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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 cocklesh...

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Main Author: Nur Izzati Zakaria
Format: UMK Etheses
Language:English
Published: 2022
Online Access:http://discol.umk.edu.my/id/eprint/13408/1/NUR%20IZZATI%20BINTI%20ZAKARIA.pdf
http://discol.umk.edu.my/id/eprint/13408/
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spelling my.umk.eprints.134082023-06-26T08:23:05Z http://discol.umk.edu.my/id/eprint/13408/ Preparation and characterisation of functionalised calcium carbonate nanoparticles from cockle shell for optical urea biosensor Nur Izzati Zakaria 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. 2022 UMK Etheses NonPeerReviewed text en http://discol.umk.edu.my/id/eprint/13408/1/NUR%20IZZATI%20BINTI%20ZAKARIA.pdf Nur Izzati Zakaria (2022) Preparation and characterisation of functionalised calcium carbonate nanoparticles from cockle shell for optical urea biosensor. Masters thesis, Universiti Malaysia Kelantan.
institution Universiti Malaysia Kelantan
building Perpustakaan Universiti Malaysia Kelantan
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaysia Kelantan
content_source UMK Institutional Repository
url_provider http://umkeprints.umk.edu.my/
language English
description 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.
format UMK Etheses
author Nur Izzati Zakaria
spellingShingle Nur Izzati Zakaria
Preparation and characterisation of functionalised calcium carbonate nanoparticles from cockle shell for optical urea biosensor
author_facet Nur Izzati Zakaria
author_sort Nur Izzati Zakaria
title Preparation and characterisation of functionalised calcium carbonate nanoparticles from cockle shell for optical urea biosensor
title_short Preparation and characterisation of functionalised calcium carbonate nanoparticles from cockle shell for optical urea biosensor
title_full Preparation and characterisation of functionalised calcium carbonate nanoparticles from cockle shell for optical urea biosensor
title_fullStr Preparation and characterisation of functionalised calcium carbonate nanoparticles from cockle shell for optical urea biosensor
title_full_unstemmed Preparation and characterisation of functionalised calcium carbonate nanoparticles from cockle shell for optical urea biosensor
title_sort preparation and characterisation of functionalised calcium carbonate nanoparticles from cockle shell for optical urea biosensor
publishDate 2022
url http://discol.umk.edu.my/id/eprint/13408/1/NUR%20IZZATI%20BINTI%20ZAKARIA.pdf
http://discol.umk.edu.my/id/eprint/13408/
_version_ 1769848733558636544
score 13.211869

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