Facilitating the indirect detection of genomic DNA in an electrochemical DNA biosensor using magnetic nanoparticles and DNA ligase

Roozbeh Hushiarian , Nor Azah Yusof , Abdul Halim Abdullah , Shahrul Ainliah Alang Ahmad , Sabo Wada Dutse
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引用次数: 19

Abstract

A common problem in applying biosensors for the detection of genomic DNA is detecting short sequences in large amounts of long double stranded DNA. A gold electrode modified with a conductive nanocomposite, poly(3,4-ethylene-dioxythiophene), and gold nanoparticles was functionalized with 2,6-Pyridinedicarboxylic acid. Immobilization of a 20-mer DNA probe as the bioreceptor was successfully carried out via a peptide bond on the surface of the modified electrode. Two segments of 15 and 20 base probes were designed and named as Capture and Reporter probes respectively. The 20-mer Reporter probe was complementary to the bioreceptor and the 15-mer Capture probe was designed to bind on to the surface of the iron oxide magnetic nanoparticles. A 35-base Target DNA complementary to the Capture and the Reporter probes was used as Template in the ligation process, with the ligation between the Reporter and Capture probes mediated by T4 ligase. Iron oxide magnetic nanoparticles functionalized with carboxylic groups on their surface synthesized in a new method were attached to the 15-mer Capture probe. After the denaturation of the final ligation product, the separation of the attached probes was carried out using 5 G permanent magnets in a three step washing procedure in TE buffer. The hybridization of the DNA bioreceptor and the Reporter probe attached to the Capture probe-Fe3O4 was monitored via oxidation and reduction of the new redox marker (ruthenium complex) intercalated into the double helix.

This technique was found to be reliably repeatable. The indirect detection of genomic DNA using this method is significantly improved and showed high efficiency in small amounts of samples with the detection limit of 5.37 × 10−14 M.

Abstract Image

利用磁性纳米颗粒和DNA连接酶在电化学DNA生物传感器中促进基因组DNA的间接检测
应用生物传感器检测基因组DNA的一个常见问题是检测大量长双链DNA中的短序列。用导电纳米复合材料聚(3,4-乙烯-二氧噻吩)和金纳米粒子修饰金电极,并用2,6-吡啶二羧酸对其进行功能化。通过修饰电极表面的肽键,成功地固定化了20-mer DNA探针作为生物受体。设计了15个碱基探针和20个碱基探针,分别命名为Capture探针和Reporter探针。20-mer的Reporter探针与生物受体互补,15-mer的Capture探针被设计用于结合到氧化铁磁性纳米颗粒的表面。在连接过程中,使用与Capture和Reporter探针互补的35碱基靶DNA作为模板,Reporter和Capture探针之间的连接由T4连接酶介导。用新方法合成了表面羧基功能化的氧化铁磁性纳米颗粒,并将其附着在15-mer Capture探针上。最终结扎产物变性后,用5g永磁体在TE缓冲液中进行三步洗涤,分离附着探针。DNA生物受体和附着在捕获探针- fe3o4上的报告探针的杂交是通过插入双螺旋的新的氧化还原标记(钌配合物)的氧化和还原来监测的。人们发现这种技术可可靠地重复使用。该方法对基因组DNA的间接检测效果显著提高,在少量样品中检测效率高,检出限为5.37 × 10−14 M。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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