Browsing by Author "Tothill, Ibtisam E."

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    Allelic variants of KLK2 gene predict presence of prostate cancer at biopsy
    (OMICS International, 2016-04-22) Nna, Emmanuel Okechukwu; Tothill, Ibtisam E.; Bailey, Tracey
    Objective: Several single nucleotide polymorphisms associated with prostate cancer risk have been reported in recent years. We evaluated polymorphisms in the human glandular kallikrein 2 (KLK2) genes because the protein product of this gene is known to be increased in prostate cancer. Materials and methods: Blood samples were collected from sixty patients who underwent prostate biopsy sectioning, and from their genomic DNA the SNPs in KLK2 gene were investigated by direct DNA sequencing. Another 138 archived prostate tissue sections were also evaluated using the TaqMan SNP genotyping assay. Results: Eighteen known SNPs were identified in the KLK2 gene. The SNPs were located in introns, coding exons and untranslated regions of the gene. Further analysis showed that two of the SNPs were associated with prostate disease. The T/T allele of rs198977 was significantly predictive of the presence of prostate cancer at biopsy and was also associated with high tumour grade. The A/A allele of rs2664155 was also significantly associated with the presence of benign hyperplasia at biopsy. Conclusion: Our results support previous reports of association of the rs198977 SNP with prostate cancer risk and also indicated a link with the disease phenotype. However, the second SNP (rs2664155) was more associated with benign hyperplasia than prostate cancer risk. The method of TaqMan SNP genotyping could be clinically useful in genetic screening and risk stratification of patients for prostate diseases.
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    Attenuation of quorum sensing using computationally designed polymers
    (Cranfield University, 2010-05) Stavroulakis, Georgios; Piletsky, Sergey A.; Tothill, Ibtisam E.; Piletsky, E.; Robinson, G.
    It is generally accepted that the majority of Gram-negative and Gram-positive bacteria communicate via production and sensing of small signal molecules, autoinducers. The ability of bacteria to sense their population density is termed quorum sensing (QS). Quorum sensing controls certain phenotypic traits, particularly virulence factors and biofilm formation. In this project a new solution for the attenuation of quorum sensing which involves selective sequestering of the signal molecules using rationally designed synthetic polymers was explored. Cont/d.
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    Biodesulfurization of dibenzothiophene by Shewanella putrefaciens NCIMB 8768
    (2007-06-01T00:00:00Z) Ansari, Farahnaz; Prayuenyong, P.; Tothill, Ibtisam E.
    The desulfurization ability of Shewanella putrefaciens strain NCIMB 8768 was studied and its activity profile was compared with the widely studied strain Rhodococcus erythropolis strain IGTS8. Dibenzothiophene (DBT) is a recalcitrant thiophenic component of fossil fuels especially among diesel blend stocks. DBT in basic salt medium (BSM) at a final concentration of 0.3, 0.6 and 0.9 mM was supplied to the microbes as the sole sulfur source. Experimental results showed that S. putrefaciens, similar to other biodesulfurization organisms, converted DBT to the end product 2-hydroxybiphenyl (HBP), as detected by the Gibbs assay and HPLC. Cells cultivated in medium containing 0.3 mM of DBT showed the highest desulfurization activity, with a maximum specific production rate 43.5 mmol/L of HBP.
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    Biodesulphurisation of coal
    (Cranfield University, 2001-07) Prayuenyong, P.; Tothill, Ibtisam E.
    The emission of sulphur oxides during the combustion of coal is one of the causes of an environmental problem known as acid rain. Biodesulphurisation technology applied as a method to remove sulphur before coal combustion was investigated in this work. The desulphurisation abilities of three specific bacterial strains including Rhodococcus erythropolis IGTS8, R. erythropolis X309 and Shewanella putrefaciens strain NCIMB 8768 have been evaluated. R. erythropolis IGTS8 and X309 were found to be able to remove both inorganic and organic sulphur from model compounds and coal samples. Their abilities to remove sulphur from benzothiophene were observed for the first time. A novel desulphurising bacterium, S. putrefaciens was also found to be able to remove inorganic and organic sulphur from coal samples. The bacterium, however, lost its ability to remove organic sulphur from model compounds during the investigation. R. erythropolis IGTS8 presented the greatest desulphurisation efficiency among the three bacterial strains. Nevertheless, the desulphurisation activity of R. erythropolis IGTS8 was too low for an economical coal biodesulphurisation process as it removed only 32.0% of total sulphur in bituminous coal, and 21.1% of total sulphur in anthracite coal. Alternatively, coal biodesulphurisation can be carried out in inexpensive conditions by using the bacteria inherent in the coal itself. The type of coal has an important effect on desulphurisation efficiency since the sulphur reduction in bituminous coal, which is in a lower rank than anthracite, was greater than the sulphur reduction in anthracite coal. This work also developed and evaluated the analytical methods used in the field. A HPLC method was developed to detect the desulphurisation metabolites of model compounds. The techniques for measuring sulphur in coal were improved.
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    Biological and artificial receptors in affinity sensor for water toxins detection
    (2003-01) Lotierzo, Manuela; Tothill, Ibtisam E.
    Molecular recognition is the basis for many of the chemical and biochemical phenomena occurring in living organisms. For example, antibodies, which are one of the different classes of natural receptor molecules, are capable of selectively recognising a specific target molecule or structure. They are therefore routinely utilised as analytical reagents in clinical and research laboratories. The design and synthesis of biomimetic recognition systems, capable of binding target molecules with affinities and specificities comparable to natural receptors, is regarded as one of the greatest challenges in bioorganic chemistry. Molecularly imprinted polymers (MIPs) have been shown to mimic the binding sites of antibodies and are, therefore, constantly gaining in interest for applications based on specific molecular recognition. This project aimed to develop affinity sensors for the detection of algal and cyanobacterial toxins such as microcystin-LR and domoic acid in water samples. Following the investigation, a heterogeneous direct competitive enzyme-linked immunosorbent assay (ELISA) format for microcystin detection was developed. The system was then transferred to an affinity membrane sorbent based ELISA. This was an amenable format for immunoassay incorporation into a disposable amperometric immunosensor device. A three-electrode system immunosensor was fabricated using thick film screenprinting technology. Amperometric HRP transduction of hydrogen peroxide catalysis, at low reducing potentials, versus Ag/AgCl reference and carbon counter electrode, was facilitated by hydroquinone mediated electron transfer. A detection limit of 0.5 pg V1 for microcystin-LR was achieved. The work undertaken also describes the design and synthesis of biomimetic recognition systems based on MIP, capable of binding target molecules with affinities and specificities on a par with natural receptors. A MIP synthetic receptor selective for microcystin-LR was studied using an enzyme-linked competitive assay and found to be comparable to polyclonal antibodies, whilst the MIP had superior stability over natural receptors. Methacrylic acid based MIP had a detection limit of 1 pg I'1, approxilately twenty times higher than that of anti microcystin-LR polyclonal antibody. A molecularly imprinted polymer was also directly synthesised by grafting on the gold chip of a surface plasmon resonance (SPR) based bioanalytical instrument system: the BIAcore 3000™. Such a chip based platform allowed a simple test of the specific MIP receptor for the marine toxin domoic acid. A fiill characterisation of the grafting procedure was initially carried out on a bare gold surface, and each step of the polymerisation was investigated by contact angle measurements and AFM imaging. The surface photo-initiated MIP film was obtained and its thickness and homogeneity evaluated. Domoic acid is a molecule that is too small for direct analysis, hence a competition reaction was performed in presence of the conjugate DA-HRP and a detection limit of 2 fig f 1 could be achieved with the BIAcore 3000™ system.
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    Biosensor development for the analysis of food quality
    (Cranfield University, 2003-09) Giakoumaki, Elsa; Tothill, Ibtisam E.; Steford, S.
    This thesis describes the development and evaluation of a number of biosensors for food applications. The first part of this thesis deals with the development of Surface Plasmon Resonance (SPR) biosensor systems, coupled with Polymerase Chain Reaction (PCR) for the detection of GMO related amplified nucleic acids in foodstuffs. The first SPR Biosensor described, used streptavidin-biotin linkage chemistry to attach a P35S nucleic acid probe on dextran-coated SPR transducer chips. Methodologies were developed for both the PCR stage and post-PCR sample preparation for the sensitive, rapid and cost-effective detection of GMO-specific amplified DNA sequences. The final embodiment of the method was an asymmetric PCR amplification system with a simple sample processing step (0.3 M NaOH for 30 min in 20 % v/v formamide). The developed PCR-SPR system was successfully applied to the screening of samples of GMO origin. The second SPR biosensor reported herein, is based on a SPR chip immobilised single-stranded thiolated DNA. The thiolated probe exhibited a hybridisation capacity of 95 RU (Resonance Units) for 100 nM of complementary DNA target and a detection limit of 5 nM. The potential of the current probe system for the detection of symmetrically amplified DNA sequences of short length was subsequently confirmed. The second part of this thesis involved preliminary studies into the development of simple, disposable screen-printed electrodes for the electrochemical determination of glucose and L-amino acids in horticultural products. The dynamic range of the developed biosensors was up to 10 mM for glucose and up to 1 mM for L-leucine determination. The developed glucose biosensor exhibited encouraging analytical performance in fresh fruit samples. However, the L-amino acid oxidase electrodes consistently underestimated the amino acid content of the fruit samples. The latter observation was found to be primarily due to inhibitory components in the matrix.
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    Biosensors and nanomaterials and their application for mycotoxin determination
    (Wageningen Academic Publishers, 2011-11-01T00:00:00Z) Tothill, Ibtisam E.
    Mycotoxin analysis and detection in food and drinks is vital for ensuring food quality and safety, eliminating and controlling the risk of consuming contaminated foods, and complying with the legislative limits set by food authorities worldwide. Most analysis of these toxins is still conducted using conventional methods; however, biosensor methods are currently being developed as screening tools for use in field analysis. Biosensors have demonstrated their ability to provide rapid, sensitive, robust and cost-effective quantitative methods for on-site testing. The development of biosensor devices for different mycotoxins has attracted much research interest in recent years with a range of devices being designed and reported in the scientific literature. However, with the advent of nanotechnology and its impact on the evolution of ultrasensitive devices, mycotoxin analysis is also benefiting from the advances taking place in applying nanomaterials in sensors development. This paper reviews the developments in the area of biosensors and their applications for mycotoxin analysis, as well as the development of micro/nanoarray transducers and nanoparticles and their use in the development of new rapid devices
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    A comparison of EIS and QCM nanoMIP-based sensors for morphine
    (MDPI, 2021-12-11) D’Aurelio, Roberta; Tothill, Ibtisam E.; Salbini, Maria; Calò, Francesca; Mazzotta, Elisabetta; Malitesta, Cosimino; Chianella, Iva
    In this work we have compared two different sensing platforms for the detection of morphine as an example of a low molecular weight target analyte. For this, molecularly imprinted polymer nanoparticles (NanoMIP), synthesized with an affinity towards morphine, were attached to an electrochemical impedance spectroscopy (EIS) and a quartz crystal microbalance (QCM) sensor. Assay design, sensors fabrication, analyte sensitivity and specificity were performed using similar methods. The results showed that the EIS sensor achieved a limit of detection (LOD) of 0.11 ng·mL−1, which is three orders of magnitude lower than the 0.19 µg·mL−1 achieved using the QCM sensor. Both the EIS and the QCM sensors were found to be able to specifically detect morphine in a direct assay format. However, the QCM method required conjugation of gold nanoparticles (AuNPs) to the small analyte (morphine) to amplify the signal and achieve a LOD in the µg·mL−1 range. Conversely, the EIS sensor method was labor-intensive and required extensive data handling and processing, resulting in longer analysis times (~30–40 min). In addition, whereas the QCM enables visualization of the binding events between the target molecule and the sensor in real-time, the EIS method does not allow such a feature and measurements are taken post-binding. The work also highlighted the advantages of using QCM as an automated, rapid and multiplex sensor compared to the much simpler EIS platform used in this work, though, the QCM method will require sample preparation, especially when a sensitive (ng·mL−1) detection of a small analyte is needed.
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    Computational design of peptide ligands for ochratoxin A
    (MDPI, 2013-06-21) Heurich, Meike; Altintas, Zeynep; Tothill, Ibtisam E.
    Abstract: In this paper, we describe a peptide library designed by computational modelling and the selection of two peptide sequences showing affinity towards the mycotoxin, ochratoxin A (OTA). A virtual library of 20 natural amino acids was used as building blocks to design a short peptide library against ochratoxin A template using the de novo design program, LeapFrog, and the dynamic modelling software, FlexiDock. Peptide sequences were ranked according to calculated binding scores in their capacity to bind to ochratoxin A. Two high scoring peptides with the sequences N'-Cys-Ser-Ile-Val-Glu-Asp-Gly-Lys-C' (octapeptide) and N'-Gly-Pro-Ala-Gly-Ile-Asp-Gly-Pro-Ala-Gly-Ile-Arg-Cys-C' (13-mer) were selected for synthesis from the resulting database. These synthesized peptides were characterized using a microtitre plate-based binding assay and a surface plasmon resonance biosensor (Biacore 3000). The binding assay confirmed that both de novo designed peptides did bind to ochratoxin A in vitro. SPR analysis confirmed that the peptides bind to ochratoxin A, with calculated KD values of ~15.7 μM (13-mer) and ~11.8 μM (octamer). The affinity of the peptides corresponds well with the molecular modelling results, as the 13-mer peptide affinity is about 1.3-times weaker than the octapeptide; this is in accordance with the binding energy values modelled by FlexiDock. This work illustrates the potential of using computational modelling to design a peptide sequence that exhibits in vitro binding affinity for a small molecular weight toxin.
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    DBT degradation enhancement by decorating Rhodococcus erythropolis IGST8 with magnetic Fe3O4 nanoparticles
    (Wiley-Blackwell, 2009-04) Ansari, Farahnaz; Grigoriev, P.; Libor, Zsuzsanna; Tothill, Ibtisam E.; Ramsden, Jeremy J.
    Biodesulfurization (BDS) of dibenzothiophene (DBT) was carried out by Rhodococcus erythropolis IGST8 decorated with magnetic Fe3O4 nanoparticles, synthesized in-house by a chemical method, with an average size of 45-50 nm, in order to facilitate the post-reaction separation of the bacteria from the reaction mixture. Scanning electron microscopy (SEM) showed that the magnetic nanoparticles substantially coated the surfaces of the bacteria. It was found that the decorated cells had a 56% higher DBT desulfurization activity in basic salt medium (BSM) compared to the nondecorated cells. We propose that this is due to permeabilization of the bacterial membrane, facilitating the entry and exit of reactant and product, respectively. Model experiments with black lipid membranes (BLM) demonstrated that the nanoparticles indeed enhance membrane permeability.
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    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
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    Detection of the inflammation biomarker C-reactive protein in serum samples: towards an optimal biosensor formula
    (MDPI, 2014-10-03) Fakanya, Wellington M.; Tothill, Ibtisam E.
    The development of an electrochemical immunosensor for the biomarker, C-reactive protein (CRP), is reported in this work. CRP has been used to assess inflammation and is also used in a multi-biomarker system as a predictive biomarker for cardiovascular disease risk. A gold-based working electrode sensor was developed, and the types of electrode printing inks and ink curing techniques were then optimized. The electrodes with the best performance parameters were then employed for the construction of an immunosensor for CRP by immobilizing anti-human CRP antibody on the working electrode surface. A sandwich enzyme-linked immunosorbent assay (ELISA) was then constructed after sample addition by using anti-human CRP antibody labelled with horseradish peroxidase (HRP). The signal was generated by the addition of a mediator/substrate system comprised of 3,3,5',5'-Tetramethylbenzidine dihydrochloride (TMB) and hydrogen peroxide (H2O2). Measurements were conducted using chronoamperometry at -200 mV against an integrated Ag/AgCl reference electrode. A CRP limit of detection (LOD) of 2.2 ng·mL-1 was achieved in spiked serum samples, and performance agreement was obtained with reference to a commercial ELISA kit. The developed CRP immunosensor was able to detect a diagnostically relevant range of the biomarker in serum without the need for signal amplification using nanoparticles, paving the way for future development on a cardiac panel electrochemical point-of-care diagnostic device.
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    Detection of β-amyloid peptide (1–16) and amyloid precursor protein (APP770) using spectroscopic ellipsometry and QCM techniques: A step forward towards Alzheimers disease diagnostics
    (Elsevier, 2010-12-15) Mustafa, M. K.; Nabok, Alexei; Parkinson, D.; Tothill, Ibtisam E.; Salam, Faridah; Tsargorodskaya, A.
    A highly sensitive method of spectroscopic ellipsometry in total internal reflection mode (TIRE) was exploited for detecting β-amyloid peptide (Aβ1–16) in the direct immune reaction with monoclonal DE2 antibodies (raised against Aβ1–16) electrostatically immobilised on the surface of gold. A rapid detection of Aβ1–16 in a wide range of concentrations from 5 μg/ml down to 0.05 ng/ml was achieved using a cost-effective and label-free direct immunoassay format. TIRE dynamic spectral measurements proved that the immune reaction between DE2 monoclonal antibodies and Aβ1–16 is highly specific with the affinity constant KD = 1.46 × 10−8 mol/l. The same DE2 antibodies were utilised for detection of amyloid precursor protein APP770, a larger protein containing Aβ1–16 domain, using the quartz crystal microbalance (QCM) measurements in liquid. A combination of QCM and TIRE kinetics results allowed the evaluation of the originally unknown concentration of APP770 in complete medium solution containing other proteins, salts, and amino acids.
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    Development fo electrochemical sensors for heavy metal ions detection in environmental samples
    (2004-01) Kadara, Rashid; Tothill, Ibtisam E.; Newman, Jeffrey D.
    The work presented in this thesis was concerned with the development of single-use drop-on sensors incorporating a three-electrode configuration (graphite carbon- working electrode, carbon-counter electrode and silver/silver chloride - reference electrode) for on-site detection of toxic heavy metals in various environmental matrices. The fabricated three-electrode configuration system was coupled with square-wave anodic stripping voltammetry (SWASV) or constant current stripping chronopotentiometry (CCSCP) in order to provide a means of a relatively inexpensive on-site detector for trace levels of lead (II), copper (II) and cadmium (II). Detections and determinations of these metals were carried out on bare screen-printed carbon electrodes (SPCEs), mercury film SPCE, bismuth film SPCE and SPCEs modified with Nafion, 2,5- Dimercapto-1, 3, 4- thiadiazole (DMTD), bismuth oxide (Bi₂O₃) and polyethyleneimine (PEI) using the optimised procedures developed for measurements. With the optimised working conditions, the results obtained indicate that the screen-printed electrochemical sensors are sensitive and reproducible enough for the CCSCP and SWASV determination of lead, copper and cadmium in the microgram per litre - milligram per litre range. Limits of detection below 20 µg I¯¹ were estimated for the trace metal detection of lead, copper and cadmium on both the bismuth and mercury film electrodes. For the bare SPCE, detection limits of 35, 45 and 59 µg I¯¹ were obtained for lead, cadmium and copper detection using CCSCP. The reproducibility of the measurements, which also contributed to the interest in developing the electrochemical sensing devices for metal ions, was below 15 % for the bare SPCE, bismuth film SPCE, and mercury film SPCE. Modifications of SPCEs with an ion-exchanger (Nafion) and a complexing agent (DMTD) provided means of increasing the sensitivity of stripping response obtained at the bare SPCE. Detection limits of 20 and 22 µg I¯¹ were estimated for lead (II) measurements at the Nafion modified SPCE and at the screen-printed DMTD modified electrode, respectively. The application of the various electrodes to real samples is demonstrated and proved successful for both water and soil extracted samples including in situ measurements at a contaminated site.
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    The development of a bacterial biosensor for the analysis of benzene in workplace air
    (2004-12) Lanyon, Yvonne H.; Tothill, Ibtisam E.
    The presence of toxic volatile organic compounds (VOCs) such as benzene in workplace air has accounted for the death of many occupationally exposed workers over the last century. The conventional gas chromatographic method of monitoring benzene is known to be costly, complex and in most cases, laboratory-based. Therefore a need exists for the development of low-cost, easy to use, portable devices that can be used on-site for the rapid evaluation of airborne benzene. In this thesis, the development of an amperometric bacterial biosensor based on Pseudomonas putida ML2 for the detection of airborne benzene is described. Benzene can be used by the bacteria as a sole carbon source, and its aerobic degradation can be measured using a dissolved oxygen electrode. In this work, P. putida ML2 cells were immobilised between two cellulose acetate membranes and fixed onto a Clark dissolved oxygen electrode. Biosensor responses were investigated in batch and kinetic (Flow Injection Analysis) mode, and also using screen-printed electrodes. In each case, the response characteristics, sensitivity, reproducibility and lifetime of the sensor were investigated, as well as construction techniques and operational parameters. The applicability of the biosensor for the analysis of air samples containing benzene was investigated. Air samples were collected from an exposure room of controlled concentration using charcoal adsorption tubes, and benzene extracted with solvent desorption using dimethylformamide (DMF). DMF proved to be compatible for use with the biosensor, causing minimal interference with the sensor response and causing no toxic effects on the bacterial cells. The biosensor displayed a linear detection range between 0.025 - 0.15 mM benzene based on standard solutions containing a maximum of 2% DMF, with a response time of 6 minutes. This linear detection range allowed the analysis of air containing between 3-16 ppm benzene, based on a 60-minute sampling period. The inter-assay reproducibility of the sensor response to standard benzene calibration curves under such conditions gave a 3% variation coefficient based on 5 separate assays (n = 17) using the same bacterial membrane. The FIA system was easily transported to an in situ location for the air sample analyses, and a correlation was obtained between the biosensor and gas chromatography (GC) results for the exposure room air samples investigated. Moreover, the biosensor displayed no interference to other benzene related compounds in the BTEX (benzene, toluene, ethylbenzene, xylene) range. Overall, this thesis has described the development of an alternative method for the monitoring of benzene in workplace air, using a bacterial biosensor based on Flow Injection Analysis. Advantages over the conventional GC methods including ease of operation, cost-effective production and portability demonstrate that the P. putida ML2 biosensor has potential applications as an alternative means for the rapid analysis of workplace air containing benzene.
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    Development of a Botrytis specific immunosensor: towards using PCR species identification
    (Cranfield University, 2014-01) Binder, Michael; Terry, Leon A.; Tothill, Ibtisam E.
    Botrytis species affect over 300 host plants in all climate areas of the world, at both pre and post-harvest stages, leading to significant losses in agricultural produce. Therefore, the development of a rapid, sensitive and reliable method to assess the pathogen load of infected crops can help to prescribe an effective curing regime. Growers would then have the ability to predict and manage the full storage potential of their crops and thus provide an effective disease control and reduce post-harvest losses. A highly sensitive electrochemical immunosensor based on a screen-printed gold electrode (SPGE) with onboard carbon counter and silver / silver chloride (Ag/AgCl) pseudo-reference electrode was developed in this work for the detection and quantification of Botrytis species. The sensor utilised a direct sandwich enzyme-linked immunosorbent assay (ELISA) format with a monoclonal antibody against Botrytis immobilised on the gold working electrode. Two immobilisation strategies were investigated for the capture antibody, and these included adsorption and covalent immobilisation after self-assembled monolayer formation with 3-dithiodipropionic acid (DTDPA). A polyclonal antibody conjugated to the electroactive enzyme horseradish peroxidase (HRP) was then applied for signal generation. Electrochemical measurements were conducted using 3,3’, 5,5’-tetramethylbenzidine dihydrochloride / hydrogen peroxide (TMB/H2O2) as the enzyme substrate system at a potential of -200 mV. The developed biosensor was capable of detecting latent Botrytis infections 24 h post inoculation with a linear range from 150 to 0.05 μg fungal mycelium ml-1 and a limit of detection (LOD) as low as 16 ng ml-1 for covalent immobilisation and 58 ng ml-1 for adsorption, respectively. Benchmarked against the commercially available Botrytis ELISA kits, the optimised immuno-electrochemical biosensor showed strong correlation of the quantified samples (R2=0.998) ... [cont.].
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    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.
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    Development of a sensitive immunosensor for the detection of cardiac Troponin T in cardiovascular disease
    (Cranfield University, 2014-12) Pawula, Maria; Tothill, Ibtisam E.; Altintas, Zeynep
    Cardiovascular disease (CVD) is currently globally the biggest cause of mortality, with rising figures, especially now in the developing world. Early and accurate diagnosis of CVD, (especially acute myocardial infarction (AMI) is important in being able to provide appropriate, timely and cost effective treatment, or to take preventative action. Biomarkers and biosensors are playing an increasingly important role in this diagnosis, especially those based on immunoassays. As technology improves and becomes cheaper, there is the potential to develop immunosensors which use optical techniques such as surface plasmon resonance (SPR) for biomarker measurement which could be used effectively in point-of-care diagnostics for real-time detection. This thesis describes the development and optimisation of a sensitive immunosensor for the AMI specific biomarker, cardiac Troponin T (cTnT), on an SPR platform. Early diagnosis of AMI requires an assay methodology which can determine very low concentrations of cTnT in human serum. The work conducted includes the development of a set of optimised conditions for the immobilisation of the capture antibody (anti-cardiac Troponin T 1C11 antibody) onto a gold surfaced SPR sensor chip, to which a self-assembled monolayer of 11-mercaptoundecanoic acid has been applied. A direct immunoassay for cTnT in buffer was examined and a limit of detection (LOD) of 25 ng ml-1 cTnT was achieved. A sandwich immunoassay format was then developed to enhance the sensitivity of the assay. The use of a detection antibody (anti-cardiac Troponin T 7G7 antibody) was shown to successfully amplify the SPR response five-fold, with the LOD improving to 5 ng ml-1 cTnT. The second stage of the project involved examining the extent of non-specific binding of the cTnT and of serum proteins, and investigating how best to minimise and control for it. Non-specific binding of cTnT was eliminated, and serum protein binding was reduced by 93% in 10% serum and 73% in 50% serum. To achieve greater sensitivity, amplification of the signal through the use of detector antibodies conjugated to gold nanoparticles (AuNPs) for the sandwich assay was investigated. The performance of the cTnT immunosensor sandwich assay in human serum was evaluated using non-modified and AuNP modified detector antibodies. The LOD of the immunosensor in 50% serum was assessed as 5 ng ml-1 cTnT for the standard sandwich assay, and 0.5 ng ml-1 cTnT when using AuNP conjugated detector antibodies to enhance the sensitivity.
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    Development of a β-Lactoglobulin sensor based on SPR for milk allergens detection
    (MDPI, 2018-03-27) Ashley, Jon; D’Aurelio, Roberta; Piekarska, Monika; Temblay, Jeff; Pleasants, Mike; Trinh, Linda; Rodgers, Thomas L.; Tothill, Ibtisam E.
    A sensitive and label-free surface plasmon resonance (SPR) based sensor was developed in this work for the detection of milk allergens. β-lactoglobulin (BLG) protein was used as the biomarker for cow milk detection. This is to be used directly in final rinse samples of cleaning in-place (CIP) systems of food manufacturers. The affinity assay was optimised and characterised before a standard curve was performed in pure buffer conditions, giving a detection limit of 0.164 µg mL−1 as a direct binding assay. The detection limit can be further enhanced through the use of a sandwich assay and amplification with nanomaterials. However, this was not required here, as the detection limit achieved exceeded the required allergen detection levels of 2 µg mL−1 for β-lactoglobulin. The binding affinities of the polyclonal antibody for BLG, expressed by the dissociation constant (KD), were equal to 2.59 × 10−9 M. The developed SPR-based sensor offers several advantages in terms of label-free detection, real-time measurements, potential on-line system and superior sensitivity when compared to ELISA-based techniques. The method is novel for this application and could be applied to wider food allergen risk management decision(s) in food manufacturing.
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    Development of affinity sensors for Microcystin-LR based on a computationally designed molecularly imprinted polymer
    (Cranfield University, 2003-01) Chianella, Iva; Piletsky, Sergey A.; Chen, B.; Tothill, Ibtisam E.
    In this work the development of affinity sensors for the detection of microcystin-LR based on a computationally designed artificial receptor is presented. Microcystin-LR is a cyclic heptapeptide hepatotoxin produced by Cyanobacteria (aquatic organisms also known as blue-green algae), which during blooms period can release toxins in water. Clinical signs of hepatotoxicosis have been observed in domestic animals and livestock and recently also in humans. At present, analysis of these toxins is achieved largely using conventional, time consuming and expensive techniques such as chromatographic methods (HPLC, TLC) and immunoassay. Therefore, the necessity of an easy and inexpensive method of analysis such a biosensor is becoming urgent. In this work an artificial receptor for microcystin-LR was synthesised using a combined approach of molecular imprinting and computer modelling. A computer-aided rational design was applied to study microcystin-LRlmonomers interactions in order to find an optimal composition for the synthesis of the receptor. The optimised composition, suggested by computer modelling, consisted in 1 mol of2-acrylamido-2-methyl-propanesulfonic acid and 6 mol ofurocanic acid ethyl ester for 1 mol of template. This monomer composition was then used to synthesise a molecularly imprinted polymer (MIP) and an enzyme- linked competitive assay was developed to characterise the computational receptor. In the assay, computational MIP was able both to detect 0.1 ~g rl of microcystin-LR and to distinguish the analyte among analogues such as microcystin-YR, microcystin-RR and nodularin. The computationally designed receptor was then used as a sensing element for the construction of sensor devices. A MIP-based piezoelectric sensor, capable of detecting 35 ~g rl of toxin in water, was developed. In order to improve the system sensitivity, the computational polymer was also used as a material in solid-phase extraction (SPE) for samples pre-concentration. The receptor was able to pre-concentrate up to 1,000 fold tap water samples spiked with only 1 J.1g rl of toxin. By combining MIP-based SPE and piezoelectric sensor an improved system with a minimum detectable concentration of toxin of 0.35 ~g rl was achieved. Encouraging preliminary results were also obtained in developing a MIP-based electrochemical sensor.