Studies On Chemiluminescence Resonance Energy Transfer System And Its Application In Tumor Marker Detection

Chemiluminescence (CL) has become a more and more popular analytical technique owing to its high sensitivity, easy operation, simple instrumentation, easy automation. This thesis studied the luminol-H₂O₂-horseradish peroxide (HRP)-fluorescein (F) chemiluminescence resonance energy transfer (CRET) system. Based on this CRET system, an aptamer-biosenser was established, achieving a high sensitivity detection of thrombin. The proposed strategy in this study has high sensitivity and high selectivity, which possessed wide prospect in vitro diagnostic, clinical research and drug screening.The main jobs of this thesis can be concluded as follows:1. The study on the luminol-H₂O₂-horseradish peroxide (HRP)-fluorescein (F) chemiluminescence resonance energy transfer (CRET) systemThis chapter studied Luminol-H₂O₂-HRP chemiluminescence system, which was enhanced by fluorescein (F). The LumAuNPs have been successfully prepared by using Luminol reduced HAuCl₄. The fluorescence spectrum of the Luminol-H₂O₂-HRP-F system at its free state and bound state were discussed, respectively. This thesis also studied the mechanism of the Luminol-H₂O₂-HRP-F CRET system. From the experimental results, Luminol as the energy donor transferred its energy to the energy acceptor fluorescence under the oxidation of H₂O₂ and the catalysis of HRP in this system. The mechanism of the CRET system is that the formation of the radical of Luminol(L·^-)was promoted by the radical of fluorescein(F·^-), then L·^- reacted with H₂O₂ and formed LO₂^2-, energy transferred from LO₂^2- to F·^- and formed FO₂^2- , finally FO₂^2- released oxygen and give out light at 510 nm.2. The study on the detection of thrombin based on CERT system by using aptamer sensorsThis chapter established a method for the detection of thrombin based on CRET system. In order to reduce the high background value, we combined the two aptamers labeled with luminol and fluorescein with Au nanoparticles-magnetic bead compounds. The aptamers were dissociated from the surface of Au nanoparticles-magnetic bead compounds taking the advantage of the specificity between aptamers and thrombin. Then, luminol-H₂O₂-horseradish peroxide (HRP)-fluorescein (F) chemiluminescence resonance energy transfer (CRET) system was adopted for the detection of thrombin. The limit of detection (LOD) for thrombin was achieved as low as 1.0×10^-9 mol/L. In addition, the proposed strategy exhibited an excellent selectivity.