Dong Shibiao, Zhao Rongtao, Zhu Jiangong, Lu Xiao, Li Yang, Qiu Shaofu, Jia Leili, Jiao Xiong, Song Shiping, Fan Chunhai, Hao RongZhang, Song HongBin
†Institute for Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
‡Institute of Applied Mechanics and Biomedical Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, China.
ACS Appl Mater Interfaces. 2015 Apr 29;7(16):8834-42. doi: 10.1021/acsami.5b01438. Epub 2015 Apr 15.
A DNA tetrahedral nanostructure-based electrochemical biosensor was developed to detect avian influenza A (H7N9) virus through recognizing a fragment of the hemagglutinin gene sequence. The DNA tetrahedral probe was immobilized onto a gold electrode surface based on self-assembly between three thiolated nucleotide sequences and a longer nucleotide sequence containing complementary DNA to hybridize with the target single-stranded (ss)DNA. The captured target sequence was hybridized with a biotinylated-ssDNA oligonucleotide as a detection probe, and then avidin-horseradish peroxidase was introduced to produce an amperometric signal through the interaction with 3,3',5,5'-tetramethylbenzidine substrate. The target ssDNA was obtained by asymmetric polymerase chain reaction (PCR) of the cDNA template, reversely transcribed from the viral lysate of influenza A (H7N9) virus in throat swabs. The results showed that this electrochemical biosensor could specifically recognize the target DNA fragment of influenza A (H7N9) virus from other types of influenza viruses, such as influenza A (H1N1) and (H3N2) viruses, and even from single-base mismatches of oligonucleotides. Its detection limit could reach a magnitude of 100 fM for target nucleotide sequences. Moreover, the cycle number of the asymmetric PCR could be reduced below three with the electrochemical biosensor still distinguishing the target sequence from the negative control. To the best of our knowledge, this is the first report of the detection of target DNA from clinical samples using a tetrahedral DNA probe functionalized electrochemical biosensor. It displays that the DNA tetrahedra has a great potential application as a probe of the electrochemical biosensor to detect avian influenza A (H7N9) virus and other pathogens at the gene level, which will potentially aid the prevention and control of the disease caused by such pathogens.
开发了一种基于DNA四面体纳米结构的电化学生物传感器，通过识别血凝素基因序列的一个片段来检测甲型H7N9禽流感病毒。基于三个硫醇化核苷酸序列与一个含有互补DNA的较长核苷酸序列之间的自组装，将DNA四面体探针固定在金电极表面，以与目标单链（ss）DNA杂交。捕获的目标序列与作为检测探针的生物素化ssDNA寡核苷酸杂交，然后引入抗生物素蛋白-辣根过氧化物酶，通过与3,3',5,5'-四甲基联苯胺底物的相互作用产生安培信号。目标ssDNA通过对cDNA模板进行不对称聚合酶链反应（PCR）获得，该cDNA模板是从咽喉拭子中甲型H7N9禽流感病毒的病毒裂解物反转录而来的。结果表明，这种电化学生物传感器能够从其他类型的流感病毒，如甲型H1N1和H3N2流感病毒，甚至从寡核苷酸的单碱基错配中特异性识别甲型H7N9禽流感病毒的目标DNA片段。其对目标核苷酸序列的检测限可达100 fM量级。此外，不对称PCR的循环次数可减少到三次以下，而电化学生物传感器仍能将目标序列与阴性对照区分开来。据我们所知，这是首次报道使用功能化的四面体DNA探针电化学生物传感器检测临床样本中的目标DNA。这表明DNA四面体作为电化学生物传感器的探针在基因水平检测甲型H7N9禽流感病毒和其他病原体方面具有巨大的潜在应用价值，这可能有助于预防和控制由此类病原体引起的疾病。
