Magnetophoretic position detection for multiplexed immunoassay using colored microspheres in a microchannel

This paper demonstrates a new magnetophoretic position detection method for multiplexed immunoassay using colored microspheres as an encoding tool in a microchannel. Colored microspheres conjugated with respective capture molecules are incubated with a mixture of target analytes, followed by reaction with the probe molecules which had been conjugated with superparamagnetic nanoparticles (SMNPs). Under the magnetic field gradient, the resulting microspheres are deflected from their focused streamlines in a microchannel, and respective colored microspheres are detected using color charge-coupled device (CCD) in a specific detection region of the microchannel. The color and position of respective colored microspheres are automatically decoded and analyzed by MATLAB program, and the position was correlated with the concentration of corresponding target analytes. As a proof-of-concept, we attempted to assay simultaneously three types of biotinylated immunoglobuline Gs (IgGs), such as goat, rabbit and mouse IgGs, using colored microspheres (red, yellow and blue, respectively). As the capture molecules, corresponding anti-IgGs were employed and target analytes were probed using streptavidin-modified superparamagnetic nanoparticles. As a result, three analytes were simultaneously assayed using colored microspheres with high accuracy, and detection limits of goat IgG, rabbit IgG and mouse IgG were estimated to be 10.9, 30.6 and 12.1 fM, respectively. In addition, with adjustment of the flow rate and detection zone, the dynamic range could be controlled by more than one order of magnitude.