Method and apparatus for the measurement of analyte concentration levels by the steady-state determination of fluorescence lifetime

摘要

The use of luminescent probe substances is detailed in methods and an apparatus for the determination of the concentration and distribution in space and time of numerous analytes of biologic and physical import in vivo and in vitro by the steady-state determination of luminescence lifetime. In the instance of analytes that quench excited states, a fluorophore whose excited state is quenched by the analyte in question is free to undergo Brownian rotation alone or when conjugated to a carrier molecule within a medium of suitable viscosity. The analysis medium is irradiated with continuous linearly polarized light at a wavelength strongly absorbed by the fluorophore. The emitted luminescence is resolved into its vector components parallel and perpendicular to the plane of polarization of the excitation light, thereby permitting the calculation of the luminescence anisotropy of the irradiated specimen. The concentration of the quencher is determined by applying a mathematical function which relates the luminescence anisotropy of the fluorophore to the concentration of the quencher. For the determination of the concentration of substances which do not themselves quench excited states a known quantity of the analyte is conjugated to a quencher molecule or an energy transfer acceptor molecule and a competition reaction is set up in which the luminescently labelled substance and the unlabelled substance within the sample compete for sites on a labelled carrier molecule. To the extent that the concentration of unlabelled substance in the sample increases and displaces labelled material on the carrier molecule the luminescence lifetime of the carrier fluorophore will increase. The luminescence anisotropy is measured at the emission band of the luminescent label on the carrier molecule and the concentration of the substance under measurement is determined by applying an empirically determined mathematical function which relates luminescence anisotropy to the concentration of the analyte.