Gold Nanocluster Containing Polymeric Microcapsules for Intracellular Ratiometric Fluorescence Biosensing

A new approach to sensing and imaging hydrogen peroxide (H₂O₂) was developed using microcapsule-based dual-emission ratiometric luminescent biosensors. Bovine serum albumin-capped gold nanoclusters (BSA-AuNCs) sensitive to H₂O₂ were coencapsulated with insensitive FluoSpheres (FSs) within polymeric capsules fabricated via the layer-by-layer method. Under single-wavelength excitation, the microcapsule-based biosensors exhibited emission bands at ∼516 and ∼682 nm resulting from the FSs and BSA-AuNCs, respectively. The polyelectrolyte multilayers lining the microcapsules were effective in protecting BSA-AuNCs from the degradation catalyzed by proteases (chymotrypsin, trypsin, papain, and proteinase K) and subsequent luminescent quenching, overcoming a key limitation of prior BSA-AuNC-based sensing systems. The luminescent response of the sensors was also found to be independent of local changes in pH (5-9). Quenching of the AuNCs in the presence of H₂O₂ enabled the spectroscopic quantification and imaging of changes in H₂O₂ concentration from 0 to 1 mM. The microcapsule sensors were easily phagocytized by murine macrophage cells (RAW 264.7), were effective as intracellular H₂O₂ imaging probes, and were successfully used to detect local release of H₂O₂ in response to an external chemical stimulus.