Interfibio Consolidated Research Group, Department of Chemical Engineering, Universitat Rovira I Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain.
Interfibio Consolidated Research Group, Department of Chemical Engineering, Universitat Rovira I Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain; ICREA, Passeig Lluís Companys 23, 08010, Barcelona, Spain.
Anal Biochem. 2020 Jun 1;598:113705. doi: 10.1016/j.ab.2020.113705. Epub 2020 Apr 1.
Genosensors for the detection of DNA via hybridisation normally require post-amplification processing such as the generation of single-stranded DNA and pre-detection labelling, complicating and lengthening the assay. A straightforward electrochemical genosensor, for the direct detection of isothermally generated nucleic acid amplicons via hybridisation is reported. The detection of Karlodinium armiger, responsible for harmful algae blooms was used as a model system to demonstrate the proof of concept. The approach exploits the use of specifically modified primers designed to generate amplicons with a central duplex flanked by a single-stranded tail at one end of the duplex and a horse-radish peroxidase on the other end. Individual gold electrodes of an array were functionalised with self-assembled monolayers of short thiolated DNA probes, designed to hybridise with the single-stranded tailed amplicon with the reporter enzyme label incorporated. The optimum amplification time was determined to be 60 min, at a fixed temperature of 37 °C. The hybridisation time to the enzyme labelled amplicon was optimised to be 10 min, but 2 min hybridisation time was also adequate. In this first example of using horse radish peroxidase-labelled primer in solution-phase recombinase polymerase amplification for subsequent detection via solid-phase hybridisation, the detection limit achieved was 0.4 fM, equivalent to 27622 cells/L, and the developed genosensor was applied to the detection of synthetic as well as genomic DNA, which had been extracted from a seawater sample.
用于通过杂交检测 DNA 的基因传感器通常需要进行扩增后处理，例如生成单链 DNA 和预检测标记，从而使检测复杂化和延长。本文报道了一种直接通过杂交检测等温生成的核酸扩增子的简单电化学基因传感器。以卡尔多林菌（Karlodinium armiger）的检测为例，该菌会导致有害藻类大量繁殖，这证明了该概念的可行性。该方法利用了经过特殊修饰的引物，这些引物设计用于生成扩增子，其中中心双链体的一端带有单链尾巴，另一端带有辣根过氧化物酶。阵列的各个金电极都经过了短硫醇化 DNA 探针的自组装单层功能化，这些探针设计用于与带有报道酶标记的单链尾巴扩增子杂交。确定最佳的扩增时间为 60 分钟，固定温度为 37°C。优化了与酶标记的扩增子的杂交时间为 10 分钟，但 2 分钟的杂交时间也足够了。在使用辣根过氧化物酶标记的引物在溶液相重组聚合酶扩增中进行后续固相杂交检测的第一个示例中，实现的检测限为 0.4 fM，相当于 27622 个细胞/L，所开发的基因传感器已应用于合成 DNA 和从海水样本中提取的基因组 DNA 的检测。
