纳米探针芯片技术用于微量乙肝病毒DNA的检测  

Detection of Hepatitis B Virus Deoxyribonucleic Acid Based on Gold Nanoparticle Probe Chip

作　　者：汪毅[1,2] 毛红菊[2] 臧国庆[1] 张宏莲[2] 金庆辉[2] 赵建龙[2] 

机构地区：[1]上海交通大学附属第六人民医院,上海200062 [2]中国科学院上海微系统与信息技术研究所,上海200050

出　　处：《分析化学》2010年第8期1133-1138,共6页Chinese Journal of Analytical Chemistry

基　　金：国家卫生部重大专项(Nos.2009ZX10004-105;2009ZX10004-301);上海市科委项目(Nos.0952nm05700;09ZR1437800)资助

摘　　要：利用两组探针修饰的微粒:(1)表面标记有可与待测乙肝病毒(HBV)DNA另一端结合的纳米金探针1(信号探针)以及可与信号探针部分结合的纳米金探针2(检测探针);(2)表面标记有可与待测HBVDNA一端结合的磁珠探针(捕捉探针1)。检测靶HBV DNA时,磁珠探针与信号探针在液相中可分别与HBV DNA靶序列一端结合最终形成三明治样结构。再以磁场将三明治样复合物从反应液中分离,以DTT溶液将信号探针从纳米金颗粒上洗脱。洗脱后的信号探针数量反映靶基因的多寡,信号探针一段与预先点样的基因芯片上的捕捉探针2结合,检测探针与信号探针另一段相结合,最后用银染液将检测探针显色从而得到靶目标DNA相对定量信息。结果表明,本检测方法的检测灵敏度达到10-15mol/L水平。检测时间少于1.5h,检测结果与HBV DNA水平呈现较好的线性关系且无假阳性结果;本方法有望用于乙肝病人血清中HBV DNA的快速筛测及其它微生物基因的检测。A nanoparticle-based bio-barcode amplification （BCA） approach was developed for the sensitive detection of hepatitis B virus （HBV） DNA. The BCA approach employed two sets of particles： （1） two-component oligonucleotide-modified gold nanoparticles （AuNPs）： one oligonuc-leotide probe called signal probe （barcode DNA） is partial complementary with the target sequence of HBV DNA and another one called detection probe is partial complementary with the signal probe; （2） single component oligonucleotide modified magnetic microparticles （MMPs）. In the presence of the target molecule,the gold nanoparticles and magnetic particles formed sandwich hybrids. Along with the isolation of sandwiched hybrids from the sample using a magnet,non-specific bound gold nanoparticles were removed,ensuring that only target-bound gold nanoparticles were collected. Subsequently,the bar-code DNAs were dehybridized from the gold nanoparticle by using the dithiothreitol （DTT）. The detection probe with AuNP mixed with bar-code DNA solution was then added to a bar-code capture DNA-modified chip,and the spots on chip were labeled with bar-code DNA strands and AuNP probes. Finally,the scanometric detection of silver stain was introduced to further amplify the signal. The results showed that it was possible in a format that offers low fmol/L （10-15 mol/L） sensitivity in the detection of HBV DNA sample and there is a good linear relationship between target concentration and spot intensity on chip. The detection of the assay could be fulfilled within 1.5 h. The method could provide a new generation of diagnostic assays for HBV or the other infectious disease.

关 键 词：乙肝病毒DNA 纳米金探针 生物条形码扩增 纳米探针芯片 

分 类 号：R440[医药卫生—诊断学]