Preparation of scFv-immobilized quartz crystal microbalance sensor by PS-tag-mediated solid-phase refolding

The activation of a single-chain Fv antibody on the surface of a quartz crystal microbalance (QCM) sensor chip was investigated in order to develop an economical and sensitive immuno-QCM sensor system for use in clinical diagnosis. On the bare gold surface of a QCM sensor chip, approximately 60 nm of hydrophilic polystyrene (phi-PS) thin film that specifically binds the polystyrene-binding peptide (PS-tag) was prepared by spin-coating and O2-plasma irradiation. When the adsorption of PS-tag-fused anti-ED-B scFv (scFv-PS) onto the phi-PS surface was directly monitored, the maximum density of scFv-PS attained was 1.56 μg/cm2, 1.6-times higher than that of scFv. The specific antigen-binding activity of scFv-PS after solid-phase refolding increased with the density of immobilized scFv-PS, and, consequently, activity 1.7 times higher than that of scFv was retained. The scFv-PS-immobilized QCM sensor chip rapidly allowed the detection of clear signals for antigen at the range of 0.1–10 μg/ml, while no signal was detectable for 10 μg/ml BSA as a negative control. The scFv-PS-immobilized QCM sensor developed in the present study will therefore be very useful for the rapid and highly sensitive detection of biomarkers, and should be applicable to clinical diagnosis.