# Study of a Miniaturizable System for Optical Sensing Application to Human Cells
## 1 Introduction  
Optical sensing applied to the human intracellular environment represents the best way for the analysis of cell functioning and its behavior in response to specific physiological conditions. Cell ultrastructure, embryogenic development [1], pH-calcium-chloride-oxygen monitoring [2] and, most importantly, metastasis proliferation [3] are a few examples of what this type of optical sensors would help to analyze. Intracellular optical sensing works by the introduction of a luminescent (fluorescent or chemiluminescent) probe inside the target cell and its subsequent detection. The configuration of an optical sensing strategy, generally, starts with the fluorophore choice (considering its optical and biochemical properties) and its characterization, mainly consisting oftesting cells survival after treatment and photostability. Once defined, the fluorescent dye must be transferred through the membrane of the investigated cell model by an uptake procedure that often requires fluorophore structural modifications, carriers and cell permeabilization [4; 5; 6]. Finally, a high performances optical sensing system for the fluorescence detection must be chosen. Nowadays, research efforts are moving towards the miniaturization, time/cost-saving and user-friendly sensing detection systems, in order to provide the basis for portable Point-of-Care platforms and patient self-monitoring. In this sense, one of the most representative examples is the use of photomultiplier-based systems having the greatest advantage, with respect to traditional detection instruments, of being highly miniaturizable, integrable and cheap hence, perfectly matching with the goal of a portable application, by using solid state photomultipliers (e.g. silicon photomultipliers, SiPM). However, this type of technology coupled with traditionally employed dyes, usually, implies the issue of the electronic management for luminescence analysis, which is quite sophisticated due to the chemical-physical properties of the luminescent probe used. Prolonged exposure times and high irradiation powers, affect conventional dyes emission efficiency, resulting in a bleaching process that reduces the fluorescence intensity and, sometimes, brings a series of toxic intracellular effects such as free radicals production [7]. Moreover, room temperature storage degrades the organic dyes normally employed for cells labeling [8; 9]. Cyanine dyes, for example, suffer from self-quenching of fluorescence [10], caused by the proximity and overlap of absorption and emission's peaks, and is photobleached after prolonged exposure to the laser beam [11; 12], hence dark conditions are required during the labeling experiment, which is problematic for fluorescence sensing, especially in time-lapse applications. Moreover, its short lifetime implies a complex electronic management of the fluorescence detection, thus increasing the complexity of sensing technology. Other evidences report a Rhodamine 6G, Fluorescein, Pyrene and Indo-1 bleaching process caused by an increase in the molecules intrinsic temperature, following the light absorption and the source irradiation power [13]. There are, also, some examples of luminescent quantum dots (nanocrystals composed by heavy metal atoms like Cd, Hg, Pb and As) causing cytotoxicity after the cell uptake, due to the release of metallic ions that can bind the sulfhydryl groups of many biomolecules, such as proteins, leading to the decrease in various sub-cellular organelles activity [14]. In order to surpass all of the limitations related to conventional optical intracellular sensing methods, we investigated an approach that used a miniaturizable photomultiplier-based system in combination with a cell labeling method performed by the use of fluorophore Tris(2,2'bipyridyl)ruthenium(II) (Ru(bpy)\({}_{3}\)\({}^{2+}\)), whose structure is schematically shown in Figure 1. This molecule is an octahedral coordination complex of transition metal ruthenium bounded to three heteroaromatic bypiridine units.