Studies On The New Methods Of Time-Resolved Fluoroimmunoassay

Time-resolved fluoroimmunoassay (TR-FIA) using lanthanide chelates as thefluorescence probes is an established immunoassay method. The most importantadvantage of TR-FIA is that it allows easy distinction of the specific fluorescencesignal of long-lived lanthanide fluorescence probe from the short-lived backgroundsignal present in most biological samples, and also obviates the problems associatedwith scattering light of the optical components, since the fluorescence of lanthanidefluorescence probe is long-lived with large Stokes shift and sharp emission band.Therefore the sensitivity of TR-FIA is higher than those of other immunoassaymethods.On the other hand, the luminescent nanomaterials have shown great promise inbioassays due to their unique luminescence properties including strong luminescence,high photostability, and adjustability of luminescence color.In the present research, a novel method of covalent binding-copolymerizationwas developed to prepare strong fluorescent silica-based europium nanoparticleshaving amino groups on the nanoparticle's surface. In the nanoparticles, theBHHCT-Eu³⁺ chelate molecules were covalently bound to silicon atoms to protectthe nanoparticles from dye leaking in bio-application. The amino groups onnanoparticle's surface make the surface modification and bioconjugation of thenanoparticles be easier. The nanoparticles were characterized and developed as a newtype of fluorescence probe for highly sensitive TR-FIA of human hepatitis B surfaceantigen (HBsAg). The method gives the detection limit of 23 pg/ml, whereas that ofthe assay using the BHHCT-Eu³⁺-labeled SA is 84 pg/ml. These results indicate thatthe sensitivity of TR-FIA can be further improved by using the new nanoparticles asa fluorescence probe. The coefficient variations of the method are less then 10%, andthe recoveries are in the range of 80-110%. The concentrations of HBsAg in 30human serum samples were determined, and the results were compared with those ofthe independently determined by an established TR-FIA method using the Eu3+chelate-labeled streptavidin. A good correlation was obtained with the correlationcoefficients of 0.993. In addition, a highly sensitive homogenous TR-FIA method for T3 based onfluorescence resonance energy transfer from DTBTA-Eu3+to Cy5 was established.The method gives the detection limit of 0.23 ng/ml. The coefficient variations of themethod are less than 2% and the recoveries are in the range of 80-110%. Theconcentrations of T3 in 30 human serum samples were determined, and the resultswere compared with those of the independently determined by a RIA method. A goodcorrelation was obtained with a correlation coefficient of 0.988. Finally, the syntheses of two novel ligands that can form fluorescent lanthanidechalets were performed.