**5. Conclusions**

A fluorescent metal ion indicator based on cross-linked pNIPAM nanoparticles was synthesized by copolymerizing fluorescein, ligand phenyl-IDA and NIPAM and cross-linker. The negative charges on the ligand make the nanoparticle swell. When Cu(II) ions are added to the system, they bind to the ligand and neutralize the negative charges, decreasing the swelling extent. The shrinkage of the particles leads to a shorter distance between adjacent fluoresceins, thus increasing self-quenching. The fluorescence intensity decreases with increasing Cu(II) concentration. Embedding the nanoparticles in the PA gel causes a larger change in the quenching due to shrinkage.

This indicator platform has several advantages over other platforms: (a) This indicator responds to Cu(II), which normally quenches fluorescence. The separation of ligand and fluorophores makes the binding site of Cu(II) separate from the fluorophore, thus decreasing the paramagnetic quenching effect on fluorescence. (b) The sensitivity and selectivity of the indicator can be modified by utilizing different ligands without changing the excitation wavelength. Theoretically, the indicator platform responds to all metal ions with appropriate ligands. (c) It was reported that a cross-linked structure improves the thermal stability of polymers [32], and in this indicator platform, it also helps to solve the problem of the stability of free-floating or end-grafted polymer chains, since the untangling of the polymer chains is not an issue. The nanoparticles are also easy to purify and recycle by centrifugation. (d) The self-quenching pNIPAM nanoparticles were embedded in a PA gel in order to prevent possible particle aggregation and increase the signal change.

The ultimate purpose of the indicator is to measure bioavailable metal ions in the environment. Future work may involve the application of a reference fluorophore in the gel to obtain ratiometric measurements, which can reduce error due to instrumental drift and simplify calibration. Meanwhile, we also plan to try an array with a donor fluorophore on one end of the pNIPAM chain and an acceptor fluorophore on the other end, to ge<sup>t</sup> a much larger signal change. The sensitivity can also be improved by using fluorophores with high quantum yield, and the limit of detection can be improved with high Cu(II) affinity ligands, which enables accurate readouts with low indicator concentration. This is beneficial to environmental monitoring since the equilibria in the environment would not be disturbed by the indicator. Moreover, the indicator is expected to selectively respond to target metal ions without the interference of other metals when metal-selective ligand systems are incorporated into the polymer.

**Author Contributions:** F.W. performed most of the experiments described in this manuscript. The manuscript was largely written by W.R.S. with input from both coauthors. R.P.P. consulted on the research and developed the ligand synthesis.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors thank Tianyu Ren for measuring the temperature coefficient for fluorescein in buffer and for confirming that 0.001 M Cu(II) does not quench the fluorescence of fluorescein in pH 6 buffer.

**Conflicts of Interest:** The authors declare no conflict of interest.
