Evaluation of surface plasmon resonance (SPR) for heparin assay .1. Protamine as a heparin affinity surface

The concentration of heparin, an anticoagulant in blood, is usually inferred from clotting type assay, which determines a parameter related to the heparin activity. Because of the heterogeneity of heparin, however, it is desirable to monitor the absolute concentration of heparin directly in the clinical range of 0-2 U/ml. Surface plasmon resonance (SPR) provides a optical direct method of monitoring binding events. Gold films, as required for SPR, were modified with protamine; the immoblized protamine interacts electrostatically with heparin so that the heparin adsorption is dependent on the absolute concentration. The ''thickness'' of the immobilized protamine layer determined the linear range of the sensor's response and the sensitivity. Less densely packed layers of protamine showed a lower detection limit for heparin, suggesting a mixing of the heparin into the incomplete protamine layer. On the other hand, thicker, denser protamine layers did not show a low concentration sensitivity to heparin although their maximum heparin binding capacity was increased. It was shown that the linear response range of the protamine modified SPR device to heparin could be modulated by altering both the protamine loading and its method of immoblization. (C) 1997 Academic Press.