DNA Fluorescent Biosensor Based On Poly(pyridinium Salt)s

Identification and detection of biological molecules are of great significance for human life and activities, especially in the diagnosis and treatment. The cationic conjugated polymers in the recognition of biological macromolecules have more obvious advantages compared with small molecule materials, When they interact with biological macromolecules, the charge and energy can transfer along the conjugated polymer rigid long-chain, and this can significantly increase a variety of opitial signal amplification. Thus, cationic polymers have been widely used to recognize biological macromolecules, such as DNA, protein and so on.Recently, the biological sensors based on cationic polymers have been extensively studied. However, the kind of the reported cationic polymers is limited. They are mainly the side-chain of cationic polymers, including polyfluorene, polythiophene derivatives.In this paper, the poly(pyridinium salt)s as novel main-chain cationic polyelectrolytes are synthesized and characterized. Furthermore, the interaction of these poly(pyridinium salt)s with calf thymus DNA (ctDNA) was investigated in detail. This thesis is divided into the following sections:First, we synthesized small molecule model compounds0015containing dipyridinium salts structure. Its chemical structure was confirmed by NMR spectroscopy, elemental analysis and other analytical tools. The interaction of0015with ctDNA was studied in phosphate buffer (pH=7.4) and neutral aqueous solution, respectively by UV-visible absorption spectroscopy and fluorescence spectroscope titration experiments. The binding constant of0015with ctDNA is estimated to be1.7×105M-1and3.22×106M-1, respectively. In addition, thermal denaturation experiments and circular dichroism spectroscopy of ctDNA in the presence of0015were also carried out. The results showed that the interaction of0015with ctDNA included mainly the electrostatic interaction and groove interaction.The cationic conjugated polyelectrolyte0019was synthesized and its chemical structure and molecular weight were confirmed by NMR spectroscopy and gel permeation chromatography (GPC). The binding constant of0019with ctDNA is estimated to be9.3x105M-1in phosphate buffer (pH=7.4) according to the UV-visible absorption spectra titration method. On the other hand, the fluorescence emission intensity of the polymer0019in phosphate buffer was significantly quenched upon the addition of ctDNA, e.g. the fluorescence emission intensity of the polymer0019was about a quarter of the initial intensity at the saturated state. According to the Stern-Volmer equation (F0/F1=1+Ksv[Q]), the calculated quenched constant Ksv was estimated to be8.79×106M-1. A linear detection range from1.5to280nM was obtained under the optimized experimental conditions with a detection limit down to the10-9M range. Furthermore, strong electrostatic attraction may be the main driving force for0019/ctDNA binding, which was proposed according to the results of circular dichroism and melting transition study of ctDNA in the presence of0019. The polymer0019/ctDNA complexes was also studied by scanning electron microscopy and electrochemistry methods, indicating that the conjugated poly(pyridinium salt)s was also expected to be used as electrochemical indicator for DNA detection.The poly(pyridinium salt)s0018and043containing calix[4]arene in the main-chain were synthesized, and their chemical structures and molecular weights were confirmed by NMR spectroscopy and gel permeation chromatography (GPC). Due to the calix[4]arene units in the polymer main-chain, the two poly(pyridinium salt)s showed good ion recognition properties towards mercury ions. The binding constants of the polymer0018and043with mercury ions are estimated to be7.86×104M-1and5.46×107M-1, respectively in ethanol/water (V/V=1:1). Moreover, the interaction of the two kinds of polymer and ctDNA was also studied by the UV-visible absorption spectroscopy and fluorescence spectrum titration method. Compared with0018, the poly(pyridinium salt)s043exhibited stronger binding with ctDNA with the binding constant of6.31×104M-1.