A self-calibrating flexible SERS substrate incorporating PB@Au assemblies for reliable and reproducible detection

The precise quantitative analysis using surface-enhanced Raman spectroscopy (SERS) in an uncontrollable environment still faces a significant obstacle due to the poor reproducibility of Raman signals. Herein, we propose a facile method to fabricate a self-calibrating substrate based on a flexible polyvinyl alcohol (PVA) film comprising assemblies of Prussian blue (PB) and Au NPs (PB@Au) for reliable detection. PB cores were coated with an Au shell through simple electrostatic interaction, forming core-shell nanostructure PB@Au assemblies within the PVA film. The outer Au layer provided identical trends in enhancement for both the PB core and neighboring targets while PB cores served as an internal standard (IS) to correct signal fluctuations. The prevention of competitive adsorption on the metal surface between targets and ISs was achieved. The proposed PVA/PB@Au film exhibited enhanced stability of Raman signals after IS correction, resulting in improved spot-to-spot and batch-to-batch reproducibility with significantly reduced standard deviation (RSD) values from 11.42% and 25.02% to 4.43% and 9.39%, respectively. Simultaneously, a higher accuracy in the quantitative analysis of 4-mercaptobenzoic acid (4-MBA) and malachite green (MG) was achieved with fitting coefficient (R²) values improving from 0.9675 and 0.9418 to 0.9974 and 0.9832, respectively. Moreover, the PVA/PB@Au film was successfully applied to detect residual MG in real fish samples. This work opens up an avenue to improve the reproducibility of Raman signals for flexible SERS substrates in the detection of residues under various complex conditions.