Evaluation of Dithio Compounds Modified Porous Silicon for Label-Free Detection and Distinction of Biothiols

This study presents a systematic evaluation of three dithio-based surface chemistries for use in a porous silicon (pSi) Fabry-Pérot photonic crystal platform, enabling label-free optical detection and differentiation of biologically relevant thiols. The sensor was functionalized with one of three dithio mediator compounds 2,2'-dithiobis(benzothiazole) (DTBT), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), or 2,2'-dithiobis(5-nitropyridine) (DTNP), each of which undergoes displacement upon exposure to biothiols such as glutathione (GSH), homocysteine (Hcy), and cysteine (Cys). A distinct optical response was observed: while all three dithio compounds exhibited a negative shift in effective optical thickness (EOT) upon exposure to Hcy and Cys, a positive EOT shift occurred in response to GSH, enabling its selective discrimination from the other biothiols. The limits of detection (LoD) varied with compound and analyte, with DTBT achieving LoDs of 3.95 μM (Hcy), 1.65 μM (Cys), and 10.47 μM (GSH); DTNB yielding 1.76 μM (Hcy), 9.20 μM (Cys), and 4.42 μM (GSH); and DTNP offering 2.38 μM (Hcy), 1.43 μM (Cys), and 5.64 μM (GSH). Among these, DTBT demonstrated the highest selectivity for GSH over competing thiols. The sensor exhibited minimal EOT changes in response to nonthiolated amino acids and maintained performance in complex matrices such as human serum. The sensors maintain stable performance with no appreciable loss in detection capability following a four-week storage period at room temperature. Finally, the dithio-functionalized pSi platform was successfully employed to distinguish between healthy and oxidatively stressed cells in a label-free manner, underscoring its potential for real-time redox state monitoring in biological systems.