Study On Detection And Dynamic Imaging Of Toxin Protein Based On Atomic Force Microscope

Background: The progress and development of life science cannot be separated from the development of imaging instruments and the breakthrough of imaging technology.How to detect and analyze the various changes of biological macromolecules by imaging technology,extract molecular information from them,and quantify these biological macromolecules qualitatively and quantitatively has important scientific significance and application value.Through AFM,the hyperfine structure and various physical and chemical properties of biological macromolecules have been visually demonstrated in mechanics,which greatly improves our understanding of protein structure and its role in biochemical reactions,and provides a new technical mean to solve the biochemical problems of biological macromolecular function and physiological behavior at the molecular level.Objective: To establish a new method for the research of biological macromolecular detection and biological macromolecular interaction using multimodal AFM.First,develop a label-free screening method for ultra-trace biotoxins.Second,provide a new platform for in situ dynamic quantification of biological effects of toxins under near physiological conditions.Third,provide a basis for further exploring toxin receptors and mechanisms by designing toxin-protein conjugates and self-assembly of DNA origami at room temperature for programmable and controllable research on toxin polymerization.Methods: 1.For the single molecule detection,ABR,Bo NT/A,RT,ETX and SEB were selected as the models,which were extracted and purified separately.Those biotoxins were further fixed on surface of APTES-mica.The optimized conditions were used to deposit the toxin protein on the mica in the form of single molecule particles.Firstly,AFM and Pi FM were used to image the toxin single-molecule particles for morphological and Pi F feedback,and to quantify the salt components and proteins qualitatively in the substrate and buffer.Secondly,the Pi F spectrum of each toxin protein particle was collected.Referring to the absorption peak of the amide band in the Fourier transform infrared absorption spectrum,10 characteristic light-induced absorption bands related to the secondary structure of the protein were screened.The peak positions and intensity of these signal bands were analyzed by Principal Component Analysis(PCA).A single molecule Pi Fspectrum database for each toxin protein was constructed based on the results of PCA.Finally,the three toxin proteins were mixed to make a simulated sample,and the simulated samples were characterized by Pi FM.The Pif spectra of the simulated samples were collected in a certain area and introduced into the previously constructed toxin PCA model to finally identify the toxin protein contained therein ingredient.2.ETX,which has a pore-forming effect on human red blood cells,is used as a research model for dynamic imaging studies.A single layer of human red blood cell outer membrane and intima was constructed by hypotonic buffer rinsing.The interaction between ETX and the inner and outer membranes was observed in situ under near physiological conditions and the characteristics of toxins at different temperatures and different membrane regions were characterized by using an environmentally controllable AFM platform.The mechanical differences between intact erythrocyte membrane and pore-formed erythrocyte membrane were characterized by AFM probe.The distribution of ETX receptors on the surface of erythrocyte membranes was analyzed by confocal reaction in combination with recombinant red fluorescent m Scar ETX.3.The recombinant expression method was used to design the c-linker-ETX which containing a cysteine residue at the N-terminus of the protein,and the corresponding prokaryotic expression vector was constructed to express the soluble protein and then purified.The c-linker-ETX forms a 1-on-1 protein-DNA conjugates with ss DNA by "Michael addition" reaction and designated as DNA-c-link-ETX.Then,to judge whether the DNA-c-link-ETX and r ETX have differences in hemolysis and structure by hemolysis test and CD experiment.The structure of rectangular origami was observed by in situ AFM at room temperature,and the efficiency of origami synthesis was discussed to lay the foundation for the study of controllable assembly of ETX.Results: 1.A label-free method for the detection of ultra-trace biotoxins was established.After surface modification and protein concentration optimization,samples of molecular surface monomolecular toxin particles were prepared using 10 n M protein solution.AFM characterization and particle analysis were consistent with the expected structure and molecular weight of the reference protein: Bo NT/A represents large molecular proteins,ABR and RT represent medium-sized molecular proteins,and ETX and SEB represent small molecular proteins,respectively.Under the air conditions,Bo NT/A has a particle size of about 10 nm,ABR and RT are about 5 nm,and ETX and SEB are about 2.5 nm.Each toxin sample was then characterized by single-wavelength Pi FM,and the protein particle distribution on the substrate was qualitatively distinguished by the inversion of the mica substrate Pi F signal.Next,the Pi F spectra of each toxin in the imaging region were collected and clustered by PCA well.ABR,RT and ETX were used as examples to prepare mixed simulation samples.The signal ratios based on the collection of Pi F spectrum and PCA of ABR: RT: ETX in the simulated sample were 9:35:55,which were located between the real value(11:38:131)and the theoretical value(2:3:5).Compared with the Fourier transform infrared spectrum,the Pi F spectrum has higher resolution in the amide band and can directly display a signal band representing the secondary structure component of the protein.By comparing the Pi FM images of each toxin,it was found that the Pi F signal feedback of ETX and SEB was much smaller than ABR,RT and Bo NT/A,indicating that the detection system is insufficient for the protein with molecular weight less than 30 k Da.However,it can be improved by replacing the mica substrate with the Au substrate.2.In situ imaging of pore-forming effect of ETX was studied.WB test and SEM image suggest that ETX forms a heptamer complex to interact with human erythrocyte membrane,forming pores on the surface of erythrocyte membrane and destroying membrane integrity.High-resolution AFM observed the morphological changes of human erythrocyte membrane after ETX incubation under near physiological conditions.The imaging of temperature controlled AFM found that 6 ?g/m L r ETX has no pore-forming effect on erythrocyte membrane when the temperature was less or equal to 23 °C.Followed by temperature increases,ETX initiates a pore-forming effect,and as the temperature drops to 23 °C,again,the pore-forming effect stops.In addition,it was observed that the membrane integrity decreased with time under the action of r ETX.The AFM results showed that the thickness of the erythrocyte membrane phospholipid layer remained unchanged during this process,demonstrating that the r ETX pore-forming effect is through disrupting integrity of phospholipid bimolecular not digestion of the phospholipid bilayer.Further experiments demonstrated that ETX can also achieve specific binding to membranes and biological effects on a single layer of erythrocyte membrane without ATP activity through preparing a partial monolayer covered ?bubble? erythrocyte membrane model.3.Preliminary study on the controllable self-assembly system of biotoxin monomer based on DNA Origami.To further verify whether the pore formation of r ETX has a heptameric structure dependence,a site-specific DNA-c-linker-ETX conjugated protein was constructed,and found that this conjugated strategy does not affect the hemolytic activity of ETX.The kinetic characteristics were performed to obtain a better understanding on the kinetics of "seam" structure.The result shows that the "seam" structure guides the correct folding of the short chain and could be growing into a complete rectangle with high yield(> 90%).Conclusion: This study focused on the exploration of multimodal AFM in biological detection and biological effect mechanism research and nanostructure assembly.Firstly,a study on the application of Pi FM to the single-molecule unlabeled trace detection method of biotoxin was carried out.And a database include ABR,Bo NT/A,RT,ETX and SEB were constructed via their own Pi F spectrum.Then,high-resolution AFM was performed to characterize pore-forming effect of ETX on erythrocyte with in situ and under near physiological condition.The results showed that ETX did not produce a pore-forming effect on the inner erythrocyte membrane during this process.In addition,ETX can also bind to the membrane on a single layer of erythrocyte membrane without depending on the live cell exists.Moreover,high resolution imaging of porous erythrocyte membranes does not provide direct evidence that ETX heptamer complex is the basis of its structure.The construction of DNA-c-linker-ETX conjugates provides a powerful tool for further quantitative and controlled study of the mechanism of ETX hemolysis and the role of heptamer in the pore-forming process.The "seam" structure effectively guides DNA origami self-assembly at room temperature and the rate of correctly fold is related to the amount of "seams".all of which has been theoretically constructing a solid basis on which the research how bio-supramolecules are structured anisotropically on origami along with its functional verification in situ can be conducted.