Development of ratiometric fluorescent Cu(II) indictors based on poly (N-isopropylacrylamide) (PNIPAM) copolymers

This dissertation describes development of ratiometric fluorescent Cu(II) indicators based on poly(N-isopropylacrylamide) (PNIPAM) copolymers. The sensing mechanism involves the phase transition and conformational change caused by the change in charge on the copolymer when it binds Cu(II). The ultimate goal is to develop ratiometric fluorescent indicators for Cu(II) ion activity measurements in water.;Two indicator designs are described: 1) copolymers of NIPAM with small percentages of a Cu(II) ligand monomer, and a pair of fluorophores dissolved in water; 2) copolymers of NIPAM with small percentages of a Cu(II) ligand monomer and a pair of fluorophores conjugated to nonporous silica nanoparticles and suspended in water. When charges on Cu(II) are introduced onto the polymer backbone by binding the ligand monomer, the polymer phase transition affects the extent of fluorescence resonance energy transfer (FRET) between the pair of fluorophores. The FRET efficiency can then be correlated to Cu(II) ion activity.;Different polymerization methods, FRET donor/acceptor pairs and ligand monomers were attempted in this research. Emulsion polymerization was applied to synthesize the type 1 indicator and Reversible Addition-Fragmentation Chain-Transfer polymerization (RAFT) was utilized to synthesize the type 2 indicator. N,N' -bis(pyridine-2-ylmethyl)prop-2-en-1-amine (DPA) and N-(4' -methyl -2,2'-bipyridin-4-yl)-N-propyl acrylamide (BP) were used as the Cu(II) ligand monomers for type 1 and type 2 indicators respectively. One fluorophore pair, Fluorescein O-acrylate/Methacryloxyethyl thiocarbonyl Rhodamine 8 (F/R) could be copolymerized directly on the polymer backbone and another fluorophore pair Alexa Fluor 555/Aiexa Fluor 647 (A/A) could be labeled by reacting with primary amine sites, which could be introduced by adding another monomer, 2-aminopropylmethacrylate hydrochloride(APMA) on the polymer backbone. 8oth pairs were labeled on type 1 indicator and the detection limit was 10-7 M Cu(II). The type 2 indicator was labeled by F/R pair only and the detection limit was 10-6 M. It was found that for the type 2 indicator, response to Cu(II) increased when the copolymer chain density was higher. The response mechanism of copolymer conjugated to silica nanoparticles appears to involve increased chain-chain repulsion when Cu(II) binds to ligand on the polymer.;In summary, novel ratiometric fluorescent Cu(II) indicators have been developed. This strategy may be extended to other metal ions.