Novel Europium Complex-Based Luminescence Biolabels For Time-Resolved Luminescence Bioassay Application

Time-resolved luminescence bioassay technique using luminescent lanthanide complexes as labels has been widely used for clinical immunoassay, DNA hybridization assay and bioimaging due to its advantages of high sensitivity, low effect of background fluorescence, and so on. As key reagents of this technique, the development of new lanthanide luminescence biolabels that have good luminescence properties is an important work. In this doctoral dissertation, several kinds of novel europium complex-based luminescence biolabels were designed and synthesized. After the characterizations of their luminescence properties in detail, the application methods of the new labels for time-resolved luminescence bioassays were established.Three new chlorosulfonylated tetradentate (3-diketonate ligands, BHHBCB, BPPBCB and BTBBCB, were designed and synthesized. After binding to BSA, the luminescence quantum yields and luminescence lifetimes of their Eu(III) complexes are40.0%,0.52ms;36.1%,0.49ms and34.0%,0.48ms, respectively. The detection limits of time-resolved luminescence measurements are2.5x10-13mol/L,2.8×10-13mol/L and4.6x10-13mol/L for the three Eu3+complexes. The BHHBCB-Eu3+-labeled streptavidin was prepared, and used for both the time-resolved luminescence immunoassay of human prostate specific antigen (PSA) and the time-resolved luminescence microscopy imaging of cryptosporidium muris. The results demonstrated the practical utility of the new Eu3+complex-based biolabel for both the time-resolved luminescence immunoassay and the imaging detection of pathogenic microorganism.By using the property that BHHBCB-Eu3+can be further coordinated by2-(N,N-dicthylanilin-4-yl)-4,6-bis(pyrazol-l-yl)-1,3,5-triazine)(BPT) to form a visible-ight-excited ternary Eu3+complex, BHHBCB-Eu3+-BPT, the visible-light-excited silica-encapsulated BHHBCB-Eu3-BPT (Eu@SiO2) nanoparticles were prepared as a nano-biolabel using BHHBCB-Eu3+-BPT conjugated3-aminopropyl(triethoxy)silane as the precursor by a water-in-oil reverse microemulsion method. The Eu@SiO2-abeled transferrin were prepared and used for the time-resolved luminescence microscopy imaging of HeLa cells. The results demonstrated the practical utility of the new nano-biolabel for the time-resolved luminescence imaging detection of living cells. By directly coupling BHHBCB-Eu3+-BPT to BSA molecules, the BSA-BHHBCB-Eu3+-BPT conjugate available for biolabeling with good water-solubility and visible-light-excitation property was prepared. The BSA-BHHBCB-Eu3+-BPT conjugate-labeled folic acid was prepared and used for the time-resolved luminescence imaging detections of living HeLa cells and RAW264.7cells. The results demonstrated the applicability of the BSA-BHHBCB-Eu3+-BPT conjugate as a visible-light-excited biolabel for the luminescence imaging recognition of cancer cells based on folic acid-folic acid receptor affinity binding interaction on the cell surface.