Ratiometric fluorescent metal ion indicators based on functionalized poly(N -isopropylacrylamide)

A novel type of ratiometric fluorescent indicator based on the phase transition of a well-known stimuli-responsive polymer poly(N-isopropylacrylamide) (PNIPAM) is presented. The sensing mechanism involves the polymer conformational change resulting from charge neutralization by the analyte metal ions. The ultimate goal is to develop ratiometric fluorescent indicators for free Cu(II) in environmental water analysis. The indicators are copolymers of N-isopropylacrylamide (NIPAM) with small percentages of fluorophores and ligand monomers. The charges on the ligands prevent PNIPAM from collapsing unless neutralized upon metal ion chelation. The polymer phase transition is transduced by either fluorescence solvatochromism or fluorescence resonance energy transfer (FRET), which forms the basis of two slightly different designs. The first design incorporates the dansyl moiety, a polarity-sensitive fluorophore which emits more strongly at a shorter wavelength when trapped in collapsed polymer chains. By separating an iminodiacetic acid (IDA)-based ligand units from the dansyl moieties, an enhanced fluorescence response was achieved with quenching metal ion Cu(II). A ratiometric readout was generated based on the fluorescence peak shift and intensity enhancement. The indicator showed a temperature-dependent response to Cu(II) but no Cu(II) selectivity against other metal ions. The log K of the indicator-Cu(II) complex at 35 °C was determined to be 4.3. Several FRET donor/acceptor pairs were attempted in the second design, which measures the average distance between the two as the chain conformation changes. Alexa Fluor 555 (donor) and 647 (acceptor) were reacted with the amine sites on the polymer either separately or simultaneously, yielding singly labeled (SL) or doubly labeled (DL) strands respectively. Through formulation optimization, both types of indicators showed satisfactory FRET intensity ratio response to Zn(II) as confirmed by the second-order scattering. Fluorescent response to Cu(II) was limited by a quenching mechanism postulated to be a cascade FRET process. The DL system also produced reasonable responses to other metal ions such as Ni(II), Hg(II) and Pb(II). It was found that the DL indicators have better sensitivity and shorter response time. Finally, the charge effect on the phase transition was studied briefly in an attempt to understand the phase transition mechanism