Quartz crystal biosensor for real-time monitoring of molecular recognition between protein and small molecular...

A quartz crystal microbalance (QCM) biosensor integrated into a flow injection analysis (FIA) system was used for the real-time investigation of molecular recognition between a protein and small molecular medicinal agents. Two sulfa-drugs, sulfamethazine (SMZ) and sulfamethoxazole (SMO), were, respectively, immobilized on the gold electrodes of the piezoelectric crystals using appropriate procedures based on self-assembly of the dithiothreitol (DTT). The binding interactions of the two immobilized drug ligands, with various proteins in solution, were followed as changes in the resonant frequency of the modified crystals. Results obtained from this rapid screen analysis clearly indicated that the two drug ligands appeared quite different in this molecular recognition procedure although their structures were similar. SMZ-immobilized sensor showed specific interaction only with IgG, while SMO-immobilized sensor showed negligible specific binding with IgG, but binding with trypsin and chymotrypsin. Further studies on the specific interaction between immobilized SMZ and three different species of IgG--human IgG, goat IgG and mouse IgG were carried out and the marked species-dependent difference was observed. The resultant sensorgrams were rapidly analyzed by using an in-house kinetic analysis software based on genetic algorithm (GA) to derive both the kinetic rate constants (kass and kdiss) and equilibrium association constants (KA) for IgG-SMZ interactions. For the interactions, KA were 5.48 x 10(5), 2.75 x 10(5) and 1.86 x 10(5) M(-1) for human IgG, goat IgG and mouse IgG, respectively. The kinetic data provided further insight into the structural/functional relationships of different IgG on a molecular level.