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.

Autor: van Bommel MR; Division of Analytical Chemistry, Leiden/Amsterdam Center of Drug Research, Leiden University, The Netherlands., de Jong AP, Tjaden UR, Irth H, van der Greef J
Jazyk: angličtina
Zdroj: Journal of chromatography. A [J Chromatogr A] 1999 Sep 10; Vol. 855 (2), pp. 383-96.
DOI: 10.1016/s0021-9673(99)00744-x
Abstrakt: 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.
Databáze: MEDLINE
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