Research On The Method And Mechanism Of Cell Membrane Penetration Based On AFM Indentation Technique

The traditional cytogenetics research is based on the statistical analysis of population cells,which ignores the effects of heterogeneity between single cells.In fact,there are genetic differences between two cells from the same area due to pathological changes.Therefore,the study of gene function at the single cell level is of great significance for the early prevention and diagnosis of diseases.With the development of Atomic Force Microscope(AFM),AFM is not only used to characterizing the morphology of nanomaterials,but also gradually used in the field of micro-nano manipulation.Since AFM's precise control of force and displacement,it has been a potential tool for non-invasive molecular transportation in the field of microinjection.However,according to the research,even if the commercial AFM probe has a tip with a nanometer radius of curvature,the probability of piercing into the cell to creat a hole in the cell membrane is very low due to the complexity of cell structure,which will seriously affect the application of AFM in the field of cell manipulation technology.Improving the penetration probability of AFM probe into cell membrane is a key step and prerequisite for the realization of AFM cell transfection technology.The key to the formation of cell membrane holes is to improve the tension of the cell membrane,but there is a lack of understanding of the tension changing caused by the penetration of the AFM probe into the cell membrane,so this paper will focus on the theme of increasing the tension of the cell membrane to improve the penetration probability of the AFM probe into the cell membrane.In this paper,the perforation of a single cell was realized by AFM indentation mode,and the morphology of cell membrane indentation was characterized in contact mode,and then it was found that the penetration probability of traditional AFM pyramid probe to cell membrane was low under fuzzy localization.Then,the methods of increasing the initial tension of cells and the external tension caused by the downward pressure of AFM probe were proposed to improve the penetration probability of AFM probe into the cell membrane,and the stress change of the probe in the process of pressing into the cell membrane was studied to explain though the finite element analysis method.Finally,the dynamic process of cell membrane pore formation and development under mechanical strain was visualized by molecular dynamics simulation.The main contents and innovative achievements are summarized as follows:(1)Study on the penetration phenomenon and efficiency of cell membrane by traditional AFM pyramid probeThe precise regional indentation experiment of mouse fibroblasts was carried out by AFM pyramid probe and we compared the morphological changes of cell membrane before and after indentation.The pores of cell membrane after indentation were characterized.It was found that the penetration behavior of cell membrane was related to the region of cell membrane.In order to explain the difference of indentation results of AFM probe in different regions,the distribution of actin microfilaments,one of the main components of cytoskeleton,was analyzed by immunofluorescence experiment,and verified that cytoskeleton played a leading role in cell mechanical properties.And it is found that the penetration rate of AFM pyramid probe to cell membrane is low under fuzzy location.(2)Increasing the penetration probability of AFM pyramid probe into cell membrane by increasing the initial tension of cell membranePolydimethylsiloxane flexible substrates with microgrooves were prepared by lithography and inversion technique,and the growth and arrangement of mouse fibroblasts on grooves of different sizes were studied.By applying strain to the flexible substrate to increase the initial tension of the cell membrane by increasing the prestress of the cell membrane,the effect of prestress on the cell stiffness is analyzed,and we found that the prestress enhances the stiffness of the cell membrane by the finite element analysis method.Through the fuzzy positioning indentation experiment of AFM pyramid probe,it was found that increasing the substrate strain of cell petri dish could increase the penetration probability of cell membrane,which was verified by dissipative dynamics simulation.(3)Increasing the penetration probability of cell membrane by increasing the external tension caused by the downpressing of AFM probeBased on the equilibrium equation of shell,the mechanical model of cylindrical probe piercing into the cell is established.Through fitting calculation,it is found that under the same indentation force,the smaller the radius of the probe is,the greater the tension of the cell membrane is.Cylindrical silicon nitride probe with diameter 200nm and carbon nanotube probe with diameter 6nm were prepared by micromanipulation and focus electron and ion beam techniques.Indentation experiments on mouse fibroblasts were carried out using the above probes.It was found that the smaller probe increased the penetration probability of cell membrane compared with the traditional larger AFM pyramid probe.Through the finite element analysis method,it is explained that the small size probe is easy to cause the stress concentration on the cell membrane surface,which leads to the destruction of the cell membrane.Furthermore,by modifying collagen on the cell membrane to strengthen the cell membrane,the stress concentration of the probe was increased and the penetration probability of the cell membrane was improved.(4)Visualization of pore formation and development of cell membrane under mechanical strain based on molecular dynamicsBased on molecular dynamics,a bilayer model of(DPPC)phospholipid was established.The configuration of phospholipid membrane with increasing area strain was studied by molecular dynamics simulation under GROMOS53A6 force field.At the same time,the changing trend of tension and stress of phospholipid membrane with the increase of strain was plot,and the key strain,tension and stress leading to cell penetration under different loading strain rates were discussed.The relationship between different loading strain rates and the number and area of pores in phospholipid molecular model was analyzed.In this paper,AFM indentation experiment and multi-scale simulation were used to study the phenomenon of pores in cell membrane under the action of mechanical force,which has a certain theoretical significance for the damage mechanism of cell membrane.In addition,since AFM has great advantages in the field of cell transfection molecular transport,increasing the prestress of cell membrane,changing the size of probe and modifying cell membrane can effectively improve the penetration probability of cell membrane,which has important guiding significance for the realization of AFM probe application in single cell detection.