Abstract
The determination of proteins with enzyme-amplified biochemical detection (EA-BCD) coupled on-line with high-performance liquid chromatography (HPLC) is demonstrated. The EA-BCD system was developed to detect biotin-containing compounds. Hemoglobin, which was used as a model compound, was biotinylated prior to sample introduction. Several biotinylation parameters, such as pH and removal of excess biotinylation reagent, were investigated. After biotinylation samples were introduced to HPLC followed by EA-BCD. To the HPLC effluent, alkaline phosphatase label streptavidin (S-AP) was added, which possesses high affinity to biotin and biotin-containing compounds. Excess S-AP was removed by means of an immobilized biotin column followed by substrate addition. The non-fluorescent substrate is converted to a highly fluorescent product by the enzyme label. A detection limit of 2 femtomol biotinylated Hb was achieved with good reproducibility and linearity. However, biotinylation at low analyte concentration suffers from low yield due to slow reaction kinetics. Finally, Hb was successfully extracted from urine with a recovery of 94%.
Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
