Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland.
Biosens Bioelectron. 2014 Jan 15;51:170-6. doi: 10.1016/j.bios.2013.07.026. Epub 2013 Jul 21.
In this work, we report on oligonucleotide probes bearing metallacarborane [3-iron bis(dicarbollide)] redox label, deposited on gold electrode for electrochemical determination of DNA sequence derived from Avian Influenza Virus (AIV), type H5N1. The oligonucleotide probes containing 5'-terminal NH2 group were covalently attached to the electrode, via NHS/EDC coupling to 3-mercaptopropionic acid SAM, previously deposited on the surface of gold. The changes in redox activity of Fe(III) centre of the metallacarborane complex before and after hybridization process was used as analytical signal. The signals generated upon hybridization with targets such as complementary or non-complementary 20-mer ssDNA or various PCR products consisting of 180-190 bp (dsDNA) were recorded by Osteryoung square-wave voltammetry (OSWV). The developed system was very sensitive towards targets containing sequence complementary to the probe with the detection limit estimated as 0.03 fM (S/N=3.0) and 0.08 fM (S/N=3.0) for 20-mer ssDNA and for dsDNA (PCR product), respectively. The non-complementary targets generated very weak responses. Furthermore, the proposed genosensor was suitable for discrimination of PCR products with different location of the complementarity region.
在这项工作中，我们报告了一种带有金属碳硼烷 [3-铁双（二碳硼烷）] 氧化还原标记的寡核苷酸探针，该探针沉积在金电极上，用于电化学测定源自禽流感病毒（AIV）、H5N1 型的 DNA 序列。含有 5'-末端 NH2 基团的寡核苷酸探针通过 NHS/EDC 偶联到先前沉积在金表面的 3-巯基丙酸 SAM 上，与电极共价连接。在杂交前后，金属碳硼烷配合物的 Fe(III)中心的氧化还原活性变化被用作分析信号。通过杂交产生的信号与互补或非互补 20 -mer ssDNA 或由 180-190 bp（dsDNA）组成的各种 PCR 产物等靶标进行记录，通过 Osteryoung 方波伏安法（OSWV）进行记录。开发的系统对与探针序列互补的靶标非常敏感，检测限估计分别为 0.03 fM（S/N=3.0）和 0.08 fM（S/N=3.0）（20-mer ssDNA）和 dsDNA（PCR 产物）。非互补靶标产生的响应非常微弱。此外，该基因传感器适用于区分互补区域位置不同的 PCR 产物。
