Injection of increasing volumes of sample solution increases sensitivity of SIA based assays, until a limit is reached, which in this case is at Sv = 100mcrL (A , B). This sample volume, equal to 5 S1/2 values (B) (2.2.6.A) yields the maximum sensitivity of the measurement when using the sandwich protocol (C,D previous page). Since the goal of this study is to establish calibration range with maximum sensitivity, sample volume of 80 mcrL was selected for calibration study.
Data collection protocol (D previous page) was set in such a way that baseline subtraction (BS) is located at 15 sec, when flow cell is filled with carrier, and the peak value is collected within 22-32 sec window (WIN). The calibration response curves (C) cover the range of 0 to 5 microM NaClO, and the calibration table (D) indicates detection limit of 0.02microM NaClO. As seen GLOCELL is extremely efficient in collecting the emitted light as the count is high, ranging from 4xexp4 to 2.3exp6. The collected data are highly reproducible (tables B,D) and the calibration is linear up to 2.5 microM NaClO. Above 10microM NaClO the PMT becomes saturated. However, as for all FIA/SIA techniques, the calibration range can be extended upwards by reducing the sample volume.
Performance of the Coiled Flowcell
2.3.13.
