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

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)/H2O2) 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 (I440) 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 (I570) 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 (I570/I440). 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.