Investigating The Antimicrobial Mechanism Of Antibacterial Material Based On Atomic Force Microscope

The abuse of antibiotics has led to the emergence of a large number of drug-resistant bacteria,the traditional antibiotic drug treatment has been difficult to meet the current needs of fighting multi-drug resistant infections.Therefore,in order to find new methods or materials that are resistant to bacterial infections and will not lead to the emergence of drug-resistant,there is an increasing demand for in-depth determination of the role of antibacterial materials such as drugs and the detailed response of bacteria to these stimuli to study their antibacterial mechanisms,it can be used to support theoretical research on new antimicrobial materials or methods.Aiming at the emerging material antibacterial methods such as photothermal antibacterial method and novel antibacterial peptide antibacterial method,they have good antibacterial ability and are not easy to cause bacteria to produce drug resistance,but people are not clear about their detailed antibacterial mechanism.Therefore,studying their antibacterial mechanism is of great help to solve the problem of bacterial resistance.In this article,atomic force microscopy is used as the main surface interface analysis method,and using a series of auxiliary analytical methods such as plate colony counting method,Live/Dead bacteria fluorescent staining,calcium ion probe,inductively coupled plasma optical emission spectrometer?ICP-5000?,molecule dynamics simulation,etc.Proposed the antibacterial mechanism of gold nanorods and self-design K₃?FA?₄K₃ antimicrobial peptides.The results of the work research are as follows:1.The study of Au nanorods triggered antibacterial photothermal lysis cell membraneAu NRs with LSPR of 740 nm were prepared by gold seed growth method,and then modifying mPEG₅₀₀₀-SH on the surface of AuNRs to improve the biocompatibility of Au nanorods.By using plate colony counting method,Live/Dead bacteria staining,calcium and magnesium ion measurement in vitro and in vivo of bacteria and molecular dynamics simulation experiment and so on.The photothermal antibacterial mechanism of the gold nanorods is as follows:The positively charged gold nanorods will adsorb on the surface of the negatively charged bacteria.The local hyperthermia will occur under the 808 nm near-infrared light,and thereby affecting the kinetic balance of calcium and magnesium inside and outside the bacteria.As the temperature rises,large amount of Ca²⁺and Mg²⁺ions will leak out,causing cell membrane disorder and affecting the stability of the cell membrane,the permeability of cell membrane will increased and lead to internal and external pressure imbalanced,finally the cell membrane will rupture to lead the death of bacterias.2.Further study on photothermal cracking mechanism of Au nanorods based on AFMFurther studies on the antibacterial mechanism of E.coli treated by PEG-AuNRs exposed to near-infrared light were carried out by AFM,and then,combined with the previous research about Ca²⁺and Mg²⁺ions,the detailed antibacterial mechanism of gold nanorod photothermal cracking was obtained:The local overheating of gold nanorods leads to a large amount Ca²⁺and Mg²⁺leak out,because of Ca²⁺Mg²⁺have the features of maintaining the stability of LPS plaques.Their loss will make the LPS plaque unstable,and increased the membrane permeability,then it will make the internal and external osmotic pressure of bacterias unbalanced and lead to membrane rupture.LPS will be collapse and since the outer membrane is linked with peptidoglycan through Braun lipoprotein,the lipoprotein folds in a different manner,so the young's modulus of bacteria is increased.At the same time,the detachment of the LPS plaque reveals substances inside the LPS layer,such as the phospholipid bimolecular layer,which will lead to the increase of adhesion force,and subsequent rupture of the membrane will cause leakage of proteins and other matter inside the bacteria,and the adhesion force will have a greatly increased.3.In situ study of antibacterial mechanism of antimicrobial peptide K₃?FA?₄K₃by using AFM under physiological conditionsThe antibacterial peptide of K₃?FA?₄K₃ sequence was designed and synthesized by KLVFFAE?A?16-22?as template.Firstly,it was proved by using the plate counting method that the antimicrobial peptide has a good ability to kill E.coli.Then,The Live/Dead bacteria staining experiment proved that the antimicrobial peptide has a membrane breaking ability.Finally,in-situ dynamic monitoring the E.coli treated with antimicrobial peptides by using AFM,a possible mechanism is proposed:the positively charged cationic antibacterial peptide K₃?FA?₄K₃ will approaches the bacterial cell membrane and accumulates through electrostatic interaction.The head of this antibacterial peptide is hydrophilic amino acid K,it can be combined with phospholipids.While the hydrophobic phenylalanine?F?and alanine?A?in the middle portion form a polypeptide/lipid complex with the phospholipid and inserted into the membrane to form a transmembrane channel.After the formation of the channel,the bacterial cell membrane structure is disordered,and the osmotic pressure inside and outside is unbalanced,thereby destroying the integrity of the cell membrane,such as cell membrane depression,cracks,etc.,making its roughness and adhesion force increased,eventually leading to the death of bacterias.