Cockle shell-derived nanoparticles for optical urea biosensor development based on reflectance transduction

An optical biosensor for urea based on urease enzyme immobilised on functionalised calcium carbonate nanoparticles (CaCO₃-NPs) was successfully developed in this study. CaCO₃-NPs were synthesised from discarded cockle shells via a simple and eco-friendly approach, followed by surface functionalisation with succinimide ester groups. The fabricated biosensor is comprised of two layers. The first (bottom layer) contained functionalised NPs covalently immobilised to urease, and the second (uppermost layer) was alginate hydrogel physically immobilised to the pH indicator phenolphthalein. The biosensor provided a colorimetric indication of increasing urea concentrations by changing from colourless to pink. Quantitative urea analysis was performed by measuring the reflectance intensity of the colour change at a wavelength of 633.16 nm. The determination of urea concentration using this biosensor yielded a linear response range of 30–1000 mM (R² = 0.9901) with a detection limit of 17.74 mM at pH 7.5. The relative standard deviation of reproducibility was 1.14%, with no signs of interference by major cations, such as K⁺, Na⁺, NH?⁺, and Mg²⁺. The fabricated biosensor showed no significant difference with the standard method for the determination of urea in urine samples.