Detection Of Protein By The Combination Of Terminal Protection And Isothermal Signal Amplification Strategy

Sensitive and selective detection of proteins plays critical roles in both molecular diagnostics and biomedical research. To achieve sensitive and selective detection of protein, affinity ligands have emerged as powerful tools. Among the affinity ligands of protein, small molecular-linked DNA has received more and more attention due to its label-free, separation-free and capable of integrated into DNA-based amplification methods. Terminal protection refers to the methods that take use of small molecular-linked DNA to recognize protein receptors, and using exonuclease to translate the recognition of protein into the presence of a specific DNA. This recognition and translation method is homogeneous and simple, without the limitation on DNA coding, which offers a powerful platform for protein detection.Taking use of signal amplification strategy to translate the presence of a target into numbers of signal output is necessary for sensitive detection of protein. With the development of technology, more and more signal amplification methods have been established. The isothermal amplification methods based on nuclease have received much attention, because they are isothermal and convenient. Among these methods, the amplification methods based on polymerase and exonuclease III have great potential in developing sensitive protein detection methods, due to its characteristic of fast and universal. By flexible design, we made some improvement on the basis of the amplification methods based on polymerase and exonuclease III to further improve the amplification efficiency. We also combined these amplification methods with terminal protection to develop sensitive and selective protein detection methods.The main researches included in the dissertation are presented as following:The first chapter is the introduction section, which includes terminal protection and amplification methods based on nucleases. Firstly, we introduced the background, principle and advantages of terminal protection. Taking the recognition probes as the basis of classification, methods based on terminal protection is divided into methods based on small-molecular linked DNA, conformational changeable aptamer, and sequence-specific duplex. The origin, development state and direction were introduced respectively. The defects of each category and their influence on the detection are discussed. The typical recognition pattern of terminal protection and reports was analyzed. Secondly, based on different nuclease used for amplification, different classifications of the commonly-used isothermal amplification methods are discussed. We also retraced the origin and development of different kinds of amplification methods, analyzed the defects on every stage and the corresponding improvement.In the second chapter, we designed a DNA machine based on circular strand-displacement polymerization reaction. The DNA machine was used to achieve dual-amplification of specific DNA sequence, and it was combined with terminal protection, finally achieved sensitive detection of protein. Based on the dual-amplification process mediated by the cooperative DNA machine, this method was able to detect folate receptor in the linear relationship between 0.80 pM-1.0 nM, and the detection limit was 0.23 pM (3σ/slope), which is comparable to most existed folate receptor detection methods. Also, this method generated G-quadruplex product, which could integrated with N-methylmorpholine and output signal,.In chapter 3, we developed a sensitive protein detection method via terminal protection and exonuclease Ⅲ-based exponential amplification. A hairpin which blocked a repeat of primer sequence in stem was used to process exponential amplification. Graphene oxide, which could distinguish the hairpin before or after exonuclease digestion and generate signal difference, was used to output signal. The feasibility of this method was verified by fluorescence method. The presence of target would end up with improvement of fluorescence signal, which proved that this method has great potential on sensitive detection of protein.