Electrochemical DNA Biosensor Based on a Tetrahedral Nanostructure Probe for the Detection of Avian Influenza A (H7N9) Virus
Electrochemical DNA Biosensor Based on a Tetrahedral Nanostructure Probe for the Detection of Avian Influenza A (H7N9) Virus
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DOI：
10.1021/acsami.5b01438
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发表时间：
2015-04-29
影响因子：
9.5
通讯作者：
Song, HongBin
中科院分区：
文献类型：
--
作者：
Dong, Shibiao;Zhao, Rongtao;Song, HongBin
建立了一种基于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片段检测甲型H7N9禽流感病毒。将DNA四面体探针固定在金电极表面，通过三个硫代核苷酸序列与含有互补DNA的更长的核苷酸序列之间的自组装与目标单链DNA杂交。将捕获的目标序列与生物素化的ssdna寡核苷酸杂交作为检测探针，然后引入亲和素-辣根过氧化物酶，通过与3,3',5,5'-四甲基联苯胺底物相互作用产生电流信号。从甲型H7N9流感病毒咽拭子病毒裂解液中逆转录cDNA模板，采用非对称聚合酶链式反应（PCR）获得目标ssDNA。结果表明，该电化学生物传感器能特异性识别甲型H1N1流感病毒和甲型H3N2流感病毒的目标DNA片段，甚至能识别寡核苷酸单碱基错配。对目标核苷酸序列的检出限可达100 fM量级。此外，不对称PCR的循环数可以减少到3个以下，电化学生物传感器仍然可以将目标序列与阴性对照区分开来。据我们所知，这是第一个使用四面体DNA探针功能化电化学生物传感器从临床样品中检测目标DNA的报告。这表明，DNA四面体作为电化学生物传感器探针在基因水平上检测甲型H7N9禽流感病毒等病原体具有很大的应用潜力，这将有助于预防和控制由这些病原体引起的疾病。
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.