A new silica-gel nanospheres (SiO(2)NPs) composition was formulated, followed by biochemical surface functionalization to examine its potential in urea biosensor development. The SiO(2)NPs were basically synthesized based on sol-gel chemistry using a modified Stober method. The SiO(2)NPs surfaces were modified with amine (-NH2) functional groups for urease immobilization in the presence of glutaric acid (GA) cross-linker. The chromoionophore pH-sensitive dye ETH 5294 was physically adsorbed on the functionalized SiO(2)NPs as pH transducer. The immobilized urease determined urea concentration reflectometrically based on the colour change of the immobilized chromoionophore as a result of the enzymatic hydrolysis of urea. The pH changes on the biosensor due to the catalytic enzyme reaction of immobilized urease were found to correlate with the urea concentrations over a linear response range of 50-500 mM (R-2 = 0.96) with a detection limit of 10 mM urea. The biosensor response time was 9 min with reproducibility of less than 10% relative standard deviation (RSD). This optical urea biosensor did not show interferences by Na+, K+, Mg2+ and NH4+ ions. The biosensor performance has been validated using urine samples in comparison with a non-enzymatic method based on the use of p-dimethylaminobenzaldehyde (DMAB) reagent and demonstrated a good correlation between the two different methods (R-2 = 0.996 and regression slope of 1.0307). The SiO(2)NPs-based reflectometric urea biosensor showed improved dynamic linear response range when compared to other nanoparticle-based optical urea biosensors.

One of the major problems in the analysis of ammonia (NH3) using spectrophotometric method is highly coloured water sample. A reflectometric NH3 sensor based on a facile single-step immobilisation of cobalt(II) (Co2+) ion onto high capacity Dowex HCR-W2 microspheres has been developed to enable the direct NH3 determination in highly coloured water samples. The combination of Co2+ ion-immobilised microspheres via cation exchange chemical reaction and reflectometric technique has improved the optode sensing performance with respect to linear response range and response time. Immobilised Co2+ ion, when complexed with NH3 gave a blue colouration of hexamminecobaltate(II) ([Co(NH3)(6)](2+)) complex at pH 13. The NH3 sensor reached maximum colour intensity in similar to 6 min at ambient conditions. The linear calibration curve was 60-290 mM NH3 with a detection limit of 16 mM NH3, which is suitable for direct determination of NH3-enriched leachate water samples. A good reproducibility between 5.4 and 5.9% relative standard deviation (RSD) was obtained with the probe and the sensing performance can be regenerated using buffer pH 1. The effect of potential interference ions has been investigated during the quantification of NH3 in heavily polluted sewage water. The results obtained were in good agreement with those produced by acidimetric reference method. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.

An amperometric electrochemical biosensor has been developed for ammonium (NH4+) ion detection by immobilising alanine dehydrogenase (AlaDH) enzyme in a photocurable methacrylic membrane made up of poly(2-hydroxyethyl methacrylate) (pHEMA) on a screen-printed carbon paste electrode (SPE). The current detected was based on the electrocatalytic oxidation of nicotinamide adenine dinucleotide reduced (NADH) that is proportional to the consumption of NH4+ ion whilst enzymatic amination of AlaDH and pyruvate is taking place. The biosensor was operated amperometrically at a potential of +0.6V and optimum pH 7. The NH4+ biosensor demonstrated linear response to NH4+ ion concentration in the range of 0.03-1.02 mg/L with a limit of detection (LOD) of 8.52 mu g/L. The proposed method has been successfully applied to the determination of NH4+ ion in river water samples without any pretreatment. The levels of possible interferents in the waters were negligible to cause any interference on the proposed method. The analytical performance of the biosensor was comparable to the colorimetric method using Nesslerisation but with much lower detection limit and linear response range at ppb level.

The use of multilayer of gold nanoparticles (AuNPs) attached on gold electrode surface via thiol chemistry to fabricate an ammonium (NH4+) ion biosensor based on alanine dehydrogenase (AlaDH) was investigated. The approach of the study was based on construction of biosensor by direct deposition of AuNPs and 1,8-octanedithiol (C8-DT) onto the gold electrode surface. For the immobilisation of enzyme, 2-mercaptoethanol (2BME) was first covalently attached to AlaDH via esther bonding and then followed by chemically attached the 2BME-modified AlaDH (2BME-AlaDH) moiety onto the AuNPs electrode via the exposed thiol group of 2BME. The resulting biosensor response was examined by means of amperometry for the quantification of NH4+ ion. In the absence of enzyme attachment, the use of three layers of AuNPs was found to improve the electrochemistry of the gold electrode when compared with no AuNPs was coated. However, when more than three layers of AuNPs were coated, the electrode response deteriorated due to excessive deposition of C8-DT. When AlaDH was incoporated into the AuNPs modified electrode, a linear response to NH4+ ion over the concentration range of 0.1-0.5 mM with a detection limit of 0.01 mM was obtained. In the absence of AuNPs, the NH4+ ion biosensor did not exhibit any good linear response range although the current response was observed to be higher. This work demonstrated that the incorporation of AuNPs could lead to the detection of higher NH4+ ion concentration without the need of dilution for high NH4+ ion concentration samples with a rapid response time of < 1 min.