基于窄内径毛细管-LIF检测系统的miR-221-3p超小体积无扩增检测

Amplification-Free Determination of miR-221-3p in Ultra-Small Volume Based on Narrow Capillary-LIF Detection System

微小核糖核酸(microRNA,miRNA)是多种疾病和生物过程的一类标志物,无扩增的miRNA检测具有保真度高以及成本低的特点。本研究采用内径为1.06μm的窄内径毛细管作为检测通道,耦联高灵敏的激光诱导荧光(LIF)检测系统用于微小核糖核酸221-3p(miR-221-3p)的超小体积无扩增检测。以miR-221-3p为靶标设计锁核酸(LNA)修饰的分子信标(MB)探针LNA/MB-221,该探针在MgCl2溶液浓度大于3 mmol/L时背景信号较弱,背景干扰更小;杂交温度为65℃时杂交样品的荧光信号更强。探针与靶标miR-221-3p的杂交样品在低浓度范围(1~10 nmol/L)内线性关系较好,样品消耗仅850fL时检出限达到8.5×10^(-22)mol(约508个分子)。该方法也可实现人胚肺成纤维细胞HFL-1和非小细胞肺癌细胞A549的无扩增检测及相对表达量分析。

Micro-ribonucleic acid(microRNA, miRNA) is a class of important biomarkers for a variety of diseases and biological processes. Amplification-free determination of miRNA has the characteristics of high fidelity and low cost. In this study, a narrow capillary with an inner diameter of 1.06 μm was used as the detection channel and coupled with a highly sensitive laser-induced fluorescence(LIF) detection system for amplification-free determination of micro-ribonucleic acid 221-3p(miR-221-3p) in ultra-small volume. The molecular beacon probe with locked nucleic acid(LNA/MB-221) modification for miR-221-3p was designed. The probe had weak background signal when the concentration of MgClsolution was greater than 3 mmol/L, providing less background interference, and the signal was better when the hybridization temperature was 65 ℃. The hybrid of miR-221-3p with LNA/MB-221probe had excellent linearity in the concentration range of 1 nmol/L to 10 nmol/L. The limit of detection with the sample consumption of 850 fL was 8.5×10^(-22)mol(approximately 508 molecules). We used this method to detect the miR-221-3p level in human fetal lung fibroblast 1(HFL-1) and non-small cell lung cancer cell(A549), and realized the non-amplification detection and relative expression analysis of miR-221-3p in cells.