Detection of single-nucleotide polymorphisms with novel leaky surface acoustic wave biosensors, DNA ligation and enzymatic signal amplification

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• 作者：Qinghua Xu^a ; ¹ ; Kai Chang^a ; ¹ ; Weiping Lu^a ; ¹ ; Wei Chen^a ; Yi Ding^b ; Shuangrong Jia^a ; Kejun Zhang^a ; Fake Li^a ; Jianfeng Shi^b ; Liang Cao^b ; Shaoli Deng^a ; ^{dengsl072@yahoo.com.cn} ; Ming Chen^a ; ^{chenming1971@yahoo.com}
• 关键词：Single nucleotide polymorphism ; Leak surface acoustic wave biosensor ; DNA ligation
• 刊名：Biosensors and Bioelectronics
• 出版年：2012
• 期刊代码：12_09565663
• 类别：ch
• 出版时间：15 March, 2012
• 卷：33
• 期：1
• 页码：274-278
• 文件大小：470 K

摘要

This manuscript describes a new technique for detecting single-nucleotide polymorphisms (SNPs) by integrating a leaky surface acoustic wave (LSAW) biosensor, enzymatic DNA ligation and enzymatic signal amplification. In this technique, the DNA target is hybridized with a capture probe immobilized on the surface of a LSAW biosensor. Then, the hybridized sequence is ligated to biotinylated allele-specific detection probe using Taq DNA ligase. The ligation does not take place if there is a single-nucleotide mismatch between the target and the capture probe. The ligated detection probe is transformed into a streptavidin-horseradish peroxidase (SA-HRP) terminal group via a biotin-streptavidin complex. Then, the SA-HRP group catalyzes the polymerization of 3,3-diaminobenzidine (DAB) to form a surface precipitate, thus effectively increasing the sensitivity of detecting surface mass changes and allowing detection of SNPs. Optimal detection conditions were found to be: 0.3 mol/L sodium ion concentration in PBS, pH 7.6, capture probe concentration 0.5 渭mol/L and target sequence concentration 1.0 渭mol/L. The detection limit was found to be 1 脳 10^鈭?2 mol/L. Using this technique, we were able to detect a single-point mutation at nucleotide A2293G in Japanese encephalitis virus.