Preparation and characterisation of functionalised calcium carbonate nanoparticles from cockle shell for optical urea biosensor

Urea in urine is commonly used as an indicator to determine kidney disease in humans. Previously, numerous sensing materials based on chemicals and artificial sources were used to immobilise urease in urea biosensors. In this study, calcium carbonate nanoparticles (CaCO₃-NPs) from discarded cockleshells were synthesised via a simple and eco-friendly approach and characterised using field-emission scanning electron microscopy (FESEM), particle size analyser (PSA), Fourier-transform infrared-attenuated total reflectance (FTIR-ATR) spectroscopy, Auger electron spectroscopy with X-ray photoelectron spectroscopy (AES-XPS), and energy-dispersive X-ray (EDX) spectroscopy. The surface of the NPs was primarily functionalised with acrylic acid N- hydroxysuccinimide ester (NAS) to provide a succinimide ester group that could covalently bind to the amine group of urease. An optical biosensor for urea based on urease immobilised on functionalised NPs (Urs/F-NPs) was successfully developed. The results showed that the NPs obtained were aragonite polymorph CaCO₃ in size 78±10.8 nm. Approximately 85.8% of the urease was successfully covalently immobilised on the surface of the NPs that had been proved by the bovine serum albumin (BSA) method, FTIR-ATR, and AES-XPS. The FTIR-ATR spectra confirmed peaks at 1120 cm-1 and 1016.63 cm⁻¹, which were due to the presence of aliphatic amine C-N and amide bonds, revealing the immobilisation of urease on functionalised NPs. Moreover, AES-XPS analysis showed changes in the binding energy (eV) before and after immobilisation with urease, with peaks at 131 eV and 170 eV representing phosphate and sulphur from urease. Since the fabricated optical biosensor involved pH changes after the addition of urea, the phenolphthalein indicator was applied. The biosensor provided a colourimetric 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 633.16 nm. Optimum activity of immobilised urease enzyme was in 25 mM phosphate buffer solution (pH 7.5) and the performance of biosensor was good using 0.04 M of phenolphthalein and 0.3 mg of urease loading. The determination of urea concentration using this biosensor yielded a linear response range of 30 to 1000 mM (R2 = 0.9901), with a detection limit of 17.74 mM. The relative standard deviation (RSD) value obtained as an assessment of the biosensor’s reproducibility was 1.14%, with no signs of interference by major cations such as K⁺, Na⁺, NH₄⁺, and Mg²⁺. The fabricated biosensor showed no significant difference from the standard method determining urea in the urine samples.