Investigation of the effect of different glassy carbon materials on the performance of Prussian Blue based sensors for hydrogen peroxide

Abstract Three different kinds of glassy carbon (GC-R, GC-K, GC-G) were equally pretreated, further modified with electrochemically deposited Prussian Blue and used as sensors for hydrogen peroxide at an applied potential of À 50 mV (vs. Ag j AgCl). Their performance was evaluated with respect to the following parameters: the coverage and electrochemistry of the electrodeposited Prussian Blue, the sensitivity and the lower limit of detection for hydrogen peroxide, and the operational stability of the sensors. GC-R showed the best behavior concerning the surface coverage and the operational stability of the electrodeposited Prussian Blue. For this electrode the sensitivity for hydrogen peroxide (10 mM) was 0.25 A/M cm 2 and the detection limit was 0.1 mM. Scanning electron microscopy was used to study the surfaces of the three electrodes before and after the electrodeposition of Prussian Blue and to search for the reason for the three different behaviors between the different glassy carbon materials. The Prussian Blue modified GC-R was also used for the construction of a glucose biosensor based on immobilizing glucose oxidase in Nafion membranes on top of electrodeposited Prussian Blue layer. The operational stability of the glucose biosensors was studied in the flow injection mode at an applied potential of À 50 mV (vs. Ag j AgCl) and alternatively injecting standard solutions of hydrogen peroxide (10 mM) and glucose (1 mM) for 3 h. For the GC-R based biosensor a 2.8% decrease of the initial glucose response was observed

Amperometric biosensors of food analysis relevance: Emphasis on the neurotoxin beta-ODAP in grass pea

Enzyme-based amperometric biosensors have been developed and characterized in flow systems for a variety of analytes such as [beta]-N-oxalyl-[alfa],[beta]-diamionpropionic acid ([beta]-ODAP), L-glutamate, glucose, lactate, and sucrose, that have food analysis relevance. The principle of enzyme "wiring" using polymeric mediators poly(1-vinylimidazole) {(Os-4, 4' dimethylbipyridyl)2Cl3+/2+} and poly(4-vinlypyridine {Os(N,N'-dimethyl-2,2'-biimidazole)3}3+/2+ has been employed in most of the sensors developed, which resulted in the fabrication of sensitive, stable and reproducible biosensors with carbonaceous electrodes (spectrographic graphite and glassy carbon). Biosensor development, application and method validation for the analysis of the neurotoxin contained in Lathyrus sativus (grass pea), [beta]-ODAP, a causative agent for the disease called neurolathyrism has been emphasized in this thesis. The hydrogel based (the above polymers) bienzyme biosensor (glutamate oxidase and horseradish peroxidase) has been applied to real samples i.e. raw grass pea seed samples collected from lathyrism prone area of Ethiopia, and processed (solid-state fermentation) grass pea. Moreover, it has been successfully applied in the estimation of [beta]-ODAP degrading abilities of microbes isolated from cow rumen, which are adapted to grow on media containing [beta]-ODAP as the soul carbon source. The validation studies showed that the biosensors developed for this analyte are practical ones. In all these analyses, the biosensors were used as end column detection in a liquid chromatographic analysis set up. Additionally an attempt was made to replace the enzyme HRP, often used in conjunction with oxidases for the development of biosensors working in low potential regime for substrates of the oxidase, with the inorganic crystalline material Prussian Blue (PB). PB can be electrochemically deposited (from a mixture of ferric chloride and ferric hexacyanoferrate) on electrode surfaces and has catalytic activity for selective electrochemical reduction of hydrogen peroxide. Different glassy carbon materials and procedure for electrochemical deposition of PB have been investigated for its optimum performance. The stability problem of PB, especially at neutral and basic pH has been improved by adding tetrabutylammonium toluene-4-sulfonate (TTS) either in the electrolytic solution (in one of the electrodepostion steps) or in the carrier buffer during flow injection analysis. A fairly sensitive and stable biosensors could be developed for sucrose, Lactate, and [beta]-ODAP based on PB modified glassy carbon electrodes further modified by the respective oxidase

Prussian blue-glutamate oxidase modified glassy carbon electrode: A sensitive L-glutamate and beta-N-oxalyl-alpha-beta-diaminopropionic acid (beta-ODAP) sensor

A sensitive biosensor has been developed for the neurotoxin beta-N-oxalyl-alpha,beta-diaminopropionic acid (beta-ODAP) contained in the seeds of grass pea (Lathyrus sativus) and for L-glutamate based on glutamate oxidase (GlOx) and a Prussian blue (PB) modified glassy carbon (GC) electrode. The configuration of the system is so as to detect the hydrogen peroxide released during the enzymatic cycle at a low applied potential, -50 mV versus Ag vertical bar AgCl, in the flow injection mode. For this purpose GlOx was coupled to PB electrodeposited onto a glassy carbon electrode and stabilised by treatment with tetrabutylammonium toluene-4-sulfonate (TTS) during one of the steps in the electrodeposition. GlOx was cross-linked with glutaraldehyde (GA), bovine serum albumin (BSA) and Tween-20 on the surface of the PB modified GC electrodes. Addition of 0.01% and 0.001% polyethyleneimine (PEI) to the immobilisation mixture resulted in an enhancement of the response signal with about 35% and 62% for glutamate and P-ODAP, respectively, when using 0.01% PEI and with 164% and 200% for glutamate and P-ODAP, respectively, when using 0.001% PEI. The linear response range for P-ODAP was extended from 0.05-0.5 mM to 0.01-1 mM, when 0.001% PEI was used. However, a higher concentration of PEI, 0.1%, caused a decrease in the sensitivity of the biosensor. (c) 2005 Elsevier B.V. All rights reserved

Electrical wiring of pyranose oxidase with osmium redox polymers

Two different flexible osmium functionalised polymers were investigated for their ability to efficiently "wire" pyranose oxidase when immobilised on graphite electrodes. The two Os-polymers differ in redox potential and the length of the side chains, where the Os2+/3+-functionalities are located. The performance of the redox polymers as mediators for the enzyme was tested with glucose. As well as analytical characteristics, the optimum operational conditions for the biosensor were investigated for both types of redox polymers: poly(I-vinylimidazole)12[osmium (4,4'-dimethyl-2,2'-bipyridyl)(2)Cl-2](2+/+) (osmium redox polymer I) and poly(vinylpyridine)-[osmium-(N,N'-methylated-2,2'-biimidalzole)(3)](2+/3 +) complex (osmium redox polymer II). All results are given as a comparison of the two systems. (c) 2005 Elsevier B.V. All rights reserved

Isolation of obligate anaerobic rumen bacteria capable of degrading the neurotoxin beta-ODAP (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) as evaluated by a liquid chromatography/biosensor analysis system

Six pure strains of obligate anaerobes capable of degrading the toxin beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (beta-ODAP) contained in grass pea (Lathyrus sativus) have been isolated from cow rumen. The new isolates were identified as Megasphaera elsdenii (five different genotypes) and Clostridium bifermentans using 16S rDNA analysis. The beta-ODAP degrading efficiency of the isolates was evaluated by measuring the amount of beta-ODAP in the growth medium, which contained beta-ODAP as the only carbon source, before and after incubation with the microbes. The method of analysis was liquid chromatography employing bioelectrochemical detection. The biosensor is based on coimmobilising two enzymes, glutamate oxidase (GlOx) and horseradish peroxidase (HRP), on the end of a spectrographic graphite electrode. beta-ODAP is oxidised by GlOx to form H2O2, which in turn is bioelectrocatalytically reduced by HRP through a mediated reaction using a polymeric mediator incorporating Os2+/3+ functionalities rapidly shuttling electrons with the electrode-giving rise to the analytical signal. On the basis of this analysis system, the new isolates are capable of utilising beta-ODAP as sole carbon source to a maximum of 90-95% within 5 days with concomitant increase in cell protein. (c) 2005 Society of Chemical Industry

Fermentation of Seeds of Teff (Eragrostis teff), Grass-pea (Lathyrus sativus), and Their Mixtures: Aspects of Nutrition and Food Safety

Fermentation of pure teff (Eragrostis teff), pure grass-pea (Lathyrus sativus), and their mixtures, 9:1 and 8:2 (teff/grass-pea) has been done at two temperatures (room temperature and 35 C) in duplicate using the strains of Lactobacillus plantarum, for bacterial fermentation, and Aspergillus oryzae and Rhizopus oligosporus in succession for solid-state fungal fermentation as inocula. In addition, the natural or spontaneous and back-slopping methods of bacterial fermentation have been done on the above four substrate groups. The pH and essential amino acid profiles of the different fermentation processes were compared. The back-slopping in teff at a temperature of 35 C gave the sharpest pH drop. All fermentations done at 35 C showed a steeper slope in their pH versus time plot compared to their room temperature counterpart. Fungal fermentation gave an improved amino acid profile for the essential ones in all of the substrate groups, except in pure grass-pea. Fermented teff/grass-pea (8:2) in this fungal fermentation has been found to be quite comparable in essential amino acid profile to an ideal reference protein recommended for children of 2-5 years of age. None of the bacterial fermentations produced a net change in their essential amino acid profile in any of the substrate groups investigated. Solid state fungal fermentation on pure grass-pea using the fungal strains R. oligosporous and A. oryzae in succession has shown that the neurotoxin -N-oxalyl-,-diaminopropionic acid (-ODAP) in grass-pea has been removed by 80% on average for the high-toxin variety and by up to 97% for the low-toxin variety as determined by an improved chromatographic method with bioelectrochemical detection coupled on-line with refractive index detection

Prussian blue modified glassy carbon electrodes - Study on operational stability and its application as a sucrose biosensor

Stabilisation of electrochemically deposited Prussian blue (PB) films on glassy carbon (GC) electrodes has been investigated and an enhancement in the stability of the PB films is reported if the electrodes are treated with tetrabutyl ammonium toluene-4-sulfonate (TTS) in the electrochemical activation step following the electrodeposition. A multi-enzyme PB based biosensor for sucrose detection was made in order to demonstrate that PB films can be coupled with an oxidase system. A tri-enzyme system, comprising glucose oxidase, mutarotase and invertase, was crosslinked with glutaraldehyde and bovine albumin serum on the PB modified glassy carbon electrode. The deposited PB operated as an electrocatalyst for electrochemical reduction of hydrogen peroxide, the final product of the enzyme reaction sequence. The electrochemical response was studied using flow injection analysis for the determination of sucrose, glucose and H2O2. The optimal concentrations of the immobilisation mixture was standardised as 8 U of glucose oxidase, 8 U of mutarotase, 16 U of invertase, 0.5% glutaraldchyde (0.025 mul) and 0.5 % BSA (0.025 mg) in a final volume of 5 mul applied at the electrode surface (0.066 cm(2)). The biosensor exhibited a linear response for sucrose (4-800 muM), glucose (2-800 muM) and H2O2 (1-800 muM) and the detection limit was 4.5, 1.5 and 0.5 muM for sucrose, glucose and H2O2, respectively. The sample throughput was ca. 60 samples h(-1). An increase in the operational and storage stability of the sucrose biosensor was also noted when the PB modified electrodes were conditioned in phosphate buffer containing 0.05 M TTS during the preparation of the PB films. (C) 2004 Elsevier B.V. All rights reserved

Glutamate oxidase advances the selective bioanalytical detection of the neurotoxic amino acid beta-ODAP in grass pea: A decade of progress

The search for an enzyme as a reagent for selective bioanalytical detection of the neurotoxic amino acid, beta-N-oxalyl-L-,alpha,beta-diaminopropionic acid, beta-ODAP (found in grass pea, Lathyrus sativus) led to its redox catalytic reaction by glutamate oxidase (GluOx). Homogeneous kinetic studies and an immobilized GluOx reactor-based flow-injection assay were initially made for beta-ODAP with small immobilized GluOx/catalase glutamate-destroying prereactors. The method was applied to examine the toxin content in processed grass pea. The kinetics and the equilibrium of the thermal isomerization of beta-ODAP to the nontoxic isomer alpha-ODAP established that GluOx is specific to the neurotoxin. The first ever GluOx-based amperometric biosensor for liquid chromatography (LC) detection was reported in 1997. This biosensor coupled with a refractive index detector improved LC performance. The most recent work with GluOx resulted in MnO2-based screen-printed amperometric biosensor, with offline elimination of glutamate interference by glutamate decarboxylase. A single-shot chemiluminescent sensor developed for hydrogen peroxide is also proposed for beta-ODAP with GluOx application. This decade of progress resulted from studies that included four Ph.D. (Ethiopia, Sweden, Austria), four M.Sc. (Ethiopia, Sweden) and Licentiate (Sweden) theses projects, plus one collaborative project in Sweden. The advances in grass pea research may be regarded as a model north-south cooperation for research and education