A steric hindrance-regulated probe with single excitation dual emissions for self-adaptive detection of biothiols and H2S in human urine samples and living cells.

Sulfur-containing small molecules, mainly including cysteine (Cys), homocysteine (Hcy), glutathione (GSH), and hydrogen sulfide (H₂S), are crucial biomarkers, and their levels in different body locations (living cells, tissues, blood, urine, saliva, etc.) are inconsistent and constantly changing. Therefore, it is highly meaningful and challenging to synchronously and accurately detect them in complex multi-component samples without mutual interference. In this work, we propose a steric hindrance-regulated probe, NBD-2FDCI, with single excitation dual emissions to achieve self-adaptive detection of four analytes. This probe was meticulously designed and constructed from a pK_a-tuned 2FDCI fluorophore and a thiol-specific recognition moiety NBD. Except for 661 nm fluorescence for indicating the total biothiols and H₂S, Cys and Hcy could trigger an additional 550 nm fluorescence. Utilizing the distinctive responses, the probe NBD-2FDCI exhibited exclusive linear ranges for GSH, Cys/Hcy, and H₂S to avoid high-level component interference. Thus, the probe was then applied for sulfur compound measurements in urine samples, indicating metabolic disorder of Cys and H₂S in bladder cancer patients. Moreover, adaptive imaging of probe NBD-2FDCI in cells was performed with the results being consistent with in vitro testing. In a word, spatial hindrance strategy-guided probes may exhibit broader prospects in the detection of similar components in complex samples.