高等学校化学学报 ›› 2011, Vol. 32 ›› Issue (10): 2274.

• 研究论文 • 上一篇    下一篇

基于二氧化硅荧光纳米颗粒与核酸染料SYBR Green Ⅰ的双色显微成像技术用于E.coli O157∶H7的检测

周丽霞1,2,4,何定庚2,3,4,何晓晓1,2,4,石慧2,3,4,王柯敏1,2,3,4,曹杰2,3,4   

  1. 1. 湖南大学生物学院,
    2. 化学生物传感与计量学国家重点实验室,
    3. 化学化工学院,
    4. 生物纳米与分子工程湖南省重点实验室, 长沙 410082
  • 收稿日期:2010-11-29 修回日期:2011-01-09 出版日期:2011-10-10 发布日期:2011-09-11
  • 通讯作者: 王柯敏;何晓晓 E-mail:kmwang@hnu.cn;xiaoxiaohe@hnu.cn
  • 基金资助:

    科技部国际合作专项(批准号: 2010DFB30300)、国家“九七三”计划项目(批准号: 2002CB513100-10)、国家自然科学基金(批准号: 90606003, 20775021)和湖南省自然科学基金创新研究群体(批准号: 10JJ7002)资助.

Detection of E. coli O157∶H7 by Fluorescent Silica Nanoparticles and SYBR Green Ⅰ Based Two\|color Microscopic Imaging Technique

ZHOU Li-Xia1,2,4, HE Ding-Geng2,3,4, HE Xiao-Xiao1,2,4*, SHI Hui2,3,4, WANG Ke-Min1,2,3,4*, CAO Jie2,3,4   

  1. 1. College of Biology,
    2. State Key Laboratory of Chemo/Biosensing and Chemometrics,
    3. College of Chemistry and Chemical Engineering,
    4. Key Laboratory for Bio\|Nanotechnology and Molecule Engineering of Hunan Province, Hunan University, Changsha 410082, China
  • Received:2010-11-29 Revised:2011-01-09 Online:2011-10-10 Published:2011-09-11
  • Contact: WANG Ke-Min;HE Xiao-Xiao E-mail:kmwang@hnu.cn;xiaoxiaohe@hnu.cn
  • Supported by:

    科技部国际合作专项(批准号: 2010DFB30300)、国家“九七三”计划项目(批准号: 2002CB513100-10)、国家自然科学基金(批准号: 90606003, 20775021)和湖南省自然科学基金创新研究群体(批准号: 10JJ7002)资助.

摘要: 将免疫荧光纳米标记技术与激光共聚焦显微成像方法相结合, 发展了一种基于二氧化硅荧光纳米颗粒和核酸染料SYBR Green Ⅰ的双色显微成像技术用于大肠杆菌O157∶H7的检测. 采用联吡啶钌(RuBpy)二氧化硅荧光纳米颗粒对羊抗大肠杆菌O157∶H7抗体进行修饰, 基于抗体\|抗原相互作用实现了其对目标大肠杆菌O157∶H7的特异性标记; 同时以核酸染料SYBR Green Ⅰ对细菌进行染色, 将细菌和纳米颗粒团聚体区分开, 实现了对大肠杆菌O157∶H7的双色标记, 并通过激光共聚焦显微镜进行免分离的荧光成像检测. 结果表明, 该方法可用于缓冲溶液体系和混合细菌样品中目标大肠杆菌O157∶H7的特异性检测, 在仅含5%目标菌的混合样品中仍能观察到具有明显黄色荧光的大肠杆菌O157∶H7, 且整个检测步骤包括样品预处理可在3 h内完成. 该方法则具有较好的灵敏度, 可检出2.6×103 Cell/mL的目标细菌样品. 若采用针对其它病原菌细胞壁抗原的特异性抗体, 则有望发展成为一种通用技术用于多种病原菌的快速和灵敏检测.

关键词: 二氧化硅荧光纳米颗粒, SYBR Green Ⅰ, 大肠杆菌O157∶H7, 双色显微成像

Abstract: A novel two-color immunofluorescent microscopic imaging technique for the rapid and sensitive detection of E. coli O157∶H7 was developed by fluorescent silica nanoparticles and SYBR Green Ⅰ. 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. The bacteria sample was then stained with a nucleic acid dye SYBR Green I and imaged with confocal microscope. 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 h, and the detection limit for E. coli O157∶H7 was 2.6×103Cell/mL. Considering the antibodies for various bacteria, this proposed method might be promising for rapid and sensitive detection of other bacteria.

Key words: Fluorescent silica nanoparticle, SYBR Green Ⅰ, E. coli O157∶H7, Two-color microscopic imaging

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