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An Enzyme-Free MicroRNA Assay Based-on Fluorescence Counting of Click Chemical Ligation-Illuminated Magnetic Nanoparticles with Total Internal Reflection Fluorescence Microscopy
摘要： 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 miRNA target in cancer cells.
关键词： click chemistry,microRNA,TIRFM,magnetic nanoparticle,fluorescence counting
更新于2025-09-23 15:21:21
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A waveguide imaging platform for livea??cell TIRF imaging of neurons over large fields of view
摘要： Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1, 2] and have also been demonstrated for short-term live-imaging of robust cell types [3-5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGCs). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications.
关键词： live-cell imaging,TIRFM,neurons,large FOV,waveguide
更新于2025-09-23 15:19:57