Phosphate Assay in Forward Flow pFI Format
120/40 s/hour, detection limit 6 ppB P
The continuous flow assay of o-phosphate (A) typically uses a constant flowrate of 2,5 ml/min yielding a sampling frequency of over 100s/hr at a range of 2 to 300 ppM P (1.2.20.)
By means of flow programming (B) an by adjusting the injected sample volume, the same flow system will accommodate concentration range from 10 to 1000 ppB P and will generate approximately 50% of waste compared to continuous flow system.
The flow programming comprises five sections within each assay cycle:
1. Priming @100%fr for 5 sec
2. Sample injection @50%fr for 1 sec
3. Incubation @2%fr for DWELL sec
4. Monitoring @50%fr for 12 sec
5. Washout @100% for 6 sec
The high concentration range (C) was run at zero seconds dwell time, while the low concentration range (D) was run using 50 seconds dwell time.
The overlaid calibration runs (E) show enhancement of the sensitivity of the assay when the incubation time was increased by slowing the flow trough the reaction coil to 80microliters/ min for 50 second interval. Thus, as sampling frequency decreased from 120 s/hr to 40s/hr, the sensitivity of the assay increased almost 10 times, due to longer incubation time. For all assays the injected sample volume was 300microliters and the flow cell light path was 10 cm long. All manifold tubing was 0.8 mm I.D.
1.2.22.
