Caspase-3 inhibitor(Chembridge Research Laboratories)

Histidine-derived carbon dots (His-CDs) were synthesized to detect staurosporine-induced apoptosis in T lymphoma (Jurkat) cells. The His-CDs were characterized for their physical and chemical properties including size, morphology, fluorescence, and surface functionality. Transmission electron microscopy (TEM) revealed a spherical morphology with an average size of 11.4 ± 3.4 nm. Fluorescence analysis showed maximum excitation at 338 nm and emission at 415 nm, attributed to the carbon dots' quantum confinement effect and surface defects. FTIR and SEM-EDS confirmed the presence of hydroxyl, amine, aromatic rings, and alkyl (C-H) functional groups and carbon, nitrogen, and oxygen elemental composition in ratios of 52%, 24.8%, and 23.3%, respectively. His-CDs were evaluated for cytotoxicity and apoptosis detection in Jurkat cells. Fluorescence microscopy and flow cytometry analysis demonstrated concentration-dependent fluorescence, suggesting effective cellular uptake of His-CDs. The apoptotic-sensing capability of His-CDs was tested using staurosporine, an apoptosis inducer. A concentration-dependent increase in fluorescence was observed with increasing staurosporine concentrations, indicating the His-CDs' sensitivity to apoptosis. The time-dependent fluorescence increases were noted with prolonged staurosporine exposure. Z-DEVD-FMK, a caspase-3 inhibitor, confirmed that the apoptosis detected by His-CDs was caspase-3 dependent, as co-treatment reduced His-CDs' fluorescence in the cell. In conclusion, these results demonstrate that His-CDs are biocompatible, sensitive apoptosis sensors and hold the potential for monitoring apoptotic pathways in cellular systems.

Caspase-3 is a pivotal enzyme in the apoptosis pathway that is responsible for executing programmed cell death through the cleavage of key cellular proteins. Existing fluorescence-based probes for caspase-3 detection suffer from limitations such as background noise from tissue autofluorescence and light scattering, reducing their sensitivity and real-time imaging capabilities. To overcome these limitations, we developed a chemiluminescent probe, Ac-DEVD-CL, that enables the highly sensitive and selective detection of caspase-3 activity. Upon caspase-3-mediated cleavage, the probe undergoes a self-immolative reaction that triggers a chemiluminescent signal, allowing real-time monitoring of the enzymatic activity. Probe Ac-DEVD-CL demonstrated an exceptionally high turn-on response, with a 5000-fold increase in the chemiluminescent signal upon enzymatic activation. The probe exhibited notable specificity for caspase-3, with minimal cross-reactivity toward other biologically relevant proteases and tumor-associated enzymes. Additionally, inhibition studies using the caspase-3 inhibitor confirmed that the probe's activation is exclusively mediated by caspase-3. A direct comparison with the commercially available fluorescent probe revealed that probe Ac-DEVD-CL offers significantly improved sensitivity, achieving a signal-to-noise ratio 380-fold higher and a limit of detection 100-fold lower. These results establish probe Ac-DEVD-CL as a highly effective tool for detecting caspase-3 activity with superior precision. Finally, we validated the probe's utility in imaging apoptosis in live cells. In 4T1 breast cancer cells treated with cisplatin, Ac-DEVD-CL generated a strong chemiluminescent signal, with a three-order-of-magnitude enhancement compared to untreated cells. Overall, the probe Ac-DEVD-CL demonstrates a significant improvement in detection sensitivity, providing a powerful and versatile chemiluminescent probe for real-time imaging of caspase-3 activity. Its exceptional sensitivity and selectivity could make it a valuable tool for cancer research, drug discovery, and therapeutic monitoring.