Gold Nanocluster Containing Polymeric Microcapsules for Intracellular Ratiometric Fluorescence Biosensing

[Image: see text] A new approach to sensing and imaging hydrogen peroxide (H(2)O(2)) was developed using microcapsule-based dual-emission ratiometric luminescent biosensors. Bovine serum albumin-capped gold nanoclusters (BSA-AuNCs) sensitive to H(2)O(2) 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 H(2)O(2) enabled the spectroscopic quantification and imaging of changes in H(2)O(2) concentration from 0 to 1 mM. The microcapsule sensors were easily phagocytized by murine macrophage cells (RAW 264.7), were effective as intracellular H(2)O(2) imaging probes, and were successfully used to detect local release of H(2)O(2) in response to an external chemical stimulus.