RESUMO
To attain the optical precision necessary to precisely quantify fluorescent or colorimetric signals, analytical systems have typically included quality-controlled cuvettes, flow cells, or dual-beam reference systems. We describe a system where a fluorescence or transmittance signal is quantified in single, standard, 12-mm-diameter polystyrene test tubes. Tube-to-tube variation is minimized by referencing the primary signal to a second reference signal. The tube is carefully oriented within a positioner that allows for the precise placement of the tube within a light path 7.6 mm in diameter. The detection system allows for use of either four pairs of fluorescence excitation/emission wavelengths or eight transmittance wavelengths, which are selected by using specific interference filters. The impact of temperature, tube imperfections, surface flaws, and distortions is minimized by using a reference ratio. Fluorescence is measured with an orthogonal photomultiplier tube, and transmittance with a photodiode; both are illuminated with an ordinary long-life tungsten-halogen lamp. This system is used with the Becton Dickinson AFFINITY system, an automated random-access analyzer with analyte-specific unit-package reagents. The polystyrene tube of the reagent package, which has an antibody-absorbed surface, serves as both the cuvette and the separation medium. Use of the reference ratio method reduces intertube imprecision of fluorometric or transmittance signals, for more precise quantification of various analytes.
