Rapid Detection Of Pathogenic Bacteria Based On Bacteriophages And Carbon Dots

The diseases caused by bacteria occur frequently,which spread widely.Although,all the countries have attached great importance to the prevention and control of pathogenic bacteria,but outbreaks of these pathogenic bacteria still occur repeatedly.These pathogenic diseases are posing a serious threat to the safety of human beings and society.The traditional culture and colony count method is reliable for detecting pathogenic bacteria,but it is timeconsuming and much laborious.The detection methods of molecular biology and immunology have greatly shortened the detection time,but they require professional equipment and technical personnel.These detection assays are prone to false positive results.Therefore,it is urgent to develop a detection method which is more efficient,rapid,highly accurate and low cost,achieving fast qualitative and quantitative detection of pathogenic bacteria.A series of sensing systems was developed based on bacteriophage and nanomaterials for qualitative and quantitative detection of E.coli and Yersinia pseudotuberculosis in this thesis.The contents are as follows:（1）Based on the principle of bacterial metabolism which produces acid metabolites,we designed and synthesized carbon quantum dots-based p H fluorescent probe,and applied it for rapid detection of Escherichia coli O157:H7.Matrix-assisted laser desorption ionization-time of flight mass spectrometry was utilized to further identify the bacteria,achieving the purpose of qualitative and quantitative detection.The developed sensing platform when applied to detect Escherichia coli O157:H7 demonstrated excellent performance with detection range 13¹.33×10⁵ CFU/m L,and detection limit 1 CFU/m L.（2）In order to enhance the detection performance,a new colorimetric method based on bacteriophage and Au Pt nanozyme was developed for the detection of Yersinia pseudotuberculosis.The specific phages toward Yersinia pseudotuberculosis were isolated from hospital waste water.The Au Pt nanoparticles exhibit excellent peroxidase-like activity.The phage conjugated magnetic beads,and the phage immobilized Au Pt nanoparticles were applied together for capturing Yersinia pseudotuberculosis.Then Au Pt catalyzed TMB-H₂O₂ to produce color reaction.Finally,the detection of Yersinia pseudotuberculosis was realized by colorimetric analysis..This sensing podium displayed detection limit of 14 CFU/m L,and detection time of 40 min.（3）For the analysis of real sample,colorimetric sensing detection method is susceptible to the interference of color samples,while electrochemical sensor is not affected.So,we designed and developed an electrochemical biosensor based on phage and poly（indole-5-carboxylic acid）/reduced graphene oxide/gold nanoparticles for the rapid and specific detection of Yersinia pseudotuberculosis.The poly（indole-5-carboxylic acid）/reduced graphene oxide/gold nanoparticles composite has high redox stability and excellent conductivity,which greatly improve the sensitivity of electrochemical biosensor.The phage based electrochemical biosensor was constructed for detection of Yersinia pseudotuberculosis with limit of detection of 3 CFU/m L,and detection time of 35 min.The biosensor revealed good storage stability and reproducibility.（4）In order to introduce enzyme having high catalyze activity into the phage-based electrochemical biosensor,T4 phage display technology was employed to immobilize β-galactosidase enzyme on the capsid surface of T4 phage.T4 phage immobilized with β-galactosidase based electrochemical biosensor was constructed to realize rapid and specific detection of Escherichia coli K12.The enzymatic property of β-galactosidase immobilized on T4 phage（β-gal T4）were improved compared with the free enzyme.The β-gal T4 was utilized to construct electrochemical biosensor for capturing Escherichia coli K12.The β-galactosidase catalyzed the substrate to produce the electroactive product.The achieved detection range was 1.50×10^{2 1}.50×10⁷ CFU/m L,limit of detection 2 CFU/m L.Antibody and T4 were used as dual recognition probe to ensure the specificity of detection.The β-galactosidase as a signal probe guaranteed the good detection sensitivity.In conclusion,this thesis illustrates successful construction of a series of sensing systems to realize the rapid detection of pathogenic bacteria.These detection methods have great potential in the analysis of food,environmental and clinical samples.