Staff publications - Cranfield Health

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Open Access
Ascorbate enhances iron uptake into intestinal cells through formation of a FeCl3-ascorbate complex
(Elsevier Science B.V., Amsterdam., 2010-11-15T00:00:00Z) Thumser, Alfred E.; Rashed, Aswir Abd; Sharp, Paul A.; Lodge, John K.
It has been well documented that ascorbate enhances iron uptake, with a proposed mechanism based on reduction to the more absorbable ferrous form. We have performed a study on the effects of ascorbate on ferric iron uptake in the human epithelial Caco-2 cell-line. Ascorbate increased uptake in a concentration- dependent manner with a significant difference between iron uptake and reduction. Uptake kinetics are characteristic of a non-essential activator and the formation of an Fe3+-ascorbate complex. This investigation provides evidence that ascorbate enhances the apical uptake of ferric iron into Caco-2 cells through the formation of a Fe3+-ascorbate complex. (C) 2010 Elsevier Ltd. All rights reserved.
Open Access
Catalytic molecularly imprinted polymer membranes: Development of the biomimetic sensor for phenols detection
(Elsevier Science B.V., Amsterdam., 2010-02-05T00:00:00Z) Sergeyeva, T. A.; Slinchenko, O. A.; Gorbach, L. A.; Matyushov, V. F.; Brovko, O. O.; Piletsky, Sergey A.; Sergeeva, L. M.; Elska, G. V.
Portable biomimetic sensor devices for the express control of phenols content in water were developed. The synthetic binding sites mimicking active site of the enzyme tyrosinase were formed in the structure of free-standing molecularly imprinted polymer membranes. Molecularly imprinted polymer membranes with the catalytic activity were obtained by co-polymerization of the complex Cu (II)–catechol–urocanic acid ethyl ester with (tri)ethyleneglycoldimethacrylate, and oligourethaneacrylate. Addition of the elastic component oligourethaneacrylate provided formation of the highly cross-linked polymer with the catalytic activity in a form of thin, flexible, and mechanically stable membrane. High accessibility of the artificial catalytic sites for the interaction with the analyzed phenol molecules was achieved due to addition of linear polymer (polyethyleneglycol Mw 20,000) to the initial monomer mixture before the polymerization. As a result, typical semi-interpenetrating polymer networks (semi-IPNs) were formed. The cross-linked component of the semi-IPN was represented by the highly cross-linked catalytic molecularly imprinted polymer, while the linear one was represented by polyethyleneglycol Mw 20,000. Extraction of the linear polymer from the fully formed semi-IPN resulted in formation of large pores in the membranes’ structure. Concentration of phenols in the analyzed samples was detected using universal portable device oxymeter with the oxygen electrode in a close contact with the catalytic molecularly imprinted polymer membrane as a transducer. The detection limit of phenols detection using the developed sensor system based on polymers–biomimics with the optimized composition comprised 0.063 mM, while the linear range of the sensor comprised 0.063–1 mM. The working characteristics of the portable sensor devices were investigated. Storage stability of sensor systems at room temperature comprised 12 months (87%). As compared to traditional methods of phenols detection the developed sensor system is characterized by simplicity of operation, compactness, and
Open Access
Characterization of Langmuir-Blodgett films of a calix[8]arene and sensing properties towards volatile organic vapors
(Elsevier Science B.V., Amsterdam., 2010-07-15T00:00:00Z) Capan, R.; Ozbek, Z.; Goktas, H.; Sen, S.; Ince, F. G.; Ozel, M. E.; Stanciu, G. A.; Davis, Frank
Within this article, we report the characterization and organic vapor sensing properties of Langmuir-Blodgett (LB) thin films of calix[8]arenes. Surface pressure-area isotherms show that very stable monolayers are formed at the air- water interface. The LB film could be deposited onto different substrates which allowed the films to be characterized by UV, quartz crystal microbalance (QCM), surface plasmon resonance (SPR) and atomic force microscopy (AFM). The results indicate that good quality, uniform LB films can be prepared with transfer ratios of over 0.95. QCM results showed that the deposited mass of calix[8]arene monolayer onto a quartz crystal decreased from 693 to 204 ng as the number of layers is increased. AFM studies showed a smooth, and void free surface morphology with a rms value of 1.202 nm. The sensing abilities of this LB film towards the development of room temperature organic vapor sensing devices are also studied. Responses of the LB films to various vapors are fast, large, and reversible. It was found that the obtained LB film is significantly more sensitive to chloroform than other vapors. It can be concluded that this molecule could have a potential application in the research area of room temperature vapor sensing devices. (C) 2010 Elsevier B.V. All rights reserved.
Open Access
Comparison of different bead-beating RNA extraction strategies: An optimized method for filamentous fungi
(Elsevier Science B.V., Amsterdam., 2012-03-01T00:00:00Z) Leite, Goncalo M.; Magan, Naresh; Medina-Vayá, Ángel
Molecular studies, especially in relation to the activity of secondary metabolite gene clusters, require the ability to extract good quality RNA from fungal biomass. This is often hindered by the cell wall structure and endogenous RNase activity in filamentous fungi. There is thus a need for rapid methods for the extraction of good quality RNA for use in microarrays and for quantitative PCR assays. The objective of this study was to examine the use of different systems for the high throughput method to extract intact RNA from filamentous fungi. Two bead beating systems with different motion patterns and speed capacities were tested in the development of the extraction protocol. They were evaluated based on the total RNA yield and overall RNA quality. The high speed bead beating with glass beads associated with an automated purification method gave more than three times higher total RNA yields with less than a quarter of the amount of mycelium required. Furthermore the integrity and overall quality was conserved, with RNA Quality Indicator (RQI) numbers consistently >7.5. This method also reduced cross contamination risks and kept RNA handling to a minimum while still being capable of multiple sample processing, reducing the time required to obtain RNA from filamentous fungi.
Open Access
Comparison of water activity and temperature impacts on growth of Fusarium langsethiae strains from northern Europe on oat-based media
(Elsevier Science B.V., Amsterdam., 2010-09-01T00:00:00Z) Medina-Vayá, Ángel; Magan, Naresh
This study has examined the effect of water activity (aw, 0.995-0.90) and temperature (10-37Â°C) on the lag phases prior to growth, growth rates and used models to develop two dimensional profiles for optimum and marginal conditions for two strains of Fusarium langsethiae from four northern European countries (UK, Norway, Sweden, and Finland) on an oat-based medium. Results showed that the optimum aw for growth was at 0.98-0.995 and 25Â°C. The limit for growth of the strains was at 0.92-0.93 aw with minima of 10Â°C. No growth occurred at 37Â°C. The lag phases prior to growth were lowest under optimum conditions and extended to >10days at marginal conditions. Statistical analyses of intra and inter- strain differences in terms of both lag phases prior to growth and growth rates were not statistically significant. However, aw and temperature were statistically significant factors. Two dimensional profiles for strains from each country of origin were built to identify optimum and marginal conditions for F. langsethiae for the first time. These environmental profiles will be beneficial for improving the ecological knowledge of this species which is able to produce trichothecene mycotoxins in a range of temperate cereals.
Open Access
Computational modeling and molecular imprinting for the development of acrylic polymers with high affinity for bile salts
(Elsevier Science B.V., Amsterdam., 2010-02-05T00:00:00Z) Yañez, Fernando; Chianella, Iva; Piletsky, Sergey A.; Concheiro, Angel; Alvarez-Lorenzo, Carmen
This work has focused on the rational development of polymers capable of acting as traps of bile salts. Computational modeling was combined with molecular imprinting technology to obtain networks with high affinity for cholate salts in aqueous medium. The screening of a virtual library of 18 monomers, which are commonly used for imprinted networks, identified N-(3-aminopropyl)-methacrylate hydrochloride (APMA·HCl), N,N-diethylamino ethyl methacrylate (DEAEM) and ethyleneglycol methacrylate phosphate (EGMP) as suitable functional monomers with medium-to-high affinity for cholic acid. The polymers were prepared with a fix cholic acid:functional monomer mole ratio of 1:4, but with various cross- linking densities. Compared to polymers prepared without functional monomer, both imprinted and non-imprinted microparticles showed a high capability to remove sodium cholate from aqueous medium. High affinity APMA-based particles even resembled the performance of commercially available cholesterol-lowering granules. The imprinting effect was evident in most of the networks prepared, showing that computational modeling and molecular imprinting can act synergistically to improve the performance of certain polymers. Nevertheless, both the imprinted and non-imprinted networks prepared with the best monomer (APMA·HCl) identified by the modeling demonstrated such high affinity for the template that the imprinting effect was less important. The fitting of adsorption isotherms to the Freundlich model indicated that, in general, imprinting increases the population of high affinity binding sites, except when the affinity of the functional monomer for the target molecule is already very high. The cross-linking density was confirmed as a key parameter that determines the accessibility of the binding points to sodium cholate. Materials prepared with 9% mol APMA and 91% mol cross-linker showed enough affinity to achieve binding levels of up to 0.4 mmol g−1 (i.e., 170 mg g−1) under flow (1 mL min−1) of 0.2 mM sodium cholate s
Open Access
Controlled release of the herbicide simazine from computationally designed molecularly imprinted polymers
(Elsevier Science B.V., Amsterdam., 2005-11-02T00:00:00Z) Piletska, Elena V.; Turner, Nicholas W.; Turner, Anthony P. F.; Piletsky, Sergey A.
The present study describes the development of materials suitable for environmental control of algae. Molecularly imprinted polymers (MIPs) were used as simazine carriers able to provide the controlled release of simazine into water. Three polymers were designed using computational modelling. The selection of methacrylic acid (MA) and hydroxyethyl methacrylate (HEM) as functional monomers was based on results obtained using the Leapfrog™ algorithm. A cross- linked polymer made without functional monomers was also prepared and tested as a control. The release of simazine from all three polymers was studied. It was shown that the presence of functional monomers is important for polymer affinity and for controlled release of herbicide. The speed of release of herbicide correlated with the calculated binding characteristics. The high-affinity MA- based polymer released 2% and the low-affinity HEM-based polymer released 27% of the template over 25 days. The kinetics of simazine release from HEM-based polymer show that total saturation of an aqueous environment could be achieved over a period of 3 weeks and this corresponds to the maximal simazine solubility in water. The possible use of these types of polymers in the field of controlled release is discusse
Open Access
Current trends in explosive detection techniques
(Elsevier Science B.V., Amsterdam., 2012-01-15T00:00:00Z) Caygill, J. S.; Davis, Frank; Higson, Seamus P. J.
The detection of explosives and explosive-related compounds has become a heightened priority in recent years for homeland security and counter-terrorism applications. There has been a huge increase in research within this area- through both the development of new, innovative detection approaches and the improvement of existing techniques. Developments for miniaturisation, portability, field-ruggedisation and improvements in stand-off distances, selectivity and sensitivity have been necessary to develop and improve techniques. This review provides a consolidation of information relating to recent advances in explosive detection techniques without being limited to one specific research area or explosive type. The focus of this review will be towards advances in the last 5 years, with the reader being referred to earlier reviews where appropriate.
Open Access
Deposition of functionalized polymer layers in surface plasmon resonance immunosensors by in-situ polymerization in the evanescent wave field
(Elsevier Science B.V., Amsterdam., 2009-01-01T00:00:00Z) Chegel, Vladimir; Whitcombe, Michael J.; Turner, Nicholas W.; Piletsky, Sergey A.
Traditionally, the integration of sensing gel layers in surface plasmon resonance (SPR) is achieved via “bulk” methods, such as precipitation, spin- coating or in-situ polymerization onto the total surface of the sensor chip, combined with covalent attachment of the antibody or receptor to the gel surface. This is wasteful in terms of materials as the sensing only occurs at the point of resonance interrogated by the laser. By isolating the sensing materials (antibodies, enzymes, aptamers, polymers, MIPs, etc.) to this exact spot a more efficient use of these recognition elements will be achieved. Here we present a method for the in-situ formation of polymers, using the energy of the evanescent wave field on the surface of an SPR device, specifically localized at the point of interrogation. Using the photo-initiator couple of methylene blue (sensitizing dye) and sodium p-toluenesulfinate (reducing agent) we polymerized a mixture of N,N-methylene-bis-acrylamide and methacrylic acid in water at the focal point of SPR. No polymerization was seen in solution or at any other sites on the sensor surface. Varying parameters such as monomer concentration and exposure time allowed precise control over the polymer thickness (from 20–200 nm). Standard coupling with 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide was used for the immobilization of protein G which was used to bind IgG in a typical biosensor format. This model system demonstrated the characteristic performance for this type of immunosensor, validating our deposition m
Open Access
Detection of Salmonella typhimurium using an electrochemical immunosensor.
(Elsevier Science B.V., Amsterdam., 2009-04-15T00:00:00Z) Salam, Faridah; Tothill, Ibtisam E.
An electrochemical immunosensor based on screen-printed gold working electrode with onboard carbon counter and silver–silver chloride pseudo-reference electrode for Salmonella typhimurium detection is described in this paper. Monoclonal anti-S. typhimurium antibody was immobilized using physical and covalent immobilization via amine coupling of carboxymethyldextran on the surface of the gold working electrode. A direct sandwich enzyme-linked immunosorbent assays (ELISA) format was then developed and optimized using a polyclonal anti-Salmonella antibodies conjugated to horseradish peroxidase (HRP) as the enzyme label. 3,3′,5,5′-Tetramethylbenzidine dihydrochloride (TMB)/H2O2 was used as the enzyme mediator/substrate system. Electrochemical detection was conducted using chronoamperometry at −200 mV vs. onboard screen-printed Ag–AgCl pseudo-reference electrode. The applied potential was selected through the study of the electrochemical behaviour of bare gold electrode with TMB–H2O2–IgG–HRP system. S. typhimurium detection of 5 × 103 cells ml−1 and 20 cells ml−1 was achieved respectively for physical and covalent antibody immobilization. The developed sensor was then compared to a commercial ELISA kit and a chromogenic agar plating method for meat samples analysis. The sensor format shows a promising technology for simple and sensitive detection system for Salmonella contamination. Rapid detection of Salmonella is a key to the prevention and identification of problems related
Open Access
Development of a sensitive detection method of cancer biomarkers in human serum (75%) using a quartz crystal microbalance sensor and nanoparticles amplification system
(Elsevier Science B.V., Amsterdam., 2010-06-30T00:00:00Z) Uludag, Yildiz; Tothill, Ibtisam E.
A simple and sensitive sensor method for cancer biomarkers [prostate specific antigen (PSA) and PSA-alpha 1-antichymotrypsin (ACT) complex] analysis was developed, to be applied directly with human serum (75%) by using antibody modified quartz crystal microbalance sensor and nanoparticles amplification system. A QCM sensor chip consisting of two sensing array enabling the measurement of an active and control binding events simultaneously on the sensor surface was used in this work. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions before applying to serum samples. Extensive interference to the QCM signal was observed upon the analysis of serum. Different buffer systems were then formulated and tested for the reduction of the non-specific binding of sera proteins on the sensor surface. A PBS buffer containing 200 mu g mL(-1) BSA, 0.5 M NaCI, 500 mu g mL(- 1) dextran and 0.5% Tween 20, was then selected which eliminated the interfering signal by 98% and enabled the biomarker detection assay to be performed in 75% human serum. By using Au nanoparticles to enhance the QCM sensor signal, a limit of detection of 0.29 ng mL(-1) PSA and PSA-ACT complex (in 75% serum) with a linear dynamic detection range up to 150 ng mL(-1) was obtained. With the achieved detection limit in serum samples, the developed QCM assay shows a promising technology for cancer biomarker analysis in patient samples. (C) 2010 Elsevier B.V. All rights reserved.
Open Access
Development of an electrochemical immunosensor for aflatoxin M1 in milk with focus on matrix interference.
(Elsevier Science B.V., Amsterdam., 2009-04-15T00:00:00Z) Parker, Charlie O.; Tothill, Ibtisam E.
A simple sensor method was developed for aflatoxin M1 analysis to be applied directly with milk by using antibody modified screen-printed carbon working electrode with carbon counter and silver–silver chloride pseudo-reference electrode. A competitive ELISA assay format was constructed on the surface of the working electrode using 3,3,5′,5′-tetramethylbenzidine dihyrochloride (TMB)/ H2O2 electrochemical detection scheme with horseradish peroxidase (HRP) as the enzyme label. The performance of the assay and the sensor was optimised and characterised in pure buffer conditions before applying to milk samples. Extensive interference to the electroanalytical signal was observed upon the analysis of milk. Through a series of chemical fractionations of the milk, and testing the electrochemical properties of the fractions, the interference was attributed to whey proteins with focus towards α-lactalbumin. A simple pre- treatment technique of incorporating 18 mM calcium chloride, in the form of Dulbucco's PBS, in a 1:1 ratio to the milk sample or standards and also to the washing buffer stabilised the whey proteins in solution and eliminate the interfering signal. The resulting immunosensor was interference free and achieved a limit of detection of 39 ng l−1 with a linear dynamic detection range up to 1000 ng l−1. The developed immunosensor method was compared to a commercial ELISA kit and an in-house HPLC method. The immunsensor was comparable, in term of sensitivity, but vastly superior in term of portability and cost therefore a key instrument for the detection of aflatoxin M1 at the source of the con
Open Access
Development of surface chemistry for surface plasmon resonance based sensors for the detection of proteins and DNA molecules
(Elsevier Science B.V., Amsterdam., 2012-01-27T00:00:00Z) Altintas, Zeynep; Uludag, Yildiz; Gurbuz, Yasar; Tothill, Ibtisam E.
The immobilisation of biological recognition elements onto a sensor chip surface is a crucial step for the construction of biosensors. While some of the optical biosensors utilise silicon dioxide as the sensor surface, most of the biosensor surfaces are coated with metals for transduction of the signal. Biological recognition elements such as proteins can be adsorbed spontaneously on metal or silicon dioxide substrates but this may denature the molecule and can result in either activity reduction or loss. Self assembled monolayers (SAMs) provide an effective method to protect the biological recognition elements from the sensor surface, thereby providing ligand immobilisation that enables the repeated binding and regeneration cycles to be performed without losing the immobilised ligand, as well as additionally helping to minimise non-specific adsorption. Therefore, in this study different surface chemistries were constructed on SPR sensor chips to investigate protein and DNA immobilisation on Au surfaces. A cysteamine surface and 1%, 10% and 100% mercaptoundecanoic acid (MUDA) coatings with or without dendrimer modification were utilised to construct the various sensor surfaces used in this investigation. A higher response was obtained for NeutrAvidin immobilisation on dendrimer modified surfaces compared to MUDA and cysteamine layers, however, protein or DNA capture responses on the immobilised NeutrAvidin did not show a similar higher response when dendrimer modified surfaces were used.
Open Access
Development of the custom polymeric materials specific for aflatoxin B1 and ochratoxin A for application with the ToxiQuant T1 sensor tool
(Elsevier Science B.V., Amsterdam., 2010-04-16T00:00:00Z) Piletska, Elena V.; Karim, K.; Coker, R.; Piletsky, Sergey A.
Two polymers were computationally designed with affinity to two of the most abundant mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) for application in the ToxiQuant T1 System. The principle of quantification of AFB1 and OTA using the ToxiQuant T1 instrument comprised of a fluorimetric analysis of mycotoxins adsorbed on the polymer upon exposure to UV light. High affinity of the developed resins allowed the adsorption of both toxins as discrete bands on the top of the cartridge with detection limit as low as 1 ng quantity of mycotoxins.
Open Access
Different sample treatment approaches for the analysis of T-2 and HT-2 toxins from oats-based media
(Elsevier Science B.V., Amsterdam., 2010-08-01T00:00:00Z) Medina-Vayá, Ángel; Valle-Algarra, Francisco M.; Jimenez, Misericordia; Magan, Naresh
A LC-DAD method is proposed for the determination of the T-2 and HT-2 toxins in cultures of Fusarium langsethiae in oat-based and other in vitro media. Test media consisted of freshly prepared milled oats to which T-2 and HT-2 toxin stock solutions were added. Different mixtures of extraction solvent (acetonitrile:water and methanol water), extraction times (30', 60' or 90') and drying methods were investigated. Results showed that extraction with methanol: water (80:20, v/v) for 90 min, drying with N-2 and subsequent analysis by LC-DAD was the fastest and most user friendly method for detecting HT-2 and T-2 toxins production by F. langsethiae strains grown on oat-based media at levels of 0.459 and 0.508 mg of toxin/kg of agar, respectively. The proposed method was used to investigate toxin production of 6 F. langsethiae strains from northern Europe and provided clear chromatograms with no interfering peaks in media with and without glycerol as water activity modifier. (C) 2010 Elsevier B.V. All rights reserved.
Open Access
DNA-based biosensor platforms for the detection of TP53 mutation
(Elsevier Science B.V., Amsterdam., 2012-07-05T00:00:00Z) Altintas, Zeynep; Tothill, Ibtisam E.
A DNA-based assay for the detection of one-point mutation in TP53 gene, responsible for lung cancer, was developed using a surface plasmon resonance (SPR) and a quartz crystal microbalance (QCM) biosensor systems. Amine coupling was employed for the immobilization of NeutrAvidin on thiol-derivatized surface to capture the biotinylated target sequence. Two targets sequences and one control DNA sequence were investigated including, a fully complementary (30 mer), one-point mutation and a non-complimentary DNA using hybridization with a detection probe strand (27 mer). The most appropriate surface coating was also examined for both sensor platforms with hybridization and single nucleotide polymorphism (SNP) detection efficiency were then compare. A 0.03-2 μM concentration range of detection probe was detected using the SPR and QCM sensors on wild and mutant type target surfaces. The linear regression analysis was performed for both sensors resulting in a R 2 value for the SPR assay of 0.985 and 0.993 for perfect and mismatch reaction and of 0.978 and 0.976 for the QCM assay, respectively. The obtained results demonstrate that the used approach represents a very promising future method for the detection of one-point mutation in genetic-based health problem with highly sensitive, specific, and real-time analysis
Open Access
Effect of fenpropimorph, prochloraz and tebuconazole on growth and production of T-2 and HT-2 toxins by Fusarium langsethiae in oat-based medium.
(Elsevier Science B.V., Amsterdam., 2011-12-15T00:00:00Z) Mateo, Eva M.; Valle-Algarra, Francisco M.; Mateo, R.; Jimenez, Misericordia; Magan, Naresh
Fusarium langsethiae has been isolated from infected cereals in central and northern Europe where it has been identified in the last decade as the main species involved in the occurrence of high levels of T-2 and HT-2 toxins, mainly in oats. The efficacy of three fungicides (prochloraz, tebuconazole, fenpropimorph) for controlling growth of two strains of F. langsethiae isolated from oats was examined at 0.96 and 0.98 a(w) at 15, 20 and 25 °C on oat-based media. The concentrations necessary for 50 and 90% growth inhibition (ED₅₀ and ED₉₀ values) were determined. The effect on the trichothecene type A mycotoxins T-2 and HT-2 was also determined. Without fungicides both strains grew faster at 0.98 than at 0.96 a(w) and the influence of temperature on growth rates was 25>20>15 °C. Prochloraz and tebuconazole were more effective than fenpropimorph against F. langsethiae. Strain, temperature and type of fungicide significantly influenced the ED₅₀ and ED₉₀ values for growth. The concentration ranges under different environmental conditions were: prochloraz (0.03-0.1 and 0.3-1.5), tebuconazole (0.06-0.9 and 1.3-8.2), and fenpropimorph (22-59 and 125-215 mg l⁻¹). Production of T-2 and HT-2 toxins was influenced by temperature, a(w), type of fungicide and dose. Levels of T-2 were usually higher than those of HT- 2 under the same conditions. The biosynthesis of T-2 toxin increased after 10 day incubation, but was reduced with decreasing temperature and increasing fungicide dose. At 0.98 a(w) T-2 levels increased in cultures containing fenpropimorph while at 0.96 a(w) the toxin concentrations increased in response to the other two fungicides. Low doses of prochloraz or tebuconazole enhanced toxin production when compared with untreated cultures for strain 2004-59 at 0.96 a(w) and 20-25 °C. HT-2 was hardly detectable in the treatments with prochloraz or tebuconazole at 0.98 a(w). This is the first study on the effect of these anti-fungal compounds on control of growth of F. langsethiae and on production of T-2 and HT-2 toxi
Open Access
Effect of solute stress and temperature on growth rate and TRI5 gene expression using real time RT-PCR in Fusarium graminearum from Spanish wheat
(Elsevier Science B.V., Amsterdam., 2010-06-15T00:00:00Z) Marin, P.; Jurado, M.; Magan, Naresh; Vazquez, C.; Gonzalez-Jaen, M. T.
The objective of this work was to study the effect of ecophysiological factors on trichothecene gene expression and growth in Fusarium graminearum. The effect of non-ionic solute water potentials and temperature was examined on in vitro mycelial growth rates and on expression of the TRI5 gene, involved in trichothecene biosynthesis, quantified by real time RT-PCR. This study showed optimal values of 25 degrees C and -2.8 MPa (0.982 a(w)) for growth. Marginal temperatures such as 15 degrees C and 30-35 degrees C, particularly in combination with water potentials below -2.8 MPa, drastically reduced growth. The expression of TRI5 was reasonably constant although some induction was observed between 20 and 30 degrees C, the most favourable temperatures for growth, depending on the water potential imposed, particularly at -7.0 MPa. A temporal kinetic experiment at 25 degrees C examined the effect of ionic solute stress on TRI5 gene expression and growth rate. The results indicated independence of growth rate and TRIS expression, as the fungal biomass increased with time while the gene expression remained constant. This suggested that favourable conditions for growth will result in higher trichothecene production, and that toxin production would always accompany the colonization process at a steady rate while the conditions for growth are permissive. Quantification of key biosynthetic toxin genes by real time RT-PCR was shown to be a valuable tool to gain knowledge of the ecophysiological basis for trichothecene biosynthesis and enable better control strategies to be developed during the life cycle of this important mycotoxigenic pathogen of cereals. (C) 2010 Elsevier B.V. All rights reserved.
Open Access
An electrochemical sensor based on carboxymethylated dextran modified gold surface for ochratoxin A analysis
(Elsevier Science B.V., Amsterdam., 2011-08-10T00:00:00Z) Heurich, Meike; Kadir, Mohamad Kamal Abdul; Tothill, Ibtisam E.
A disposable electrochemical immunosensor method was developed for ochratoxin A analysis to be applied for wine samples by using a screen-printed gold working electrode with carbon counter and silver/silver chloride pseudo-reference electrode. An indirect competitive enzyme-linked immunosorbent assay (ELISA) format was constructed by immobilising ochratoxin A conjugate using passive adsorption or covalent immobilisation via amine coupling to a carboxymethylated dextran (CMD) hydrogel on the gold working electrode. Electrochemical detection was performed using 3,3′,5,5′-tetramethylbenzidine dihyrochloride (TMB) and hydrogen peroxide with horse radish peroxidase (HRP) as the enzyme label. Chronoamperometry at -150mV vs. onboard screen-printed Ag-AgCl pseudo-reference electrode was then used to detect the generated signal. The performance of the assay and the sensor was optimised and characterised in pure buffer conditions before applying to wine samples. The resulting immunosensor for ochratoxin A in buffer achieved a limit of detection of 0.5μgL-1 with a linear dynamic detection range of 0.1-10μgL-1 for passive adsorption of the toxin conjugate. While for covalent immobilisation through CMD-modified gold electrode, a limit of detection of 0.05μgL-1 was achieved with a linear dynamic detection range of 0.01-100μgL-1. The CMD-modified gold immunosensor was then evaluated in spiked and affinity purified wine samples achieving a detection limit comparable to buffer solutions (0.05μgL-
Open Access
Electrochromic effects from a simple commercial polymer membrane.
(Elsevier Science B.V., Amsterdam., 2007-08-01T00:00:00Z) Davis, Frank; Law, Karen A.; Bridge, Kerry; Higson, Seamus P. J.
A simple commercial polyester polymer membrane has been found to exhibit an intense electrochromic effect. Most polymers which undergo electrochromic effects contain either transition metals or extensive conjugated systems. We have found that a simple commercial polyester membrane when coated with gold and polarised to −4 V (versus Ag) in aprotic organic solvents displays an electrochromic colour change from a colourless to an intense red stat