A microsphere coupled micropatterning method for cytokine detection

A protein micropatterning method that coupled a microsphere-based immunoassay was used for the detection of cytokines IL-6 and interferon-γ. Carboxylated polystyrene microspheres were first coated with antibodies specific for cytokines (capture antibody), incubated with target antigens, and then mixed with fluorescent Cy3 labeled antibodies (detection antibody), forming a sandwich structure (capture antibody/cytokines/detection antibody) on their surfaces. The microspheres were placed into an array of microwells fabricated on a solid substrate, and microsphere arrays were obtained. Currently the most widely used microarrays all involve multi-step surface modifications directly on a planar substrate. Using our method, surface modifications were performed on the microspheres in a solution with enhanced chemical reaction efficiency. The microwells served to trap and concentrate microspheres, which results in uniform spot size. Microspheres of different sizes were used, and their effect on the detection was studied. The microsphere arrays were characterized using a normal fluorescence microscope with a mercury lamp. The detection ranges for cytokines IL-6 and IFN-γ were 7.8–500 pg/ml and 15–500 pg/ml, respectively. This method is extendable to the detection of other biomolecules.