| Among different analysis approaches, signal amplification is one of the most popular methods because one target can generate multiple signal after amplification, which promises a higher sensitivity. So far, more and more signal amplification techniques have been developed. Among these techniques, enzyme-assisted amplification attracts much attention because of the high signal amplification efficiency. Exonuclease III (Exo III)-based target recycling is a newly developed enzyme-assisted amplification approach. It has certain advantages including isothermality, high efficiency, and universality. Thus, Exo Ⅲ-based target recycling holds a great promise for the establishment of highly sensitive analysis method.Label-free detection is another important development tendency of fluorescence analysis methods. Most of current fluorescence analysis approaches rely on the fluorophore-label probes to generate fluorescent signals. These labelled probes always brought about high cost and complex operation. Moreover, the labeled fluorophore might not be completely quenched, which increased the background. In contrast, label-free methods are usually low-cost, simplify, and avoid the high background caused by fluorophore-label probes.In order to construct biosensors that have advantages including high detection sensitivity and label-free analysis, we combine Exo Ⅲ-based signal amplification with the formation of G-quadreplex DNA structure. In this thesis, we construct a series of label-free and amplifying detection technology platforms for the quantitative analysis of three different targets including DNA, biological small molecule, and metal ion. The main researches included in the dissertation are presented as following:The first chapter is the introduction section to summarize characters of different enzymes including endonuclease, exonuclease, ligase, and polymerase. The applications of these enzymes in fluorescence analysis and signal amplification are also summarized. The formation mechanism and analytical application of G-quadreplex DNA structure are also summarized.In the second chapter, a sensitive and label-free fluorescence assay for DNA detection has been developed based on cascade signal amplification combining Exo Ⅲ-catalyzed recycling with rolling circle amplification (RCA). Under the optimal conditions, the fluorescence intensity increased proportionally to the concentration of target DNA within the range from8－10-12mol L-1to100×10-12mol L-1, with a detection limit of3.2×10-12mol L-1(3σ/slope). Compared with traditional Exo Ⅲ-based signal amplification methods, this cascade signal amplification strategy exhibited higher signal amplification efficiency and improved the detection sensitivity significantly. Moreover, the G-quadruplex RCA product could produce fluorescence signal by simply adding NMM, without the need of fluorophore-labeled oligonucleotide probes.In the third chapter, we have developed a highly sensitive and label-free fluorescence signal amplification strategy for the detection of NAD+based on ligation-triggered and Exo Ⅲ-catalyzed signal amplification. A trigger DNA was split into two separate fragments as the building blocks for the DNA ligation reaction, which was used to recognize target NAD+. Next, trigger DNA could be hybridized with G4/template DNA duplex and trigger the recycling process in the present of Exo III, releasing lots of G4DNA. N-methyl mesoporphyrin IX (NMM) was employed to interact with G4DNA and generated label-free fluorescence signal. The fluorescence intensity increased proportionally to the concentration of target DNA within the range from5.0×10-12mol L-1to1.0×10-9mol L-1,with a detection limit of3.0×10-12mol L-1(3σ/slope). This signal amplification strategy offered significant advantages compared with previous methods, including higher sensitivity, better selectivity and performance in complex samples. These advantages endow our signal amplification strategy with a promising potential in biological small molecules quantification. |
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