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The detection of HBV DNA with gold nanoparticle gene probes

查看全文 作　　者：Dong [1]Xia;Xiaoping [2]Luo;Qin [1,3]Ning;Qianghua [4]Lu;Kailun [4]Yao;Zuli [4]Liu 高影响力作者 机构地区：[1]Laboratory of Infectious Immunology,Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China;[2]Department of Pediatrics,Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China;[3]Department of Infectious Disease, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China;[4]Department of Physics, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China高影响力机构 出　　处：《Journal of Nanjing Medical University》索引2007年第21卷第4期,共6页高影响力期刊 基　　金：This project was supported by National High Technology Program( No.2002AA302202);International S&T Cooperation Program of China(2003DF000034);S&T Program of Wuhan(20041003068-05) 摘　　要：Objective: Gold nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA mea-surement was studied. Methods: Alkanethiol modified oligonucleotide was bound with self-made Au nanoparticles to formnanoparticle HBV DNA gene probes, through covalent binding of Au-S. By using a fluorescence-based method, the number ofthiol-derivatized, single-stranded oligonucleotides and their hybridization efficiency with complementary oligonucleotides in solutionwas determined. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotides serving as detection probes, andoligonucleotides immobilized on a nylon membrane surface acting as capturing probes, HBV DNA was detected visually by sand-wich hybridization based on highly sensitive aggregation and silver staining. The modified nanoparticle HBV DNA gene probeswere also used to detect the HBV DNA extracted from serum in patients with hepatitis B. Results: Compared with bare Aunanoparticles, oligonucleotide modified nanoparticles had a higher stability in NaCl solution or under high temperature environ-ment and the absorbance peak of modified Au nanoparticles shifted from 520nm to 524nm. For Au nanoparticles, the maximaloligonucleotide surface coverage of hexaethiol 30-mer oligonucleotide was (132 ±10) oligonucleotides per nanoparticle, and thepercentage of hybridization strands on nanoparticles was (22 ±3%). Based on a two-probe sandwich hybridization/nanoparticleamplification /silver staining enhancement method, Au nanoparticle gene probes could detect as low as 10-11 mol/L composite HBVDNA molecules on a nylon membrane and the PCR products of HBV DNA visually. As made evident by transmission electron mi-croscopy, the nanoparticles assembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to de-tect HBV DNA molecules in liquid. Conclusion:Our results showed that successfully prepared Au nanoparticle HBV DNA geneprobes could be used to detect HBV DNA directly. The detection-visuallized method has many advantages, including high sensitivity,simple operation and low cost. This technique has potential applications in many fields, especially in multi-gene detection chips. 关 键 词：乙型肝炎 DNA 病毒检测 毫微粒基因
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