The Research Of DNA Biological Sensor Based On The Molecular Rotor Mechanism

With the development of the human Genome project and the further study of the epidemic infectious disease, the application of DNA sensors in gene detection analysis, contaminant detection and microbiology is more and more widely. Compared with the traditional technology, DNA biosensor has the advantage that rapid, sensitive, simple operation, no pollution, and with the function of molecular recognition, gene purification. As DNA not only has the ability that identify complementary nucleic acid, but also can identify metal ions, small molecules, proteins and cells, which were developed as a biological universal molecules. DNA biosensor has been widely used in genetic disease research, its main application are thermal type, electrochemical, optical, quality sensitive type and so on. The fluorescent DNA biosensor due to its simple operation, rapid, high sensitivity, good selectivity, and mostly in homogeneous solution the advantages of the reaction, more and more received extensive attention of research workers. Based on this, this thesis chooses the cavity DNA and G-quadruplex DNA two special DNA to design research type fluorescent DNA biosensor. Using the advantage of this method that rapid, sensitive, low background, we utilize the molecular rotor mechanism realize SNP detection, and specificity, high sensitivity detection of potassium ion, mercury ions. The main research contents as follows:1. Selective recognition of ds-DNA cavities by a molecular rotor:switched fluorescence of thioflavin TThioflavin T (ThT) has been widely utilizing as a fluorescent marker for amyloid fibrils. However, ThT as an efficient reporter for a specific DNA structure still remains in question. In this work, we found that ThT experiences an obvious enhancement in fluorescence intensity when binding to DNAs containing cavity structures such as abasic site, gap site, and mismatch site. Such enhancement in fluorescence can not be realized for DNA without these cavity structures. The DNA cavities provide appropriate spaces to accommodate ThT and allow for occurrence of some specific interactions. The stacking interaction of the bound ThT with the cavity context bases is the main driving force for ThT binding to the cavities. This interaction restricts the excited state’rapid torsional rotation around the single C-C bond between benzothiazole (BZT) and dimethylaminobenzene (DMAB) moieties and thus results in a decreased population of the nonradiative twisted internal charge-transfer (TICT) state. This stacking interaction is impossible to occur for the DNA without these cavities. This properties can be used to detect the DNA cavities with a high selectivity and sensitivity. We expect that this virtue of ThT in targeting these DNA structures very likely to be developed into practical and functional DNA-based sensors or devices.2. A molecular rotor-based fluorescent probe for selective recognition of hybrid G-quadruplex and a K+sensorDevelopments in probes having binding specificity for a G-quadruplex structure and adaptive recognition without disturbance to the DNA native conformation is still a challenge. The usually used G-tetrad-size-alike macrocyclic fluorophores can not meet this requirement. Herein we demonstrate that thioflavin T (ThT) as a novel fluorescent probe can selectively target G-quadruplexes especially having a hybrid structure. UV-vis absorption spectra, fluorescence spectra, and Tm experiments were used to confirm the binding specificity. The ThT binding has no disturbance on the native G-quadruplex structure in Na+and K+solutions. The fluorescence enhancement is believed to be caused by rotation restriction of benzothiazole (BZT) and dimethylaminobenzene (DMAB) rings in the ThT excited state upon its G-quadruplex binding. This molecular rotor mechanism in terms of fluorescence enhancement was confirmed using a non-rotor analogue of ThT. On the basis of these properties, a selective and label-free fluorescent K+sensor was developed. A detection limit of1mM for K+even in presence of100mM Na+was easily achieved. The coexistence of the other metal ions produces a fluorescence response comparable to K+alone. We believe that ThT will have potential applications in the structure identification of hybrid G-quadruplexes and construction of G-quadruplex-based sensors.3. Highly selective detection of mercury ions with benzothiazole derivatives as fluorescent probesA label-free, rapid, sensitive and selective DNA-based fluorescent sensor was presented for determination of Hg2+. We selected a specific sequence oligonucleotide, Tel22(5’-AGGGTTAGGGTTAGGGTTAGGG-3’), which can form a G-quadruplex structure upon the addition of K+. Without Hg2+in the sample solution, Tel22could form hybrid structures, resulting in the fluorescence of ThT being increased sharply. When Hg2+is present in the sample solution, Hg2+can damage the fluorescent system. The fluorescence signal is then decreased obviously compared with that without Hg2+. Meanwhile, a detection limit of500nM is attained. Selectivity experiments reveal that the fluorescent sensor is specific for Hg2+even with interference by high concentrations of other metal ions. This strategy provided a promising alternative to Hg2+determination in the presence of other metal ions. With excellent sensitivity and selectivity, this sensor is potentially suitable for monitoring of Hg2+in environmental applications.