Electrochemical quantification of DNA amplicons via the detection of non-hybridised guanine bases on low-density electrode arrays.
Electrochemical quantification of DNA amplicons via the detection of non-hybridised guanine bases on low-density electrode arrays.
复制标题
DOI：
10.1016/j.bios.2008.10.021
复制
发表时间：
2009-03
影响因子：
12.6
通讯作者：
O. Henry;J. L. A. Sanchez;D. Latta;C. O’Sullivan
中科院分区：
文献类型：
--
作者：
O. Henry;J. L. A. Sanchez;D. Latta;C. O’Sullivan
介绍了一种新的电化学检测和DNA在金电极上的信号放大策略。目前基于电化学检测电活性碱基特异性标签（如亚甲基蓝（MB））的DNA生物传感方法存在着冗长的孵育和洗涤步骤。针对这些限制，我们报道了一种新的电化学定量方法，使用107个碱基长的控制基因LTA作为模型靶标。用15个金电极阵列检测非杂化鸟嘌呤碱基与溶液中存在的MB相互作用后杂化双相的形成。杂交后，电极表面的游离鸟嘌呤数量从8个增加到25个，这是由于靶序列中的鸟嘌呤碱基没有参与杂交，仍然可以自由地与亚甲基蓝直接相互作用。游离鸟嘌呤的增加使MB直接集中在电极表面。我们发现互补LTA在100nM处记录的MB信号通常比非杂化状态高2.14倍。非互补探针的交叉反应性极低（<7%）。对亚甲蓝浓度、杂交时间和再生能力进行了优化。该方法在6.25 ~ 50 nm范围内呈定量线性，LOD为17.5nM。该分析快速且易于执行，不需要长时间的亚甲基蓝标签孵育或洗涤步骤的要求。正在进行的工作解决了鸟嘌呤位置对信号的影响，以便定制设计PCR产品的特定信号域。
A new strategy for the electrochemical detection and signal amplification of DNA at gold electrodes is described. Current methodologies for DNA biosensing based on the electrochemical detection of electroactive base-specific labels such as methylene blue (MB) suffer from lengthy incubation and washing steps. Addressing these limitations, we report a novel approach for the electrochemical quantification of surface hybrid, using the control gene LTA, 107 bases long, as a model target. An array of 15 gold electrodes was used to detect the formation of hybridised duplex following interaction of non-hybridised guanine bases with MB present in solution. Upon hybridisation the number of free guanines present at the electrode surface increased from 8 to 25 due to guanine bases present in the target sequence which did not participate in hybridisation and remained free to interact directly with methylene blue. This increase in free guanines consequently concentrated MB directly at the electrode surface. We found that the MB signal recorded for 100nM of the complementary LTA was typically 2.14 times higher than that of the non-hybridised state. Very low cross-reactivity (<7%) with a non-complementary probe was recorded. The assay was optimised with regards to methylene blue concentration, hybridisation time and regeneration. The assay was quantitative and linear in the range of 6.25–50nM target DNA exhibiting an LOD of 17.5nM. The assay was rapid and easy to perform, with no need for lengthy incubations with the methylene blue label or requirement for washing steps. Ongoing work addresses the impact of guanine location on the signal in order to tailor design specific signalling domains of PCR products.