The emission spectrum of the mg ml sample in cuvette
The emission spectrum of the 0.7mg/ml sample in cuvette is shown in Fig. 2 (black solid line). The curve perfectly mirrors the literature emission spectra, exhibiting a peak at about 630nm . Quite different is the case of the fluorophore dried form. The emission spectra are, for such form, quite different from literature data . The curve morphology changed and a new and dominant peak around 590nm appeared (red line in Fig. 2). In order to exclude any contribution given by the solid surface used for deposition, we spotted Ru(bpy)32+ on aluminum (Al), silicon (Si), and glass surfaces as well as on the grids used for TEM analysis (see experimental). The emission data are also shown in Fig. 2 (blue, green and orange line, respectively): they exhibit the same morphological alteration of the curve (the new dominant peak at 590nm) already observed for the dried sample on glass slide, only the relative height are different, but no conclusion can be drown from the PL intensity at room temperature. It should be underlined we used an insulator (glass), semiconductor (Si, to be sure that the surface was Si, a sample deep in HF was performed just before fluorophore deposition), metal (Al) and C coated grids as deposition surfaces. The goal was to determine if the surface electronic properties could modify the fluorophore emission properties. The data clearly show that the surface role is not the dominant effect ruling the Ru(bpy)32+ emission properties, at the deposition conditions used. We believe that the strong blue shift of the emission peak is due to a cooperative interaction of the molecules, as already observed in the orexin receptor antagonist measurements. In fact, the shift may be attributed to a HOMO (highest occupied molecular orbital) – LUMO (lowest unoccupied molecular orbital) distance shift. According to literature , such shift could be originated by a strong interaction with the substrate, as we observed analyzing dried Cy5 emission. Cy5 showed different peaks and emission curves depending of which type of surface (insulating or not) is used for spotting (see Fig. 1 in Supplemental materials). In Cy5 the stabilization of the HOMO orbital, which could cause the shift of the emission peak, is given by the insulating substrate presence. For dried Ru(bpy)32+, instead, we believe that the stabilizing interaction occurs not with the substrate but among the molecules themselves. Two main evidences allow us to support such conclusion. First, in Ref. , the substrate was powdered and mixed to the fluorophore in order to enhance the interaction. The full mix was dried. In our case the solution is just spotted on the substrate, hence, the interaction with the substrate is only due to the molecular layer at the interface and many layer are deposited on top of it. Second, in our case the emission blue shift occurs regardless of the substrate characteristics. The same peak occurs if the fluorophore is deposited on an insulator (glass), a semiconductor (Si) or a conductor (Al) surface. The only difference being the peak intensity. We believe in our experimental set-up, is the molecule-to-molecule interaction to dominate the emission properties, suggesting the molecules, if available in a suitable concentration, tend to interact, even clustering. The last optical characterization was the Ru(bpy)32+ lifetime using a SiPM detector. As already observed for the emission, the lifetime value changed depending on the fluorophore physical state (dissolved or dried), as shown in Fig. 3. The experimental data (points in figure) were fitted (dashed red lines) to obtain the lifetime (τ) values. To perform the analysis we used a multi-exponential as indicated by the following equation: The lifetime measured in solution (0.7mg/ml of fluorophore in Milli-Q water) was 358±0.9ns (see Fig. 3 black line and Table 1), according to literature , within the experimental errors. To validate SiPM and whole experimental system’s efficiency we measured also Cy5’s lifetime, which was 2.15±0.06ns as reported in other works  (see Table 1).