A New Method Based On Fluorescence Quenching Effect Of Nanomaterials In MicroRNA Detection

MicroRNAs (miRNAs) are a group of small noncoding18-25nucleotide-long RNAs which can regulate the gene expression by complementary binding to the3’-untranslated regions of their target messenger RNAs. Recently, aberrant expression of miRNAs is commonly observed in cancer initiation, oncogenesis, and tumor response to treatment, therefore miRNAs emerge as tissue-specific biomarkers for diagnosis and prognosis and as targets for new drug discovery. The efficient use of miRNA-biomarker requires precise analysis of miRNAs, so the aim of this thesis is to develop a rapid, sensitive, and selective fluorescence approach used for detection of miRNAs. This assay can be used for estimating the miRNA expression in cells with good reliability, and could provide comprehensive and dependable information for the early detection of miRNA-related cancer. The main results are as follows:1. We report a new strategy for microRNA analysis based on the fluorescence quenching of gold nanoparticles (AuNPs) integrating with a conformation-switched hairpin-structured oligonucleotide probe for improving selectivity. The probe, which was labeled with a fluorophore (FITC, fluorescein isothiocyanate) at its5’-terminus, was assembled on the AuNPs surface. The assay is based on the fluorescence recovery of the FITC after the FITC-labeled probe is hybridized with the target. The plot of the background-subtracted fluorescence intensity vs. the logarithm value of miR-122concentration displays a linear relationship in the range from0.05to50pM with a limit of detection (LOD) of-0.01pM (at a signal/noise of3). The proposed’ approach displays a good selectivity by discriminating one-based mismatched target sequence, and has a potential application in single nucleotide polymorphism analysis. Moreover, the proposed assay method has a promise in practical application with great accuracy and reliability for miRNA detection, and can be a potential tool for sensitive analysis of miRNAs (biomarkers) in tissues or cells and supplies valuable information for biomedical research and early clinical diagnosis.2. This chapter reports a rapid, sensitive, and selective miRNA assay by coupling the graphene oxide (GO) fluorescence quenching with site-specific cleavage of an endonuclease. The method is developed by designing a single-stranded probe that carries both a binding region (facilitates the interaction of the probe with GO, inducing fluorescence quenching of the5’-terminus-labeled fluorophore, FAM) and a sensing region (specifically recognizes the target (miR-126) and hybridizes with it to form a duplex, which contains the RsaI endonuclease-recognized sequence). The assay is based on the fluorescence recovery of the FAM after the duplex was cleaved by RsaI. It can determine as low as～5fM (at a signal/noise of3) miR-126with a linear range of4orders of magnitude and has an ability to discriminate the target sequence from even single-base mismatched sequence and other miRNA sequences. Moreover, it can also be used for rapid estimation of the miR-126expressions in several different type of cells (including HMVEC, H226, A549, H358, MCF-10A, and MDA-MB231cells). The advantages of this assay is that it operates via detecting the recovered fluorescence signal, which is a combined result of the specific hybridization and the site-specific cleavage, thus should be impervious to false signals arising due to the nonspecific adsorption of interferants. Therefore, the approach could provide comprehensive and dependable information for the early detection of miRNA-related cancer.