Engineering a BSA-templated Iridium Oxide Nanozyme for Colorimetric Evaluation of Total Antioxidant Capacity in Food Samples

Engineering a BSA-templated Iridium Oxide Nanozyme for Colorimetric Evaluation of Total Antioxidant Capacity in Food Samples | 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 Engineering a BSA-templated Iridium Oxide Nanozyme for Colorimetric Evaluation of Total Antioxidant Capacity in Food Samples Shuangshuang Yan, Dongying An, Yutian Zhou, Xinli Guo, Zhihang Qin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8592177/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Antioxidants are crucial for capturing and neutralizing free radicals to prevent oxidative damage, which implies assessing the antioxidant content in foods and medications is essential. In order to quantify the total antioxidant capacity (TAC) of food samples, a straightforward and sensitive colorimetric sensing method utilizing an iridium oxide (IrOx) nanozyme was developed. By employing bovine serum albumin (BSA) as a structural template and iridium as the precursor, a biomimetic mineralization strategy was utilized to synthesize b-IrOx nanozymes exhibiting intrinsic peroxidase (POD)-like activity, enabling their effective substitution of natural peroxidases in catalyzing redox reactions. Through single electron transfer (SET) and hydrogen atom transfer (HAT) processes, antioxidants in samples can compete with substrates for •OH, causing a hue shift that depends on the antioxidant concentration. The proposed b-IrOx-based colorimetric assay was successfully used to measure the TAC of mango juice (948.31 ± 0.83 µM), yellow peach juice (776.72 ± 2.47 µM), and apple juice (154.77 ± 1.70 µM) with good accuracy (recovery rate: 90.49%-110.62%) and reproducibility (relative standard deviation, RSD < 3.0%). The b-IrOx nanozymes were compared with a commercial TAC standard kit to further confirm the reliability of the TMB colorimetric assay. The results showed no significant statistical difference between the two methods (p > 0.05). Therefore, the b-IrOx colorimetric assay offers a simple, low-cost approach for the rapid and precise determination of TAC in food samples. food analysis total antioxidant capacity peroxidase-like nanozyme colorimetric sensor Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Up to now, several analytical techniques have been explored for TAC determination, including the use of 2,2′-azino-bis(3-ethylbenzothiazole-6-sulfonate) (ABTS) assay, oxygen radical absorption capacity (ORAC) assay, ferric reducing antioxidant capacity (FRAP) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (Li et al. 2021 ; Munteanu et al. 2021; Rumpf et al. 2023 ; Egu et al. 2023 ). These methods construct colorimetric or fluorescent detection systems to quantify TAC by relying on single electron transfer (SET) and hydrogen atom transfer (HAT) as their core reaction mechanisms (Nakano et al. 2019 ; Zeb et al. 2020). In addition, instrumental analytical techniques including high performance liquid chromatography (HPLC) (Yalcin et al. 2021 ), gas chromatography (Duarte et al. 2023 ) and electrochemical analysis (Ishida et al. 2022 ) have also been widely used for TAC detection. Although these techniques may be well applicable for TAC assays, most of them suffer from obvious disadvantages such as expensive equipment, time-consuming experimental processes, the requirement for highly specialized skills, and some specific reaction conditions (Acidri et al. 2020 ). Therefore, there is an urgent need to develop a simple, convenient, and rapid alternative method for TAC determination to fulfill the demands of widespread practical applications in food science and related fields. Nanozymes, which possess inherent enzyme-like catalytic properties, have gained significant interest in recent years because they offer benefits compared to natural enzymes, including lower production costs, greater stability in various environments, and ease of large-scale manufacturing (Yang et al. 2021 ; Liang et al. 2019; Mou e al. 2022). Colorimetry offers a solution to current technological constraints in antioxidant analysis due to its cost-effectiveness, simplicity, and faster analysis time (Chen et al. 2023 ; Deshwal et al. 2024 ; Baranwal et al. 2024 ). For 3,3',5,5'-tetramethylbenzidine (TMB)-based colorimetric TAC detection, the typical reaction principle is as follows: in the presence of H 2 O 2 , a catalyst can promote the oxidation of the chromogenic substrate TMB, resulting in its transformation from a colorless state to a blue oxidized product (oxTMB). In the presence of antioxidants within the system, these compounds compete with TMB for the hydroxyl radicals (•OH) produced during the reaction. This competitive interaction inhibits the formation of oxidized TMB, resulting in a reduction in the intensity of the blue signal. This alteration in the signal exhibits a positive correlation with the concentration of antioxidants, thereby serving as the fundamental principle for the quantification of TAC (Chen et al. 2025 ). In conventional colorimetric assays for the determination of TAC, natural peroxidase is commonly employed as the catalytic agent to facilitate the aforementioned chromogenic reaction, owing to its high catalytic efficiency, favorable biocompatibility, and pronounced substrate specificity. However, natural peroxidase exhibits significant limitations in practical applications: its extraction and purification are both time-consuming and expensive, and its catalytic activity is highly susceptible to environmental conditions, often resulting in enzyme denaturation and subsequent loss of function. These challenges hinder its widespread use in large-scale TAC detection and underscore the need for alternative catalytic agents, such as nanozymes (Zhang et al. 2024 ). In this study, we have innovatively designed a sensitive colorimetric sensor for TAC determination by exploiting the peroxidase (POD)-like activity of bovine serum albumin (BSA)-templated iridium oxide nanozyme (b-IrOx). b-IrOx efficiently catalyses chromogenic substrate (TMB) in the presence of H 2 O 2 to produce colored oxidation products (oxTMB). When antioxidants are present in the solution, they compete with substrates for •OH through HAT and SET mechanisms, causing a decrease in the colored products (Halliwell et al. 1987 ; Hu et al. 2024 ). The enzyme-like activity of the nanozyme was utilized to develop a colorimetric assay for the determination of TAC in three different types of commercial fruit juices. The experimental results showed that the sensor has good accuracy (recovery rate: 90.49%-110.62%) and reproducibility (RSD < 3.0%) in TAC detection for mango juice (948.31 ± 0.83 µM TE), yellow peach juice (776.72 ± 2.47 µM TE), and apple juice (154.77 ± 1.70 µM TE). To further validate the reliability of this sensor, a comparative test was conducted with a commercial TAC standard kit, and statistical analysis indicated no significant difference between the TAC values measured by the two methods (p > 0.05), which further confirms the accuracy and feasibility of the b-IrOx-based TMB colorimetric sensor for practical TAC detection. These results indicate that the sensor exhibits strong practical utility in the detection of TAC in food samples and establish a basis for future developments in food monitoring and public health protection. Materials and methods Reagents and chemicals. Iridium (III) chloride, 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and 3,3′,5,5′-tetramethyl-benzidine (TMB) were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Labgic Technology Co., Ltd. (Beijing, China). Glutathione (GSH), cysteine (Cys), gallic acid (GA), caffeic acid (CA), and terephthalic acid (TA) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Ascorbic acid (AA) and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The total antioxidant capacity assay kit (ABTS method) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Apparatus. Fourier transform infrared (FTIR) spectroscopy measurements were conducted using a Nicolet 6700 spectrometer (Thermo Nicolet Corporation, USA) with a wavenumber range of 400–4000 cm⁻¹. X-ray photoelectron spectroscopy (XPS) data were acquired using a Thermo Scientific ESCALAB 250Xi spectrometer. Morphological and microstructural analyses were performed using a high-resolution transmission electron microscope (HRTEM, FEI Tecnai G2 F30) and a scanning electron microscope (SEM, Hitachi SU8010). Ultraviolet-visible (UV-vis) spectroscopic analysis was carried out using a PERSEE F-7000 UV-vis spectrophotometer and a BioTek CYTATION 5 Microplate Reader. Fluorescence analysis was performed using a fluorescence spectrophotometer F-7000 (Hitachi, Japan). Synthesis of b-IrOx nanozymes. In this work, the b-IrOx with POD-like activity was synthesized through a biomineralization process under alkaline conditions. BSA (150 mg) was dissolved in 10 mL of deionized water. Subsequently, an aqueous solution of Iridium(III) chloride (4 mL, 25 mM) was added dropwise to the BSA solution. The mixture was stirred continuously for 30 min, followed by the addition of 2 M NaOH solution to adjust the pH to 12.0. The reaction was allowed to proceed for 24 h at 80°C with vigorous stirring. The resulting product was collected, washed three times alternately with ethanol and deionized water, and then centrifuged at 10000 rpm for 10 min. The collected product was then freeze-dried and stored at 4°C for further use. POD-like activity of b-IrOx nanozymes. The POD-like activity of b-IrOx was assessed by its catalytic oxidation of TMB in the presence of H 2 O 2 , which results in the formation of blue oxTMB. The experimental protocol entailed the sequential addition of 30 µL of TMB (1 mM), 30 µL of b-IrOx (50 µg/mL), and 30 µL of H 2 O 2 (5 mM) solutions, with the total volume adjusted to 600 µL using a NaAc-HAc buffer (pH 4.0). After incubating the mixture at room temperature for 30 min, UV-visible spectra were measured over the wavelength range of 450–750 nm. To optimize the catalytic efficiency, several key parameters of the catalytic reaction were investigated, including pH (3.0–10.0), temperature (0–60°C), b-IrOx concentration (5-250 µg/mL), and catalytic reaction time (1–60 min). Research on kinetics. Steady-state kinetic experiments were performed to evaluate the POD-like catalytic behavior of b-IrOx nanozymes, using TMB and H 2 O 2 as the oxidant. All experiments were conducted under the optimized reaction conditions (pH 4.0, 25°C, 50 µg/mL of b-IrOx concentration) to ensure consistency and reliability. The Michaelis-Menten equation (Eq. ( 1 )) was used to the substrate concentration ( [S] )-initial reaction velocity ( V ) data, and the Lineweaver-Burk double reciprocal plot was applied for verification. Kinetic assays with variable TMB and H 2 O 2 concentration and kinetic parameter calculation were provided in the Supplementary Information. Detection of antioxidants and establishment of TAC quantification method . To detect target antioxidants and quantify TAC, a unified TMB-based colorimetric biosensing system using b-IrOx nanozyme was adopted. The analytes included three individual antioxidants (AA, caffeic acid, and GSH) and Trolox, which served as the standard for TAC quantification. For TAC quantification, a series of standard aqueous solutions of Trolox were prepared to construct the standard curve for TAC quantification. For the TMB-based system, 30 µL of antioxidants with gradient concentrations, including Trolox (0.1–100 µM), AA (0.5–20 µM), caffeic acid (0.1–40 µM), and GSH (0.2–90 µM) standard solution, 30 µL of b-IrOx nanozyme solution (50 µg/mL), 30 µL of H 2 O 2 solution (5 mM) and 30 µL of TMB chromogenic solution (1 mM) were mixed sequentially. The total volume was adjusted to 600 µL using NaAc-HAc buffer (pH 4.0). After incubation at 25°C for 30 min, the absorbance at wavelength range of 500–750 nm was measured. All results are presented as mean ± SD (n = 3). A standard curve was plotted with the absorbance decrease value as the ordinate and Trolox concentration as the abscissa, and a regression equation was established to quantify the TAC in samples. The TAC of samples was expressed as Trolox equivalent concentration. Selectivity Assay. To evaluate the selectivity of the b-IrOx-based sensing method for antioxidants, interference experiments were conducted using common interfering substances in fruit juices (glucose, amino acids, metal ions) and various antioxidants (AA, GA, CA, GSH, Cys). The reaction system for TMB-based assay contained 50 µg/mL b-IrOx, 1 mM TMB, 5 mM H 2 O 2 , and pH 4.0 buffer. Interfering substances were introduced at concentrations of 20 µM for glucose and amino acids, or 100 µM for metal ions, while antioxidants were added at a concentration of 10 µM. The TMB-based system was incubated at room temperature for 30 min with absorbance measured at 652 nm. Sample treatment and validation assay. Three types of commercially available non-concentrated reduced (NFC) juices (mango juice, yellow peach juice, and apple juice) from popular brands in supermarkets were selected as samples to assess the accuracy and practical applicability of the established b-IrOx-based sensing method. The NFC juice samples were first mixed thoroughly to ensure homogeneity, and then directly analyzed using the sensing system. Their TAC was determined via two approaches for cross-validation: 1) the b-IrOx-based TMB colorimetric assay; 2) a commercially available standard TAC assay kit (ABTS method), where the detection was performed strictly according to the kit instruction manual to obtain the reference TAC values of the fruit juice samples. These two approaches were designed to verify the reliability of the self-established b-IrOx system against mature commercial detection tools. The test results were reported as the mean ± SD of three independent parallel measurements. In cases where the sample contained a higher concentration of antioxidants than the linear range of the Trolox standard curve (0.1–100 µM Trolox equivalent), it was further diluted with deionized water. The TAC of the original NFC juice sample (T, µM) was calculated using the formula (1): $$\:\text{T}\text{=}{\text{C}}_{\text{Trolox}}\text{×}\frac{{\text{V}}_{\text{total}}}{{\text{V}}_{\text{sample}}}\text{×}\text{D}\text{}\text{}$$ 1 Where: C Trolox is Trolox equivalent concentration of the diluted sample, obtained from the standard curve (µM); V total is total volume of the detection reaction system (µL, 200 µL in this work); V sample is volume of the diluted sample added to the reaction system (µL, 10 µL in this work); D is dilution ratio of the original NFC juice. Results and Discussion Synthesis and characteristics of b-IrOx. In this study, biomineralization of b-IrOx nanoparticles commenced with the adsorption of Ir 3+ ions onto BSA, facilitated by the carboxy and amino groups' affinity towards metal ions. The TEM images of b-IrOx nanoparticles exhibited a spherical morphology as shown in Fig. 1 A, with an average diameter of 34.56 nm. The elemental analysis of b-IrOx nanoparticles in Fig. 1 B and C clearly identified the presence of Ir, C, N, S, and O elements, which aligns with the composition of BSA and IrOx. Additionally, the elemental mapping images (Fig. 1 C) showed that these elements are evenly distributed throughout the nanoparticles, confirming that BSA has been effectively combined with the IrOx core to create b-IrOx. As shown in Fig. 1 D, the characteristic peaks of b-IrOx corresponding to functional groups of BSA were existed, and subtle shifts in partial peak positions indicated interactions between BSA and IrOx, confirming the integration of the two components. XPS was employed to analyze the surface chemical state of b-IrOx nanoparticles. In Fig. 1 E, the XPS full spectrum confirmed the presence of Ir, C, N, O, and S elements on the nanoparticle surface, aligning with EDS results and ruling out significant contamination from other elements. In Fig. 1 F, the high-resolution Ir 4f XPS spectrum showed distinct spin-orbit splitting peaks. The peaks observed at 61.8 eV and 64.7 eV correspond to the 4f₇/₂ and 4f₅/₂ states of Ir⁴⁺, respectively, providing direct evidence that iridium in the nanoparticles predominantly exists in the + 4 oxidation state. The deconvolution of the high-resolution O 1s spectrum (Fig. 1 G) identified two principal peaks: one located at 532.6 eV, which is ascribed to the -COOH and -C = O functional groups originating from bovine serum albumin (BSA), and another at 531.0 eV, corresponding to Ir–O bonds characteristic of IrO 2 . This result further confirmed the formation of IrO 2 -related species in the b-IrOx nanoparticles, completing the characterization of their surface chemical structure. Taken together, the comprehensive characterizations collectively confirmed the successful synthesis of b-IrOx nanoparticles with well-defined spherical morphology, uniform BSA combination, and dominant Ir⁴⁺ oxidation state within IrO₂-related species, providing a comprehensive structural basis for exploring their functional properties in subsequent studies. POD-like activity of b-IrOx. For the purpose of examining the enzyme-mimetic properties of the prepared b-IrOx, the oxidation reaction of TMB chromogenic substrate was carried out in the presence of b-IrOx and H 2 O 2 by using a UV-vis spectrophotometer to monitor the formation of oxidized products. As shown in Fig. 2 A, the conversion of TMB substrate to oxTMB was monitored at λ max = 652 nm. The results confirm the generation of oxTMB product, demonstrating the POD-like catalytic activity of b-IrOx towards TMB, accompanied by a significant colorimetric response. To elucidate the primary factors influencing TMB oxidation, various operational parameters were assessed, including b-IrOx concentration, reaction time, temperature, and pH. In the TMB system, as shown in Figure S1 A, the catalytic activity of b-IrOx increases from pH 3.0 to 10.0, peaking at pH 4.0, and then sharply decreases under weakly acidic and neutral conditions. Subsequent experiments used acetic acid buffer at pH 4.0. The effect of reaction temperature (Figure S1 B) shows that the catalytic activity of b-IrOx significantly increases from 10°C to 60°C; however, considering the limitation of high temperature on practical application scenarios, 25°C was selected as the optimal reaction temperature. Furthermore, the concentration and incubation time (1–60 min) were investigated for its impact on UV signal under the optimal pH and temperature conditions. b-IrOx concentration optimization (Figure S2A) demonstrated that the catalytic activity increased with the nanozyme concentration from 5 to 250 µg/mL. To balance catalytic efficiency and practical considerations, 50 µg/mL was determined as the optimal b-IrOx concentration for all subsequent experiments. As shown in Figure S2B, the UV signal significantly increases from 1.0 to 30 min and gradually stabilizes. For experimental convenience, the incubation time was 30 min. To quantitatively assess the POD-like activity of b-IrOx, steady-state kinetics were studied by independently varying the concentrations of H 2 O 2 or TMB. For the TMB substrate system, when H 2 O 2 was fixed, the Michaelis-Menten curve for TMB and its corresponding Lineweaver-Burk plot (Fig. 2 B-C) yielded K m = 0.34 mM and V max = 0.36×10 − 8 M/s. When TMB was fixed, the Michaelis-Menten curve for H 2 O 2 and its corresponding Lineweaver-Burk plot (Fig. 2 E-F) gave K m = 2.19 mM and V max = 0.36×10 − 8 M/s. Notably, high affinity of b-IrOx for H 2 O 2 not only boosts its catalytic performance but also supports the upcoming sensitive TAC colorimetric assay. The mechanism of the b-IrOx nanozyme-based colorimetric method involves a competitive reaction between TMB and antioxidants for •OH on nanozymes. To confirm •OH generation, TA reacts with •OH to form fluorescent 2-hydroxy terephthalic acid (TAOH). As shown in Fig. 2 D, a strong fluorescent signal at 430 nm was observed in the IrOx + TA + H 2 O 2 group, while control groups (IrOx + TA, H 2 O 2 + TA, IrOx + H 2 O 2 ) had weak or no fluorescence. It is demonstrated that b-IrOx catalyzes H 2 O 2 to generate •OH, enabling SET and HAT reactions for the detection of antioxidants. b-IrOx-based colorimetric methods for antioxidants. Under the optimal conditions, the performance of the b-IrOx-based sensing system was analyzed by determining target antioxidants, including Trolox, GSH, AA, and caffeic acid. In Fig. 3 A-D, the concentration-dependent absorption changes directly reflect the competitive reaction between TMB and antioxidants for •OH on the nanozyme surface: antioxidants scavenge •OH, reducing oxTMB formation and decreasing the 652 nm absorption intensity. This mechanism is further supported by the linear quantitative relationships in Fig. 3 E-H, where both HAT-type (Trolox, caffeic acid) and SET-type (GSH, AA) antioxidants show reliable linear responses. Four linear calibration curves were obtained in the TMB-based colorimetric strategy (Table S1 ). The high R 2 values (> 0.99) confirm the good linear relationship between ΔA and antioxidant concentration, laying a foundation for accurate quantitative detection. Additionally, selectivity studies (Figure S3) revealed that common interfering substances like glucose, amino acids, and various ions had negligible effects on the absorbance intensity, further ensuring the specificity of the b-IrOx nanozyme-based TMB colorimetric sensing system. Real sample analysis. The practical applicability was confirmed through standard addition experiments on real food samples. NFC juices, including yellow peach juice, apple juice, and mango juice, were purchased from a local supermarket. In Fig. 4 A and Table S2, for yellow peach juice, spiked Trolox at 10 µM, 20 µM, and 30 µM showed recoveries of 90.49%-101.78%. Apple juice exhibited recoveries of 98.97%-110.62% for the same spiked levels. Mango juice had recoveries of 99.62%-110.21%. All recoveries fell within the range of 90.49%-110.62%, and the relative standard deviations (RSD) for all measurements were less than 3%. These results indicate minimal matrix interference and validate that the detection system is reliable for real sample analysis. To verify the practical applicability of the developed b-IrOx-based sensing system, this study determined the TAC of real samples (mango juice, yellow peach juice, apple juice) using the core detection mode—b-IrOx-based TMB colorimetric method. A commercial ABTS kit was used as the reference standard, and the results were retained for accuracy comparison. All experiments used Trolox as the standard substance to ensure consistency in TAC quantification, with results expressed as µM TE. The TAC detection results of the two methods for each juice sample have been summarized in Fig. 4 B and Table 1 . These results confirm the reliability and consistency of the developed b-IrOx-based TMB colorimetric method and ABTS kit in TAC measurement. Notably, the b-IrOx nanozyme, which exhibits POD-like activity and serves as the centerpiece of this innovative colorimetric approach, offers clear benefits compared to natural enzymes, including easy synthesis and improved stability. Compared to traditional POD-based commercial ABTS kits, the developed TMB colorimetric method shows comparable TAC detection capabilities for the tested fruit juices, while avoiding over-reliance on complex detection platforms. Table 1 Comparative study of TAC detection in fruits using the proposed b-IrOx-based TMB colorimetric strategies and a standardized ABTS kit. Samples b-IrOx-based TMB colorimetric assay (µM, Mean ± SD) ABTS kit (µM, Mean ± SD) Yellow peach juice 776.72 ± 2.47 801.10 ± 2.58 Apple juice 154.77 ± 1.70 165.30 ± 2.24 Mango juice 948.31 ± 0.83 987.03 ± 1.75 Overall, the b-IrOx-based TMB colorimetric method presents a promising alternative for TAC detection and analysis in practical scenarios such as small-scale fruit juice processing plants and routine quality control. It maintains accuracy comparable to commercial ABTS kits and provides a stable, easy-to-operate solution for TAC quantification in fruit juice samples. Conclusions The study demonstrated a one-step self-assembly method to produce a novel b-IrOx nanozyme, which effectively substitutes natural enzymes for TAC detection in food samples. It was specifically validated in mango juice, yellow peach juice and apple juice. This colorimetric sensing approach offers advantages over natural enzymes by eliminating the high costs and time required for enzyme production, preventing enzyme activity denaturation under harsh conditions, enabling the widespread use of the TMB method for TAC measurement, and presenting a feasible TAC detection and analysis alternative. Evaluation using the three fruit juice samples confirms the promising practical application potential of this b-IrOx-based sensing strategy. It avoids reliance on complex detection platforms, making it suitable for scenarios like small-scale fruit juice processing plants or routine quality control. This method thus presents a feasible, stable alternative for TAC detection and analysis in food samples. Declarations The authors declare no competing interests. Author Contribution Shuangshuang Yan: Writing-review and editing, Characterization. Dongying An: Writing-original draft, Validation, Data curation. Yutian Zhou: Visualization, Investigation. 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Oxid Med Cell Longev 2020(1):9568278 Yalcin S, Karakas O, Okudan ES, Baskan KS, Cekic SD, Apak R (2021) HPLC Detection and Antioxidant Capacity Determination of Brown, Red and Green Algal Pigments in Seaweed Extracts. J Chromatogr Sci 59(4):324–337 Yang W, Yang X, Zhu L, Chu H, Li X, Xu W (2021) Nanozymes: Activity Origin, Catalytic Mechanism, and Biological Application. Coord Chem Rev 448:214170 Zeb A (2020) Concept, Mechanism, and Applications of Phenolic Antioxidants in Foods. J Food Biochem 44(9):13394 Zhang X, Wang J, Chang N, Yang Y, Li Y, Wei Q, Ni C, Song W, Ma M, Feng X, Fan R (2024) Cu-BTC Derived Mesoporous CuS Nanomaterial as Nanozyme for Colorimetric Detection of Glutathione. Molecules 29(9):2117 Additional Declarations No competing interests reported. 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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-8592177","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":583508376,"identity":"bef5688a-5b59-4b97-923e-2c274fe4e437","order_by":0,"name":"Shuangshuang Yan","email":"","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Shuangshuang","middleName":"","lastName":"Yan","suffix":""},{"id":583508377,"identity":"cc377169-dca9-454d-84eb-0cff2a14cb49","order_by":1,"name":"Dongying An","email":"","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Dongying","middleName":"","lastName":"An","suffix":""},{"id":583508378,"identity":"8b8288da-1ed5-4ac1-a545-8438bc635558","order_by":2,"name":"Yutian Zhou","email":"","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yutian","middleName":"","lastName":"Zhou","suffix":""},{"id":583508379,"identity":"1ab6fce8-6872-4168-b917-880f897bfa27","order_by":3,"name":"Xinli Guo","email":"","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xinli","middleName":"","lastName":"Guo","suffix":""},{"id":583508380,"identity":"e4a58b0d-3e6d-41ab-bece-61a075c798ea","order_by":4,"name":"Zhihang Qin","email":"","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zhihang","middleName":"","lastName":"Qin","suffix":""},{"id":583508381,"identity":"92e384a4-f489-4f04-9083-d290330294d7","order_by":5,"name":"Li Yuan","email":"","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Yuan","suffix":""},{"id":583508382,"identity":"846b24f7-7f47-4efa-bba6-112b85803563","order_by":6,"name":"Mengmeng Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYNCDD1BagmgdjDNI1sLMQ4wWg+NnD7+6UXHHbm177+HXNn8OR/M3MB+8zcNgl4dTy5m8NOucM8+St505l2ad23Y4d8YBtmRrHobkYlxazA7kmBkDVSab3QAxGg7nbmDgMZPmYTiQ2IBLy/k3QJX/gFruAxkWf0Ba+L/h13Ijx/gx0HA7sxs8xo8Z2MC2sOHVYn/jjRlzzrHDCWZncswYe9vSc2ccZjO2nGOQjFOLZH+O8eecmsP2ZsfPGH/48cc6t7+9+eGNNxV2OLUAARsoFkAKQIxmYOyABA1wqwcCZlAysYcy6vAqHQWjYBSMgpEJALjUXxjLsSe+AAAAAElFTkSuQmCC","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":true,"prefix":"","firstName":"Mengmeng","middleName":"","lastName":"Wang","suffix":""},{"id":583508383,"identity":"60fd1d49-0130-427c-ae76-e335ee4b7e15","order_by":7,"name":"Yang Zhang","email":"","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-01-13 12:53:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8592177/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8592177/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101655612,"identity":"eaf5b583-4680-4252-8206-cc0873d9e6ec","added_by":"auto","created_at":"2026-02-02 09:57:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":153485,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of b-IrOx nanoparticles. (A) TEM images. (B) EDS analysis. (C) Elemental mapping images. (D) FT-IR spectra of BSA and b-IrOx. (E) XPS full-spectrum of b-IrOx. (F) High-resolution Ir 4f XPS spectrum of b-IrOx nanoparticles. (G) High-resolution O 1s XPS spectrum of b-IrOx nanoparticles.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8592177/v1/3808ba34dabfa635a087f947.jpeg"},{"id":101753093,"identity":"2981d9c4-d407-40d4-b3e5-7125c86ea6aa","added_by":"auto","created_at":"2026-02-03 10:39:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":225105,"visible":true,"origin":"","legend":"\u003cp\u003e(A) POD-like activity: typical absorption spectra of TMB, oxidation catalyzed by b-IrOx and control groups in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at pH 4.0. (B) The concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was 5 mM and the TMB concentration varied. (C) Double reciprocal plots of the \u003cem\u003eMichaelis-Menten\u003c/em\u003e equation from the activity date of the concentration of TMB. (D) Fluorescent spectra of TA after reaction with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the absence and presence of b-IrOx. Concentration of b-IrOx was 50 µg/mL, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was 10 mM. (E) The concentration of TMB was 1 mM and the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration varied. (F) Double reciprocal plots of the \u003cem\u003eMichaelis-Menten \u003c/em\u003eequation from the activity date of the concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8592177/v1/1a087a7ea8c76423de940cef.png"},{"id":101655608,"identity":"9e3bbfc4-ea6c-444a-8f9f-b12343aacfcc","added_by":"auto","created_at":"2026-02-02 09:57:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":292232,"visible":true,"origin":"","legend":"\u003cp\u003eOptical density values of the b-IrOx nanozyme-based sensing strategy as a function of HAT species antioxidants (A) Trolox and (D) caffeic acid, and SET working species antioxidants (B) GSH and (C) AA. (E-H) Linear calibration plots corresponding to (A-D) with TMB as the substrate, respectively.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8592177/v1/dd8804b37f26e232b75c9ad3.png"},{"id":101753887,"identity":"5ab4b8db-732e-4079-89d0-065b4abddc46","added_by":"auto","created_at":"2026-02-03 10:41:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59702,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Repeatability (n=3) measurements of the nanozyme-based assay for different Trolox standards recovery study in different fruit samples in TMB systems. (B) Comparison of TAC detection in three samples using the commercial ABTS kit and the b-IrOx-based TMB based colorimetric assay. Data are shown as mean ± SD ((ns: p \u0026gt; 0.05, *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8592177/v1/4df5f943c67f83cbcb79e2fc.png"},{"id":104813226,"identity":"611a0324-5cfb-4137-9a1a-275ae60652b2","added_by":"auto","created_at":"2026-03-17 13:06:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1252177,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8592177/v1/f7644dae-33ed-4e3d-9ba8-3631e6c37a9a.pdf"},{"id":101655609,"identity":"b4262d7e-1ba4-46a2-b612-7ea5a4be0e71","added_by":"auto","created_at":"2026-02-02 09:57:46","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":130048,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-8592177/v1/835853ecb678762c9130b67f.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Engineering a BSA-templated Iridium Oxide Nanozyme for Colorimetric Evaluation of Total Antioxidant Capacity in Food Samples","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUp to now, several analytical techniques have been explored for TAC determination, including the use of 2,2\u0026prime;-azino-bis(3-ethylbenzothiazole-6-sulfonate) (ABTS) assay, oxygen radical absorption capacity (ORAC) assay, ferric reducing antioxidant capacity (FRAP) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Munteanu et al. 2021; Rumpf et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Egu et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These methods construct colorimetric or fluorescent detection systems to quantify TAC by relying on single electron transfer (SET) and hydrogen atom transfer (HAT) as their core reaction mechanisms (Nakano et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zeb et al. 2020). In addition, instrumental analytical techniques including high performance liquid chromatography (HPLC) (Yalcin et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), gas chromatography (Duarte et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and electrochemical analysis (Ishida et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) have also been widely used for TAC detection. Although these techniques may be well applicable for TAC assays, most of them suffer from obvious disadvantages such as expensive equipment, time-consuming experimental processes, the requirement for highly specialized skills, and some specific reaction conditions (Acidri et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, there is an urgent need to develop a simple, convenient, and rapid alternative method for TAC determination to fulfill the demands of widespread practical applications in food science and related fields.\u003c/p\u003e \u003cp\u003eNanozymes, which possess inherent enzyme-like catalytic properties, have gained significant interest in recent years because they offer benefits compared to natural enzymes, including lower production costs, greater stability in various environments, and ease of large-scale manufacturing (Yang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liang et al. 2019; Mou e al. 2022). Colorimetry offers a solution to current technological constraints in antioxidant analysis due to its cost-effectiveness, simplicity, and faster analysis time (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Deshwal et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Baranwal et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For 3,3',5,5'-tetramethylbenzidine (TMB)-based colorimetric TAC detection, the typical reaction principle is as follows: in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, a catalyst can promote the oxidation of the chromogenic substrate TMB, resulting in its transformation from a colorless state to a blue oxidized product (oxTMB). In the presence of antioxidants within the system, these compounds compete with TMB for the hydroxyl radicals (\u0026bull;OH) produced during the reaction. This competitive interaction inhibits the formation of oxidized TMB, resulting in a reduction in the intensity of the blue signal. This alteration in the signal exhibits a positive correlation with the concentration of antioxidants, thereby serving as the fundamental principle for the quantification of TAC (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In conventional colorimetric assays for the determination of TAC, natural peroxidase is commonly employed as the catalytic agent to facilitate the aforementioned chromogenic reaction, owing to its high catalytic efficiency, favorable biocompatibility, and pronounced substrate specificity. However, natural peroxidase exhibits significant limitations in practical applications: its extraction and purification are both time-consuming and expensive, and its catalytic activity is highly susceptible to environmental conditions, often resulting in enzyme denaturation and subsequent loss of function. These challenges hinder its widespread use in large-scale TAC detection and underscore the need for alternative catalytic agents, such as nanozymes (Zhang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we have innovatively designed a sensitive colorimetric sensor for TAC determination by exploiting the peroxidase (POD)-like activity of bovine serum albumin (BSA)-templated iridium oxide nanozyme (b-IrOx). b-IrOx efficiently catalyses chromogenic substrate (TMB) in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to produce colored oxidation products (oxTMB). When antioxidants are present in the solution, they compete with substrates for \u0026bull;OH through HAT and SET mechanisms, causing a decrease in the colored products (Halliwell et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The enzyme-like activity of the nanozyme was utilized to develop a colorimetric assay for the determination of TAC in three different types of commercial fruit juices. The experimental results showed that the sensor has good accuracy (recovery rate: 90.49%-110.62%) and reproducibility (RSD\u0026thinsp;\u0026lt;\u0026thinsp;3.0%) in TAC detection for mango juice (948.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 \u0026micro;M TE), yellow peach juice (776.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47 \u0026micro;M TE), and apple juice (154.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 \u0026micro;M TE). To further validate the reliability of this sensor, a comparative test was conducted with a commercial TAC standard kit, and statistical analysis indicated no significant difference between the TAC values measured by the two methods (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), which further confirms the accuracy and feasibility of the b-IrOx-based TMB colorimetric sensor for practical TAC detection. These results indicate that the sensor exhibits strong practical utility in the detection of TAC in food samples and establish a basis for future developments in food monitoring and public health protection.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eReagents and chemicals.\u003c/b\u003e Iridium (III) chloride, 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and 3,3\u0026prime;,5,5\u0026prime;-tetramethyl-benzidine (TMB) were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Labgic Technology Co., Ltd. (Beijing, China). Glutathione (GSH), cysteine (Cys), gallic acid (GA), caffeic acid (CA), and terephthalic acid (TA) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Ascorbic acid (AA) and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The total antioxidant capacity assay kit (ABTS method) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eApparatus.\u003c/b\u003e Fourier transform infrared (FTIR) spectroscopy measurements were conducted using a Nicolet 6700 spectrometer (Thermo Nicolet Corporation, USA) with a wavenumber range of 400\u0026ndash;4000 cm⁻\u0026sup1;. X-ray photoelectron spectroscopy (XPS) data were acquired using a Thermo Scientific ESCALAB 250Xi spectrometer. Morphological and microstructural analyses were performed using a high-resolution transmission electron microscope (HRTEM, FEI Tecnai G2 F30) and a scanning electron microscope (SEM, Hitachi SU8010). Ultraviolet-visible (UV-vis) spectroscopic analysis was carried out using a PERSEE F-7000 UV-vis spectrophotometer and a BioTek CYTATION 5 Microplate Reader. Fluorescence analysis was performed using a fluorescence spectrophotometer F-7000 (Hitachi, Japan).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of b-IrOx nanozymes.\u003c/b\u003e In this work, the b-IrOx with POD-like activity was synthesized through a biomineralization process under alkaline conditions. BSA (150 mg) was dissolved in 10 mL of deionized water. Subsequently, an aqueous solution of Iridium(III) chloride (4 mL, 25 mM) was added dropwise to the BSA solution. The mixture was stirred continuously for 30 min, followed by the addition of 2 M NaOH solution to adjust the pH to 12.0. The reaction was allowed to proceed for 24 h at 80\u0026deg;C with vigorous stirring. The resulting product was collected, washed three times alternately with ethanol and deionized water, and then centrifuged at 10000 rpm for 10 min. The collected product was then freeze-dried and stored at 4\u0026deg;C for further use.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePOD-like activity of b-IrOx nanozymes.\u003c/b\u003e The POD-like activity of b-IrOx was assessed by its catalytic oxidation of TMB in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which results in the formation of blue oxTMB. The experimental protocol entailed the sequential addition of 30 \u0026micro;L of TMB (1 mM), 30 \u0026micro;L of b-IrOx (50 \u0026micro;g/mL), and 30 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (5 mM) solutions, with the total volume adjusted to 600 \u0026micro;L using a NaAc-HAc buffer (pH 4.0). After incubating the mixture at room temperature for 30 min, UV-visible spectra were measured over the wavelength range of 450\u0026ndash;750 nm. To optimize the catalytic efficiency, several key parameters of the catalytic reaction were investigated, including pH (3.0\u0026ndash;10.0), temperature (0\u0026ndash;60\u0026deg;C), b-IrOx concentration (5-250 \u0026micro;g/mL), and catalytic reaction time (1\u0026ndash;60 min).\u003c/p\u003e \u003cp\u003e \u003cb\u003eResearch on kinetics.\u003c/b\u003e Steady-state kinetic experiments were performed to evaluate the POD-like catalytic behavior of b-IrOx nanozymes, using TMB and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as the oxidant. All experiments were conducted under the optimized reaction conditions (pH 4.0, 25\u0026deg;C, 50 \u0026micro;g/mL of b-IrOx concentration) to ensure consistency and reliability. The \u003cem\u003eMichaelis-Menten\u003c/em\u003e equation (Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)) was used to the substrate concentration (\u003cem\u003e[S]\u003c/em\u003e)-initial reaction velocity (\u003cem\u003eV\u003c/em\u003e) data, and the \u003cem\u003eLineweaver-Burk\u003c/em\u003e double reciprocal plot was applied for verification. Kinetic assays with variable TMB and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration and kinetic parameter calculation were provided in the Supplementary Information.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of antioxidants and establishment of TAC quantification method\u003c/b\u003e. To detect target antioxidants and quantify TAC, a unified TMB-based colorimetric biosensing system using b-IrOx nanozyme was adopted. The analytes included three individual antioxidants (AA, caffeic acid, and GSH) and Trolox, which served as the standard for TAC quantification. For TAC quantification, a series of standard aqueous solutions of Trolox were prepared to construct the standard curve for TAC quantification. For the TMB-based system, 30 \u0026micro;L of antioxidants with gradient concentrations, including Trolox (0.1\u0026ndash;100 \u0026micro;M), AA (0.5\u0026ndash;20 \u0026micro;M), caffeic acid (0.1\u0026ndash;40 \u0026micro;M), and GSH (0.2\u0026ndash;90 \u0026micro;M) standard solution, 30 \u0026micro;L of b-IrOx nanozyme solution (50 \u0026micro;g/mL), 30 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution (5 mM) and 30 \u0026micro;L of TMB chromogenic solution (1 mM) were mixed sequentially. The total volume was adjusted to 600 \u0026micro;L using NaAc-HAc buffer (pH 4.0). After incubation at 25\u0026deg;C for 30 min, the absorbance at wavelength range of 500\u0026ndash;750 nm was measured. All results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). A standard curve was plotted with the absorbance decrease value as the ordinate and Trolox concentration as the abscissa, and a regression equation was established to quantify the TAC in samples. The TAC of samples was expressed as Trolox equivalent concentration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSelectivity Assay.\u003c/b\u003e To evaluate the selectivity of the b-IrOx-based sensing method for antioxidants, interference experiments were conducted using common interfering substances in fruit juices (glucose, amino acids, metal ions) and various antioxidants (AA, GA, CA, GSH, Cys). The reaction system for TMB-based assay contained 50 \u0026micro;g/mL b-IrOx, 1 mM TMB, 5 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and pH 4.0 buffer. Interfering substances were introduced at concentrations of 20 \u0026micro;M for glucose and amino acids, or 100 \u0026micro;M for metal ions, while antioxidants were added at a concentration of 10 \u0026micro;M. The TMB-based system was incubated at room temperature for 30 min with absorbance measured at 652 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSample treatment and validation assay.\u003c/b\u003e Three types of commercially available non-concentrated reduced (NFC) juices (mango juice, yellow peach juice, and apple juice) from popular brands in supermarkets were selected as samples to assess the accuracy and practical applicability of the established b-IrOx-based sensing method.\u003c/p\u003e \u003cp\u003eThe NFC juice samples were first mixed thoroughly to ensure homogeneity, and then directly analyzed using the sensing system. Their TAC was determined via two approaches for cross-validation: 1) the b-IrOx-based TMB colorimetric assay; 2) a commercially available standard TAC assay kit (ABTS method), where the detection was performed strictly according to the kit instruction manual to obtain the reference TAC values of the fruit juice samples. These two approaches were designed to verify the reliability of the self-established b-IrOx system against mature commercial detection tools. The test results were reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent parallel measurements. In cases where the sample contained a higher concentration of antioxidants than the linear range of the Trolox standard curve (0.1\u0026ndash;100 \u0026micro;M Trolox equivalent), it was further diluted with deionized water. The TAC of the original NFC juice sample (T, \u0026micro;M) was calculated using the formula (1):\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{T}\\text{=}{\\text{C}}_{\\text{Trolox}}\\text{\u0026times;}\\frac{{\\text{V}}_{\\text{total}}}{{\\text{V}}_{\\text{sample}}}\\text{\u0026times;}\\text{D}\\text{}\\text{}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere: \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003eTrolox\u003c/em\u003e\u003c/sub\u003e is Trolox equivalent concentration of the diluted sample, obtained from the standard curve (\u0026micro;M); \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003etotal\u003c/em\u003e\u003c/sub\u003e is total volume of the detection reaction system (\u0026micro;L, 200 \u0026micro;L in this work); \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003esample\u003c/em\u003e\u003c/sub\u003e is volume of the diluted sample added to the reaction system (\u0026micro;L, 10 \u0026micro;L in this work); D is dilution ratio of the original NFC juice.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eSynthesis and characteristics of b-IrOx.\u003c/b\u003e In this study, biomineralization of b-IrOx nanoparticles commenced with the adsorption of Ir\u003csup\u003e3+\u003c/sup\u003e ions onto BSA, facilitated by the carboxy and amino groups' affinity towards metal ions. The TEM images of b-IrOx nanoparticles exhibited a spherical morphology as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, with an average diameter of 34.56 nm. The elemental analysis of b-IrOx nanoparticles in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C clearly identified the presence of Ir, C, N, S, and O elements, which aligns with the composition of BSA and IrOx. Additionally, the elemental mapping images (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) showed that these elements are evenly distributed throughout the nanoparticles, confirming that BSA has been effectively combined with the IrOx core to create b-IrOx. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, the characteristic peaks of b-IrOx corresponding to functional groups of BSA were existed, and subtle shifts in partial peak positions indicated interactions between BSA and IrOx, confirming the integration of the two components.\u003c/p\u003e \u003cp\u003eXPS was employed to analyze the surface chemical state of b-IrOx nanoparticles. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, the XPS full spectrum confirmed the presence of Ir, C, N, O, and S elements on the nanoparticle surface, aligning with EDS results and ruling out significant contamination from other elements. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, the high-resolution Ir 4f XPS spectrum showed distinct spin-orbit splitting peaks. The peaks observed at 61.8 eV and 64.7 eV correspond to the 4f₇/₂ and 4f₅/₂ states of Ir⁴⁺, respectively, providing direct evidence that iridium in the nanoparticles predominantly exists in the +\u0026thinsp;4 oxidation state. The deconvolution of the high-resolution O 1s spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) identified two principal peaks: one located at 532.6 eV, which is ascribed to the -COOH and -C\u0026thinsp;=\u0026thinsp;O functional groups originating from bovine serum albumin (BSA), and another at 531.0 eV, corresponding to Ir\u0026ndash;O bonds characteristic of IrO\u003csub\u003e2\u003c/sub\u003e. This result further confirmed the formation of IrO\u003csub\u003e2\u003c/sub\u003e-related species in the b-IrOx nanoparticles, completing the characterization of their surface chemical structure. Taken together, the comprehensive characterizations collectively confirmed the successful synthesis of b-IrOx nanoparticles with well-defined spherical morphology, uniform BSA combination, and dominant Ir⁴⁺ oxidation state within IrO₂-related species, providing a comprehensive structural basis for exploring their functional properties in subsequent studies.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePOD-like activity of b-IrOx.\u003c/b\u003e For the purpose of examining the enzyme-mimetic properties of the prepared b-IrOx, the oxidation reaction of TMB chromogenic substrate was carried out in the presence of b-IrOx and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by using a UV-vis spectrophotometer to monitor the formation of oxidized products. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the conversion of TMB substrate to oxTMB was monitored at λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;652 nm. The results confirm the generation of oxTMB product, demonstrating the POD-like catalytic activity of b-IrOx towards TMB, accompanied by a significant colorimetric response.\u003c/p\u003e \u003cp\u003eTo elucidate the primary factors influencing TMB oxidation, various operational parameters were assessed, including b-IrOx concentration, reaction time, temperature, and pH. In the TMB system, as shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, the catalytic activity of b-IrOx increases from pH 3.0 to 10.0, peaking at pH 4.0, and then sharply decreases under weakly acidic and neutral conditions. Subsequent experiments used acetic acid buffer at pH 4.0. The effect of reaction temperature (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB) shows that the catalytic activity of b-IrOx significantly increases from 10\u0026deg;C to 60\u0026deg;C; however, considering the limitation of high temperature on practical application scenarios, 25\u0026deg;C was selected as the optimal reaction temperature. Furthermore, the concentration and incubation time (1\u0026ndash;60 min) were investigated for its impact on UV signal under the optimal pH and temperature conditions. b-IrOx concentration optimization (Figure S2A) demonstrated that the catalytic activity increased with the nanozyme concentration from 5 to 250 \u0026micro;g/mL. To balance catalytic efficiency and practical considerations, 50 \u0026micro;g/mL was determined as the optimal b-IrOx concentration for all subsequent experiments. As shown in Figure S2B, the UV signal significantly increases from 1.0 to 30 min and gradually stabilizes. For experimental convenience, the incubation time was 30 min.\u003c/p\u003e \u003cp\u003eTo quantitatively assess the POD-like activity of b-IrOx, steady-state kinetics were studied by independently varying the concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or TMB. For the TMB substrate system, when H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was fixed, the \u003cem\u003eMichaelis-Menten\u003c/em\u003e curve for TMB and its corresponding \u003cem\u003eLineweaver-Burk\u003c/em\u003e plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C) yielded \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e = 0.34 mM and V\u003csub\u003emax\u003c/sub\u003e = 0.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e M/s. When TMB was fixed, the \u003cem\u003eMichaelis-Menten\u003c/em\u003e curve for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and its corresponding \u003cem\u003eLineweaver-Burk\u003c/em\u003e plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F) gave \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e = 2.19 mM and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e = 0.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e M/s. Notably, high affinity of b-IrOx for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e not only boosts its catalytic performance but also supports the upcoming sensitive TAC colorimetric assay.\u003c/p\u003e \u003cp\u003eThe mechanism of the b-IrOx nanozyme-based colorimetric method involves a competitive reaction between TMB and antioxidants for \u0026bull;OH on nanozymes. To confirm \u0026bull;OH generation, TA reacts with \u0026bull;OH to form fluorescent 2-hydroxy terephthalic acid (TAOH). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, a strong fluorescent signal at 430 nm was observed in the IrOx\u0026thinsp;+\u0026thinsp;TA\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, while control groups (IrOx\u0026thinsp;+\u0026thinsp;TA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;TA, IrOx\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) had weak or no fluorescence. It is demonstrated that b-IrOx catalyzes H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate \u0026bull;OH, enabling SET and HAT reactions for the detection of antioxidants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eb-IrOx-based colorimetric methods for antioxidants.\u003c/b\u003e Under the optimal conditions, the performance of the b-IrOx-based sensing system was analyzed by determining target antioxidants, including Trolox, GSH, AA, and caffeic acid. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D, the concentration-dependent absorption changes directly reflect the competitive reaction between TMB and antioxidants for \u0026bull;OH on the nanozyme surface: antioxidants scavenge \u0026bull;OH, reducing oxTMB formation and decreasing the 652 nm absorption intensity. This mechanism is further supported by the linear quantitative relationships in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H, where both HAT-type (Trolox, caffeic acid) and SET-type (GSH, AA) antioxidants show reliable linear responses. Four linear calibration curves were obtained in the TMB-based colorimetric strategy (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The high R\u003csup\u003e2\u003c/sup\u003e values (\u0026gt;\u0026thinsp;0.99) confirm the good linear relationship between ΔA and antioxidant concentration, laying a foundation for accurate quantitative detection. Additionally, selectivity studies (Figure S3) revealed that common interfering substances like glucose, amino acids, and various ions had negligible effects on the absorbance intensity, further ensuring the specificity of the b-IrOx nanozyme-based TMB colorimetric sensing system.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReal sample analysis.\u003c/b\u003e The practical applicability was confirmed through standard addition experiments on real food samples. NFC juices, including yellow peach juice, apple juice, and mango juice, were purchased from a local supermarket. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Table S2, for yellow peach juice, spiked Trolox at 10 \u0026micro;M, 20 \u0026micro;M, and 30 \u0026micro;M showed recoveries of 90.49%-101.78%. Apple juice exhibited recoveries of 98.97%-110.62% for the same spiked levels. Mango juice had recoveries of 99.62%-110.21%. All recoveries fell within the range of 90.49%-110.62%, and the relative standard deviations (RSD) for all measurements were less than 3%. These results indicate minimal matrix interference and validate that the detection system is reliable for real sample analysis.\u003c/p\u003e \u003cp\u003eTo verify the practical applicability of the developed b-IrOx-based sensing system, this study determined the TAC of real samples (mango juice, yellow peach juice, apple juice) using the core detection mode\u0026mdash;b-IrOx-based TMB colorimetric method. A commercial ABTS kit was used as the reference standard, and the results were retained for accuracy comparison. All experiments used Trolox as the standard substance to ensure consistency in TAC quantification, with results expressed as \u0026micro;M TE. The TAC detection results of the two methods for each juice sample have been summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These results confirm the reliability and consistency of the developed b-IrOx-based TMB colorimetric method and ABTS kit in TAC measurement. Notably, the b-IrOx nanozyme, which exhibits POD-like activity and serves as the centerpiece of this innovative colorimetric approach, offers clear benefits compared to natural enzymes, including easy synthesis and improved stability. Compared to traditional POD-based commercial ABTS kits, the developed TMB colorimetric method shows comparable TAC detection capabilities for the tested fruit juices, while avoiding over-reliance on complex detection platforms.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative study of TAC detection in fruits using the proposed b-IrOx-based TMB colorimetric strategies and a standardized ABTS kit.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eb-IrOx-based TMB colorimetric assay\u003c/p\u003e \u003cp\u003e(\u0026micro;M, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eABTS kit\u003c/p\u003e \u003cp\u003e(\u0026micro;M, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYellow peach juice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e776.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e801.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApple juice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e154.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e165.30\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMango juice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e948.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e987.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOverall, the b-IrOx-based TMB colorimetric method presents a promising alternative for TAC detection and analysis in practical scenarios such as small-scale fruit juice processing plants and routine quality control. It maintains accuracy comparable to commercial ABTS kits and provides a stable, easy-to-operate solution for TAC quantification in fruit juice samples.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe study demonstrated a one-step self-assembly method to produce a novel b-IrOx nanozyme, which effectively substitutes natural enzymes for TAC detection in food samples. It was specifically validated in mango juice, yellow peach juice and apple juice. This colorimetric sensing approach offers advantages over natural enzymes by eliminating the high costs and time required for enzyme production, preventing enzyme activity denaturation under harsh conditions, enabling the widespread use of the TMB method for TAC measurement, and presenting a feasible TAC detection and analysis alternative. Evaluation using the three fruit juice samples confirms the promising practical application potential of this b-IrOx-based sensing strategy. It avoids reliance on complex detection platforms, making it suitable for scenarios like small-scale fruit juice processing plants or routine quality control. This method thus presents a feasible, stable alternative for TAC detection and analysis in food samples.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eShuangshuang Yan: Writing-review and editing, Characterization. Dongying An: Writing-original draft, Validation, Data curation. Yutian Zhou: Visualization, Investigation. Xinli Guo: Visualization, Investigation. Zhihang Qin: Visualization, Investigation. Li Yuan: Supervision. Mengmeng Wang: Writing-review and editing, Project administration. Yang Zhang: Conceptualization, Funding acquisition, Supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors appreciate financial supports from the Shenyang Science and Technology Talent Special Project (RC230022), Natural Science Foundation of Liaoning Province (2023-MSLH-288), Scientific and Technological Innovation Fund for Master's Degree Postgraduates of Shenyang Medical College (Y20240505) and Innovation and Entrepreneurship Training Program of Shenyang Medical College (20259210).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcidri R, Sawai Y, Sugimoto Y, Handa T, Sasagawa D, Masunaga T, Yamamoto S, Nishihara E (2020) Phytochemical Profile and Antioxidant Capacity of Coffee Plant Organs Compared to Green and Roasted Coffee Beans. 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Molecules 29(9):2117\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"food analysis, total antioxidant capacity, peroxidase-like nanozyme, colorimetric sensor","lastPublishedDoi":"10.21203/rs.3.rs-8592177/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8592177/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntioxidants are crucial for capturing and neutralizing free radicals to prevent oxidative damage, which implies assessing the antioxidant content in foods and medications is essential. In order to quantify the total antioxidant capacity (TAC) of food samples, a straightforward and sensitive colorimetric sensing method utilizing an iridium oxide (IrOx) nanozyme was developed. By employing bovine serum albumin (BSA) as a structural template and iridium as the precursor, a biomimetic mineralization strategy was utilized to synthesize b-IrOx nanozymes exhibiting intrinsic peroxidase (POD)-like activity, enabling their effective substitution of natural peroxidases in catalyzing redox reactions. Through single electron transfer (SET) and hydrogen atom transfer (HAT) processes, antioxidants in samples can compete with substrates for \u0026bull;OH, causing a hue shift that depends on the antioxidant concentration. The proposed b-IrOx-based colorimetric assay was successfully used to measure the TAC of mango juice (948.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 \u0026micro;M), yellow peach juice (776.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47 \u0026micro;M), and apple juice (154.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 \u0026micro;M) with good accuracy (recovery rate: 90.49%-110.62%) and reproducibility (relative standard deviation, RSD\u0026thinsp;\u0026lt;\u0026thinsp;3.0%). The b-IrOx nanozymes were compared with a commercial TAC standard kit to further confirm the reliability of the TMB colorimetric assay. The results showed no significant statistical difference between the two methods (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Therefore, the b-IrOx colorimetric assay offers a simple, low-cost approach for the rapid and precise determination of TAC in food samples.\u003c/p\u003e","manuscriptTitle":"Engineering a BSA-templated Iridium Oxide Nanozyme for Colorimetric Evaluation of Total Antioxidant Capacity in Food Samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 09:57:41","doi":"10.21203/rs.3.rs-8592177/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d6015e45-623b-4175-b233-2b148d5a271e","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-17T13:05:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 09:57:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8592177","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8592177","identity":"rs-8592177","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}