Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain.
Canaan RI & Facultad Medicina, Universidad Alfonso X el Sabio, E-28691 Madrid, Spain.
Biosens Bioelectron. 2016 Dec 15;86:516-521. doi: 10.1016/j.bios.2016.07.003. Epub 2016 Jul 6.
A novel electrochemical approach for determination of miRNAs involving a sandwich hybridization assay onto streptavidin-magnetic beads (Strep-MBs), hybridization chain reaction (HCR) amplification and amperometric detection at disposable screen-printed carbon electrodes is reported. Using miRNA-21 as the target analyte, a dynamic linear range from 0.2 to 5.0nM with a 60pM (1.5fmol in 25μL) detection limit was obtained. The achieved sensitivity is 24-fold higher than a non-HCR amplification approach involving conventional sandwich type assay onto MBs. Moreover, the whole assay time lasted 1h 45min which is remarkably shorter than other reported methodologies. The methodology exhibited full selectivity against other non-complementary miRNAs as well as an acceptable discrimination between homologous miRNA family members. The applicability of this novel approach was demonstrated by determining mature miRNA-21 in total RNA (RNAt) extracted from tumor cells and human tissues.
一种新型电化学方法用于 miRNA 的测定，该方法涉及将夹心杂交反应固定在链霉亲和素磁珠（Strep-MBs）上，然后进行杂交链式反应（HCR）扩增，最后在可抛弃的丝网印刷碳电极上进行安培检测。以 miRNA-21 为目标分析物，得到了从 0.2 到 5.0nM 的动态线性范围，检测限为 60pM（25μL 中 1.5fmol）。与涉及 MBs 上常规夹心型测定的非 HCR 扩增方法相比，该方法的灵敏度提高了 24 倍。此外，整个测定时间仅为 1 小时 45 分钟，明显短于其他已报道的方法。该方法对其他非互补 miRNA 具有完全选择性，并且能够在同源 miRNA 家族成员之间进行良好的区分。该新方法的适用性通过测定从肿瘤细胞和人体组织中提取的总 RNA（RNAt）中的成熟 miRNA-21 得到了验证。
