title: Indirect immunofluorescence detection of E. coli O157:H7 with fluorescent silica nanoparticles.
abstract: A method of fluorescent nanoparticle-based indirect immunofluorescence assay using either fluorescence microscopy or flow cytometry for the rapid detection of pathogenic Escherichia coli O157:H7 was developed. The dye-doped silica nanoparticles (NPs) were synthesized using W/O microemulsion methods with the combination of 3-aminopropyltriethoxysilane (APTES) and fluorescein isothiocyanate (FITC) and polymerization reaction with carboxyethylsilanetriol sodium salt (CEOS). Protein A was immobilized at the surface of the NPs by covalent binding to the carboxyl linkers and the surface coverage of Protein A on NPs was determined by the Bradford method. Rabbit anti-E. Coli O157:H7 antibody was used as primary antibody to recognize E. coli O157:H7 and then antibody binding protein (Protein A) labeled with FITC-doped silica NPs (FSiNPs) was used to generate fluorescent signal. With this method, E. Coli O157:H7 in buffer and bacterial mixture was detected. In addition, E. coli O157:H7 in several spiked background beef samples were measured with satisfactory results. Therefore, the FSiNPs are applicable in signal-amplified bioassay of pathogens due to their excellent capabilities such as brighter fluorescence and higher photostability than the direct use of conventional fluorescent dyes.
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, PR China; College of Chemistry and Environmental Engineering, Shanxi Datong University, Datong 037009, PR China.
authors.forename: Ze-Zhong
authors.initials: ZZ
authors.lastname: Chen
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Cai
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, PR China.
authors.forename: Min-Yan
authors.initials: MY
authors.lastname: Chen
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, PR China.
authors.forename: Dai-Wen
authors.initials: DW
authors.lastname: Pang
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, PR China. Electronic address: hwtang@whu.edu.cn.
authors.forename: Hong-Wu
authors.initials: HW
authors.lastname: Tang
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D004655:Emulsions / Q000737:chemistry; D004927:Escherichia coli Infections / Q000175:diagnosis* / Q000382:microbiology; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D011437:Propylamines; D011817:Rabbits; D012821:Silanes / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry*; D013205:Staphylococcal Protein A / Q000737:chemistry
chemical_list: D004655:Emulsions; D005456:Fluorescent Dyes; D055767:Immobilized Proteins; D011437:Propylamines; D012821:Silanes; D013205:Staphylococcal Protein A; D012822:Silicon Dioxide; D016650:Fluorescein-5-isothiocyanate; C477625:amino-propyl-triethoxysilane
keywords: Bacteria; Bioassay; Flow cytometry; Fluorescent nanoparticles; Water-in-oil microemulsion