Browsing by Author "Turner, Anthony P. F."

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    Amperometric biosensor for formic acid in air
    (Elsevier, 2000-11-01) Sandström, K. J. Mattias; Newman, Jeffrey D.; Sunesson, Anna-Lena; Levina, Jan-Olof; Turner, Anthony P. F.
    The possibility of developing a simple, inexpensive and specific personal passive ”real-time” air sampler based on biosensor technology was investigated. Formic acid was used as a model substance. The sensor is based on the enzymatic reaction between formic acid and formate dehydrogenase with NAD+ as a cofactor and Meldola’s blue as mediator. An effective way to immobilise the enzyme, cofactor and Meldola’s blue on screen-printed electrodes was found to be in a mixture of glycerol and phosphate buffer covered with a gas-permeable membrane. When the sensor was introduced into an atmosphere containing formic acid, it gave a distinct and rapid amperometric response.
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    An amperometric enzyme electrode for the detection of L-lactate
    (Cranfield University, 1997-06) Selkirk, Jane Yvonne; Turner, Anthony P. F.; Saini, S.
    The main tasks of this thesis were to evaluate a number of amperometric enzyme electrode chemistries for the selective and sensitive detection of L-lactate, and apply mass fabrication technologies to reproducibly manufacture sensors in a controllable manner. The sensors studied were based on the use of lactate oxidase with a range of modified-carbon electrodes. Noble metals, hexacyanoferrate (111) or Prussian Blue were used to modify carbon electrodes for the electro-catalytic determination of hydrogen peroxide, the product of the reaction of lactate oxidase with L-lactate. Tetrathiafulvalene was employed as an artificial mediator between the enzyme and the electrode. Polypyrrole was tested as a means of immobilising lactate oxidase and to achieve direct charge transfer to the underlying carbon electrode. The characteristics of the sensor responses to hydrogen peroxide, L-lactate and ascorbate were compared, in relation to the electrochemical electrode area. From this investigation, it was confirmed that screen-printed electrodes were more reproducible to manufacture than hand-fabricated electrodes. For screen-printed rhodinised-carbon electrodes, an operating potential of +400 mV (SCE) was selected. Interference from ascorbic acid and sensitivity to hydrogen peroxide were deten-nined to be 26 gA. mM-' CM-2 and 27 gA. mM-'. CM-2, respectively. Screen-printed carbon electrodes modified with platinum, rhodium or palladium were selected for further investigation. Rhodium on carbon performed the best in ten-ns of sensitivity and selectivity at low potentials, and different formations of rhodium-carbon complexes were studied. Although rhodium electroplated onto carbon screen-printed electrodes was examined, printing inks made from a preformed powder of rhodium on carbon-graphite proved to be the preferred route of electrode fabrication. Screen printing, ink-jet printing and Cavro solution deposition were employed to fabricate the amperometric enzyme electrodes. These sensors were composed of rhodinised carbon and lactate oxidase in a water-based electrode ink with a protective outer membrane layer. Each stage, from ink preparation to membrane composition, was developed empirically. The sensitivity, stability and reproducibility of the working electrode was improved by altering it to a homogeneous ink, consisting of carbon graphite powder, rhodinised carbon powder (5% Rh by weight), hydroxyethyl cellulose (2% w/v) and lactate oxidase in the weight ratio of 2: 8: 18: 1. A layer of cellulose acetate (2% w/v in a 1: 1 solution of acetone to cyclohexanone) and an outer coating of a polyurethane called Pellethane (I% to 4% w/v in dimethyl formarnide and tetrahydrofuran) improved the selectivity, sensitivity and detection range of the sensor, allowing it to operate in physiological solutions with reduced passivation from protein adsorption. The sensor design was revised to allow its passage through a catheter and operation within a blood vessel; it was manufactured on flexible material using screen printing and Cavro solution deposition techniques. These miniature sensors, with a working surface of 0.5 x 15 mm, were capable of linearly measuring lactate up to 3 mM in buffer solutions with an average sensitivity of 44.8 nA. mM-1 L- actate. To test the sensor operation in physiological solutions, a flow injection system was employed. A planar three-electrode card used in this system was manufactured using screen printing and Cavro solution deposition techniques. L-lactate concentrations up to 6.4 mM were sensitively and, after minor correction, accurately determined in undiluted plasma and whole blood samples. This thesis has therefore made progress toward mass fabricating an amperometric enzyme electrode device suitable for the deten-nination of L-lactate concentrations in vitro.
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    Approaches to allergy detection using aptasensors
    (Taylor and Francis, 2007) Brys, Emile; Tombelli, Sara; Minunni, Maria E.; Mascini, Marco; Turner, Anthony P. F.
    1. Immunoglobulins and IgE Antibodies are glycoprotein molecules which are produced by plasma cells in response to an immunogen (1). They belong to a class of spherical proteins called globulins and are hence known as immunoglobulins (Igs). Their primary function is to mediate the host immune response by binding to antigens.
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    Aptamers for biosensors
    (Cranfield University and University of Firenze., 2008) Bini, Alessandra; Mascini, Marco; Turner, Anthony P. F.
    Aptamers are single-stranded DNA or RNA molecules isolated in vitro by a selection and amplification method. Aptamers bind with high specificity and affinity to a wide range of target molecules, with dissociation constant comparable to antibodies. In this work aptamers were employed as a new kind of bio-recognition element in affinity biosensors for the detection of clinically relevant proteins in heterogeneous assay, using Piezoelectric Quartz Crystal Microbalance and Surface Plasmon Resonance as transducers. The work was focused on two case studies, i.e. the Thrombin-binding aptamer and the aptamer against C-Reactive Protein. From an analytical point of view, the work was devoted to the optimisation of the analytical performance of a piezoelectric and an optical aptasensor for Thrombin and C-Reactive Protein detection, respectively. Efforts towards the application of these aptasensors in complex matrices, such as human plasma and serum, were also undertaken, in order to demonstrate the wide applicability of aptamers, as an alternative to antibodies. In this work, the possibility of introducing a computationally-assisted method to study aptamer-protein interaction and aptamer selection was also evaluated. For this purpose, the Thrombin-binding aptamer was chosen as a model and a retrospective docking study was performed by comparing the affinity of mutated sequences for thrombin with that of the Thrombin-binding aptamer, on the basis of a computationally-derived binding score. Finally, the reliability of computational results was tested by experimental measurements. For this purpose, the Thrombin-binding aptamer and other mutated sequences, selected on the basis of their binding score, were employed for the development of optical biosensors and the resulting analytical performances were compared. Even if further studies should be carried out in order to validate the proposed computational approach to aptamer selection, this work can have a significant impact on future aptamers selection for sensors and diagnostics.
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    Artificial muscles powered by glucose
    (Wiley, 2019-06-19) Mashayekhi Mazar, Fariba; Martinez, Jose G.; Tyagi, Manav; Alijanianzadeh, Mahdi; Turner, Anthony P. F.; Jager, Edwin W. H.
    Untethered actuation is important for robotic devices to achieve autonomous motion, which is typically enabled by using batteries. Using enzymes to provide the required electrical charge is particularly interesting as it will enable direct harvesting of fuel components from a surrounding fluid. Here, a soft artificial muscle is presented, which uses the biofuel glucose in the presence of oxygen. Glucose oxidase and laccase enzymes integrated in the actuator catalytically convert glucose and oxygen into electrical power that in turn is converted into movement by the electroactive polymer polypyrrole causing the actuator to bend. The integrated bioelectrode pair shows a maximum open‐circuit voltage of 0.70 ± 0.04 V at room temperature and a maximum power density of 0.27 µW cm−2 at 0.50 V, sufficient to drive an external polypyrrole‐based trilayer artificial muscle. Next, the enzymes are fully integrated into the artificial muscle, resulting in an autonomously powered actuator that can bend reversibly in both directions driven by glucose and O2 only. This autonomously powered artificial muscle can be of great interest for soft (micro‐)robotics and implantable or ingestible medical devices manoeuvring throughout the body, for devices in regenerative medicine, wearables, and environmental monitoring devices operating autonomously in aqueous environments
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    Bi-functional sulphonate-coupled reduced graphene oxide as an efficient dopant for a conducting polymer with enhanced electrochemical performance
    (Royal Society of Chemistry, 2020-06-25) Meng, Lingyin; Dagsgård, Frida; Turner, Anthony P. F.; Mak, Wing Cheung
    The rapidly emerging field of organic bioelectronics has witnessed the wide use of conducting polymers (CPs) to fabricate advanced chemically modified electrodes (CMEs) for biosensors and biomedical devices. The electrochemical performance of the CPs in such devices is closely related to the quality and physiochemical nature of the dopants. A bi-functional graphene oxide derivative with high reduction degree and negatively-charged sulphonate functionality, i.e. sulphonate-coupled reduced graphene oxide (S-RGO), was developed and used as an efficient dopant for a CP with enhanced electrochemical performance. The S-RGO was synthesised via a facile one-pot hydrothermal reaction using 4-hydrazinobenzosulphonic acid (4-HBS) as reductant and sulphonate precursor simultaneously. The resulting S-RGO possesses high aqueous dispersion stability (more than 6 months), high electrical conductivity (1493.0 S m−1) and sulphonate functionality. Due to these specific properties, S-RGO demonstrated improved electropolymerisation efficiency for poly(3,4-ethylenedioxythiophene) (PEDOT) proving an effective dopant for the preparation of a PEDOT:S-RGO film (5 mC) with faster polymerisation time (37 s) compared to the conventional 2D dopants GO (PEDOT:GO, 129 s) and RGO (PEDOT:RGO, 66 s). The resulting PEDOT:S-RGO appeared as a homogenous film with uniformly distributed S-RGO dopant, low equivalent series resistance and low charge transfer resistance. Moreover, the electrochemical transduction performance of the PEDOT:S-RGO interface was evaluated with 4 different analytes, including ferric/ferrocyanide redox probe, dopamine, nicotinamide adenine dinucleotide and hydrogen peroxide. As a result of the synergistic effect of S-RGO and PEDOT, the PEDOT:S-RGO demonstrated enhanced electrochemical performance with respect to faster electrode kinetics (smaller ΔEp), ∼2 and ∼4 times increased current responses, and lower peak potentials compared to PEDOT:GO and PEDOT:RGO. This bi-functional S-RGO dopant combined the advantages of conventional GO and RGO to deliver sulphonate functionality and high conductivity for the preparation of advanced PEDOT interface with improved electrochemical performance, that could potentially be applied for applications in electrochemical sensors, biosensors and bioelectronic devices
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    Biomimetic sensors for HbA1c
    (Cranfield University, 2010-07) Biela, Anna; Piletsky, Sergey A.; Turner, Anthony P. F.
    Diabetes mellitus is a growing health problem worldwide. Suitable long-term control and management of this disease are enabled by determination of glycated haemoglobin (HbA1c) in blood. The results are given as %HbA1c of total haemoglobin. Presently available tests vary in cost and convenience and there is an identified need to introduce improved equipment for self-monitoring. This dissertation focuses on fast and straightforward detection of glycated haemoglobin (HbA1c) using cyclic voltammetry and chronoamperometry. Haemoglobin was determined by monitoring its reaction with potassium ferricyanide on screen printed electrodes at an oxidative potential +500 mV. A working electrode was modified with carbon nanotubes to enhance electron transfer. A calibration curve was linear in a range from 0.83 to 83 mg/mL. Another innovative approach to detecting haemoglobin using its enzymatic activity was also developed. Detection of haemoglobin was performed with hydroquinone and hydrogen peroxide on screen printed electrodes at a potential -400 mV in a Flow Injection Analysis system (FIA). Cont/d.
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    Biosensor and bioelectrocatalysis studies of enzymes immobilized on graphite electrode materials
    (1987-05) Schneider, B. H.; Higgins, I. J.; Turner, Anthony P. F.
    The immobilization of glucose oxidase and lipoamide dehydrogenase on graphite electrodes was studied for their application in mediated bioelectrochemical systems. These are techniques where the electrons involved in an enzymatic redox reaction are either supplied by, or given to an electrode from the enzyme. In the biosensor mode, where a current is measured due to product oxidation, the immobilized enzyme may be used to monitor its substrate, for example a biosensor for glucose based on the enzyme glucose oxidase. In the bioelectrosynthesis mode, when a cathodic potential is applied to the electrode, electrons may be transferred to the enzyme via a mediator thereby driving a thermodynamically difficult reaction, such as the reduction of the coenzyme NAD+ . A glucose sensor based on glucose oxidase immobilized onto graphite foil electrodes which had been doped with the mediator dimethylferrocene was found to produce both a linear and rapid response to changes in glucose concentration in solution. The kinetics of this particular system were studied in depth in order to explain its behaviour. Results showed that the porous nature of the electrode material resulted in immobilization of the enzyme throughout a number of layers• Consequently, diffusion of glucose into the electrode became the rate limiting process at low glucose concentrations, which resulted in linear calibration curves. This was characterized by a substrate modulus, which was found to be sufficiently large to predict diffusional limitations. From this the effective rate of glucose diffusion within the electrode was estimated, and found to be up to 5 times smaller than that in free solution. The rapid response of the sensor, on the other hand, was due to the absence of any mass transfer limitations external to the electrode surface. When lipoamide dehydrogenase was employed in the same system it was found that complications arose due to coupling of NADH oxidation with the adsorbed mediator. By employing a less anodic potential of +150 mV vs. Ag/AgCl at NADH concentrations below 1 mM the detection of enzymatic oxidation of the coenzyme was achieved. Enzymatic reduction of NAD+ was achieved using lipoamide dehydrogenase immobilized onto graphite felt electrodes. This reaction was mediated by methyl viologen, with the enzyme-modified electrode used to reduce the mediator at a potential of -0.7 V ( vs. SCE ). In this fashion the reduced form of methyl viologen was recycled, thereby driving the reduction of NAD . By coupling this system to lactate dehydrogenase it was possible to effect the reduction of pyruvate.
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    Biotin–specific synthetic receptors prepared using molecular imprinti
    (Elsevier Science B.V., Amsterdam., 2004-02-16T00:00:00Z) Piletska, Elena V.; Piletsky, Sergey A.; Karim, K.; Terpetschnig, E.; Turner, Anthony P. F.
    The composition of new molecularly imprinted polymers (MIPs) specific for biotin was optimised using molecular modelling software. Three functional monomers: methacrylic acid (MAA), 2-(trifluoromethyl)acrylic acid (TFAA) and 2-acrylamido- 2-methyl-propanesulfonic acid (AMPSA), which demonstrated the highest binding scores with biotin, were tested on their ability to generate specific binding sites. The imprinted polymers were photografted to the surface of polystyrene microspheres in water. The affinity of the synthetic "receptor" sites was evaluated in binding experiments using horseradish peroxidase-labelled biotin. A good correlation was found between the modelling results and the performance of the materials in the template rebinding study. The dissociation constants for all MIPs were 1.4-16.8 nM, which is sufficient for most analytical applications where biotin is used as a label.
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    Conducting polymer-reinforced laser-irradiated graphene as a heterostructured 3D transducer for flexible skin patch biosensors
    (American Chemical Society, 2021-11-02) Meng, Lingyin; Turner, Anthony P. F.; Mak, Wing Cheung
    Flexible skin patch biosensors are promising for the noninvasive determination of physiological parameters in perspiration for fitness and health monitoring. However, various prerequisites need to be met for the development of such biosensors, including the creation of a flexible conductive platform, bending/contact stability, fast electrochemical kinetics, and immobilization of biomolecules. Here, we describe a conducting polymer-reinforced laser-irradiated graphene (LIG) network as a heterostructured three-dimensional (3D) transducer for flexible skin patch biosensors. LIG with a hierarchically interconnected graphene structure is geometrically patterned on polyimide via localized laser irradiation as a flexible conductive platform, which is then reinforced by poly(3,4-ethylenedioxythiophene) (PEDOT) as a conductive binder (PEDOT/LIG) with improved structural/contact stability and electrochemical kinetics. The interconnected pores of the reinforced PEDOT/LIG function as a 3D host matrix for high loading of “artificial” (Prussian blue, PB) and natural enzymes (lactate oxidase, LOx), forming a compact and heterostructured 3D transducer (LOx/PB-PEDOT/LIG) for lactate biosensing with excellent sensitivity (11.83 μA mM–1). We demonstrated the fabrication of flexible skin patch biosensors comprising a custom-built integrated three-electrode system achieve amperometric detection of lactate in artificial sweat over a wide physiological linear range of 0–18 mM. The advantage of this facile and versatile transducer is further illustrated by the development of a folded 3D wristband lactate biosensor and a dual channel biosensors for simultaneous monitoring of lactate and glucose. This innovative design concept of a heterostructured transducer for flexible biosensors combined with a versatile fabrication approach could potentially drive the development of new wearable and skin-mountable biosensors for monitoring various physiological parameters in biofluids for noninvasive fitness and health management.
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    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
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    Custom synthesis of molecular imprinted polymers for biotechnological application: preparation of a polymer selective for tylosin
    (Elsevier Science B.V., Amsterdam., 2004-02-16T00:00:00Z) Piletsky, Sergey A.; Piletska, Elena V.; Karim, K.; Foster, G.; Legge, C.; Turner, Anthony P. F.
    A molecularly imprinted polymer (MIP) selective for tylosin was designed and synthesised using a computational method (MIP “dialling”). In re-binding experiments the MIP demonstrated high affinity for tylosin in aqueous solutions and in organic solvents. The synthesised polymer was tested for re-binding with the template and related metabolites such as tylactone, narbomycin and picromycin. The HPLC analysis showed that the computationally designed polymer is specific and capable of separating the template from its structural analogues. The MIP was capable of recovering tylosin from broth samples. The polymer capacity for tylosin was estimated as 6.4 mg/g for MIP, which was suitable for practical application and tylosin recovery from broth samples. Among the advantages of this was the possibility to adsorb tylosin from a complex media with easy removal of oils and other impurities which are present in significant quantities, which can create problems for its chromatographic purification procedure. The MIP “dialling” procedure can have a general significance for the fast preparation of specific adsorbents for biotechnological appli
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    Design of molecularly imprinted polymers for sensors and solid phase extraction
    (Cranfield University, 2002-04) Subrahmanyam, Sreenath; Turner, Anthony P. F.; Piletsky, Sergey A.
    This thesis presents broadly the applications of molecularly imprinted polymers in sensors and solid phase extraction. Sensors for creatine and creatinine have been reported using a novel method of rational design of molecularly imprinted polymers (MIPs), and solid phase extraction of aflatoxin-B 1 has also been described in the thesis. A method for the selective detection of creataine and creatinine is reported in this thesis, which is based on the reaction between polymerised hemithioacetal, formed by allyl mercaptan, o-phthalic aldehyde, and primary amine leading to the formation of fluorescent isoindole complex. This method was demonstrated for the detection of creatine using creatine-imprinted MIPs. Since MIPs created using traditional methods were unable to differentiate between creatine and creatinine, a new approach to the rational design of a MIP selective for creatinine was developed using computer simulation. A virtual library of functional monomers was assigned and screened against the target molecule, creatinine, using molecular modeling software. The monomers giving the highest binding score were further tested using simulated annealing in order to mimic the complexation of the functional monomers with template in the monomer mixture. The result of this simulation gave an optimised MIP composition. The computationally designed polymer demonstrated superior selectivity in comparison to the polymer prepared using traditional approach, a detection limit of 25 μM and good stability. The 'Bite-and- Switch' approach combined with molecular imprinting can be used for the design of assays and sensors, selective for amino containing substances. MEP for the selective binding properties for aflatoxin-B 1 was prepared using the computational approach. The results obtained demonstrate that the MISPE offers a simple, convenient and a rapid methodology for solid phase extraction of aflatoxin-B 1 even at very low concentrations of 2 ppb. The commercially available C-18 cartridges were able to recover only about 52% of aflatoxin-B 1 at concentrations of 2 ppb when compared with almost complete recovery by the MIP. We have proved here that, MIPs as a solid phase extraction materials offer important and practical advantages with respect to other solid phase extraction methodologies.
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    Detection of TP53 mutation using a portable Surface Plasmon Resonance DNA-based biosensor
    (Elsevier, 2005-02-02T17:36:22Z) Jiang, Tieshan; Minunni, Maria E.; Wilson, P. K.; Zhang, Jian; Turner, Anthony P. F.; Mascini, Marco
    A DNA-based Surface Plasmon Resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETATM SPR-EVM-BT, from Texas Instruments. A direct immobilization procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridization reactions between the immobilized probe and a short sequence (26 mer) complementary, non-complementary and one point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR) amplified real samples, DNA extracted from both normal, wild type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base and hence it appears to be a strong candidate technique for the detection of gene mutation.
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    Development of a rapid immunoassay for human pathogenic markers
    (Cranfield University, 2006-09) Lawton, Nicola Jane; Turner, Anthony P. F.; Morgan, Sarah; Burgess, Paul J.
    A demand exists for a fast, sensitive, reliable and economical test for pyogenic sepsis that provides a “real time” bed-side assessment. Detection of significant intra-abdominal sepsis can be particularly problematic in the ICU setting and in patients with multi-system organ failure. Lysozyme, first reported by Fleming (1922), is a bacteriolytic enzyme released during phagocytosis. Previous studies have shown significant correlation between lysozyme levels and the presence of intra-abdominal abscess in both animals and humans. A method which determines and quantifies lysozyme as part of an assessment of an acutely ill patient in whom major sepsis is suspected; would significantly aid diagnosis and prescription of the most effective form of treatment. To date measurement of lysozyme has been by turbidometry, with consequent poor sensitivity and reliability. Other methods of assay include fluorescence, radial immunodiffussion and enzyme linked immunosorbent assay (ELISA). This study reports a modified ELISA technique which provides a cheap, sensitive and reliable method of lysozyme determination, producing results in <100 minutes. The ELISA has been tested with ~200 clinical samples provided by the patients at the Great Western Hospital, Swindon. Two ELIFA techniques were also developed for lysozyme and E.coli detection. These techniques also provide a cheap and rapid alternative to the more traditional immunoassays. Results from the ELIFA and mini-ELIFA were obtained qualitatively after only 10 minutes. An SPR detection technique was also devised. The BIAcore 3000 was used to create a biosensor for serum lysozyme using an artificial receptor in the form of an aptamer. This system was tested with clinical serum samples, is reusable and took <80 minutes to immobilise a ligand on a blank sensor and analyse a serum sample for lysozyme. Although further research and development is required on the mini-ELIFA and lysozyme biosensor, the ELISA detection system may prove a useful tool in the diagnosis of sepsis in critically ill patients.
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    Electrochemical Immunoassay for Free Prostate Specific Antigen (f-PSA) Using Magnetic Beads
    (Wiley InterScience, 2008) Sarkar, P.; Ghosh, D.; Bhattacharyay, D.; Setford, S. J.; Turner, Anthony P. F.
    Prostate specific antigen (PSA) is a prominent marker for the prostate carcinoma. It is found in human blood in free (f-PSA) and complex forms. These two forms together are called total PSA (t-PSA). Estimation of both forms is essential to predict malignancy. In this study we report a unique and effective technique of electrochemical detection of f-PSA using magnetic beads on a three-electrode screen-printed sensor. A magnetic bead enzyme linked immunosorbent assay (ELISA) was performed in a cuvette. Following the immunoassay, magnetic beads were recovered by a magnetic concentrator and transferred on the working electrode of the 3-electrode assembly. The amperometric response, a measure of the amount of residual enzyme activity on the beads and hence the concentration of analyte in solution, was determined by addition of enzyme substrate. The device has a detection limit of <0.1 ng mL-1 f-PSA and a linear range of 0 to 1 ng mL-1 f-PSA.
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    Evaluation of an FIA Operated Amperometric Bacterial Biosensor, Based on Pseudomonas Putida F1 for the Detection of Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX)
    (Taylor and Francis, 2005) Rasinger, Josef D.; Marrazza, Giovanna; Briganti, Fabrizio; Scozzafava, Andrea; Mascini, Marco; Turner, Anthony P. F.
    Recently, the development and optimization of a flow injection analysis (FIA) operated bacterial biosensor based on the aerobic catabolism of Pseudomonas putida ML2 was reported in the literature (Lanyon et al. 2004, 2005). By adapting information from these reports, we investigated whether operating parameters and procedures of the benzene biosensor could be directly applied to a new system based on a different bacterial strain for the detection of the whole benzene, toluene, ethylbenzene, and xylenes range. Cells of the investigated bacterial strain, Pseudomonas putida F1, were immobilized between two cellulose acetate membranes and fixed onto a Clark dissolved oxygen electrode. The P. putida F1 aerobically degrades benzene, toluene, and ethylbenzene (BTE) (Cho et al. 2000). The BTE biosensor in kinetic mode FIA displayed a linear range of 0.02-0.14 mM benzene (response time: 5 min, base-line recovery time: 15 min), 0.05-0.2 mM toluene (response time: 8 min, baseline recovery time: 20 min), and 0.1-0.2 mM ethylbenzene (response time: 12 min, baseline recovery time: 30 min), respectively. Due to the differences in sensitivity, response, and baseline recovery times for BTE, it was possible to differentiate each compound in mixtures of these volatile organic compounds (VOCs). No response for xylenes could be obtained since they cannot be completely metabolized by this bacterial strain. However, it was reported that the range of compounds degradable by P. putida F1 can possibly be expanded by cultivating the cells on different carbon sources (Choi et al. 2003). The sensor showed good intra- and interassay reproducibility, and all obtained results were comparable with those reported in the literature. The demonstrated reproducibility and the simplicity and ease of use as well as the portability for in situ measurements indicates that the biosensor could be suitable as a reliable initial warning device for elevated BTE levels in indoor and outdoor environments.
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    Fluorescence-based optical biosensors for clinical and environmental applications
    (Cranfield University, 1996-12) Psoma, Sotiria D.; Turner, Anthony P. F.
    The aim of this thesis was to investigate the feasibility of simultaneous utilisation of pH and oxygen-dependent fluorescent indicators for the development of a novel fibre-optical fluorescence-based bio sensor. This approach would be used to measure simultaneously changes in the two indicator species generated by a single enzyme-catalysed reaction in response to one analyte where both the indicators and the enzyme are immobilised in the same sol-gel matrix, and to offer more accurate and reliable results using this portable optical biosensor in the clinical and environmental fields. HPTS (1-hydroxypyrene-3,6,8-trisulfonic acid) and tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate, respectively, were used as the target fluorescent indicators; these two indicators had no cross sensitivity separate or in the same solution and well-separated emission bands at 510 nm and 610 mn, respectively. The catalytic oxidation of glucose by the enzyme glucose oxidase was initially investigated using the two indicators, and subsequently the same principle was applied in other biocatalysed oxidations such as of lactate, xanthine and phenol. Substrate concentration was assessed by simultaneously measuring two parameters: oxygen consumption, through the reduction of the fluorescence intensity of tris(2,2'-bipyridyl) ruthenium(II) chloride hexahydrate; and the production of acid, through pH changes affecting the fluorescence intensity of HPTS.A thorough spectroscopic study of the enzymatic oxidation of glucose was performed using glucose oxidase in solution in a cuvette, in the presence of both indicators. A number of combinations of wavelengths of the indicators for excitation and fluorescence were utilised in order to establish calibration curves with the optimum performance for glucose detection in the diabetic range. Similarly results were taken from the kinetic studies of lactate oxidase, xanthine oxidase and polyphenol oxidase for the detection of lactate and xanthine in blood and phenol in water at ppb-levels, using the above principle. The application and characterisation of immobilisation techniques for the fluorescence-based blood-glucose b iosenor were carried out. The advantages of the microcapsulation sol-gel method over conventional immobilisation techniques for application in an optical biosensor, were elucidated and this immobilisation technique was implemented for glucose and phenol detection. Finally, additional solution studies were conducted and used to evaluate the implementation and performance of the above method when used for the detection and measurement of glucose concentration in biological samples such as human serum.
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    A glucose sensor for fermentation monitoring
    (Cranfield University, 1987-10) Brooks, Steven; Turner, Anthony P. F.; Ashby, R. E; Clarke, D. J
    The evaluation, analysis and development of an oxygen-insensitive amperometric glucose biosensor and its application in microbial batch culture are described. The biosensor consisted of a graphite foil electrode modified with glucose oxidase and 1,1'-dimethylferrocene, and operated via mediated electron transfer from the enzyme to the electrode. Initial evaluations illustrated several operating characteristics which would be expected to cause problems in continuous monitoring applications, most notably sensor instability and a progressive increase in response time. The main underlying causes of these unfavorable characteristics were identified as enzyme loss, mediator loss and substrate diffusion limitation within the electrode. As a consequence of these insights, further development of the sensor was undertaken. A number of different electrode materials and enzyme immobilization techniques were tested, resulting in the development of a novel immobilization procedure using a hexadecylamine coating to bind 'the activated carbohydrate residues of periodate-oxidized glucose oxidase. This improved the sensor lifetime and response time under continuous operation. Strategies for the reliable application of the biosensor in fermentation monitoring were evaluated. In-line flow cell and in_§itu membrane probe approaches were considered, and the latter approach was preferred: Considerable attention was devoted to optimising the design of such probes. The best design accommodated a three electrode configuration with a multiple biosensor array. It was found necessary to allow for periodic on-line calibration within the aseptically operating probe. This configuration was successfully applied on-line to monitor glucose in batch cultures of Escherichia coli.
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    Home blood glucose biosensors: a commercial perspective
    (Elsevier, 2005-06-15) Newman, Jeffrey D.; Turner, Anthony P. F.
    Twenty years on from a review in the first issue of this journal, this contribution revisits glucose sensing for diabetes with an emphasis on commercial developments in the home blood glucose testing market. Following a brief introduction to the needs of people with diabetes, the review considers defining technologies that have enabled the introduction of commercial products and then reviews the products themselves. Drawing heavily on the performance of actual instruments and publicly available information from the companies themselves, this work is designed to complement more conventional reviews based on papers published in scholarly journals. It focuses on the commercial reality today and the products that we are likely to see in the near future.