# Study of a Miniaturizable System for Optical Sensing Application to Human Cells
## 4 Discussion  
We investigated a strategy aimed for the implementation of a miniaturized intracellular optical sensing for human cells. The approach is based on the use of a cell labeling process, performed through the fluorophore Tris(2,2'-bipyridyl) ruthenium(II), in combination with a miniaturizable detection system suitable for portable optical sensing applications. Ru(bpy)\({}_{3}\)\({}^{2+}\) fluorophore for cell labeling brings a series of optimizations to both sample preparation and optical detection. Conventional dyes share some chemical-physical properties that are quite limiting towards a possible integration and miniaturization of the sensing system. Literature report some evidences of photobleaching and phototoxicity processes for some dyes stressed by prolonged laser exposures, so that dark condition and low energy laser beam are needed. Other evidences highlight, also, the risk of self-quenching for most of the conventional dyes, due to their absorption and emission peaks short gap and overlap, which imply a complex architecture and electronic management of the luminescence detection system. The use of Ru(bpy)\({}_{3}\)\({}^{2+}\) for cell labeling allows overpassing all these limitations, thanks to its structural and optical properties. The fluorophore, in fact, is robust and able to keep its structural and functional integrity under very unsustainable chemical-physical conditions [12], and exhibits long gap absorption/emission peaks, that avoid the risk of self-quenching, thus simplifying the detection  
Figure 10: LoVos fluorescence as a function of their number for 0.14 \(\upmu\)M Ru(bpy)\({}_{3}\)\({}^{2+}\) labeling across cell generations (600 cells correspond to 5 days recovery time). Fluorescence was acquired with a 488 nm light source and collected at 620–630 nm. technology. Moreover, the molecule is commercially available, thus reducing costs and time related to structural modifications, carriers functionalization and/or cell permeabilization. The characterization of the sensing strategy, in terms of labeling efficiency and detection sensitivity, was performed on HDFs, representative of the human cell model. FM and CLSM revealed that Ru(bpy)\({}_{3}\)\({}^{2+}\) passed through the HDFs membrane to reach the intracellular environment with a cytosolic localization. The specific uptake mechanism was not characterized, since there are some studies in the literature [17]. The optical characterization of labeled HDFs by the unconventional photoluminescence system (Figure 2) proved the suitability of our method for the integration in an optical biosensor device; the use of miniaturized photomultipliers, already employed in sensing applications [25; 26; 27] may result in the fabrication of a portable sensing system that could be used also within incubators. Results from optical testing reported a sensitive Ru(bpy)\({}_{3}\)\({}^{2+}\) fluorescence detection, highlighting a strong correlation with both fluorophore concentration and labeling duration. Finally, cell vitality was not affected by the labeling technique. In fact, MTT assays revealed that HDF cells treated with different fluorophore concentrations (0.14 uM to 1.4 uM) kept their vitality, if compared to controls. Once characterized and validated, we investigated the versatility of this strategy studying other cell models such as the human colon adenocarcinoma cells, LoVo. We performed the uptake of Ru(bpy)\({}_{3}\)\({}^{2+}\), as reported in Figure 8, and tested the intracellular fluorescence of LoVo cells and their vitality at 1, 2, and 5 days after uptake, emulating a fluorescent time-lapse analysis of tumor cell proliferation. Results reported in Figure 9 proved that the labeling method did not affect LoVo viability, since there were no divergences between control and treated samples, and the fluorescence signal was perfectly detectable across time and cell generations, thanks to the robustness of Ru(bpy)\({}_{3}\)\({}^{2+}\).