Homogeneous immunoassay for the detection of trinitrotoluene (TNT) based on the reactivation of apoglucose oxidase using a novel FAD-trinitrotoluene conjugate

摘要

A flavin adenine dinucleotide-trinitrotoluene derivative (FAD-TNT) was synthesized by coupling N-6-(2-aminoethyl)-FAD covalently to the N-hydroxysuccinimidyl ester of trinitrophenyl-gamma-aminobutyric acid and characterized by negative-ion electrospray-ionization mass spectrometry (ESI-MS) after purification by reversed-phase HPLC. Free FAD-TNT can be detected at very low levels by recombination with apoglucose oxidase, since the FAD-TNT-glucose oxidase complex is enzymatically active. On the contrary, if FAD-TNT has been bound by an anti-TNT antibody, the conjugate cannot recombine with apoglucose oxidase any more. Based on these two phenomena, a homogeneous apoenzyme reactivation immunoassay system (ARIS) was developed for the detection of TNT. No separation step is needed in this assay. Proportionality between the TNT concentration and enzyme activity was demonstrated with a detection limit of 5 mu g/L TNT. [References: 23]