Balvedi Renata P A, Castro Ana C H, Madurro João M, Brito-Madurro Ana G
Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia 38400-902, Brazil.
Institute of Chemistry, Federal University of Uberlândia, Uberlândia 38400-902, Brazil.
Int J Mol Sci. 2014 May 21;15(5):9051-66. doi: 10.3390/ijms15059051.
This paper describes methodology for direct and indirect detections of a specific oligonucleotide for Epstein-Barr virus (EBV) using electrochemical techniques. The sequence of oligonucleotide probe (EBV1) revealed a high sequence identity (100%) with the EBV genome. For the development of the genosensor, EBV1 was grafted to the platform sensitized with poly(4-aminothiophenol). After that, the hybridization reaction was carried out with the complementary target (EBV2) on the modified electrode surface using ethidium bromide as DNA intercalator. The oxidation peak currents of ethidium bromide increased linearly with the values of the concentration of the complementary sequences in the range from 3.78 to 756 µmol·L⁻¹. In nonstringent experimental conditions, this genosensor can detect 17.32 nmol·L⁻¹ (three independent experiments) of oligonucleotide target, discriminating between complementary and non-complementary oligonucleotides, as well as differentiating one-base mismatch, as required for detection of genetic diseases caused by point mutations. The biosensor also displayed high specificity to the EBV target with elimination of interference from mix (alanine, glucose, uric acid, ascorbic acid, bovine serum albumin (BSA), glutamate and glycine) and good stability (120 days). In addition, it was possible to observe differences between hybridized and non-hybridized surfaces through atomic force microscopy.
本文描述了使用电化学技术直接和间接检测爱泼斯坦-巴尔病毒（EBV）特定寡核苷酸的方法。寡核苷酸探针（EBV1）的序列与EBV基因组显示出高度的序列同一性（100%）。为了开发基因传感器，将EBV1嫁接到用聚（4-氨基硫酚）敏化的平台上。之后，使用溴化乙锭作为DNA嵌入剂，在修饰电极表面与互补靶标（EBV2）进行杂交反应。溴化乙锭的氧化峰电流在3.78至756 µmol·L⁻¹范围内与互补序列浓度值呈线性增加。在非严格实验条件下，这种基因传感器可以检测17.32 nmol·L⁻¹（三个独立实验）的寡核苷酸靶标，区分互补和非互补寡核苷酸，以及区分单碱基错配，这是检测由点突变引起的遗传疾病所必需的。该生物传感器对EBV靶标也表现出高特异性，消除了混合物（丙氨酸、葡萄糖、尿酸、抗坏血酸、牛血清白蛋白（BSA）、谷氨酸和甘氨酸）的干扰，并且具有良好的稳定性（120天）。此外，通过原子力显微镜可以观察到杂交和未杂交表面之间的差异。
