| Within the recent century, there are four large global eruptions of influenza viruses. One of them caused about 7 million casualties. The recent outbreak of avian flu virus in China has received widespread attention around the world because of its highly pathogenic nature. At present, the main detection methods of avian influenza virus include quantitative PCR based on fluorescence, RT-PCR detection method, nucleic acid sequence-based amplification (NASBA) and DNA biosensors. Because of the importance of DNA biosensors in the diagnosis of major diseases, more attention has been attracted in biological and medicinal fields. In particular, the DNA biosensors are developing very quickly in recent years due to the evolvement of electrochemical detection technologies. Many kinds of electrodes have been used in this method. Gold electrodes often give the highest sensitivity and lowest detection limits among them. But because of its high production costs, they are not widely used in the normal detection. To extend their application, we developed a new type of gold electrode fabrication method for electrochemical DNA biosensors, which effectively reduces the cost of the gold electrodes for electrochemical DNA biosensors. Electrochemical detection of DNA specific for H7N9 virus was realized by combining the method with a kind of nucleic acid amplification. There are four chapters in this thesis:Chapter Ⅰ. DNA electrochemical biosensor technology was briefly discussed, including its working principle, the fabrication process, detection method and application in various fields. Isothermal nucleic acid amplification and signal amplification method that used for improving the detection sensitivity of DNA electrochemical biosensors were introduced. Current research status of H7N9 type of avian influenza virus and its detection technologies were described. The aim of the present work was proposed.Chapter Ⅱ. A DNA biosensor with low-cost patterned gold film electrodes on polystyrene substrates was developed and the conditions of electrode fabrication were optimized. To compare with the commercial gold film electrodes on the glass substrate, the fabricated electrodes were characterized by using the images of atomic force microscopy and the signal of electrochemical hybridization. The Result showed that this new type of gold electrode is more ideal than the commercial gold electrode in terms of detection. A process of the electrochemical detection of DNA using the fabricated gold electrode was established. The feasibility of the electrochemical detection by this new type of DNA biosensor was confirmed.Chapter III. Because the gene of H7N9 specific virus is RNA, we applied the proposed gold electrode DNA biosensor for microRNA to get high sensitivity, high specificity. MicroRNA was selected as the model compounds. And we also studied in the time of the RCA amplification and the sensitivity of micro-143 in human blood samples. RCA reaction was observed to occur at a rapid rate before 3 hours, while after three-hour amplification the products detected by chronocoulometry did not increase any more. And the concentration of micro-143 in human blood samples placed on the gold electrodes was 500 pg/μL. The work was an important base of the detection of H7N9 type of bird flu virus described in the next chapter.Chapter IV. We have established a successful method for the electrochemical detection of H7N9 virus DNA, based on work described in the previous two chapters. Blank signal was greatly reduced by PBS buffer containing 0.5% BSA, make gold electrode DNA biosensor for detecting single sample become a reality, and it will be beneficial to the simple and efficient electrochemical detection of specific DNA sequences in human bodies, various viruses and bacteria in the future. This will provide a potential way for DNA based clinical testing. |
PDF Full Text Request