Research On Novel Rhodamine-based Spectral Probes For Hg²⁺ With Excellent Performance

Mercury is one of the most toxic heavy metals and Hg²⁺ is the most common species of mercury pollutants. Thus, monitoring Hg²⁺ rapidly and sensitively is of great importance to the environmental and life science. Spectral probes are maneuverable, highly sensitive and suitable for in-situ and real-time monitoring without any destruction, so they are attracted much attention in variety of detection methods for Hg²⁺. Rhodamine moiety becomes one of the most popular chromophores and fluorophores for spectral probes due to its long absorption and emission wavelengths, high fluorescence quantum yield and easily modified structure. Although many rhodamine-based spectral probes have been reported, there are still some problems in terms of raw materials, preparation and performance of the probes, and the sensing mechanism has yet to be studied further.Therefore, in this paper four new rhodamine-based spectral Hg²⁺ probes with excellent properties were developed, their structures were fully characterized, and their sensing mechanism were explored. The detailed contents include:（1） Two novel fluorescent probes（RTTU and RPTU） for Hg²⁺ were synthesized by rhodamine B as reporter, triethylenetetramine（or tetraethylenepentamine） as linker and phenyl isothiocyanate as receptor. The optimal detection media for RTTU and RPTU were CH3CN/HEPES buffer solution（pH = 7.2） 9/1 and 10/1（v/v） respectively. They responded to Hg²⁺ by 20-fold and 15-fold fluorescence enhancement, their Hg²⁺ linear concentration ranges were 25–200 μM and 30–210 μM, and their detection limits were 3.04×10-7 and2.12×10-7 mol/L, respectively. RTTU and RPTU showed high interference immunity, could work in a physiological pH span and could be applied in real sample assay successfully.1:3 RTTU-Hg²⁺ and 1:4 RPTU-Hg²⁺ complexes formed with the binding constants of9.88×1011 M-3 and 1.00×1016 M-4, respectively. The sensing mechanism of RTTU for Hg²⁺was that RTTU bound Hg²⁺ to S atoms, followed by HgS and some other fragments left irreversibly to produce some new structures. Our sensing mechanism is very different from those described in the literatures and it was basically confirmed by LC-MS and IR analysis.（2） A new colorimetric and fluorescent probe（RATU） for Hg²⁺ was synthesized from rhodamine B, tris（2-aminoethyl）amine and phenyl isothiocyanate. In CH3CN/H2O（1/99,v/v）, Hg²⁺ aroused an emerging absorption peak at 565 nm with an obvious color change from colorless to red and a 32-fold fluorescence enhancement at 586 nm for the RATU solution. The absorbance and fluorescence maxima increased linearly with the concentration of Hg²⁺ in the range of 0–90 μM with the detection limit of 6.36 ×10-6 mol/L and 6.08×10-8 mol/L, respectively. RATU exhibited low cytotoxicity and highly interference immunity. RATU could work in a physiological pH span and could be employed to monitor Hg²⁺ in environmental water samples and living cells. Moreover,RATU could be supported in cellulose discs for detecting Hg²⁺ in 100% aqueous solution.A 1:2 RATU-Hg²⁺ complex formed with a binding constant of 2.89×108 M-2. The response mechanism of RATU to Hg²⁺ was similar to that of RTTU, and here it was supported by LC-MS, IR and 1H NMR analysis results further.（3） A new structurally simple colorimetric and fluorescent probe for Hg²⁺ was obtained by reacting rhodamine B with thiobisethylamine. Upon addition of Hg²⁺ in RMTE CH3CN/HEPES buffer（1/99, v/v, pH=7.05）, an emerging absorption peak at 561 nm with a clear color change from colorless to red and a 170-fold fluorescence enhancement at 578 nm occurred. In the scope of 0–120 μM of Hg²⁺, the absorbance and fluorescence intensity of the RMTE solution kept good linear relationship with the Hg²⁺ concentration. The colorimetric and fluorescent detection limits were 2.08×10-6 mol/L and 1.44×10-7 mol/L respectively. Just like RATU, RMTE also displayed low cytotoxicity and highly interference immunity. It could also work in a physiological pH span and successfully monitor Hg²⁺ in water samples and living cells, and also could be supported in cellulose discs for detecting Hg²⁺ in 100% aqueous solution. But different from RATU, the bindingmode of RMTE toward Hg²⁺ is 1:1 with a binding constant of 5.39 ′ 103 M-1. RMTE chelated Hg²⁺ reversibly through N, O and S atoms. This mechanism was verified by LC-MS and 1H NMR analysis results.