Yook Heng Lee

Use of Fe3O4 Nanoparticles for Enhancement of Biosensor Response to the Herbicide 2,4-Dichlorophenoxyacetic Acid.

Magnetic nanoparticles of Fe3O4 were synthesized and characterized using transmission electron microscopy and X-ray diffraction. The Fe3O4 nanoparticles were found to have an average diameter of 5.48 ±1.37 nm. An electrochemical biosensor based on immobilized alkaline phosphatase (ALP) and Fe3O4 nanoparticles was studied. The amperometric biosensor was based on the reaction of ALP with the substrate ascorbic acid 2-phosphate (AA2P). The incorporation of the Fe3O4 nanoparticles together with ALP into a sol gel/chitosan biosensor membrane has led to the enhancement of the biosensor response, with an improved linear response range to the substrate AA2P (5-120 μM) and increased sensitivity. Using the inhibition property of the ALP, the biosensor was applied to the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The use of Fe3O4 nanoparticles gives a two-fold improvement in the sensitivity towards 2,4-D, with a linear response range of 0.5-30 μgL-1. Exposure of the biosensor to other toxicants such as heavy metals demonstrated only slight interference from metals such as Hg2+, Cu2+, Ag2+ and Pb2+. The biosensor was shown to be useful for the determination of the herbicide 2, 4-D because good recovery of 95-100 percent was obtained, even though the analysis was performed in water samples with a complex matrix. Furthermore, the results from the analysis of 2,4-D in water samples using the biosensor correlated well with a HPLC method.

Determination of ammonium ion using a reagentless amperometric biosensor based on immobilized alanine dehydrogenase.

The use of the enzyme alanine dehydrogenase (AlaDH) for the determination of ammonium ion (NH4+) usually requires the addition of pyruvate substrate and reduced nicotinamide adenine dinucleotide (NADH) simultaneously to effect the reaction. This addition of reagents is inconvenient when an enzyme biosensor based on AlaDH is used. To resolve the problem, a novel reagentless amperometric biosensor using a stacked methacrylic membrane system coated onto a screen-printed carbon paste electrode (SPE) for NH4+ ion determination is described. A mixture of pyruvate and NADH was immobilized in low molecular weight poly(2-hydroxyethyl methacrylate) (pHEMA) membrane, which was then deposited over a photocured pHEMA membrane (photoHEMA) containing alanine dehydrogenase (AlaDH) enzyme. Due to the enzymatic reaction of AlaDH and the pyruvate substrate, NH4+ was consumed in the process and thus the signal from the electrocatalytic oxidation of NADH at an applied potential of +0.55 V was proportional to the NH4+ ion concentration under optimal conditions. The stacked methacrylate membranes responded rapidly and linearly to changes in NH4+ ion concentrations between 10–100 mM, with a detection limit of 0.18 mM NH4+ ion. The reproducibility of the amperometrical NH4+ biosensor yielded low relative standard deviations between 1.4–4.9%. The stacked membrane biosensor has been successfully applied to the determination of NH4+ ion in spiked river water samples without pretreatment. A good correlation was found between the analytical results for NH4+ obtained from the biosensor and the Nessler spectrophotometric method.

Poly(hydroxyl ethyl methacrylate) hydrogel matrix for phenol biosensor

This paper discusscs the use of poly(hydroxy1 ethyl methacrylate). @oly(HEMA)) hydmgel as a mamx for tyrosinase hmobilization in a biosensor for the detection of phenolic compounds. A two-electrode system, i.e. a combination of working electrode and a Ag/AgCI (3M KCI) reference electrode was used for the consrm~tion of the biosensor. Screen primed electrodes were used as working electrodes for these smdy. The responses of the biosensor containing poly(HEM.4) membranes with tyrosinase immobilized towards phenol, catechol and m-cresol werc ~xamined.

An ultrasensitive hollow-silica-based biosensor for pathogenic Escherichia coli DNA detection

AbstractA novel electrochemical DNA biosensor for ultrasensitive and selective quantitation of Escherichia coli DNA based on aminated hollow silica spheres (HSiSs) has been successfully developed. The HSiSs were synthesized with facile sonication and heating techniques. The HSiSs have an inner and an outer surface for DNA immobilization sites after they have been functionalized with 3-aminopropyltriethoxysilane. From field emission scanning electron microscopy images, the presence of pores was confirmed in the functionalized HSiSs. Furthermore, Brunauer–Emmett–Teller (BET) analysis indicated that the HSiSs have four times more surface area than silica spheres that have no pores. These aminated HSiSs were deposited onto a screen-printed carbon paste electrode containing a layer of gold nanoparticles (AuNPs) to form a AuNP/HSiS hybrid sensor membrane matrix. Aminated DNA probes were grafted onto the AuNP/HSiS-modified screen-printed electrode via imine covalent bonds with use of glutaraldehyde cross-linker. The DNA hybridization reaction was studied by differential pulse voltammetry using an anthraquinone redox intercalator as the electroactive DNA hybridization label. The DNA biosensor demonstrated a linear response over a wide target sequence concentration range of 1.0×10-12–1.0×10-2 μM, with a low detection limit of 8.17×10-14 μM (R2 = 0.99). The improved performance of the DNA biosensor appeared to be due to the hollow structure and rough surface morphology of the hollow silica particles, which greatly increased the total binding surface area for high DNA loading capacity. The HSiSs also facilitated molecule diffusion through the silica hollow structure, and substantially improved the overall DNA hybridization assay. Graphical abstractStep-by-step DNA biosensor fabrication based on aminated hollow silica spheres

A New Laccase Based Biosensor for Tartrazine

Laccase enzyme, a commonly used enzyme for the construction of biosensors for phenolic compounds was used for the first time to develop a new biosensor for the determination of the azo-dye tartrazine. The electrochemical biosensor was based on the immobilization of laccase on functionalized methacrylate-acrylate microspheres. The biosensor membrane is a composite of the laccase conjugated microspheres and gold nanoparticles (AuNPs) coated on a carbon-paste screen-printed electrode. The reaction involving tartrazine can be catalyzed by laccase enzyme, where the current change was measured by differential pulse voltammetry (DPV) at 1.1 V. The anodic peak current was linear within the tartrazine concentration range of 0.2 to 14 μM (R2 = 0.979) and the detection limit was 0.04 μM. Common food ingredients or additives such as glucose, sucrose, ascorbic acid, phenol and sunset yellow did not interfere with the biosensor response. Furthermore, the biosensor response was stable up to 30 days of storage period at 4 °C. Foods and beverage were used as real samples for the biosensor validation. The biosensor response to tartrazine showed no significant difference with a standard HPLC method for tartrazine analysis.

Pemegunan Enzim Urease dalam Bahan Hidrogel Metakrilat untuk Menghasilkan Membran Biosensor Urea

Bahan hidrogel metakrilat yang disediakan secara foto–pempolimeran telah dikaji sebagai satu matriks untuk pemegunan enzim urease. Tujuan utama penyediaan membran metakrilat untuk pemegunan enzim urease adalah untuk menilai sama ada membrane jenis ini boleh digunakan dalam reka bentuk biosensor urea. Sifat kehidrofilikan/kehidrofobikan hidrogel dimanipulasikan melalui komposisi monomer 2–hidroksil etil metakrilat (HEMA) dan metil metakrilat (MMA). Kajian pemegunan enzim yang telah dilakukan termasuk pemegunan urease secara foto–pempolimeran, kajian larut resap urease daripada membran dan penilaian ke atas sifat keaktifan enzim yang terpegun. Tiga jenis membran polimer telah dikaji, iaitu dua jenis membran yang disediakan secara foto–pempolimeran (fotoHEMA iaitu 100% HEMA dan MH28, nisbah MMA : HEMA = 2 : 8) dan juga polimer poli(2–hidroksietil metakrilat) (pHEMA) yang boleh didapati secara komersial dan digunakan sebagai perbandingan. Hasil kajian menunjukkan bahawa urease boleh di pegunkan dalam bahan hidrogel metakrilat secara foto–pempolimeran terus tanpa enzim dinyahaktifkan walaupun terdedah kepada cahaya ultra–lembayung. Penggunaan komposisi MMA : HEMA dan proses penyediaan membran secara foto–pempolimeran dapat menghasilkan sifat kehidrofilikan/kehidrofobikan bahan membran hidrogel yang dikehendaki dan seterusnya kelarutresapan enzim juga dikurangkan berbanding membran daripada pHEMA komersial. Bahan hidrogel metakrilat foto–polimer yang paling sesuai sebagai matriks pemegunan enzim urease untuk tujuan kegunaan biosensor urea ialah hidrogel MH28 yang mempunyai peratus serapan air <8% dalam masa sejam. Bahan ini juga didapati boleh memberikan keaktifan enzim terpegun yang lebih tinggi berbanding dengan bahan hidrogel lain yang dikaji. Kajian ini telah menunjukkan bahawa kaedah mudah seperti proses foto–pempolimeran boleh digunakan dalam penyediaan membran untuk sesuatu biosensor dan ia dapat menangani masalah kehilangan enzim dari pada membran biosensor yang nipis. Kata kunci: Bahan hidrogel metakrilat; foto–pempolimeran; serapan air; hidrofilik; hidrofobik; enzim; larut resap Methacrylate hydrogel materials prepared by photopolymerisation were used as matrices for urease enzyme immobilization for the purpose of assessing their possible application as biosensor membranes for urea. The hydrophilic/hydrophobic properties of the hydrogel were manipulated by changing the composition of the monomers 2–hydroxyl ethyl methacrylate (HEMA) and methyl methacrylate (MMA). Studies on enzyme immobilization included immobilization by photocuring, enzyme leaching and acitivity evaluation. Three types of polymeric membranes were studied, i.e. two of them were prepared by photocuring (fotoHEMA, i.e. 100% HEMA and MH28 with MMA : HEMA ratio = 2 : 8) and polymer poly(2-hydroxylethyl methacrylate) (pHEMA), which can be obtained commercially and used for comparison purposes. The results indicated that urease was not deactivated even exposed to ultra–violet light during photopolymerisation. The use of the MMA : HEMA composition and photocuring process is able to produce membrane of the desired hydrophilicity/ hydrophobicity and reduced the leaching of the enzyme compared with commercially available pHEMA. The most suitable methacrylate hydrogel material for urease immobilization for biosensor membrane was polymer MH28 that had water absorption <8% within an hour. This hydrogel material was also observed to retain higher activity for the immobilized urease when compared with the other hydrogel materials investigated. This work has demonstrated that a simple method such as photocuring can be used in the fabrication of a biosensor membrane and the problem of enzyme leaching can be prevented from a thin biosensor membrane. Key words: Methacrylic hydrogel materials; photo–polymerisation; water aborption; hydrophilic; hydrophobic; enzyme; leaching