Wu Zai-Sheng, Jiang Jian-Hui, Fu Li, Shen Guo-Li, Yu Ru-Qin
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, People's Republic of China.
Anal Biochem. 2006 Jun 1;353(1):22-9. doi: 10.1016/j.ab.2006.01.018. Epub 2006 Jan 30.
A novel system for the detection of DNA hybridization in a homogeneous format is developed. This method is based on fluorescence quenching by gold nanoparticles used as both nanoscaffolds for the immobilization of capture sequences and nanoquenchers of fluorophores attached to detection sequences. The oligonucleotide-functionalized gold nanoparticles are synthesized by derivatizing the colloidal gold solution with 5'-thiolated 12-base oligonucleotides. Introduction of sequence-specific target DNAs (24 bases) into the mixture containing dye-tagged detection sequences and oligonucleotide-functionalized gold nanoparticles results in the quenching of carboxytetramethylrhodamine-labeled DNA fluorescence because DNA hybridization occurs and brings fluorophores into close proximity with oligonucleotide-functionalized gold nanoparticles. The quenching efficiency of fluorescence increases with the target DNA concentration and provides a quantitative measurement of sequence-specific DNA in sample. A linearity is obtained within the range from 1.4 to 92 nM. The target sequence is detected down to 2 nM. This new system not only overcomes many of the drawbacks inherent in radioisotopic measurement or enzyme-linked assay but also avoids the requirement for the stem-loop structure compared with conventional molecular beacons. Furthermore, the background signal that is defined as fluorescence quenching arising from electrostatic attraction between positively charged fluorophores and negatively charged gold nanoparticles is comparatively low due to electrostatic repulsion between negatively charged oligonucleotides. In addition, this is a homogeneous assay that can offer the potential to be monitored in real time, be amenable to automation, eliminate washing steps, and reduce the risk of contamination.
开发了一种用于检测均相形式DNA杂交的新型系统。该方法基于金纳米颗粒的荧光猝灭，金纳米颗粒既用作固定捕获序列的纳米支架，又用作连接到检测序列的荧光团的纳米猝灭剂。通过用5'-硫醇化的12碱基寡核苷酸衍生化胶体金溶液来合成寡核苷酸功能化的金纳米颗粒。将序列特异性靶DNA（24个碱基）引入含有染料标记的检测序列和寡核苷酸功能化金纳米颗粒的混合物中，会导致羧基四甲基罗丹明标记的DNA荧光猝灭，因为发生了DNA杂交，使荧光团与寡核苷酸功能化金纳米颗粒紧密靠近。荧光猝灭效率随靶DNA浓度增加而增加，并能对样品中的序列特异性DNA进行定量测量。在1.4至92 nM范围内获得线性关系。能检测到低至2 nM的靶序列。这个新系统不仅克服了放射性同位素测量或酶联测定中固有的许多缺点，而且与传统分子信标相比，避免了对茎环结构的要求。此外，由于带负电荷的寡核苷酸之间的静电排斥，由带正电荷的荧光团与带负电荷的金纳米颗粒之间的静电吸引引起的背景信号（定义为荧光猝灭）相对较低。此外，这是一种均相测定法，具有实时监测、易于自动化、无需洗涤步骤以及降低污染风险的潜力。
