The vital role of trace metals in cellular functions at biological, chemical and molecular levels is undeniable. The binding of metal ions to the bioavailable sites provides the structural framework and facilitates significant biological processes such as cell metabolism, hemoglobin synthesis and bone development in living organisms. In aquatic ecosystem, the accumulation of trace metals can occur naturally by weathering or as the result of human action of mining, use of pesticides and unmanaged disposal of industrial wastes into the water resources. These actions deteriorate the existing water quality and impose a greater threat to the existence of living organisms in such environment. Hence, these metal ions should be monitored regularly to maintain a tolerable water quality and a healthy ecosystem. The goal of our research is to develop a stable indicator platform to detect Cu(II) ion activity in water.

Our indicator platform comprises of a pNIPAm backbone, an anionic ligand as a binding site, donor fluorophore in the polymer chain and acceptor fluorophore encapsulated in a nanoparticle. The polymer chain was synthesized, characterized and then grafted to an acceptor impregnated nanoparticle. Cu(II) is introduced to the system, the metal-ligand complexation neutralizes the negative charge induced by the anion in polymer backbone and brings the fluorophores closer allowing an efficient energy transfer by Forster Resonance Energy Transfer (FRET). The process consists of two synthetic routes. The first synthesis follows the conventional Stober's method to produce dye-doped silica nanoparticles (SiNPs) in suitable size range. The acceptor fluorophore, Rhodamine isothiocyanate (RITC) is impregnated into silica nanoparticles to preserve the fluorescence of our indicator. These particles were characterized by highly-resolved SEM images and the size was averaged to be 45 nm as measured in those images. The core-shell structure and end-group functionalization were confirmed by FT-IR analysis. The second synthesis is Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization of a thermo-responsive, poly N-isopropylacrylamide (pNIPAm), copolymerized with a low mole percent (~2--4 %) of an anionic iminodiacetate ligand and fluorescein-o acrylate (FoA). The donor fluorophore (FoA) was added towards the end of synthesis to selectively locate it at the opposite end of the polymer chain. This was characterized by 1H NMR and end-group analysis was performed to determine polymer chain length and get average molecular weight estimation. These FNPs were then assembled together by graft to method to create a FRET-based indicator platform.

The optical measurement was done in Fluorescence spectrophotometer (FS) where the change in fluorescence intensity of acceptor and donor fluorophores was studied. The result revealed that even though the fluorescence increased gradually overtime, the intensity gap minimized and eventually closed as more time passed. Based on Cu-anIDA complexation, the range of detection for Cu(II) ion activity by this indicator was found in lower ppm range (0.331--6.61). The data collected for the indicator system using Dynamic Light Scattering (DLS) was in good agreement with FS data and supported the fact that fluorescence fluctuation was due to polymer entanglement which was untangling overtime. The A/D ratio plotted against time proved this ratiometric fluorescent indicator platform was stable to some extent and with thoughtful assemblage could be applied to measure metal ion activity under normal environmental condition.