Silk-Hydrogel-Immobilized Gold-Nanocluster-Seeded Catalase Protein as a Pre-Plasmonic Probe for Colorimetric Peroxide Sensing

Protein-stabilized gold nanoclusters (AuNCs) are emerging as luminescent probes for various sensing applications. However, the technical difficulty of designing solid analytical platforms with these probes impedes their sensor applications. Herein, blue fluorescent (emission ∼ λ460nm) catalase protein (Cat)-AuNCs with peroxidase-mimicking activity were synthesized through an aurichlorohydric acid-led reaction process that concurrently released the heme prosthetic group from the protein matrix, turning off the catalase activity. A significant structural transition with a ∼1.9-fold increase in the β-sheets of the protein occurred during the process of seeding AuNCs of size ∼1.5 nm (dia) in protein molecules, as revealed from the respective circular dichroism and scanning electron microscopy studies. An atomic force microscopy study revealed the presence of AuNCs in the surface periphery of the catalase protein. Upon interaction with the substrate H2O2, these nanoclusters were destabilized and transformed into free plasmonic gold nanoparticles (AuNPs) with an average size of 10 nm through an internal aggregation process. The plasmonic signal (absorbance λ520nm) intensity of the released AuNPs was increased with the increasing concentration of H2O2, offering a linear detection range of 20–200 mM (R2 = 0.99) and a limit of detection of 1 mM for the peroxide. The phenomenon was utilized to develop a colorimetric sensor by immobilizing Cat-AuNCs in silk fibroin hydrogels, which offered stability to the clusters for up to 3 months. The plasmonic response signal from the sensor surface could be captured as a red pixel intensity and color change (yes/no format) for the respective quantitative and qualitative detections of H2O2 for diverse applications.