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 H 2 O 2 . 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) H 2 O 2 , 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 10 0 to 10 2 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.

Smartphone-Based Immunochemical Sensor Exploiting Peroxidase-Like Activity of Ligand-Capped Gold Nanostars: A Proof-of-Concept Detection of Mycobacterium Bovis

“…The dithiothreitol (DTT) reduction method was used to prepare IgG antibodies containing sulfhydryl groups, with minor modifications from previously reported protocols (Ji et al, 2010). In short, a 200 μL solution of IgG antibody (QUBMA-Bov) produced previously (Das et al, 2022;Stewart et al, 2012) was prepared at a concentration of 1 mg/mL in PBS 0.1 M -EDTA 2 mM buffer. 8 μL of DTT 0.5 M prepared in aqueous solution was then added and the mixture incubated under gentle stirring for 25 min at RT.…”

“…Their assay also showed positive results for samples containing high numbers of M. bovis cells (≥10 4− 5 CFU/g) in line with the sensitivity values reported herein. More recently, ferromagnetic AuNP (Gilbride et al, 2021) and biogenic AuNP (Das et al, 2022) have been used to develop colourimetric assays, improving the LOD values reported above. These systems, however, still rely on expensive lab-based readout equipment, while at the same time cannot escape the limited robustness of immunoassays for detecting small bacterial concentrations.…”

Nanoparticles Mimicking Oxidase Activity and their Application in Synthesis of Neurodegenerative Therapeutic Drug L‐DOPA

“…Liu et al (2015) reported that platinum NPs exhibited catechol oxidase like activity [11] . Das et al (2022) synthesized gold NPs using Prunus nepalensis fruit extract which can mimic peroxidase like activity and used for the detection of Mycobacterium bovis [12] . Rasheed et al (2020) reported catalase like activity of iron oxide and manganese oxide NPs [13] .…”

“…[11] Das et al (2022) synthesized gold NPs using Prunus nepalensis fruit extract which can mimic peroxidase like activity and used for the detection of Mycobacterium bovis. [12] Rasheed et al (2020) reported catalase like activity of iron oxide and manganese oxide NPs. [13] Nanoparticles such as gold, Fe 3 O 4, ZnO, TiO 2 show peroxidase activity in the presence of hydrogen peroxide via electron transfer method.…”

A template-free assembly of Cu,N-codoped hollow carbon nanospheres as low-cost and highly efficient peroxidase nanozymes

“…Nanozymes are a type of nanomaterial with enzyme-like properties, which combines the advantages of nanomaterials with natural enzymes, and thus have attracted widespread attention. [1][2][3] Since Fe 3 O 4 as a nanozyme was reported for the first time, 2 various nanomaterials with peroxidase-like activities have been developed, 3 including noble metals, [4][5][6] transition metal oxides [7][8][9] and carbon-based materials. [10][11][12] However, many shortcomings hinder the application of metalbased nanozymes, such as high cost (noble metals), lack of specificity (transition metal oxide) and health risk (V 2 O 5 ).…”