Sensitivity of a majority of reagent based assays is enhanced by increasing of the incubation time. In SI format, stopping the flow is the convenient way of adjusdting  
the length of  incubation  time, during which  the reacting components are arrested within a chosen section of the flow manifold. The SI assay protocol yields two types
of readout, depending on  where the reacting components are held during the incubation time:

  • In the HOLDING COIL (SHC protocol)
  • In the FLOW  CELL (SFC protocol)

Selecting Assay Protocol
Stopping  flow in order to capture the sample in the holding coil (SHC protocol) yields response in form of a peak, similar to Flow Injection response curve, the height of which is proportional to the concentration of the analyte. This is because  the reaction mixture is monitored, while it flows through the flow cell.  SHC protocol is easy to optimize, since besides sample and reagent sequencing, the only variable is the length of the incubation time, which determines sensitivity and throughput of the assay. 2.2.18.

Stopping  flow while the sample is in the flow cell (SFC protocol) yields reaction rate curve, recorded while  the course of the chemical reactions is monitored in real time. By zeroeing the baseline and starting data collection  right at the beginning of the stop flow period, background color of sample and  of reagents, as well as the influence of the refractive index are eliminated. This is why the SFC protocol yields better LOD values than SHC protocol, but it is more difficult to optimize, since it requires a precise positioning of the zone of the reactants in the flow cell. 2.2.36.