Schmieder Stefan, Weißpflog Janek, Danz Norbert, Hübner Max, Kreth Simone, Klotzbach Udo, Sonntag Frank
Micro- and Biosystems Engineering Fraunhofer Institute for Material and Beam Technology IWS Dresden Germany.
Microoptical Systems Fraunhofer Institute for Applied Optics and Precision Engineering IOF Jena Germany.
Eng Life Sci. 2017 Sep 25;17(12):1264-1270. doi: 10.1002/elsc.201700104. eCollection 2017 Dec.
MiRNAs are endogenous noncoding RNA molecules. They play important gene-regulatory roles by binding to the mRNA of target genes thereby leading to either transcript degradation or translational repression. In virtually all diseases, distinct alterations of miRNA expression profiles have been found thus suggesting miRNAs as interesting biomarkers. Here, we present an SPR biosensor that utilizes disposable, injection-molded sensor chip/microfluidic hybrids combined with a lateral imaging optical system for parallel analysis of three one-dimensional spot arrays to detect miRNA-93. To increase the sensitivity of the biosensor we used two different amplification strategies. By adding an RNA-DNA-hybrid antibody for primary signal amplification, a limit of detection of 10 pmol/L was achieved. Based on that method we demonstrate the detection of miRNA-93 in total RNA lysate from HEK-293 cells. Utilizing an enzymatic signal amplification with Poly(A) polymerase, the sensitivity could be increased even further leading to a limit of detection of 1 fmol/L.
微小RNA（miRNAs）是内源性非编码RNA分子。它们通过与靶基因的信使核糖核酸（mRNA）结合发挥重要的基因调控作用，从而导致转录本降解或翻译抑制。几乎在所有疾病中，都发现了miRNA表达谱的明显改变，因此表明miRNAs是有趣的生物标志物。在此，我们展示了一种表面等离子体共振（SPR）生物传感器，该传感器利用一次性注塑成型的传感器芯片/微流体混合体与横向成像光学系统相结合，用于对三个一维斑点阵列进行平行分析，以检测miRNA-93。为了提高生物传感器的灵敏度，我们使用了两种不同的扩增策略。通过添加一种用于一级信号放大的RNA-DNA杂交抗体，实现了10皮摩尔/升的检测限。基于该方法，我们展示了在人胚肾293（HEK-293）细胞的总RNA裂解物中对miRNA-93的检测。利用聚腺苷酸（Poly(A)）聚合酶进行酶促信号放大，灵敏度可以进一步提高，导致检测限达到1飞摩尔/升。
