Two typed bio-conjugated soft-interface for highly sensitive immunoassay was developed by integrating a phospholipid polymer. Nano-sphered surface with poly [2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)]: PMBN) was prepared by electrospray deposition (ESD) method. The three dimensional nano-sphered surface can be captured an antibody with high density around 860 ng/cm2. The theoretical amount of closest packed immobilized antibodies on flat surface is around 650 ng/cm2, thus large amount of antibodies were immobilized on the nano-sphere surface. The water stability of PMBN nanostructure was improved by crosslinking with 1,4-butylenediamine and by heating. Both heated and cross-linked PMBN nanostructure was not changed at all remaining high porosity after immersing in water. The specific signal in the immunoassay was enhanced with both heated and cross-linked PMBN nanostructure. The PMBN nanostructure which has high porosity and high water stability realized highly sensitive immunoassay.
As the other platform, we developed a novel soft-interface consisting of a well-defined phospholipid polymer surface on which Staphylococcal Protein A (SpA) was site-selectively immobilized. The phospholipid polymer platform was prepared on silicon substrates using the surface-initiated atom transfer radical polymerization (SI-ATRP) technique. Orientation-controlled antibodies were achieved using enzymatic reactions, and these antibodies captured 1.8 ± 0.1 antigens on average, implying that at least 80% of immobilized antibodies reacted with 2 antigens. Theoretical multivalent binding analysis further revealed that orientation-controlled antibodies had antigen-antibody reaction equilibrium dissociation constants (Kd) as low as 8.6 × 10-10 mol/L, whereas randomly oriented and partially oriented antibodies showed Kd values of 2.0 × 10-7 mol/L and 1.2 × 10-7 mol/L, respectively. These findings support the significance of antibody orientation because controlling the orientation resulted in high reactivity and theoretical binding capacity.