Materials

leaves were collected from Bu Gia Map National Park, Binh Phuoc Province, Vietnam. Chemicals including silver nitrate (AgNO), 3,3’,5,5’-tetramethylbenzidine (CHN), hydrogen peroxide (HO), acetic acid (CHCOOH) and sodium acetate (CHCOONa), which were of analytical grade without further purification, were purchased from Acros Co., Belgium. Deionized water was thoroughly utilized in all the experiments.

Methods

Synthesis of AgNPs from leaf extract

The synthesis of silver nanoparticles (AgNPs) was performed via leaf extract, following previous reports2, 5. Specifically, 0.25 mL of leaf extract was mixed with 5 mL of an aqueous AgNO₃ solution under reaction conditions of 2.0 mM silver ion concentration, 80°C, and a reaction time of 70 minutes. The formation of AgNPs was verified via UV–Vis spectrophotometry. The synthesized AgNP solution was subsequently stored at 8°C for future applications.

Determination of the optimal temperature for the TMB and HO reactions with the AgNP catalyst

To optimize the reaction temperature, 400 µL of 5 mM TMB, 2 mL of acetate buffer solution (pH 5), 600 µL of 400 µM H₂O₂, and 150 µL of the synthesized AgNP solution were each carefully added to a 10 mL bottle. The bottle was covered with foil to protect it from light and placed in a thermostatic bath at various temperatures (30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C) for 30 minutes. Following incubation, UV‒Vis spectroscopy was used to measure the absorbance of the solutions to determine the optimal temperature for the reaction of TMB and H₂O₂ in the presence of the AgNP catalyst.

Determination of the optimal time for the TMB and H₂O₂ reactions with the AgNP catalyst

To determine the optimal reaction time, 400 µL of 5 mM TMB, 2 mL of acetate buffer solution (pH 5), 600 µL of 400 µM H₂O₂, and 150 µL of the AgNP solution were added to a 10 mL container, which was carefully covered with foil. This container was then placed in a thermostatic reaction tank set to the optimal temperature. UV‒Vis spectroscopy measurements were taken at 10-minute intervals to identify the optimal reaction time for the interaction between TMB and H₂O₂ in the presence of the AgNP catalyst.

Determination of the optimal pH for the TMB and H₂O₂ reactions with the AgNP catalyst

To optimize the pH for the reaction, 400 µL of 5 mM TMB, 2 mL of acetate buffer solution at various pH values, 600 µL of 400 µM H₂O₂, and 150 µL of the AgNP solution were added to 10 mL bottles and covered with foil. These bottles were then placed in a thermostatic bath set to the optimal temperature and time. UV‒Vis spectroscopy measurements were conducted to identify the optimal pH for the TMB and H₂O₂ reactions in the presence of the AgNP catalyst.

Determination of the optimal AgNP concentration for the TMB and HO reactions with the AgNP catalyst

To optimize the concentration of AgNPs, 400 µL of 5 mM TMB, 2 mL of acetate buffer solution (adjusted to the optimal pH), 600 µL of 400 µM H₂O₂, and varying volumes of the AgNP solution (60 µL, 70 µL, 80 µL, 90 µL, 100 µL, 110 µL, 120 µL, and 130 µL) were added to separate 10 mL bottles, each covered with foil. These bottles were then placed in a thermostatic bath set to the optimal temperature and time. UV‒Vis spectroscopy was used to determine the optimal concentration of AgNPs for the TMB and H₂O₂ reactions.

Determination of the optimal TMB concentration for TMB and HO reactions with AgNP catalysts

To determine the optimal concentration of TMB, 2 mL of acetate buffer solution (adjusted to the optimal pH), 600 µL of 400 µM H₂O₂, the optimal volume of AgNP solution (determined ), and varying concentrations of TMB (400 µL of 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, or 6.5 mM) were added to separate 10 mL bottles, which were covered with foil. These bottles were then placed in a thermostatic bath set to the optimal temperature and time. UV‒Vis spectroscopy was performed to determine the optimal TMB concentration for the TMB and H₂O₂ reactions catalyzed by the AgNPs.

Determination of the linear range and limit of detection (LOD) value

To determine the optimal concentration of H₂O₂, 400 µL of TMB, 2 mL of acetate buffer solution (adjusted to the optimal pH), and the optimal volume of AgNP solution were combined with varying concentrations of H₂O₂ (600 µL of 10 µM, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM, and 400 µM) in separate 10 mL bottles, each covered with foil. These bottles were placed in a thermostatic bath set to the optimal temperature and time. UV‒Vis spectroscopy was conducted on the solutions to identify the optimal concentration of H₂O₂ for the reaction with TMB catalyzed by AgNPs. The limits of detection (LODs) and limits of values were calculated via Equations (1) and (2), respectively:

where SD and a are the standard deviation and slope of the linear regression line, respectively.

Synthesis of AgNPs from leaf extract

Figure 2a shows that the peak absorption of the AgNPs synthesized from leaf extract was in the wavelength range of 400–500 nm. The UV‒Vis spectrum of the AgNPs synthesized from leaf extract exhibited a surface plasmon resonance (SPR) peak at 427 nm. Microscopy images (Figure 2b and Figure 2c) revealed that the structure of the AgNPs consisted mostly of spheres with an average size of 13 nm, which aligns with the findings of a previous study 2.

Optimization of the TMB and HO reactions with the AgNP catalyst

Figure 3 shows the UV‒Vis spectra of TMB oxidation as a function of reaction temperature. The absorption spectra were recorded over a temperature range from 20°C to 60°C. The data indicated that the absorbance at a wavelength of 654 nm increased progressively with temperature, reaching a maximum at 35°C. Beyond this temperature, a decrease in absorbance was observed, which was likely attributed to the thermal instability of ox-TMB at higher temperatures. Accordingly, 35°C was identified as the most favorable temperature for the efficient oxidation of TMB by H₂O₂, facilitated by the AgNP catalyst.

Results of the optimal time for the reaction of TMB and H₂O₂ with the AgNP catalyst

The effect of reaction time on TMB oxidation was evaluated at the optimal temperature of 35°C, with measurements taken at 10-minute intervals to identify the optimal reaction duration. The absorbance data obtained via UV‒Vis spectroscopy are presented in Figure 4. Superimposing the absorption spectra for various time points revealed that the reaction conducted for 30 minutes presented the highest absorbance intensity at 654 nm. This observation suggests that extended reaction times under these conditions may lead to the degradation of -TMB. Therefore, a reaction time of 30 min was determined to be the most suitable condition for the oxidation of TMB by H₂O₂ in the presence of the AgNP catalyst, starting from the moment the sample was introduced into the thermostatic bath.

Results of the optimal pH for the reaction of TMB and H₂O₂ with the AgNP catalyst

pH plays a principal role in regulating the oxidation rate of TMB. UV‒Vis spectra were recorded and compared for samples prepared under various pH conditions, as illustrated in Figure 5. The data revealed that samples with a pH of 4 presented the highest absorbance intensity at 654 nm, while this intensity progressively decreased for samples with low or high pH values. This trend can be attributed to the reaction equilibrium under highly acidic conditions (pH < 4.0) shifting toward the formation of -TMB. Conversely, at higher pH values, the decreasing formation efficiency of the colored product at 654 nm may result from the elevated redox potential of the substrates, leading to decreased susceptibility to oxidation22. Consequently, pH 4 was identified as the optimal condition and was selected for further exploration of the factors influencing the TMB redox process with H₂O₂ in the presence of an AgNP catalyst.

The effects of the optimal AgNP concentration on the TMB and HO reactions

A survey was conducted to investigate the influence of the AgNP catalyst volume on the absorption intensity of the samples in solution. The absorption spectra of the solutions containing TMB, acetate buffer (pH 4), H₂O₂, and various volumes of the AgNP catalyst were analyzed (Figure 6). At a wavelength of 654 nm, the absorption intensity increased progressively as the AgNP volume increased from 60 µL to 120 µL under constant conditions of TMB concentration, acetate buffer, and H₂O₂. However, a significant decrease in the absorption intensity was observed in the sample containing 130 µL of the AgNP catalyst. This indicated that increasing the catalyst volume increased the surface area, thereby improving the catalytic efficiency of the oxidation‒reduction reaction. Nevertheless, an excessive amount of AgNPs likely resulted in the decomposition of H₂O₂, reducing the reaction yield. Therefore, 120 µL of AgNP catalyst was identified as the optimal volume and was selected for subsequent investigations of other reaction parameters.

The effects of the optimal TMB concentration on the TMB and HO reactions with the AgNP catalyst

The TMB concentration is a critical parameter for evaluating the reaction efficiency between TMB and HO in the presence of the synthesized AgNP catalyst. Figure 7 shows the dependence of the reaction efficiency on the TMB concentration through changes in the absorption intensity observed in the UV‒Vis spectra at 654 nm. The results revealed that the absorption intensity increased with increasing TMB concentration, reaching a maximum at 5.5 mM. Above this concentration, the absorbance decreased significantly, likely due to the oxidation of monoamine groups in TMB at higher concentrations. Consequently, a TMB concentration of 5.5 mM was selected as the optimal condition for the redox reaction between TMB and HO in the presence of the AgNP catalyst.

Influence of the HO concentration on the TMB and HO reactions with the AgNP catalyst

Figure 8 shows the dependence of the absorbance values on the HO concentration. The absorbance intensity at 654 nm increased with increasing HO concentration, reaching a maximum at 350 µM. Beyond this concentration, the TMB concentration appeared insufficient to react fully with HO. A linear relationship was observed within the range of 100–300 µM, described by the regression equation y = 0.91574 + 3.10484×10 C​​, with a standard deviation (SD) of 0.00438. The limit of detection (LOD) for HO, which is based on the colorimetric reaction between oxidized TMB (ox-TMB) and HO catalyzed by AgNPs, was determined to be 46.55 µM, with a limit of quantification (LOQ) of 141.07 µM. These findings, combined with the optimal conditions for TMB oxidation shown in Table 1, underscore the potential of AgNPs as effective catalysts in the TMB-HO redox reaction, demonstrating their applicability as sensitive HO sensors.