【作者】 秦迪岚；

【导师】 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2008， 博士

【摘要】 病原菌检测涉及到食品安全检验、疾病诊断、环境监测以及反生物恐怖等领域,与人类健康、社会安定和国家安全息息相关。传统的病原菌检测方法灵敏度低、费时耗力。各种建立在现代分子生物学、免疫学技术和现代分析仪器基础上的病原菌快速检测方法,为提高检测的灵敏度和可靠性做出了重要贡献,然而这些方法仍然存在各种局限性。功能化纳米材料具有许多普通材料无可比拟的优良特性,在病原菌的快速、灵敏检测上显示出广阔的应用前景。其中,硅壳荧光纳米材料是荧光最强的纳米材料之一,基于硅壳荧光纳米材料建立的荧光纳米标记技术比传统荧光标记技术具有更高的灵敏度和更好的光稳定性,为样品中少量病原菌的超灵敏检测提供了新的契机。本论文瞄准病原菌检测这一研究方向,在对各种病原菌检测方法进行简要综述的基础上,以荧光纳米标记与编码技术用于结核杆菌、大肠杆菌O157:H7等重要病原菌的快速、灵敏和多元检测为主线,主要开展了以下几个方面的研究工作:一、基于硅壳荧光纳米颗粒的间接免疫荧光显微镜法(FNP-IIFM)用于结核杆菌快速检测的研究。将荧光纳米标记技术与免疫荧光分析法相结合,发展了一种新型的基于硅壳荧光纳米颗粒的间接免疫荧光显微镜法用于结核杆菌的快速检测。通过采用兔抗结核杆菌抗体对目标结核杆菌进行特异性识别后,以修饰了葡萄球菌蛋白A的联吡啶钌(RuBpy)硅壳荧光纳米颗粒作为信号指示,利用葡萄球菌蛋白A与抗体之间的特异性结合,实现对结核杆菌的间接标记,通过荧光显微镜对标记的结核杆菌进行荧光成像检测。结果表明,该方法可以成功应用于对缓冲液体系、混合细菌样品和掺杂痰液样品中结核杆菌的检测,整个检测步骤包括样品预处理可在4h内完成;并且,采用硅壳荧光纳米颗粒作为荧光标记物用于结核杆菌的检测比传统荧光染料具有更高的信号强度与更好的光稳定性,有望应用于临床痰液标本中结核杆菌的快速检测。二、基于硅壳荧光纳米颗粒和SYBR Green I的双色流式细胞术(FSiNP@SG-FCM)用于结核杆菌检测的研究。将基于硅壳荧光纳米颗粒的免疫荧光分析法与流式细胞术相结合,发展了一种新型的基于功能化RuBpy硅壳荧光纳米颗粒和SYBR Green I的改良双色流式细胞术用于结核杆菌的检测。采用RuBpy硅壳纳米颗粒标记的抗体对样品中目标结核杆菌进行免疫荧光染色后,以核酸染料SYBR Green I对样品中细菌的核酸进行染色,从而将细菌与样品中的碎片杂质区分,通过多参数流式细胞术对样品中双染色的结核杆菌进行测定与分析。将该方法用于对缓冲液体系和掺杂尿液样品中的结核杆菌进行检测。结果表明,该方法显著地减小了由纳米颗粒团聚以及纳米颗粒对杂质碎片的非特异性吸附所引起的假阳性信号,对缓冲液体系和掺杂尿液样品的检测下限分别为3.5×10~3和3.0×10~4 cells/ml结核杆菌,并且比基于FITC标记的传统流式细胞术具有更高的灵敏度,整个检测步骤包括样品预处理可在2h内完成,有望应用于临床尿液标本中结核杆菌的快速检测。三、多色光学编码硅壳荧光纳米材料的制备研究。基于荧光共振能量转移(FRET)与反相微乳液法硅壳荧光纳米材料制备技术,发展了一种简易的多色光学编码硅壳荧光纳米材料的制备新方法。采用两种FRET供受体荧光染料按不同的比例与载体材料人免疫球蛋白(或多聚赖氨酸)作用制备荧光编码的载体材料,以荧光编码的载体材料为内核材料,利用氨水催化硅烷化试剂在反相微乳液中水解包裹内核材料制备成一系列多色光学编码硅壳纳米材料。对两类光学编码硅壳纳米材料的形貌和荧光性质进行考察,结果表明,以人免疫球蛋白为载体材料制备的光学编码硅壳纳米材料为球形,而以多聚赖氨酸作为载体材料制备的光学编码硅壳纳米材料为棒状,通过改变供受体染料的标记比,可以获得一系列具有不同荧光发射光谱性质的纳米球或纳米棒,由于荧光共振能量转移效应,这些材料能以同一波长的光激发出不同颜色的荧光,而且荧光强、性质稳定、制备方法简便易行,在病原菌多元检测、多基因表达分析、蛋白质多元分析、多通道生物学测定、医学诊断学等方面都有广阔的应用前景。四、多色光学编码硅壳荧光纳米棒用于病原菌DNA片断多元检测的研究。基于多色光学编码硅壳荧光纳米棒建立多元荧光标记技术并与三明治固相核酸杂交检测技术相结合,发展了一种可用于对大肠杆菌O157:H7、单核细胞增生性李斯特菌和金黄色葡萄球菌三种常见食源性病原菌DNA片断同时检测的多元分析方法。针对三种目标菌编码特异性毒素的基因序列合成目标DNA片断以及特异性的捕获探针和信号探针,在微珠上分别修饰三种捕获探针,对三种细菌目标DNA片断进行杂交捕获后,以分别修饰了三种信号探针的三种不同颜色的光学编码硅壳纳米棒作为信号指示对目标DNA进行杂交检测。利用该方法对单一目标细菌DNA片断检测性能的考察表明,方法的检测下限为0.3 nM DNA、线性范围为0.3-1.9 nM DNA、可在2 h内完成分析;在此基础上进一步利用三种不同颜色的光学编码硅壳纳米棒作为信号指示与三明治核酸杂交相结合成功地实现了对大肠杆菌O157:H7、单核细胞增生性李斯特菌和金黄色葡萄球菌三种食源性病原菌DNA片断的同时检测,极大地提高了检测的效率。该方法可望用于病原菌基因的多元分析中。更多还原

【Abstract】 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.更多还原