Near-Simultaneous and Real-Time Detection of Multiple Analytes in Affinity Microcolumns Menake E. Piyasena, † Tione Buranda,* ,‡ Yang Wu, § Jinman Huang, § Larry A. Sklar,* ,‡,§ and Gabriel P. Lopez* ,†,§ Cancer Center and Department of Pathology, University of New Mexico School of Medicine, Department of Chemical and Nuclear Engineering, and Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131 A miniaturized immunoassay system based on beads in poly(dimethylsiloxane) microchannels for analyzing mul- tiple analytes has been developed. The method involves real-time detection of soluble molecules binding to recep- tor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer. A preliminary account of this work based on single-analyte format has been published in this journal (Buranda, T.; Huang, J.; Perez-Luna, V. H.; Schreyer, B.; Sklar, L. A.; Lopez, G. P. Anal. Chem. 2002, 74, 1149 -1156). We have extended the work to a multianalyte model system composed of discrete segments of beads that bear distinct receptors. Near-simultaneous and real-time detection of diverse analytes is demonstrated. The importance of this work is established in the exploration of important factors related to the design, assessment, and utility of affinity microcolumn sensors. First, beads derivatized with sur- face chemistry suitable for the attachment of fluorescently labeled biomolecules of interest are prepared and char- acterized in terms of functionality and receptor site densities by flow cytometry. Second, calibrated beads are incorporated in microfluidic channels. The analytical device that emerges replicates the basic elements of affinity chromatography with the advantages of microscale and real-time direct measurement of bound analyte on beads rather than the indirect determination from eluted sample typical of affinity chromatography. In addition, the two-compartment analysis of the assay data as demon- strated in single-analyte columns provides a template upon which the dynamics of multiple-analyte assays can be characterized using existing theoretical models and be tested experimentally. The assay can potentially detect subfemtomole quantities of protein with high signal-to- noise ratio and a large dynamic range spanning nearly 4 orders of magnitude in analyte concentration in microliter to submicroliter volumes of analyte fluid. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes (e.g., biomolecular indicators of deseases). Proof-of- principle analytes include FLAG peptide and carcinoem- bryonic antigen detected at physiologically relevant con- centration levels. Assays derived from the molecular recognition interactions of biological molecules have been the mainstay of many clinical, biochemical, and environmental research efforts. 1-13 A variety of approaches have been developed for performing immunoassays in single as well as multianalyte format. 8,14-22 Besides the con- ventional sandwich, direct binding, and competition-based assays, a number of approaches such as flow injection analysis 21,23-32 and those based on small-volume microfluidic devices have emerged 33-41 Common themes in the design and optimal use of these microanalytical devices have emerged: 42 (a) The presence of beads * To whom correspondence should be addressed. 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