Abstract
Surface plasmon resonance (SPR) is highly sensitive to biomolecular interactions and refractive index (RI) changes. SPR biosensor has advantages of label-free with analyte, real-time and specific interaction detections, and high throughput measurement. Through immobilization of biomolecules on the sensor chip as a probe, SPR is widely used in antibody-antigen binding, DNA hybridization, enzyme-substrate interaction, and etc. There are four kinds of SPR detection: angular interrogation, wave- length interrogation, intensity measurement, and phase measurement. Among these methods, phase measurement is the most sensitive one. Since Nelson et al. (Nelson et al., 1996) proposed the phase measurement method, to our knowledge, the latter published work have focused mainly on improving detection limit and other bio-applications and none of them has applied to dynamic measurement in biomolecular interactions. Here, I present the study by using a homemade phase-detection based SPR system of measuring the kinetic parameters from antibody-antigen binding. First, I tested the repeatability of kinetics by six samples in the same concentration; the results show a good repeatability of 6.76% in CV value. Second, I tested the velocity effect to antibody binding ability by varying the flow rate from 150 to 600 μl/min; the results indicate that the difference of binding rate is small (within 8.84%), implying that we can fix at any velocity in the tested range to conduct our experiment. Finally, I conducted the dynamically binding test of antigen-antibody using both the homemade SPR system and a commercial Biacore X SPR system. The association (ka) and dissociation (kd) rate constants obtained from the homemade system were (1.02±0.17) × 104M-1s-1 and (3.01±1.43) × 10-3s-1, respectively. Comparing to the Biacore X, I found that the ka value obtained from the home-made system was smaller than that obtained from the Biacore X. This discrepancy was considered mainly from the dilution effect, in which the adjacent running buffer diluted the sample solution adjacent to the running buffer; mass transport effect was considered to be the second reason to affect the association rate constant. Phase-based SPR system has characteristics of high sensitivity and short linear range in measurement whereas commercial instrument, based on the angular interrogation, has relative poor sensitivity and longer measurement range. Therefore, the phase-based SPR system would reveal more powerful in the low concentration-end of dynamic biomolecular interaction measurement.