Study On The Monitoring Of Biochemical Porcess Based On Surface-enhanced Raman Spectroscopy

From the second half century to the present,life sciences were developing at an impressive speed.Especially the breakthroughs in molecular biology,which has brought life sciences into the molecular field.As a result,the monitoring requirements for biological samples has raised,especial for biochemical processes.Surface-enhanced Raman scattering(SERS)has become one of the powerful tools due to its high detection sensitivity,short detection time,mild test conditions,small water-based interference,and rich fingerprint information from the molecular level.However,there are still many difficulties and challenges in the dynamic analysis of biological samples using surface-enhanced Raman scattering.For example,there is a lack of SERS substrate with high detection sensitivity,good test reproducibility,and relatively low preparation difficulty and cost.And it is difficult to accurately indicate the source of SERS signals because of the complexity of biochemical reaction system,the uncertainty of instability and biocompatibility of nano materials,and problems such as difficulty in studying the mechanism of action.In addition,Raman quantification is still a problem,since the poor detection reproducibility caused by the heterogeneity in biochemical reaction systems(such as bacteria and cell culture).In this thesis,electrochemical preparation technology,nanotechnology,surface plasmon resonance(SPR)technology,MEMS processing technology and so on were combined with SERS analysis to monitoring of the biochemical processes and analysis of reaction mechanism.We focused on and broke through the problems of poor sensitivity of SERS materials,low signal reproducibility,poor biocompatibility,signals interference by impurities,and unclear signal sources.We have established methods and techniques for SERS monitoring of biochemical process,and reveal the mechanism in the biochemical processes at the molecular level.The oxidation of phospholipids and DNA,and the bacteriostatic processes were studied.The main research work of this paper is as follow:Part ?:Dynamic monitoring of oxidation process of phosphatidylcholine based on silver micro/nano composite SERS substratePhospholipid is one of the main components of cell membranes.Its oxidation process is not only a necessary detection in food safety,but also closely related to the occurrence of many diseases.In order to monitor the process of phospholipid oxidation,a silver micro/nano composite SERS substrate was prepared on the surface of ITO glass by electrochemical deposition.Its apparent enhancement factor was up to 7.8×10⁷,and with excellent detection reproducibility.The substrate perpared could meet the real-time detection of phospholipid oxidation process.Then the oxidation process of phospholipid was detected in 0,0.25,0.5,1,2,5,10,15,and 20 days by SERS.The peak intensity at1441 cm^-1(CH₂bending vibration)was used as the internal standard of SERS analysis.By investigating the changes of relative intensity of I₈₇₂/I₁₄₄₁,I₁₂₆₆/I₁₄₄₁,I₁₃₀₀/I₁₄₄₁,and I₁₆₅₁/I₁₄₄₁ with time,the corresponding relationship between the relative peak intensity ratio of SERS and the change of phospholipid structure during oxidation was established.And it was found that the oxidation process of phospholipid in aqueous ethanol solution accorded with the second-order kinetic equation of the reaction.By analysing the SERS spectrum of the oxidation process of phospholipid in 0?20 days,and the newly formed peak had a clear correlation with the oxidation intermediate glycerol and malondialdehyde(MDA)of phospholipid.In addtion,the peak intensity at872 cm^-1 gradually increased,while the peak intensity at 1272 cm^-1 increased firstly and then decreased with the oxidation process proceeding.The oxidation intermediate glycerol and MDA were also measured by phospholipid acid value test,oxidation product mass spectrometry,these results were quite consistent with SERS analysis.Part ?:Monitoring and Mechanism Analysis of DNA Oxidation Process by the Nanobowl-shaped Silver Array SERS SubstrateReactive oxygen species(ROS)generated by normal cell metabolism or exogenous compounds can cause phospholipid peroxidation in cell membranes and intracellular DNA oxidative damage,leading to a variety of diseases.Hence,monitoring the DNA oxidation process is of great significance.Since DNA is photosensitive,the surface plasmon resonance(SPR)technology and SERS spectroscopy were combined and the silver nano-bowl array SERS substrate was thus designed and prepared to amplify the SERS signals.Then the SERS analysis of DNA oxidation process was carried out under the detection conditions of low detection power and short integration time.After characterising by SEM and simulating by finite time domain difference(FDTD)software,it was found that the silver nano-bowl array SERS substrate could improve the detection efficiency of DNA samples by 11.8 times compared with ordinary silver nano-substrate.By SERS analysis of DNA samples of supercoiled,open-loop and linear conformations,we drew a conclusion that the strong change of 838 cm^-1 peak intensity was closely related to DNA conformation.Fenton reagent was used as oxidizing reagent and Na₂SO₄ characteristic peak intensity was apply for the SERS analysis internal standard,the oxidation process of plasmid DNA was monitored in 0-24 h,and the changes of I₆₅₇/I₉₉₈,I₇₂₃/I₉₉₈,I₁₅₈₇/I₉₉₈,I₁₆₄₈/I₉₉₈ and DNA oxidation product 8-OHd G was also monitored over time.Then the relationship between the relative peak intensity ratios and the base structure change in DNA and 8-OHd G(the oxidation end product)was established,which extended the application of SERS technology in the process monitoring of photosensitive biological samples.Furthermore,the conclusion of SERS spectral analysis was verified by the DNA conformation and oxidative damage gel detection and colorimetric detection of 8-OHd G.Part ?:Study of Antimicrobial Process and Mechanism of Curcumin Liposome&Au NPs Complex by SERSThe interaction mechanism between drugs and pathogenic microorganisms plays a vital role in the development of new antibacterial drugs.The monitoring of antibacterial processes is also one of the current research hotspots.In this chapter,the curcumin liposome&Au NPs complex was designed and prepared according to the monitoring requirements of the complex biochemical process in bacteriostatic systems.The SEM and TEM characterization results showed that after encapsulated by drug-loaded liposomes,the curcumin liposome&Au NPs complex could be used as a bacteriostatic agent to show faster onset time and better antibacterial effect on MRSA.At the same time,it could also be used as a SERS reinforcing material because of the inclusion of gold nanoparticles.Dropping this complex on the surface of silver nano substrates could achieve better SERS detection and reproducibility.Based on this detection mode,the SERS spectra of three mixtures of methicillin-resistant Staphylococcus aureus(MRSA)and curcumin,curcumin liposome and the complex were analyzed with the characteristic peak(672 cm^-1)of DMSO as a internal standard.It was found that I₇₂₈/I₆₇₂,I₉₅₄/I₆₇₂,and I₁₃₃₁/I₆₇₂ were decreasing with time,while I₁₆₄₈/I₆₇₂ showed a tendency to increase in the short term and then disappear.The antibacterial effect of curcumin and complex on MRSA was also investigated.Based on the SERS detection mode and the microstructure of MRSA,the intrinsic relationship between the characteristic peak intensity ratios and the inhibition process and bacteriostatic mechanism were established.Through analysing the SERS peaks and monitoring the important intermediates MDA and 8-OHd G by SERS,it was found that in the system of curcumin,complex and MRSA,excessive ROS was produced,and curcumin could destroy the cell wall and cell membrane structure of MRSA,while phospholipid oxidation product in the process of curcumin liposome&Au NPs complex and MRSA could produce more ROS,which could accelerate the destruction of cell wall and cell membrane.More DNA oxidation product 8-OHd G was also produced at the same time.Therefore,the complex showed better antibacterial effect.The antibacterial process and mechanism based on SERS spectral analysis were confirmed by active oxygen detection kit,apoptosis detection kit and flow cytometry.In short,SERS technology can not only be used for real-time detection of complex biochemical samples,but also a powerful tool for biochemical reaction mechanism analysis.Part ?:Real-time detection of endotoxin in the antibacterial process based on integrated array three-dimensional biomimetic array SERS microstructure and SERS tagsEndotoxin,one of the important components of the cell wall of gram-negative bacteria,is usually released after the death and disintegration of bacteria.It enters the organism and produce an immune response,which is of great significance in the safety evaluation of biological agents and clinical diagnosis.Based on the difficulty of endotoxin monitoring in the bacteriostasis process and the blank of SERS quantitative detection of endotoxin,a novel SERS substrate was designed and prepared with MEMS processing technology,which took cicada wings as a natural template and a three-dimensional biomimetic array SERS microstructure was thus obtained.The SERS substrate had good homogeneity and reproducibility of detection.The 3d bionic SERS substrate was integrated in SERS chip to produce the 3d bionic SERS microstructure with array structure.Due to the small volume of the culture microcavity,the problem of uneven distribution of bacteria liquid phase could be partly solved.With the help of SERS tags modified by DNA aptamer,endotoxin could be specifically identified and detected,which solved the problem of impurity interference.By shortening the binding time of ligand and endotoxin to 100 s,a new method for real-time in situ detection of endotoxin in bacteriostasis process was established.DMSO was added into the system of pseudomonas aeruginosa and bacteriostatic agent as the internal standard for SERS analysis.The concentration and relative peak strength ratio of the standard toxin was used to establish the fitting curve:y=0.0034x+0.2534,R²=0.9748.Detection limit is as low as 6.125 ng/m L.Through the analysis of the fitting curve and SERS spectrogram in the bacteriostatic system,the real-time monitoring study of endotoxin content in the bacteriostatic system at various times was carried out.The results were verified by the endotoxin ELISA test kit,and showed a good consistency.The SERS detection method can be used for in situ,real-time and quantitative detection of specific samples in complex biochemical processes.