A 2D microfluidic paper sensor for mercury speciation in fish based on the peroxidase-mimicking activity of...

A novel, field-deployable colorimetric assay based on a two-dimensional microfluidic paper-based analytical device (2D μPAD) was developed for on-site mercury speciation in fish samples. The device integrates l-Cysteine (L-Cys) functionalized iron oxide nanoparticles (Fe₃O₄@L-Cys NPs) that act as mercury-responsive nanozyme. In the presence of mercury species, L-Cys could be displaced from the Fe₃O₄ NPs' surface by a stronger L-Cys-Hg-L-Cys coordination complex. The uncoated Fe₃O₄ NPs reactivates the peroxidase-like activity of the nanozyme catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide into blue-colored TMBox for quantitative readout. Speciation was performed directly on the 2D μPAD using a selective ethylenediaminetetraacetic acid (EDTA) masking step. The pre-deposited EDTA in the sample addition zone complexes inorganic mercury (Hg²⁺), preventing its interaction with the NPs, allowing the colorimetric signal to reflect only methylmercury (MeHg⁺). Under optimal conditions, the 2D μPAD enabled to monitor total mercury in a linear range of 25 to 200 μg L^-1 and MeHg⁺ in the range of 12.5 - 200 μg L^-1. The developed assay showed limits of detection as low as 7.3 μg L^-1 and 3.6 μg L^-1 for total mercury and MeHg⁺, respectively, with a repeatability lower than 3.7 % (N = 5) in all cases. The developed 2D μPAD was applied to determine total mercury and MeHg⁺ in fish samples, yielding results consistent with the centralized Direct Mercury Analyzer method. The method was further validated using two certified reference materials, BCR-464 (tuna muscle) and DOLT-4 (dogfish liver), with measured values in good agreement with certified concentrations (t-test; p < 0.05).