| Chemiluminescence (CL) has become a more and more popular analytical technique owing to its high sensitivity, simple instrumentation, wide linear range, automatic, simplicity and rapidity. This thesis studied a CL enhancer Fluorescein (F), which has an enhanced effect on the Luminol-H2O2-HRP system, and proposed the chemiluminescence resonance energy transfer (CRET) mechanism of the Luminol-H2O2-HRP-F system. Based on the system, a detection for the DNA has been achieved with high sensitivity. Further more, this new enhanced CL system was also used to fabricate a CRET molecular beacon probe, CRET-BMBPs, and this new molecular beacon probes has been successfully used for the fast detection of ATP in K562 cells and 4T1 cells, the results shows high coherence with the HPLC method for comparison. So, with high sensitivity, wide linear range, simplicity, this method can be used for the detection of many kinds of tumor markers.The main jobs of this thesis can be concluded as follows:1. The studies on the new enhanced CL system of Luminol-H2O2-HRP-FThis thesis studied a CL enhancer, Fluorescein (F), which has a remarkable enhanced effect on the Luminol-H2O2-HRP system, and proposed the possible mechanism of the Luminol-H2O2-HRP-F system. The kinetic property and the fluorescence spectrum have been studied for the Luminol-H2O2-HRP system. This thesis also studied the concentration effect of fluorescein on Luminol-H2O2-HRP-F system's fluorescence spectrum. Based on this, the Luminol-H2O2-HRP-F CRET mechanism has also been preliminary proposed.2. The studies of LumAuNPs composite's preparation using Luminuol reduction of HAuCl4 methodThe LumAuNPs have been successfully prepared using the Luminuol reduction of HAuCl4 method. The effect of the reactant's amount on the size of the LumAuNPs has been discussed. The TEM images show that uniformly LumAuNPs with excellent dispersivity and chemiluminescence property were prepared using this method. Combining the LumAuNPs's UV-Vis and fluorescence spectrum, the optimum preparation conditions were chosen, the HAuCl4 solution: 0.02% (w/w), 100 mL; the Luminuol solution: 0.01 mol/L, 1.8 mL.3. The studies of the detection for the DNA using a new molecular beacon probe based on the CRET system.Based on the Luminol-H2O2-HRP-F CRET mechanism, a new molecular beacon probe (CRET-BMBPs) has been fabricated for the DNA detection. the linear range of DNA was 1.0×10-9 ~ 1.0×10-8 mol·L-1, the regression equation could be expressed as y = 11.57679 + 60.09556 x, with the correlation coefficient of 0.99935. Based on this, Au@Fe3O4 has been applied for decreasing the CL background. After the improvement, the linear range of DNA was 1.0×10-10 ~ 1.0×10-9mol·L-1, the regression equation could be expressed as y = -9.69283 + 73.61775 x, with the correlation coefficient of 0.99975. Furthermore, the selectivity of the CRET-BMBPs has also been discussed.4. The studies of the detection for the ATP in cancer cells using new molecular beacon aptamer probe based on the CRET system.Based on the Luminol-H2O2-HRP-F CRET mechanism, a new molecular beacon aptamer probe, CRET-BMBP has been designed for the Adenosine Triphosphate (ATP) detection in cancer cells. For the CRET-BMBP, the linear range of ATP was 1.0×10-6 M ~ 1.0×10-5 M, the regression equation could be expressed as I = 56.0273 C + 17.4505. A detection limit of 1.1×10-7 M can be estimated for the CRET-BMBPs. The reaction temperature between ATP and its aptamer has influences on the experiment results, and the influences have also been discussed. This new probe has been successfully used for the ATP detection in K562 and 4T1 cancer cells, the concentration of ATP in the K562 cell is 3.7 (±0.3) mM, (n = 3) and 2.4 (±0.3) mM for 4T1 cells. For comparison, HPLC was conducted for the analysis of the same sample, relatively good correlations was obtained as R = 0.9997 for CRET-BMBPs. |
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