Fluorescent Nanoparticle-Based Labeling And Encoding Technology For Detection Of Some Important Pathogenic Bacteria

Detection of pathogenic bacteria is vital in food and environment safty,clinical diagnosis and anti-bioterrorism.Traditional methods for the detection of pathogenic bacteria either lack sensitivity or take a long time for analysis.Current rapid methods based on the modern molecular biology,immunology and analytical instruments have contributed much on the improvement of sensitivity and accuracy of the bacterial detection but still exhibit deficiencies in some extent.Recently,functionalized nanomaterials with unique chemical and physical properties have been successfully applied in the rapid and sensitive detection of pathogenic bacteria.Fluorescent silica nanoparticles are one of the most luminescent nanomaterials,which have gained increasing attention these years.Due to the superiority over conventional fluorophores in terms of fluorescence intensity and photostability,fluorescent silica nanoparticles will open new opportunity in ultrasensitive detection of trace amounts of pathogenic bacteria.Aiming at the important aspect of pathogenic bacterial detection,this thesis mainly focuses on development of rapid,sensitive and multiplexed detection methods for several important pathogenic bacteria by using fluorescent nanoparticle-based labeling and encoding technology.1.Fluorescent nanoparticle-based indirect immunofluorescence microscopy (FNP-ⅡFM) for detection of Mycobacterium tuberculosis.A method of fluorescent nanoparticle-based indirect immunofluorescence microscopy(FNP-ⅡFM) has been developed for the rapid detection of M.tuberculosis. An anti-Mycobacterium tuberculosis antibody was used as primary antibody to recognize M.tuberculosis,and then an antibody binding protein(Protein A) labeled with Tris(2,2-bipyridyl)dichlororuthenium(Ⅱ) hexahydrate(RuBpy)-doped silica nanoparticles was used to generate fluorescent signal for microscopic examination. With this method,M.tuberculosis in bacterial mixture as well as in spiked sputum was detected.Total assay time including sample pretreatment was within 4h.The use of the fluorescent nanoparticles revealed amplified signal intensity and higher photostability than the direct use of conventional fluorescent dye as label.This proposed FNP-ⅡFM method will be promising for rapid detection of M.tuberculosis in clinical sputum samples.2.Fluorescent nanoparticles and SYBR GreenⅠbased two-color flow cytometry (FSiNP@SG-FCM) for detection of M.tuberculosis. A method using an improved two-color flow cytometric analysis by a combination of bioconjugated fluorescent silica nanoparticles and SYBR GreenⅠ(FSiNP@SG-FCM) has been developed for the rapid detection of M.tuberculosis.M. tuberculosis was specially labeled with antibody-conjugated RuBpy-doped silica nanoparticles,then stained with a nucleic acid dye SYBR GreenⅠto exclude background detrital particles,followed by multiparameter determination with flow cytometry.With this method,false positives caused by aggregates of nanoparticle-bioconjugates and nonspecific binding of nanoparticle-bioconjugates to background debris could be significantly decreased.This assay allowed for detection of as low as 3.5×10~3 and 3.0×10~4 cells/ml M.tuberculosis in buffer and spiked urine respectively with higher sensitivities than the FITC-based conventional flow cytometry.The total assay time including sample pretreatment was within 2 h.This proposed FSiNP@SG-FCM method will be promising for rapid detection of M. tuberculosis in clinical urine samples.3.Synthesis of the optically encoded silica nanomaterials.We have synthesized optically encoded silica nanomaterials with an easy water-in-oil microemulsion method.A pair of FRET(frequency resonance energy transfer) donor-acceptor chromophores were simultaneously labeled on immunoglobulin G(or poly lysine) at varied ratios to prepare the spectroscopically encoded core materials,which were then housed inside a silica shell by the hydrolysis and polycondensation of tetraethoxysilane(TEOS) in water-in-oil microemulsion to synthesize the optically encoded silica nanomaterials.The studies of the morphologies and fluorescence properties of the two kinds of optically encoded silica nanomaterials showed that different morphologies of encoded silica nanomaterials with the regular sphere-shape or rod-shape could be synthesized by using immunoglobulin G or poly lysine respectively as core materials in the preparation process.By varying the labeling ratios of the two chromophores,nanospheres or nanorods with varied emission spectra were obtained.FRET-mediated emission signatures could be tuned to have these nanomaterials exhibit multiple colors under one single wavelength excitation.These nanomaterials also possessed unique properties of intense fluorescence,high photostability and easy bioconjugation.These optically encoded silica nanomaterials are potential to be used as barcoding tags for multiplexed signaling and bioassays.4.Multiplexed detection of pathogenic bacteria DNA by using the optically encoded silica nanorods. A multiplexed microbead-based sandwich DNA hybridization assay by using the optically encoded silica nanorods as identification tags has been developed for detection of oligonucleotide sequences specific to three food-borne pathogenic Escherichia coli O157:H7,Listeria monocytogenes and Staphylococcus aureus simultaneously.For each bacteria strain,the capture and signal probes were selected within the sequence of a gene encoding a strain-specific toxin.The three capture probes were respectively immobilized onto different microbeads,and the encoded nanorods with three colors were each functionalized with signal probes for one of the pathogens.A mix of the capture probes functionalized microbeads were used to capture the complementary objective sequences,and then were exposed to a cocktail of the fluorescent nanorods labeled signal probes to provide detection signals.The microbead-based sandwich DNA hybridization assay was employed initially for single pathogenic bacteria DNA detection.Results showed that the detect limit of the assay was 0.3 nM DNA with a linear range of 0.3-1.9 nM DNA.The assay could be achieved in 2 h.Following the single-color study multiplexed microbead-based sandwich DNA hybridization assay was successfully used in the detection of the three target pathogenic bacteria DNA sequences simultaneously with encoded nanorods. This assay will be promising for multiplexed detection of nucleic acids of pathogenic bacteria.