Studies Of Novel Biosensing Technology Based On Nanomaterials And Rolling Circle Amplification

The activities of enzymes, nucleic acids and small molecules play irreplaceable roles inlife entity. Therefore, the development of strategies with high sensitivity, selectivity andaccuracy has great significance for vital research and clinical diagnosis and therapy.Fortunately, biosensing technique affords powerful means for these requirements. In recentyears, the development of nanotechnology and rolling circle amplification provides a newplatform for biosensors with high sensitivity and selectivity. In this thesis, a series of novelbiosensing strategies based on nanomaterials and rolling circle amplification (RCA) weredeveloped for the detection of microRNA, DNA methylation and histone deacetylation,respectively. The detailed content was described as follows：The ability to detect spatial and temporal microRNA (miRNA) distribution at thesingle-cell level is essential for understanding the biological roles of miRNAs andmiRNA-associated gene regulatory networks. In Chapter2, we report for the first time thedevelopment of a target-primed rolling circle (TPRCA) amplification strategy for highlysensitive and selective in situ visualization of miRNA expression patterns at the single-celllevel. This strategy uses a circular DNA as the probe for in situ hybridization (ISH) with thetarget miRNA molecules, and the free3′terminus of miRNA then initiates an in situ RCAreaction to generate a long tandem repeated sequence with thousands of complementarysegment. After hybridization with fluorescent detection probes, target miRNA molecules canbe visualized with ultrahigh sensitivity. Because the RCA reaction can only be initiated bythe free3′end of target miRNA, the developed strategy offers the advantage over existingISH methods in eliminating the interference from precursor miRNA or mRNA. This strategyis demonstrated to show high sensitivity and selectivity for the detection of miR-222expression levels in human hepatoma SMMC-7721cells and hepatocyte L02cells. Moreover,the developed TPRCA-based ISH strategy is successfully applied to multiplexed detectionusing two-color fluorescent probes for two miRNAs that are differentially expressed in thetwo cell lines.DNA methylation is an important epigenetic event for transcriptional regulation, beingregarded as a biomarker for cancer. In Chapter3, we present a facile detection of DNAmethylation by RCA coupled with fluorescent DNA-scaffolded silver nanoclusters (AgNCs).After bisulfite treatment of methylated DNA, padlock probe was hybridized onto the targetbisulfte treated and formed a circular probe by the E.coli DNA ligase if it was a perfect match between them. The oligonucleotides as scaffolds for the synthesis of AgNCs servesubsequently as a template for RCA. After HhaI cleavage reaction, the resultant reporteroligonucleotides can act as scaffolds for the synthesis of fluorescent AgNCs functioning assignal indicators in a label-free and environmental-friendly format. This RCA-based methodexhibits excellent specificity and high sensitivity with a detection limit of6.4fM.Histone deacetylase is high expression in cancer cells and involve in many processessuch as tumor cell growth, proliferation and expression regulation. In recent years, it has gotincreasingly attraction from academic attention as a target for cancer therapy. In Chapter4,we chose histone deacetylase1as analysis object and establish a new histone deacetylationdetection method based on graphene oxide platform. In this method, a polypeptide sequencewas designed to have two parts of functions, a portion of the sequence can adsorb on thesurface of the graphene oxide through the π-π stacking so that the graphene oxide can quenchthe fluorescence of fluorophore through fluorescence resonance energy transfer; anotherportion contains a fluorophore and the lysine acetylation site. In the presence of the histonedeacetylase, acetyl on the lysine will be dislodged. Thus the site of lysine can be recognizedand cleavaged by trypsin, the fluorophore is free to avoid the graphene oxide quenching. Atlast, the intensity of fluorescent signal is detected after the reaction to achieve the purpose toquantitative the histone deacetylase1.