| Triplex DNA is formed on the basis of the duplex DNA. The structure of triple-helix is with great potential in the development of new biological molecule tools and treatment reagents. It can identify specific sequences of duplex DNA as well as on gene regulation, including interference with transcription, replication and reorganization. The research of triplex DNA will help further understanding cellular processes, reveal the formation mechanism of genetic diseases, and provide a new method to new treatments for the establishment of these diseases.The main contents of this paper were to combine sensitive signal conversion function of ECL technology, triple helix DNA hybridization technique with the aptamer probes for protein and molecule specific recognition, and prepared a new ECL biosensor, which was designed by DNA sequence to identify and selectively detect the concentration of adenosine or thrombin. This method was simple and flexible. It provided new tools and ideas for protein and small molecule research. This thesis included the following three parts:1 A novel triplex electrochemiluminescence biosensor based on the ferrocene quenching of tris(2,2'-bipyridyl)ruthenium luminescence was developed. First the DNA tagged with tris(2,2'-bipyridyl)ruthenium was self-assembled onto the surface of gold electrodes. And then it hybrided with a specific sequence of DNA and ferrocene (Fc) labeled DNA, in the presence of binding agent to form the structure of triplex DNA. The concentration of the synthesized of luminous markers was calibrated, and the probe solution was characterized, and the binding agent was choosed. Finally a viable triplex DNA biosensor was constructed for the next detection of thrombin and adenosine.2 On the basis of preliminary work, a novel ECL-triplex aptamer-based biosensing method for the determination of thrombin was developed based on a structure-switching ECL-quenching mechanism, so that for the detection of thrombin concentrations. In the presence of thrombin, the aptamer sequence prefered to form the aptamer-thrombin complex and the switch of the binding partners occured in conjunction with the generation of a strong ECL signal owing to the dissociation of FcDNA. The luminous intensity changed with the concentration of thrombin can be tested, the linear range was 4.0×10-15 to 3.0×10-14mol L-1. A detection limit of 1.0 f mol L-1 of thrombin was achieved with high sensitivity. At the same time, a duplex ECL contrast test was designed, the results showed that the sensitivity of our triplex biosensor increased more than 100-fold. The method provided an effective way to improve the specificity and sensitivity for protein detection.3 On the base of the above two sensors, a novel ECL-triplex aptamer-based biosensing method for the determination of adenosine was developed. In the presence of adenosine, the tris(2,2'-bipyridyl)ruthenium labeled DNA-1 probe combined with adenosine to form adenosine - aptamer hairpin complex structure, which is closer to the surface of electrode and away from the ferrocene to increase the luminous intensity. The ECL intensity was increased with the increase of the concentration of adenosine in the range of 1.0×10-9 to 1.0×10-8 mol L-1. The detection limit is 2.7×10-10mol L-1. Meanwhile, we made a duplex contrast test, the sensitive of triplex biosensor increased about 10-fold. We used the cytidine and uridine for its selectivity, and we found that our triplex biosensor had good selectivity. The method provided a new thought for the detection of small molecules with electrochemical biosensor.
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