Deciphering the Mechanism of Carbon Dots toward Sequential Detection of Mercury and l -Cysteine: Excitation Wavelength Matters

Excitation wavelength-dependent emission is a unique photophysical channel with significant implications in analytical and biological applications; however, such phenomena remain rarely explored. To address this, carbon dots (CDs) were synthesized via a microwave-assisted method and systematically characterized using SEM, TEM, FTIR, XPS, UV-visible absorption, and fluorescence spectroscopy. Spectroscopic studies reveal that the emission from CDs strongly depends on the excitation wavelength, confirming the presence of energetically distributed and heterogeneous emissive surface states. These surface states enable a selective population of distinct excited states, thereby modulating the emission characteristics. The global analysis also identified three emissive pathways in the CDs. These pathways have a short lifetime of about 1 ns, an intermediate lifetime that corresponds to the intrinsic citrazinic acid emission (6.3 ns), and a long lifetime that can extend up to 10.8 ns. This provides further support for the presence of multiple surface states that contain rapidly decaying traps and emissive traps that have longer radiative lifetimes compared to free citrazinic acid. On the other hand, the sensing performance was also governed by excitation wavelength, indicating that different excited states possess varying electron transfer quenching efficiencies toward Hg2+ ions. The Stern-Volmer analysis and time-resolved fluorescence decay measurements reveal an electron transfer quenching mechanism, where reduced fluorescence lifetimes and negative free energy change values confirm thermodynamically favorable electron transfer from excited CDs to Hg2+ ions. Furthermore, the CDs exhibit reversible sensing behavior with l-cysteine, restoring fluorescence via the competitive chelation of Hg2+ ions, as supported by steady-state and time-resolved fluorescence measurements. Cytotoxicity and bioimaging studies have been performed on human lung carcinoma (A549) cells that reveal negligible toxicity and enable intracellular imaging of Hg2+ quenching and l-cysteine-mediated recovery. Overall, the obtained results on CDs establish a direct correlation between excitation-dependent photophysical properties and sensing mechanisms, providing a rational framework for the design of wavelength-dependent metal-ion detection and live-cell imaging applications.