The Construction Of Fluorescence Amplification Strategy Based On Hybridization Chain Reaction And The Research Of Its Application

Protein medicines can be devided into polypeptides, genetic engineering drugs, monoclonal antibodies and recombinant vaccines, etc. Compared with traditional small molecule drugs, protein medicines possess the unique characteristic, such as, high activity, strong specificity, low toxicity, clearly biological function and conducive to be applied in clinical application. The dosage of protein medicine in the process of taking will directly influence the effect of the treatment and prevention, and is related to people’s health and safety. In addition to the therapeutic effect, some protein medicine is also the component of normal secretion in the body, and it can be used as diagnostic markers. Thus, the strategy that is able to detect the trace amount of drug ingredients in complex sample with simplicity, fastness, sensivity and reliability is of great importance to adjust the dosage, monitor the development degree of disease and the early diagnosis of some diseases.In recent years, the sensitive method that is based on nucleic acid amplification for determination of protein medicine in complex samples provides powerful tools and methods. Specially, hybridization chain reaction (HCR) has attracted much attention. HCR is an isothermal and enzyme-free signal amplification method, which is powered by the free energy of base pair formation. Compared with traditional amplification methods, the DNA fragments in HCR are not duplicated but act as initiator temples to trigger self-assemble into polymers. Various biosensors based on HCR strategy for different targets, such as DNA, protein and polysaccharide has been demonstrated, even for in vivo imaging. Taking advantage of HCR method and the fessibility of its applification, we proposed the novel and sensitive method based on HCR signal amplification for the detection of protein medicine. There are fous parts in this article and the main contents are as follows:The first chapter is the introduction section, and this article summarizes the significance, background, principle, the testing method and the existing problems of the detection of protein medicines at the present stage. In addition, the single molecule detection in the quantitative detection is described.In the second chapter, an ultrasensitive fluorescence method for determination of protein is developed based on HCR. Because the nanobead probe carries with a large number of oligonucleotides per protein binding event, there is obvious amplification in the nicked polymers. Then, numerous SYBR Green I molecules were intercalated into the grooves of the long dsDNA polymers, generating a substantially apparent increase in the corresponding fluorescence intensity. With HCR amplification and magenetic nanobead to preamplify the fluorescence signal and reduce the background signal, the detection range of this method was from5.0×10-17mol/L to1.0×10-12mol/L, and the detection limit of this assay was1.4×10-17mol/L. Compared with the reported protein detection methods, our method exhibited ultrahigh sensitivity. What is more, the assay was also studied for clinical application in human serum with a satisfactory and reliable result.In the third chapter, a novel and sensitive quantitative method based on single molecule counting and HCR is constructed. The streptavidin acted as a bridge bounded to the biotinylated immunocomplex, which provided three sites to fixate the biotinylated initiator strands. The initiator strands triggered the chain reaction of hybridization to form a long double-helix polymer and SYBR Green I, acted as the fluorescence label, intercalated into the grooves of the long dsDNA polymer. Then, the quantitative detection of TNF-a was realized by single molecule counting. According to the relationship between the number of bright dots and the concentration of targets, the quantitative detection of protein medicine is realized. These results greatly demonstrated that the proposed method possessed the potentiality in clinical application and it was suitable for quantification of biomarker under low concentration.