A protein A-based orientation-controlled immobilization strategy for antibodies using nanometer-sized gold particles...

A novel protein A (PA)-based strategy for the orientation-controlled immobilization of antibodies using nanometer-sized gold (Nano-gold) particles and an amine-terminated plasma-polymerized film (PPF) has been proposed. A quartz crystal microbalance was fabricated, accordingly, coupling with haptoglobin (HP) antibody followed by HP immunoassay, as a model test system. The crystal was modified with plasma-polymerized n-butyl amine film to deposit Nano-gold particles and PA, on which HP antibodies were immobilized. The surface topology of the as-prepared crystal was characterized by use of scanning electron microscopy. In contrast to the traditional flat gold surface, the assembled Nano-gold particle monolayer could allow PA molecules bound with higher bioactivity and loading amount (density), achieving better antibody-binding capabilities. Results indicate that immunosensors prepared using the developed PPF-Nano-gold-PA binding procedure exhibit increased analytical performance compared with those produced using the direct PA binding procedure and the PPF-based glutaraldehyde cross-linking procedure. A HP serum concentration as low as 0.41 nM can be determined by this new system. Regenerated simply by rinsing in the acid buffer, the proposed sensor can achieve up to 11 assay cycles without significant loss of sensitivity.