Construction And Fluorescence Imaging Of Heavy Metal Ion-targeting Peptide Probes

The extensive use of heavy metal ions in industry,agriculture,medical and other industries has caused serious environmental pollution.Heavy metal pollution,characterized by toxicity,persistence,and non-biodegradability,has an increasingly negative impact on ecosystems and human health.Studies have found that the deposition of Gd³⁺and La³⁺in the body can cause damage to the liver and nervous system,and the abnormal content of Cu²⁺in the body is closely related to neurodegenerative diseases.Therefore,there is an urgent need to establish efficient heavy metal ion detection methods for environmental monitoring,disease diagnosis and other fields.Peptide probes are widely used in the detection of metal ions,proteins,and nucleic acids,due to their remarkable advantages such as simple synthesis,good water solubility,and easy modification.In this thesis,we intend to construct novel peptide probes for specific detection of Gd³⁺,La³⁺and Cu²⁺,and apply them to fluorescence imaging in cells and organisms.The main contents are as follows:（1）A novel TPE（tetraphenylethylene）-modified peptide probe F1（TPE-Glu-Glu-Glu-Pro-Gly-Glu-Glu-Glu）was constructed,which targets Gd³⁺in a"Turn-On"mode.In the presence of Gd³⁺,the fluorescence of the probe F1 was significantly enhanced due to aggregation-induced emission.The probe F1 specifically binds to Gd³⁺in a 1:3ratio without interference from other common metal ions.The binding constant was2.34×10¹⁵（mol/L）^-3,which indicated that the binding stability of F1-Gd³⁺was good.The probe F1 can detect Gd³⁺with high sensitivity in buffer solution and saliva,and the detection limit reaches 39 nmol/L and 53 nmol/L,respectively.The probe has good water solubility and biocompatibility,and has been successfully applied to the imaging of Gd³⁺in cells and zebrafish,and has wide application potential in biological imaging and disease diagnosis.（2）Two TPE-modified peptide probes F2（TPE-Glu-Glu-Pro-Gly-Glu-Glu）and F3（TPE-Glu-Pro-Gly-Glu）were designed and synthesized.Responsiveness experiments found that in the presence of La³⁺,both probes F2 and F3 would produce aggregation-induced emission,resulting in a significant increase in fluorescence;at the same time,the number of glutamic acid in the peptide probe significantly affected the ability of the probe to target and recognize La³⁺,and the probe F2 showed better specific binding ability to La³⁺.The titration experiments and Job’s working curve showed that the binding ratio of probe F2 to La³⁺was 1:2;the binding stability of F2-La³⁺was better,and the binding constant was 1.29×10¹⁰（mol/L）^-2.Computer simulation results show that La³⁺specifically binds to the amide bond in glutamic acid and the oxygen of hydroxyl in the carboxyl group of the peptide probe.The sensitivity of probe F2 to detect La³⁺in buffer solution and saliva was better,and the detection limit reached 120 nmol/L and 320 nmol/L,respectively.The probe has low cytotoxicity and has been successfully applied to the imaging of La³⁺in cells and zebrafish,and has good application prospects in the detection of medical and environmental samples.（3）The Dansyl-modified peptide probe F4（Dansyl-Gly-Ser-Ser-Gly）was constructed.The probe F4 can target and recognize Cu²⁺and S^2-in the"On-Off-On"mode.The fluorescence of the probe F4 was significantly quenched in the presence of Cu²⁺,indicating that the probe F4 could respond to Cu²⁺in the"Turn-Off"mode.The probe F4 specifically binds to Cu²⁺in a 1:1 ratio without interference from other common metal ions.The binding constant was 8.60×10~4（mol/L）^-1.The probe F4 can detect Cu²⁺with high sensitivity in buffer solution,and the detection limit reaches 114nmol/L.When S^2-was added to the complex of F4-Cu,the fluorescence intensity would be significantly enhanced,indicating that the probe F4-Cu could respond to S^2-in the"Turn-On"mode,and the detection process was not disturbed by other common anions.The sensitivity of probe F4-Cu to detect S^2-in buffer solution was better,and the detection limit reached 357 nmol/L.The probe has good biocompatibility and was successfully applied to the imaging of Cu²⁺,S^2-in cells and zebrafish.The novel peptide probe provides a new tool for the specific detection of Cu²⁺and S^2-,and has good application potential in disease diagnosis and environmental protection.