| Being one of the most infectious viruses, the human hepatitis B virus (HBV) can induce chronic hepatitis and hepatocirrhosis; therefore, considerable attention has been paid to developing sensitive and selective methods for its detection. Thrombin is a common protein that has great importance in molecular biology, and a lot of efforts have been done to develop sensitive sensors to detect thrombin. The molecular recognition of aptamer-target and antibody-antigen are very similar. As a novel molecule recognition tool, the aptamers have several advantages over antibodies. First, aptamers have been developed for a large range of targets, including substances with low or no immunogenicity and toxins. Second, the aptamers with good stability can be easily regenerated in minutes after denaturizing as nucleic acids, conveniently labeled or modified by functional groups, and suitably adopted in nanodevices. So in analytical techniques, aptamers are emerging as a kind of novel recognition molecules. This masteral dissertation constitutes by four parts besides the preface:(1) The digestion and degradation of the dsDNA to a single chain by exonucleaseⅢwere studied by fluorescence spectroscopy. HBV can be detected by measuring the fluorescence intensity of the solution analyzed. The best reaction conditions were obtained through the study of the effect of different buffer solution, different pH and different reaction time on the system's fluorescent intensity. TT buffer (pH 8.0,250 mM Tris-HCl,0.1% (V/V) Tween 20) was chosen as the reaction buffer, the pH of TT buffer was pH 9.0, the reaction time of biotin and streptavidin was 25 min, 20 min was chosen as the hybridization time of capture probes and target DNA, 1.1 h was the enzyme incubation time. The effect of HBV concentration on the system′s fluorescent intensity under the best reaction conditions was also studied. The fluorescence intensity of the system increased when the concentration of HBV increases based on which the concentration of HBV can be detected. In the system of fluorescence spectroscopy based on exonucleaseⅢ, the HBV could be quantified in the ranges of 5.0×10-13～5.0×10-12 M and the detection limit is 3.35×10-13 M. (2) Using a highly ultrasensitive fluorescence detection method based on gold nanoparticles and magnetic beads assisted by dithiothreitol (DTT) could enable the detection of target DNA. The detection of HBV is achieved by monitoring fluorescence signals by liberating the adsorbed thiolated oligonucleotides modified with fluorophore from the gold nanoparticle surface. In addition, MBs are used to permit low nonspecific binding of DNA-conjugated AuNPs and to take advantage of easy magnetic separation. At the same time, AuNPs could offer large signal amplification. The nonspecific binding of detection-probe-conjugated AuNPs is lowered by washing DNA-linked MB-AuNP assemblies. The high signal amplification and low background are capable of the detection of 2.18×10-14 M target DNA.(3) A sensitive fluorescence assay for hepatitis B virus (HBV) DNA was developed based on streptavidin-coated 96-well microplates and GoldMag-CS nanoparticles (NPs). Streptavidin-coated 96-well microplates permitted -conjugated GoldMag-CS nanoparticles, and GoldMag-CS nanoparticles could offer significant signal amplification and low nonspecific binding of DNA because of easy separation. DTT was introduced to displacing fluorophore modified DNA from the surface of the GoldMag-CS nanoparticles to the solution, and then quantified. The high signal amplification and low background were capable of the detection limit of 7.52 fM.(4) The assay utilized sandwich binding of two affinity aptamers for increasing specificity. Recognition DNA (S3) loaded on the gold nanoparticles (AuNPs) partly hybridized with initiator DNA (S4) which was capable of being displaced by survivin DNA (S5). So initiator DNA was released into the solution and it was able to open the hairpin structure S6, which in turn opened the other hairpin structure S7. A third strand-displacement reaction completed the duplex DNA, and released the initiator DNA. Then the released initiator DNA could open the hairpin structure S6 to initiate another autocatalytic strand-displacement reaction. A sophisticated network of three such duplex-formation cycles was designed to amplify fluorescence signal. Other proteins, such as bovine serum albumin (BSA) and lysozyme did not have much interference in the assay above for thrombin detection. The detection limit of thrombin was as low as 4.3×10-13 M based on the AuNPs amplification and the autocatalytic strand-displacement cycle reaction. This method could be used in biological samples with excellent selectivity. |
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