The optimization of gold nanoparticles–horseradish peroxidase as peroxidase mimic using central composite design for the detection of hydrogen peroxide

The oxidizing agent, hydrogen peroxide (H2O2), which is a part of reactive oxygen species (ROS) is well-known to contribute to oxidative stress-induced damage to biological molecules. An excess of free radicals can harm health and is associated with human diseases. Gold nanotechnology, a highly relevant nanomaterial, has been utilized as a new material in advanced sensor detection. In this study, colorimetric methods based on peroxidase enzymes were developed for measuring H2O2. The synthesized gold nanoparticles (AuNPs) showed a concentration of approximately 1.73 nM at a wavelength of 520 nm. The average diameter displayed a uniform size distribution, estimated at 18 nm, and an increase in the shell thickness of AuNPs-horseradish peroxidase (HRP) was observed in the TEM images. The AuNPs-HRP system demonstrated remarkable catalytic activity in the reaction of the chromogenic substrate tetramethylbenzidine (TMB) with H2O2, resulting in the production of an oxide product. The optimal conditions for the AuNPs-HRP system, as determined by central composite design (CCD), were a temperature of 25 °C and a pH of 7 within an 8 h period. A strong linear relationship was observed between different absorbance values and the H2O2 concentration, with a coefficient of determination of 0.9956. A portable platform was successfully used to determine H2O2 levels in beverages with recoveries ranging from 95.51% to 118.85%. These findings suggest that the AuNPs-HRP system could be applied to detect H2O2 in beverages.


Introduction
Hydrogen peroxide (H 2 O 2 ) is an oxidizing agent and is widely recognized as a cytotoxic agent whose levels must be minimized through the action of antioxidant defense enzymes [1].It exerts a wide range of physiological and pathological effects within animal, plant, and bacterial cells in culture [2].Additionally, H 2 O 2 is utilized in numerous processes, including oxidation, disinfection, and bleaching [3].Furthermore, several beverages contain H 2 O 2 , which quickly diffuses into the cells of the oral cavity and the upper part of the gastrointestinal tract, leading to damage to various organs [4].These harms are linked to human health and diseases such as Parkinson's and Alzheimer's disease [5].
Over the previous decades, expensive equipment and complex operating processes have been established for H 2 O 2 detection using chemiluminescence, electrochemical, and enzymatic methods [6,7].Due to H 2 O 2 being the typical oxidizing substrate for horseradish peroxidase (HRP), there have been significant advancements in enzyme immunoassay-driven technology [8].A previous study introduced a novel voltammetric enzyme-linked immunoassay system using 3,3′-diaminobenzidine (DAB), H 2 O 2 , and HRP for sensitive detection in human serum.DAB was employed as the electroactive substrate in the HRP-catalyzed oxidation reaction in the presence of H 2 O 2 .The proposed electrochemical enzyme-linked immunosorbent assay method is characterized by its simplicity, cost-effectiveness, reproducibility, and high sensitivity.This method holds promise for the clinical diagnosis of carcinoembryonic antigen (CEA) [9].The study of Chen et al (2021) developed a localized surface

Chemicals and reagents
Hydrochloric acid (HCl), nitric acid (HNO 3 ), a gold (III) chloride (AuCl 3 ) solution, and citric acid (C 6 H 8 O 7 ) were purchased from Sigma-Aldrich (Missouri, United States).All chemicals used in this study are of analytical grade.

Synthesis of gold nanoparticle
To ensure the removal of any contaminants and residues from the equipment, the volumetric flask and magnetic bar were cleaned with aqua regia that a mixture of 3 ml of HCl was added to 1 ml of HNO 3 and washed with distilled water (DW).A modified literature approach was used to synthesize citrate-capped AuNPs [26].In brief, the gold solution which was diluted in HCl (1.637 g ml −1 ; 3.4 μl) was mixed with a boiling aqueous solution (13ml).The mixture was stirred with a magnetic stirrer at 120 °C.Citric acid (10 mg ml −1 ; 500 μl) was added to the mixed solution.The solution was stirred continuously in the dark until the color changed from light yellow to red wine, at which point the stirring was stopped.The solution was cooled to room temperature for 10 min.The gold nanoparticle solution was stored at 4 °C until use.

UV-visible spectroscopic characterization
An AuNPs was characterized using a UV-vis spectrophotometer (SpectraMax M3, Molecular Devices, Sunnyvale, California, USA) within the wavelength range of 400-700 nm at room temperature.The highest peak value was used to calculated the relationship between the attenuation of light passing through a substance and the properties of that substance, following the Beer-Lambert Law with the following formula: bC, where A represents absorbance, ε is the molar absorption coefficient (M −1 cm −1 ), b is the optical path length (cm), and C is the molar concentration (M).

Functionalization of AuNPs-HRP system
Following established procedures, a highly pure suspension of HRP in aqueous ammonium sulfate was dialyzed and filtered [27].The HRP stock solution was diluted with distilled water to a final concentration of 1.6 μg ml −1 and stored at cold temperatures.HRP enzyme was conjugated to the surface of citrate-stabilized AuNPs.The reaction mixture was stirred and incubated for 30 min at room temperature.Subsequently, AuNPs were formed through electrostatic interactions with the HRP enzyme.As a result, AuNPs modified with an HRP enzyme through passive absorption (AuNPs-HRP) were obtained.The AuNPs-HRP particle solution was stored at 4 °C and used for further experiments.

SDS-PAGE analysis of AuNPs-HRP system
The binding of HRP with AuNPs was further investigated using SDS-PAGE analysis, following standard procedures.Twenty microliters of the AuNPs, AuNPs-HRP, and HRP solutions with varying concentrations (0.8-6.4 μg ml −1 ) were loaded into individual gel lanes.The gel was stained using a 0.15% (v/v) Coomassie Brilliant Blue R-250 solution in 45% (v/v) methanol and 10% (v/v) glacial acetic acid for 1 h.Subsequently, the gel was washed with a destaining solution containing 10% (v/v) methanol and 7.5% (v/v) glacial acetic acid until clear bands were visible.An Amersham ECL Rainbow marker (Merck; Darmstadt, Germany) served as the prestained protein standard marker.The bands were visualized using the Davinch-chemi™ Chemiluminescence Imaging system (Davinch Mini Chemi Q6; Davinch-K, Seoul, South Korea), and the gel image was captured with a digital camera.

TEM analysis
The spherical morphology of the AuNPs and AuNPs-HRP was characterized through functionalization using a high-resolution field emission transmission electron microscope, FE-TEM (Model JEM-F200; JEOL, Tokyo, Japan), along with selected-area electron diffraction (SAED) at an acceleration voltage of 200 kV.The average physical diameters were determined using ImageJ software (version 1.52a; National Institutes of Health, United States).

Synthesis of AuNPs-HRP system with TMB as peroxidase-mimic catalytic activity
The protocol for the TMB/E Ultra-Sensitive, Blue, Horseradish Peroxidase Substrate (soluble) of the ELISA test kit (Merck; Darmstadt, Germany) was adapted.The AuNPs-HRP system used to detect H 2 O 2 with TMB as a peroxidase-mimicking catalyst was synthesized.TMB was prepared in a 100 mM citric acid/sodium acetate buffer, at pH 5.4 [28].This mixed solution consisted of 75 μl of AuNPs-HRP and 10 μl of H 2 O 2 .After vortexing, the solution was incubated for 5 min at room temperature.A final volume of 15 μl of TMB was added in a 1.5ml microtube.The resulting solution was transferred to a 96-well plate.Image analysis was performed using a UVvis spectrophotometer, recording values from 400 to 700 nm.To stop the reaction, 100 μl of 0.3M sulfuric acid was added, yielding a yellow product [29].Finally, the analytical signal at 450 nm was used to calibrate the H 2 O 2 values.

Gel staining analysis of AuNPs-HRP system
Concisely, gelatin powder and low-melting agarose (in a 1:10 ratio) were mixed in collagenase buffer (100 ml).The solution was heated for 2 min and poured into a 10-cm dish.After the solution cooled, 1.5ml of TMB solution was added.The gel dish was stored at 4 °C until use.Filter paper discs (size 6 mm; Advantec MFS, Inc., California, USA) were placed on the gel and then loaded with AuNPs, HRP, and AuNPs-HRP.The gel was left at room temperature until the solution dried.Subsequently, H 2 O 2 was applied to the filter paper, and the color change was observed.

The regression analysis and optimization experimental design of the AuNPs-HRP system
The Design-Expert statistical software (version 6.0.2,Stat-Ease, Inc., Minnesota, USA) was utilized for regression analysis and optimizing of the AuNPs-HRP system to understand the influence of independent variables on H 2 O 2 detection.The experiment was based on a CCD with three axial points and an alpha (α) value of 1.2.To determine optimal conditions, three independent variables were considered: temperature (°C, X 1 ), the incubation time (h, X 2 ), and pH (X 3 ).The levels and ranges of these independent variables for H 2 O 2 detection are presented in table 1.The complete quadratic equation, derived using multiple regression for these three factors was used to describe the response surface as the regression model: , where Y represents measured response variable; β 0 is a constant; β i , β ii and β ij are respective linear, quadratic, and interactions coefficients, respectively; X i and X j are coded variables; k is the number of studied factors; e is the random error.
The variance of the regression model and R 2 were examined.The optimum conditions were based on optical density, performed in triplicate and calculated to validate the model.

Peroxidase activity using an AuNPs-HRP system for H 2 O 2 detection
The slope of the linear relationship and the coefficient of determination (R 2 ) were determined for H 2 O 2 levels ranging from 25 μM to 1600 μM through sensitivity performance experiments conducted under optimal parameters.The analytical signal was detected at 450 nm using a UV-vis spectrophotometer.

Evaluation of H 2 O 2 determination for beverage
To develop H 2 O 2 detection in beverages, mineral water and sparking water purchased from a local convenience store (Busan, South Korea) were used in the AuNPs-HRP system.Non-diluted aliquots (10 μl) of the samples were directly added to the AuNPs-HRP system.Different concentrations (20, 40, and 80μM) of H 2 O 2 were added to the solution to maintain a total volume of 100 μl.The solution was then transferred to a 96-well plate and detected with a UV-vis spectrophotometer at 450 nm.The percentages of recoveries and relative standard deviations (RSD) were calculated using the following formulas: Average 100

Statistical analysis
All experiments were conducted in triplicate (n = 3).The results were presented as averages and standard deviations (SD).Statistical analysis was performed using SPSS software (Version 16; SPSS Inc., Illinois, United States).Statistically significant differences were considered for p-values of 0.05, 0.01, and 0.001, respectively.

Characterization of AuNPs and AuNPs-HRP system
According to the Beer-Lambert Law, the synthesized AuNPs at λ = 520 nm using an extinction coefficient of 3.67 × 10 8 M −1 cm −1 showed a concentration of approximately 1.73 nM (figure 1(A)).Additionally, a conjugated HRP enzyme on the surface of the AuNPs was observed with an approximate redshift of about 10 nm in absorption wavelength after functionalization.It was found that the size of the AuNPs-HRP complex increased with surface modification.As shown in figure 1(B), these results was confirmed that the AuNPs-HRP system was successfully produced, indicated by the presence of a single protein band with a molecular weight of 40 kDa in lane 3. The presence of HRP protein levels depended on HRP concentration and was revealed in lanes 4 to 7. Furthermore, figure 2 displayed TEM images of the AuNPs and AuNPs-HRP systems (20 nm and 50 nm), respectively followed by the calculation of the size distribution.The average diameter of both the AUNPs and AuNPs-HRP system was estimated to be 18 nm based on particle image captured in the TEM image.TEM images confirmed the physical diameter and size distribution were 17.6 ± 1.9 nm (figures 2(A)), 17.5 ± 1.8 nm (figures 2(B)), 17.7 ± 1.4 nm (figure 2(C)), and 18.2 ± 2.3 nm (figure 2(D)), respectively.It is evident that AuNPs and AuNPs-HRP systems exhibited a uniform size distribution.Notably, the increase in shell thickness observed in the AuNPs-HRP system compared to to AuNPs indicates that the AuNPs were coated by HRP after modification.These results confirm the successful incorporation of HRP on the surface of the AuNPs.3.2.The catalytic activity of the AuNPs-HRP/H 2 O 2 /TMB system Based on catalytic activity, a simple colorimetric assay was developed for the determination of H 2 O 2 detection.The performance of the AuNPs-HRP/H 2 O 2 /TMB system was examined as the catalytic system (figure 3(A)).The colorimetric assay based on the AuNPs-HRP system, resulted in a remarkable redshift in the absorption peak to 640 nm after the combination of AuNPs-HRP and H 2 O 2 with the TMB system, indicating enzyme activity.This finding demonstrats that the AuNPs-HRP/H 2 O 2 /TMB system exhibites a catalytic activity in the presence of H 2 O 2 .
As shown in figure 3(B), after the AuNPs-HRP/H 2 O 2 /TMB system was treated with sulfuric acid (H 2 SO 4 ) to stop the acidification reaction, an oxidation process occurred due to the catalytic effect.The color of the product changed from blue to yellow, and the absorbance displayed a significant shift in the absorption peak from 640 nm to 450 nm.

AuNPs-HRP system on gel staining analysis
To examine the catalytic performance of the AuNPs-HRP/H 2 O 2 /TMB system in gel staining analysis, the oxidation of TMB in the presence of the AuNPs-HRP system and corresponding to H   system exhibited greater catalytic activity than other systems, owing to the synergistic effects of oxidizing TMB by a catalytic reagent and generating a color product.These findings suggest that the AuNPs-HRP/H 2 O 2 system indeed demonstrates catalytic activity in the presence of H 2 O 2 .

Regression and optimization of AuNPs-HRP/H 2 O 2 /TMB system conditions
Based on the model summary statistics of the AuNPs-HRP/H 2 O 2 /TMB system, a quadratic model relating independent factors to the response value was suggested by CCD.The experimental matrix was designed using the software.The actual and predicted absorbance values of catalytic activity achieved with the coded variables, were summarized in table 2 for a total of 20 treatments, which included 14 combinations with 6 replications of the center points.The Analysis of Variance (ANOVA) was used to determine whether the input factors had an impact on the response in terms of color intensity.As shown in table 3, a full quadratic model was employed to analyze the AuNPs-HRP/H 2 O 2 /TMB system.Color intensity served as the response variable.The positive coefficient value for pH indicated that an increase in pH tended to raise color intensity.Conversely, the coefficients for temperature and time were negative, suggesting that higher values for temperature and time tended to decrease color intensity.Variance inflation factor (VIF) values were equal to one, indicating that the factors were independent and not affected by the lack of orthogonality in the design.A small standard error (SE) for the model represented greater experimental precision.The highest F-value, observed for temperature (1077.68)was considered the most significant parameter.The lack-of-fit test by ANOVA revealed that the P-value was not significant relative to the pure error, indicating that the fits the AuNPs-HRP/H 2 O 2 /TMB system well.The adequate precision value was 52.989, signifying a sufficiently positive signal.The adjusted R-squared value of 0.9917 closely approximated the expected coefficient of determination R-squared value of 0.9956 as established by the model.This suggests that the three factors (temperature, time, and pH) and their respective levels exhibited a strong correlation with the color intensity of the AuNPs-HRP/H 2 O 2 /TMB system.
As shown in figure 4, the contour and 3D surface plot of absorbance of color intensity illustrate the effect of the combination of three factors within the AuNPs-HRP/H 2 O 2 /TMB system.The relationship between temperature and time indicats that an increase in temperature and time results in a decrease in the absorbance response (figure 4(A)).These findings suggest that the AuNPs-HRP/H 2 O 2 /TMB activity is reduced by higher temperature longer durations.In figure 4(B), it is evident that absorbance tends to increase as the pH parameter rises, indicating that the oxidation process readily occurs under basic conditions.However, a reduction in absorbance is observed as the duration time decreases.Conversely, an increase in both time and a concurrent decrease in pH led to an elevation in absorbance.This suggests that the expression of the oxidation process has greater stability under acidic conditions (figure 4(C)).The variation in temperature, time, and pH has a reasonably significant impact on the absorbance capacity.
The estimated equation of the model for absorbance of color intensity showed significant positive linear effects for temperature, time, and pH.The quadratic terms indicated that the addition of these variables significantly affects absorbance for H 2 O 2 detection.The interaction effects between temperature and time, as well as between time and pH displayed highly significant positive effects, suggesting that interactions among these three factors are essential for H 2 O 2 detection.The model is represented by the equation below:

( )
Therefore, these findings suggest that the catalytic activity of the AuNPs-HRP/H 2 O 2 /TMB system is reduced by high temperature and time-dependent factors but is increased by a higher pH.The optimal process parameters were determined to be at 25 °C with a pH solution of 7.7, and a duration of 8 h was selected to achieve the highest absorbance for H 2 O 2 detection.These optimum values were subsequently employed in realtime detection for a confirmation test to obtain results in further experiments.

Peroxidase activity using an AuNPs-HRP system for H 2 O 2 detection
As depicted in figure 5, the absorbance at 450 nm was examined for the quantitative estimation of H 2 O 2 concentration under optimal conditions, and the absorbance value increased with different concentrations of H 2 O 2 .This indicated that as the H 2 O 2 concentration increased, the absorbance gradually rose.According to the calibration plot, a significant corelation between the color parameter and H 2 O 2 levels was observed, ranging from 25 to 1600 M. Due to H 2 O 2 generation, the AuNPs-HRP system catalyzed the oxidation of TMB, resulting in the characteristic blue coloration of the aqueous solution [14].After the addition of H 2 SO 4 to halt the acidifying reaction, a yellow product color was produced, corresponding to the diamine form [29].This variation was visually observeble, as shown in the images of colored products corresponding to various H 2 O 2 concentrations.Additionally, the equation of the linear regression was defined as = + y x 0.0013 0.2132, and the positive correlation (R 2 = 0.9983) confirmed a strong linear relationship.These results suggest that the AuNPs-HRP/H 2 O 2 /TMB system holds promise for further research in the field of beverages.

The application of AuNPs-HRP system in beverage
To assess the efficacy of the developed methodology, the catalytic activity of the AuNPs-HRP/H 2 O 2 /TMB system for detecting H 2 O 2 concentration was applized to mineral water and sparkling water.Since no H 2 O 2 was initially present in the sameples, H 2 O 2 was added to the beverage samples at concentrations of 20, 40, and 80 μM under optimal conditions.The catalytic activity of the AuNPs-HRP/H 2 O 2 /TMB system in beverage applications is summarized in table 4. The results from UV-vis spectrophotometer detection showed recovery values ranging from 95.51% to 118.85% with a relative standard deviation of less than 10%.These findings indicate that the developed method, based on the AuNPs-HRP/H 2 O 2 /TMB system with UV-vis spectrophotometer application, is reliable, functional, and suitable for sample analysis.

Discussion
Reactive oxygen species (ROS) are known to play a role in neuronal damage caused by oxidative stress [30].One of the important biological molecules associated with oxidative biosynthetic reactions, metabolism, and cell signaling in host defense is H 2 O 2 [31].Free radicals rapidly interact with enzymes, membrane lipids, and other vital cellular components, ultimately leading to cell death [32].ROS-related diseases can be attributed to an excess of ROS such as cardiovascular and neurodegenerative diseases [33].However, H 2 O 2 finds utility in various industries, including the food, pharmaceuticals, medicine, and chemicals [14,34].To expedite control and implement corrective measures regrading products containing H 2 O 2 , technologies that can identify its unintended presence in food and beverages prove advantageous.Due to their excellent biocompatibility properties, AuNPs have garnered increasing attention in advanced sensor detection [18].Effective binding between AuNPs and peroxidase enzymes has been reported.Employing enzymatically active HRP-coated AuNPs, they catalyzed polymerization, resulting in thermoresponsive polymers, and fully recovered without significant loss of their catalytic activity [35].Tannic acid-modified gold nanoparticles exhibit superior nanozyme activity and catalyze the oxidation reaction of TMB in the presence of H 2 O 2 [36].Positively charged gold nanoparticles possess intrinsic peroxidase-like activity and can catalyze the oxidation of the peroxidase substrate, TMB, by H 2 O 2 , providing a straightforward approach to colorimetric detection [23].In this study, the AuNPs-HRP system as catalytic activity using an operational reaction system comprising TMB to determine H 2 O 2 with statistical optimization via CCD.This biosensor system was applied to the detection of H 2 O 2 in beverages.
The characterization of the synthesized AuNPs and the AuNPs-HRP system was performed at 520 nm with a concentration of 1.73 nM.Surface modification after the synthesis of the AuNPs-HRP system resulted in an increased the absorption wavelength.In line with a previous study, Emami et al (2015) reported that gold nanoparticle size was 16 nm with a typical wavelength value of 526 nm.Spectra analysis following the interaction between AuNPs and antibodies (Ab) showed a shift from 526 nm to 532 nm, indicating the binding of Ab to AuNPs [37].Additionally, gold nanoparticles synthesized by borohydride reduction of aurate salt were confirmed by a single peak in the absorbance spectra at 530 nm [38].The molecular weight of the AuNPs-HRP system was confirmed by the presence of a single protein band at 40 kDa using SDS-PAGE.HRP exists in at least 15 different isoenzyme forms in horseradish root.The molecular mass of HRP expressed in plants typically ranges from 34 to 44 kDa [39].However, the molecular mass of purified HRP was determined to be 40 kDa [40].TEM analysis of the AUNPs and AuNPs-HRP systems revealed that the size distribution of AUNPs and the AuNPs-HRP system was estimated to be 18 nm.The increase in shell thickness in the AuNPs-HRP system confirmed the successful incorporation on the surface of AuNPs after modification.Different size distributions of gold nanoparticles have been reported in previous studies.Forinstance, regarding the active site of gold nanoparticles, Liu et al (2019) reported that the largest unmodified AuNPs were estimated to be 9.4 nm [41].Meanwhile, Emami et al (2015) reported that the gold nanoparticle particles were almost faceted spherical and had an average particle diameter of 16 nm [37].However, Zhang et al (2015) revealed that the average size of AuNPs after conjugation with HRP and Ab was about 33 nm.The size increase indicated that AuNPs had been surface-tagged with HRP and Ab [42].
The performance of the AuNPs-HRP/H 2 O 2 /TMB system was examined as a catalytic system for determining H 2 O 2 , and it demonstrated a catalytic effect in the presence of H 2 O 2 .Similarly, Hu et al (2020)  reported that the resulting a deoxyribonucleic acid (DNA)-HRP-AuNP probes exhibited HRP-dependent chromogenic reactions based on the catalytic decomposition of H 2 O 2 , resulting in a blue color and an absorption peak centered at 652 nm [43].This interaction enhanced the catalytic activity of the AuNPs, acting as nanozymes related to the oxidized product of TMB transformation, and the solution's colot turned bright blue [36].After the AuNPs-HRP/H 2 O 2 /TMB system was treated with H 2 SO 4 , the blue color changed to yellow.This indicated an oxidation process occurring due to the presence of a catalytic effect [36].The color product transitioned from blue to yellow, and the absorbance exhibited an extraordinary shift in the absorption peak to 450 nm.Additionally, initially, we verified the oxidation of TMB by the AuNPs-HRP system in the presence of H 2 O 2 through gel staining analysis.The AuNPs-HRP/H 2 O 2 system exhibited catalytic activity, attributable to the synergistic effects of oxidizing TMB as a catalytic reagent, resulting in the formation of a blue-colored product.This finding suggests that the AuNPs-HRP/H 2 O 2 system indeed demonstrated catalytic activity in the presence of TMB.
The RSM based on the CCD was employed to design experiments and develop quadratic equation models that predict the optimal conditions for desirable responses.The regression and optimization of the AuNPs-HRP/H 2 O 2 /TMB system involved comparing actual and predicted response absorbance values for catalytic activity using coded variables for treatments.The ANOVA analysis indicated that the designed model was well-suited to the developed system.The adjusted R 2 and the R 2 value of the model suggested that all factors had a high correlation with the AuNPs-HRP/H 2 O 2 /TMB system.These factors were found to be independent and temperature was identified as the most significant parameter.It's worth noting that peroxidase catalyst performance is known to be influenced by the reaction temperature [44].In a study by Peng et al (2020), they reported that color intensity decreased as the temperature approached 40 °C during the optimization of parameters for TMB [15].The investigation of the thermal stability of HRP was reported by Zhong et al (2016), where it was found that HRP was completely inhibited at temperatures exceeding 40 °C due to structural damage.These findings demonstrate that natural enzymes can rapidly lose their catalytic activity when the environmental conditions change [45].The pH value directly impacts the results.In a prior study, it was observed that the nanoparticles were well dispersed at pH 7.0 indicating pH-dependent precipitation of nanoparticles.As the solution became more acidic, the peak broadened, signifying the precipitation of nanoparticles [38].Additionally, time exhibited an inverse relationship with the absorbance of color intensity.Wu et al (2019) reported that HRP activity diminished within a day, hilighting its low stability susceptibility to inactivation in its surroundings [46].
The linear relationship between color intensity, as a color parameter and the absorbance of H 2 O 2 levels in the range from 25 to 1600 μM demostrated a strong correlation.As the H 2 O 2 concentration increased, the absorbance consistently increased.The R 2 value, which represents the proportion of the variation in the dependent variable that can be predicted from the independent variable, confirmed a reasonable level of agreement [47].
The practical applications of the AuNPs-HRP/H 2 O 2 /TMB system were validated in beverages.Under optimal conditions, mineral water and sparkling water were supplemented with H 2 O 2 at concentrations of 20, 40, and 80 μM.The catalytic activity of the AuNPs-HRP/H 2 O 2 /TMB system yielded a reasonable percentage recovery.It was observed that the H 2 O 2 concentration and the percent concentration recoveries were consistent and exhibited low variability.These findings suggest that the developed system was precise and reliable for sample analysis.Previous studies have also reported on the application of AuNPs technology for sample detection.For instance, the practicability of a nanozyme-based colorimetric sensor was evaluated using the Lead (II) ion or Pb 2+ at different concentrations (25, 50, and 100 ng × ml −1 ), which were added to spring, bottled drinking, and tap water samples.The obtained recoveries with associated RSD, indicated good precision and accuracy in the detection of Pb 2+ in real water samples [36].Ni et al (2014) reported the use of AuNPs for detecting melamine in raw milk and milk powder.Melamine was added at concentrations of 6 and 200 nM.The favorable recoveries demonstrated the reliability of the proposed method for the pratical detection of melamine [48].The applicability of the gold enzyme-based paper chip in practical scenarios was also assessed.Different concentrations of Mercuric ion (Hg 2+ ) (0.05, 0.5, 5, and 50 mg L −1 ) were introduced into tap water.The recoveries fell within an acceptable range with the associated RSD indicating that the system is suitable for the practical analysis of Hg 2+ in real samples [49].Moreover, Zhou et al (2019) applied nanoparticles for glucose determination in human serum samples.The results of determination and recovery displayed good precision, suggesting that our detection system holds pratical potential for biological analysis and clinical diagnosis [50].

Conclusion
In this study, a simple and sensitive AuNPs-HRP system was developed as a colorimetric method based on peroxidase enzymes for H 2 O 2 measurement.The AuNPs-HRP system successfully demonstrated functionalization.This one-step AuNPs-HRP system exhibited remarkable activity in catalyzing the reaction of TMB with H 2 O 2 to produce an oxidized product.The AuNPs-HRP system showed good agreement with their corresponding peroxidase-like activities.Our findings indicate that the developed system is precise and sensitive.Additionally, this system could be alternatively applied for visual colorimetric detection, further enhancing its utility for future applications, especially in H 2 O 2 detection in beverages.

Figure 2 .
Figure 2. TEM images and size distribution histograms of (A-B) AuNPs and (C-D) the AuNPs-HRP system for 20 nm and 50 nm, respectively.The TEM images display higher magnification views of the physical diameter and size distribution histogram.

Figure 3 .
Figure 3. (A) UV-visible absorption spectra of AuNPs-HRP, AuNPs/H 2 O 2 , AuNPs-HRP/H 2 O 2 , and AuNPs-HRP/H 2 O 2 /TMB systems with the characteristic absorption peaks of the red and blue products at 520 nm and 640 nm, respectively.(B) UV-visible absorption spectra of the AuNPs-HRP/H 2 O 2 /TMB system and AuNPs-HRP/H 2 O 2 /TMB system after the addition of H 2 SO 4 , displaying characteristic absorption peaks of the blue and yellow products at 640 nm and 450 nm, respectively.The inserted photograph demonstrates the catalytic enzyme activity system with color changing.(C) A comparison of images before and after H 2 O 2 treatment of AuNPs, HRP, H 2 O 2 , HRP/H 2 O 2 , AuNPs-HRP, AuNPs/H 2 O 2 , and AuNPs-HRP/H 2 O 2 systems on a gel staining plate.

Figure 4 .
Figure 4.The effect of different factor combinations, including (A) Temperature × Time, (B) Temperature × pH, and (C) Time × pH, on the absorbance of color intensity within the AuNPs-HRP/H 2 O 2 /TMB system.

Figure 5 .
Figure 5.The linear equation and correlation coefficient for color parameters between absorbance and H 2 O 2 concentration detection using a UV-vis spectrophotometer.The inserted photograph shows the AuNPs-HRP/H 2 O 2 /TMB system with color changes corresponding to different of H 2 O 2 concentrations (25-1600 μM).

Table 1 .
Levels of independent variables in CCD for AuNPs-HRP/H 2 O 2 /TMB system.

Table 2 .
Treatments of CCD and response values of AuNPs-HRP/H 2 O 2 /TMB system.