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-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.
van Bommel
M R
MR
Division of Analytical Chemistry, Leiden/Amsterdam Center of Drug Research, Leiden University, The Netherlands.
de Jong
A P
AP
Tjaden
U R
UR
Irth
H
H
van der Greef
J
J
D001710:Biotin / Q000737:chemistry; D004798:Enzymes / Q000737:chemistry*; D017022:Flow Injection Analysis / Q000379:methods*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D004798:Enzymes; D001710:Biotin