Research On Rapid Detection Of Pathogen And Interaction Between Bacteria And Cells Based On Fluorescent Labeling

Foodborn pathogen usually caused infection in human along with the distribution of food.So the efficient detection of pathogenic bacteria was an important guarantee for human health.In addition,the emergence of antibiotic-resistant bacterial strains created a true need to search for new antimicrobial drugs.To meet the problems of complex matrix,longer consuming time,few pathogens in bacterial detection,and deficiency that effects of potential anti-bacterial drugs were usually evaluated through time consuming and complicated methods which were unsuitable for rapid screening.Fluorescent detection with high sensitivity and excellent selectivity was induced in this work.Quantitative analytical methods which reduced influence from background and excess labeling reagent were developed and a microfluidic chip integrated magnetic enrichment,fluorescent labeling and detection was designed to detect pathogenic bacteria.Based on the research of fluorescent probes and labeling for bacteria,fluorescent detection was used to evaluate effects of quorum sensing inhibitors rapidly and efficiently.The work offered new methods for bacteria detection in complex samples and effect detection of antimicrobial drugs.All above had essential research value and potential applications in food safety,human health and infectious treatment.The main research work and results are as follows:(1)A rapid and sensitive analytical method was developed to detect bacteria which combined magnetic enrichment,PEG chromatography with fluorescent labeling.The amino-modified magnetic nanoparticles could efficiently capture E.Coli by electrostatic interaction,which decreased the interference from complex matrix and enriched E.Coli in samples.Acridine orange(AO)with high quantum yield was used to label the captured E.Coli rapidly.In order to removal the free magnetic nanoparticles and AO,the suspension was poured into PEG separation column and separated.The presence of 100 cfu·m L-1 E.Coli could be detected for semi-quantitative analysis with naked eye,and the concentration could be further evaluated by fluorescent detection.It was demonstrated that a good linear relationship existed between the fluorescence intensity with the concentration of E.coli ranging from 102 to 106 cfu·m L-1,with a detection limit of 100 cfu·m L-1.Cooked meat samples were detected by this method within 80 min and the results coincided with conventional plate count method.(2)A method was developed to detect bacteria in a microfluidic chip which integrated magnetic capture,fluorescent labeling and detection.The chip was consisted of six serpentine structures and detection zones.AO and magnetic captured bacteria mixed well and achieved labeling in serpentine channels and were magnetic captured in the detection zones.Fluorescence signals obtained from E.Coli in detection zones by self-designed fluorescent analyzer were a trend of linear correlationship with the concentrations of E.Coli in range of 102-106 cfu·m L-1.The limit of detection was 100 cfu·m L-1 and was finished within 50 min,six samples could be detected simultaneously.(3)Multifunctional magnetic carbon quantum dots were designed and prepared to capture and label salmonella simultaneously.Furthermore,a fluorescent detection method which had good selectivity and little background interference was proposed to detect salmonella.Carbon dots were linked with amino-modified magnetic nanoparticles to form magnetic carbon quantum dots(Fe3O4@CQDs).To achieve selective labeling,Fe3O4@CQDs were modified with Aptamers.Fluorescence intensity of Aptamers modified magnetic carbon quantum dots decreased due to the aggregation after label.The change values were linearly associated with the concentrations of Salmonella within the range of 103-106 cfu·m L-1.The existence of 103 cfu·m L-1 Salmonella in milk and chicken breast could be detected within 60 min,which indicated the proposed method had potential in practical samples.(4)The interaction between bacteria and cells was studied via labeling bacteria with carbon quantum dots.A fluorescent detection method was developed to evaluate the effects of quorum sensing(QS)inhibitors to bacterial infection.The inhibition effects of tannic acid,magnolol and kaempferol in salmonella infection were detected while furanone C30,an acknowledged quorum sensing inhibitor was used as a positive control drug.The inhibiting effects of those drugs in adhesion were: furanone C30> magnolol>tannic acid>kaempferol,while furanone C30>tannic acid>magnolol> kaempferol in invasion.Besides,the all drugs could inhibit QS which regulated via AHL and suppress swarming motility of salmonella,and only magnolol suppress swimming.Above results indicated those drugs inhibited bacterial infection via the suppression of QS and motility in salmonella.The proposed fluorescence detection could be used in antibacterial drugs research and provided a new method to novel antibacterial infection drug screening.