| The study of cell-microenvironment interactions is of great significance in the fields of wound healing,tissue repair,and disease prevention.In vitro,micro/nano structures with unique physical characteristics can be manufactured through micro/nano fabrication technology to simulate various mechanical stimuli generated by the cellular microenvironment in vivo.Among them,laser interference lithography(LIL)has become the most commonly used technical means for preparing micro/nano structures due to its large exposure area within the laser coherence range,short experimental period and relatively simple operation.However,the structures obtained only with LIL are high structural similarities,and cannot construct complex multi-dimensional micro/nano structures.In addition,the complexity of cell response to the microenvironment also indicates that the regulation of micro/nano structures on cell behavior remains to be further studied.Therefore,it is a very meaningful topic to combine LIL with other micro/nano fabrication methods to obtain structures with rich morphology and stiffness for the study of cell behavior.This thesis focuses on the preparation method of micro/nano structures based on LIL,and studies the regulation effect of micro/nano structures on cell behavior.The following are the main studies that have carried out in this work:1)Analysis of the two-beam laser interference light field and research on the interaction mechanism between the laser and the materialOn the basis of laser interference theory,the parameters such as light intensity,contrast,period and polarization state combination of two-beam laser interference were theoretically deduced,and the light field distribution of two-beam interference under different incident angles was obtained by MATLAB simulation.Based on the analysis of the interaction mechanism between the laser and the material,the mechanism of the micro/nano structure prepared by LIL was studied when the metal film was used as the "photoresist".According to the requirements of the thesis,the design and physical construction of the two-beam laser interference lithography system were completed,and two micro/nano structure preparation methods based on two-beam laser interference lithography were proposed.2)Study on the regulation of cell behavior by different stiffness surface Pt nanoparticle arraysA nano-transfer method based on two-beam laser interference lithography was proposed to obtain platinum nanoparticles(Pt NPs)arrays on glass and polydimethylsiloxane(PDMS)surfaces.The Young’s modulus of Pt NPs with different stiffness surfaces was determined by analyzing the characteristic parameters such as substrate morphology and stiffness.Based on the "motor-clutch" model,an improved cell-substrate equivalent coupling model was established,and the assumption of viscoelastic base was proposed.By studying the effect of substrate on the behavior of mouse brain microvascular endothelial cells(b End.3),it was found that the significant difference in Young’s modulus between the Pt NPs and the substrate is the key to the orderly arrangement of cells induced by Pt NPs array.Compared with the Pt NPs array on the surface of glass substrate,the Pt NPs array on the surface of PDMS can not only promote cell adhesion and growth,but also induce the orderly arrangement of cells.In addition,the appearance of wrinkles around cells on the surface of PDMS substrate has confirmed the hypothesis of viscoelastic substrate.This work demonstrates that the discontinuous nanostructures regulate the orderly arrangement of cells,and provides an effective method for the design of biological functional substrates for preparing rigid patterns on the surface of flexible materials,and also helps to understand the cell durotaxis.3)Study on the regulation of cell behavior by nanopillars and nanoholesA metal-assisted chemical etching(MACE)method based on two-beam laser interference lithography was proposed,and the preparation of silicon nanopillar(Si NP),silicon nanohole(Si NH),silicon nanopillar/silicon nanohole(Si NP/Si NH)hybrid arrays were obtained.By analyzing the characteristic parameters such as the morphology,roughness,structural height and element content of the substrate,it was confirmed that the above substrate did not contain silver particles,which eliminated the interference of silver particles on the cell experiment.In addition,by studying the effect of substrate on the behavior of human lung adenocarcinoma cells(A549),it was found that the Si NP array restricted the cell spreading,and the cells were round and the filopodia were dominant.The Si NH array promoted the cell spreading,the cells spread freely and the lamellipodia and filopodia were dominant.The Si NP area in the Si NP/Si NH hybrid array had a capture effect on the cells,and about 80% of the cells selectively adhered to this area,and the lamellipodia were dominant.Furthermore,the sub-surface characteristics of cell-substrate were revealed through atomic force acoustic microscopy,which made up for the defects of atomic force microscopy topography and provided more comprehensive information for the study of cell morphology.This work provides a simple method for the design of nanohybrid structures for capturing tumor cells,and demonstrates the regulation of nanohybrid structures on cell spread and pseudopodia distribution,and helps to understand the capture of cells by nanohybrid structures.4)Study on the regulation of cell behavior by nanopillar/nanohole grid hybrid arrayA MACE method based on two-beam double-exposure laser interference lithography was proposed to realize the preparation of Si NP/Si NH grid hybrid array.By analyzing the characteristic parameters such as the morphology,structural height and element content of the substrate,it was confirmed that the above substrate does not contain silver particles,which eliminated the interference of silver particles on the cell experiment.In addition,by studying the effect of substrate on the growth of human neuroblastoma cells(SHSY5Y),it was found that the Si NP area of the Si NP/Si NH grid hybrid array has a trapping effect on cells,when the array period was 14 μm,the localization ratio of cell was about 83%.By analyzing the cell growth and localization probability on the Si NP/Si NH grid hybrid array with different periods,it was found that the reduction of the period was not conducive to the accurate localization of cells and would reduce the localization ratio of cells,when the period was 7 μm,the ratio of cell localization was about 67%.However,when the period was increased,the ratio of cell localization remained around 84%±1%,and when the period was too large(48 μm),multiple cells would adhere to a nanopillar area,which was not conducive to the independent growth of cells.This study has proved that the physically modified nanostructures are effective in regulating the localized growth of cells.In practical applications,the size of the structure can be reasonably designed based on the cell size to improve the ratio of cell localization.These findings provide an effective surface physical modification method for the study of single cell patterning,and also contribute to a better understanding of the law of localized cell growth. |
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