Effecets Of Hematoporphvrin Monomethyl Ether Against Bacteria And CD44 Single-molecule Recognition Events On Mesenchymal Stem Cells Surface Studied By Atomic Force Microscopy

Because of the nano-meter/subnano-meter resolution and simple sample preparation procedure,atomic force microscopy(AFM) has been proved a powerful tool to obtain topography and biomechanical properties of various materials.In author's experiments, AFM was used as an important means to detect the photoactivation effects of hematoporphvrin monomethyl on Gram-positive bacteria -Staphylococcus aureus and Gram-negative bacteria -Escherichia coli and the CD44 single-molecule recognition events on mesenchymal stem cell surface.In the first part,the photodynamic inactivation of Gram-positive bacteria-Staphylococcus aureus and Gram-negative bacteria- Escherichia coli by hematoporyrin monomethyl ether was investigated by the reduction of colony unit and AFM.Results indicated that 90%of Staphylococcus aureus was photoinactivated by illumination with 30 min visible light(power density 200 mW/cm~2) in the presence of 50μg/mL HMME. The antiactivation efficiency to Staphylococcus aureus with light irradiation was much obvious than that in dark at the same concentration of HMME,but there was not noticeable damage to E.coli with illumination or in dark.AFM ultrastructure images showed that the cell surface of photodynamic inactivated bacteria was all damaged seriously but there's no cell contents leaked.So we concluded that the attacked sites to bacteria cells by hematoporyrin monomethyl ether were bacteria membrane structure. And atomic force microscopy provides us a visual technique to study the mechanism of bacteria reacted with photosensitizers.In the second part,Functionalized AFM was used to study the single-molecule recognition events on the surface of the rat mesenchymal stem cells(MSCs).Molecular recognition plays an important role in biological systems and is observed between receptor-ligand,antigen-antibody,DNA-protein,sugar-lectin,RNA-ribosome,etc.To assess the specificity of recognition events,control experiments have been performed by recording force-distance curves using modified and unmodified AFM tips,respectively. Under these conditions,thousands of force-curves have been acquired over different sites of the sample and the unbinding force of the observed events has been measured.In the results,we obtained the CD44 molecule distribution on the surface of MSCs which indicated the CD44 molecule distribution was not homogeneous.Simultaneously,from force-curve analysis we estimated the interaction force is around 250 pN.In the present, AFM is the only force-measuring technique that can map the nanoscale lateral distributon of single molecular recognition sites on biosurfaces.In the third part,AFM was used to measure the morphology and nanomechanical properties(elasticity and adhesion) of erythrocyte taken from type 2 mellitus patients and healthy people seperately.The results showed that stiffness and adhesion of diabetes patients' erythrocyte were higher than healthy people's.In morphology,the patient' erythrocyte was much lower in cell median height(Mh)and cell peak to valley(Rp-v), but bigger higher in diameter than those of the erythrocyte of healthy people.AFM provides us more detailed data for better understanding the pathologic characteristics of erythrocyte and has been proved to be a powerful tool in disease detecting and diagnosis.