Chiang Mai Journal of Science

Conversion of Fe-MOF to Peroxidase-like Prussian Blue Nanozymes for Hydrogen Peroxide Detection

1. INTRODUCTION

     Hydrogen peroxide (H₂O₂) is a reactive oxygen species with disinfectant, antiviral, and antibacterial properties, and serves as a common byproduct of numerous biochemical reactions [1]. Therefore, it is a common analyte employed for the indirect detection of cellular signaling, aging mechanisms, and specific oxidases, such as glucose oxidase and lactate oxidase [2,3]. Thus, the detection of H₂O₂ is crucial in various fields, including biosensing and food industry [4,5]. Currently, a range of techniques have been successfully employed for H₂O₂ detection, including chromatography, titration, and electrochemical methods, yet each suffers from inherent limitations [6-8]. The titration method suffers from low efficiency and poor sensitivity, while chromatography is limited by complex operational procedures and derivatization requirements, hindering its widespread application [9,10]. Electrochemical sensors offer simple operation, short response time, and high sensitivity, but exhibit insufficient specificity [11]. In contrast, colorimetric methods, which induce a color change upon interaction with target analytes, have gained widespread popularity. In particular, the use of enzymes, such as HRP, which can be applied to the detection of H₂O₂ [12], confers high efficiency and specificity on colorimetric methods.
     Since Fe₃O₄ nanoparticles (Fe₃O₄ NPs) were first reported as nanozymes with peroxidase-like activity [13], nanozymes have garnered significant attention in recent years for applications in biosensing, biomedical engineering, and environmental protection [14-16]. Compared with natural enzymes, nanozymes offer distinct advantages such as lower cost in preparation, enhanced stability, superior tolerance to harsh conditions, and high catalytic efficiency [17]. Therefore, nanomaterial-based nanozymes have emerged as a new research frontier. To date, numerous nanomaterials, including metal-organic frameworks (MOFs), covalent organic frameworks (COFs), single-atom catalysts, transition metals and their oxides, and carbon-based nanostructures have been shown to exhibit enzyme-like catalytic activity [18]. Among these nanozymes, nanoscale peroxidase-like and oxidase-like mimics have garnered considerable interest. Specifically, a variety of nanomaterials, including Cu-N@C, Au clusters, AuNPs, Co₃O₄ NPs, and FeZn SAC-N@C, have been demonstrated to possess enzyme-like activity [19-22].
     Prussian blue (PB) is a common inorganic coordination compound featuring mixed iron valence states. In the face-centered cubic lattice of PB, Fe(III) is coordinated to nitrogen atoms in octahedral coordination environments, while Fe(II) is coordinated to carbon atoms [23]. Therefore, PBNPs exhibit efficient peroxidase-like activity, owing to the presence of Fe³⁺/Fe²⁺ redox pairs [24]. However, PBNPs synthesized by conventional methods exhibit poor dispersibility and insufficient stability in aqueous media, which compromises their nanozyme catalytic activity [25].
     Among various synthetic methods for nanoparticles, nanoparticle-to-nanoparticle crystalline transformation has emerged as an attractive strategy. This transformation refers to a process in which one type of nanoparticle serves as the precursor, by regulating reaction conditions, the precursor undergoes changes in crystal structure and phase composition while maintaining its nanoscale morphology, ultimately yielding a different type of nanoparticle [26]. For instance, Wu et al. synthesized FeS-Ni₃S₂/NF nanosheets via partial chemical etching (with Fe(CN)₆ 3- replacing OH-), followed by chemical etching/anion exchange of Fe(CN)₆ 3- and OH- by S2- [27]. Additionally, Xu et al. prepared Co₂[Fe(CN)₆] with a mesoporous double-shelled hollow structure through a facile ligand exchange reaction between the solid cuboid-shaped cobalt precursors and K₃[Fe(CN)₆] [28]. However, most processes for preparing nanomaterials via anion exchange exhibit low reaction rates and require harsh reaction conditions such as high temperatures or organic solvents [29].
     Metal-organic frameworks (MOFs) are a novel class of crystalline porous hybrid materials featuring infinite lattices constructed from metal cations/clusters and multidentate organic linkers [30]. Notably, many MOFs exist as nanoparticles, with diameters typically ranging from several nanometers to hundreds of nanometers, and represent a unique class of porous nanoparticles. Compared with traditional porous materials, MOFs offer distinct advantages including a large specific surface area, ultra-high porosity, tunable pore size, facile post-synthetic modifications, and excellent thermal stability [31,32]. With the continuous advancement of MOF research, Fe-based metal-organic frameworks (Fe-MOFs), as an important type of MOFs, have gained increasing traction in bio-related fields for their advantages, such as low toxicity, good stability, and flexible structures [33].
     To the best of our knowledge, anion exchange-based nanoparticles synthesis under room temperature aqueous conditions has not been reported to date. In this study, PBNPs were prepared via a nanoparticle-to-nanoparticle crystalline transformation method using iron(III) trimesate (Fe-BTC MOF) as the precursor in aqueous media at room temperature. Subsequently, the peroxidase-like activity of the PBNPs was systematically investigated. The PBNPs were successfully applied as peroxidase-like nanozymes for the colorimetric detection of H₂O₂.

2. MATERIALS AND METHODS

2.1 Chemicals and Materials
     2,2'-Azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). 1,3,5-Benzenetricarboxylic acid (BTC) was purchased from J&K Scientific (Beijing, China), and Potassium hexacyanoferrate(II) was purchased from Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Iron(III) chloride hexahydrate and all other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2 Preparation of Fe-BTC MOF
     H₃BTC (trimesic acid) was selected as the organic ligand for the Fe-BTC MOF precursor. The well-established synthesis protocols and extensive literature characterization of Fe-BTC make it a reliable and convenient precursor for the subsequent preparation of PBNPs [34,43,44].
     Fe-BTC MOF was prepared following the method reported by Victoria Gascón [34]. Briefly, the solution 1 was prepared by dissolving 0.263 g of trimesic acid (H₃BTC) in 3.685 mL of 1.06 M NaOH solution (pH 8.0), followed by the addition of 6.388 mL of deionized water. The solution 2 was prepared by dissolving 0.761 g of FeCl₃∙6H₂O in 10 mL of deionized water. Then, the solution 2 was added dropwise to the solution 1 with continuous stirring. The resultant suspension was stirred at 25 °C for 10 min. The resulting mixture was centrifuged at 8,000 × g for 5 min. The collected precipitate was washed three times with deionized water.

2.3 Preparation of Prussian Blue Nanoparticles (PBNPs)
     The Fe-BTC MOF was dispersed in 2 mL deionized water to a final concentration of 15 mg/mL, followed by the addition of 1 mL of 0.1 M K₄Fe(CN)₆ solution. The mixture was incubated at 25 °C for 24 h. The resulting precipitate (PBNPs) was centrifuged at 10,000 × g for 10 min, washed three times with deionized water, and subsequently redispersed in 3 mL deionized water. The PBNPs were stored at room temperature for further use.

2.4 Characterization
     Scanning electron microscopy (SEM) (ZEISS GeminiSEM 300, Germany) was used to characterize the morphologies of the Fe-BTC MOF and the PBNPs. Their microstructures were observed via transmission electron microscopy (TEM) (FEI Talos F200x, USA) while their elemental composition was analyzed using energy dispersive spectroscopy (EDS) attached to the SEM. The crystal structure of PBNPs were investigated by X-ray diffraction (XRD) (Rigaku Ultima IV, Japan).

2.5 Assay of Nanozyme Activity
     Peroxidase-like activity of PBNPs was explored by examining four different reaction systems in citric acid-Na₂HPO₄ buffer (200 mM, pH 2.2): (1) ABTS (10 mM); (2) ABTS (10 mM) and PBNPs (30 mg/mL); (3) ABTS (10 mM) and H₂O₂ (10 mM); (4) ABTS (10 mM), PBNPs (30 mg/mL), and H₂O₂ (10 mM). Solution mixtures were incubated at 40 °C for 10 min. Then, test samples were centrifuged at 10,000 × g for 10 min, and supernatants were collected. Absorbance spectra of the supernatants were recorded in the 350−500 nm range with a UV-visible spectrophotometer (U-1800, Hitachi, Japan).
     Therefore, the peroxidase-like activity of PBNPs was evaluated using H₂O₂ and ABTS as substrates: 2 mL of citric acid-Na₂HPO₄ buffer (200 mM, pH 2.2), 200 µL of H₂O₂ solution (10 mM), 100 µL of ABTS solution (10 mM), and 50 µL of the PBNPs suspension (30 mg/mL) were mixed and incubated at 40 °C for 10 min. Subsequently, the reaction mixture was centrifuged at 10,000 × g for 10 min, and the supernatants were collected. The absorbance of the collected supernatants was measured at 420 nm. The control experiment was performed by replacing the PBNPs suspension with an equal volume of deionized water. The activity of PBNPs was defined as the amount of nanozyme required to consume 1 µmol H₂O₂ per minute as one unit of activity (U).

2.6 Effects of pH, Temperature and Incubation Time on the Peroxidase-like Activity of PBNPs
     The effect of pH on the peroxidase-like activity of the PBNPs was investigated over a pH range of 2.0–6.0 at 25 °C. Similarly, the effect of temperature on the peroxidase-like activity of the PBNPs was investigated over a range of 25–70 °C at the optimal pH. To assess the influence of incubation time, the reaction system was incubated at the optimal temperature and pH for different times (5, 10, 15, and 20 min). The relative activity of the PBNPs was calculated via the following equation (1):

where A denotes the absorbance measured for each test sample at 420 nm; A₀ is the absorbance of the blank control sample (without H₂O₂); and A_max represents the maximum absorbance value obtained from all tested samples.

2.7. Stability of PBNPs
     For determination of the pH stability, the nanozyme was incubated in 50 mM phosphate-citrate buffer (pH 2.2) and carbonate buffer (pH 7.0, pH 9.0) for 2 h, 3 h, 18 h and 42 h. For determination of the thermal stability, the nanozyme was incubated at 4 °C, 25 °C, and 40 °C in deionized water for 1 h, 2 h and 3 h. Aliquots of the nanozyme solution were collected, and their residual activities were measured under optimal conditions. The initial activity was defined as 100%.

2.8. Steady-State Kinetic Assay of Nanozyme
     To determine the kinetic parameters of the PBNPs, different concentrations of H₂O₂ (1-10 mM) and different concentrations of ABTS (1-10 mM) were used. The nanozyme activity was measured by the method described in Section 2.5 under optimal reaction conditions. The V_max and K_m of the nanozyme were calculated by the Lineweaver−Burk plot [35].

2.9. Storage Stability of PBNPs
     The storage stability of the PBNPs was evaluated by incubating in deionized water at 4 °C. The PBNPs were taken out every 5 days and the residual activity was determined as described above. The initial activity was defined as 100%.

2.10. Colorimetric Detection of H₂O₂
     A dose-response curve for H₂O₂ detection was obtained by adding 50 µL PBNPs (30 mg/mL), 100 µL ABTS (10 mM), and 200 µL different concentrations of H₂O₂ (0−200 μM) to 2 mL citric acid-Na₂HPO₄ buffer (200 mM, pH 2.2). The mixture was incubated at 40 °C for 10 min. Then, the supernatants were collected after centrifugation at 10,000 × g for 10 min. The absorbance of the supernatants was recorded at 420 nm. The limit of detection (LOD) for H₂O₂ was calculated using the following equation.

where σ represents the standard deviation of ten blank tests and s is the slope of the calibration curve.

3. RESULTS AND DISCUSSION

3.1 Preparation of PBNPs
     The preparation of PBNPs involved two basic steps, with the core mechanism relying on coordination reaction and post-synthetic ligand exchange [36]. Firstly, Fe-BTC MOF was synthesized via a one-pot rapid coordination reaction that occurred immediately and completed within several minutes. The reaction was driven by the coordination between Fe³⁺ and the carboxylate groups of H₃BTC. Subsequently, post-synthetic ligand exchange was conducted to replace -BTC ligands with -CN ligands and the heterogeneous ligand exchange proceeded when Fe-BTC MOF was suspended in K₄Fe(CN)₆ solution, with the driving force being the stronger coordination affinity of -CN ligands for Fe³⁺ compared with -BTC ligands. The reaction was carried out at room temperature for 24 h to ensure sufficient substitution of -BTC ligands by -CN ligands, ultimately forming PBNPs. The molar yield of the Fe-BTC MOF synthesized in the first step was estimated to be 81.1% based on the molar amount of the initial Fe³⁺ added. The molar yield of PBNPs obtained from the subsequent ligand exchange step was calculated to be 55.1% based on the molar amount of Fe in the initial Fe-BTC MOF.

3.2 Characterization of PBNPs
     The morphologies of the Fe-BTC MOF and the PBNPs were characterized by SEM. TEM was used to characterize the microstructure of the PBNPs. As presented in Fig. 2a, the Fe-BTC MOF exhibited aggregates of many small nanocrystals, with an average particle size of approximately 50 nm. The material did not have well-defined edges/corners or regular shapes morphologically, consistent with the results reported in multiple studies [34,37]. The PBNPs displayed highly uniform morphologies—specifically, well-defined regular cubic structures—with an average particle size of approximately 40 nm (Fig. 2b). To analyze the Fe-BTC MOF and the PBNPs, energy dispersive spectroscopy (EDS) was performed. The mass fractions of the Fe-BTC MOF (Fig. 2a) and PBNPs (Fig. 2b) indicated that the Fe-BTC MOF contained only three elements: Fe, O, and C, with the N content being merely 0.07%. This confirmed that the N element observed in the Fe-BTC MOF was background noise. After the formation of PBNPs mediated by Fe-BTC MOF, the content of N increased from 0.07% to 7.49%, and the content of Fe rose from 16.70% to 56.69%. These results demonstrate that the PBNPs were successfully formed under the mediation of Fe-BTC MOF.
     The TEM image (Fig. S1) showed that the Fe-BTC MOF was converted into the PBNPs. As shown in Fig.S1 b, the PBNPs exhibited relatively distinct cubic lattice fringes with an interplanar spacing of 5.063 Å within the PBNPs, which matched well with that of the (200) plane of the face-centered cubic PBNPs, confirming the successful formation of PBNPs mediated by Fe-BTC MOF.
     XRD patterns of the Fe-BTC MOF and PBNPs were depicted in Fig. 3. For the Fe-BTC MOF, the diffraction pattern was consistent with that of commercial Basolite® F300 and that reported in the previous literature [38], confirming the successful synthesis of the target Fe-BTC MOF with high crystallinity. For the PBNPs, the diffraction peak positions were in good agreement with those documented in previous studies on PBNPs [39] and matched the standard JCPDS card (No. 73–0687), indicating the formation of a well-defined PB crystal structure.

3.3 Intrinsic Peroxidase-like Activity
     As shown in Figure S5, the characteristic maximum absorbance peak of ABTS·⁺ at 420 nm (OD_420nm) was not observed when neither H₂O₂ nor the PBNPs was present. A weak increase in absorbance was detected when the reaction system contained both ABTS and PBNPs, revealing a modest oxidation capacity of the PBNPs toward ABTS in the absence of H₂O₂. A slightly higher OD_420nm was visible in the presence of both ABTS and H₂O₂. However, a significantly higher OD_420nm was detected only when the reaction system contains ABTS, H₂O₂, and the PBNPs, attesting to the intrinsic peroxidase-like activity of the as-synthesized PBNPs in the presence of H₂O₂.

3.4 Enzymatic Properties of Nanozyme
     Analogous to natural enzymes, nanozymes exhibit intrinsic activity that is significantly affected by experimental conditions. Hence, the effects of pH and temperature on the intrinsic peroxidase-like activity of the PBNPs were investigated to identify the optimal reaction conditions. As shown in Fig. 4a, the optimal pH was 2.2, and the nanozyme retained only approximately 25% relative activity at pH 3.0, 9% at pH 4.0, 5% at pH 5.0, and even 2% at pH 6.0. The results showed that the nanozyme exhibited high activity under acidic condition, and the enzymatic activity decreased rapidly with increasing pH. The reason might be attributed to the decomposition of H₂O₂ under near-neutral or weakly acidic conditions [40]. The optimal temperature for the nanozyme was 40 °C (Fig. 4b). The nanozyme retained over 80% of the relative activity at 60 °C.
     The effect of incubation time was also investigated (Fig. S4). The relative activity increased from 5 min to 10 min, reaching the maximum at 10 min. With further extension of incubation time to 15 min and 20 min, the relative activity slightly still remained at a high level (above 95%). Therefore, 10 min was selected as the optimal incubation time for detection experiments.
     The pH tolerance of the PBNPs was depicted in Fig. 4c. The nanozyme exhibited the best stability at the optimal pH and retained 85.8% residual activity after being incubated at pH 2.2 for 42 h. Although the nanozyme exhibited extremely low catalytic activity under neutral and alkaline conditions, it maintained good stability at the corresponding pH values. The nanozyme retained 68.8% residual activity at pH 7.0, and even maintained 84.1% residual activity at the same pH after 42 h. The thermal tolerance of the PBNPs was presented in Fig. 4d. After incubation at 40 °C for 3 h, the nanozyme still maintained 96% residual activity. At 4 °C and 25 °C for 3 h, the nanozyme still retained detectable enzymatic activity. The PBNPs were very stable across a relatively wide temperature range.

3.5 Kinetic Parameters of the PBNPs
     To investigate the peroxidase-mimetic catalytic mechanism of PBNPs, steady-state kinetic analysis was performed using H₂O₂ and ABTS as substrates. Fig. S2 shows Lineweaver-Burk double-reciprocal plots obtained for PBNPs. As shown in Fig. S2, K_m (H₂O₂ substrate) for PBNPs was 1.09 mM and K_m (ABTS substrate) was 0.10 mM. In addition, V_max (H₂O₂ substrate) for PBNPs was 1.71 μM·s^-1 and V_max (ABTS substrate) was 0.14 μM·s^-1.
     As summarized in Table 1, the K_m (H₂O₂ substrate) value was lower than that of most other nanozymes, attesting to a relatively higher affinity for H₂O₂ compared with both HRP and other nanozymes [13,20,25,41,42]. In addition, the V_max (H₂O₂ substrate) value for PBNPs was higher than that of HRP and other nanozymes, revealing a faster reaction for the nanozyme [13,20,25,41-43]. Notably, the K_m value of the PBNPs (1.09 mM) was higher than that reported for the Fe-BTC MOF (0.38 mM) [43], indicating a lower substrate affinity. This difference might be attributed to the porous structure of the Fe-BTC MOF, which allowed for easier substrate diffusion to the active sites compared to the PBNPs.

3.6 Storage Stability of PBNPs
     Storage stability is an important aspect in the development of reliable biocatalysts for commercial-scale applications. The storage stability of the PBNPs was evaluated by assaying the residual activity over a 20-day incubation period in deionized water at 4 °C. As shown in Fig. S3, the PBNPs exhibited strong storage stability. The nanozyme still retained 73.6% of its initial activity after 20 days.

3.7 Colorimetric Detection of H₂O₂
     Because of the remarkable affinity for H₂O₂ shown by PBNPs, the nanozyme was applied for the colorimetric detection of H₂O₂. Figure 5 shows the calibration curve for the absorbance at 420 nm versus the concentration of H₂O₂. The regression equation was obtained as y = 0.00813x + 0.03635 (R² = 0.9943) with a linear range of 2.5–20 µM. The limit of detection (LOD) at a signal-to-noise ratio (S/N) of 3 was calculated to be 1.61 µM.
     Table 2 summarized the performance of various peroxidase-like nanozymes reported in the literature for the H₂O₂ detection [25,41-45]. The PBNPs exhibited a limit of detection (LOD) of 1.61 μM and a linear range of 2.5-20 μM. Compared to previously reported nano-materials, the sensitivity of the PBNPs was superior to those of CoFe₂O₄ [41], Fe-BTC MOF [44], and Co-Fe/MOF [45], while being slightly higher than those of mSiO₂@PB [25], GNC900 [42], and Fe-BTC MOF [43]. Noteworthily, the PBNPs showed a wider linear range compared to the precursor (Fe-BTC), making them suitable for the detection of H₂O₂ [43,44]. Furthermore, the PBNPs required only 10 minutes of reaction time (Table 1), which was considerably faster than mSiO₂@PB (15 min), GNC900 (20 min) and Fe-BTC MOF (60 min) [25,42,43].
     Interference experiments depicted in Fig. S6 revealed no significant alteration in the absorbance at 420 nm in the presence of other interfering ions, amino acids, glucose, maltose, or sucrose. Notably, even when the concentration of the interfering substances was 10-fold higher than that of H₂O₂, no discernible impact on the absorbance signal was observed. This observation indicated that the assay demonstrated excellent anti-interference performance for H₂O₂.
     Subsequently, the practical applicability of PBNPs was assessed for the detection of H₂O₂ in three commercial drink samples. No endogenous H₂O₂ was detected in two of the drink samples (below the limit of detection, LOD), while the third sample was found to contain 3.36 μM of H₂O₂. As indicated in Table S1, the recoveries of spiked H₂O₂ from the samples ranged between 96 % and 110 %, with a relative standard deviation (RSD) of less than 4 %, thereby affirming the high precision and great potential of the ABTS + PBNPs system for accurate H₂O₂ detection in real-world scenarios.

4. CONCLUSIONS

     In summary, we successfully prepared peroxidase-like Prussian Blue nanoparticles (PBNPs) using Fe-BTC MOF as the precursor for the colorimetric detection of H₂O₂. The resulting PBNPs exhibited excellent catalytic activity, small particle size, strong tolerance to extreme conditions, and good storage stability. Additionally, the preparation method of the PBNPs featured high yield, simple conditions, and easy operation. Furthermore, compared with other previously reported nanozymes and HRP, the PBNPs exhibited a shorter response time and higher sensitivity for H₂O₂ detection [20,25,41-45]. These properties rendered the PBNPs a reliable candidate for H₂O₂ detection under harsh conditions.

ACKNOWLEDGEMENTS

     This research was supported by the Nanjing University of Science and Technology and Dalian Minzu University. Mr. Liu was supported by the Nanjing University of Science and Technology Undergraduate Training Program for Innovation and Entrepreneurship (Item Number 202410288015Z).

AUTHOR CONTRIBUTIONS

     Qing Liu: Validation, Formal analysis, Investigation, Writing-original draft. Baoquan Liu: Conceptualization, Resources, Project administration. Yuanrui Gao: Visualization. Chengli Yang: Data curation. Ruofu Shi: Software. Dali Li: Methodology, Writing-review & editing, Supervision, Funding acquisition.

CONFLICT OF INTEREST STATEMENT

     The authors declare that they hold no competing interests.

DECLARATION OF USE OF GENERATIVE AI

     During the preparation of this work, the authors used ChatGPT (GPT-4, OpenAI) to improve the readability and language of the manuscript. After using this tool, the authors reviewed and edited the content as necessary and take full responsibility for the publication's content.

FUNDING

     This research was financially supported by the Nanjing University of Science and Technology Undergraduate Training Program for Innovation and Entrepreneurship. Item Number: 202410288015Z.

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