Multiplex immunoassays on size-categorized individual beads using time-resolved fluorescence Original Research Article

Miniaturized multiplex immunoassays were studied on individual beads to detect analytes in the same reaction mixture. With this approach hands-on time, cost, and amount of reagents as well as waste produced in the assays were reduced. Particles were categorized according to size for immunoassays of prostate specific antigen (PSA) and acute myocardial infarction (AMI) markers. Additionally, free and dual PSA assays were carried out on a single bead. The analyte concentration was detected directly on the surface of the beads using stable, intrinsically fluorescent europium and terbium chelates, and time-resolved fluorometry. Less than 0.4 ng ml−1 PSA (corresponding to ca. 10 amol) was detected in free, dual and multiplex PSA assays and 0.1 and 2.4 ng ml−1 of myoglobin (Mb) and creatine kinase MB (CK-MB), respectively, were monitored in the multiplex AMI marker assay. The effect of bead size and material on free PSA detection was also investigated. The beads were detected three times under different conditions; in liquid, after drying and after dissociating europium ions from the chelate into the DELFIA® fluorescence enhancement solution. The same detection sensitivity was found for the dissociative and non-dissociative methods indicating that the current labeling technology for the surface detection is comparable to the commercial DELFIA system.