pFI: Optimizing SFC Assay
Two reagents and two transfers
As discussed previously, the precision of SFC method critically depends on the position of the reacting sample zone within the flow cell. Therefore not only the volumes and flowrates generated by the pumps must be strictly reproducible, but also the action of the pumps must be perfectly synchronized else otherwise the position of sample zone in the flow cell will vary. There is, inevitably, a pause when, for example (B): P2,150,75, 0 starts to aspirate, while P1,100,50,1 starts to deliver, but as long as this pause is reproducible the SFC method will be sufficiently precise. Therefore this page examines reproducibility of two reagent assay protocols in SFC protocol, becaust the microfluidic manipulations are more complex and therefore more likely to fail.
Two reagent assay SFC assay protocol (A) comprises four steps of which the last one, the transfer of reaction mixture into flow cell by means of return volume Rv, is important to identify.
The reproducibility of microfluidic manipulations was examined by using a dye (bromothymol blue, BTB) prepared in a well buffered solution, and by using DI water as a carrier, as and for “reagents” #1 and #2. By changing the return volume Rv, the thus obtained, superimposed stop flow responses (B) show that Rv 120 mcrL places the centroid of sample zone in the middle of the light path of the 13cm long flow cell. It also yields better reproducibility than lower Rv values (C), which leave part of sample zone upstream, thus yielding spikes at 44 second position ( Rv 40,60,80 mcrL). By reducing the injected sample volume Sv from 80mcrL to 40 mcrL the centroid position appears to move to Rv 100mcrL. Reproducibility of measurement for Sv of 80 mcrL rsd. of 1.0%, is comparable to that single transfer SFC method and confirms reliability of more complex microfluidic manipulations.
Additional dye tests that offer insight into mixing of reactants at confluence point within LOV manifold are summarized in (2.2.6.F.)
1.2.31.
