Determination of immunoglobulin M concentration by piezoelectric crystal immunobiosensor coated with protamine

In the present study, the specific binding between protamine and immunoglobulin M (IgM) has been exploited to construct a piezoelectric crystal based immunobiosensor for the determination of concentration of IgM. The system consisted of highly stable IC based oscillator, 8-digit frequency counter and modified piezoelectric crystal device. The crystal surface was physically modified and chemically treated (refluxed) with strong acid to produce stable hydroxylic groups of silicon oxide. This modified surface reacted strongly with coupling reagents for binding of protein molecules. The protamine was immobilized by using either gamma-aminopropyltriethoxy silane (gamma-APTES) or 2.2.2-trifluoroethanesulfonyl chloride (tresyl chloride). Scanning electron microscope images of piezo crystal revealed that tresyl activated surface presented more surface area for binding than gamma-APTES modified surface and showed better sensitivity. This immobilization technique also improved the reproducibility and long term stability of the detection system. Using the system described, the IgM concentration up to the level of 10 ng/ml could be detected without interference of IgG.