Physical adsorption of protamine for heparin assay using a quartz crystal microbalance and electrochemical impedance spectroscopy Original Research Article

This study attempted to determine absolute heparin concentration in phosphate buffer solution (PBS, pH 7.4) by using quartz crystal microbalance (QCM) as an affinity biosensor. Electrochemical impedance spectroscopy (EIS) was also used to investigate immobilization of protamine and heparin assay. In addition, the effectiveness of physical adsorption in immobilizing protamine was confirmed by examining the preparation conditions, including the incubation time and protamine concentration. It induced maximum decrease (ca. −100 Hz) in oscillating frequency of QCM by applying 20 mg/ml protamine and 20 min for incubation in PBS. Heparin adsorption onto protamine-modified electrode in PBS revealed an exponential-like binding curve and long duration for reaching the steady state in frequency response of QCM. Moreover, two linear calibration curves were obtained judging from the initial slope (df/dt) and the frequency change (Δf) of QCM obtained after a binding interval (600 s) for heparin concentrations from 0 to 3.0 and 7.0 U/ml, respectively. In EIS analysis, calibration curves with linear concentration range of 0–3.0 U/ml were obtained for heparin in PBS when ferrocyanide was used as an electroactive marker.