Sensing And Imaging Of The Metal Ions In Environment And Living Cells With Functional Nucleic Acid

Metal element has a dual role in the system of living organisms. On the one hand, it maintains life activities of animal and plant, but it is also likely to endanger their health. Therefore, it's very important to effectively detect the metal ions and study their content and distribution in the environment and vivo, which is helpful to protect the environment and health of human. Functional nucleic-acid refers to the nucleic-acid molecule which is highly capable of binding a specific target or has some catalytic function. The functional nucleic-acid probe has some unique advantages:It can be obtained through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) screening, theoretically, the functionalized nucleic acids of any targets could be screened out; the screened functional nucleic acid can be obtained by synthesis, which is suitable for large-scale production; some functional nucleic acid, such as L-nucleic acids (L-DNA), is more stable under physiological environment and possesses better anti-enzyme degradation effect, providing the feasibility to detect metal ions in vivo; and finally, functional nucleic acid can keep its activity even after being denatured and activated for several times, which makes it easier to be saved, reducing the consumption of the facilities. More importantly, some functional nucleic acids display high degree of specificity to metal ions, and the response signal is related with the content of metal ion, which provides a possibility to simply, conveniently and effectively detect metal ions. Therefore, with the advantage of functional nucleic acids, we have designed a series of biosensors for the detection of metal ions in the environment and vivo.In the second chapter, we built a biosensor based on potassium ion sensing oligonucleotide (Potassium Sensing Oligonucleotide, PSO). First, we used DNA strand of the modified fluorescence resonance energy transfer (FRET) reporter group to optimize the terminal of PSO to obtain higher sensitivity, and then diacyl lipid was assembled onto PSO by DNA synthesizer. In the presence of potassium ions, the configuration of G-tetramer will make the two fluorophores (FAM and TAMRA) which produced FRET effect get close, resulting in change of FRET efficiency. In addition, with the change of the concentration of potassium ion, the fluorescence intensity ratio is reversible. Based on this experimental program, we assembled this kind of sensor on the cell membrane, and for the first time applied this in the real-time detection of dynamic changes of K+ concentration in the cellular microenvironment.In the third chapter, we built the L-type thrombin binding aptamer (L-TBA) K+ sensor used in homogeneous solution and living cells. By utilizing the advantage of predictable properties of L-TBA, the K+ sensor based on L -TBA can overcome the intrinsic defects of D-PSO, enabling K+ imaging in living cells and exhibiting the advantages of precision, safety and bio-orthogonal to the PSO.In the fourth chapter, with the knowledge of Pb2+ DNAzymes and that L-DNAzyme and D-DNAzyme have similar catalytic activity in the presence of non-chiral metal ion cofactor but L-DNAzyme is more stable in biological sample, we designed the biological sensor with L-DNAzyme recognition unit using the reported D-DNAzyme sequence. The sensor is used in environmental water testing, such as water from Xiangjiang River and fetal bovine serum (FBS), to detect Pb2+. Up to time now, sorts of DNAzymes have been selected to recognize various kinds targets, and that is highly possible to provide other new L-DNAzyme-based sensing systems for sensitive and selective detection of numerous targets and find extensive applications in the biomedical and environmental fields.