Yang Bing, Huang Le-qun
Medical School, Nanjing University, Nanjing 210093, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2010 May;39(3):296-304. doi: 10.3785/j.issn.1008-9292.2010.03.014.
To investigate the specificity of the dual-functionalized nanoparticles probes (NPs) self-assembled with colloidal gold.
13-nm gold nanoparticles were prepared with citrate reduction of HAuCl(4). 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 nano-water (as a blank control) and the different target DNAs including complementary DNA,non-complementary DNA and two DNAs of one base mismatch, respectively.
The red formation and the positive signal in MS detection of reporter mass code 693 (M+Na) 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 (nano-pure 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.
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.
研究用胶体金自组装的双功能化纳米颗粒探针（NPs）的特异性。
用柠檬酸盐还原氯金酸（HAuCl₄）制备13纳米的金纳米颗粒。这些金纳米颗粒依次用甲型流感病毒（H1N1）HA基因的特异性单链寡核苷酸和质荷比为693的二硫分子进行功能化。NPs溶液呈现红色。磁性微粒（MPs）用H1N1的HA基因中的另一种特异性单链寡核苷酸进行修饰。夹心复合物（MP-靶标-NPs）由靶标DNA与MPs和NPs形成。观察溶液中的颜色变化，并用基质辅助激光解吸电离飞行时间质谱（MALDI TOF MS）检测解杂交产物。此外，分别用纳米水（作为空白对照）和不同的靶标DNA（包括互补DNA、非互补DNA和一个碱基错配的两种DNA）研究了探针的特异性。
观察到红色形成以及在质谱检测中报告质量数693（[M+Na]⁺）处的阳性信号，这表明仅当溶液中存在完全互补的靶标DNA时才形成夹心复合物。未观察到颜色形成变化，且MALDI TOF MS未检测到峰信号，表明系统中不存在感兴趣的靶标（纳米纯水）、非互补DNA和一个碱基错配的两种DNA，这表明靶标DNA与两种探针之间未形成夹心复合物。
考虑到制备过程简单且特异性高，双功能化金纳米颗粒探针将在核酸分析中得到广泛且越来越多的应用。特别是，它在疾病早期诊断、单核苷酸多态性（SNP）分型等方面将具有广阔的应用前景。
