGO NCs (Fig. 1 m), confirming effective GO reduction, reduced oxygenated functional groups, and partial restoration of graphitic structure [17]. Peak position shifts of the D and G bands further support GO reduction. FT-IR spectroscopy was performed to verify the incorporation and reduction of GO in the composites (Fig. S3). For pure Bi₂Fe₄O₉ NPs, the dominant infrared absorption peaks are located in 400–850 cm − ¹, assigned to the stretching/bending vibrations of Fe-O and Bi-O bonds [19]. Upon incorporation with GO, new absorption peaks emerge at ~ 1030 and ~ 1340 cm − ¹, attributed to the C-H bending vibration and C = C stretching vibration in the benzene-like ring structure of GO, respectively. The marked attenuation of oxygen-containing groups in the composite confirms the effective reduction of GO to rGO. Further TG and DTG analyses (Fig. S4) corroborate these findings: Bi 2 Fe 4 O 9 /rGO NCs exhibit more substantial weight loss than pure Bi 2 Fe 4 O 9 NPs over 30–600℃, with pronounced DTG peaks attributed to the thermal decomposition of rGO [35], directly evidencing the successful incorporation and reduction of GO into the Bi 2 Fe 4 O 9 matrix. 3.2 POD-like activity of Bi 2 Fe 4 O 9 /rGO NCs As shown in Fig. 2 a (lines 1–4), neither the individual TMB (line 1) nor the Bi 2 Fe 4 O 9 +TMB binary system (line 2) showed obvious absorption across the wavelength range of 400–800 nm. For the TMB+H 2 O 2 binary system (line 3), only weak absorption was observed, confirming spontaneous H 2 O 2 decomposition to trace ROS. In contrast, the ternary system (Bi 2 Fe 4 O 9 +TMB+H 2 O 2 , line 4) exhibited a distinct absorption peak at 652 nm after 20 min, arising from oxTMB formation. These results demonstrate that Bi 2 Fe 4 O 9 NPs exhibit POD-like activity but no oxidase (OXD)-like activity [36]. Under identical conditions, the POD-like activities of Bi 2 Fe 4 O 9 /rGO NCs with varying GO contents were assessed (Fig. 2 a, lines 5–8). Bi 2 Fe 4 O 9 /rGO NCs exhibited higher POD-like activity than pure Bi 2 Fe 4 O 9 NPs, with activity dependent on GO loading: the highest activity was observed at 100 µL GO dispersion, while excess GO reduced activity, likely due to Bi 2 Fe 4 O 9 aggregation and morphological heterogeneity, which diminish accessible active sites. For cyclic reuse, the TMB+H 2 O 2 +Bi 2 Fe 4 O 9 /rGO reaction mixture was centrifuged at 10000 rpm for 5 min. The precipitate was rinsed with deionized water, then reintroduced into fresh TMB+H 2 O 2 solution under the same conditions. The variation of the UV absorbance intensity of oxTMB at 652 nm is depicted in Fig. S5. After five consecutive cycles, the relative activity remained above 90%. This result confirms that Bi 2 Fe 4 O 9 /rGO NCs exhibit excellent enzyme-mimetic catalytic reproducibility and stability, which support their potential for practical sensing applications. To elucidate the POD-like catalytic mechanism of Bi 2 Fe 4 O 9 /rGO NCs, radical trapping experiments were performed employing IPA (•OH scavenger), NaN 3 ( 1 O 2 scavenger), and p-BQ (O 2 •− scavenger). As shown in Fig. 2 b, both the addition of IPA and p-BQ could significantly reduce the absorbance of the TMB+H 2 O 2 +Bi 2 Fe 4 O 9 /rGO system at 652 nm, whereas NaN 3 had no notable effect, demonstrating that •OH and O 2 •− are the primary reactive species, consistent with observations for pure Bi 2 Fe 4 O 9 (Fig. 2 c) [29]. The influence of •OH on the catalytic effect has also been analyzed by fluorescence spectrum. As shown in Fig. 2 d, TA exhibits negligible fluorescence in the absence of •OH, but reacts with •OH to form 2-hydroxyterephthalic acid, which emits characteristic fluorescence at 435 nm [30]. The results of lines 1–4 indicate that neither H 2 O 2 alone nor Bi-based materials alone can trigger •OH generation. In contrast, a distinct 435 nm emission peak emerged in the system containing TA, H 2 O 2 , and Bi 2 Fe 4 O 9 NPs (line 5). This confirms that Bi 2 Fe 4 O 9 NPs can activate H 2 O 2 to produce •OH. Notably, the fluorescence intensity of the system with Bi 2 Fe 4 O 9 /rGO NCs (line 6) was significantly higher than that with pure Bi 2 Fe 4 O 9 NPs. This verifies that Bi 2 Fe 4 O 9 /rGO NCs have superior POD-like activity. They can more efficiently activate H 2 O 2 to generate •OH radicals, thus facilitating TMB oxidation. In order to understand the reason why the addition of graphene oxide improves the catalytic performance at the atomic level, we conducted DFT calculations. A heterostructure was constructed by combining the (121) crystal plane of Bi 2 Fe 4 O 9 with reduced graphene oxide, and the adsorption energies ( \(\:{\text{E}}_{\text{ads}}\) ) of TMB and H 2 O 2 molecules were calculated on both the heterojunction surface and the pure Bi 2 Fe 4 O 9 surface. The \(\:{\text{E}}_{\text{ads}}\) was calculated according to Eq. 1: \(\:{\text{E}}_{\text{ads}}\text{=}{\text{E}}_{\text{t}\text{otal}}\text{−}{\text{E}}_{\text{mol}}\text{−}{\text{E}}_{\text{sub}}\) (Eq. 1) where \(\:{\text{E}}_{\text{t}\text{otal}}\) denotes the total energy of the slab with the adsorbed molecule, \(\:{\text{E}}_{\text{sub}}\) represents the total energy of the pristine slab, and \(\:{\text{E}}_{\text{mol}}\) is the total energy of the isolated H 2 O 2 or TMB molecule in a large vacuum box. A negative \(\:{\text{E}}_{\text{ads}}\) value indicates an exothermic adsorption process, implying a stronger binding interaction between the molecule and the surface. The optimized molecular structures and the data used to calculate the adsorption energy were described in Fig. 3 and Table 1 . The calculation results show that the E ads of TMB on Bi 2 Fe 4 O 9 NPs and Bi 2 Fe 4 O 9 /rGO NCs were − 2.18 and − 5.67 eV respectively. The E ads of the Bi 2 Fe 4 O 9 /rGO NCs and H 2 O 2 (-3.44 eV) are also lower than that between the Bi 2 Fe 4 O 9 NPs and H 2 O 2 (-1.49 eV). The lower adsorption energies indicate that Bi 2 Fe 4 O 9 /rGO NCs are more likely to bind to the substrates (TMB and H 2 O 2 ). Table 1 Adsorption energy-related parameters obtained by DFT calculations. E (eV) TMB H 2 O 2 Bi 2 Fe 4 O 9 NPs Bi 2 Fe 4 O 9 /rGO NCs Bi 2 Fe 4 O 9 NPs Bi 2 Fe 4 O 9 /rGO NCs E sub -583.24 -1484.17 -583.24 -1484.17 E mol -236.14 -236.14 -18.10 -18.10 E total -821.56 -1725.98 -602.83 -1505.71 E ads -2.18 -5.67 -1.49 -3.44 3.3 Optimization of experimental conditions Experimental conditions were optimized to determine the steady-state kinetic parameters of Bi 2 Fe 4 O 9 /rGO, with the effects of pH, buffer concentration, catalyst loading, substrate concentration, reaction temperature and incubation time on the absorbance at 652 nm (A 652 ) of the TMB+H 2 O 2 +Bi 2 Fe 4 O 9 /rGO system systematically investigated (Fig. 4 ). For pH optimization, A 652 increased from pH 3.5 to 4.5 and then decreased at higher pH. This was attributed to the enhanced polarity of protonated TMB, which facilitates binding to the nanozyme active center and promotes oxidation via charge effects. For HAc-NaAc buffer concentration, A 652 reached its maximum when the concentration of the buffer is 0.1 M because the lower concentrations failed to maintain the optimal pH 4.5, while excessive ionic strength disrupted surface electrostatic interactions via charge screening effects [37]. Catalyst loading exhibited a concentration-dependent effect for A 652 increased with Bi 2 Fe 4 O 9 /rGO concentration up to 0.75 µg/mL, after which activity plateaued due to catalyst flocculation and solution darkening at higher concentrations. Thus, 0.75 µg/mL was selected as the optimal catalyst concentration. For substrate concentrations, A 652 increased with TMB and H 2 O 2 concentrations until reaching plateaus at 0.096 mM TMB and 0.9 mM H 2 O 2 , respectively, where further increases did not significantly enhance absorbance. The activity increased from 25 to 40℃ as reaction temperature due to enhanced molecular collision, while higher temperatures reduced activity via by-product formation. Incubation time was optimized to 20 min, as A 652 increased with time up to 20 min and then plateaued, indicating reaction equilibrium. 3.4 Steady-state kinetics studies of Bi 2 Fe 4 O 9 /rGO NCs To further characterize the catalytic performance of Bi 2 Fe 4 O 9 /rGO NCs, enzymatic kinetic analyses were performed to determine the Michaelis-Menten kinetic parameters K m and V max [28]. Under the optimal experimental conditions, based on the Michaelis-Menten equation (Eq. 2), steady-state kinetic analysis was conducted by changing the substrate concentration. \(\:\text{V}\text{=}\text{(}{\text{V}}_{\text{max}}\bullet\text{[}\text{S}\text{])}/\text{(}{\text{K}}_{\text{m}}\text{+[}\text{S}\text{])}\) (Eq. 2) where V is the initial reaction rate, V max is the maximum reaction rate, [ S ] is the substrate concentration, K m is the Michaelis-Menten constant, which can determine the affinity between the enzyme and the substrate. The calculation of V can be done by Eq. 3: \(\:\text{V}\text{=}\text{∆}\text{C}/\text{∆}\text{t}\text{=}\text{(}\text{∆}\text{A}/\text{ε}\text{b}\text{)}/\text{∆}\text{t}\) (Eq. 3) where \(\:\text{C}\) represents the concentration of oxTMB and t represents the reaction time. The change in oxTMB concentration Δ C can be calculated according to the Lambert-Beer law, where A refers to the absorbance at 652 nm in the absorption spectrum, the corresponding molar absorptivity ε = 3.9×10 4 M − 1 ·cm − 1 [19], b is the width of the cuvette (1 cm). Taking the reciprocal of both sides of the Michaelis-Menten equation, the Lineweaver-Burk double reciprocal equation is obtained in Eq. 4: \(\:\text{1}/\text{V}\text{=}\text{(}{\text{K}}_{\text{m}}/{\text{V}}_{\text{max}}\text{)}\bullet\text{(}\text{1}/\text{[}\text{S}\text{]}\text{)}\text{+}\text{(}\text{1}/{\text{V}}_{\text{max}}\text{)}\) (Eq. 4) Plotting 1/ V against 1/[ S ] results in a straight line. The intercept of this line on the x-axis is the absolute value of 1/ K m , while the intercept on the y-axis is 1/ V max . The Michaelis-Menten equation curves and corresponding Lineweaver-Burk plots of Bi 2 Fe 4 O 9 /rGO for TMB and H 2 O 2 as substrates are shown in Fig. 5 , the K m values of for TMB and H 2 O 2 are 0.09 mM and 0.24 mM respectively. The V max values for TMB and H 2 O 2 were 3.23×10 − 8 M/s and 1.93×10 − 8 M/s, respectively. Compared to Fig. S6 and Table S1 , the K m of Bi 2 Fe 4 O 9 /rGO is lower than that of pure pure Bi 2 Fe 4 O 9 , indicating improved affinity for TMB and H 2 O 2 , which aligns well with DFT calculations. While its V max is significantly higher, confirming a faster catalytic reaction rate. Notably, while the K m of Bi 2 Fe 4 O 9 /rGO for TMB is slightly higher than the reported Bi 2 Fe 4 O 9 values [19], this discrepancy likely stems from differences in material morphology. Comparison of the kinetic parameters of various catalysts in Table S1 also demonstrates that Bi 2 Fe 4 O 9 /rGO exhibits superior catalytic performance. 3.5 Colorimetric sensing of GSH GSH exhibits potent regulatory effects on redox reactions and can inhibit the oxidation of TMB by H 2 O 2 (Fig. 6 a). Under optimized conditions, the performance of the Bi 2 Fe 4 O 9 /rGO NCs-based colorimetric sensor for GSH detection was evaluated. As depicted in Fig. 6 b, increasing GSH concentration led to a gradual fading of the blue color of the detection system, accompanied by a decrease in A 652 . A good linear relationship was observed between GSH concentration and A 652 , with the inset of Fig. 6 b showing a linear response over 10–250 nM (y= -0.0013x + 0.6373, R 2 = 0.9930). The limit of detection (LOD) was calculated as 3.1 nM, determined by LOD=3 σ / S , where σ is the standard deviation (SD) of eight replicate blank measurements and S is the slope of the calibration curve. As summarized in Table S2, the proposed sensor exhibits a wider linear range and lower LOD compared to most previously reported colorimetric GSH sensors, demonstrating its competitive analytical performance for GSH quantification. To assess the selectivity of the sensor, 13 potential interfering small molecules and ions (Ala, Cys, Arg, Lys, DA, UA, AA, Lac, Glu, Fru, K + , Na + , Mg 2+ , Ca 2+ ) were individually introduced into the reaction system at a concentration twice that of GSH. Notably, interfering substances including Cys、DA、UA and AA also exhibit inherent reducing properties, which could theoretically compete with GSH to inhibit TMB oxidation by H 2 O 2 . However, as shown in Fig. 6 c, GSH exerted the most pronounced inhibitory effect on TMB oxidation, resulting in the largest decrease in A 652 . This distinct difference in inhibitory potency confirms the superior selectivity of the Bi 2 Fe 4 O 9 /rGO-based colorimetric method for GSH detection. The reproducibility of the sensor was evaluated by performing five replicate measurements at GSH concentrations of 50, 100, and 200 nM, yielding relative standard deviations (RSDs) of 1.1–2.5% (n = 5), which confirms the good reproducibility of the method. 3.6 Detection of spiked GSH in FBS To verify the applicability of the Bi 2 Fe 4 O 9 /rGO-based probe in complex biological matrices, GSH detection was performed in deproteinated FBS using the standard addition method. As shown in Fig. 6 d, the calibration curve for FBS matrix (y = 0.0009x + 0.0868, R 2 =0.9964) exhibits a slightly lower slope and higher intercept than the buffer system (y = 0.0012x + 0.0849, R 2 =0.9960), reflecting the biological matrix effect caused by endogenous reducing interferents in FBS [38]. Based on the FBS standard addition curve, the endogenous GSH concentration in FBS was calculated to be 96.4 nM. Spiked recovery experiments were further performed to evaluate the accuracy of the method (Table 2 ). The results showed that the recovery rates of spiked GSH in FBS ranged from 94.4% to 99.3%, which is within the acceptable range for biological sample quantification. These results confirm that the Bi 2 Fe 4 O 9 /rGO-based colorimetric method can effectively correct for matrix effects in FBS and achieve accurate and reliable detection of GSH, supporting its practical application in complex biological samples. Table 2 Spiked recovery analysis of GSH in FBS Sample Spiked (nM) Found (nM) Recovery (%) RSD (%, n = 5) FBS 0 96.4 - - 20 116.0 98.0 2.3 50 145.7 98.6 2.2 100 195.7 99.3 1.5 150 239.4 95.3 1.8 200 293.1 98.3 1.4 250 332.4 94.4 1.6 4 Conclusions In this work, Bi 2 Fe 4 O 9 /rGO NCs were successfully synthesized via a hydrothermal method. The introduction of rGO effectively improved the dispersion of Bi 2 Fe 4 O 9 nanoparticles and significantly enhanced their POD-like activity. Steady-state kinetic analysis and DFT calculations revealed that the nanocomposites exhibited stronger substrate affinity and lower adsorption energies for TMB and H 2 O 2 , accounting for the improved catalytic performance. Leveraging the inhibitory effect of GSH, a colorimetric sensing platform was constructed, achieving a linear range of 10–250 nM and a limit of detection of 3.1 nM. Satisfactory spiked recoveries (94.4%-99.3%) in FBS samples demonstrated its potential for practical detection in complex biological matrices. Declarations Competing interests The authors declare no competing interests. Funding This work was funded by the National Natural Science Foundation of China (11805154, 21804135), Lanzhou Youth Science and Technology Talent Innovation Project (No. 2025-QN-085), Longyuan Youth Project in Gansu Province (Jinxiu Guo), Key Laboratory of Eco-functional Polymer Materials (Northwest Normal University, KF-24-05). Author Contribution Jinxiu Guo: Data curation, Investigation, Methodology, Writing-original draft. Haozhe Jia: Data curation, Investigation. Runtian Ma: Visualization, Writing - review & editing, Funding acquisition. Zuoxiang Ma: Formal analysis, Methodology, Supervision. Yanjun Cui: Funding acquisition, Supervision. Bing Hu: Investigation, Validation, Supervision. Zhifang Zhang: Writing-review & editing. Haijiao Xie: Methodology, Software. Acknowledgement This work was financially supported by the National Natural Science Foundation of China (11805154, 21804135), Lanzhou Youth Science and Technology Talent Innovation Project (No. 2025-QN-085), Longyuan Youth Project in Gansu Province (Jinxiu Guo), Key Laboratory of Eco-functional Polymer Materials (Northwest Normal University, KF-24-05). Data availability All data supporting the findings of this study are available within the paper and its Supplementary Information. References Li W, Li M, Qi J (2021) Nano-drug design based on the physiological properties of glutathione. Molecules 26(18): 5567. https://doi.org/10.3390/molecules26185567 Ketterer B, Coles B, Meyer DJ (1983) The role of glutathione in detoxication. Environmental Health Perspectives 49: 59–69. https://doi.org/10.2307/3429581 Gasmi A, Nasreen A, Lenchyk L, et al (2025). An update on glutathione's biosynthesis, metabolism, functions, and medicinal purposes. Current Medicinal Chemistry 31(29): 4579–4601. https://doi.org/10.2174/0109298673251025230919105818 Ma F, Li L, Xu Z, et al (2025) Prognostic modeling and immune infiltration analysis in hepatocellular carcinoma using glutathione metabolism-associated genes. European Journal of Medical Research 30: 1141. https://doi.org/10.1186/s40001-025-03403-8 Albiach-Delgado A, Moreno-Casillas JL, Ettabaa-Bahji Y, et al (2024) Portable point-of-care surface enhanced Raman scattering spectroscopy for the quantification of glutathione in whole blood microsamples. Talanta 279: 126566. https://doi.org/10.1016/j.talanta.2024.126566 Tsiasioti A, Tzanavaras PD (2023) Determination of glutathione and glutathione disulfide using liquid chromatography: a review on recent applications. Microchemical Journal 193: 109157. https://doi.org/10.1016/j.microc.2023.109157 Xie J, Cheng D, Li P, et al (2021) Au/metal-organic framework nanocapsules for electrochemical determination of glutathione. ACS Applied Nano Materials 4(5): 4853–4862. https://doi.org/10.1021/acsanm.1c00394 Xi S, Wang L, Cheng M, et al (2022) Developing a DNA logic gate nanosensing platform for the detection of acetamiprid. RSC Advances 12 (42): 27421–27430. https://doi.org/10.1039/D2RA04794B Jiang WC, Shi YH, Li XM, et al (2026) Conjugated precursors of isomers to enhance the peroxidase-like activity of carbon dots nanozymes for constructing multi-scenario sensing systems for glutathione and glutathione S-transferase. Journal of Colloid and Interface Science 701: 138768. https://doi.org/10.1016/j.jcis.2025.138768 Nguyen VAN, Vu TH, Nguyen PT, et al (2025) Dual-mode optical detection of sulfide ions using copper-anchored nitrogen-doped graphene quantum dot nanozymes. Biosensors 15(8): 528. https://doi.org/10.3390/bios15080528 Gao L, Zhuang J, Nie L, et al (2007) Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nature Nanotechnology 2(9): 577–583. https://doi.org/10.1038/nnano.2007.260 Fu R, Ma Z, Zhao H, et al (2023) Research progress in iron-based nanozymes: catalytic mechanisms, classification, and biomedical applications. Analytical Chemistry 95(29): 10844–10858. https://pubs.acs.org/doi/10.1021/acs.analchem.3c01005 Han J, Chen Y, Xiang X, et al (2024) A comparative analysis of the antibacterial spectrum of ultrasmall manganese ferrite nanozymes with varied surface modifications. ACS Applied Materials & Interfaces 16(12): 14385–14404. https://doi.org/10.1021/acsami.3c16490 Murugan C, Park S (2023) Cerium ferrite@ molybdenum disulfide nanozyme for intracellular ROS generation and photothermal-based cancer therapy. Journal of Photochemistry and Photobiology A: Chemistry 437: 114466. https://doi.org/10.1016/j.jphotochem.2022.114466 Feng L, Liu B, Xie R, et al (2021) An ultrasmall SnFe 2 O 4 nanozyme with endogenous oxygen generation and glutathione depletion for synergistic cancer therapy. Advanced Functional Materials 31(5): 2006216. https://doi.org/10.1002/adfm.202006216 Yang J, Gao J, Fu P, et al (2019) Synthesis and gas sensing properties of Bi 2 Fe 4 O 9 nanosheets. Materials Research Express 6(9): 095083. https://doi.org/10.1088/2053-1591/ab31b3 Sun H, Liu Y, Zhang Y, et al (2014) Synthesis of Bi 2 Fe 4 O 9 /reduced graphene oxide composite by one-step hydrothermal method and its high photocatalytic performance. Journal of Materials Science: Materials in Electronics 25(10): 4212–4218. https://doi.org/10.1007/s10854-014-2151-4 Zeng T, Liu Z, Huang G, et al (2022) Visible-light photovoltaic effect in multiferroic Bi 2 Fe 4 O 9 thin film. Materials Letters 309: 131411. https://doi.org/10.1016/j.matlet.2021.131411 Razavi M, Barras A, Ifires M, et al (2022) Colorimetric assay for the detection of dopamine using bismuth ferrite oxide (Bi 2 Fe 4 O 9 ) nanoparticles as an efficient peroxidase-mimic nanozyme. Journal of Colloid and Interface Science 613: 384–395. https://doi.org/10.1016/j.jcis.2022.01.041 Wang Z, Zhang R, Yan X, et al (2020) Structure and activity of nanozymes: Inspirations for de novo design of nanozymes. Materials Today 41: 81–119. https://doi.org/10.1016/j.mattod.2020.08.020 Neal C J, Kolanthai E, Wei F, et al (2024) Surface chemistry of biologically active reducible oxide nanozymes. Advanced Materials 36(10): 2211261. https://doi.org/10.1002/adma.202470073 Yang J, Dai H, Sun Y, et al (2022) 2D material-based peroxidase-mimicking nanozymes: catalytic mechanisms and bioapplications. Analytical and Bioanalytical Chemistry 414(9): 2971–2989. https://doi.org/10.1007/s00216-022-03985-w Rajarathinam T, Jayaraman S, Kim CS, et al (2025) Two-dimensional nanozyme nanoarchitectonics customized electrochemical bio diagnostics and lab-on-chip devices for biomarker detection. Advances in Colloid and Interface Science 341: 103474. https://doi.org/10.1016/j.cis.2025.103474 Gao H, Yu H, Yang S, et al (2024) Ultrasensitive detection of H 2 O 2 via electrochemical sensor by graphene synergized with MOF-on-MOF nanozymes. Microchimica Acta 191(8): 482. https://doi.org/10.1007/s00604-024-06541-8 Qi Y, Li B, Song D, et al (2023) Ultrafast colorimetric detection of Cr (VI) based on competition of 8-HQ to Cr (VI) and TMB oxides using GO/AuNPs nanocomposites as peroxidase mimic. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 297: 122722. https://doi.org/10.1016/j.saa.2023.122722 Peng D, Que M, Deng X, et al (2023) Mn 3 O 4 nanoparticles decorated porous reduced graphene oxide with excellent oxidase-like activity for fast colorimetric detection of ascorbic acid. Microchimica Acta 190(6): 243. https://doi.org/10.1007/s00604-023-05822-y Grimme S., Antony J., Ehrlich S., et al (2010) A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. Journal of Chemical Physics 132 (15): 154104. https://doi.org/10.1063/1.3382344 Rodriguez JMG, Hux NP, Philips SJ, et al (2019) Michaelis-Menten graphs, Lineweaver-Burk plots, and reaction schemes: Investigating introductory biochemistry students’ conceptions of representations in enzyme kinetics. Journal of Chemical Education 96(9): 1833–1845. https://doi/10.1021/acs.jchemed.9b00396 Chang Y, Zhang Y, Hu T, et al (2023) Carbonaceous material modified MoO 2 nanospheres with oxygen vacancies for enhanced visible-light photocatalytic oxidative coupling of benzylamine. Molecules 28(12): 4739. https://doi.org/10.3390/molecules28124739 Charbouillot T, Brigante M, Mailhot G, et al (2011) Performance and selectivity of the terephthalic acid probe for •OH as a function of temperature, pH and composition of atmospherically relevant aqueous media. Journal of Photochemistry and Photobiology A: Chemistry 222(1): 70–76. https://doi.org/10.1016/j.jphotochem.2011.05.003 Kresse GG, Furthmüller JJ (1996) Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set. Physical Review B 54: 11169. https://doi.org/10.1103/PhysRevB.54.11169 Blochl PE (1994) Projector augmented-wave method. Physical Review B 50: 17953–17979. https://doi.org/10.1103/PhysRevB.50.17953 Perdew JP, Burke K, Ernzerhof M (1996) Generalized gradient approximation made simple. Physical Review Letters 77(18): 3865. https://doi.org/10.1103/PhysRevLett.77.3865 Crowe KM (2014) Optimizing protein precipitation efficiency for assessing the contribution of low molecular weight compounds to serum antioxidant capacity. Clinical biochemistry 47(15): 116–118. http://dx.doi.org/10.1016/j.clinbiochem.2014.06.021 Patel J, Parmar M, Shahabuddin S, et al (2025) Designing a Z-scheme rGO-SnS 2 synergistic photocatalyst for photocatalytic mineralization of atrazine and 2,4-dichlorophenoxyacetic acid and applying machine learning for predictive modelling of photocatalytic performance. Nanoscale Advances 7(11): 3485–3507. https://doi.org/10.1039/d5na00143a Wang X, Li QZ, Zhao Y, et al (2025) Recent advances in oxidase-like nanozymes: mechanisms, prediction models, and applications. ACS Applied Materials & Interfaces 17(49) 66110–66150. https://doi.org/10.1021/acsami.5c15196 Xu AY, Blanco MA, Castellanos MM, et al (2023) Role of domain-domain interactions on the self-association and physical stability of monoclonal antibodies: effect of pH and salt. The Journal of Physical Chemistry B 127 (39): 8344–8357. https://doi.org/10.1021/acs.jpcb.3c03928 Dega NK, Ganganboina AB, Tran HL, et al (2022) BSA-stabilized manganese phosphate nanoflower with enhanced nanozyme activity for highly sensitive and rapid detection of glutathione. Talanta 237: 122957. https://doi.org/10.1016/j.talanta.2021.122957 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx graphicalabstract.jpg Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 24 Apr, 2026 Reviews received at journal 23 Apr, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviews received at journal 30 Mar, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers invited by journal 28 Mar, 2026 Editor assigned by journal 25 Mar, 2026 Submission checks completed at journal 25 Mar, 2026 First submitted to journal 20 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9180770","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":616748146,"identity":"a8c6316a-ca90-4349-84db-164686774761","order_by":0,"name":"Jinxiu Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYJACA4YKKIuHeC1nSNXCwNhGihb5iOQDxbzzau35ZyQwPnjbxiBvTkiL4Y20BMOZ244nzriRwGw4t43BcGcDIS0zcgwMPm47lsBwI4FNmreNIcHgADFaEuccs5e/kcD+mygt8hIgWxpqGDcAbWEmSosBz7MEwxnHDiRuPPOwWXLOOQnDDQRtaU8+ZsxTU2cvdzz54Ic3ZTbyhG05wMBmwMBwGMhkbAASEgTUg2xpYGB+wMBQR1jlKBgFo2AUjFwAADv/QHHUE5q4AAAAAElFTkSuQmCC","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Jinxiu","middleName":"","lastName":"Guo","suffix":""},{"id":616748147,"identity":"fc50a99d-4d71-4951-849d-116da3b0f5ee","order_by":1,"name":"Haozhe Jia","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Haozhe","middleName":"","lastName":"Jia","suffix":""},{"id":616748150,"identity":"e301c7b5-e7b6-47d3-be83-3987fa508309","order_by":2,"name":"Runtian Ma","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Runtian","middleName":"","lastName":"Ma","suffix":""},{"id":616748152,"identity":"e56ef1b5-12a4-4ea0-a43f-546dbbd86e95","order_by":3,"name":"Zuoxiang Ma","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zuoxiang","middleName":"","lastName":"Ma","suffix":""},{"id":616748159,"identity":"0386c315-bf77-4760-971b-6d75708b4f04","order_by":4,"name":"Yanjun Cui","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanjun","middleName":"","lastName":"Cui","suffix":""},{"id":616748162,"identity":"810d219e-e269-4979-9cdd-90229016159d","order_by":5,"name":"Bing Hu","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Hu","suffix":""},{"id":616748163,"identity":"f2dfff52-e54f-4185-9e2f-a9b02ff70408","order_by":6,"name":"Zhifang Zhang","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhifang","middleName":"","lastName":"Zhang","suffix":""},{"id":616748164,"identity":"1d6c8768-fdec-4eb4-a61f-22f8a40d7888","order_by":7,"name":"Haijiao Xie","email":"","orcid":"","institution":"Hangzhou Yanqu Information Technology Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Haijiao","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2026-03-20 16:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9180770/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9180770/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106085748,"identity":"ed98b8f9-a888-469e-a589-c6a7217ab9b4","added_by":"auto","created_at":"2026-04-03 09:37:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1174849,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic illustration of the synthetic route of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs; FESEM images of (b) pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e and (c-f) Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs prepared with varying volumes of GO dispersion (50, 100, 200, 400 μL); (g) TEM image of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-50; (h) XRD pattern, (i) XPS survey spectra, (k-l) high-resolution XPS spectra and (m) Raman spectra of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9180770/v1/919f1de103027ef9de5c3229.png"},{"id":106085749,"identity":"44142d68-84fa-4bad-9974-17f46ca215ec","added_by":"auto","created_at":"2026-04-03 09:37:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":248944,"visible":true,"origin":"","legend":"\u003cp\u003e(a) UV-vis absorption spectra of different reaction systems in HAc-NaAc buffer (pH 4.5) after incubation at 40 ℃ for 20 min; (b) effects of radical scavengers with varying concentrations on the absorbance of TMB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO system at 652 nm, (c) effects of 1 mM radical scavengers on the absorbance of TMB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e system at 652 nm; (d) fluorescence spectra of TA as a fluorescent probe (λ\u003csub\u003eex\u003c/sub\u003e = 315 nm)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9180770/v1/bb0019da26f44cc0cb10d5d0.png"},{"id":106094655,"identity":"40e221ca-6cfa-441e-ae69-ce2aaddffa59","added_by":"auto","created_at":"2026-04-03 11:43:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126582,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the interaction between catalyst and substrates\u003c/p\u003e\n\u003cp\u003e(a) Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e and TMB, (b) Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, (c) Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO and TMB, (d) Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9180770/v1/1baf57075ca26b6f6fe18473.png"},{"id":106094402,"identity":"d39764b2-0bb6-4c25-95a7-6dcde9db50e6","added_by":"auto","created_at":"2026-04-03 11:42:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1040713,"visible":true,"origin":"","legend":"\u003cp\u003eA\u003csub\u003e652\u003c/sub\u003e of the TMB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO system at different (a) pH values (3.5-6.0), (b) concentrations of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO (0-1.35 μg/mL), (c) concentrations of TMB (0-0.128 mM), (d) concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0-1.8 mM), (e) temperature (25-70 ℃) and (f) incubation time (0-60 min)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9180770/v1/6e7794aed31a2ba5bbbf092d.png"},{"id":106085750,"identity":"a7fdd14d-bc49-4da5-8e99-fb945eb86ac3","added_by":"auto","created_at":"2026-04-03 09:37:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1159512,"visible":true,"origin":"","legend":"\u003cp\u003eMichaelis-Menten curves of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO with (a) TMB and (b) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e used as substrate, Lineweaver-Burk plots of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO from the activity data of the varied concentration of (c) TMB and (d) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9180770/v1/a96c5b0b8e229be2760fcd70.png"},{"id":106085752,"identity":"9b79937b-1fed-4aa1-93f4-9ac6e64475e4","added_by":"auto","created_at":"2026-04-03 09:37:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":295362,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic diagram of a colorimetric sensor GSH based on Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO; (b) A\u003csub\u003e652\u003c/sub\u003e of TMB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO solutions in presence of various GSH concentrations; (c) selectivity of the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-based colorimetric sensor for GSH over 13 potential interfering species (the interferents were tested at 400 nM, while GSH was at 200 nM); (d) calibration curves for GSH detection in buffer and FBS using the standard addition method\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9180770/v1/e8add7ed671eef84c003ed50.png"},{"id":106402007,"identity":"8b4795d2-19a6-4cbd-a56d-1b75c3241c79","added_by":"auto","created_at":"2026-04-08 09:10:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4805171,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9180770/v1/81789c12-d9d1-44dc-98db-5d6a6dea2091.pdf"},{"id":106094996,"identity":"6f559588-ba6d-4ab7-bfdf-94c62b9a31ac","added_by":"auto","created_at":"2026-04-03 11:43:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2790708,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9180770/v1/28f6dae164341736934276f8.docx"},{"id":106094609,"identity":"bfefc87a-2e6c-4f51-a26d-90bd39ad9dcc","added_by":"auto","created_at":"2026-04-03 11:42:58","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":110424,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180770/v1/9613bb8ddc57529229d7d535.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bi2Fe4O9/reduced graphene oxide nanocomposites with enhanced peroxidase activity for sensitive glutathione detection","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eGlutathione (GSH) is a tripeptide consisting of glutamic acid, cysteine, and glycine condensed by peptide bonds [1]. GSH has become an important metabolic regulators and biomarkers due to its vital physiological functions, such as detoxication [2], protection against oxidative and free radical damage [3] and defense against liver injury [4]. Therefore, it is indispensable to establish an efficient and sensitive method to determine GSH. Spectroscopy, chromatography and electrochemical methods have been applied in the detection of GSH [5\u0026ndash;7]. These traditional analytical methods each have their inherent advantages, but they are plagued by some limitations involving expensive equipment, cumbersome sample pretreatment, time-consuming procedures, excessive solvent usage, laborious electrode modification and sensitivity to matrix interference [8]. In contrast, ultraviolet-visible (UV-vis) spectrophotometry based on the inhibition of nanozyme-catalyzed 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by GSH has emerged as a promising alternative. This method combines the advantages of low cost, simple operation, rapid readout, and visual color change, making it suitable for on-site and large-scale sample analysis [9].\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eNatural enzymes, represented by horseradish peroxidase (HRP), have long been the gold standard for catalytic reactions in biosensing. However, their practical application is severely hindered by inherent limitations: complex and costly preparation/purification processes, poor stability under extreme conditions, and difficulty in recycling and reuse. For instance, HRP loses over 50% of its activity below pH 3.0 or at temperatures above 50 ℃ [10]. Iron-based nanozymes, as one of the earliest developed synthetic enzyme mimics, have successfully addressed these drawbacks [11]. They feature facile synthesis, low toxicity, excellent stability, and tunable catalytic performance, making them ideal candidates for replacing natural enzymes in various fields [12].\u003c/p\u003e \u003cp\u003eAmong iron-based nanozymes, metal ferrite nanomaterials have garnered extensive attention due to their versatile enzyme-mimetic activities and structural adjustability. Han et al. synthesized MnFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes modified with different functional groups (-COOH, -NH\u003csub\u003e2\u003c/sub\u003e, -PEG) and found that the surface functionalization not only enhanced their peroxidase activity but also endowed them with tailored antibacterial properties. MnFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-COOH showed preferential inhibition of Gram-positive bacteria, while MnFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NH\u003csub\u003e2\u003c/sub\u003e exhibited the most significant and broad-spectrum antibacterial activity [13]. Lu and colleagues constructed MoS\u003csub\u003e2\u003c/sub\u003e-bPEI-CeFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoflowers with a core-shell structure, where MoS\u003csub\u003e2\u003c/sub\u003e served as the photothermal core and branched polyethylenimine (bPEI)-modified CeFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles (NPs) acted as the catalytic shell. The CeFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs exhibited superior Fenton-like activity, efficiently generating intracellular reactive oxygen species (ROS), while the MoS\u003csub\u003e2\u003c/sub\u003e core enhanced photothermal therapy efficacy, achieving a synergistic effect in tumor treatment [14]. Feng et al. further reported that SnFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs possess dual catalase-like and peroxidase-like activities, showcasing promising potential for tumor therapy [15].\u003c/p\u003e \u003cp\u003eBi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs, a functional metal oxide with excellent gas sensing, photocatalytic, and photoelectric conversion properties [16\u0026ndash;18], have been identified as promising peroxidase mimics and successfully used for the colorimetric detection of dopamine [19]. Fe active sites and the d-orbitals of Fe\u003csup\u003e3+\u003c/sup\u003e in Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs was considered to operate in the catalytic oxidation of TMB in presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [19]. However, pristine Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs tend to form irregular sheet-like aggregates due to van der Waals forces and hydrogen bonding interactions. This aggregation not only reduces their specific surface area but also shields the active Fe\u003csup\u003e3+\u003c/sup\u003e sites, limiting the exposure of catalytic centers and thus compromising their enzyme-mimetic activity. Therefore, optimizing the morphology and dispersion of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs is essential to enhance their catalytic performance for practical applications.\u003c/p\u003e \u003cp\u003eModulation of properties of nanozymes can be performed by controlling the structure, morphology, surface properties, and combining with other materials [20, 21]. Among various modifiers, two-dimensional (2D) nanomaterials stand out as ideal carriers or synergistic components for nanozyme optimization. Their unique planar structure provides a large specific surface area, which promotes nanozyme dispersion and increases the exposure of active sites; meanwhile, the abundant functional groups on their surface enhance the affinity between the nanozyme and substrates, and the high electron conductivity accelerates charge transfer during catalytic reactions [22, 23]. Graphene, graphene oxide (GO), and reduced graphene oxide (rGO) are typical 2D materials that have been widely used in the construction of TMB/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-based biosensors [24\u0026ndash;26].\u003c/p\u003e \u003cp\u003eTo address the aggregation issue of pristine Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs and further enhance their peroxidase (POD)-like activity, we fabricated Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO nanocomposites by integrating Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e with GO via a hydrothermal method. The nanocomposites can efficiently catalyze the oxidation of TMB to form deep-blue oxTMB in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, enabling quantitative evaluation of peroxidase activity through absorbance measurement. Density functional theory (DFT) calculations were employed to gain mechanistic insights into the rGO-mediated enhancement of enzyme-mimetic activity [27]. Based on the competitive inhibition of GSH on the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-TMB-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e system, a highly sensitive and selective colorimetric method for GSH detection was constructed. Its applicability was validated in complex biological samples such as fetal bovine serum (FBS), providing a reliable analytical tool for GSH-related biological research and clinical diagnosis.\u003c/p\u003e"},{"header":"2 Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals and reagents\u003c/h2\u003e \u003cp\u003eBi(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO (\u0026ge;\u0026thinsp;99%), Fe(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;9H\u003csub\u003e2\u003c/sub\u003eO (98%), GO solution (\u0026ge;\u0026thinsp;98%, 1\u0026ndash;5 \u0026micro;m, 1\u0026ndash;2 layers, 1\u0026ndash;3 wt% dispersion in H\u003csub\u003e2\u003c/sub\u003eO, pH\u0026thinsp;=\u0026thinsp;1\u0026ndash;2), TMB (\u0026ge;\u0026thinsp;98%), p-Benzoquinone (p-BQ, 99%), terephthalic acid (TA, 99%), L-glutathione (GSH, 99%), D-(+)-glucose (Glu, 99%), lactose (Lac, 98%), fructose (Fru, 99%), uric acid (UA, \u0026ge;\u0026thinsp;98.0%), D-alanine (Ala, 98%), D-cysteine (Cys, 97%), L-arginine (Arg, \u0026ge;\u0026thinsp;99%), L(+)-ascorbic acid (AA, 99%), KCl (\u0026ge;\u0026thinsp;99.8%), NaCl (99.99%), MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO (\u0026ge;\u0026thinsp;98%), CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO (98%), FBS were purchased from Shanghai Titan Scientific Co., Ltd. (Shanghai, China). HNO\u003csub\u003e3\u003c/sub\u003e (65\u0026ndash;68% w/w), acetone and dopamine hydrochloride (DA, 98.0%) were bought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). NaAc (99%) and ethanol were gotten from Guangdong Guanghua Sci-Tech Co., Ltd. (Shantou, China). H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30 wt%) was purchased from Fuchen Chemical Reagent Factory (Tianjin, China). HAc (\u0026ge;\u0026thinsp;99.8%) was obtained from Chengdu Kelong Chemical Co., Ltd (Chengdu, China). NaOH was bought from Damao Chemical Reagent Co., Ltd. (Tianjin, China). Isopropanol (IPA) was bought by Shanghai Jianxin Chemical Co., Ltd. (Shanghai, China). NaN\u003csub\u003e3\u003c/sub\u003e (\u0026ge;\u0026thinsp;99.5%) was gotten from Bomei Biotechnology Co., Ltd. (Hefei, China). Unless stated otherwise, all other reagents were of analytical grade, deionized water (18.2 MΩ\u0026middot;cm at 25\u0026deg;C) was used throughout the experiments, and all reagents were used as received without further purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs were synthesized via a slightly modified hydrothermal method reported previously [17]. Briefly, 0.485 g of Bi(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO and 0.404 g of Fe(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;9H\u003csub\u003e2\u003c/sub\u003eO were first dissolved in a mixture of 13 mL of deionized water and 2 mL of HNO₃, followed by magnetic stirring at room temperature for 30 min. An 8 M NaOH solution was added dropwise to the above mixture until the pH reached 10\u0026ndash;11, resulting in the formation of a red-brown suspension. Subsequently, 100 \u0026micro;L of GO dispersion was introduced under continuous stirring. The resulting suspension was filtered and rinsed with deionized water until the filtrate reached neutral pH. The obtained brown precipitate was mixed with 40 mL of 12 M NaOH solution, and the mixture was vigorously stirred for 1 h. The suspension was then transferred to a Teflon-lined stainless-steel autoclave, which was heated at 200 ℃ for 24 h. After cooling to room temperature naturally, the precipitate was collected via centrifugation, rinsed three times alternately with deionized water and ethanol, and dried at 60 ℃ to obtain the final product. For comparison, pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs were synthesized via the identical procedure but without the addition of the GO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs\u003c/h2\u003e \u003cp\u003eThe morphologies and elemental compositions of the samples were characterized using a field-emission scanning electron microscope (FESEM, Regulus 8100, Hitachi, Japan), a transmission electron microscope (TEM, Talos F200x, FEI, USA), and energy dispersive X-ray spectroscopy (EDS) mapping. The chemical states of elements were analyzed via X-ray photoelectron spectroscopy (XPS, K-Alpha, Thermo Scientific, USA). Fourier transform infrared spectroscopy (FT-IR) was used to identify the functional groups in the materials by a FT-IR-650 spectrometer (Gangdong Co. Ltd. Tianjin, China). The rGO was characterized by Raman spectroscopy (LabRAM HR Evolution, Horiba, Japan) using a laser with an excitation wavelength of 633 nm. Thermogravimetric (TG) and derivative thermogravimetric (DTG) analyses were performed on a thermal analyzer (STA 449 F5 Jupiter, NETZSCH, Germany) at a heating rate of 10\u0026deg;C/min. The crystal structures were determined via X-ray diffraction (XRD, XD-3, Purkinje General Instrument Co., Ltd., Beijing, China) with Cu Kα radiation, at a scanning rate of 2\u0026deg;/min from 10\u0026deg; to 80\u0026deg;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Catalytic activity of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs and steady-state kinetic study\u003c/h2\u003e \u003cp\u003eIn order to evaluate the catalytic activity of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO, we performed the catalytic experiment of the composites for the oxidation of TMB in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The typical assay was carried out by adding 96 \u0026micro;L of 1 mM TMB solution (in acetone), 60 \u0026micro;L of 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution (in H\u003csub\u003e2\u003c/sub\u003eO) and 25 \u0026micro;L of 30 \u0026micro;g/mL catalyst into 819 \u0026micro;L of 0.1 M acetic acid-sodium acetate (HAc-NaAc) buffer solution (pH 4.5) with reacted at 40 ℃ for 20 min. All chemical reagents were freshly prepared and all reactions were monitored at 652 nm by a Ultraviolet-visible (UV-vis) spectrophotometer (T2602S, YOKE, Shanghai, China). The steady-state kinetic values were determined by changing the concentration of TMB (0-0.128 mM) or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0-1.8 mM) as substrate in 0.1 M acetic acid buffer (pH\u0026thinsp;=\u0026thinsp;4.5) with 0.75 \u0026micro;g/mL catalyst. The Michaelis-Menten parameters were calculated by using the Lineweaver-Burk double reciprocal plot according to the Michaelis-Menten model [28].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Detection of ROS\u003c/h2\u003e \u003cp\u003eTo clarify the POD-like catalytic mechanism, radical trapping experiments were conducted in 0.1 M HAc-NaAc buffer (pH 4.5) at 40\u0026deg;C for 20 min. The reaction system contained the catalyst at 0.75 \u0026micro;g/mL, TMB at 0.096 mM, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at 0.9 mM. Specific scavengers were introduced at optimized concentrations to selectively quench distinct reactive species: IPA for hydroxyl radicals (\u0026bull;OH), NaN\u003csub\u003e3\u003c/sub\u003e for singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e), and p-BQ for superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) [29]. Furthermore, the generation of \u0026bull;OH was verified using TA as a fluorescent probe [30]. 0.5 mM TA, 0.9 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 0.75 \u0026micro;g/mL catalyst were mixed in the above buffer, followed by incubation at 40℃ for 20 min. Fluorescence emission spectra were recorded in the range of 400\u0026ndash;550 nm at the maximum excitation wavelength (λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;315 nm) using a Shimadzu RF-600 fluorometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 DFT Calculations\u003c/h2\u003e \u003cp\u003eAll theoretical calculations were carried out with the Vienna Ab Initio Simulation Package (VASP) [31] integrated with the projector-augmented-wave (PAW) method [32]. The simulations were grounded in spin-polarized DFT within the generalized gradient approximation (GGA) framework using the Perdew-Burke-Ernzerhof (PBE) functional [33]. The on-site Coulomb interaction of Fe 3d electrons was described using the DFT\u0026thinsp;+\u0026thinsp;U method (Dudarev scheme) with an effective U value (Ueff) of 3.0 eV. Long-range van der Waals interactions were incorporated via the DFT-D3 method [27], the plane-wave cutoff energy was set to 500 eV, and the energy convergence criterion was fixed at 1 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e eV. The first Brillouin zone was sampled with a 1\u0026times;1\u0026times;1 k-point Monkhorst-Pack grid for all calculations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 GSH detection assay\u003c/h2\u003e \u003cp\u003eFor colorimetric detection, GSH at varied concentrations were added to the solution containing 0.096 mM TMB, 0.9 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 0.75 \u0026micro;g/mL Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs. The final volume of the solution was adjusted to 1.0 mL with 0.1 M acetic acidic buffer (pH 4.5). The reaction mixture was incubated at 40 ℃ for 20 min, then analyzed at 652 nm to obtain the calibration curve and the limit of detection (LOD\u0026thinsp;=\u0026thinsp;3σ/K) of GSH, where σ is the standard deviation of the blank sample measurements (n\u0026thinsp;=\u0026thinsp;8) and K is the slope of the calibration curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Preparation of FBS\u003c/h2\u003e \u003cp\u003eFBS Samples were stored at -18\u0026deg;C until analysis. In order to eliminate the influence of proteins in the serum on the test results, a protein precipitant needs to be used. Noticing that using other reagents might affect the detection conditions, protein precipitation was conducted by adding acetone to serum in a ratio of 1:10 (v/v) [34]. Then the mixture was vortexed for 30 s, followed by incubation in an ice bath for 10 min, and then centrifuged at 4000 rpm for 5 min. The content and recovery rate of GSH in the FBS samples were determined using the standard addition method. Instead of GSH, the pure FBS and spiked samples were added to the above reaction solution in section \u003cspan refid=\"Sec9\" class=\"InternalRef\"\u003e2.7\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Preparation and characterization of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs\u003c/h2\u003e\n\u003cp\u003eDilute nitric acid effectively addresses the poor solubility of bismuth nitrate, but markedly lowers the pH of the precursor solution. Since the formation of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e requires a relatively high pH environment, the use of high-concentration NaOH as a mineralizer is necessitated for the successful synthesis of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e crystals [14]. To prepare Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs with varying GO mass fractions (0.375%, 0.75%, 1.5%, and 3%), different volumes of GO dispersion (50, 100, 200, and 400 \u0026micro;L) were added to the mixed solution of Bi(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO and Fe(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;9H\u003csub\u003e2\u003c/sub\u003eO, which were designated as Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-50, Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-100, Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-200, and Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-400, respectively.\u003c/p\u003e\n\u003cp\u003ePhotographs of the as-prepared samples are displayed in Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. As the GO content increases, the color of the final product deepens gradually. Comparison of FESEM images in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb-f reveals that pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs exhibit aggregated block-like morphologies, whereas Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs transform into nanosheet structures with the introduction of GO. When the GO addition volume reaches 100 \u0026micro;L (corresponding to a mass fraction of 0.75%), the sheet-like Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs exhibit excellent dispersibility and uniformity, with a length of 500\u0026ndash;1000 nm with a thickness of 100\u0026ndash;200 nm. Further increasing the GO addition leads to a reduction in the material\u0026rsquo;s uniformity. Thus, Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO specifically refers to Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-100 in the following text. As displayed in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg, the TEM image of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-50 clearly reveals thin and transparent lamellar structures assigned to GO nanosheets, which act as a supporting matrix to facilitate the uniform dispersion of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e sheets on its surface. The results of EDS elemental distribution mapping and analysis are presented in Fig. S2 clearly illustrate of Bi, Fe, C and O elements, which display the presence and homogeneous distribution of mentioned elements in the entire of the nanohybrids.\u003c/p\u003e\n\u003cp\u003eThe phase purity of the as-prepared Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs was examined using XRD (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eh). All characteristic diffraction peaks could be indexed to the pure mullite phase Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e of the orthorhombic structure (JCPDS No. 00-025-0090). Among them, the diffraction peaks at 61.8\u0026deg;, 56.6\u0026deg;, 47.0\u0026deg;, 39.0\u0026deg;, 37.5\u0026deg;, 33.7\u0026deg;, 29.8\u0026deg;, 28.9\u0026deg;, 28.2\u0026deg; and 14.8\u0026deg;correspond to the crystal planes (004), (332), (141), (212), (202), (130), (002), (211), (121) and (001), respectively [19]. No impurity peaks were detected, confirming that GO addition did not alter the crystal structure or phase purity of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e. Additionally, the characteristic GO (001) diffraction peak near 11.1\u0026deg; was not observed, likely due to its low content in the composite [17].\u003c/p\u003e\n\u003cp\u003eXPS was employed to analyze the chemical states of elements in the composite. The XPS survey spectrum (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ei) confirms the presence of Bi, Fe, O, and C, with a higher carbon content than pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e, verifying GO incorporation. The trace carbon in pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e likely arises from residual impurities. Bismuth elements are split into Bi 4f\u003csub\u003e7/2\u003c/sub\u003e and Bi4f\u003csub\u003e5/2\u003c/sub\u003e peaks at 164.8 eV and 159.5 eV, and these binding energy positions indicate that bismuth elements in the composite material exist in the form of Bi\u003csup\u003e3\u003c/sup\u003e⁺. High-resolution XPS spectra of Fe 2p, O 1s and C 1s are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ej-l. The Fe 2p spectrum displays peaks at 710.9 eV (Fe 2p\u003csub\u003e3/2\u003c/sub\u003e) and 724.8 eV (Fe 2p\u003csub\u003e1/2\u003c/sub\u003e), which deconvolve into Fe\u003csup\u003e2+\u003c/sup\u003e and Fe\u003csup\u003e3+\u003c/sup\u003e respectively. In addition, the two satellite peaks at 717.9 eV (Sat. I) and 732.2 eV (Sat. II) further confirm Fe\u003csup\u003e3+\u003c/sup\u003e species. The O 1s spectrum deconvolves two peaks with binding energies of 530.3 eV (lattice oxygen) and 532.0 eV (adsorbed oxygen) [19]. The C 1s spectrum shows peaks at 284.5 eV (C-C\u0026thinsp;=\u0026thinsp;C groups) and 288.6 eV (O-C\u0026thinsp;=\u0026thinsp;O groups) [17].\u003c/p\u003e\n\u003cp\u003eRaman spectroscopy was used to investigate structural changes in GO before and after composite formation. GO exhibits two characteristic Raman peaks: the D peak (1332 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, defect-induced) and G peak (1590 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicative of graphitic order). GO typically has a high I\u003csub\u003eD\u003c/sub\u003e/I\u003csub\u003eG\u003c/sub\u003e ratio due to abundant oxygenated groups and defects. However, the I\u003csub\u003eD\u003c/sub\u003e/I\u003csub\u003eG\u003c/sub\u003e ratio decreases significantly in Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003em), confirming effective GO reduction, reduced oxygenated functional groups, and partial restoration of graphitic structure [17]. Peak position shifts of the D and G bands further support GO reduction.\u003c/p\u003e\n\u003cp\u003eFT-IR spectroscopy was performed to verify the incorporation and reduction of GO in the composites (Fig. S3). For pure Bi₂Fe₄O₉ NPs, the dominant infrared absorption peaks are located in 400\u0026ndash;850 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;, assigned to the stretching/bending vibrations of Fe-O and Bi-O bonds [19]. Upon incorporation with GO, new absorption peaks emerge at ~\u0026thinsp;1030 and ~\u0026thinsp;1340 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;, attributed to the C-H bending vibration and C\u0026thinsp;=\u0026thinsp;C stretching vibration in the benzene-like ring structure of GO, respectively. The marked attenuation of oxygen-containing groups in the composite confirms the effective reduction of GO to rGO. Further TG and DTG analyses (Fig. S4) corroborate these findings: Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs exhibit more substantial weight loss than pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs over 30\u0026ndash;600℃, with pronounced DTG peaks attributed to the thermal decomposition of rGO [35], directly evidencing the successful incorporation and reduction of GO into the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e matrix.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 POD-like activity of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs\u003c/h2\u003e\nAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea (lines 1\u0026ndash;4), neither the individual TMB (line 1) nor the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e+TMB binary system (line 2) showed obvious absorption across the wavelength range of 400\u0026ndash;800 nm. For the TMB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e binary system (line 3), only weak absorption was observed, confirming spontaneous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition to trace ROS. In contrast, the ternary system (Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e+TMB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, line 4) exhibited a distinct absorption peak at 652 nm after 20 min, arising from oxTMB formation. These results demonstrate that Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs exhibit POD-like activity but no oxidase (OXD)-like activity [36]. Under identical conditions, the POD-like activities of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs with varying GO contents were assessed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, lines 5\u0026ndash;8). Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs exhibited higher POD-like activity than pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs, with activity dependent on GO loading: the highest activity was observed at 100 \u0026micro;L GO dispersion, while excess GO reduced activity, likely due to Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e aggregation and morphological heterogeneity, which diminish accessible active sites.\u003cbr /\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor cyclic reuse, the TMB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO reaction mixture was centrifuged at 10000 rpm for 5 min. The precipitate was rinsed with deionized water, then reintroduced into fresh TMB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution under the same conditions. The variation of the UV absorbance intensity of oxTMB at 652 nm is depicted in Fig. S5. After five consecutive cycles, the relative activity remained above 90%. This result confirms that Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs exhibit excellent enzyme-mimetic catalytic reproducibility and stability, which support their potential for practical sensing applications.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo elucidate the POD-like catalytic mechanism of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs, radical trapping experiments were performed employing IPA (\u0026bull;OH scavenger), NaN\u003csub\u003e3\u003c/sub\u003e (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e scavenger), and p-BQ (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e scavenger). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, both the addition of IPA and p-BQ could significantly reduce the absorbance of the TMB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO system at 652 nm, whereas NaN\u003csub\u003e3\u003c/sub\u003e had no notable effect, demonstrating that \u0026bull;OH and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e are the primary reactive species, consistent with observations for pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec) [29]. The influence of \u0026bull;OH on the catalytic effect has also been analyzed by fluorescence spectrum. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, TA exhibits negligible fluorescence in the absence of \u0026bull;OH, but reacts with \u0026bull;OH to form 2-hydroxyterephthalic acid, which emits characteristic fluorescence at 435 nm [30]. The results of lines 1\u0026ndash;4 indicate that neither H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e alone nor Bi-based materials alone can trigger \u0026bull;OH generation. In contrast, a distinct 435 nm emission peak emerged in the system containing TA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs (line 5). This confirms that Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs can activate H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to produce \u0026bull;OH. Notably, the fluorescence intensity of the system with Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs (line 6) was significantly higher than that with pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs. This verifies that Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs have superior POD-like activity. They can more efficiently activate H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate \u0026bull;OH radicals, thus facilitating TMB oxidation.\u003c/p\u003e\n\u003cp\u003eIn order to understand the reason why the addition of graphene oxide improves the catalytic performance at the atomic level, we conducted DFT calculations. A heterostructure was constructed by combining the (121) crystal plane of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e with reduced graphene oxide, and the adsorption energies (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{E}}_{\\text{ads}}\\)\u003c/span\u003e\u003c/span\u003e) of TMB and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e molecules were calculated on both the heterojunction surface and the pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e surface. The \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{E}}_{\\text{ads}}\\)\u003c/span\u003e\u003c/span\u003e was calculated according to Eq.\u0026nbsp;1:\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{E}}_{\\text{ads}}\\text{=}{\\text{E}}_{\\text{t}\\text{otal}}\\text{\u0026minus;}{\\text{E}}_{\\text{mol}}\\text{\u0026minus;}{\\text{E}}_{\\text{sub}}\\)\u003c/span\u003e\u003c/span\u003e (Eq.\u0026nbsp;1)\u003c/p\u003e\n\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{E}}_{\\text{t}\\text{otal}}\\)\u003c/span\u003e\u003c/span\u003e denotes the total energy of the slab with the adsorbed molecule, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{E}}_{\\text{sub}}\\)\u003c/span\u003e\u003c/span\u003e represents the total energy of the pristine slab, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{E}}_{\\text{mol}}\\)\u003c/span\u003e\u003c/span\u003e is the total energy of the isolated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or TMB molecule in a large vacuum box. A negative \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{E}}_{\\text{ads}}\\)\u003c/span\u003e\u003c/span\u003e value indicates an exothermic adsorption process, implying a stronger binding interaction between the molecule and the surface.\u003c/p\u003e\n\u003cp\u003eThe optimized molecular structures and the data used to calculate the adsorption energy were described in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The calculation results show that the E\u003csub\u003e\u003cem\u003eads\u003c/em\u003e\u003c/sub\u003e of TMB on Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs and Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs were \u0026minus;\u0026thinsp;2.18 and \u0026minus;\u0026thinsp;5.67 eV respectively. The E\u003csub\u003e\u003cem\u003eads\u003c/em\u003e\u003c/sub\u003e of the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (-3.44 eV) are also lower than that between the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (-1.49 eV). The lower adsorption energies indicate that Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs are more likely to bind to the substrates (TMB and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eAdsorption energy-related parameters obtained by DFT calculations.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eE\u003c/em\u003e (eV)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eTMB\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003esub\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-583.24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-1484.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-583.24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-1484.17\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003emol\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-236.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-236.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-18.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-18.10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003etotal\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-821.56\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-1725.98\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-602.83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-1505.71\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003eads\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-2.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-5.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-1.49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-3.44\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Optimization of experimental conditions\u003c/h2\u003e\nExperimental conditions were optimized to determine the steady-state kinetic parameters of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO, with the effects of pH, buffer concentration, catalyst loading, substrate concentration, reaction temperature and incubation time on the absorbance at 652 nm (A\u003csub\u003e652\u003c/sub\u003e) of the TMB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO system systematically investigated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). For pH optimization, A\u003csub\u003e652\u003c/sub\u003e increased from pH 3.5 to 4.5 and then decreased at higher pH. This was attributed to the enhanced polarity of protonated TMB, which facilitates binding to the nanozyme active center and promotes oxidation via charge effects. For HAc-NaAc buffer concentration, A\u003csub\u003e652\u003c/sub\u003e reached its maximum when the concentration of the buffer is 0.1 M because the lower concentrations failed to maintain the optimal pH 4.5, while excessive ionic strength disrupted surface electrostatic interactions via charge screening effects [37]. Catalyst loading exhibited a concentration-dependent effect for A\u003csub\u003e652\u003c/sub\u003e increased with Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO concentration up to 0.75 \u0026micro;g/mL, after which activity plateaued due to catalyst flocculation and solution darkening at higher concentrations. Thus, 0.75 \u0026micro;g/mL was selected as the optimal catalyst concentration. For substrate concentrations, A\u003csub\u003e652\u003c/sub\u003e increased with TMB and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations until reaching plateaus at 0.096 mM TMB and 0.9 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, respectively, where further increases did not significantly enhance absorbance. The activity increased from 25 to 40℃ as reaction temperature due to enhanced molecular collision, while higher temperatures reduced activity via by-product formation. Incubation time was optimized to 20 min, as A\u003csub\u003e652\u003c/sub\u003e increased with time up to 20 min and then plateaued, indicating reaction equilibrium.\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Steady-state kinetics studies of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs\u003c/h2\u003e\n\u003cp\u003eTo further characterize the catalytic performance of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs, enzymatic kinetic analyses were performed to determine the Michaelis-Menten kinetic parameters \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e [28]. Under the optimal experimental conditions, based on the Michaelis-Menten equation (Eq.\u0026nbsp;2), steady-state kinetic analysis was conducted by changing the substrate concentration.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{V}\\text{=}\\text{(}{\\text{V}}_{\\text{max}}\\bullet\\text{[}\\text{S}\\text{])}/\\text{(}{\\text{K}}_{\\text{m}}\\text{+[}\\text{S}\\text{])}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;2)\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003eV\u003c/em\u003e is the initial reaction rate, \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e is the maximum reaction rate, [\u003cem\u003eS\u003c/em\u003e] is the substrate concentration, \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e is the Michaelis-Menten constant, which can determine the affinity between the enzyme and the substrate. The calculation of \u003cem\u003eV\u003c/em\u003e can be done by Eq.\u0026nbsp;3:\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{V}\\text{=}\\text{∆}\\text{C}/\\text{∆}\\text{t}\\text{=}\\text{(}\\text{∆}\\text{A}/\\text{\u0026epsilon;}\\text{b}\\text{)}/\\text{∆}\\text{t}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;3)\u003c/p\u003e\n\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{C}\\)\u003c/span\u003e\u003c/span\u003e represents the concentration of oxTMB and \u003cem\u003et\u003c/em\u003e represents the reaction time. The change in oxTMB concentration \u0026Delta;\u003cem\u003eC\u003c/em\u003e can be calculated according to the Lambert-Beer law, where \u003cem\u003eA\u003c/em\u003e refers to the absorbance at 652 nm in the absorption spectrum, the corresponding molar absorptivity \u003cem\u003e\u0026epsilon;\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.9\u0026times;10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e[19], \u003cem\u003eb\u003c/em\u003e is the width of the cuvette (1 cm). Taking the reciprocal of both sides of the Michaelis-Menten equation, the Lineweaver-Burk double reciprocal equation is obtained in Eq.\u0026nbsp;4:\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{1}/\\text{V}\\text{=}\\text{(}{\\text{K}}_{\\text{m}}/{\\text{V}}_{\\text{max}}\\text{)}\\bullet\\text{(}\\text{1}/\\text{[}\\text{S}\\text{]}\\text{)}\\text{+}\\text{(}\\text{1}/{\\text{V}}_{\\text{max}}\\text{)}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;4)\u003c/p\u003e\n\u003cp\u003ePlotting 1/\u003cem\u003eV\u003c/em\u003e against 1/[\u003cem\u003eS\u003c/em\u003e] results in a straight line. The intercept of this line on the x-axis is the absolute value of 1/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, while the intercept on the y-axis is 1/\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eThe Michaelis-Menten equation curves and corresponding Lineweaver-Burk plots of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO for TMB and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as substrates are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values of for TMB and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are 0.09 mM and 0.24 mM respectively. The \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e values for TMB and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were 3.23\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e M/s and 1.93\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e M/s, respectively. Compared to Fig. S6 and Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO is lower than that of pure pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e, indicating improved affinity for TMB and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which aligns well with DFT calculations. While its \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e is significantly higher, confirming a faster catalytic reaction rate. Notably, while the \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO for TMB is slightly higher than the reported Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e values [19], this discrepancy likely stems from differences in material morphology. Comparison of the kinetic parameters of various catalysts in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e also demonstrates that Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO exhibits superior catalytic performance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 Colorimetric sensing of GSH\u003c/h2\u003e\n\u003cp\u003eGSH exhibits potent regulatory effects on redox reactions and can inhibit the oxidation of TMB by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). Under optimized conditions, the performance of the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs-based colorimetric sensor for GSH detection was evaluated. As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb, increasing GSH concentration led to a gradual fading of the blue color of the detection system, accompanied by a decrease in A\u003csub\u003e652\u003c/sub\u003e. A good linear relationship was observed between GSH concentration and A\u003csub\u003e652\u003c/sub\u003e, with the inset of Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb showing a linear response over 10\u0026ndash;250 nM (y= -0.0013x\u0026thinsp;+\u0026thinsp;0.6373, R\u003csup\u003e2\u003c/sup\u003e= 0.9930). The limit of detection (LOD) was calculated as 3.1 nM, determined by LOD=3\u003cem\u003e\u0026sigma;\u003c/em\u003e/\u003cem\u003eS\u003c/em\u003e, where \u003cem\u003e\u0026sigma;\u003c/em\u003e is the standard deviation (SD) of eight replicate blank measurements and \u003cem\u003eS\u003c/em\u003e is the slope of the calibration curve. As summarized in Table S2, the proposed sensor exhibits a wider linear range and lower LOD compared to most previously reported colorimetric GSH sensors, demonstrating its competitive analytical performance for GSH quantification.\u003c/p\u003e\n\u003cp\u003eTo assess the selectivity of the sensor, 13 potential interfering small molecules and ions (Ala, Cys, Arg, Lys, DA, UA, AA, Lac, Glu, Fru, K\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e) were individually introduced into the reaction system at a concentration twice that of GSH. Notably, interfering substances including Cys、DA、UA and AA also exhibit inherent reducing properties, which could theoretically compete with GSH to inhibit TMB oxidation by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. However, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec, GSH exerted the most pronounced inhibitory effect on TMB oxidation, resulting in the largest decrease in A\u003csub\u003e652\u003c/sub\u003e. This distinct difference in inhibitory potency confirms the superior selectivity of the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-based colorimetric method for GSH detection. The reproducibility of the sensor was evaluated by performing five replicate measurements at GSH concentrations of 50, 100, and 200 nM, yielding relative standard deviations (RSDs) of 1.1\u0026ndash;2.5% (n\u0026thinsp;=\u0026thinsp;5), which confirms the good reproducibility of the method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e3.6 Detection of spiked GSH in FBS\u003c/h2\u003e\n\u003cp\u003eTo verify the applicability of the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-based probe in complex biological matrices, GSH detection was performed in deproteinated FBS using the standard addition method. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed, the calibration curve for FBS matrix (y\u0026thinsp;=\u0026thinsp;0.0009x\u0026thinsp;+\u0026thinsp;0.0868, R\u003csup\u003e2\u003c/sup\u003e =0.9964) exhibits a slightly lower slope and higher intercept than the buffer system (y\u0026thinsp;=\u0026thinsp;0.0012x\u0026thinsp;+\u0026thinsp;0.0849, R\u003csup\u003e2\u003c/sup\u003e =0.9960), reflecting the biological matrix effect caused by endogenous reducing interferents in FBS [38].\u003c/p\u003e\n\u003cp\u003eBased on the FBS standard addition curve, the endogenous GSH concentration in FBS was calculated to be 96.4 nM. Spiked recovery experiments were further performed to evaluate the accuracy of the method (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The results showed that the recovery rates of spiked GSH in FBS ranged from 94.4% to 99.3%, which is within the acceptable range for biological sample quantification. These results confirm that the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-based colorimetric method can effectively correct for matrix effects in FBS and achieve accurate and reliable detection of GSH, supporting its practical application in complex biological samples.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eSpiked recovery analysis of GSH in FBS\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSample\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSpiked (nM)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFound (nM)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRecovery (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRSD (%, n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"7\" align=\"left\"\u003e\n\u003cp\u003eFBS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e96.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e116.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e98.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e145.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e98.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e195.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e99.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e239.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e293.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e98.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e332.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e94.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eIn this work, Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs were successfully synthesized via a hydrothermal method. The introduction of rGO effectively improved the dispersion of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e nanoparticles and significantly enhanced their POD-like activity. Steady-state kinetic analysis and DFT calculations revealed that the nanocomposites exhibited stronger substrate affinity and lower adsorption energies for TMB and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, accounting for the improved catalytic performance. Leveraging the inhibitory effect of GSH, a colorimetric sensing platform was constructed, achieving a linear range of 10\u0026ndash;250 nM and a limit of detection of 3.1 nM. Satisfactory spiked recoveries (94.4%-99.3%) in FBS samples demonstrated its potential for practical detection in complex biological matrices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the National Natural Science Foundation of China (11805154, 21804135), Lanzhou Youth Science and Technology Talent Innovation Project (No. 2025-QN-085), Longyuan Youth Project in Gansu Province (Jinxiu Guo), Key Laboratory of Eco-functional Polymer Materials (Northwest Normal University, KF-24-05).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJinxiu Guo: Data curation, Investigation, Methodology, Writing-original draft. Haozhe Jia: Data curation, Investigation. Runtian Ma: Visualization, Writing - review \u0026amp; editing, Funding acquisition. Zuoxiang Ma: Formal analysis, Methodology, Supervision. Yanjun Cui: Funding acquisition, Supervision. Bing Hu: Investigation, Validation, Supervision. Zhifang Zhang: Writing-review \u0026amp; editing. Haijiao Xie: Methodology, Software.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (11805154, 21804135), Lanzhou Youth Science and Technology Talent Innovation Project (No. 2025-QN-085), Longyuan Youth Project in Gansu Province (Jinxiu Guo), Key Laboratory of Eco-functional Polymer Materials (Northwest Normal University, KF-24-05).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Li W, Li M, Qi J (2021) Nano-drug design based on the physiological properties of glutathione. Molecules 26(18): 5567. https://doi.org/10.3390/molecules26185567\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Ketterer B, Coles B, Meyer DJ (1983) The role of glutathione in detoxication. Environmental Health Perspectives 49: 59\u0026ndash;69. https://doi.org/10.2307/3429581\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gasmi A, Nasreen A, Lenchyk L, et al (2025). An update on glutathione's biosynthesis, metabolism, functions, and medicinal purposes. Current Medicinal Chemistry 31(29): 4579\u0026ndash;4601. https://doi.org/10.2174/0109298673251025230919105818\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Ma F, Li L, Xu Z, et al (2025) Prognostic modeling and immune infiltration analysis in hepatocellular carcinoma using glutathione metabolism-associated genes. European Journal of Medical Research 30: 1141. https://doi.org/10.1186/s40001-025-03403-8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Albiach-Delgado A, Moreno-Casillas JL, Ettabaa-Bahji Y, et al (2024) Portable point-of-care surface enhanced Raman scattering spectroscopy for the quantification of glutathione in whole blood microsamples. Talanta 279: 126566. https://doi.org/10.1016/j.talanta.2024.126566\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Tsiasioti A, Tzanavaras PD (2023) Determination of glutathione and glutathione disulfide using liquid chromatography: a review on recent applications. Microchemical Journal 193: 109157. https://doi.org/10.1016/j.microc.2023.109157\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Xie J, Cheng D, Li P, et al (2021) Au/metal-organic framework nanocapsules for electrochemical determination of glutathione. ACS Applied Nano Materials 4(5): 4853\u0026ndash;4862. https://doi.org/10.1021/acsanm.1c00394\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Xi S, Wang L, Cheng M, et al (2022) Developing a DNA logic gate nanosensing platform for the detection of acetamiprid. RSC Advances 12 (42): 27421\u0026ndash;27430. https://doi.org/10.1039/D2RA04794B\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Jiang WC, Shi YH, Li XM, et al (2026) Conjugated precursors of isomers to enhance the peroxidase-like activity of carbon dots nanozymes for constructing multi-scenario sensing systems for glutathione and glutathione S-transferase. Journal of Colloid and Interface Science 701: 138768. https://doi.org/10.1016/j.jcis.2025.138768\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Nguyen VAN, Vu TH, Nguyen PT, et al (2025) Dual-mode optical detection of sulfide ions using copper-anchored nitrogen-doped graphene quantum dot nanozymes. Biosensors 15(8): 528. https://doi.org/10.3390/bios15080528\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gao L, Zhuang J, Nie L, et al (2007) Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nature Nanotechnology 2(9): 577\u0026ndash;583. https://doi.org/10.1038/nnano.2007.260\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Fu R, Ma Z, Zhao H, et al (2023) Research progress in iron-based nanozymes: catalytic mechanisms, classification, and biomedical applications. Analytical Chemistry 95(29): 10844\u0026ndash;10858. https://pubs.acs.org/doi/10.1021/acs.analchem.3c01005\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Han J, Chen Y, Xiang X, et al (2024) A comparative analysis of the antibacterial spectrum of ultrasmall manganese ferrite nanozymes with varied surface modifications. ACS Applied Materials \u0026amp; Interfaces 16(12): 14385\u0026ndash;14404. https://doi.org/10.1021/acsami.3c16490\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Murugan C, Park S (2023) Cerium ferrite@ molybdenum disulfide nanozyme for intracellular ROS generation and photothermal-based cancer therapy. Journal of Photochemistry and Photobiology A: Chemistry 437: 114466. https://doi.org/10.1016/j.jphotochem.2022.114466\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Feng L, Liu B, Xie R, et al (2021) An ultrasmall SnFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozyme with endogenous oxygen generation and glutathione depletion for synergistic cancer therapy. Advanced Functional Materials 31(5): 2006216. https://doi.org/10.1002/adfm.202006216\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Yang J, Gao J, Fu P, et al (2019) Synthesis and gas sensing properties of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e nanosheets. Materials Research Express 6(9): 095083. https://doi.org/10.1088/2053-1591/ab31b3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sun H, Liu Y, Zhang Y, et al (2014) Synthesis of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/reduced graphene oxide composite by one-step hydrothermal method and its high photocatalytic performance. Journal of Materials Science: Materials in Electronics 25(10): 4212\u0026ndash;4218. https://doi.org/10.1007/s10854-014-2151-4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Zeng T, Liu Z, Huang G, et al (2022) Visible-light photovoltaic effect in multiferroic Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e thin film. Materials Letters 309: 131411. https://doi.org/10.1016/j.matlet.2021.131411\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Razavi M, Barras A, Ifires M, et al (2022) Colorimetric assay for the detection of dopamine using bismuth ferrite oxide (Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e) nanoparticles as an efficient peroxidase-mimic nanozyme. Journal of Colloid and Interface Science 613: 384\u0026ndash;395. https://doi.org/10.1016/j.jcis.2022.01.041\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wang Z, Zhang R, Yan X, et al (2020) Structure and activity of nanozymes: Inspirations for de novo design of nanozymes. Materials Today 41: 81\u0026ndash;119. https://doi.org/10.1016/j.mattod.2020.08.020\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Neal C J, Kolanthai E, Wei F, et al (2024) Surface chemistry of biologically active reducible oxide nanozymes. Advanced Materials 36(10): 2211261. https://doi.org/10.1002/adma.202470073\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Yang J, Dai H, Sun Y, et al (2022) 2D material-based peroxidase-mimicking nanozymes: catalytic mechanisms and bioapplications. Analytical and Bioanalytical Chemistry 414(9): 2971\u0026ndash;2989. https://doi.org/10.1007/s00216-022-03985-w\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Rajarathinam T, Jayaraman S, Kim CS, et al (2025) Two-dimensional nanozyme nanoarchitectonics customized electrochemical bio diagnostics and lab-on-chip devices for biomarker detection. Advances in Colloid and Interface Science 341: 103474. https://doi.org/10.1016/j.cis.2025.103474\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gao H, Yu H, Yang S, et al (2024) Ultrasensitive detection of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e via electrochemical sensor by graphene synergized with MOF-on-MOF nanozymes. Microchimica Acta 191(8): 482. https://doi.org/10.1007/s00604-024-06541-8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Qi Y, Li B, Song D, et al (2023) Ultrafast colorimetric detection of Cr (VI) based on competition of 8-HQ to Cr (VI) and TMB oxides using GO/AuNPs nanocomposites as peroxidase mimic. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 297: 122722. https://doi.org/10.1016/j.saa.2023.122722\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Peng D, Que M, Deng X, et al (2023) Mn\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles decorated porous reduced graphene oxide with excellent oxidase-like activity for fast colorimetric detection of ascorbic acid. Microchimica Acta 190(6): 243. https://doi.org/10.1007/s00604-023-05822-y\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Grimme S., Antony J., Ehrlich S., et al (2010) A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. Journal of Chemical Physics 132 (15): 154104. https://doi.org/10.1063/1.3382344\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Rodriguez JMG, Hux NP, Philips SJ, et al (2019) Michaelis-Menten graphs, Lineweaver-Burk plots, and reaction schemes: Investigating introductory biochemistry students\u0026rsquo; conceptions of representations in enzyme kinetics. Journal of Chemical Education 96(9): 1833\u0026ndash;1845. https://doi/10.1021/acs.jchemed.9b00396\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chang Y, Zhang Y, Hu T, et al (2023) Carbonaceous material modified MoO\u003csub\u003e2\u003c/sub\u003e nanospheres with oxygen vacancies for enhanced visible-light photocatalytic oxidative coupling of benzylamine. Molecules 28(12): 4739. https://doi.org/10.3390/molecules28124739\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Charbouillot T, Brigante M, Mailhot G, et al (2011) Performance and selectivity of the terephthalic acid probe for \u0026bull;OH as a function of temperature, pH and composition of atmospherically relevant aqueous media. Journal of Photochemistry and Photobiology A: Chemistry 222(1): 70\u0026ndash;76. https://doi.org/10.1016/j.jphotochem.2011.05.003\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Kresse GG, Furthm\u0026uuml;ller JJ (1996) Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set. Physical Review B 54: 11169. https://doi.org/10.1103/PhysRevB.54.11169\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Blochl PE (1994) Projector augmented-wave method. Physical Review B 50: 17953\u0026ndash;17979. https://doi.org/10.1103/PhysRevB.50.17953\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Perdew JP, Burke K, Ernzerhof M (1996) Generalized gradient approximation made simple. Physical Review Letters 77(18): 3865. https://doi.org/10.1103/PhysRevLett.77.3865\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Crowe KM (2014) Optimizing protein precipitation efficiency for assessing the contribution of low molecular weight compounds to serum antioxidant capacity. Clinical biochemistry 47(15): 116\u0026ndash;118. http://dx.doi.org/10.1016/j.clinbiochem.2014.06.021\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Patel J, Parmar M, Shahabuddin S, et al (2025) Designing a Z-scheme rGO-SnS\u003csub\u003e2\u003c/sub\u003e synergistic photocatalyst for photocatalytic mineralization of atrazine and 2,4-dichlorophenoxyacetic acid and applying machine learning for predictive modelling of photocatalytic performance. Nanoscale Advances 7(11): 3485\u0026ndash;3507. https://doi.org/10.1039/d5na00143a\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wang X, Li QZ, Zhao Y, et al (2025) Recent advances in oxidase-like nanozymes: mechanisms, prediction models, and applications. ACS Applied Materials \u0026amp; Interfaces 17(49) 66110\u0026ndash;66150. https://doi.org/10.1021/acsami.5c15196\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Xu AY, Blanco MA, Castellanos MM, et al (2023) Role of domain-domain interactions on the self-association and physical stability of monoclonal antibodies: effect of pH and salt. The Journal of Physical Chemistry B 127 (39): 8344\u0026ndash;8357. https://doi.org/10.1021/acs.jpcb.3c03928\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Dega NK, Ganganboina AB, Tran HL, et al (2022) BSA-stabilized manganese phosphate nanoflower with enhanced nanozyme activity for highly sensitive and rapid detection of glutathione. Talanta 237: 122957. https://doi.org/10.1016/j.talanta.2021.122957\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bi2Fe4O9, reduced graphene oxide, peroxidase-like activity, glutathione, colorimetric assay","lastPublishedDoi":"10.21203/rs.3.rs-9180770/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9180770/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e nanoparticles (Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs) and Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/reduced graphene oxide nanocomposites (Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs) were successfully synthesized via a hydrothermal method. Morphological and compositional characterizations revealed that the introduction of graphene oxide (GO) not only modifies the lamellar morphology of Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs but also enhances their dispersion, thereby increasing the specific surface area of the composites. Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs exhibited excellent peroxidase (POD)-like activity, efficiently catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to form a blue oxidation product (oxTMB) with a characteristic absorption peak at 652 nm. Steady-state kinetic analysis confirms Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO NCs have superior catalytic activity and substrate affinity to pure Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs, and density functional theory calculations reveal the atomic-level mechanism for the enhanced POD-like activity, as the nanocomposites show much lower adsorption energies for TMB (-5.67 eV) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (-3.44 eV) than Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e NPs (TMB: -2.18 eV, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: -1.49 eV). Leveraging the competitive inhibitory effect of glutathione (GSH) on the Bi\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e/rGO-TMB-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e catalytic system, a sensitive and selective colorimetric platform for GSH detection was established, achieving a linear range of 10\u0026ndash;250 nM and a limit of detection of 3.1 nM. Furthermore, the platform exhibited satisfactory recovery rates (94.4%-99.3%) in fetal bovine serum samples, demonstrating its great potential for practical application in complex biological matrices and analytical sensing fields.\u003c/p\u003e","manuscriptTitle":"Bi2Fe4O9/reduced graphene oxide nanocomposites with enhanced peroxidase activity for sensitive glutathione detection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-03 09:36:38","doi":"10.21203/rs.3.rs-9180770/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-24T20:23:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-23T08:09:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276389382725553234478460955508633903381","date":"2026-04-06T05:46:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-30T12:07:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89916905472688356588835508248342187510","date":"2026-03-30T07:14:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-28T13:52:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-25T22:52:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-25T22:51:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2026-03-20T16:19:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"210bad57-21a7-4b63-99a8-cc1c46124729","owner":[],"postedDate":"April 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T20:38:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-03 09:36:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9180770","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9180770","identity":"rs-9180770","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}