Browsing by Author "Tothill, Ibtisam E."
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Item Open Access Allelic variants of KLK2 gene predict presence of prostate cancer at biopsy(OMICS International, 2016-04-22) Nna, Emmanuel Okechukwu; Tothill, Ibtisam E.; Bailey, TraceyObjective: 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.Item Open Access 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.Item Open Access 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.Item Open Access 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.Item Open Access 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.Item Open Access 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 devicesItem Open Access 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, IvaIn 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.Item Open Access Detection of Salmonella typhimurium using an electrochemical immunosensor.(Elsevier Science B.V., Amsterdam., 2009-04-15T00:00:00Z) Salam, Faridah; Tothill, Ibtisam E.An electrochemical immunosensor based on screen-printed gold working electrode with onboard carbon counter and silver–silver chloride pseudo-reference electrode for Salmonella typhimurium detection is described in this paper. Monoclonal anti-S. typhimurium antibody was immobilized using physical and covalent immobilization via amine coupling of carboxymethyldextran on the surface of the gold working electrode. A direct sandwich enzyme-linked immunosorbent assays (ELISA) format was then developed and optimized using a polyclonal anti-Salmonella antibodies conjugated to horseradish peroxidase (HRP) as the enzyme label. 3,3′,5,5′-Tetramethylbenzidine dihydrochloride (TMB)/H2O2 was used as the enzyme mediator/substrate system. Electrochemical detection was conducted using chronoamperometry at −200 mV vs. onboard screen-printed Ag–AgCl pseudo-reference electrode. The applied potential was selected through the study of the electrochemical behaviour of bare gold electrode with TMB–H2O2–IgG–HRP system. S. typhimurium detection of 5 × 103 cells ml−1 and 20 cells ml−1 was achieved respectively for physical and covalent antibody immobilization. The developed sensor was then compared to a commercial ELISA kit and a chromogenic agar plating method for meat samples analysis. The sensor format shows a promising technology for simple and sensitive detection system for Salmonella contamination. Rapid detection of Salmonella is a key to the prevention and identification of problems relatedItem Open Access 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.Item Open Access 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.Item Open Access 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.Item Open Access 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.].Item Open Access Development of a sensitive detection method of cancer biomarkers in human serum (75%) using a quartz crystal microbalance sensor and nanoparticles amplification system(Elsevier Science B.V., Amsterdam., 2010-06-30T00:00:00Z) Uludag, Yildiz; Tothill, Ibtisam E.A simple and sensitive sensor method for cancer biomarkers [prostate specific antigen (PSA) and PSA-alpha 1-antichymotrypsin (ACT) complex] analysis was developed, to be applied directly with human serum (75%) by using antibody modified quartz crystal microbalance sensor and nanoparticles amplification system. A QCM sensor chip consisting of two sensing array enabling the measurement of an active and control binding events simultaneously on the sensor surface was used in this work. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions before applying to serum samples. Extensive interference to the QCM signal was observed upon the analysis of serum. Different buffer systems were then formulated and tested for the reduction of the non-specific binding of sera proteins on the sensor surface. A PBS buffer containing 200 mu g mL(-1) BSA, 0.5 M NaCI, 500 mu g mL(- 1) dextran and 0.5% Tween 20, was then selected which eliminated the interfering signal by 98% and enabled the biomarker detection assay to be performed in 75% human serum. By using Au nanoparticles to enhance the QCM sensor signal, a limit of detection of 0.29 ng mL(-1) PSA and PSA-ACT complex (in 75% serum) with a linear dynamic detection range up to 150 ng mL(-1) was obtained. With the achieved detection limit in serum samples, the developed QCM assay shows a promising technology for cancer biomarker analysis in patient samples. (C) 2010 Elsevier B.V. All rights reserved.Item Open Access 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, ZeynepCardiovascular 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.Item Open Access 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.Item Open Access 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.Item Open Access Development of an affinity sensor for Ochratoxin A(Cranfield University, 2008-01) Heurich, Meike; Tothill, Ibtisam E.Ochratoxin A is a contaminant in wine and known to be immunosuppressive and possibly carcinogenic. Therefore, the development of a rapid and sensitive method for field analysis is required for risk assessment and management. The work presented in this thesis reports the construction of a sensor platform capable of fulfilling these requirements. As a sensor platform, screen-printed thick film electrodes and microelectrodes on a silicone support were investigated for sensor development. As biological recognition elements, an antibody specifically binding ochratoxin A and a peptide receptor that was designed using computational modelling were examined. A disposable immunosensor for ochratoxin A was developed based on screen-printing technology. An indirect competitive immunoassay format was used on bare screen printed gold electrode (SPGE). The performance of this sensor was compared to carboxmethylated dextran (CMD) modified SPGE. Detection was performed by chronoamperometry monitoring the reaction of tetramethylbenzidine and hydrogen peroxide catalysed by horseradish peroxidase. The SPGE-based immunosensor achieved a detection limit of 100 ng L-1 and the CMD-modified SPGE immunosensor 10 ng L-1. The latter has been used for ochratoxin A determination in wine samples and was validated against standard HPLC and a commercial immunoassay test kit. Wine sample analysis involved the sample pre-treatment using immunoaffinity chromatography, electrochemical wine component characterisation and interference control. The immunosensor format was transferred to a gold microelectrode array based on a silicone support for the purpose of signal sensitivity enhancement and miniaturisation in the prospect of field analysis. Preliminary data showed the characterisation of the microelectrode array immunosensor construction and characterisation. Further optimisation is needed to establish a calibration curve with the required sensitivity. The second part of the work comprised the design of a peptide receptor for ochratoxin A using computational methods by screening de novo designed peptide libraries. An octapeptide (CSIVEDGL) and a 13-peptide (GPAGIDGPAGIRC) were selected for synthesis and affinity characterised for ochratoxin A recognition using a surface plasmon resonance biosensor (BiacoreTM). The peptide receptors showed good sensitivity for ochratoxin A of 10 μg L-1. Preliminary affinity characterisation resulted in KA = 63 mM-1 for the 13-mer peptide and KA = 84 mM-1 for the octapeptide, which appears to be binding with higher strength to ochratoxin A. The affinity values correspond to the binding score (binding energy) calculated by computational modelling. This work shows the potential of designing peptide receptors for small molecules (e.g. ochratoxin A) and suggests their application in affinity sensors for detecting ochratoxin A contamination.Item Open Access Development of an electrochemical immunosensor for aflatoxin M1 in milk with focus on matrix interference.(Elsevier Science B.V., Amsterdam., 2009-04-15T00:00:00Z) Parker, Charlie O.; Tothill, Ibtisam E.A simple sensor method was developed for aflatoxin M1 analysis to be applied directly with milk by using antibody modified screen-printed carbon working electrode with carbon counter and silver–silver chloride pseudo-reference electrode. A competitive ELISA assay format was constructed on the surface of the working electrode using 3,3,5′,5′-tetramethylbenzidine dihyrochloride (TMB)/ H2O2 electrochemical detection scheme with horseradish peroxidase (HRP) as the enzyme label. The performance of the assay and the sensor was optimised and characterised in pure buffer conditions before applying to milk samples. Extensive interference to the electroanalytical signal was observed upon the analysis of milk. Through a series of chemical fractionations of the milk, and testing the electrochemical properties of the fractions, the interference was attributed to whey proteins with focus towards α-lactalbumin. A simple pre- treatment technique of incorporating 18 mM calcium chloride, in the form of Dulbucco's PBS, in a 1:1 ratio to the milk sample or standards and also to the washing buffer stabilised the whey proteins in solution and eliminate the interfering signal. The resulting immunosensor was interference free and achieved a limit of detection of 39 ng l−1 with a linear dynamic detection range up to 1000 ng l−1. The developed immunosensor method was compared to a commercial ELISA kit and an in-house HPLC method. The immunsensor was comparable, in term of sensitivity, but vastly superior in term of portability and cost therefore a key instrument for the detection of aflatoxin M1 at the source of the conItem Open Access Development of an impedimetric biosensor for lung cancer detection.(2018-06) Arabnejad, Mahdi; Tothill, Ibtisam E.; Chianella, IvaThe promise of biosensors offering attractive features has kept the field active and growing. The aim is often to develop a device which is sensitive, specific, rapid, portable, cheap, and with the ability to capture an analyte in different matrices without cross-reactivity. The challenges increase when the aim is simultaneous multi-analyte detection on a single platform, without the need for complex procedures and expensive instruments. Such a system is invaluable in many clinical settings, where disease diagnosis and progression are multifactorial. Cancer is a good example of complex diagnosis requirements. This project is aimed at the development of biosensing platform to overcome the aforementioned problems and allow direct, simple analysis with the minimum of sample pre-treatment. The early detection of lung cancer has been chosen as there is no commercial biosensor available for detection of this disease and for lung cancer diagnosis multi-analyte recognition is necessary. This project presents the development of impedimetric and magnetic sensing platform for early detection of lung cancer via detecting the neuron-specific enolase (NSE) and carcinoembryonic antigen (CEA) which are known as potential lung cancer biomarkers. The sensing platform developed here comprises of magnetic manipulation, screen printed electrode (SPE), and magnetic nanobeads. The magnetic nanobeads (MBs) were functionalised with antibodies to fish the analyte from the sample, and to move them over the sensing area. Moreover, magnetic nanobeads were used to increase the chance of antigen-antibody complex formation. After cleaning the surface of electrodes with 50 mM KOH in 25% H₂O₂ (for 10 minutes), immunosensors were developed by immobilising the antibodies on the gold working electrode of SPEs through formation of self-assembled monolayer (SAM layer) due to its simplicity, stability, well-organised structure and low background noise. The optimised NSE immunosensor with 10 µg/ml of 10-7937 antibody was successfully tested to measure various concentrations of NSE protein (0 – 100 ng/ml) in both PBS buffer and 100 % serum using functionalised MBs with 2.4 mg/ml of 10-7938 antibody. The optimised sensor achieved detection limit of 0.18 ng/ml (R²= 0.9848) in buffer and 0.52 ng/ml (R² = 0.9977) in 100 % serum with via EIS with the use of 10 mM potassium ferri/ferrocyanide as a redox probe. The impedimetric CEA immunosensor was developed and optimised by use of 20 µg/ml of 12-140-01 antibody and was used to measure analyte with functionalised MBs with 2.4 mg/ml of 12-140-10 antibody. The CEA immunosensor was also able to measure various CEA concentrations (0 – 100 ng/ml) in both PBS buffer and 100 % serum in presence of 10 mM potassium ferri/ferrocyanide. The sensor achieved low limit of detection as 0.26 ng/ml (R² = 0.9924) and 0.76 ng/ml (R²= 0.9839) for CEA detection in buffer and 100 % serum, respectively. In conclusion, both immunosensors developed here, using EIS and the magnetic sensing platform, were capable of detecting their corresponding biomarkers in serum in relatively short time (40 minutes) and in the appropriate concentration range, as serum concentrations higher than 12.5 ng/ml and 7 ng/ml for NSE and CEA respectively can indicate presence of cancer. To the best of our knowledge, no one has reported a use of magnetic platform as the one developed in this thesis.Item Open Access Development of electrochemical immunosensors for HER-1 and HER-2 analysis in serum for breast cancer patients(MDPI, 2023-03-07) Wignarajah, Shayalini; Chianella, Iva; Tothill, Ibtisam E.In this work, two human epidermal growth factor receptors, HER-1 and HER-2, were selected as biomarkers to enable the detection of breast cancer. Therefore, two biosensors were developed using gold sensor chips coupled with amperometric detection of the enzyme label horse radish peroxidase (HRP). The biosensors/immunosensors relied on indirect sandwich enzyme-linked immunosorbent assays with monoclonal antibodies (Ab) against HER-1 and HER-2 attached to the sensors to capture the biomarkers. Detection polyclonal antibodies followed by secondary anti-rabbit (for HER-1) and anti-goat (for HER-2) IgG antibody-HRP were then applied for signal generation. In buffer, the developed sensors showed limits of detections (LOD) of 1.06 ng mL−1 and 0.95 ng mL−1 and limits of quantification (LOQ) of 2.1 ng mL−1 and 1.5 ng mL−1 for HER-1 and HER-2, respectively. In 100% (undiluted) serum, LODs of 1.2 ng mL−1 and 1.47 ng mL−1 and LOQs of 1.5 ng mL−1 and 2.1 ng mL−1 were obtained for HER-1 and HER-2, respectively. Such limits of detections are within the serum clinical range for the two biomarkers. Furthermore, gold nanoparticles (AuNP) labelled with secondary anti-rabbit and anti-goat IgG antibody-HRP were then used to enhance the assay signal and increase the sensitivity. In buffers, LODs of 30 pg mL−1 were seen for both sensors and LOQs of 98 pg mL−1 and 35 pg mL−1 were recorded for HER-1 and HER-2, respectively. For HER-2 the AuNPs biosensor was also tested in 100% serum obtaining a LOD of 50 pg mL−1 and a LOQ of 80 pg mL−1. The HER-2 AuNP electrochemical immunosensor showed high specificity with very low cross-reactivity to HER-1. These findings demonstrate that the two developed sensors can enable early detection as well as monitoring of disease progression with a beneficial impact on patient survival and clinical outcomes.
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