pFI: Optimizing SHC Assay
Two reagents and two transfers

Flow Injection is based on two simultaneously ongoing kinetic processes; mutual dispersion of sample with reagents and a the resulting chemical reactions. More often than not, neither of these processes reaches completion during an assay cycle, and therefore it is of paramount importance that these processes take place in a strictly reproducible fashion. Therefore, although plausible, the concept of mini pFI may not be feasible, unless all components of the instrument perform in strictly reproducible fashion.  Performance of one pump system for  miniSI applications (Chapter 2) confirms reliability of miliGAT drive, but for mini pFI application an additional requirement has to be fulfilled, i.e. reproducible synchronization of the two pumps. Therefore we shall examine reproducibility of two reagent assay protocols that also can serve as  templates for  single and multiple reagent assays in SHC and SFC format.

Two reagent assay SHC assay protocol (A) comprises
1) sample aspiration by P1
2) sample transfer and simultaneous aspiration of the first  reagent by synchronized action of P2 and P1,
3) transfer of reaction mixture and simultaneous aspiration of the second reagent by synchronized action of P1 and P2, 
4) stop flow period (WAIT},
5) monitoring of reacted sample as it is transported through flow cell by P1.

Reproducibility of microfluidic manipulations was examined by using a dye (bromothymol blue, BTB ) in a well buffered solution, and by using DI water as a carrier, as well as reagent #1 and #2.. By increasing the injected sample volume (Sv), the thus obtained and superimposed response curves (B) exhibit an increasing peak height in a fashion typical for all FI techniques.  The peak height for construction of the graph (C) was obtained by subtracting baseline (BS) from peak maxima located within the window (WIN). Reproducibility of absorbencies (C) was in average 3.3% comparable conventional cFi.

The dye test is a very useful diagnostic tool and should be used any time when instrument is assembled, or reconfigured. Obviously, presence of air bubles anywhere in the flow path will adversely affect reproducibility of measurement. Indeed, poor reproducibility of microfluidic manipulations is rarely due to pump or valve failure, but almost always due to presence air, or due to backpressure in obstructed flow line. De aeration of DI used as a carrier, and for preparation of reagents is often the efficient  remedy.