Ratiometric copper(II) indicators based on poly(n-isoproprylacrylamide) and fluorescence resonance energy transfer

An approach to ratiometric fluorescence detection of Cu(II) ions was devised by copolymerizing N-isopropylacrylamide with small percentages of bipyridine and amine monomers. The copolymer was divided into two portions. The amine group on one portion was functionalized with AlexaFluor555 (donor fluorophore) and the other with AlexaFluor647 (acceptor fluorophore). The indicator consists of a mixture of these two portions. Aggregation above the lower critical solution temperature (LCST) of this copolymer brings about a large increase in fluorescence resonance energy transfer (FRET). Addition of Cu(II) to the aggregated copolymer introduces charge onto the backbone, causing the copolymer to deaggregate with a resulting decrease in FRET. The ratio of acceptor to donor fluorescence varies with Cu(II) concentration. The intermolecular FRET coupled to copolymer aggregation results very large change on the fluorescence intensity ratio of acceptor to donor when Cu(II) binds to the indicator. A plot of intensity ratio vs. pCu is sigmoidal with a log Kf of 6.1 for the Cu(II)--bipyridine complex. The data are consistent with the formation of a 1 : 1 complex. The copolymer responds to higher concentrations of other transition metal ions. The selectivity for Cu(II) is consistent with the literature values for 1 : 1 formation constants for bipyridine with metal ions. Another approach to ratiometric detection of Cu(II) involves intramolecular polymer conformational change resulting from charge repulsion induced by binding Cu(II). Indicator used for this approach is a PNIPAM copolymer strand labeled with both donor and acceptor fluorophore. The copolymer remains random coil confirmation without the presence of Cu(II). The density of labeled AlaFluor dyes can be manipulated to establish a FRET between donor and acceptor fluorophores. The established FRET can be reversed when Cu(II) binds to the indicator because charge repulsion expands the copolymer strand and result a larger average distance between donor and acceptor. Therefore Cu(II) can be detected by measuring the fluorescence intensity ratio of acceptor to donor. The intramolecular indicator has been used to measure the ratio change in response to Cu(II) addition in complicated matrix, including calf serum, processed waste water and river water. The advantage of the intramolecular FRET strategy is that this approach requires very low indicator concentration since the mechanism does not involve polymer aggregation. Moreover, the measured can be realized at room temperature, instead of above the LCST. However, the ratio change in response to Cu(II) binding is much lower than the intermolecular FRET approach