Enzyme amplification as detection tool in continuous-flow systems II. On-line coupling of liquid chromatography to enzyme-amplified biochemical detection after pre-column derivatization with biotin

Enzyme-amplified biochemical detection (EA-BCD) was used as a post-column d etection technique, coupled on-line with high-performance liquid chromatogr aphy (HPLC). The enzyme detection system was developed to detect biotin or biotin containing compounds. Biotinylation is widely used to label analytes of interest ranging from small molecules to proteins and DNA. Naphthalene aldehyde and anthracene aldehyde were used as model compounds. Both compoun ds were biotinylated off-line with biotin aminocaproic hydrazide (BACH), On -column biotinylation was performed by preconcentration of anthracene aldeh yde on copper phthalocyanine. After biotinylation, samples were introduced to the HPLC system. Enzyme-labeled streptavidin, which possesses high affin ity to biotin, was added post-column to the HPLC effluent. Excess of enzyme -labeled affinity protein was removed by means of an immobilized biotin col umn. After separation of free and bound fraction, substrate was added, whic h was converted to a fluorescent product by the enzyme label. Using alkalin e phosphatase as an enzyme label, a mass detection limit after on-column pr econcentration and biotinylation of 250 fmol was achieved. (C) 1999 Elsevie r Science B.V. All rights reserved.