The Traditional Approach
SPR experiments for the determination of kinetic rate constants have often been run sequentially, using two flow cells, one as a reference and one for the binding interaction (Figure 1). The reference cell did not contain any ligand and served to measure nonspecific binding of the analyte to the surface of the chip, bulk effects, and signal drift.
A single concentration of analyte was flowed over the ligand of interest bound to the surface in the sample flow cell, and the corresponding response data were collected. The reference cell signal was subtracted from the signal in the sample flow cell. The surface was then regenerated (analyte removed) to prepare the ligand for the next concentration of analyte.
This sequence was repeated until a full analyte concentration series was measured. Such a sequential approach necessitates that one flow cell always be used as a reference, rather than to produce additional binding interaction data. The end result is reduced sample throughput, as well as the risk of loss of ligand activity due to repeated regeneration cycles.