In SIA flow manifold (A), incubation of sample with reagent can be carried out either
in a holding coil (SHC protocol) or
in a flow cell (SFC protocol)
by stopping the flow for a desired time interval at controlled temperature. The advantage of SHC protocol is its speed and simplicity, while SFC protocol offers higher sensitivity and selectivity, since it is free from influence of background color of sample and reagents.
In order to optimize any reagent based assay, its kinetics should be investigated by using SHC protocol. As shown on the following example of glucose assay, the data obtained by SHC technique are then used to optimize the SFC protocol. 2.2.11.A.
In SFC protocol the mixture of sample with reagent is arrested within a flow cell and therefore changes of absorbance or fluorescence are only due to chemical reactions taking place. Therefore increasing response in time is recorded only in the presence of ANALYTE, while in the absence of analyte, a horizontal line is recorded (BLANK).
Since sample (S) and reagent (R) zone overlap (B), maximum sensitivity is obtained by adjusting the flow reversal volume that positions peak maximum within the flow cell (C). This flow reversal volume can be identified either by
running a series of experiments using the same analyte (2.2.9.) concentration and by varying the flow reversal volume or,
by identifying flow reversal volume from peak maximum, obtained by SHC method.
Since the SHC method is more convenient, it is preferable to use this approach, detailed in Section 2.2.11.A.