One-Step Ligand Immobilization and Single Sample Injection for Regeneration-Free Surface Plasmon Resonance Measurements of Biomolecular Interactions.

Surface plasmon resonance (SPR) has been well established as a method of choice for label-free kinetic measurements of biomolecular interactions. The conventional approach involves multiple injections of an analyte of different concentrations into a fluidic channel covered with a fixed ligand density. Optimization of the experimental conditions and assessment of the data quality can be complicated by issues such as disruption of the ligand structure by the regeneration step and the limited availability of the sample solution. By sequentially closing fluidic channels on a five-channel SPR instrument, different densities of a ligand can be immobilized and determined in one step. With a subsequent injection of a single sample solution, SPR sensorgrams can be simultaneously collected to yield binding and dissociation rate constants (ka and kd) and dissociation constant (KD) between the ligand and analyte. For biomolecular interactions that obey the Langmuir isotherm, we show that the fidelity of the kinetic data can only be reliably confirmed when there exists a strong linear correlation between the SPR signals and the ligand densities. The use of a multichannel SPR instrument also obviates the regeneration step, allowing the binding kinetics between the green fluorescent protein and its antibody to be measured. In comparison to the conventional approach, the method simplifies the experimental procedure, reduces costs associated with sensor chips and biological samples, expedites kinetic measurements, and allows affinity constants to be determined more straightforwardly.