Fluorescent silicon nanoparticles-based ratiometric fluorescence immunoassay for sensitive detection of ethyl carbamate in red wine

Abstract

Abstract Herein, a ratiometric fluorescence (RF) enzyme-linked immunosorbent assay (RF-ELISA) for sensitive detection of ethyl carbamate (EC) was developed by introducing fluorescent silicon nanoparticles (Si NPs) into the chromogenic substrate system (o-phenylenediamine (OPD)/H 2 O 2 ) of a conventional horseradish peroxidase (HRP)-based ELISA platform to assemble a RF-based signal output system. For this system, the fluorescence of Si NPs at 440 nm (I 440 ) was acted as the reference signal which could be efficiently quenched by 2,3-diaminophenazine (DAP), the HRP-catalyzed oxidation product of OPD; Meanwhile, the fluorescence of DAP at 570 nm (I 570 ) was served as response signal. Therefore, variation in the amount of HRP labeled secondary antibody bound on the microplate which is associated with antibody-antigen recognition events in conventional HRP-based ELISA could be transferred into a more sensitive RF signal (I 570 /I 440 ). On the basis of monoclonal antibody (mAb) which could specifically recognize EC derivative, xanthyl ethyl carbamate (XEC), a Si NPs-based RF-ELISA for EC via a simple pre-analysis derivatization was developed. When detecting the EC content in red wine, this method exhibits a working range from 3.9 to 105.0 μg/L and a limit of detection (LOD) of 2.6 μg/L with excellent specificity, accuracy and reproducibility. The sensitivity is approximately 33-fold higher than that of traditional colorimetric ELISA. The proposed Si NPs-based RF-ELISA is not only highly suitable for screening EC in a large number of samples, but also provides a potential platform for high-throughput and sensitive determination of other analytes for food safety monitoring.