Nitrogen-doped carbon nanodots as a biocompatible FRET donor for caspase-3 detection and chemotherapy response evaluation in osteosarcoma cell lysates.

Osteosarcoma is a highly aggressive bone malignancy requiring precise tools for early diagnosis and effective evaluation of chemotherapy efficacy. Here, we report an "off-on" fluorescent nanosensor based on nitrogen-doped carbon nanodots (N-CNDs) for the sensitive and selective detection of caspase-3 activity, a key apoptosis executor. The sensor is constructed by conjugating carboxyl-functionalized N-CNDs with a BHQ1-labeled peptide substrate containing the caspase-3-specific DEVD cleavage motif. In the absence of caspase-3, fluorescence is quenched via FRET. Upon caspase-3 activation, the peptide is cleaved, releasing BHQ1 and restoring fluorescence. The nanosensor exhibits high sensitivity (detection limit of 0.0106 U/mL), excellent selectivity, and robust performance in complex biological media. As a proof-of-concept application, it detects elevated caspase-3 activity in osteosarcoma cell lysates (MG-63) compared to normal osteoblast lysates (hFOB1.19) and enables quantitative, dose-dependent assessment of apoptosis induced by cisplatin and doxorubicin directly in cancer cell lysates. With its current format, this cell-lysate-based platform provides a practical and sensitive tool for preclinical drug screening and rapid comparison of chemotherapeutic potency, while laying a foundation for future adaptation toward live-cell and in vivo imaging applications.