Integrated paper|transition metal dichalcogenides colorimetric device for the nanozymatic-sensing of glutathione in saliva

Herein, a colorimetric paper-based device (PAD) for the direct glutathione (GSH) determination in saliva is proposed. The device sensing ability is based on the GSH-induced inhibition of the peroxidase (POD)-like activity of paper-integrated transition metal dichalcogenides 2D-nanoflakes (TMDs). TMDs (i.e., MoS2, WS2, MoSe2, and WSe2) were nanostructured via sonochemical exfoliation in water using sodium cholate as stabilizing agent, and for the first time, their POD-like activity was comprehensively studied on paper towards TMB (3,3',5,5'-tetramethylbenzidine) oxidation. The nanozymatic activity of the four TMDs was carefully tested on six different papers under varying TMD loading conditions. MoS2 resulted in the most performing 2D-TMD in combination with the Whatman 602H paper. The MoS2-PAD was thus used to build up a smartphone-based colorimetric strategy that relies on the GSH-mediated inhibition of catalytic TMB oxidation, resulting in a blue color switch-off. The MoS2-PAD, shown in Figure 1, was conceived to host a 3-point 'control calibration' and sample analysis in triplicate. Its manufacturing relies on the printing of hydrophobic barriers on paper to define the sensing spots, acting also as MoS2 and TMB reservoirs; this was performed using a simple portable wax printer. The MoS2-PAD smartphone-based colorimetric assay consists of two straightforward steps, i.e., (i) saliva and H2O2 loading, (ii) smartphone readout after 10 min. The GSH matrix matched calibration returned a linear range enclosed between 0.5 and 10 μM (R² = 0.991) with a LOD of 0.14 μM, showing a useful inter-batch reproducibility (RSD ≤ 4%, n = 3). The device reliability was demonstrated through GSH determination in 17 real saliva samples, obtaining accurate (recovery: 81-117%) and reproducible data (RSD ≤ 16%, n=3), correlated with the Ellman-reference method (r = 0.972). Summing up, the proposed PAD enables GSH determination at physiological relevant levels, requiring only 3 μL of sample, representing a step beyond toward point-of-care device based on nanozymatic matter.