Study On Fluorescence Biosensor Based On Nucleic Acid

The design of biosensors is an important and interesting subject in analytical chemistry and biochemistry and has been extensively studied. Fluorescent chemical sensor relys on fluorescence signals as the detection methods, including fluoscence enhancement, quenching or migration of emission wavelength. With convention, high sensitivity, selectivity and real-time detection making use of optical fiber technology, this kind of biosensor has become a field of interest of photochemical sensor researchers.Protein is a type of important biomarkers. For example, many tumor markers are enzyme, antigen, global protein, ferritin and protein like. The hazards caused by illegally adding melamine into food have been the concern of the community in recent years. Because melamine contains66%nitrogen by mass, there are frequent reports that melamine has been illegally added into milk products and feeds in order to increase apparent protein content. Therefore, it is very useful to develop simple, rapid and sensitive methods for the detection of melamine in real samples, Heavy metals in our environment can hardly be degradated, accumulating in human’s body via various approaches, doing harm to human, even endangering when seriously. Therefore, it is of great significance to establish simple, fast, sensitive detection methods. In this research paper, we develop for the detection of several fluorescent chemical sensor using fluorescence signal as detection signal. Details are as follows:(1) Here we report the principle of a terminal protection assay of small-molecule-linked DNA for protein. First, we use small molecules to modify DNA chains, through the special recognition between small molecules and the corresponding combination proteins, rolling circle amplification reaction and ExoIII amplification to achieve the detection of folate binding protein.. Reactions are carried out in homogeneous. After reaction between small molecules and proteins, we add Exo I which can digest a single stranded DNA into the solution, then protection of the DNA can not be hydrolyzed; Second, introduction of the ring probe (ligation probe) into the solution, after template chain and ring probe have hybridizated, we add E-coli DNA ligation and ligation buffer to induce link reaction; With primer, Bst DNA polymerase, deoxyribonucleic acid and circle template, rolling circle amplification can perform, which produce a long single chain DNA which repetitive sequence is complementary with circle template DNA sequence. Finally, we add a taqman probe (fluorophore and quencher groups are close enough, so there is no fluorescence signal when the probe exits lonely) which can hybrid with RCA product and ExoⅢ to the system. The RCA product hybridize with taqman probe and then ExoⅢ strats to digest the probe, so that the fluorophore and quencher groups are separated. Fluorescence detection was performed. Fluorescence intensity can be used for quantitative the concentration of folate binding protein.(2) A highly sensitive and selective fluorescence sensor for the detection of melamine using Sybr Green I(SG) is presented in this paper. It is based on the melamine-induced conformational change of a polythymine single stranded DNA (ssDNA) and the difference fluorescence intensity between ssDNA and double stranded DNA (dsDNA) with SG. As we know, SG is a double-strand-chelating dye and the interaction between SG and ssDNA is weak, so the fluorescence intensity is very low when the random-coil polyT24strand was mixed with only SG. In the presence of melamine, however, T-melamine-T bonding, because of the formation of triple hydrogen-bonding between melamine and thymine, results in polyT24strand conformational switching from a random coil to a hairpin structure, then SG perfectly binds to the double stranded hairpin. Thus, we can observe a remarkable increase in the fluorescent intensity. The detection limit is lower than the international standard melamine content in food. This method is simple and fast.(3) Monitoring the level of Ag+in an aquatic ecosystem continues to be of interest because Ag+is highly toxic to aquatic organisms and is widely used in the electrical industry, photography and imaging industry, and pharmacy. Numerous chemosensors have been developed for the sensitive, simple, and rapid detection of Ag+in an aqueous solution, based on organic fluorophores and semiconductor quantum dots. However, these chemosensors have some limitations such as low water solubility, poor selectivity toward Ag+. In the fourth chapter, we reported a maker-free fluorescence method for silver ions which is based on specific recognition between silver ions and cytosine. The C-rich DNA would be fold when cytosine and silver ions form the conformation of C-Ag+-C. We can obtained the significantly enhanced fluorescence signal when adding the Sybr Green I. The method is simple, fast and low cost.