Single reagent SHC protocol (A) comprises the following steps:
1) sample (red) is aspirated into holding coil (HC1)
2) sample is transferred into HC2 simultaneously with reagent (green), that is added at the confluence point. The mixing ratio of sample with reagent is controlled by flowrates of the pumps. The reaction mixture is arrested in HC2 by stopping the flow for desired incubation time,
3) the reaction product (orange) is moved trough the flow cell. The response curves of all SHC protocols are similar to the conventional cFI peaks, their height being proportional to concentration of the target analyte (B). Single reagent SHC protocol, can be expanded to two or multiple reagent assays as shown in examples on the following pages. (1.2.36. and 1.2.38.)
The single reagent SFC protocol (C) comprises the following steps:
1) sample (red) is aspirated into HC1,
2) reagent (GREEN) is aspirated into HC2
3) sample and reagent are simultaneously propelled trough confluence point towards the detector,
4) the resulting product (orange) is arrested in flow cell for reaction rate monitoring. The response (D), are reaction rate curves, the slope of which is proportional to the concentration of the target analyte. Examples of SFC based assays are (1.2.32. and 1.2 38.)
The temperature of the reacting mixture can be elevated , in order to accelerate reaction rate. For SHC technique, holding coils are thermostated. For SFC technique the flow cell is thermostated.
Assay protocol for SHC is easy to optimize, because the sample zone flows through the flow cell.
Because SFC protocol is based on selection of a portion of dispersed sample zone, it has to be optimized as described in (1.2.30.)