Vitamin B3 as a high acid-alkali tolerant peroxidase mimic for colorimetric detection of hydrogen peroxide and glutathione

2.1 Chemicals and materials

Nicotinic acid (VB3), D(+)-Lactose, D(+)-Glucose, and superoxide dismutase (SOD) were purchased from Yuanye Biological Technology Co., Ltd. (Shanghai, China). TMB and o-phenylenediamine (OPD) were obtained from Titan Scientific Co., Ltd. (Shanghai, China). L-Lysine, L-Arginine, and L-Histidine were obtained from Chengdu Huaxia Chemical Reagent Co., Ltd. (Chengdu, China). 2,2-azinobis (3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS), isonicotinamide, and nicotinamide were obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). Anhydrous ethanol was obtained from Chongqing Chuandong Chemical Co., Ltd. (Chongqing, China). Isonicotinic acid was obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). H₂O₂ (30%), isopropanol, fructose, sodium chloride, and potassium chloride were purchased from Chengdu Chron Chemicals Co., Ltd. (Chengdu, China). Calcium chloride anhydrous was purchased from Damao Chemical Reagent Factory (Tianjin, China). The glutathione buccal tablet was purchased from Fuan Pharmaceutical Group Qingyutang Pharmaceutical Co., Ltd. (Chongqing, China). The water used in the whole process was purified by the water purification system (ATSelem 1820A, Antesheng Environmental Protection Equipment Co., Ltd., Chongqing, China).

2.2 Investigation on the peroxidase mimicking activity of VB3

To investigate the peroxidase-like activity of VB3, the catalytic oxidation experiments using different typical chromogenic substrates (TMB, OPD or ABTS) were performed in the presence of H₂O₂ and detected by a spectrophotometric method. In brief, the catalytic reaction was carried out in the presence of 50.0 mM of VB3, 4.0 mM of chromogenic substrates, and 0.1 mM of H₂O₂ in phosphate buffer (10.0 mM, pH 6.0) in an Eppendorf tube. The total reaction volume was 400.0 μL. After incubation for 40 min at 60 °C, the absorption spectra were monitored in the range of 400–750 nm by a UV-5500 UV/VIS spectrophotometer (Metash, Shanghai, China).

2.3 Kinetic study of VB3

The kinetic experiments were carried out using 50.0 mM of VB3 with a fixed concentration of H₂O₂ and varied concentrations of TMB or vice versa. Based on the Lineweaver-Burk double reciprocal plot (Eq. (1)) derived from the Michaelis-Menten equation, the kinetic parameters were obtained.

2.4 Detection of H₂O₂ and glutathione

The detection method for H₂O₂ was performed as follows. Various reactants, including TMB (4.0 mM), different concentrations of H₂O₂ (from 5.0 to 100.0 μM), and VB3 solution (50.0 mM), were homogeneously mixed. The absorbance at 652 nm of the solution was measured after incubation at 60 °C for 40 min. As for the practical sample analysis, three brands of disinfectants were diluted appropriately with phosphate buffer to conform to the linear range of calibration plot, followed by spiking with different concentrations of H₂O₂ (10.0, 20.0, and 40.0 μM). The detection method is the same as above.

Glutathione detection procedure through a typical colorimetric analysis was as follows. Various reactants, including TMB (4.0 mM), H₂O₂ (0.1 mM), VB3 solution (50.0 mM), and varied concentrations of glutathione solutions (from 5.0 to 50.0 μM), were homogenously mixed. The absorbance at 652 nm of the mixture solution was measured after incubation at 60 °C for 40 min. For the determination of real glutathione sample, commercial glutathione tablets were dissolved in phosphate buffer, followed by filtration through a 0.22 μm membrane filter (Titan, Shanghai). Then, appropriate dilution of the clarified filtrate was carried out to conform to the linear range of calibration plot to perform the analysis. The accuracy of the proposed method can be evaluated by the sample spiked recovery and relative standard deviation (RSD) between the determined amount and the specification.

3.1 Peroxidase mimicking activity of VB3

The Fig. 1 demonstrates the peroxidase-like activity of VB3 in the presence of H₂O₂. The peroxidase mimicking activity of VB3 was characterized by monitoring the catalytic oxidation of the chromogenic substrates (TMB, ABTS, and OPD) at the existence of H₂O₂. As shown in Fig. S1a-c, in the presence of H₂O₂, VB3 can catalyze the oxidation of chromogenic substrates to obtain corresponding colored products that exhibit obvious absorption peaks at 652 nm, 450 nm, and 420 nm, respectively. Notably, when VB3, H₂O₂ or different chromogenic substrates exist in pairs, the characteristic absorption peaks are negligible, suggesting the inherent peroxidase mimicking property of VB3. In addition, as illustrated in the insets of Fig. S1, since only integrate VB3-H₂O₂-substrate reaction system exhibits apparent color variance, the catalytic activity of VB3 can also be verified by naked eyes. Moreover, to explore the functional group effect, the relative activity of VB3 and its structure analogues (Fig. 2c) was further compared under different concentrations. As shown in Fig. 2a-b, nicotinic acid (VB3) and isonicotinic acid exhibit obvious concentration-dependent activity, while nicotinamide and isonicotinamide only show a weak activity though the concentrations are increased, which indicates that the carboxyl group of VB3 and isonicotinic acid plays an important role in their peroxidase mimicking activity, which is in consistent with previous literature (Liu et al., 2017).

Close

Fig. 1

Schematic illustration of peroxidase-like activity of nicotinic acid and the detection mechanism of glutathione.

Export to PPT

Close

Fig. 2

The peroxidase-like activity of different small molecules. (A) The relative peroxidase-like activity of different small molecules under varied concentrations; (B) The absorption spectra of different peroxidase mimic catalyzed system; (C) The chemical structure of different small molecules.

Export to PPT

3.2 Reaction conditions investigation

Like natural horseradish peroxidase and most enzyme mimics, the catalytic performance of VB3 is easily affected by several reaction conditions such as pH, temperature, VB3 concentration, and reaction time. As shown in Fig. 3a, with the increase in pH value, the relative activity of VB3 keeps relatively stable and reaches the maximum at pH 6.0. Within the pH range of 3.0–9.0, the catalytic activity of VB3 remains over 80% of that at optimal pH. The carboxyl group of VB3 may play a significant role in buffering the influence of acidity and alkalinity in the reaction system, thus leading to the excellent tolerance in a broad pH range of its peroxidase-like performance. Fig. 3b demonstrates the influence of temperature on the peroxidase mimicking activity of VB3, the temperature range (20–70 °C) was evaluated under optimal pH condition. Unlike most other peroxidase mimics, VB3 exhibits higher catalytic activity as temperature is increased, which may be ascribed to the inherent thermostability of VB3. Therefore, the excellent heat resistance and acid-alkali tolerance of VB3 make it possible for potent applications under extreme conditions. Furthermore, Fig. 3c indicates that the increasing concentration of VB3 can accelerate the catalytic reaction velocity, which levels off at 50 mM. Moreover, as the time-dependent plot shown in Fig. 3d, the catalytic reaction rate slows down at 40 min. Therefore, the optimal experimental conditions for subsequent analysis were selected as pH 6.0, 60 °C of reaction temperature, 20.0 mM of VB3 concentration, and 40 min of reaction time, respectively. Additionally, to assess the storage stability, the peroxidase-like activity of VB3 was measured under optimal conditions at 10-day intervals for 70 days. As shown in Fig. S2, the catalytic performance of VB3 keeps almost the same of its initial catalytic activity even after 70 days, exhibiting excellent storage stability.

Close

Fig. 3

Effects of (A) pH, (B) temperature, (C) VB3 concentration and (D) incubation time on the peroxidase mimetic activity of VB3. Experiments were carried out using 50 mΜ of VB3 dissolved in PBS buffer (10 mM, pH = 6.0) at 60 °C with 4.0 mΜ of TMB and 0.1 mM of H₂O₂ as substrates. The maximum point in each curve was set as 100%. The error bars represent the standard deviations derived from three independent measurements.

Export to PPT

3.3 Kinetic analysis

The catalytic behaviors of VB3 with H₂O₂ and TMB as individual substrates were further investigated by virtue of steady-state kinetic experiments. Kinetic experiments were carried out by fixing the concentration of H₂O₂ and tuning the TMB concentration under the optimal conditions or vice versa. Fig. S3a and c demonstrate the Michaelis-Menten plots with substrate concentrations as independent variables. Fig. S3b and d represent the correlations between the initial velocity and substrate concentration, which are well-fitted to Lineweaver-Burk plots. Generally, the K_m value indicates the affinity between the substrate and the enzyme, and a smaller K_m represents a stronger affinity. The obtained K_m and v_max values of VB3 toward substrates are summarized in Table S1, and kinetic parameters of some other peroxidase mimics are also listed for comparisons. The K_m of VB3, using H₂O₂ as the substrate, is 0.037 mM. This value is approximately 32 times lower than that of HRP and some organic small-molecule peroxidase simulants, indicating a strong affinity between VB3 and H₂O₂. In addition, the K_m parameter of VB3 for TMB is 3.28 mM, implying that only minute quantity of TMB is necessary for maximum catalytic activity. However, the k_cat value of VB3 is only close to acidic amino acids, and lower than that of natural horseradish peroxidase, which is similar to previously reported small-molecule peroxidase mimics. Therefore, it is still a meaningful and challenging topic of discovery more small-molecule peroxidase mimics with higher catalytic activity.

3.4 Catalytic mechanism

To understand the catalytic mechanism of the peroxidase mimic, ROS involved in VB3-H₂O₂-TMB chromogenic system were further investigated. As illustrated in Fig. S4, the increasing addition amount of common hydroxyl radical (^•OH) scavenger isopropanol results in obvious larger absorbance decline of VB3-H₂O₂-TMB chromogenic system (Bandi et al., 2020). On the other hand, the addition of superoxide anion (O₂^•−) scavenger (superoxide dismutase) only contributes to a slight absorbance decrease (Shi et al., 2018a, 2018b). These results indicate that the production of ^•OH plays a major role in the catalytic oxidation process to obtain the blue product TMB oxide and the O₂^•− plays a partial role.

3.5 Detection of H₂O₂

Hydrogen peroxide is a common intermediate product in extensive biochemical processes, so the accurate determination of H₂O₂ is increasingly significant. Based on the H₂O₂ concentration dependent peroxidase mimicking property of VB3, as a proof-of-concept implementation, a facile colorimetric method was fabricated to determine H₂O₂. As shown in Fig. 4a, the H₂O₂ dose–response plot indicates that the absorbance at 652 nm of reaction mixture raises along with the increase in H₂O₂ concentration. Fig. 4b demonstrates that the absorbance at 652 nm of reaction mixture exhibits a good linear correlation with H₂O₂ concentration between 5.0 μM and 100.0 μM with the linear regression equation of A = 0.0087 [H₂O₂] + 0.2323 (R² = 0.9950), and the limit of detection (LOD) toward H₂O₂ is calculated to be 3.0 μM (signal-to-noise ratio, S/N = 3). The LOD value is lower than several nanomaterials-based peroxidase mimics in previous reports (Table 1). Moreover, as illustrated in Fig. 4c, the color variance of the reaction mixtures of different H₂O₂ concentrations can be easily distinguished by naked eyes. Thus, these results demonstrate that the proposed approach can be applied in H₂O₂ analysis.

Close

Fig. 4

(A) The absorption spectra of VB3-TMB system upon adding different concentrations of H₂O₂ (5–100.0 μM, from bottom to top); (B) Calibration plots of the absorbance versus the concentrations of H₂O₂ under the optimal conditions; (C) The corresponding digital photographs. The error bars represent the standard deviation of three repeated measurements.

Export to PPT

The colorimetric procedure was further used for the determination of H₂O₂ in different brands of medical liquid disinfectants. The real samples of medical liquid disinfectant solutions were diluted appropriately to conform to the linear range of calibration plot. The results are listed in Table S2. The VB3-based detection method allows the detection of H₂O₂ in practical samples with satisfactory spiked recoveries within the range of 94.3–106.0% and RSD values within the range of 1.0–6.1% with different spiked concentrations (10.0 μM, 20.0 μM, and 40.0 μM), which indicates the reliability and accuracy of the established method.

3.6 Detection of glutathione

Glutathione, which is a sulfur tripeptide composed of L-cysteine, L-glutamic acid, and glycine, widely exists in living organisms (Wang et al., 2020). As an essential bioactive molecule, it is involved in diverse physiological processes, such as anti-oxidation, immunity, and detoxification effects (Kong et al., 2021). However, unbalanced glutathione level is closely associated with many diseases, such as cancer, stroke, liver damage, and heart problems (Zhu et al., 2021). Thus, it is meaningful for developing sensitive method for glutathione quantification in clinical diagnosis. As illustrated in Fig. 1, the blue TMB oxide can be reduced to colorless TMB by glutathione owing to its efficient reduction capacity. Therefore, a convenient method was fabricated for glutathione detection based on the excellent peroxidase-mimicking property of VB3. The UV–Vis spectra were recorded by adding glutathione at different concentrations to VB3-H₂O₂-TMB reaction system. Fig. 5a illustrates that the absorbance at 652 nm decreases gradually with the increase in glutathione concentration. Fig. 5b shows that the absorbance intensity at 652 nm gives a good linear correlation (A = -0.0123 [glutathione] + 0.843, R² = 0.9949) to glutathione concentration in the range of 5.0–50.0 μM, with the LOD value of 2.9 μM (S/N = 3). The comparisons of different nanomaterials-based peroxidase mimics for glutathione detection were summarized in Table 2. The data reveal that the proposed method based on VB3 is a competitive one for the sensitive detection of glutathione of a broad linear range and low detection limit.

Close

Fig. 5

(A) The absorbance spectra of VB3-TMB-H₂O₂ solution in the presence of various concentrations of glutathione and (B) the linear calibration plots of absorbance against concentrations of glutathione; (C) Selectivity of glutathione colorimetric sensor with interferences by monitoring the absorbance at 652 nm, including 0.05 mM of glutathione and 2.5 mM of interferences (K⁺, Na⁺, Ca²⁺, Ni²⁺, lactose, fructose glucose, lysine, histidine, and arginine); (D) Detection of glutathione in commercial glutathione tablets (the specifications are also shown for comparison). The error bars represent the standard deviation of three repeated measurements.

Export to PPT

The selectivity and interference assay of the detection method was further performed at the existence of several interferences including common sugars (lactose, fructose, and glucose), various amino acids (lysine, histidine, and arginine), and metal ions (K⁺, Na⁺, and Ca²⁺). As can be seen in Fig. 5c, even the interferences’ concentrations are 50 times higher than that of glutathione, there are still no obvious signal response variance (A/A0), which indicates the colorimetric sensor based on the peroxidase mimicking activity of VB3 exhibits a good selectivity for glutathione detection. Additionally, the inset of Fig. 5c also provides verification for the selectivity of the proposed colorimetric method by naked eyes. Finally, the proposed colorimetric sensor was used for glutathione detection in commercial glutathione buccal tablets. As shown in Fig. 5d, the detection results fit well with the tablet specifications with the recoveries in the range of 99.0%–102.4% and RSD within the range of 1.5%–3.0%. These results validate the practicability and accuracy of the established method for glutathione determination in practical specimens.