Institute of Animal Reproduction and Food Research, Polish Academy of Sciences , Tuwima 10, Olsztyn, Warmian-Masurian, 10-747 Poland.
Anal Chem. 2013 Nov 5;85(21):10167-73. doi: 10.1021/ac401547h. Epub 2013 Oct 9.
The duo-genosensor consisting of two different oligonucleotide probes immobilized covalently on the surface of one gold electrode via Au-S bond formation was used for simultaneous determination of two different oligonucleotide targets. One of the probes, decorated on its 5'-end with ferrocene (SH-ssDNA-Fc), is complementary to the cDNA representing a sequence encoding part of H5 hemagglutinin from H5N1 virus. The second probe, decorated on its 5'-end with methylene blue (SH-ssDNA-MB), is complementary to cDNA representing the fragment of N1 neuraminidase from the same virus. The presence of both probes on the surface of gold electrodes was confirmed with Osteryoung square-wave voltammetry (OSWV). The changes in redox activity of both redox active complexes before and after the hybridization process were used as analytical signal. The peak at +400 ± 2 mV was observed in the presence of 40 nM ssDNA used as a target for SH-ssDNA-Fc probe. This peak increased with the increase of concentration of target ssDNA. It indicates the "signal on" mode of analytical signal generation. The peak at -250 ± 4 mV, characteristic for SH-ssDNA-MB probe, was decreasing with the increase of the concentration of the complementary ssDNA target starting from 8 to 100 nM. This indicates the generation of electrochemical signal according to the "signal off" mode. The proposed duo-genosensor is capable of simultaneous, specific, and good sensitivity probing for the sequences derived from genes encoding two main markers of the influenza virus, hemagglutinin and neuraminidase.
由通过 Au-S 键形成共价固定在一个金电极表面的两个不同寡核苷酸探针组成的双基因传感器用于同时测定两种不同的寡核苷酸靶标。其中一个探针在其 5'-末端用二茂铁（SH-ssDNA-Fc）修饰，与 cDNA 互补，该 cDNA 代表编码 H5N1 病毒 H5 血凝素部分序列的序列。第二个探针在其 5'-末端用亚甲蓝（SH-ssDNA-MB）修饰，与 cDNA 互补，该 cDNA 代表来自同一病毒的 N1 神经氨酸酶的片段。金电极表面存在两种探针的情况通过 Osteryoung 方波伏安法（OSWV）得到证实。杂交前后两种氧化还原活性复合物的氧化还原活性变化被用作分析信号。在存在用作 SH-ssDNA-Fc 探针的靶标 ssDNA 40 nM 的情况下，观察到+400±2 mV 的峰。该峰随着靶标 ssDNA 浓度的增加而增加。这表明分析信号生成的“信号开启”模式。对于 SH-ssDNA-MB 探针，特征性的-250±4 mV 峰随着互补 ssDNA 靶标浓度从 8 到 100 nM 的增加而减小。这表明根据“信号关闭”模式生成电化学信号。所提出的双基因传感器能够同时、特异性和良好灵敏度地探测来自编码流感病毒两种主要标志物血凝素和神经氨酸酶的基因的序列。
