Target-Triggered Enzyme-Free Amplification Strategy for Sensitive Detection of MicroRNA in Tumor Cells and Tissues

Abstract

MicroRNAs (miRNAs) participate in important processes of life course. Because of their characters of small sizes, vulnerable degradabilities, and sequences similarities, the existing detection technologies mostly contain enzymatic amplification reactions for acquisition of high sensitivities and specificities. However, specific reaction conditions and time-dependent enzyme activities are caused by the accession of enzymes. Herein, we designed a target-triggered enzyme-free amplification platform that is realized by circulatory interactions of two hairpin probes and the integrated electrochemiluminescence (ECL) signal giving-out component. Benefiting from outstanding performances of the enzyme-free amplification system and ECL, this strategy is provided with a simplified reaction process, high sensitivity, and operation under isothermal conditions. Through detection of the miRNA standard substance, the sensitivity of this platform reached 10 fmol, and a splendid specificity was achieved. We also analyzed three tumor cell lines (human lung adenocarcinoma, breast adenocarcinoma, and hepatocellular liver carcinoma cell lines) through this platform. The sensitivities of 10(3) cells, 10(4) cells, and 10(4) cells were, respectively, achieved. Furthermore, clinical tumor samples were tested, and 21 of 30 experimental samples gave out positive signals. Thus, this platform possesses potentials to be an innovation in miRNA detection methodology.

Detection of microRNA in clinical tumor samples by isothermal enzyme-free amplification and label-free graphene oxide-based SYBR Green I fluorescence platform

“…They involve in many important life activities and they have higher expression levels in many kinds of cancers (Hwang and Mendell, 2006;Yue and Tigyi, 2006;Khan et al, 2011;Liao et al, 2014). Therefore, quantification of miRNAs is great import in clinical diagnosis of cancer.…”

“…The usage of enzymes will need particular reaction time and conditions to maintain the enzyme's activities. Recently, an enzyme-free amplification (EFA) method has been developed (Yin et al, 2008;Li et al, 2011;Chen et al, 2013;Jiang et al, 2013;Liao et al, 2014). It overcomes the disadvantages of enzymatic amplification.…”

“…However, these methods all used luminophore labeled probes as signal probes. (Yin et al, 2008;Li et al, 2011;Chen et al, 2013;Jiang et al, 2013;Liao et al, 2014).…”

Development of an indirect competitive ELISA based on immunomagnetic beads’ clean-up for detection of maduramicin in three chicken tissues

“…It has been widely used for cell separation (Šafařı´k & Šafařı´ková, 1999), protein or gene analysis (Liao et al, 2014;Zhu, Zhou, & Xing, 2012) and pathogen detection (Diler, Obst, Schmitz, & Schwartz, 2011). MBs with a large surface area have good biocompatibility and fast reactability to couple with antibodies or small molecules .…”

A glassy carbon electrode modified with in-situ generated chromium-loaded CdS nanoprobes and heparin for ultrasensitive electrochemiluminescent determination of thrombin

“…Due to the unique quantum size dependent electrochemical properties of semiconductor nanocrystals (NCs) and controllable ECL merits [2], NCs, have become fascinating luminophores for the fabrication of biosensors. The prepared ECL biosensors have been widely applied in immunoassay of protein, DNA detection, carbohydrate analysis and cytosensing [3][4][5][6][7][8][9]. However, compared with conventional luminescent reagents like luminal or Ru(bpy) 3 2+ , NCs usually suffer from relatively weaker ECL emissions.…”