| Based on the intercalation binding between ethidium bromide and herring sperm DNA (hsDNA), a novel biosensor utilizing fluorescent reversible regulation of cysteamine-capped ZnSe quantum dots (CA-capped ZnSe-QDs) in QDs-DNA-EB system, and the interaction of hsDNA with diquat dibromide and malachite green were experimentally and theoretically studied by fluorescent spectrometry, UV-vis absorbance spectrophotometry and molecular docking. The main results include the following parts:1. Two types of single nuclear ZnSe-QDs capped with cysteamine and reduced glutathione, respectively, were synthesized in aqueous. The characterization results showed that the positively charged CA-capped ZnSe-QDs with particle size of 3.5 nm, ultraviolet absorption spectrum between 240-300 nm, excitation wavelength of 287 nm, the emission wavelength of 576 nm and full width half maximum fluorescence intensity of 20 nm; while the negatively charged GSH-ZnSe-QDs with particle size of 4.0 nm, ultraviolet absorption peak of 347 nm, excitation wavelength of 300 nm, the emission wavelength of 380 nm and full width half maximum fluorescence intensity of 70 nm.2. It was proved that the positively charged CA-capped ZnSe-QDs can be used for the biosensor based on fluorescent reversible regulation of the CA-capped ZnSe-QDs in the QDs-DNA-EB system, while the negatively charged GSH-ZnSe-QDs can not. The hsDNA bond with the QDs through electrostatic interaction and quench the fluorescent of the QDs due to the photoinduced electron transfer from hsDNA to QDs and formed QDs-hsDNA complexes under 0.01 mol/L PBS buffer with pH 6.0. When different concentrations of EB were added to QDs-DNA complexes, hsDNA was released from the complexes with the fluorescence of CA-capped ZnSe-QDs recovery.The results indicate that the content of EB could be determined by the fluorescent reversible regulation of the CA-capped ZnSe-QDs in the QDs-DNA-EB system. The result of molecular docking directly displayed that the phenanthridine plain of EB insert into the adjacent base pairs of DNA indicating the classical intercalation between EB and hsDNA.A relative theoretical model established in this study to quantitatively present the fluorescent reversible regulation processes were used to calculate the characteristic binding parameters. During the fluorescence quenching process of CA-capped ZnSe-QDs induced by hsDNA the equilibrium constant K1 and the binding point numbers n under various temperatures of:288.15 K (the corresponding ?=2.78×104 L/mol, n=1.39),298.15 K (the corresponding K1=4.25×104 L/mol, n=1.48) and 308.15 K (the corresponding K1=6.80×104 L/mol, n=1.56) were calculated, while in the fluorescence recovery processes of QDs-DNA complexes induced by EB, the apparent equilibrium constant K2 is 8.25×1012 with binding point numbers m of 0.53 at 298.15 K. When the fluorescent switch was used for the determination of EB, the linear range was of 1.4×10-6-1.07×10-7mol/L with detection limit of 1.22×10-7mol/L.3. The fluorescence of EB-DNA complexes can be partly quenched by diquat and malachite green in a certain concentration. Meanwhile, hypochromic effect of hsDNA was detected by comparing the ultraviolet absorption spectrum of diquat-hsDNA complexes and malachite green-hsDNA compelexes with hsDNA at 260 nm. These results suggest that both diquat and malachite green interaction with DNA by intercalation model. Besides, the results of molecular docking showed that the diquat insert into the adjacent base pairs of DNA with the entire molecule while malachite green with its amino benzene.The association constant of DNA with diquat, malachite green and EB were 5.38x104 L/mol,6.78×104 L/mol and 2.86×105 L/mol, respectively. According to the results of molecular docking, the minimum free energy of the interaction of DNA with diquat, malachite green and EB were -6.20,-8.39 and -8.53 kJ/mol, separately. Therefore, the binding capacity between DNA and the three kinds of ligands (from strong to weak):ethidium bromide> malachite green> diquat. |
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