Construction,Characterization,and Bioimaging Of Genetically-encoded Fluorescent Probes For Heavy Metal Detection

With the sharp blossoming of industry and agriculture,heavy metal pollution incidents are common,which badly threatens the physical health of citizens.In particular,heavy metal ions headed by Hg²⁺,Cd²⁺,Pb²⁺,etc.are the most serious hazards to life.Currently,the detailed mechanism of the overall cytotoxicity of heavy metal ions Hg²⁺,Cd²⁺,and Pb²⁺stress has not been entirely explained.Therefore,it is very important to construct fluorescent probes with high sensitivity,high selectivity,and high spatial and temporal resolution to monitor toxic heavy metal ions in real time and in situ,and to interpret the chemical mechanism of toxicity and the regulation mechanism of cellular self-protection stress at the cellular level.According to the needs of the above research,based on the three-dimensional structure information of the Mer R family metal regulatory proteins and their complexes with target metal ions,this paper selects the residues in the key structural and functional regions of metal ion binding for splicing.The metal ions Hg²⁺and Cd²⁺maintain the original coordination configuration to bind the metal-binding peptides.Subsequently,the small peptide was embedded into the loop of the fluorescent protein barrel structure,aiming to utilize the excellent binding properties of the constructed metal-binding peptide to the target metal ion and the conformational change induced by binding to the target metal ion.The energy information of the conformational change is effectively converted into the visual change information of the fluorescence intensity of the coupled fluorescent protein,thereby realizing the efficient design of gene-encoded fluorescent probes with different sensing mechanisms.The specific research work and research results are as follows:（1）Based on the above methods and strategies,we designed a gene-encoded fluorescent probe SFGFP-MerBD for the detection of Hg²⁺.First,we constructed and optimized the fluorescent probe SFGFP-MerBD recombinant plasmid vector by molecular cloning technology,and characterized the expression of the probe protein by SDS-PAGE,which proved that the constructed fluorescent probe can be expressed in large quantities in bacteria.Second,a series of in vitro performance characterization experiments were performed on the purified protein.For fluorescent probe sensitivity and selectivity characterization experiments,First,through the fluorescence intensity changes of the designed probe and skeleton protein SFGFP in response to Hg²⁺,it was found that only the fluorescent probe inserted into the MerBD small peptide could respond to Hg²⁺,and the sensitivity of the detection range of Hg²⁺response was 1.26 n M～2500n M,and at a certain There is a linear relationship between the fluorescence quenching efficiency and the logarithm of Hg²⁺concentration.Second,through the competition experiment analysis of Hg²⁺with some main group metal ions and transition metal ions,it is found that the probe has strong specificity for Hg²⁺and can resist the interference of other metal ions.Thirdly,through the competitive UV titration experiment of fluorescent probes,the affinity constant of fluorescence quenching caused by Hg²⁺binding to the probes was fitted,and the Kd was2.00±0.08×10^-16.Characterization experiments for the mechanism of fluorescent probe response to Hg²⁺.First,through the mutation experiment of the metal ion binding site and the observation of the ultraviolet spectrum in the wavelength range of 230 nm to 290 nm,it is proved that the metal binding peptide MerBD and Hg²⁺on the fluorescent probe still maintain a special triangular coordination configuration.environment.Second,the comparative analysis of UV-Vis spectrophotometer and circular dichroism spectrum in the wavelength range of 340 nm～520 nm found that with the increase of Hg²⁺concentration in the protein solution,the chromogenesis of protonated and deprotonated fluorescent proteins changed.relative proportions of the group.And a significant increase in ellipticity was found in the CD spectral wavelength range.The above clearly shows that the Hg²⁺binding metal ion domain successfully caused the conformational change of the small peptide,and the conformational change was effectively transmitted to theβ-barrel structure of SFGFP,thereby realizing the high-sensitivity detection of Hg²⁺.Thirdly,it is proved by the high-sensitivity stable transient fluorescence spectrometer that the addition of Hg²⁺has little effect on the fluorescence lifetime,which belongs to the static quenching process.Finally,research experiments on intracellular imaging of fluorescent probes show that the designed fluorescent probes can respond dynamically,reversibly and rapidly to changes in Hg²⁺concentration in the cellular environment.（2）Combined with the experience in the design of mercury ion fluorescent probes,we designed a gene-encoded fluorescent probe SFGFP-CadBD for the detection of Cd²⁺.First,we used molecular cloning technology to construct the fluorescent probe SFGFP-CadBD recombinant plasmid vector,and characterized by SDS-PAGE to prove that the fluorescent probe can be expressed in large quantities in bacteria,and can effectively fold to produce green fluorescence at room temperature.Second,a series of in vitro performance characterization experiments were performed on the purified protein.First,the performance comparison between the designed probe and the backbone protein SFGFP in response to Cd²⁺was characterized by fluorescence-visible spectrophotometer,which fully proved that the designed probe’s response to Cd²⁺was mainly caused by the insertion of the small peptide CadBD.The concentration range of Cd²⁺was 0.1μM/L～24μM/L.Second,by mutating the binding site,it was found that the fluorescence intensity in response to Cd²⁺decreased to varying degrees after any single mutation.At the same time,after adding Cd²⁺,the corresponding spectral peaks in the wavelength range of200 nm to 280 nm in the ultraviolet spectrum were also raised.More precisely,the four binding sites are still involved in the coordination of the metal ion Cd²⁺.Third,when a high concentration of 20μM single metal was added,the designed probe could not only quench Cd²⁺,but also had certain responses to Cu²⁺,Fe³⁺,and Ni²⁺.It is speculated that the constructed metal-binding peptide lacks the synergistic effect of site II,resulting in a difference in the selectivity of fluorescent probes from the original Cad R.Therefore,there is still a lot of room for optimization to meet the specificity of the probe SFGFP-CadBD in response to metal ion Cd²⁺.Finally,fluorescent probes obtained through in vitro characterization screening were successfully imaged in HEK 293T cells and could indicate changes in Cd²⁺concentration present in the cells.According to the research resμLts of this paper,the design method described therein provides a feasible direction for better exploring the significance of metal ions in biology based on genetically encoded fluorescent probes.It also provides a design idea for the indication function of other metal ions,amino acids,organic substances,etc.in the organism.