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ACS Sens. 2018 Dec 28;3(12):2667-2674. doi: 10.1021/acssensors.8b01169. Epub 2018 Dec 3.

An Enzyme-Free MicroRNA Assay Based On Fluorescence Counting of Click Chemical Ligation-Illuminated Magnetic Nanoparticles with Total Internal Reflection Fluorescence Microscopy.

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Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an , Shaanxi Province 710119 , P. R. China.


MicroRNAs (miRNAs) have been considered as promising cancer biomarkers. However, the simple but sensitive detection of low levels of miRNAs in biological samples still remains challenging. Herein, we wish to report an entirely enzyme-free, simple, and highly sensitive miRNA assay based on the counting of cycling click chemical ligation (3CL)-illuminated fluorescent magnetic nanoparticles (MNPs) with a total internal reflection fluorescence microscopy (TIRFM). In this strategy, each miRNA molecule can trigger many cycles of click chemical ligation reactions to produce plentiful ligated oligonucleotides (ODNs) with both 5'-biotin and 3'-fluorophore, resulting in efficient signal amplification. It is worth noting that only the ligated ODNs can bring fluorophores onto streptavidin-functionalized MNPs (STV-MNPs). Notably, merely 10 fluorescent molecules on each 50 nm MNP can make it bright enough to be clearly visualized by the TIRFM, which can significantly improve the detection sensitivity for miRNA. Through fluorescence counting of individual MNPs and integrating their fluorescence intensities, the amount of target miRNA can be quantitatively determined. This miRNA assay can be accomplished in a mix-and-read manner just by simply mixing the enzyme-free 3CL reaction system with the MNPs before TIRFM imaging, which avoids tedious immobilization, washing, and purification steps. Despite the extremely simple operation, this strategy exhibits high sensitivity with a quite low detection limit of 50 fM target miRNA as well as high specificity to well discriminate miRNA sequences with a single-base variation. Furthermore, the applicability of this method in real biological samples is also verified through the accurate detection of the miRNA target in cancer cells.


TIRFM; click chemistry; fluorescence counting; magnetic nanoparticle; microRNA


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