双功能化胶体金纳米探针的制备及其特异性考察  

Self-assembly of dual-functionalized gold nanoparticle probe and its specificity

作　　者：杨冰[1] 黄乐群[1] 

机构地区：[1]南京大学医学院,江苏南京210093

出　　处：《浙江大学学报（医学版）》2010年第3期296-304,共9页Journal of Zhejiang University(Medical Sciences)

基　　金：国家教育部归国华侨学者科研基金(No.0214168001)

摘　　要：目的:制备一种新型的双功能化胶体金纳米探针,并考察其特异性。方法:选择二硫化物分子和甲型流感病毒(H1N1亚型)HA基因中特异性的23 mer寡核苷酸片段组装到胶体金颗粒表面,制成双功能化胶体金纳米探针(识别探针,颜色呈酒红色);利用磁性微球作为固相支持物,偶联另一段特异性H1N1寡核苷酸片段制成捕捉探针;两者与靶DNA(完全匹配DNA)结合,形成无色的捕捉探针-靶DNA-识别探针("三明治"夹心式复合物),并采用基质辅助激光解析离子化飞行时间质谱(MALDI TOF MS)测定。同时,在选择向体系中分别加入超纯水(作为空白对照)、完全匹配DNA、不完全匹配DNA及2条仅有1个碱基错配的DNA片段作为靶目标进行实验,观察体系颜色变化及质谱测定,考察探针的特异性。结果:捕捉探针和识别探针只能与完全匹配DNA结合,形成夹心式复合物,反应体系颜色由酒红色转变为无色,充分洗涤后加热去杂交,则由无色复呈酒红色,质谱检测出现信号(m/z 693);而其他靶目标(不完全匹配DNA及2条仅有1个碱基错配的DNA片段)和空白对照实验,不能与2种探针形成夹心式复合物,反应体系均没有颜色变化(仍呈现酒红色),充分洗涤后体系呈无色,加热去杂交体系仍呈无色,质谱检测未见信号(m/z 693)。这说明胶体金纳米探针的特异性很高,可区分仅有1个碱基错配的靶目标。结论:本研究建立了一种新型的自组装双功能化胶体金纳米探针,其制备过程简单、特异性很高。此类型胶体金纳米探针在核酸分析,尤其是疾病早期诊断,寡核苷酸多态性分型等方面将有广泛的应用前景。Objective： To investigate the specificity of the dual-functionalized nanopaticle probes（NPs） self-assembled with colloidal gold.Methods： 13-nm gold nanoparticles were prepared with citrate reduction of HAuCl4.These gold nanoparticles were sequentially functionalized with the specific single-strand oligonucleotide of HA gene of influenza A virus（H1N1） and disulfide molecules of m/z at 693.The NPs solution showed the red formation.The magnetic microparticles（MPs） were modified with another specific single-strand oligonucleotide in HA gene of H1N1.The sandwich complexes（MP-Target-NPs） were formed by the target DNA with the MPs and the NPs.The color change in the solution was observed and the dehybridization product was detected by MALDI TOF MS.Moreover specificity of the probes was investigated with nanowater（as a blank control） and the different target DNAs including complementary DNA,non-complemetary DNA and two DNAs of one base mismatch,respectively.Results： The red formation and the positive signal in MS detection of reporter mass code 693（＋） were observed,which indicated the formation of sandwich complexes formed only when the completely complementary target DNAs were presented in the solution.No color formation changes and no peak signal detected by MALDI TOF MS were observed,showing that none of target of interest（nanopure water）,non-complementary DNA and two DNAs of one base mismatch existed in the systems,which indicated no sandwich complexes formed between the target DNAs and the two probes.Conclusions： Considering the simple preparation procedure and high specificity,the dual-functionalized gold nanoparticle probes would be widely and increasingly used in nucleic acid analysis.In particular,it would have broad application prospects in early diagnosis of diseases,single nucleotide polymorphism（SNP） typing and so on.

关 键 词：核酸探针 胶体金 磁力学 微球体 光谱法 质量 基质辅助激光解吸电离 纳米技术 

分 类 号：Q503[生物学—生物化学]