The heliX device is equipped with two spots allowing for simultaneous measurements. One of the two spots may be used for the actual binding kinetics experiment (“measurement spot”) while the other one serves as a ligand-free reference (“reference spot”). In this configuration, the reference spot signal may be used to reference-correct the measurement spot signal in temporal allocation. This is called real-time referencing.
Blank measurements are control measurements without analyte, conducted before, in between or after actual measurements. Real-time referenced blank signals should appear as flat baseline signals. However, small artifacts may remain. In this case, or in the lack of real-time references, blank signals may be subtracted from the measurement signals for a (second-order) reference-correction. This is called blank referencing.
The combination of real-time referencing and blank referencing is referred to as double referencing.