A new bioanalytical method for urea determination by using the immobilized urease on the highly luminescent ZnS quantum dots (QDs), which functioned as the pH fluorescent label has been developed. Bioconjugation of ZnS QDs to urease via amide bond was performed to obtain the ZnS QDs-urease bioconjugate. Upon enzymatic hydrolysis of urea by the immobilized urease, a pH change to a more alkaline condition has led to the deprotonation of ZnS QDs, and an increase in the fluorescence intensity can be observed. The fluorescence intensity of the urease conjugated ZnS QDs changed linearly with the urea concentrations between 4 � 10?9 M and 4 � 10?3 M (R2 = 0.992) at pH 6 with a calibration sensitivity of 179.46 intensity/decade. No noticeable influence by the Ca2+, Mg2+, K+ and Na+ ions on the response of the fluorescent pH bioprobe. Urea determination in soil sample by using the urea bioprobe was in good agreement with the standard DMAB (p-dimethylaminobenzaldehyde) UV�vis spectrophotometric method. By employing the strategy of ZnS QDs-urease bioconjugation, the stable covalent link between the two substances has appeared to widen the dynamic range and lower the detection limit for urea compared to free enzyme and QDs in solution for bioassay of urea concentration. � 2016

A new silica-gel nanospheres (SiO2NPs) composition was formulated, followed by biochemical surface functionalization to examine its potential in urea biosensor development. The SiO2NPs were basically synthesized based on sol-gel chemistry using a modified Stober method. The SiO2NPs 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 SiO2NPs 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 +, Mg 2+ 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 SiO2NPs-based reflectometric urea biosensor showed improved dynamic linear response range when compared to other nanoparticle-based optical urea biosensors.

A regenerable electrochemical DNA biosensor based on a new type of acrylic microspheres and gold nanoparticles (AuNPs) composite coated onto a screen printed electrode (SPE) has been successfully developed for specific determination of the 35 S promoter from cauliflower mosaic virus (CaMV 35S) gene in soybean. DNA probe was immobilised onto acrylic microspheres via covalent bonding. The presence of modified gene in soybean can be detected via hybridisation of CaMV 35S gene-modified DNA with immobilised DNA probe, which was monitored by differential pulse voltammetry of anthraquinone-2-sulfonic acid monohydrate sodium salt (AQMS) as redox indicator during hybridisation event. The peak current signal of AQMS was linearly related to the target CaMV 35S gene concentration over the range of 2 × 10-15 to 2 × 10 -9 M (R2 = 0.982) with a very low concentration detect limit (7.79 × 10-16 M). The recovery test showed satisfactory results of 94.6 ± 5.1-105.4 ± 4.9% (n = 5) when the biosensor was used for the determination of genetically modified (GM) DNA sequences extracted from GM soybean samples. The DNA biosensor showed good reproducibility (relative standard deviation (RSD) below 5.0%, n = 5) and regenerability (RSD below 5.0%, n = 7). The biosensor response was stable up to 45 days of storage period at 4 C. The main advantages of this biosensor design are very low detection limit and capability of reusing the biosensor for at least seven times after regeneration with mild sodium hydroxide.