Retrieving Peroxidase-like Activity of Ligand-capped Gold Nanostars for the Detection of Mycobacterium bovis

Tuning and controlling size, shape and surface characteristics of plasmonic gold nanoparticles can help define their photonic, catalytic, and spectroscopic properties, and therefore are crucial for the development of biosensors. This strategy is demonstrated in the present study. Gold nanostars (Au NST) chemically synthesized through a seeding approach exhibited not only Vis-NIR tunable localized surface plasmon resonance (LSPR), but also strong peroxidase-mimicking activity which can catalyse the oxidation of tetramethylbenzidine (TMB) into an oxidized chromogenic product (oxTMB). The catalytic activity of gold nanostars was found to correlate well with their size and LSPR mode, and to be strongly affected by capping ligands (i.e. poly and oligo ethylene glycol, 11-mercaptoundecanoic acid, 4-mercaptobenzoic acid). When capped with molecular binders (i.e. peptides and antibodies), the peroxidase-mimicking activity was suppressed by almost 100% under the examined conditions. However, a gold deposition reaction catalysed by the nanostars themselves (so-called autocatalytic enlargement) was not affected by the capping, resulting in increased particle size and increased numbers of newly formed nuclei acting as independent centres for the catalysis. The autocatalytic enlargement thus overcame the barrier of bioconjugation-induced suppression and paved the way for biosensor development. The particle characterizations and catalytic processes were thoroughly analysed by absorption spectroscopy, HR-TEM, ζ-potential measurements.
To prove its applicability, the sensing approach was employed to develop a colorimetric biosensor for the detection of Mycobacterium bovis. M. bovis has been described as one of the main hazards of raw drinking milk in the EU, leading to more than 1,000 cases of zoonotic tuberculosis each year. More importantly, due to their slow-growth nature, the detection of pathogenic Mycobacterium species using culture methods is very time-consuming and thus extremely challenging. To date, no effective sensors have been developed to detect the presence of this bacteria in food samples, as most regulations only apply to the presence of the bacteria in the animal.
Towards this end, a binding immunoassay was implemented to select the antibody with the highest avidity for M. bovis. The selected antibody was then immobilized onto the Au NST, using thioctic acid-N-hydroxysuccinimide ester as a heterobifunctional crosslinker. This bionanoconjugate was enlarged by adding Au3+ ions to a final concentration of 10-4 M, thus restoring bare gold surfaces available for TMB oxidation. This step has resulted in the potential to reach higher sensitivity through signal amplification compared with conventional approaches. Integration of this colorimetric biosensing approach with a smartphone reader to allow the development of a semi-quantitative and user-friendly biosensor for in-field applications will also be reported