Study On The Detection Of E. Coli O157:H7 Using Fluorescent Nanoparticle-Based Labeling And Hydrogel Technology

Detection of pathogenic bacteria is vital in food and environment safty, clinical diagnosis and anti-bioterrorism. A fast and sensitive method for pathogenic bacteria detection is very important. E. coli O157:H7 has aroused serious attention as a public health problem because it has caused a number of human infections through the consumption of foods and water. Illnesses caused by E. coli O157:H7 can range from mild, watery diarrhea to life-threatening conditions, such as hemolytic uremic syndrome and hemorrhagic colitis. Aiming at the important aspect of pathogenic bacterial detection, this thesis mainly focuses on development of rapid and sensitive detection methods for E. coli O157:H7 by using fluorescent nanoparticle-based labeling and hydrogel technology.1. Detection of E. coli O157:H7 using fluorescent silica nanoparticles and SYBR GreenⅠbased two-color microscopic imaging techniqueA novel two-color immunofluorescent microscopic imaging technique for the rapid and sensitive detection of E. coli O157:H7 has been developed based on fluorescent silica nanoparticles and SYBR GreenⅠ. Based on antigen-antibody interaction, the anti-E. coli O157:H7 antibody conjugated RuBpy-doped silica nanoparticles were used to recognize E. coli O157:H7 specifically. Then, the bacteria sample was stained with a nucleic acid dye SYBR GreenⅠ, thus resulting in the two-color labeling of the target E. coli O157:H7. By using the confocal microscope imaging technique, the separation-free detection of E. coli O157:H7 was successfully carried out in buffer and bacterial mixture, respectively. The whole detection process could be finished within 3 hours. And this technique exhibited the sensitivity with the detection limit of 2.6×10~3cells/mL for E. coli O157:H7. Considering the antibodies for various bacteria, this proposed method might be promising for rapid and sensitive detection of other bacteria.2. Rapid and ultrasensitive E. coli O157:H7 quantitation by combination of ligandmagnetic nanoparticles enrichment with fluorescent nanoparticles based two-color flow cytometryA novel, fast and sensitive determination strategy for E. coli O157:H7 has been developed by combination of ligandmagnetic nanoparticles (LMNPs) enrichment with fluorescent nanoparticles (FNPs) based two-color flow cytometry assay (LMNP-FNP@SG-FCM). E. coli O157:H7 was first captured and enriched through the lectin concanavalin A (Con A) favored strong adhesion of E. coli O157:H7 to the mannose-conjugated magnetic nanoparticles (LMNPs). The enriched E. coli O157:H7 was further specially labeled with goat anti-E. coli O157:H7 antibody-conjugated RuBpy-doped FNPs, and then stained with a nucleic acid dye SYBR GreenⅠ(SYBR-I) to exclude background detrital particles. After dual-labeled with FNPs and SYBR GreenⅠ, the enriched E. coli O157:H7 was determined using multiparameter flow cytometry analysis. With this method, the detection sensitivity was greatly improved due to the LMNPs enrichment and the signal amplification of fluorescent nanoparticles label method. Furthermore, the false positives caused by aggregates of nanoparticle-bioconjugates and nonspecific binding of nanoparticle-bioconjugates to background debris could be significantly decreased. This assay allowed the detection of E. coli O157:H7 in PB buffer as low as 7cells/mL. The total assay time including E. coli O157:H7 sample enrichment and detection was within 4 hours. And artificially contaminated bottled mineral water sample with concentration of 6cells/mL can be detected by this method. It is believed that the proposed LMNP-FNP@SG-FCM method will find wide applications in biomedicine fields demanding higher sensitive bacterial identification.3. Detection of E. coli O157:H7 using the mannose-functionalized hydrogelsBy combining the hydrogel with the nucleic acid dye SYBR GreenⅠ, we have engineered mannose-functionalized hydrogel for E. coli O157:H7 detection based on multivalent binding of Con A. In this method, the hydrogel was first synthesized by using acrylamide, N, N-dimethyl acrylamide and N-acryloxysuccinimide, employing ammonium persulfate (APS) as the catalyst and etramethylethylenediamine (TEMED) as the accelerator. Then, the mannose was conjugated to the surface of the prepared hydrogel. After preparing the functional hydrogel, the sample was incubated with the hydrogel for 1h and then stained with a nucleic acid dye SYBR GreenⅠ. Finally the labeled E. coli O157:H7 was determined through the fluorescence in vivo imaging. The detection of E. coli O157:H7 was successfully carried out in buffer and bacterial mixture, respectively by using this method. This assay allowed the detection of E. coli O157:H7 in PB buffer as low as 3.7×10~1cells/mL. The total assay time was less than 2h.