The preparation of a fluorescent dual-modality nanosensor for the discrimination and determination of biothiols in real samples and its practical detection kit†

Biothiols widely exist in living organisms and have a crucial function of maintaining redox balance in the human body. It is vital yet difficult to develop probes that can simultaneously detect and distinguish biothiols. In this study, a highly sensitive dual-modality nanosensor, NBD-Nap@NCC, was developed for the discrimination and determination of biothiols in real samples, and its practical application was elucidated based on RGB analysis using a smartphone. The sensitive nanosensor was successfully prepared through the surface modification of nanocrystalline cellulose (NCC), combining NBD and naphthalene fluorophores. Owing to the high electron-withdrawing behavior of the NBD group, which led to a PET mechanism between the fluorophores, the prepared NBD-Nap@NCC nanosensor had a very weak fluorescence response. However, after treatment with Hcy or Cys, NBD-Nap@NCC quickly provided remarkable and different rates of fluorescence “turn-on” responses in both blue and green channels, which was attributed to naphthalene and NBD fluorophores as a result of the inhibition of the PET mechanism. However, after treatment with GSH, only a significant blue-channel emission, which was attributed to the naphthalene fluorophore was obtained, indicating the inhibition of the PET mechanism. Furthermore, the NCC platform demonstrated improved sensitivity and selectivity because of the increased surface area and higher number of binding sites due to modification of the NBD group on the surface. The detection limit ranged from 0.910 to 1.150 μmol L⁻¹ for biothiols with a large dynamic response range. The accuracy of the sensor in determining the concentrations of Hcy, Cys, and GSH in real samples was evaluated via HPLC and spike/recovery analysis. Additionally, paper-based analysis kits were fabricated for the practical detection of biothiols based on RGB changes using a smartphone application.