1,720,985 research outputs found
Alcohol Dehydrogenase Biosensor based on Poly (Aniline)-Poly (Vinylsulfonate) modified electrode and enhancement effect of CA2+ ions on the Electrocatalytic oxidation of NADH at Poly(Aniline)-Poly (Vinylsulfonate) and Poly (Aniline)-Poly (Styrenesulfonate) modified Electrodes
No full textA membrane enzyme electrode sensitive towards ethanol was fabricated based on poly(aniline)-poly(vinylsulfonate) modified electrodes. Using the membrane electrode design and by varying the physical parameters, we established that the membrane enzyme electrode current response was consistent with a reversible enzyme kinetic model. Under conditions in which the product concentration was negligible within the enzyme layer of the enzyme electrode, the substrate-dependent current response could be described using a coupled reaction-diffusion model based on irreversible enzyme kinetics. This is the first report on the use of poly(aniline) modified electrodes as amperoraetric biosensors for the detection of ethanol. In the second part of this work, we investigated the enhancement of steady-state current towards NADH at poly(aniline)-poly(vinylsulfonate) and poly(aniline)- poly(styrenesulfonate) modified electrodes in the presence of calcium ions, using electrochemical methods, ^'P NMR and kinetic modelling. We observed reversible binding between Ca^^ and poly(aniline)-poly(vinylsulfonate) from cyclic voltammetry and steady- state experiments. The enhancement of electrocatalytic current towards NADH in the presence of between 20 and 40 mM Ca^^ were about 12 and 27 times for thin films of poly(aniline)-poly(vinylsulfonate) and poly(aniline)-poly(styrenesulfonate), respectively. This enhancement effect of Ca^^ ions on the electrocatalytic oxidation of NADH at poly(aniline) modified electrodes was much greater than those observed by workers using other mediators. From kinetic modelling of the experimental data, we found that the enhancement effect of Ca^^ ions was due to a large change in the polymer binding affinity for NADH or partitioning of NADH into the polymer film. The binding energy gain was estimated to be about 14 kJ m o f' in the presence of 25 mM Ca^^. This was confirmed by measurements using solid state ^'P NMR which indicated that NADH accumulated in the polymer film only in the presence of Ca^^.</p
Immobilisation of enzymes on poly(aniline)-poly(anion) composite films. Preparation of bioanodes for biofuel cell applications
No full textImmobilisation of enzymes is important for applications such as biosensors or biofuel cells. A poly(histidine) tag had been introduced on the C terminus of a lactate dehydrogenase enzyme. This mutant enzyme was then immobilised onto poly(aniline) (PANi)-poly(anion) composite films, PANi-poly(vinylsulfonate) (PVS) or PANi-poly(acrylate) (PAA). The NADH produced by the immobilised enzyme in the presence of ?-nicotinamide adenine dinucleotide (NAD+) and lactate is oxidised at the poly(aniline)-coated electrode at 0.05 to 0.1 V vs. saturated calomel electrode (SCE) at 35 °C
Electrochemical syntheses of highly ordered macroporous conducting polymers grown around self-assembled colloidal templates
No full textThree-dimensional highly ordered macroporous conducting polymer films were prepared using a self-assembled colloidal template based on poly(styrene) latex spheres. Poly(pyrrole), poly(aniline) and poly(bithiophene) were polymerised electrochemically and the polymer grown through the interstitial spaces between poly(styrene) latex spheres (0.5 mum or 0.75 mum in diameter) self-assembled in a close-packed array on gold substrates. The latex sphere template was subsequently removed by dissolution in toluene. Regular pore sizes and interconnected channels within the conducting polymer films were evident from scanning electron microscopy studies. The pore sizes for the conducting polymers studied were related to the dimensions of poly(styrene) spheres used as the template. Evidence for shrinkage of the structure was found for some polymers studied
A method for the determination of enzyme mass loading on an electrode surface through radioisotope labelling
No full textA direct method has been developed for the quantitation of the amount of immobilised enzymes on biosensor surfaces. This quantity is of key importance in establishing the activity, kinetics and optimal immobilisation conditions in the construction of both amperometric and optical biosensors. Recombinant L-lactate dehydrogenase incorporating both a biosynthetically introduced radiolabel, H-3-leucine, and a hexahistidine peptide tag was immobilised on a poly(aniline) composite film and then quantitated by liquid scintillation counting. It was found that enzyme mass loading was proportional to the concentration of LDH in solution, and also depended on the morphology of the composite film. The LDH mass loading on the composite film doubled when a surface cysteine containing variant was used, possibly due to the covalent attachment of the cysteine to the diiminoquinoid rings of the poly(aniline)
The design of dehydrogenase enzymes for use in a biofuel cell: the role of genetically introduced peptide tags in enzyme immobilization on electrodes
No full textThe immobilization of the mutants of L-lactate dehydrogenase (LDH) on poly(aniline) (PANi) composite films has been investigated. Mutants possessing peptide tags of varying charge and nucleophilicity were created to probe the nature of the interaction between the protein and PANi. These results are significant for the development of a 'generic' approach to the immobilization of enzymes and other proteins
Oxidation of NADH produced by a lactate dehydrogenase immobilised on poly(aniline)-poly(anion) composite films
No full textThe immobilisation of enzymes is important for applications in bioelectrochemistry such as biosensors or biofuel cells. In this paper we report the immobilisation of lactate dehydrogenase (LDH) on poly(aniline)–poly(acrylate) [PANi–PAA] and poly(aniline)–poly(vinylsulfonate) [PANi–PVS] composite films. Two genetically engineered forms of LDH (E.C.1.1.1.27) from Bacillus stearothermophilus, one with a poly(histidine) tag on the C-Terminus (LDH-CHis) the other with a poly(histidine) tag on the N-terminus (LDH-NHis), together with the wild type enzyme (WT-LDH) were studied. The LDH-CHis and LDH-NHis both have better affinity for the poly(aniline)–poly(anion) composite films than the WT-LDH. The immobilised LDH reduces the coenzyme NAD+ to NADH and oxidises the substrate, -lactate, to pyruvate. The NADH produced is then oxidised at the poly(aniline)–poly(anion) composite films. The effects of buffer concentration, temperature, NAD+ concentration, enzyme immobilisation conditions, film thickness and electrode rotation rate on the catalytic current were all investigated
The effect of calcium ions on the electrocatalytic oxidation of NADH by poly(aniline)-poly(vinylsulfonate) and poly(aniline)-poly(styrenesulfonate) modified electrodes
No full textAddition of Ca2+ ions increases the electrocatalytic response towards NADH at poly( aniline) poly(vinylsulfonate) and poly( aniline) poly( styrenesulfonate) modified electrodes by up to 12 and 25 times, respectively. This enhancement effect is reproducible and reversible. Measurement of the amount of charge passed through the polymer film in the presence of Ca2+ indicates a correlation between the total amount of Ca2+ incorporated within the polymer film and the magnitude of the enhancement of the electrocatalytic current for NADH oxidation at the polymer film. In addition, P-31-NMR studies show that the amount of NADH bound within the poly( aniline) film is increased significantly when Ca2+ is present. These results suggest that the enhancement of the NADH oxidation current at poly( aniline) modified electrodes caused by Ca2+ is due to an increase in the partition of NADH into the polymer film and/or an increase in the binding affinity of the polymer for NADH
Immobilisation of lactate dehydrogenase on poly(aniline)-poly(acrylate) and poly(aniline)-poly-(vinyl sulphonate) films for use in a lactate biosensor
No full textThe immobilisation of enzymes on an electrode surface, in such a manner that they retain both substrate specificity and high levels of catalytic activity, is of great importance in bioelectrochemistry. This includes areas such as the development of enzyme-catalysed fuel cell electrodes, biosensors and other biotechnological applications. We have investigated the catalytic activity of hexahistidine tagged variants of lactate dehydrogenase (EC 1.1.1.27) from the thermophile Bacillus stearothermophilus both in solution and when immobilised on poly(aniline)-poly(acrylate) (PANi-PAA) or poly(aniline)-poly(vinyl sulphonate) (PANi-PVS) composite films. Both the C- and N-terminally tagged enzymes are readily immobilised on the modified electrode and catalyse the conversion of lactate and NAD+ to pyruvate and NADH. The NADH that is generated can be readily oxidised at the PANi-modified electrode surface.In solution, the activity of the C-tagged enzyme (LDH-CHis) was some 30% less that of the wild-type under comparable conditions, whereas the N-tagged enzyme was found to possess essentially the same activity as the wild-type. However, when the enzymes were immobilised on PANi-PAA and PANi-PVS the C-tagged enzyme films showed a higher NADH-dependent current than the wild-type LDH whilst the N-tagged enzyme had the highest of the three. In addition, the C-tagged enzyme film appeared more stable than the wild-type LDH-PANi film. A novel immobilisation chemistry of the enzyme is proposed to account for these observations
Planar electrochromotography and electromembrane protein separation using nanoporous alumina materials
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Development of electroanalytical methods for studying microbes
No full textThis thesis explores the utility of electrochemical biosensors for studying and detecting microbes. It consists of five chapters: a mini-review and four independent experimental works. Chapter 1 reviews the current state of novel biosensing techniques for ultrasensitive detection of microbes with highly promising application for disease diagnosis. Because of their remarkable specificity, sensitivity and response time, biosensors often present potential and powerful tools for quantitative and accurate detection of microbes. Recent advancements in nanotechnology, transduction system and genetic engineering provide various strategies to improve the detection performance of biosensors. In this chapter, I summarize concise and comprehensive lists of the recent bacteria and virus biosensors, and their analytical performance. In addition, I also include a brief summary of state-of-the-art molecular technologies for the detection of microbes.
In Chapter 2, a sensitive and specific electrochemical membrane-based nanobiosensor is reported for quantitative and label-free detection of Escherichia coli cells and analysis of the viable but nonculturable state of E. coli cells that remain mostly undetected using conventional methods. The sensing mechanism depends on the blocking of nanochannels of an alumina-modified platinum wire electrode coated with a layer of anti-E. coli polyclonal antibody (isotype: IgG), upon the formation of immunocomplexes at the nanoporous alumina membrane. The resulting obstacle to diffusive mass transfer of a neutral redox probe, ferrocenemethanol, toward the underlying platinum electrode decreases the Faradaic current response of the nanobiosensor, measured using cyclic voltammetry. Experimental parameters including loading amount of antibody and pH are optimized. The membrane-based nanobiosensor gives a low limit of detection of 22 cfu mL-1 over a wide linear working range from 10 to 106 cfu mL-1 (R2 = 0.999). The nanobiosensor is specific toward E. coli with negligible cross reactivity to two other Gram-negative bacteria, Serratia marcescens and Salmonella typhimurium. Relative standard deviation for triplicate analysis of 2.5 % indicates good reproducibility. Differentiation of live, viable-but-non-culturable and dead E. coli cells are performed by monitoring of the bacterial enzyme catalytic activity using ferrocenemethanol as the alternative electron acceptor to oxygen, in the presence of glucose. In Chapter 3, the same design of membrane-based nanobiosensor is employed for ultrasensitive detection of dengue type 2 virus (DENV-2). Anti-DENV-2 monoclonal antibody (clone 3H5, isotype IgG) is used as the biorecognition probe in this work. The stepwise construction of nanobiosensor and detection of DENV-2 are characterized using differential pulse voltammetry. A low limit of detection of 1 pfu mL-1 with linear working range from 1 to 103 pfu mL-1 (R2 = 0.976) can be achieved by the nanobiosensor. The nanobiosensor is specific toward DENV-2 with minimal cross reaction with other non-specific viruses such as Chikungunya virus, West Nile virus, and dengue type 3 virus. Relative standard deviation for triplicate analysis of 5.9 % reveals reasonably useful level of reproducibility. Additionally, I demonstrate the direct quantification of DENV-2 load in whole mosquito vector, Aedes aegypti using the nanobiosensor.
Chapter 4 reports the real-time monitoring of filamentous bacteriophage M13 infection of E. coli cells using an impedimetric microbial biosensor constructed from a gold electrode covalently coated with a self-assembled monolayer of anti-E. coli polyclonal antibody. After phage infection, damage to the lipopolysaccharide layer on the outer membrane surface of E. coli cells causes changes to its morphology and surface charge, resulting in the aggregation of anionic redox probe, ferri/ferrocyanide at the electrode surface and thereby increases the electron-transfer rate. The consequent decrease in electron-transfer resistance in the presence of phage is monitored using the electrochemical impedance spectroscopy. The filamentous phage-bacterium interaction, which is hardly observable using the conventional microscopic methods, is detected within 5 h using this impedimetric microbial sensor, thus demonstrates its superior performance in terms of analysis time, ease and reduced reliance on the labeling steps during in-situ monitoring of phage infection process.
In Chapter 5, an impedimetric cell-based biosensor is fabricated from a poly-L-lysine-modified screen-printed carbon electrode to monitor the real-time DENV-2 infection of surface-immobilized baby hamster kidney fibroblast cells. Based on the described platform, DENV-2 induced cytopathic or cytopathogenic effects (degenerative morphological change, detachment, membrane degradation and death of host cells), which are indicated by a drastic decrease in impedance signal response, can be detected within ~ 35 hours post infection. A parameter that describes the kinetics of cytopathogenesis, CIT50 (time taken for 50 % decrease in impedance signal response) reveals an inverse linear relationship to the logarithm of virus titer. It is also reported that CIT50 values are delayed by 31.5 h for each order of magnitude decrease in multiplicity of infection. Therefore, the virus titer of given samples can be determined based on the measurement of impedance signal response and analysis of CIT50. Finally, the concluding remarks of these experimental works and the future outlook for biosensing techniques are included in the conclusion section.DOCTOR OF PHILOSOPHY (SPMS
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