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.

Catalytic ferromagnetic gold nanoparticle immunoassay for the detection and differentiation of Mycobacterium tuberculosis and Mycobacterium bovis.

A ferromagnetic gold nanoparticle based immune detection assay, exploiting the enhanced signal amplification of inorganic nanozymes, was developed and evaluated for its potential application in the detection of Mycobacterium tuberculosis complex (MTBC) organisms, and simultaneous identification of Mycobacterium bovis. Ferromagnetic gold nanoparticles (Au-Fe3O4 NPs) were prepared and their intrinsic peroxidase-like activity exploited to catalyse 3,3',5',5-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). When the Au-Fe3O4 NPs were functionalised by direct coupling with MTBC-selective antibodies, a nanoparticle based immune detection assay (NPIDA) was developed which could detect Mycobacterium tuberculosis (MTB) and differentiate M. bovis. In the assay, the intrinsic magnetic capability of the functionalised Au-Fe3O4 NPs was used in sample preparation to capture target bacterial cells. These were incorporated into a novel immunoassay which used species selective monoclonal antibodies (mAb) to detect bound target. The formation of a blue TMB oxidation product, with a peak absorbance of 370 nm, indicated successful capture and identification of the target. The detection limit of the NPIDA for both MTB and M. bovis was determined to be comparable to conventional ELISA using the same antibodies. Although limited matrix effects were observed in either assay, the NPIDA offers a reduced time to confirmatory identification. This novel NPIDA was capable of simultaneous sample concentration, purification, immunological detection and speciation. To our knowledge, it represents the first immune-based diagnostic test capable of identifying MTBC organisms and simultaneously differentiating M. bovis.