In this work, we developed a microfluidic system for immunoassays where we combined the use of magnetic nanoparticles as immunosupport, a microfluidic magnetic trap, and a fluorogenic substrate in continuous flow for detection which, together with the optimization of the functionalization of surfaces to minimize nonspecific interactions, resulted in a detection limit in the order of femtomolar and a total assay time of 40 min for antibiotin antibody detection. A magnetic trap made of carbonyl-iron microparticles packaged inside a 200 μm square microchannel was used to immobilize and concentrate nanoparticles. We functionalized the surface of the iron microparticles with a silica-polyethylene glycol (PEG) shell to avoid corrosion and unspecific protein binding. A new one-step method was developed to coat acrylic microchannels with an organofunctional silane functionalized with PEG to minimize unspecific binding. A model immunoassay was performed using nanoparticles decorated with biotin to capture antibiotin rabbit Immunoglobulin G (IgG) as target primary antibody. The detection was made using antirabbit IgG labeled with the enzyme alkaline phosphatase as a secondary antibody, and we measured fluorescence with a fluorescence microscope. All steps of the immunoassay were performed inside the chip. A calibration curve was obtained in which a detection limit of 8 pg/ml of antibiotin antibody was quantified. The simplicity of the device and the fact that it is made of acrylic, which is compatible with mass production, make it ideal for Point-Of-Care applications.
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