Exploiting the intrinsic Michael-acceptor reactivity of osimertinib via thiol-functionalized carbon dots: a...

Osimertinib (OMB) is a third-generation EGFR-tyrosine kinase inhibitor used as a first-line therapy for EGFR-mutant non-small cell lung cancer, but its narrow margin between effective and toxic exposure, substantial inter-patient pharmacokinetic variability, and susceptibility to covalent degradation in serum make sensitive, selective methods for its detection essential for therapeutic drug monitoring and pharmacokinetic evaluation. In this work, thiol-functionalized carbon dots (SH@CDs) were synthesized by a one-pot hydrothermal method and developed as a fluorometric "turn-off" probe for OMB. The sensing mechanism relies on a dual covalent-electrostatic interaction: OMB's electrophilic acrylamide group undergoes thiol-Michael addition with the probe's surface -SH groups, while its protonated tertiary amine is simultaneously drawn to the anionic carbon-dot surface, together cross-linking neighboring dots and triggering aggregation-caused fluorescence quenching, as confirmed by lifetime, FTIR, zeta-potential, and TEM analyses. Under optimized conditions, the fluorescence quenching ratio (F ₀/F) showed a linear response toward OMB over the range 1.0-60.0 ng mL^-1, with a limit of detection of 0.35 ng mL^-1-outperforming previously reported spectrophotometric, electrochemical, and even several chromatographic methods. The probe showed selective recognition of cationic, acrylamide-functionalized covalent kinase inhibitors, and was successfully applied to the determination of OMB in spiked and real human serum samples, with results statistically comparable to a reference method, supporting its use in pharmacokinetic studies and routine clinical monitoring of OMB in NSCLC patients.