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

• Published in: Journal of Chromatography A 1999-09, Vol.855 (2), p.383-396
• Main Authors: van Bommel, M.R, de Jong, A.P.J.M, Tjaden, U.R, Irth, H, van der Greef, J
• Format: Article
• Language: English
• Subjects: Alkaline phosphatase; Analytical biochemistry: general aspects, technics, instrumentation; Analytical chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biotin; Biotin - chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Enzymes - chemistry; Exact sciences and technology; Flow Injection Analysis - methods; Fundamental and applied biological sciences. Psychology; Other chromatographic methods; Reproducibility of Results; Spectrometry, Fluorescence
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(99)00744-X

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.