Enzyme amplification as detection tool in continuous-flow systems I. Development of an enzyme-amplified biochemical detection system coupled on-line to flow-injection analysis

The on-line coupling of flow-injection analysis (FIA) to an enzyme-amplifie d biochemical detection (EA-BCD) system is described. The aim of this study is the development of a detection system able to detect biotin-containing compounds at low concentration levels. The detection system is based on the interaction of biotin with enzyme-labeled affinity proteins. Biotin posses ses a high affinity to both streptavidin and anti-biotin Fab fragments, whi ch are both tested. Several biotin derivatives are available with different reactive probes, which can be used to label analytes of interest. Therefor e, biotin acts as a universal probe for the enzyme-amplified biochemical de tection. Alkaline phosphatase (AP) was used as enzyme label. Several parame ters, such as substrate type and concentration, concentration of enzyme-lab eled affinity protein, reaction time and reaction temperature were examined . Biotin aminocaproic acid was used as a model compound. In addition to bio tin aminocaproic hydrazide, other biotinylation reagents were also examined . With fluorescence detection of the enzyme-generated product, a mass detec tion limit of 1 fmol was achieved. (C) 1999 Elsevier Science B.V. All right s reserved.