Fe_3O_4(核)/Au(壳)纳米颗粒探针的制备及其在检测乙型肝炎病毒DNA中的应用  

HBV DNA detection with oligodeoxynucleotide modified Fe_3O_4(core)/Au(shell) nanoparticle probes

作　　者：习东[1] 宁琴[1] 卢强华[2] 姚凯伦[2] 刘祖黎[2] 罗小平[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院感染免疫研究室,武汉430030 [2]华中科技大学物理系

出　　处：《中华检验医学杂志》2006年第4期339-345,共7页Chinese Journal of Laboratory Medicine

基　　金：国家863高技术计划资金资助项目(2002AA302202);科技部"国际科技合作重点项目计划"(2003DF000034);武汉市科技计划资助项目(20041003068-05)

摘　　要：目的制备Fe3O4(核)/Au(壳)纳米颗粒乙型肝炎病毒脱氧核糖核酸(hepatitisBvirusdeoxynucleicacid,HBVDNA)基因探针,研究其在检测HBVDNA中的应用。方法分别用枸橼酸还原金氯酸法、化学共沉淀法制备Au、Fe3O4纳米颗粒,二法结合制备Fe3O4(核)/Au(壳)纳米颗粒。通过金-硫(Au-S)共价键,将烷氢硫醇修饰的寡核苷酸结合在Fe3O4(核)/Au(壳)纳米颗粒上,制备纳米颗粒HBVDNA基因探针。用荧光标记法检测纳米颗粒探针表面寡核苷酸的覆盖率和与其互补的寡核苷酸的杂交效率。在尼龙膜上,使用Fe3O4(核)/Au(壳)纳米颗粒基因探针通过斑点杂交法、Ag染色法检测HBVDNA。在纳米颗粒基因探针液相检测体系中加入HBVDNA,透射电镜下观察。结果制备的Fe3O4(核)/Au(壳)纳米颗粒粒径均匀,为(15±5)nm,水相分散良好,具有磁性。Fe3O4(核)/Au(壳)纳米颗粒基因探针表面寡核苷酸的最大覆盖率为(120±8)条,最大杂交效率为(14±2)%。斑点杂交法可检出低至1010copies/ml的合成靶DNA,可目视化检出乙肝患者血清中HBVDNA的PCR产物。在液相检测体系中加入HBVDNA,透射电镜观察到纳米颗粒组装成大的网状结构的聚集体。结论成功制备了Fe3O4(核)/Au(壳)纳米颗粒HBVDNA基因探针,它可直接用于HBVDNA的检测。Objective To increase the sensitivity and specifity of HBV DNA measurement by using oigodeoxynucleotide modified Iron oxide （ Fe3O4, core ）/Au （ shell ） nanoparticle probes. Methods Au nanoparticles were produced via citrate reduction of tetrachloroauric acid （ HAuCl4）. Superparamagnetic iron oxide nanoparticles （ SPION ） were produced with chemical precipitation. The coating of Au on magnetic nanoparticles was performed via citrate reduction of HAuCl4 on the surface of SPION that served as seeds. Alkanethiol modified oligonucleotides were bound with self-made Fe3O4 （core）/Au （shell） nanoparticles to form nanoparticle HBV DNA gene probes through covalent binding of Au-S. By using a fluorescence-based method, the number of thiol-derivatized single-stranded oligonucleoticles on nanoparticle and their hybridization efficiency with complementary oligonucleotides in solution were determined. With the aid of Fe3O4 （ core ）/Au （ shell ） nanoparticle-supported mercapto-modified oligonucleotides serving as detection probes, and oligonucleotides immobilized on nylon membrane surface acting as capturing probes, HBV DNA was detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. The modified nanoparticle HBV DNA gene probes were also used to detect the HBV DNA extracted from serum in patients with hepatitis B. Four irrelevant DNA fragments with the same length as composite HBV DNA were used as negative controls for their specificity evaluation. Results Transmission electron microscopy（TEM） showed prepared Fe3O4 （ core）/Au（shell） nanoparticles were well-dispersed, and their diameter were （15± 5 ） nm. In comparison with bare Fe3O4 （core）/Au（shell） nanoparticles, the absorbance peak of oligonucleotide modified Fe3O4 （ core）/Au（shell） nanoparticles shifted from 522 nm to 525 nm. For Fe3O4 （core）/Au（shell） nanoparticles, the maximal oligonucleoticle surface coverage of hexaethiol 30-mer oligonucleoticles is （1

关 键 词：肝炎病毒 乙型 DNA DNA探针 

分 类 号：R450[医药卫生—治疗学]