Microfluidic chip for fast bioassays-evaluation of binding parameters

A seven channel polystyrene (PS) microchip has been constructed using a micromilling machine and a high-temperature assembling. Protein A (PA) has been immobilized by a passive sorption on the microchannel walls. Two bioaffinity assays with human immunoglobulin G (hIgG) as a ligand have been carried out. (i) PA as the receptor and fluorescently labeled hIgG (FITC-hIgG) as the ligand, (ii) PA as the receptor with hIgG as the quantified ligand and fluorescently labeled goat anti-human IgG (FITC-gIgG) as the secondary ligand. One incubation step of the assays took only 5 min instead of hours typical for enzyme-linked immunosorbent assay applications. Calibration curves of the dependence of a fluorescence signal on the hIgG concentration in a sample have been obtained in one step due to a parallel arrangement of microchannels. A mathematical model of the PA-FITC-hIgG complex formation in the chip has been developed. The values of the kinetic constant of the PA-FITC-hIgG binding (k(on)=5.5 m(3) mol(-1) s(-1)) and the equilibrium dissociation constant of the formed complex (K(d)