Evaluation of progesterone content in saliva using magnetic particle-based immuno supported liquid membrane assay...

Progesterone in saliva was monitored using a new method called magnetic particle-based immuno supported liquid membrane assay (m-ISLMA) in a sequential injection (SI) setup, allowing automatic sample cleanup, analyte enrichment, and detection in a single analysis unit. Progesterone (Ag) diffuses from a continuous flowing sample - the donor - into a supported organic liquid membrane (SLM), based on analyte partitioning (solubility) between the aqueous donor and the organic phase. The Ag is re-extracted from the SLM into a second stagnant aqueous acceptor, containing antibodies (Ab) immobilized on magnetic beads, held at the bottom of the acceptor by a magnet. Due to the formation of strong Ag-Ab-bead complexes and a large excess of Ab-beads, the Ag is accumulated and selectively enriched in the acceptor. The extracted progesterone was quantified by injecting into the acceptor a horseradish peroxidase (HRP) labeled analyte tracer, the substrate (luminol, H(2)O(2), and p-iodophenol), and finally detection of the generated chemiluminescence by a photomultiplier tube. After optimization of experimental parameters (e.g., sample flow rate, extraction time, type of organic solvent and antibody-bead concentration in the acceptor), a detection limit of 8.50+/-0.17 fgL(-1) and a dynamic range between 35 fgL(-1) and 10 pgL(-1) was reached. The progesterone level of saliva for three subjects (women in different period of ovarian cycle) was investigated, and the corresponding progesterone concentrations detected with m-ISLMA coincided well with the expected values.