Silicon oxide microchips functionalized with fluorescent probes for quantitative real-time glutathione sensing in living cells

Glutathione (GSH) plays a vital role in the regulation of intracellular functions which alterations in physiological glutathione levels are associated to various diseases. Molecular bioimaging is a sensitive method for GSH detection, but challenges persist in the development of fluorescent probes, mainly concerning long-term tracking of intracellular GSH concentration because of aggregation of molecular probes and their washout in cells. Engineered nanomaterials have shown great promise for increasing the disease diagnosis accuracy. Microchips generated by advanced microfabrication techniques can be applied in designing biomedical devices due to control over size, shape, and bioactive coatings utilization. In the current work, the synthesis and characterization of two GSH probes, Bdpy1 and Bdpy2, is reported, each offering irreversible and reversible GSH reactions, respectively. These GSH probes are immobilized on silicon oxide microchips (SOμC), micro-fabricated using photolithographic techniques, to give SOμC-Bdpy1 and SOμC-Bdpy2. Both functionalized microchips exhibited sensitivity to GSH, and, notably, the reversible SOμC-Bdpy2 showed less time dependency, making it more suitable for long-term intracellular GSH sensing. <i>In vitro</i> experiments in HeLa cells reveal both SOμC-Bdpy1 and SOμC-Bdpy2 were internalized in living cells, showing SOμC-Bdpy2 more reliable results (due to its less time dependency) for quantifying intracellular GSH. Remarkably, the intracellular GSH measurement was monitored by SOμC-Bdpy2 for 48 h, indicating the functionalized microchips capability to detect GSH amount in different time intervals. This study introduces a promising approach for long term quantification of intracellular GSH, overcoming the limitation of fluorescent probes and offering valuable insights into microchip-based sensing methodologies.