Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain.
IRTA, Ctra. Poble Nou, km 5.5, 43540 Sant Carles de la Ràpita, Tarragona, Spain.
Biosens Bioelectron. 2019 Jun 1;134:76-82. doi: 10.1016/j.bios.2019.03.060. Epub 2019 Mar 30.
An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2'-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples.
本文提出了一种用于检测和定量检测 Karlodinium armiger 的电化学生物传感器。该传感器利用长尾引物和标记有二茂铁的 dATP 类似物来产生 PCR 产物，这些产物可以直接在金电极阵列上杂交，并使用方波伏安法进行定量测量。长尾引物由目标物的特异性序列组成，后面跟着一个碳间隔序列和一个专门设计的不与基因组 DNA 结合的序列，从而形成由单链结合引物包围的双链体。通过杂交来实现电化学检测，优化了 7-(二茂铁乙炔基)-7-脱氮-2'-脱氧腺苷三磷酸的掺入，以达到最大 PCR 效率和通过杂交实现电化学检测可达到的检测限和灵敏度之间的折衷。以 315aM 起始 DNA 浓度的线性范围从 315aM 到 10 fM，检测限为 277aM，灵敏度为 122 nA 十年。该系统成功应用于检测实际海水中的基因组 DNA。
