Two reagent assays are based on a sequence, where sample is sandwiched between two reagents, within a holding coil (A). Next the reacting sample is transported into the flow cell by a flow reversal. In order to maximize the sensitivity of the assay, the volume of flow reversal has to be determined by injecting a dye, simulating the sample, while water is used instead of reagents. By using a blue dye the peak maximum (B), was found to be at 240μL of flow reversal, and this value was applied for the assay of phosphate, when applying molybdate and ascorbic acid as reagents, and the thus optimized assay protocol.
The apparatus was a Global FIA miniFloPro system, customized to accommodate lab-on-valveŽ module supplied by FIAlab. (C). Ocean Optics spectro-photometer and tungsten lamp were used for referenced absorbance measurement. The stop flow reaction rate measurement, carried out for 50 seconds (D), yielded linear calibration within the range of 0,125, 250 and 500 ppBP with r =0.99861. (E). The assay cycle lasted 67 seconds yielding sampling
rate of 54 s/hr.
Note that this assay protocol can be used for any two reagent assay, provided that the flow channel will have the same volume and flow geometry as the one used in this experimental setup.
Phosphate Assay
Development of Two Reagent Assay
2.2.18.
