Novel Lanthanide Fluorescence Probes And Their Application To Time-Resolved Fluorescence Bioassay

Time-resolved fluorescence (TRF) bioassay technique based on long-livedfluorescence of lanthanide probes is an ultrasenstive bioanalytical method, and hasbeen widely used in clinical diagnostics and biotechnology. In this doctoraldissertation, several kinds of fluorescent lanthanide chelates and nanoparticles weredesigned, prepared, characterized, and developed as new fluorescence probes forhighlysensitive TRF bioassays.A new ligand, N,N,N1,N1-[4'-(2'''-thienyl)-2,2':6',2"-terpyridine-6,6"-diyl]bis(methylenenitrilo) tetrakis (acetic acid) (TTTA) was designed and synthesized. TheEu³⁺ chelate of the ligand is strongly fluorescent with quantum yield of 0.15 andlifetime of 1.284 ms. The TTTA-Eu³⁺ chelate was used for streptavidin (SA) labelingand the labeled SA was used for time-resolved fluoroimmunoassay (TR-FIA) ofhuman prostate-specific antigen (PSA) and insulin in human sera. The method givesdetection limits of 33 pg·ml^-1 and 44 pg·ml^-1 for PSA and insulin, respectively,together with good accuracy and precision.The TTTA-Eu³⁺-doped and 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexan-edion-6"-yl)chlorosulfo-o–terphenyl (BHHCT)-Eu³⁺ covalently bound silica-basedfluorescent nanoparticles with primary amino groups on their surface were preparedby using a water-in-oil (W/O) microemulsion method. Characterizations bytransmission electron microscopy and fluorometric methods indicate that thenanoparticles are spherical and uniform in size, highly photostable than the freeeuropium chelates. The covalent binding fluorescent nanoparticles are stronglyfluorescent with a fluorescence quantum yield of 50%. After the nanoparticles werelabeled to SA, the nanoparticle-labeled SA was used in sandwich-type TR-FIAs ofhuman a-fetoprotein (AFP), carcinoembryonic antigens (CEA) and hepatitis Bsurface antigens (HBsAg) in human sera. The methods give detection limits of 85pg·ml-1 for AFP, 1.9 pg·ml-1 for CEA, and 23 pg·ml-1 for HBsAg. The concentrationsof HBsAg in 30 human serum samples were determined, and the results werecompared with those independently determined by an established TR-FIA methodusing the BHHCT-Eu3+-labeled SA. A good correlation was obtained, which showsthat the new method would be useful for highly sensitive TR-FIAs. Inorganic-organic hybrid titania-based nanoparticles covalently bound toBHHCT-Eu3+ were synthesized by a sol-gel technique. The effects of solvent's type andEuCl3 concentration on size and fluorescence lifetime of the nanoparticles wereinvestigated. The characterizations by transmission electron microscopy andfluorometric methods indicate that the nanoparticles are near spherical and stronglyfluorescent having a fluorescence quantum yield of 11.6% and a long fluorescencelifetime of⁰.4 ms. The nanoparticle-labeled SA was also successfully employed as afluorescent probes for TR-FIA of PSA. A new terbium chelate-based fluorescence probe specific for singlet oxygen,N,N,N1,N1-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-(9"-anthryl)pyridine] tetrakis(acetic acid) (PATA)-Tb3+ (PATA-Tb3+) was designed, synthesized and characterized.The fluorescence quantum yield of PATA-Tb3+ chelate can be increased about 20 timeswith a long lifetime of 2.76 ms after it is reacted with O2. The probe was used for the 1quantitative detection of O2 generated from a MoO4 /H2O2 system. The method 1 2-gives a detection limit of 10.8 nmol·L-1, which shows that the detection of O2 by 1using the new probe is highly sensitive.