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

Abstract

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 (K_M) 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.

Keywords: Mycobacterium bovis; biogenic nanoparticles; biosensing; green synthesis; indirect enzyme-linked immunosorbent assay (iELISA); peroxidase-mimicking.

Figure 1

Characterization of biogenic Au NPs. (A) UV–vis spectrum of biologically synthesized Au NPs using P. nepalensis fruit extract. The inset shows the color formation of the reaction mixture due to the reduction of the gold salt to form Au NPs: (i) gold salt solution, (ii) P. nepalensis fruit extract, (iii) crude Au NPs, and (iv) washed Au NPs. (B) XRD analysis of Au NPs synthesized by P. nepalensis fruit extract.

Figure 2

TEM images. (A) Bright field images (inset: SAED pattern). (B) Dark field image. (C) High-angle annular dark field image. (D) Particle size analysis performed on approximately 100 particles using ImageJ software and (E) EDX analysis of biogenic Au NPs synthesized from P. nepalensis fruit extract.

Figure 3

(A) UV–vis spectroscopic analysis of the catalyzed reaction of TMB/H₂O₂ in the absence and presence of Au NPs. Figure 3A inset: (i) TMB, (ii) TMB + H₂O₂, (iii) Au NPs, and (iv) TMB + H₂O₂ +Au NPs. (B) UV–vis full spectral analysis of the catalyzed reaction of 1 mM TMB/6% H₂O₂ in the presence of 100 pM Au NPs.

Figure 4

Typical kinetic analysis of TMB oxidation. (A) Kinetic analysis of TMB oxidation by Au NPs in the presence of varying TMB (0–1 mM) and H₂O₂ concentrations (1–10%). The concentration of the Au nanozyme was fixed at 100 pM. (B) Kinetic analysis of TMB oxidation by the HRP enzyme in the presence of varying TMB (0–1 mM) and H₂O₂ concentrations (1–10%). Samples were analyzed in triplicate (n = 3), and the standard deviation was deduced from these data. (C) Kinetic analysis of the HRP enzyme along with Au NPs at different pH values (2–12). (D) Kinetic analysis of the HRP enzyme along with Au NPs at different temperatures (20–60 °C). All the absorbance values were measured at 370 nm.

Figure 5

Comparison of the catalytic efficiency of HRP and Au NPs. The reaction was carried out in the presence of 0.1 mM TMB and various H₂O₂ concentrations for 10 min at RT. Concentrations of the natural enzyme and nanozyme were fixed at 100 pM, and the absorbance was measured at 370 nm.

Figure 6

FTIR spectra of biogenic Au NPs synthesized by using P. nepalensis extract.

Figure 7

(A) UV–vis spectrum of biogenic Au NPs-QUBMA-Bov conjugates in comparison with that of Au NPs only (B) kinetic analysis of the catalyzed reaction of 1 mM TMB/6% (v/v) H₂O₂ in the presence of 100 pM Au NPs and Au NPs-QUBMA-Bov conjugates.

Figure 8

iELISA sensitivity assay using AuNPs-QUBMA-Bov conjugates to detect M. bovis (A) in a laboratory buffer solution (PBS, pH 7.4, Tween-20 0.05% v/v, PBST) and (B) in a solid cheese matrix solution. (C) iELISA specificity assay using AuNPs-QUBMA-Bov conjugates to detect M. bovis in a laboratory buffer (PBST) solution.