Studies Of Novel Biosensing Technology Based On Silver Nanoclusters And Nucleic Acid Probe

Biosensor holds great potential in practical analysis and scientific research because it has advantages such as low cost, high sensitivity, high selectivity and the ability to be used in complex system. Silver nanoclusters and nucleic acid probes have been widely used in the field of biosensors and exhibit great achievements. Therefore, they can provide more sensor design strategies and platforms. This doctoral thesis is based on advantages of silver nanoclusters and nucleic acid probes in biosensing and biochemical analysis applications, combining with the sophisticated signal amplification methods, focusing on the research hotspots, establishing several biosensing and biochemical analysis methods for mRNA quantitative analysis and sensitive detection of small molecules. The developed methods are simple, high sensitive, specific, low cost, and can also be applied in complex system. The detailed contents are described as follows.Oligonucleotide-templated AgNCs have attracted special attention in chemical-sensing and biomedical imaging, but low sensitivity have limited its development in biosensor. So,we used hybridization chain reaction as a signal amplification. In chapter 1, we developed a strategy based on hybridization chain reaction induced fluorescence enhancement of silver clusters for mRNA detection. Different from traditional HCR, four hairpin probes were designed here, in order to avoid the guanine-rich tail and the DNA-AgNCs capturing strand to be designed in the same hairpin probe and to induce non-specific fluorescence activation. While hairpin probes HI has been extended with G-rich sequence at its 3'end, and hairpin probes H3 has been extended 28 nt segment at its 5'end for hybridization with a part of NC probe which contains DNA template for silver clusters preparation. The presence of mRNA triggers orderly catalyzed hybridization among hairpin probes HI, H2, H3 and H4. As a result, long chain DNA polymer structures were generated. The hybridization with presynthesis DNA-AgNCs brings the G-rich overhang close to AgNCs, which greatly enhances the fluorescent intensity of AgNCs. This assay is capable of effectively detecting target mRNA. Compared with previously reported mRNA detection methods, this method does not need any chemical modifications. In adition, the method has other advantages like high sensitivity, and can be used for the determination of mRNA in complex cellular extracts.Split aptamer represents one of the major mechanisms in developing sensors toward varying analytes. Current split aptamer assays have been limited by their inferior sensitivity, while novel split aptamer-based strategies with efficient signal amplification has not been reported. The challenge for combining signal amplification approaches with split aptamer lies in the lacking of downstream biochemical reactions that are specific to the assembly of split aptamer fragments. In chapter 3, we developed a novel, highly sensitive split aptamer mediated endonuclease amplification (SAMEA) strategy for the construction of aptameric sensors. We realize that the assembly of split aptamer fragments in response to its target actually brings two tail sequences in close proximity, forming a three-way junction structure that is essential for proximity-dependent hybridization. This realization motivates us to investigate the hypothesis of using the three-way junction structure as the substrate of DNA endonuclease IV (Endo IV). This investigation reveals a new finding that Endo IV efficiently cleaves the substrate in a three-way junction structure with substantial signal amplification. Based on this new finding, a new split aptamer-based signal amplification strategy is then developed via a combination of split aptamer with proximity-dependent hybridization mediated Endo IV amplification. To our knowledge, this is the first time that split aptamer has been coupled to a signal amplification method. Therefore, it may provide a new paradigm for the design of ultrasensitive aptameric sensors, because incorporation of efficient signal amplification in split aptamer can substantially enhance the detection sensitivity. To demonstrate the concept, we choose small molecules cocaine as the model targets in this study. The results revealed that the developed strategy offers ?105-fold sensitivity improvement as compared to previous amplification-free split aptamer assays.