A portable microfluidic device-based Fe₃O₄–urease nanoprobe-enhanced colorimetric sensor for the detection of heavy metals in fish tissue

A portable microfluidic device with highly sensitive enzyme nanoprobe (Fe₃O₄ MNPs–urease, average size 34.6 nm) was demonstrated for the analysis of heavy metals ions (Hg²⁺, Cd²⁺ and Pb²⁺) in fish gill and muscle tissue. The immobilized urease nanoprobe (K_m = 0.05 mM) exhibited twofold sensitivity over the free enzyme assay (apparent K_m = 0.1 mM). The nanoprobe was characterized using SEM, EDAX, PSA and FT-IR. The inhibition measurements were carried out for individual as well as the mixture of metal ions (CRM standards of 9 elements (CRM_mix-9)). The lower limit of quantification (LOQ) (0.5, 0.1, and 0.1 ng L⁻¹ for Hg²⁺, Cd²⁺, and Pb²⁺) and lower limit of detection (LOD) was achieved at 0.1 ng L⁻¹ with sensitivity 8–14% per decade for Hg²⁺, Cd²⁺, and Pb²⁺ ions. A visual result can be observed by the naked eye through the microfluidic device as well as with 96 transparent microwell plates. The order of relative inhibition was found to be CRM_mix-9 > (Hg²⁺ + Cd²⁺ + Pb²⁺) > (Cd²⁺ + Pb²⁺) > (Pb²⁺ + Hg²⁺) > (Hg²⁺ + Cd²⁺) > Pb²⁺ > Cd²⁺ > Hg²⁺, respectively. The recovery % in fish tissues were found to be 88–98% for Hg²⁺, Cd²⁺ and Pb²⁺ ions.