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
Mr. Van Bommel et al., 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, J CHROMAT A, 855(2), 1999, pp. 383-396
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.