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

The essential information on small molecules, nucleic acids, protein and enzymes obtained accurately and sensitively has great significance for biological medicine study, clinical diagnosis and therapy. However, with the development of the scientific research, getting the dynamic information of these life processes sensitively and accurately in real time is still great challenge to analysts. Therefore, the development of strategies with high sensitivity, selectivity and accuracy is important for biomedical research and clinical diagnosis. In this thesis, a series of novel biosensing strategies based on rolling circle amplification and nanomaterials were developed for small molecule, nucleic acid, proteins and enzyme activity detection, respectively. These results primarily proved that the proposed technologies were feasible, reliable and accurate. The detailed content was described as follows：In Chapter2: The ability to detect spatial and temporal microRNA (miRNA) distribution at the single-cell level is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. We report for the first time the development of a target-primed RCA (TPRCA) strategy for highly sensitive and selective in situ visualization of miRNA expression patterns at the single-cell level. This strategy uses a circular DNA as the probe for in situ hybridization (ISH) with the target miRNA molecules, and the free3′terminus of miRNA then initiates an in situ RCA reaction to generate a long tandem repeated sequence with thousands of complementary segment. After hybridization with fluorescent detection probes, target miRNA molecules can be visualized with ultrahigh sensitivity. Because the RCA reaction can only be initiated by the free3′end of target miRNA, the developed strategy offers the advantage over existing ISH methods in eliminating the interference from precursor miRNA or mRNA. This strategy is 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 detection using two-color fluorescent probes for two miRNAs that are differentially expressed in the two cell lines. The results reveal that the developed strategy may have great potential for in situ miRNA expression analysis for basic research and clinical diagnostics.In Chapter3: DNA methylation is an important epigenetic event for transcriptional regulation, being regarded as a biomarker for cancer. Sensitive and specific detection of DNA methylation in CpG sites of genomic DNA is imperative to DNA methylation discovery, study, and clinical diagnosis. Herein, we present a facile detection of DNA methylation by RCA coupled with fluorescent DNA-scaffolded AgNCs. After bisulfite treatment of methylated DNA, padlock probe was hybridized onto the target bisulfte 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 serve subsequently as a template for RCA. After HhaI cleavage reaction, the resultant reporter oligonucleotides can act as scaffolds for the synthesis of fluorescent AgNCs functioning as signal indicators in a label-free and environmental-friendly format.This RCA-based method exhibits excellent specificity and high sensitivity with a detection limit of6.4fM.In Chapter4: we presented a novel biosensing technology for the detection of telomerase activity in cancer cells based on the fact that guanine can trigger transformation of Ag NCs from a dark species to a bright red-emitting species. In this assay, the primer contains oligonucleotides as scaffolds for the synthesis of AgNCs. This primer can act as scaffolds for the synthesis of dark AgNCs without telomerase. The bright red-emitting clusters AgNCs can be obtained when the primer adds hexameric repeats (TTAGGG)n by the action of telomerase.The results indicated that the method could be used for sensitive determination of telomerase in a concentration range from500to50000HeLa cells. Given the simplicity, convenience of this approach, the proposed method may provide an alternative approach for the study of the telomerase activity.In Chapter5: A novel biosensor was designed for sensing of targets such as protein and small molecule based on the self assembled aptamer–MoS2nanosheets architecture. This DNA-MoS2nanosheet was constructed with aptamer labeled with fluorophore only at one end can self assembled onto the surface of MoS2nanosheet to form stable aptamer–MoS2nanosheet architecture, still keeping the binding affinity and specificity of the aptamer. DNA-MoS2nanosheets can act as a low background signal platform was used for the small molecule (Adenosine triphosphate) or protein (human α-thrombin) detection based on long-range resonance energy transfer. In the absence of target, the adsorption of the aptamer labeled with fluorophore on MoS2nanosheets makes the dyes approaching closely toward the proximity to MoS2nanosheets surface resulting in high efficiency quenching of fluorescence of the dyes and shows very low background. With the addition of target, binding of the aptamer probes to the target can release the aptamer away from the MoS2nanosheet, the quenched fluorescence is recovered significantly. This biosensor has the advantages in its superb specificity, being rapid, and convenient. Morover, aptamer–MoS2aptasensor design can be easily extended to develop a variety of probes for detection of a wide range of targets by simply changing the fluorophores and altering aptamer sequences.In Chapter6: DNA phosphorylation, catalyzed by polynucleotide kinase (PNK), plays significant regulatory roles in many biological events. Here, a novel fluorescent nanosensor based on phosphorylation-specific exonuclease reaction and efficient fluorescence quenching of single-stranded DNA (ssDNA) by WS2nanosheet has been developed for monitoring the activity of PNK using T4polynucleotide kinase (T4PNK) as a model target. The fluorescent dye-labeled double-stranded DNA (dsDNA) remains highly fluorescent when mixed with WS2nanosheets because of the weak adsorption of dsDNA on WS2nanosheets. While dsDNA is phosphorylated by T4PNK, it can be specifically degraded by λ exonuclease, producing ssDNA strongly adsorbed on WS2nanosheets with greatly quenched fluorescence. Because of the high quenching efficiency of WS2nanosheets, the developed platform presents excellent performance with a wide linear range, low detection limit and high signal-to-background ratio, the detection limit of T4PNK was0.01U mL-1. Additionally, inhibition effects from adenosine diphosphate, ammonium sulfate, and sodium chloride have been investigated. The method may provide a universal platform for PNK activity monitoring and inhibitor screening in drug discovery and clinic diagnostics.