Study Of Thrombin Detection Based On Aptamer And Molecular Beacons

Protein is one of the important parts of organism. Protein detection, which is more direct and accurate than DNA detection, plays an important role in diagnosis. While the tedious operations such as the radiolabeling, electrophoresis and radio autography in the traditional methods blocked the deep study in bioscience; it's very urgent to develop rapid, simple and high efficient protein detection methods. In this thesis, thrombin was used as a model protein to develop new protein detection methods using aptamers and molecular beacons. This thesis is composed of the following three parts:1. Thrombin detection based on double-hairpin aptamer probeAptamer and cDNA of molecular beacon were combined into one no labeling aptamer probe for thrombin detection. The probe had two hairpin structures, in which the cDNA of molecular beacon was sealed in its two stems. Therefore, molecular beacon was hard to hybridize with the probe without thrombin; when thrombin bound with the aptamer part of probe, the outside stem would be opened and another unstable single stem would make the probe easier to hybridize with molecular beacon, then obvious fluorescence signal was generated. Thrombin could be detected according to the increase of fluorescence intensity. This method was sensitive and specific. The influence of heparin on the interaction of thrombin and anti-thrombin was also investigated by this method. Therefore, this aptamer probe based method could be extended to other protein detection, as well as the study of interaction between protein and other correlative molecules.2. Thrombin detection based on the aptamer probe containing cDNA of molecular beaconA new no labeling aptamer probe was developed by combining aptamer and cDNA of molecular beacon. Aptamer probes and molecular beacons were used as recognizer and reporter, respectively. When there was no thrombin, aptamer probes could hybridize with molecular beacons in the highest degree, generating the largest fluorescence signal; when thrombin was introduced, the hybridization of molecular beacons and probes was depressed, and the depressing degree was associable with the concentration of thrombin. According to the fluorescence signal change, thrombin could be detected rapidly and sensitively. The influence of heparin on the...