Smart sensor for assessment of oxidative/nitrative stress biomarkers using a dual-imprinted electrochemical paper-based analytical device

We present a novel dual-imprinted electrochemical paper-based analytical device (Di-ePAD) to simultaneously determine 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 3-nitrotyrosine (3-NT) and assess oxidative and nitrative biomarkers in urine and plasma samples. The Di-ePAD was designed with hydrophobic barrier layers formed on filter paper to provide three-dimensional circular reservoirs and assembled electrodes. The molecularly imprinted polymer (MIP) was synthesized using a silica nanosphere decorated with silver nanoparticles (SiO₂@AgNPs) as a core covered with dual-analyte imprinted sites on the polymer to recognize selectively and bind the target biomarkers. This strategy drives monodispersity and enhances the conductivity of the resulting MIP core-shell products. 3-NT-MIP and 8-OHdG-MIP were synthesized by successively coating the surface of SiO₂@AgNPs with L-Cysteine via the thiol group, then terminating with MIP shells. The dual imprinted core-shell composites possess attractive properties for the target biomarkers' sensing, including catalytic activity, selectivity, and good conductivity. The Di-ePAD revealed excellent linear dynamic ranges of 0.01–500 μM for 3-NT and 0.05–500 μM for 8-OHdG, with detection limits of 0.0027 μM for 3-NT and 0.0138 μM for 8-OHdG. This newly developed method based on the synergistic effects of SiO₂@AgNPs combined with promising properties of MIP offers outstanding selectivity, sensitivity, reproducibility, simplicity, and low cost for quantitative analysis of 3-NT and 8-OHdG. The proposed Di-ePAD showed good accuracy and precision when applied to actual samples, including urine and serum samples validated by a conventional HPLC method.