# Study of a Miniaturizable System for Optical Sensing Application to Human Cells
## 1 Introduction
Its optical properties would allow for the solving of some issues, related to the use of the conventional fluorophores described before. Ru(bpy)\({}_{3}\)\({}^{2+}\) has two absorption peaks at 290 nm and 450 nm, ligand-center (LC) and metal-ligand (MLCT) electronic transitions, respectively, and a lifetime  
Figure 1: Structure of Ru(bpy)\({}_{3}\)\({}^{2+}\). of 360 ns suspended in water. Absorption peaks are far away from the emission one at 630 nm [15; 16] and 100 nm to the closest absorption peak, thus strongly reducing the self-quenching of fluorescence since the absorption and emission curves do not overlap. Therefore, the incident radiation can be shielded using a simple and cheap band-pass filter. Moreover, the molecular properties do not change even in stressful environmental conditions [12]. We already proved the Ru(bpy)\({}_{3}\)\({}^{2+}\) stability by monitoring its absorption, emission and lifetime under very unsustainable chemical-physical conditions [12]. More precisely, we tested fluorophore in a variable range of temperatures, from RT to 100 \({}^{\circ}\)C; pH, from 1 to 11; light irradiation, up to 24 hours. All of the experiments that were performed indicated that Ru(bpy)\({}_{3}\)\({}^{2+}\) could be used in a wide range of conditions, therein including the environmental light. Most of the evidences of Ru-based complexes for intracellular labeling report examples other than the commercial Ru(bpy)\({}_{3}\)\({}^{2+}\), as a result of long and expensive chemical functionalization [17; 18; 19; 20] including, sometimes, carrier mediations [21]. As an example, in [17] uptake in cells was performed using a Ru(bpy)\({}_{2}\)dpp2\({}^{2+}\) molecule modified by changing bypiridine with 4,7-diphenyl-1, 10-phenanthroline ligand in order to make the fluorophore more hydrophobic for uptake, but strongly increasing the costs and dye preparation time. Moreover, characterizations of cell uptake processes of modified Ru-based complexes in human cells revealed that the bigger the aromatic surface of the N-N ligand in the [Ru(bpy)\({}_{2}\) (N-N)]\({}^{2+}\) starting molecule the higher the cytotoxicity of the final compound was, thus affecting the cell viability [22]. For these reasons, we introduced a different approach to the intracellular analysis of human cells, based on the Ru(bpy)\({}_{3}\)\({}^{2+}\) performances and stability and its suitability for the development of an innovative intracellular optical sensing system. We performed a full characterization of the labeling and sensing approaches by introducing the fluorophore Ru(bpy)\({}_{3}\)\({}^{2+}\) in human dermal fibroblasts (HDF) and, subsequently, detected its fluorescence by a sensitive and miniaturizable laser beam-photomultiplier based system. HDFs uptake of Ru(bpy)\({}_{3}\)\({}^{2+}\) was confirmed by Fluorescence Microscopy (FM) and Confocal Laser Scanning Microscopy (CLSM). Cells survival to the labeling method was checked by MTT assays of cells exposed to the fluorophore. A further investigation of cell sensitivity to Ru(bpy)\({}_{3}\)\({}^{2+}\) was performed on a human tumor cell model, performing the fluorophore uptake within the human colon adenocarcinoma (LoVo) cells and monitoring their vitality and fluorescence intensity across time.