Peroxidase-Mimicking Activity of Biogenic Gold Nanoparticles Produced from Prunus nepalensis Fruit Extract: Characterizations and Application for the Detection of Mycobacterium bovis

In the present study, a facile, eco-friendly, and controlled synthesis of gold nanoparticles (Au NPs) using Prunus nepalensis fruit extract is reported. The biogenically synthesized Au NPs possess ultra-active intrinsic peroxidase-like activity for the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. Chemical analysis of the fruit extract demonstrated the presence of various bioactive molecules such as amino acids (l-alanine and aspartic acids), organic acids (benzoic acid and citric acid), sugars (arabinose and glucose), phenolic acid, and bioflavonoids (niacin and myo-inositol), which likely attributed to the formation of stable biogenic Au NPs with excellent peroxidase-mimicking activity. In comparison with the natural horseradish peroxidase (HRP) enzyme, the biogenic Au NPs displayed a 9.64 times higher activity with regard to the reaction velocity at 6% (v/v) H2O2, presenting a higher affinity toward the TMB substrate. The Michaelis–Menten constant (KM) values for the biogenic Au NPs and HRP were found to be 6.9 × 10–2 and 7.9 × 10–2 mM, respectively, at the same concentration of 100 pM. To investigate its applicability for biosensing, a monoclonal antibody specific for Mycobacterium bovis (QUBMA-Bov) was directly conjugated to the surface of the biogenic Au NPs. The obtained results indicate that the biogenic Au NPs-QUBMA-Bov conjugates are capable of detecting M. bovis based on a colorimetric immunosensing method within a lower range of 100 to 102 cfu mL–1 with limits of detection of ∼53 and ∼71 cfu mL–1 in an artificial buffer solution and in a soft cheese spiked sample, respectively. This strategy demonstrates decent specificity in comparison with those of other bacterial and mycobacterial species. Considering these findings together, this study indicates the potential for the development of a cost-effective biosensing platform with high sensitivity and specificity for the detection of M. bovis using antibody-conjugated Au nanozymes.


Optimization study of biogenic synthesis process of Au nanoparticles
Four parameters were monitored for optimizing the process of Au nanoparticle synthesis: temperature, reaction pH, reductant dilution factor and concentration of gold salt. The process was first optimized for the reaction pH and temperature. Four different reaction pH: 4

Free radical scavenging activity and antioxidant potential of P. nepalensis fruit extract
To understand the chemical characteristics of the P. nepalensis fruit extract, and estimate the antioxidant potential and reducing ability, different assays were performed. Likewise, total phenolic content and flavonoid content responsible for the antioxidant and reduction potency were also assessed.

Reducing power assay
Reducing power assay was performed by adapting the protocol described by Yildirim et al. 1 .
P. nepalensis fruit extract (20 µL) was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) followed by 2.5 mL of potassium ferricyanide as described by Yang et al. 2 . After 30 min incubation at 50 ºC, 2.5 mL of trichloroacetic acid was added and centrifuged at 1008 g force for 10 min 3 . The supernatant (2.5 mL) was mixed properly by pipetting with 2.5 mL of distilled water followed by 0.5 mL of ferric chloride. Finally, the absorbance was measured at 700 nm using a UV-Visible spectrophotometer where the reducing power is directly proportional to the absorbance value 4,5 .

DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) scavenging ability
DPPH scavenging ability was determined according to the method reported by Blois (1958) with slight modifications 6 . Two hundred microliters of P. nepalensis fruit extract was mixed with 2 mL of 0.1 mM DPPH and 50 mM Tris-HCl buffer (800 µL, pH 7.4). After 30 min incubation at RT, the reduction of DPPH was measured at 517 nm. Tubes without the samples were taken as control, whereas an ascorbic acid (1 mg/mLl) solution was taken as the standard 4,5 . This is represented as % DPPH radical scavenging activity and is calculated using SEq. (1).

Determination of total phenolic content
Total phenolic content was calculated with a spectrophotometer by using Folin-Ciocalteu reagent 7 . P. nepalensis fruit extract (2 mL) was mixed with 10 mL of Folin-Ciocalteu diluted reagent (1/10 with double-distilled deionized water). After 2 min incubation, 8 mL of sodium carbonate was added and the reaction solutions were incubated in dark conditions for 2 h at room temperature. The absorbance was measured at 765 nm 4,5 . Gallic acid standard curve was prepared with the concentration range from 50 to 500 µg/mL. Results are articulated in micrograms (µg) of gallic acid equivalent (GAE) per mL of P. nepalensis fruit extract 8 .

Total flavonoid content
Total flavonoid content was determined by the aluminium chloride colorimetric method 9 . One mL of P. nepalensis fruit extract was mixed with 4 mL of distilled water, followed by the addition of 0.3% (v/v) NaNO 2 solution. After incubation (5 min), 0.3% AlCl 3 was added and the solution incubated for a further 6 min. Two mL of 1 M NaOH solution was added and the final volume of the reaction mixture was prepared to 10 mL by adding distilled water. After incubation (15 min), the absorbance was read at 570 nm. The total flavonoid content present in the solution was calculated from the calibration curve, and the result expressed as mg catechin equivalent per g dry weight 9 .

Characterization of peroxidase mimicking activity of biogenic Au NPs
Peroxidase like activity of biogenic Au NPs was determined using TMB as a chromogenic substrate. Experiments were carried out using 100 pM of biogenic synthesized Au NPs in a S4 final reaction volume of 1 mL, with 1 mM of TMB and 6% (v/v) H 2 O 2 . The reaction mixture was incubated at RT for 10 minutes, and full-spectrum analysis was carried out using UV-Visible spectrophotometer. Time-dependent UV-Visible full spectral analysis of the catalyzed reaction using the same conditions was done in 1 mL reaction volume for 90 min 10 .

Understanding the reaction mechanism of TMB oxidation catalyzed by peroxidase mimicking biogenic Au NPs
To understand the reaction mechanism of the TMB oxidation using biogenic Au NPs, 1 mM TMB was catalyzed using the same conditions, mentioned earlier (section 1.3). The reaction kinetics were monitored at three different wavelengths: 370 nm, 450 nm and 650 nm, using a UV-Visible spectrophotometer for 20 min and samples readings were recorded at one-minute intervals 11 .

Determining the role of P. nepalensis fruit extract for the catalysis of TMB oxidation
To understand the role of P. nepalensis fruit extract for the oxidation of chromogenic substrate TMB in the presence of H 2 O 2 a simple experiment was carried out by using 10 µL of fruit extract, 1 mM of TMB, 6 % H 2 O 2 in a reaction volume of 1 mL. For a comparative study, 100 pM of biogenic Au NPs was taken as a positive control. For blank, only TMB along with H 2 O 2 was taken without any NPs or fruit extract 12 .

Optimization study
The UV-visible readings for the effect of different parameters in the biogenic synthesis of Au nanoparticles can be seen in Figure S1. Figure S1A represents the optimization study of Au nanoparticles synthesis using different pH levels where natural pH was measured as 4.3 (a mixture of gold salt and prunus extract). From this figure, it is clearly visible that at natural pH rapid synthesis of Au nanoparticles took place with a SPR maximum at 520 nm. Additionally, an increase in the yield of the NPs is also noticed as compared to pH 4. A further increase in the pH to 7.0 and pH 10 has resulted in a rapid damping in the intensity of the SPR band. This suggests that the formation of Au NPs is favoured under acidic conditions (2 to 7). An increase in the pH to basic condition either has decreased the formation of initial metal nuclei or may have resulted in the agglomeration of the NPs. Figure S1B shows the effect of reaction temperature for the synthesis of Au NPs where increase in the temperature from 4°C to 90°C S5 has shown a slight variation in the SP band. At RT (room temperature) yield of the synthesized nanoparticles has been considered as highest with an SP maximum at 522 nm. A decrease in the reaction temperature has resulted in the low-intensity SP band, which is possibly due to the formation of less number of nuclei during the reduction process. Figure S1C shows the effect of concentration of the gold salt solution on the synthesis of Au NPs. Reaction with 1 mM gold salt has shown a notable peak with a SPR maximum at 519 nm. It suggests that with an increase in the concentration from 2 mM to 4 mM, SP peak has shown redshift along with a significant amount of damping. The presence of a higher concentration of gold salt may have led to rapid nucleation and simultaneous growth (heterogeneous process), which might have resulted in the formation of particles with different morphology and size. . Figure S1D shows the effect of the rate of dilution of prunus extract on the synthesis of Au NPs. Low dilution has resulted in aqueous NP dispersion with a broad SP peak, which centers at 550 nm. Increasing the dilution factor to 1:10 has produced particles with a sharp SP peak at 510 nm. But, further increasing the dilution factor, 1:100 and 1:1000, has not produced any Au NPs; this is probably due to the insufficient amount of reducing agents required for the synthesis of Au NPs.

Field Emission Scanning Electron Microscope (FESEM) analysis
Morphological characterization of biogenic Au nanoparticles was done by using field emission scanning electron microscope (FESEM). Agglomerated Au nanoparticles with a particles size range from 20-35 nm can be observed in Figure S2. Particles are present in polydisperse form due to the agglomeration (probably occurred during the sample preparation). Uniformity in the size of Au NPs can be observed. This observation was in accordance with the data obtained from UV-visible spectrum of Au nanoparticles.

Characterization of peroxidase mimicking activity of biogenic Au NPs
To understand the full reaction mechanism of the TMB oxidation process catalyzed by biogenic Au NPs, a kinetic analysis of the TMB oxidation was performed with 1 mM TMB, 6% H 2 O 2, and 100 pM biogenic Au NPs at set conditions for 20 min. Figure S4A demonstrates the reaction mechanism of the TMB oxidation process where TMB, parent diamine (TMB 0 ) gets oxidized by one-electron oxidation to form cation radical (TMB +1 ) (λ max 370 nm) giving vivid blue color, which is in equilibrium with a meriquinoid complex or charge transfer complex (CTC) (λ max 650 nm) displaying green color. The reaction is further catalyzed by peroxidase mimicking biogenic Au NPs in the presence of H 2 O 2 to form diamine oxidation product (TMB +2 ) or quinodiimine of yellow color (λ max 450 nm) 13 . Figure S4 B-D, demonstrate the S6 UV-Visible spectral analysis of TMB oxidation at different stages of the TMB oxidation process at three different wavelengths such as 370, 650, and 450 nm. From previous studies, it has been observed that the peak absorbance at 650 nm has been utilized widely as a diagnostic peak to investigate the peroxidase mimicking activity of Au NPs 10,14-16 . However, interference of physicochemical reaction parameters plays an imperative role in inducing aggregation of Au NPs in the high electrolyte reaction medium, attributing a red-shift of the plasmon peak around 600-700 nm. Thus, the absorption peak around 650 nm, representing the formation of oxidized TMB products is potentially less acceptable due to the potential overlapping of peaks generated from the aggregated nanoparticles. Additionally, Figure S4B  Biogenic Au NPs showed excellent intrinsic peroxidase mimicking activity in presence of H 2 O 2 indicating the ability to oxidize TMB with higher efficiency. For biological synthesis process, P. nepalensis fruit extract acted as a reducing as well as a capping agent for the synthesis of stable Au NPs, indicating the probable presence of biological components from S7 the fruit extract over the surface of the nanoparticles. Hence, the role for fruit extracts in possessing intrinsic peroxidase-like activity of Au NPs remains unknown. As a preliminary test of the ability of fruit extract to oxidize TMB, a simple reaction was carried out with the similar protocol outlined above. Figure