For Ru bpy dried samples the lifetime
For Ru(bpy)32+ dried samples, the lifetime was given by two components. These data confirmed that the fluorophore interactions modified the fluorescent properties; in fact, we observed a second emission peak with a shorter lifetime. The lifetime was measured for all the surfaces used to deposit the fluorophore: glass, Si and Al. The data, shown in Fig. 3, are also summarized in Table 1. In the table are reported three lifetime values to make easier the comparison. In fact, all the samples exhibit the τ2 component, typical of the suspended form. The strong difference observed is Ru(bpy)32+ deposited on both glass and Al, an insulator and a conductor surface, respectively, exhibit a shorter lifetime component.
In all measurements, the lifetime is always over 100ns, a promising feature for our applications.
All results here reported show that Ru(bpy)32+ has biochemical and optical properties useful for target gene labeling in miniaturized DNA-chip. In fact, the fluorophore emission peak is far away from the Asiaticoside one. It makes possible to avoid the self-quenching. Ru(bpy)32+ exhibits a quite long lifetime, which would allow us to use a simple electronic system to drive the source and detectors in an integrated DNA-chip. Moreover, it is photostable, as shown in Fig. 2 of Supplemental materials.
In literature, there are some evidences about spotted surface effects on photochemical properties of Ru(bpy)32+, in terms of structural sensitivity to O2 and “rigidochromism”  if fluorophore is encapsulated in polymers. However, the optical characterization we performed, clearly indicated a Ru(bpy)32+ sensitivity to its physical state and environment, independently from the surface. Its excitation/emission properties changes with the state transition from dissolved to dried form, probably due to molecule–molecule aggregations, rather than surface-molecule interaction. This hypothesis was verified by a careful study using TEM analysis.
We used TEM microscopy to observe the fluorophore molecules in dried form, in order to verify if the absorption/emission changes of Ru(bpy)32+ could be related to molecular cooperation. Like many carbon based molecules, even the Ru(bpy)32+ is very sensitive to the electron beam and very light to have a high contrast on the image. In order to identify and study the fluorophore dried steric conditions, we performed a phase contrast imaging with the HRTEM. The analysis clearly showed a thin crystalline layer coming from the negative contrast element (uranyl acetate) deposited on the TEM grid, as shown by the image in Fig. 4A. This layer surrounds regions having a different contrast. Chemical analysis through EDX spectroscopy (reported in Fig. 4B) confirmed that within these regions there was ruthenium, unlike in the rest of the grid.
These results allowed us to conclude that Ru(bpy)32+ in dried form may collapse in clusters but a certain percentage of single molecules is also visible. See, as an example, the impressive image (within the red circle) showing the crystallographic planes of uranyl acetate arranged around a region perfectly resembling the Ru(bpy)32+ features. Both the hexagonal geometry and the diameter (1nm) resemble the fluorophore single molecule. The presence of lattice fringes in correspondence of the region occupied by the Ru(bpy)32+, however, is a defocus artifacts that can arise from phase contrast imaging from the neighborhood uranium atoms. Wider regions surrounded by the acetate are also visible in Fig. 4A, suggesting that the molecules can agglomerate and may interact, probably causing the small changes of their fluorescent properties previously detected.
Conclusion Photochemical properties of metal transition complex tris(2,2′-bipyridyl)ruthenium(II), for optical sensing application, have been studied. The analysis showed that this molecule is a viable alternative to the conventional fluorophore CY5 for target gene labeling in optical DNA-chip. In fact, Ru(bpy)32+ excludes the risk of fluorescence self-absorption, thanks to the large distance between the absorption/emission peaks, and allows the use of simple electronics for the fluorescence analysis, thanks to the long lifetime. At the same time, optical studies revealed a dependence of both fluorophore properties, emission and lifetime, on the environmental conditions. Finally, Ru(bpy)32+ is photostable unlike Cy5.