Design Of Composite-structured Substrate Via Liquid Interface Self-assembly Based On SERS And Its Rapid Detecting Performance To Foodborne Bacterial Spores

Foodborne pathogenic bacteria spores are major potential threats to food safety and human health due to their extreme resistance and not easily eliminating.The related research of prevention and control has received much attention from the academic study and industrial production in recent years.Detection is the prerequisite of prevention and control,but traditional detecting technology is limited by timeliness,thus developing sensitive and efficient rapid-detection technology of spores is an urgent problem in the field of food safety.Surface-enhanced Raman spectroscopy(SERS)as a bio-fingerprint differentiating spectroscopy technology,has been reported to exhibit better performance in acquiring more characteristic information of biomolecular structure and components,with advantages of non-specific labels,convenience,high-efficiency and strong signals,which can meets the demand of rapid detection of bacterial spores in the field of food safety.Because of high quality substrate material is crucial to the good performance of SERS technology,this study is carried out around the optimization and preparation of good performance substrate.The traditional microbial detection method PCR was firstly compared with SERS technology for mutual imprinting,highlighting its detection advantages and qualitative ability,then SERS substrates of two kinds of multi hot spots uniformly arranged structure and novel composite-structure were prepared,which combined with multivariate statistical analysis methods and chemometrics to construct recognition models and quantitative relationships,achieving sensitive and efficient rapid detection of bacterial spores.This study was divided into three parts,which were as follows:(1)Comparative study on the investigation and identification of foodborne pathogenic spores based on PCR technology and the rapid identification of SERS technology.In this part,8 typical bacterial colonies were isolated and cultured from randomly selected ham sausages on the market,through(non)heat-activiation treatment and based on different selective culture media.After microscopic examination of Gram stain and PCR identification,the results were Bacillus licheniformis,Bacillus amylolyticus,Clostridium perfringens,Bacillus subtilis,Bacillus cereus,Klebsiella,Raoulia plantarum,and Raoulia ornithinolyticus.The SERS spectra of 8 bacterial strains were collected and the spectral analysis was carried out in the main characteristic peak area of 400-1800cm-1,The different food-borne pathogens were successfully distinguished based on the significant or subtle spectral characteristic peak differences.In addition,the results of multivariate statistical analysis show that the cumulative contribution rate of PCA is more than 95% and the SERS spectra of each strain can be classified from each other in independent clusters,realizing the classification and recognition of different bacterial strains and the differentiation of gram-negative and gram-positive bacteria.It provides a fast,effective and reliable way for the field of food safety and microbial rapid detection,also provides theoretical basis and foundation for the subsequent qualitative and quantitative analysis of bacterial spores based on SERS technology.(2)Preparation of Ag NPs self-assembled solid-phase substrate via seed-mediated growth for rapid identification of different bacterial spores based on SERS.In this part,the size and shape of nanostructures were optimized by the seed-mediated growth method,and a kind of solid-phase substrate(Ag NPs Self-assembled solid-phase,Ag NPs-SASP)with closely-packed and ordered hot spots was prepared based on the liquid-phase interface self-assembly technology.This film-like uniform two-dimensional array is attached to the silicon wafer with good SERS reproducibility and enhancement effect,and the enhancement-factort is up to 1.79 ×104.The results of SERS analysis of three different bacterial spores showed that the number and intensity of characteristic peaks(Ca DPA)were dominant in the SERS spectra of spores,and exits differences in the SERS spectra of different spores.The specific characteristic peaks of different spores belong to different biological structures and chemical bond vibration forms.The application of multivariate statistical analysis not only effectively achieved the identification of three spores,but also realized the distinction between Clostridium and Bacillus.Linear discrimination can distinguish SERS spectra of three spores with 100% good sensitivity and specificity.Overall,SERS technology based on Ag NPs-SASP substrate developed in this part provides an effective tool for foodborne pathogens spore detection and food safety risk control.(3)Construction and optimization of Graphene@Ag-MLF composite-structure SERS platform based on liquid-interface self-assembly and its rapid-detecting performance to different foodborne bacterial spores.In this part,we loaded Ag NPs on graphene film based on SERS technology and prepared a new kind of SERS biosensor with graphene@Ag-MLF composite structure.Through FE-SEM and EDS elemental mapping,we characterized the uniform morphology of substrate and the distribution of different elements.SERS measurements of three different spores showed that the enhancement factor reached above 107.Analysis of abundant characteristic peak attribution can also provide support for the preliminary identification of spores.In addition,Graphene@Ag-MLF has stable enhancement performance and good reproducibility(RSD<3%).Combined with multivariate statistics analysis and chemometrics,the results show that the cumulative variance contribution rate of PCA was 96.35%,and HCA can achieve independent cluster with non-interference of different spores’ spectra.Based on the optimal principal component,the KNN and LS-SVM were applied to construct a fast qualitative identification model for further analysis,in which the prediction set and training set of LS-SVM are 100% to achieve complete differentiation.Finally,the LOD of three different spores is under102 CFU/m L,and there is a good quantitative linear relationship between SERS intensity and spore concentration.This new Graphene@Ag-MLF composite structure SERS platform has fast,sensitive and stable performance,providing a strong technical support for the application of SERS technology in the field of food safety and bacterial spores detection.