Gokce Gultekin, Erdem Arzum, Ceylan Cagdas, Akgöz Muslum
Cumhuriyet University, Education Faculty, Department of Elementary Science, 58140 Sivas, Turkey; Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, Bornova, 35100 Izmir, Turkey.
Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, Bornova, 35100 Izmir, Turkey.
Talanta. 2016;149:244-249. doi: 10.1016/j.talanta.2015.11.071. Epub 2015 Nov 28.
This work describes the single-use electrochemical DNA biosensor technology developed for voltammetric detection of sequence selective DNA hybridization related to important human and veterinary pathogen; Toxoplasma gondii. In the principle of electrochemical label-free detection assay, the duplex of DNA hybrid formation was detected by measuring guanine oxidation signal occured in the presence of DNA hybridization. The biosensor design consisted of the immobilization of an inosine-modified (guanine-free) probe onto the surface of pencil graphite electrode (PGE), and the detection of the duplex formation in connection with the differential pulse voltammetry(DPV) by measuring the guanine signal. Toxoplasma gondii capture probe was firstly immobilized onto the surface of the activated PGE by wet adsorption. The extent of hybridization at PGE surface between the probe and the target was then determined by measuring the guanine signal observed at +1.0V. The electrochemical monitoring of optimum DNA hybridization has been performed in the target concentration of 40µg/mL in 50min of hybridization time. The specificity of the electrochemical biosensor was then tested using non-complementary, or mismatch short DNA sequences. Under the optimum conditions, the guanine oxidation signal indicating full hybridization was measured in various target concentration from 0.5 to 25µg/mL and a detection limit was found to be 1.78µg/mL. This single-use biosensor platform was successfully applied for the voltammetric detection of DNA hybridization related to Toxoplasma gondii in PCR amplicons.
这项工作描述了一种一次性电化学DNA生物传感器技术，该技术用于伏安法检测与重要的人类和兽医病原体——刚地弓形虫相关的序列选择性DNA杂交。在电化学无标记检测分析原理中，通过测量DNA杂交时发生的鸟嘌呤氧化信号来检测DNA杂交形成的双链体。生物传感器的设计包括将肌苷修饰（无鸟嘌呤）的探针固定在铅笔石墨电极（PGE）表面，并通过测量鸟嘌呤信号结合差分脉冲伏安法（DPV）检测双链体的形成。首先通过湿吸附将刚地弓形虫捕获探针固定在活化的PGE表面。然后通过测量在+1.0V观察到的鸟嘌呤信号来确定探针与靶标在PGE表面的杂交程度。在杂交时间为50分钟、靶标浓度为40µg/mL的条件下进行了最佳DNA杂交的电化学监测。然后使用非互补或错配的短DNA序列测试了电化学生物传感器的特异性。在最佳条件下，在0.5至25µg/mL的各种靶标浓度下测量了指示完全杂交的鸟嘌呤氧化信号，检测限为1.78µg/mL。这个一次性生物传感器平台成功应用于PCR扩增子中与刚地弓形虫相关的DNA杂交的伏安法检测。
