Flow field‐flow fractionation with chemiluminescence detection for flow‐assisted, multianalyte assays in heterogeneous phase

On‐line, continuous chemiluminescence detection has been recently applied to gravitational field‐flow fractionation as a new method suitable for flow‐assisted assays based on the separation between the analyte in solution and the analyte bound to micrometer‐sized particles. In this work, flow field‐flow fractionation coupled with off‐line chemiluminescence detection is exploited for the development of flow‐assisted, multianalyte assays. Micrometer‐sized, polystyrene beads coated with enzymes suitable for chemiluminescence detection, such as horseradish peroxidase and alkaline phosphatase, are used as model samples. The high size‐based selectivity of flow field‐flow fractionation and specificity of chemiluminescence detection make possible, in a single run with very short analysis time, the simultaneous, high sensitivity detection of the different enzymes linked to beads of different size.