Cu²⁺,Hg²⁺and Zn²⁺ Fluorescent Sensors Based On Interactions Of Metal Ions And Peptides

As modern science and technology and social industrialization continuously develop,certain industrial production processes have illegal heavy metal waste discharge,causing serious heavy metal pollution.Furthermore,metal ions have important physiological functions in living organisms.Thus,it is of vital importance to know how to conduct rapid and effective detection of heavy metal ions to related fields such as life sciences,environmental sciences,and medicine.Among the current detection methods,the fluorescence chemosensor has attracted much attention due to its advantages such as good selectivity,high sensitivity and fast response.And peptide have the advantages of mature synthetic methods,good water solubility,biocompatibility and low toxicity.Therefore,three peptide-based fluorescent sensors were synthesized by solid-state synthesis,and the sensing properties and applications of these three sensors to Cu²⁺,Hg²⁺and Zn²⁺were studied in detail.The main contents are as follows:1.The research background and design principle of peptide-based chemical sensors for heavy metal ions are briefly introduced,and the research progress of peptide-based chemical sensors in recent years is reviewed.The research purpose and significance of this topic are proposed.2.Fluorescent pentapeptides D-HTEHW-NH₂（D-P5）were synthesized by Fmoc solid-state peptide synthesis using Dansyl group as the fluorophores.The interaction between D-P5 and 15 metal ions was studied by using ultraviolet absorption spectrum,fluorescence spectrum and circular dichroism spectrum.The results showed that D-P5 had a strong binding ability to Cu²⁺,Hg²⁺and Zn²⁺.The binding ratios of D-P5 to Cu²⁺,Hg²⁺and Zn²⁺were calculated by Job’s plot to be 2:1,1:1 and 2:1,respectively.And the binding constants calculated by the Benesi-Hildebrand formula were 2.1×10⁵ M^-1/2,6.4×10⁵ M^-1,and 8.6×10⁴ M^-1/2,respectively.The fluorescence sensing properties of D-P5 on Cu²⁺,Hg²⁺and Zn²⁺and its influencing factors were studied.The detection limits were 38.0 n M,42.0n M and 60.0 n M,respectively,and they have been successfully used for the detection of Cu²⁺,Hg²⁺and Zn²⁺in actual water samples.3.Since the Pro-Gly structure can form a stableβ-corner secondary structure,and His is a good active coordination group,in this study,a Dansyl-based fluorescence chemical sensor D-HPGD-NH₂（D-P4）with Pro-Gly structure was synthesized,and the interaction between the tetrapeptides D-P4 and various metal ions was studied by fluorescence spectroscopy.The results showed that D-P4 had a strong specific binding ability to Hg²⁺.The addition of Hg²⁺causes the fluorescence of D-P4 to be quenched,and the color changes from yellow to colorless under a 365 nm ultraviolet lamp.The interaction between D-P4 and Hg²⁺was further studied by ultraviolet absorption spectrum,circular dichroism spectroscopy,X-ray photoelectron spectroscopy,and molecular docking.The binding ratio of D-P4 to Hg²⁺was calculated by Job’s plot to be 1:1.And the binding constants calculated by the Benesi-Hildebrand formula was 1.1×10⁵M^-1.The fluorescence sensing properties of D-P4 for Hg²⁺and its influencing factors were studied.The detection limit was 61.0 n M,and it can also be used to detect the concentration of Hg²⁺in actual water samples.In addition,we found that the D-P4-Hg system has a highly sensitive and selective fluorescence enhancement effect on biological sulfhydryl groups（GSH,Hcy and Cys）,and can be applied to the sensitive detection of Cys with a detection limit of 80.0n M.4.A fluorescence chemical sensor D-HQRTHW-NH₂（D-P6）was designed and synthesized based on zinc finger protein with high selectivity and sensitivity to Zn²⁺.Based on the previous work,this research deeply explored the interaction mode between Zn²⁺and D-P6.D-P6 has high selectivity for Zn²⁺based on the energy resonance transfer mechanism.The energy resonance transfer mechanism was discussed by fluorescence lifetime experiment,and the binding mode of D-P6 and Zn²⁺was studied by NMR and molecular docking.The binding ratio of D-P6 to Zn²⁺was calculated by Job’s plot to be 2:1.The dissociation constant was 9.0×10¹² M by nonlinear least squares fitting.The detection limit of D-P6 for Zn²⁺was 33.0 n M,and it was successfully applied to cell imaging using a confocal microscope.The results showed that D-P6 can penetrate living cells and can be used to detect Zn²⁺ in HeLa cells.