title: 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.
abstract: The on-line coupling of flow-injection analysis (FIA) to an enzyme-amplified 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 possesses a high affinity to both streptavidin and anti-biotin Fab fragments, which are both tested. Several biotin derivatives are available with different reactive probes, which can be used to label analytes of interest. Therefore, biotin acts as a universal probe for the enzyme-amplified biochemical detection. Alkaline phosphatase (AP) was used as enzyme label. Several parameters, such as substrate type and concentration, concentration of enzyme-labeled affinity protein, reaction time and reaction temperature were examined. Biotin aminocaproic acid was used as a model compound. In addition to biotin aminocaproic hydrazide, other biotinylation reagents were also examined. With fluorescence detection of the enzyme-generated product, a mass detection limit of 1 fmol was achieved.
authors.affiliation: Division of Analytical Chemistry, Leiden/Amsterdam Center of Drug Research, Leiden University, The Netherlands.
authors.forename: M R
authors.initials: MR
authors.lastname: van Bommel
authors.forename: A P
authors.initials: AP
authors.lastname: de Jong
authors.forename: U R
authors.initials: UR
authors.lastname: Tjaden
authors.forename: H
authors.initials: H
authors.lastname: Irth
authors.forename: J
authors.initials: J
authors.lastname: van der Greef
mesh_terms: D001710:Biotin / Q000737:chemistry; D004798:Enzymes / Q000737:chemistry*; D017022:Flow Injection Analysis / Q000379:methods*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
chemical_list: D004798:Enzymes; D001710:Biotin