Horseradish Peroxidase-Encapsulated Fluorescent Bio-Nanoparticle for Ultra-Sensitive and Easy Detection of Hydrogen Peroxide

Hydrogen peroxide (H2O2) has been a fascinating target in various chemical, biological, clinical, and industrial fields. Several types of fluorescent protein-stabilized gold nanoclusters (protein-AuNCs) have been developed for sensitive and easy detection of H2O2. However, its low sensitivity makes is difficult to measure negligible concentrations of H2O2. Therefore, to overcome this limitation, we developed a horseradish peroxidase-encapsulated fluorescent bio-nanoparticle (HEFBNP), comprising bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) and horseradish peroxidase-stabilized gold nanoclusters (HRP-AuNCs). The fabricated HEFBNP can sensitively detect H2O2 owing to its two properties. The first is that HEFBNPs have a continuous two-step fluorescence quenching mechanism, which comes from the heterogenous fluorescence quenching mechanism of HRP-AuNCs and BSA-AuNCs. Second, the proximity of two protein-AuNCs in a single HEFBNP allows a reaction intermediate (•OH) to rapidly reach the adjacent protein-AuNCs. As a result, HEFBNP can improve the overall reaction event and decrease the loss of intermediate in the solution. Due to the continuous quenching mechanism and effective reaction event, a HEFBNP-based sensing system can measure very low concentrations of H2O2 up to 0.5 nM and show good selectivity. Furthermore, we design a glass-based microfluidic device to make it easier use HEFBNP, which allowed us to detect H2O2 with the naked eye. Overall, the proposed H2O2 sensing system is expected to be an easy and highly sensitive on-site detection tool in chemistry, biology, clinics, and industry fields.