纳米颗粒基因探针的制备及其在检测肝炎病毒中的应用
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摘要
【背景及目的】
乙型、丙型病毒性肝炎是分别由乙型肝炎病毒(hepatitis B virus, HBV)、丙型肝炎病毒(hepatitis C virus, HCV)引起的危害严重的传染病。我国是乙型肝炎和丙型肝炎高发区。乙型肝炎和丙型肝炎的合并感染率高,其中尤以药瘾者及输血者的合并感染率为最高。目前,检测HBV脱氧核糖核酸(deoxynucleic acid, DNA)、HCV核糖核酸(nucleic acid, RNA)是判断病毒复制水平,观察疗效和评估预后的主要指标。病毒核酸的检测方法主要有两类,一类是聚合酶链反应(PCR),另一类是核酸杂交。临床检验的常规方法是PCR,PCR对实验室环境要求高,结果可出现假阳性,作为临床常规检验手段尚有争议。目前常规检测HBV和HCV的PCR技术是用不同的反应体系分别扩增血清标本中病毒核酸,既繁琐又增加污染机会。为实现同步检测HBV和HCV,研究人员用多重PCR在一个反应体系内同时扩增HBV和HCV的病毒核酸,已取得了一定成效,但用多重引物对多个模板同时扩增具有较大难度,特别是HCV感染者血清中病毒浓度较低。核酸杂交需要检测核酸的基因探针,如放射性标记基团、荧光基团、化学发光基团。目前,放射性标记探针已被非放射性标记取代,但荧光基团、化学发光基团易于淬灭,应用具有局限性。因此有必要研发特异、敏感、经济、安全并方便于临床使用的检测探针和技术。
DNA分子的大小在纳米尺度内。近年来兴起的DNA纳米技术是将DNA结合到纳米颗粒上构成纳米颗粒基因探针,通过与靶DNA之间的互补实现纳米颗粒的组装,已成为一种新型的DNA检测系统。具有高电子密度的金(Au)纳米颗粒在透射电镜下易于观察,Au纳米颗粒聚集为微米大小即呈现肉眼可见的红色,银染色可放大检测信号。除Au之外,多种纳米材料已被开发,其中核/壳结构的纳米颗粒备受瞩目。本课题通过化学方法制备Au、Fe_3O_4(核)/Au(壳)纳米颗粒HBV DNA、HCV cDNA基因探针,通过尼龙膜上斑点杂交或透射电镜观察液相中杂交聚集体的方法检测病毒核酸,以期建立纳米颗粒直接检测病毒基因的方法。同时,利用Fe_3O_4(核)/Au(壳)纳米颗粒基因探针在磁场中的特殊性质,通过高梯度磁场进行杂交聚集体的分离,探索Fe_3O_4(核)/Au(壳)纳米颗粒基因探针通过磁场检测病毒基因的新方法。进而,我们以检测HBV、HCV双重感染为目的,对纳米金比色芯片同步目视化检测方法的可行性和有效性进行研究,并探讨用Au纳米颗粒基因探针在透射电镜下同步检测HBV、HCV双重感染的方法。
【方法】
分别用柠檬酸还原金氯酸法、化学共沉淀法制备Au、Fe_3O_4纳米颗粒,二法结合制备Fe_3O_4(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Au、Fe_3O_4(核)/Au(壳)纳米颗粒上,制备Au、Fe_3O_4(核)/Au(壳)纳米颗粒HBV DNA、HCV cDNA基因探针,紫外-可见分光光度计进行波长扫描。用荧光标记法检测纳米颗粒基因探针表面寡核苷酸的覆盖数和与其互补的寡核苷酸的杂交效率。提取乙型肝炎病人血清中HBV DNA,PCR扩增。同时提取乙型、丙型肝炎合并感染病人血清中的病毒DNA和RNA,利用多重PCR方法扩增HBV DNA和HCV cDNA。纳米颗粒基因探针检测病毒核酸的方法有三种:方法一为检测探针与固定在尼龙膜上的捕捉链构成双探针,用斑点杂交法检测HBV DNA的PCR产物或者HBV DNA和HCV cDNA的多重PCR产物,加入银离子(Ag+)-对苯二酚液染色观察结果;方法二为在含有Au或者Fe_3O_4(核)/Au(壳)纳米颗粒基因探针的液相检测体系中加入HBV DNA和/或HCV cDNA,透射电镜下观察;方法三是将Fe_3O_4(核)/Au(壳)纳米颗粒基因探针杂交体系通过高梯度磁场,检测磁场处理前后Au特征性吸收峰值的改变。
【结果】
制备的Au纳米颗粒、Fe_3O_4(核)/Au(壳)纳米颗粒粒径分别为(10±5)nm、(15±5)nm,水相分散良好。Au、Fe_3O_4(核)/Au(壳)纳米颗粒探针表面寡核苷酸的最大覆盖数分别为(132±10)条、(120±8)条,最大杂交效率分别为(22±3)%、(14±2)%。Au、Fe_3O_4(核)/Au(壳)纳米颗粒分别在520nm、522 nm有最大吸收峰,经寡核苷酸修饰后,最大吸收峰分别改变为524nm、525 nm。Au、Fe_3O_4(核)/Au(壳)纳米颗粒在尼龙膜上用斑点杂交法可分别检出低至10fmol、100 fmol的合成靶DNA,可目视化检出乙型肝炎患者血清中HBV DNA的PCR产物。在纳米颗粒基因探针液相检测体系中加入HBV DNA,透射电镜观察到纳米颗粒组装成大的网状结构的聚集体。通过高梯度磁场可以将磁性探针与靶DNA形成的杂交聚集体与对照进行区分。同时提取获得乙型、丙型肝炎合并感染病人血清中病毒RNA及DNA,多重PCR同时扩增HBV DNA和HCV cDNA,纳米金比色芯片可以同步目视化检出乙、丙型肝炎合并感染病人血清中HBV DNA和HCV RNA的PCR产物。使用Au纳米颗粒基因探针通过透射电镜可同步检出乙、丙型肝炎合并感染患者血清中提取的HBV DNA和HCV RNA。
【结论】
1、成功制备了Au、Fe_3O_4(核)/Au(壳)纳米颗粒基因探针,它们可直接用于肝炎病毒的检测。
2、通过芯片分析技术可实现目视化检测HBV DNA的PCR产物或乙、丙型肝炎合并感染患者的HBV DNA和HCV RNA的多重PCR产物,纳米金比色芯片诊断方法操作简单、特异性好、成本低廉、结果判定指标客观,可望在许多领域、尤其是病毒基因检测领域有广泛用途,此类基因芯片在多基因检测芯片上有潜在的应用价值。
3、通过透射电镜,使用纳米颗粒基因探针可直接检测乙肝病人阳性血清中提取的HBV DNA,可同步检出乙、丙型肝炎合并感染患者血清中提取的HBV DNA和HCV cDNA,甚至可达到无需PCR水平,此方法具有敏感性高、特异性好的特点。
4、Fe_3O_4(核)/Au(壳)纳米颗粒基因探针杂交体系通过高梯度磁场可以将磁性探针与靶DNA形成的杂交聚集体与对照进行区分,表明磁性纳米颗粒基因探针结合磁场应用进行核酸检测简单、易行、实用。
Sequence-specific methods for detecting polynucleotides are critical to the diagnosis of genetic and pathogenic diseases. Most detection systems make use of the hybridization of a target polynucleotide with oligo- or polynucleotide probes containing covalently linked reporter groups, which include radioactive labels, fluorescent labels and chemilumine- scence schemes, etc. Each of these strategies has advantages and disadvantages and no single method has gained supremacy. During the last decade, there has been an increasing interest in using nanoparticles in DNA detection. In the present paper, we prepared gold or gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes and using these probes we established a methology to detect HBV DNA molecule extracted from hepatitis B patient on nylon membrane by blot-hybridization or in liquid media by TEM. We have examined the feasibility of the detection of HBV DNA with gold-coated iron oxide nanoparticles and magnetic separator. And we further established a simple and rapid visual method based on either DNA microarray or TEM with nanogold supported probes for detecting HBV and HCV simultaneously.
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 oligonucleotide was bound with self-made Au and Fe_3O_4(core)/Au(shell)nanoparticles to form nanoparticle HBV DNA gene probes through covanlt binding of Au-S. With a fluorescence-based method, for Au nanoparticles and Fe_3O_4(core)/Au(shell) nanoparticles, the maximal surface coverage of hexaethiol 30-mer oligonucleotides was (132±10) and (120±8) oligonucleotides per nanoparticle, and the maximal percentage of hybridization strands on nanoparticles was (22±3)% and (14±2)%, respectively. Based on a two-probe sandwich hybridization/ nanoparticle amplification/ silver staining enhancement method, Au nanoparticle and Fe_3O_4(core)/Au(shell) nanoparticle gene probes could detect as low as 10-11mol/L and 10-10mol/L composite HBV DNA molecules on nylon membrane, respectively, and the PCR products of HBV DNA visually. As evidenced by transmission electron microscopy, the nanoparticles assembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to detect HBV DNA molecules.
HBV DNA and HCV RNA extracted from serum in patients with HBV and HCV coinfection can be specifically amplified simultaneously in one system by multi-primer PCR. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotide serving as detection probe, and oligonucleotide immobilized on nylon membrane surface acting as capturing probe, HBV and HCV PCR products were detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. As evidenced by transmission electron microscopy, large network aggregates assembled by nanoparticles identifying HBV and HCV simultaneously in serum in patients with HBV and HCV coinfection were seen clearly.
Our results suggested that DNA modified nanoparticle probe can be used to detect virus nucleic acid. The established oligonucleotide array for the detection of HBV and HCV coinfection is convenient and efficient with high specificity. 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. It provides a nationale for the measurement of other viruses coinfection. Transmission electron microscopy can detect HBV or HBV and HCV simultaneously in serum in patients. Detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method.
乙型、丙型病毒性肝炎是分别由乙型肝炎病毒(hepatitis B virus, HBV)、丙型肝炎病毒(hepatitis C virus, HCV)引起的危害严重的传染病。我国是乙型肝炎和丙型肝炎高发区。乙型肝炎和丙型肝炎的合并感染率高,其中尤以药瘾者及输血者的合并感染率为最高。目前,检测HBV脱氧核糖核酸(deoxynucleic acid, DNA)、HCV核糖核酸(nucleic acid, RNA)是判断病毒复制水平,观察疗效和评估预后的主要指标。病毒核酸的检测方法主要有两类,一类是聚合酶链反应(PCR),另一类是核酸杂交。临床检验的常规方法是PCR,PCR对实验室环境要求高,结果可出现假阳性,作为临床常规检验手段尚有争议。目前常规检测HBV和HCV的PCR技术是用不同的反应体系分别扩增血清标本中病毒核酸,既繁琐又增加污染机会。为实现同步检测HBV和HCV,研究人员用多重PCR在一个反应体系内同时扩增HBV和HCV的病毒核酸,已取得了一定成效,但用多重引物对多个模板同时扩增具有较大难度,特别是HCV感染者血清中病毒浓度较低。核酸杂交需要检测核酸的基因探针,如放射性标记基团、荧光基团、化学发光基团。目前,放射性标记探针已被非放射性标记取代,但荧光基团、化学发光基团易于淬灭,应用具有局限性。因此有必要研发特异、敏感、经济、安全并方便于临床使用的检测探针和技术。
DNA分子的大小在纳米尺度内。近年来兴起的DNA纳米技术是将DNA结合到纳米颗粒上构成纳米颗粒基因探针,通过与靶DNA之间的互补实现纳米颗粒的组装,已成为一种新型的DNA检测系统。具有高电子密度的金(Au)纳米颗粒在透射电镜下易于观察,Au纳米颗粒聚集为微米大小即呈现肉眼可见的红色,银染色可放大检测信号。除Au之外,多种纳米材料已被开发,其中核/壳结构的纳米颗粒备受瞩目。本课题通过化学方法制备Au、Fe_3O_4(核)/Au(壳)纳米颗粒HBV DNA、HCV cDNA基因探针,通过尼龙膜上斑点杂交或透射电镜观察液相中杂交聚集体的方法检测病毒核酸,以期建立纳米颗粒直接检测病毒基因的方法。同时,利用Fe_3O_4(核)/Au(壳)纳米颗粒基因探针在磁场中的特殊性质,通过高梯度磁场进行杂交聚集体的分离,探索Fe_3O_4(核)/Au(壳)纳米颗粒基因探针通过磁场检测病毒基因的新方法。进而,我们以检测HBV、HCV双重感染为目的,对纳米金比色芯片同步目视化检测方法的可行性和有效性进行研究,并探讨用Au纳米颗粒基因探针在透射电镜下同步检测HBV、HCV双重感染的方法。
【方法】
分别用柠檬酸还原金氯酸法、化学共沉淀法制备Au、Fe_3O_4纳米颗粒,二法结合制备Fe_3O_4(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Au、Fe_3O_4(核)/Au(壳)纳米颗粒上,制备Au、Fe_3O_4(核)/Au(壳)纳米颗粒HBV DNA、HCV cDNA基因探针,紫外-可见分光光度计进行波长扫描。用荧光标记法检测纳米颗粒基因探针表面寡核苷酸的覆盖数和与其互补的寡核苷酸的杂交效率。提取乙型肝炎病人血清中HBV DNA,PCR扩增。同时提取乙型、丙型肝炎合并感染病人血清中的病毒DNA和RNA,利用多重PCR方法扩增HBV DNA和HCV cDNA。纳米颗粒基因探针检测病毒核酸的方法有三种:方法一为检测探针与固定在尼龙膜上的捕捉链构成双探针,用斑点杂交法检测HBV DNA的PCR产物或者HBV DNA和HCV cDNA的多重PCR产物,加入银离子(Ag+)-对苯二酚液染色观察结果;方法二为在含有Au或者Fe_3O_4(核)/Au(壳)纳米颗粒基因探针的液相检测体系中加入HBV DNA和/或HCV cDNA,透射电镜下观察;方法三是将Fe_3O_4(核)/Au(壳)纳米颗粒基因探针杂交体系通过高梯度磁场,检测磁场处理前后Au特征性吸收峰值的改变。
【结果】
制备的Au纳米颗粒、Fe_3O_4(核)/Au(壳)纳米颗粒粒径分别为(10±5)nm、(15±5)nm,水相分散良好。Au、Fe_3O_4(核)/Au(壳)纳米颗粒探针表面寡核苷酸的最大覆盖数分别为(132±10)条、(120±8)条,最大杂交效率分别为(22±3)%、(14±2)%。Au、Fe_3O_4(核)/Au(壳)纳米颗粒分别在520nm、522 nm有最大吸收峰,经寡核苷酸修饰后,最大吸收峰分别改变为524nm、525 nm。Au、Fe_3O_4(核)/Au(壳)纳米颗粒在尼龙膜上用斑点杂交法可分别检出低至10fmol、100 fmol的合成靶DNA,可目视化检出乙型肝炎患者血清中HBV DNA的PCR产物。在纳米颗粒基因探针液相检测体系中加入HBV DNA,透射电镜观察到纳米颗粒组装成大的网状结构的聚集体。通过高梯度磁场可以将磁性探针与靶DNA形成的杂交聚集体与对照进行区分。同时提取获得乙型、丙型肝炎合并感染病人血清中病毒RNA及DNA,多重PCR同时扩增HBV DNA和HCV cDNA,纳米金比色芯片可以同步目视化检出乙、丙型肝炎合并感染病人血清中HBV DNA和HCV RNA的PCR产物。使用Au纳米颗粒基因探针通过透射电镜可同步检出乙、丙型肝炎合并感染患者血清中提取的HBV DNA和HCV RNA。
【结论】
1、成功制备了Au、Fe_3O_4(核)/Au(壳)纳米颗粒基因探针,它们可直接用于肝炎病毒的检测。
2、通过芯片分析技术可实现目视化检测HBV DNA的PCR产物或乙、丙型肝炎合并感染患者的HBV DNA和HCV RNA的多重PCR产物,纳米金比色芯片诊断方法操作简单、特异性好、成本低廉、结果判定指标客观,可望在许多领域、尤其是病毒基因检测领域有广泛用途,此类基因芯片在多基因检测芯片上有潜在的应用价值。
3、通过透射电镜,使用纳米颗粒基因探针可直接检测乙肝病人阳性血清中提取的HBV DNA,可同步检出乙、丙型肝炎合并感染患者血清中提取的HBV DNA和HCV cDNA,甚至可达到无需PCR水平,此方法具有敏感性高、特异性好的特点。
4、Fe_3O_4(核)/Au(壳)纳米颗粒基因探针杂交体系通过高梯度磁场可以将磁性探针与靶DNA形成的杂交聚集体与对照进行区分,表明磁性纳米颗粒基因探针结合磁场应用进行核酸检测简单、易行、实用。
Sequence-specific methods for detecting polynucleotides are critical to the diagnosis of genetic and pathogenic diseases. Most detection systems make use of the hybridization of a target polynucleotide with oligo- or polynucleotide probes containing covalently linked reporter groups, which include radioactive labels, fluorescent labels and chemilumine- scence schemes, etc. Each of these strategies has advantages and disadvantages and no single method has gained supremacy. During the last decade, there has been an increasing interest in using nanoparticles in DNA detection. In the present paper, we prepared gold or gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes and using these probes we established a methology to detect HBV DNA molecule extracted from hepatitis B patient on nylon membrane by blot-hybridization or in liquid media by TEM. We have examined the feasibility of the detection of HBV DNA with gold-coated iron oxide nanoparticles and magnetic separator. And we further established a simple and rapid visual method based on either DNA microarray or TEM with nanogold supported probes for detecting HBV and HCV simultaneously.
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 oligonucleotide was bound with self-made Au and Fe_3O_4(core)/Au(shell)nanoparticles to form nanoparticle HBV DNA gene probes through covanlt binding of Au-S. With a fluorescence-based method, for Au nanoparticles and Fe_3O_4(core)/Au(shell) nanoparticles, the maximal surface coverage of hexaethiol 30-mer oligonucleotides was (132±10) and (120±8) oligonucleotides per nanoparticle, and the maximal percentage of hybridization strands on nanoparticles was (22±3)% and (14±2)%, respectively. Based on a two-probe sandwich hybridization/ nanoparticle amplification/ silver staining enhancement method, Au nanoparticle and Fe_3O_4(core)/Au(shell) nanoparticle gene probes could detect as low as 10-11mol/L and 10-10mol/L composite HBV DNA molecules on nylon membrane, respectively, and the PCR products of HBV DNA visually. As evidenced by transmission electron microscopy, the nanoparticles assembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to detect HBV DNA molecules.
HBV DNA and HCV RNA extracted from serum in patients with HBV and HCV coinfection can be specifically amplified simultaneously in one system by multi-primer PCR. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotide serving as detection probe, and oligonucleotide immobilized on nylon membrane surface acting as capturing probe, HBV and HCV PCR products were detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. As evidenced by transmission electron microscopy, large network aggregates assembled by nanoparticles identifying HBV and HCV simultaneously in serum in patients with HBV and HCV coinfection were seen clearly.
Our results suggested that DNA modified nanoparticle probe can be used to detect virus nucleic acid. The established oligonucleotide array for the detection of HBV and HCV coinfection is convenient and efficient with high specificity. 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. It provides a nationale for the measurement of other viruses coinfection. Transmission electron microscopy can detect HBV or HBV and HCV simultaneously in serum in patients. Detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method.
引文
1张志馄,崔作林.《纳米材料与纳米技术》.第四版.北京.国防工业出版社,2001年7月. 4-6
2张立德,牟季美.《纳米材料与纳米结构》.第二版.北京.科学出版社,2001年3月. 2-3
3徐辉碧.《纳米医药》.第一版.北京.清华大学出版社,2004年1月. 299-301
4 R. E. Cavicchi, R. H. Silsbee. Coulomb Suppression of Tunneling Rate from Small Metal Particles. Phys. Rev. Lett. 1984, 52(5): 1453-1457
5 Lopez Quintela, M. A., Rivas, J., Liz, L., Winter, I. Quantum effects in ultrafine neodymium-iron-boron particles. NATO ASI Series, Series B: Physics . 1991, 259(Sci. Technol. Nanostruct. Magn. Mater.), 567-572
6 Ball P, Garwin L, Matrix-mediated synthesis of nanocrystallineγ-Fe2O3, a new optically transparent magnetic material. Nature. 1992, 355: 761-771
7 Christof M. Niemeyer. Nanoparticles, Proteins, and Nucleic Acids: Biotechnology Meets Materials Science. Angew. Chem. Int. Ed. 2001, 40: 4128-4158
8 Bawendi MG, Steigerwald M. L., Brus L E. The quantum mechanicsof larger semiconductor clusters (“quantum dots”). Annu. Rev. Phys. Chem. 1990 , 41: 477-496
9 Junu Chatterjee, Yousef Haik, Ching-Jen Chen. Polythylene magnetic nanoparticle: a new magnetic material for biomedical applications. Journal of Magnetism and Magnetic Materials. 2002, 246: 382-391
10 R. S. Molday, L .L. Molday. Separation of cells labeled with immunospecific iron dextran microspheres using high magnetic chromatography. FEBS. 1984, 170(2): 232-237
11 N.Cem Balci, Mustafa Sirvanci, Cihan Duran, et al. Hepatic adenomatosis MRI demonstration with the use of superparamagnetic iron oxide. Journal of Clinical Imaging 2002, 26: 35-38
12 Maire-France Bellin, Catherine Beigelman, Sophie Precetti-Morel. Iron oxide-enhanced MR lymphography: initial experience. European Journal of Radiology. 2000, 34: 257- 264
13 James J. Storhoff, Robert Elghanian, Robert C. Mucic, Chad A. Mirkin, Robert L.Letsinger. One-Pot Colorimetric Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle Probes. J. Am. Chem. Soc. 1998, 120: 1959-1964
14 T. Andrew Taton, Chad A. Mirkin, Robert L. Letsinger. Scanometric DNA Array Detection with Nanoparticle Probes. Science 2000, 289:1757-1760
15 So-Jung Park, T. Andrew Taton, Chad A. Mirkin. Array-Based Electrical Detection of DNA with Nanoparticle Probes. Science 2002, 295:1503-1506
16 YunWei Charles Cao, Rongchao Jin, Chad A. Mirkin. Nanoparticles with Raman Spectroscopic Fingerprints for DNA and RNA Detection. Science 2002, 297:1536-1540
17 J. Manuel Perez, Lee Josephson, Terence O’Loughin, Dagmar Hogemann, Ralph Weissleder. Magnetic relaxation switches capable of sensing molecular interactions. Nature Biotechnology, 2002, 20: 816-820
18 Lee Josephson, J. Manuel Perez, Ralph Weissleder. Magnetic Nanosensors for the Detection of Oligonucleotide Sequences. Angew. Chem. Int. Ed. 2001, 40: 3204-3206
19 Lin He, Michael D. Musick, Sheila R. Nicewarner, Frank G. Silinas, Stephen J. Benkovic, Michael J. Natan, Christine D. Keating. Colloidal Au-Enhanced Surface Plasmon Resonance for Ultrasensitive Detection of DNA Hybridization. J. Am. Chem. Soc. 2000, 122: 9071-9077
20 Yossi Weizmann, Fernando Patolsky, Eugenii Katz, Itamar Willner. Amplified DNA Sensing and Immunosensing by the rotation of Functional Magnetic Particles. J. Am. Chem. Soc. 2003, 125: 3452-3454
21 Joseph Wang, Ronen Polsky, Arben Merkoci, Kathryn L. Turner.“Electroactive Beads”for Ultrasensitive DNA Detection. Langmuir 2003, 19: 989-991
22 Joseph Wang, Danke Xu, Ronen Polsky. Magnetically-Induced Solid-State Electrochemical Detection of DNA Hybridization. J. Am. Chem. Soc. 2002, 124: 4208-4209
23 Alexandre I, Hamels S, Dufour S, et al. Colorimetric silver detection of DNA microarrays. Anal Biochem, 2001, 295(1):1~8.
24 Shubo Han, Jianqiao Lin, Feimeng Zhou, Robert Luis Vellanoweth. Oligonucleotide -Capped Gold Nanoparticles for Improved Atomic Force Microscopic Imaging and Enhanced Selectivity in Polynucleotide Detection. Biochemical and Biophysical Research Communications. 2000, 279: 265-269
25 Mirkin CA, Letsinger RL, Mucic RC, et al. A DNA-based method for rationallyassembling nanoparticle into macroscopic materials. Nature, 1996, 382:607~609.
26 A. P. Alivisatos, K. P. Johnsson, X. Peng, et al. Organization of Nanocrystal Molecules Using DNA. Nature 1996, 382: 609-611
27 G. Mie. Beitrage zur Optik Truber Medien, Speziell Kolloidaler Metallosungen. Ann. Phys. 1908, 25: 377-445
28 Frank Caruso. Nanoengineering of Particle Surfaces. Adv. Mater. 2001, 13: 11-22
29 E. E. Carpenter, A. Kumbhar, J. A. Wiemann, et al. Synthesis and magnetic properties of gold-iron-gold nanocomposites. Materials Science and Engineering A 2000, 286: 81-86
30 J. Lin, W. Zhou, A. Kumbhar, et al. Gold-Coated Iron (Fe@Au) Nanoparticles: Synthesis, Characterization, and Magnetic Field-Induced Self-Assembly. J. Solid State Chem. 2001, 159: 26-31
31 H. Yu, M. Chen, P. M. Rice, et al. Dumbbell-Like Bifunctional Au-Fe3O4 Nanoparticles. Nano Lett. 2005, 5: 379-382
32 T. Kinoshita, S. Seino, K. Okitsu, et al. Magnetic evaluation of nanostructure of gold-iron composite particles synthesized by reverse micelle method. J. Alloys & Compounds 2003, 359: 46-50
33 J. L. Lyon, D. A. Fleming, M. B. Stone, et al. Synthesis of Fe Oxide Core/Au Shell Nanoparticles by Iterative Hydroxylamine Seeding. Nano Lett. 2004, 4: 719-723
34 S-J. Cho, J-C. Idrobo, J. Olamit, et al. Growth Mechanisms and Oxidation Resistance of Gold-Coated Iron Nanoparticles. Chem. Mater. 2005, 17: 3181-3186
35 S-J. Cho, S. M. Kauzlarich, J. Olamit, et al. Characterization and magnetic properties of core-shell structured Fe/Au nanoparticles. J. Appl. Phys. 2004, 95: 6804-6806
36 M. Chen, S. Yamamuro, D. Farrell, et al. Gold-coated iron nanoparticles for biomedical applications. J. Appl. Phys. 2003, 93: 7551-7553
37 L. Wang, J. Luo, Q. Fan, et al. Monodispersed Core-Shell Fe3O4@Au Nanoparticles. J. Phys. Chem. B 2005, 109: 21593-21601
38 Demers LM, Mirkin CA, Mucic RC, et al. A fluorescence-based method for determing the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles. Anal Chem, 2000, 72:5535~5541.
39 L. Babes, B. Denizot, G. Tanguy, et al. Synthesis of Iron Oxide Nanoparticles Used as MRI Contrast Agent: A Parametric Study. J. Colloid and Interface Sci. 1999, 212: 474 -482
40 Pena SRN, Raina S, Goodrich GP, et al. Hybridization and enzymatic extension of Au nanoparticle-bound oligonucleotide. J Am Chem Soc. 2002,124(25):7314-7323.
41 Z.L. Liu, H.B. Wang, Q.H. Lu, et al. Synthesis and characterization of ultrafine well-dispersed magnetic nanoparticles. Journal of Magnetism and Magnetic Materials, 2004, 283: 258–262
42 J. Turkevitch, P. C. Stevenson, J. Hillier. Nucleation and Growth Process in the Synthesis of Colloidal Gold. Discuss. Faraday Soc. 1951, 11: 55-75
43 G. Frens. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions. Nature: Phys. Sci. 1973, 241:20-22
44 T. Yonezawa, T. Kunitake. Practical Preparation of Anionic Mercapto- Ligand-Stabilized Gold Nanoparticles and Their Immobilization. Colloids Surf. A: Physicochem. Eng. Asp. 1999, 149: 193-199
45 S. Bucak, D. A. Jones, P. E. Laibinis, T. A. Hatton. Protein Separations Using Colloidal Magnetic Nanoparticles. Biotechnol. Prog. 2003, 19: 477-484
46 Jennifer LL, David AF, Mattw BS, et al. Synthesis of Fe Oxide core /Au shell nanoparticles by iterative hydroxylamine seeding. Nano Letters, 2004, 4:719-723.
47 Maxwell DJ, Taylor JR, Nie S. Self-assembled nanoparticle probes for recognition and detection of biomolecules. J Am Chem Soc, 2002, 124:9606~9612.
48 Lee CM, Ong GY, Lu SN, et al. Durability of lamivudine-induced HBeAg seroconversion for chronic hepatitis B patients with acute exacerbation. J Hepatol, 2002, 37:669~674.
49 Saldanha J. Validation and standardisation of nucleic acid amplification technology assays for the detection of viral contamitation of blood and blood products. J Clin Virol, 2001, 20:7~13.
50 Elghanian R, Storhoff JJ, Mucic RC, et al. Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles. Science,1997, 277:1078-1081.
51 Wang YF, Pang DW, Zhang ZL, et al. Visual gene diagnosis of HBV and HCV based on nanoparticle probe amplification and silver staining enhancement. J Med Virol, 2003, 70: 205-211.
52中华医学会传染病与寄生虫病学分会、肝病学分会,病毒性肝炎防治方案.中华肝脏病杂志,2000,8:249-250.
53刘锡光,祁自柏,熊诗松.病毒性肝炎实验诊断学.人民卫生出版社.北京.1999,23-76.
54 Hacia JG, Brody LC, Chee MS, et al. Detection of heterozygous mutations in BRCA1using high density oligonucleotide arrays and two-colour fluorescence analysis. Nature Genet. 1996, 14:441~447.
55 Mansfield ES, Worley JM, McKenzie SE, et al. Nucleic acid detection using non-radioactive labelling methods. Mol Cell Probes, 1995, 9(3):145-156.
56 Micales BK, Lyons GE. In situ hybridization: use of 35S-labeled probes on paraffin tissue sections. Methods, 2001, 23(4):313-323.
57 Chen JW, Wu R, Yang PC, et al. Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection Genomics, 1998, 51(3): 313~324.
58 Yuan J, Wang G. Lanthanide complex-based fluorescence label for time-resolved fluorescence bioassay. J Fluoresc, 2005, 15(4):559-68.
59 Weng J, Ren J. Luminescent quantum dots: a very attractive and promising tool in biomedicine, 2006,13(8): 897-909.
60 Thaxton CS, Georganopoulou DG, Mirkin CA. Gold nanoparticle probes for the detection of nucleic acid targets. Clinica Chimica Acta, 2006, 363(1): 120~126.
1.宁琴,杨东亮,罗小平,等.暴发型病毒性肝炎小鼠模型的研究及应用.中华肝脏病杂志,2002,10:224-226.
2. Ning Q, Liu M, Kongkham P, et al.The nucleocapsid protein of murine hepatitis virus type 3 induces transcription of the novel fgl2 prothrombinase gene. J Biol Chem,1999, 274: 9930-9936.
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6. Levy GA, Liu MF, Ding JW, et al. Molecular and functional analysis of the human prothrombinase gene(hfgl2) and its role in viral hepatitis. Am J Pathol 2000, 156: 1217- 1225.
7. Yuwaraj S, Ding JW, Liu MF, et al. Genomic characterization, localization, and functional expression of fgl2, the human gene encoding fibroleukin: a novel human procoagulant. Genomics 2001, 71: 330-338.
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9.陈悦,宁琴,王宝菊等.重型乙型肝炎患者肝组织中人纤维介素基因的表达及意义.中华医学杂志,2003,6:446-450.
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2张立德,牟季美.《纳米材料与纳米结构》.第二版.北京.科学出版社,2001年3月. 2-3
3徐辉碧.《纳米医药》.第一版.北京.清华大学出版社,2004年1月. 299-301
4 R. E. Cavicchi, R. H. Silsbee. Coulomb Suppression of Tunneling Rate from Small Metal Particles. Phys. Rev. Lett. 1984, 52(5): 1453-1457
5 Lopez Quintela, M. A., Rivas, J., Liz, L., Winter, I. Quantum effects in ultrafine neodymium-iron-boron particles. NATO ASI Series, Series B: Physics . 1991, 259(Sci. Technol. Nanostruct. Magn. Mater.), 567-572
6 Ball P, Garwin L, Matrix-mediated synthesis of nanocrystallineγ-Fe2O3, a new optically transparent magnetic material. Nature. 1992, 355: 761-771
7 Christof M. Niemeyer. Nanoparticles, Proteins, and Nucleic Acids: Biotechnology Meets Materials Science. Angew. Chem. Int. Ed. 2001, 40: 4128-4158
8 Bawendi MG, Steigerwald M. L., Brus L E. The quantum mechanicsof larger semiconductor clusters (“quantum dots”). Annu. Rev. Phys. Chem. 1990 , 41: 477-496
9 Junu Chatterjee, Yousef Haik, Ching-Jen Chen. Polythylene magnetic nanoparticle: a new magnetic material for biomedical applications. Journal of Magnetism and Magnetic Materials. 2002, 246: 382-391
10 R. S. Molday, L .L. Molday. Separation of cells labeled with immunospecific iron dextran microspheres using high magnetic chromatography. FEBS. 1984, 170(2): 232-237
11 N.Cem Balci, Mustafa Sirvanci, Cihan Duran, et al. Hepatic adenomatosis MRI demonstration with the use of superparamagnetic iron oxide. Journal of Clinical Imaging 2002, 26: 35-38
12 Maire-France Bellin, Catherine Beigelman, Sophie Precetti-Morel. Iron oxide-enhanced MR lymphography: initial experience. European Journal of Radiology. 2000, 34: 257- 264
13 James J. Storhoff, Robert Elghanian, Robert C. Mucic, Chad A. Mirkin, Robert L.Letsinger. One-Pot Colorimetric Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle Probes. J. Am. Chem. Soc. 1998, 120: 1959-1964
14 T. Andrew Taton, Chad A. Mirkin, Robert L. Letsinger. Scanometric DNA Array Detection with Nanoparticle Probes. Science 2000, 289:1757-1760
15 So-Jung Park, T. Andrew Taton, Chad A. Mirkin. Array-Based Electrical Detection of DNA with Nanoparticle Probes. Science 2002, 295:1503-1506
16 YunWei Charles Cao, Rongchao Jin, Chad A. Mirkin. Nanoparticles with Raman Spectroscopic Fingerprints for DNA and RNA Detection. Science 2002, 297:1536-1540
17 J. Manuel Perez, Lee Josephson, Terence O’Loughin, Dagmar Hogemann, Ralph Weissleder. Magnetic relaxation switches capable of sensing molecular interactions. Nature Biotechnology, 2002, 20: 816-820
18 Lee Josephson, J. Manuel Perez, Ralph Weissleder. Magnetic Nanosensors for the Detection of Oligonucleotide Sequences. Angew. Chem. Int. Ed. 2001, 40: 3204-3206
19 Lin He, Michael D. Musick, Sheila R. Nicewarner, Frank G. Silinas, Stephen J. Benkovic, Michael J. Natan, Christine D. Keating. Colloidal Au-Enhanced Surface Plasmon Resonance for Ultrasensitive Detection of DNA Hybridization. J. Am. Chem. Soc. 2000, 122: 9071-9077
20 Yossi Weizmann, Fernando Patolsky, Eugenii Katz, Itamar Willner. Amplified DNA Sensing and Immunosensing by the rotation of Functional Magnetic Particles. J. Am. Chem. Soc. 2003, 125: 3452-3454
21 Joseph Wang, Ronen Polsky, Arben Merkoci, Kathryn L. Turner.“Electroactive Beads”for Ultrasensitive DNA Detection. Langmuir 2003, 19: 989-991
22 Joseph Wang, Danke Xu, Ronen Polsky. Magnetically-Induced Solid-State Electrochemical Detection of DNA Hybridization. J. Am. Chem. Soc. 2002, 124: 4208-4209
23 Alexandre I, Hamels S, Dufour S, et al. Colorimetric silver detection of DNA microarrays. Anal Biochem, 2001, 295(1):1~8.
24 Shubo Han, Jianqiao Lin, Feimeng Zhou, Robert Luis Vellanoweth. Oligonucleotide -Capped Gold Nanoparticles for Improved Atomic Force Microscopic Imaging and Enhanced Selectivity in Polynucleotide Detection. Biochemical and Biophysical Research Communications. 2000, 279: 265-269
25 Mirkin CA, Letsinger RL, Mucic RC, et al. A DNA-based method for rationallyassembling nanoparticle into macroscopic materials. Nature, 1996, 382:607~609.
26 A. P. Alivisatos, K. P. Johnsson, X. Peng, et al. Organization of Nanocrystal Molecules Using DNA. Nature 1996, 382: 609-611
27 G. Mie. Beitrage zur Optik Truber Medien, Speziell Kolloidaler Metallosungen. Ann. Phys. 1908, 25: 377-445
28 Frank Caruso. Nanoengineering of Particle Surfaces. Adv. Mater. 2001, 13: 11-22
29 E. E. Carpenter, A. Kumbhar, J. A. Wiemann, et al. Synthesis and magnetic properties of gold-iron-gold nanocomposites. Materials Science and Engineering A 2000, 286: 81-86
30 J. Lin, W. Zhou, A. Kumbhar, et al. Gold-Coated Iron (Fe@Au) Nanoparticles: Synthesis, Characterization, and Magnetic Field-Induced Self-Assembly. J. Solid State Chem. 2001, 159: 26-31
31 H. Yu, M. Chen, P. M. Rice, et al. Dumbbell-Like Bifunctional Au-Fe3O4 Nanoparticles. Nano Lett. 2005, 5: 379-382
32 T. Kinoshita, S. Seino, K. Okitsu, et al. Magnetic evaluation of nanostructure of gold-iron composite particles synthesized by reverse micelle method. J. Alloys & Compounds 2003, 359: 46-50
33 J. L. Lyon, D. A. Fleming, M. B. Stone, et al. Synthesis of Fe Oxide Core/Au Shell Nanoparticles by Iterative Hydroxylamine Seeding. Nano Lett. 2004, 4: 719-723
34 S-J. Cho, J-C. Idrobo, J. Olamit, et al. Growth Mechanisms and Oxidation Resistance of Gold-Coated Iron Nanoparticles. Chem. Mater. 2005, 17: 3181-3186
35 S-J. Cho, S. M. Kauzlarich, J. Olamit, et al. Characterization and magnetic properties of core-shell structured Fe/Au nanoparticles. J. Appl. Phys. 2004, 95: 6804-6806
36 M. Chen, S. Yamamuro, D. Farrell, et al. Gold-coated iron nanoparticles for biomedical applications. J. Appl. Phys. 2003, 93: 7551-7553
37 L. Wang, J. Luo, Q. Fan, et al. Monodispersed Core-Shell Fe3O4@Au Nanoparticles. J. Phys. Chem. B 2005, 109: 21593-21601
38 Demers LM, Mirkin CA, Mucic RC, et al. A fluorescence-based method for determing the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles. Anal Chem, 2000, 72:5535~5541.
39 L. Babes, B. Denizot, G. Tanguy, et al. Synthesis of Iron Oxide Nanoparticles Used as MRI Contrast Agent: A Parametric Study. J. Colloid and Interface Sci. 1999, 212: 474 -482
40 Pena SRN, Raina S, Goodrich GP, et al. Hybridization and enzymatic extension of Au nanoparticle-bound oligonucleotide. J Am Chem Soc. 2002,124(25):7314-7323.
41 Z.L. Liu, H.B. Wang, Q.H. Lu, et al. Synthesis and characterization of ultrafine well-dispersed magnetic nanoparticles. Journal of Magnetism and Magnetic Materials, 2004, 283: 258–262
42 J. Turkevitch, P. C. Stevenson, J. Hillier. Nucleation and Growth Process in the Synthesis of Colloidal Gold. Discuss. Faraday Soc. 1951, 11: 55-75
43 G. Frens. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions. Nature: Phys. Sci. 1973, 241:20-22
44 T. Yonezawa, T. Kunitake. Practical Preparation of Anionic Mercapto- Ligand-Stabilized Gold Nanoparticles and Their Immobilization. Colloids Surf. A: Physicochem. Eng. Asp. 1999, 149: 193-199
45 S. Bucak, D. A. Jones, P. E. Laibinis, T. A. Hatton. Protein Separations Using Colloidal Magnetic Nanoparticles. Biotechnol. Prog. 2003, 19: 477-484
46 Jennifer LL, David AF, Mattw BS, et al. Synthesis of Fe Oxide core /Au shell nanoparticles by iterative hydroxylamine seeding. Nano Letters, 2004, 4:719-723.
47 Maxwell DJ, Taylor JR, Nie S. Self-assembled nanoparticle probes for recognition and detection of biomolecules. J Am Chem Soc, 2002, 124:9606~9612.
48 Lee CM, Ong GY, Lu SN, et al. Durability of lamivudine-induced HBeAg seroconversion for chronic hepatitis B patients with acute exacerbation. J Hepatol, 2002, 37:669~674.
49 Saldanha J. Validation and standardisation of nucleic acid amplification technology assays for the detection of viral contamitation of blood and blood products. J Clin Virol, 2001, 20:7~13.
50 Elghanian R, Storhoff JJ, Mucic RC, et al. Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles. Science,1997, 277:1078-1081.
51 Wang YF, Pang DW, Zhang ZL, et al. Visual gene diagnosis of HBV and HCV based on nanoparticle probe amplification and silver staining enhancement. J Med Virol, 2003, 70: 205-211.
52中华医学会传染病与寄生虫病学分会、肝病学分会,病毒性肝炎防治方案.中华肝脏病杂志,2000,8:249-250.
53刘锡光,祁自柏,熊诗松.病毒性肝炎实验诊断学.人民卫生出版社.北京.1999,23-76.
54 Hacia JG, Brody LC, Chee MS, et al. Detection of heterozygous mutations in BRCA1using high density oligonucleotide arrays and two-colour fluorescence analysis. Nature Genet. 1996, 14:441~447.
55 Mansfield ES, Worley JM, McKenzie SE, et al. Nucleic acid detection using non-radioactive labelling methods. Mol Cell Probes, 1995, 9(3):145-156.
56 Micales BK, Lyons GE. In situ hybridization: use of 35S-labeled probes on paraffin tissue sections. Methods, 2001, 23(4):313-323.
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58 Yuan J, Wang G. Lanthanide complex-based fluorescence label for time-resolved fluorescence bioassay. J Fluoresc, 2005, 15(4):559-68.
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