Browsing by Author "Altintas, Zeynep"

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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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    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 functionalized nanostructured polymeric membranes for water purification
    (Elsevier, 2016-04-24) Altintas, Zeynep; Chianella, Iva; Da Ponte, Gabriella; Paulussen, Sabine; Gaeta, Soccorso; Tothill, Ibtisam E.
    Pharmaceuticals specific molecularly imprinted polymers nanoparticles (MIPNPs) were synthesized and applied onto the polyvinylidene fluoride (PVDF) membranes previously subjected to the plasma treatment. Diclofenac-, metoprolol- and vancomycin-MIPs were applied onto the membranes and scanning electron microscopy was employed to visualize MIPNPs on the membrane. After functionalization of the membranes with target-specific MIPs the molecularly imprinted membranes (MIMs) affinity against their targets was evaluated using solid phase extraction (SPE) technique coupled with high performance liquid chromatography (HPLC). MIMs were used as filters to load the target solutions through employing a vacuum pump to evaluate the amount of pharmaceuticals in filtrate. Moreover, a comparative study was performed by comparing the efficiency of MIMs functionalized either by adsorption or covalent immobilization. The capacity analysis of MIPNPs by SPE–HPLC revealed 100%, 96.3%, and 50.1% uptake of loaded solution of metoprolol, diclofenac and vancomycin, respectively. MIMs showed 99.6% uptake with a capacity of 60.39 ng cm2 for metoprolol; 94.7% uptake with a capacity of 45.09 ng cm2 for diclofenac; and 42.6% uptake with a capacity of 16.9 ng cm2 for vancomycin. HPLC detection limits of targets were found as 3.7, 7.5 and 15 ng mL−1 for diclofenac, metoprolol and vancomycin respectively. A small scale pilot test was also conducted which indicates the promising future applications of the developed MIMs for high volume of filtrates especially in the case of the plasma-treated PVDF membranes prepared by covalent immobilization of the MIPs.
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    Development of surface chemistry for surface plasmon resonance based sensors for the detection of proteins and DNA molecules
    (Elsevier Science B.V., Amsterdam., 2012-01-27T00:00:00Z) Altintas, Zeynep; Uludag, Yildiz; Gurbuz, Yasar; Tothill, Ibtisam E.
    The immobilisation of biological recognition elements onto a sensor chip surface is a crucial step for the construction of biosensors. While some of the optical biosensors utilise silicon dioxide as the sensor surface, most of the biosensor surfaces are coated with metals for transduction of the signal. Biological recognition elements such as proteins can be adsorbed spontaneously on metal or silicon dioxide substrates but this may denature the molecule and can result in either activity reduction or loss. Self assembled monolayers (SAMs) provide an effective method to protect the biological recognition elements from the sensor surface, thereby providing ligand immobilisation that enables the repeated binding and regeneration cycles to be performed without losing the immobilised ligand, as well as additionally helping to minimise non-specific adsorption. Therefore, in this study different surface chemistries were constructed on SPR sensor chips to investigate protein and DNA immobilisation on Au surfaces. A cysteamine surface and 1%, 10% and 100% mercaptoundecanoic acid (MUDA) coatings with or without dendrimer modification were utilised to construct the various sensor surfaces used in this investigation. A higher response was obtained for NeutrAvidin immobilisation on dendrimer modified surfaces compared to MUDA and cysteamine layers, however, protein or DNA capture responses on the immobilised NeutrAvidin did not show a similar higher response when dendrimer modified surfaces were used.
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    DNA-based biosensor platforms for the detection of TP53 mutation
    (Elsevier Science B.V., Amsterdam., 2012-07-05T00:00:00Z) Altintas, Zeynep; Tothill, Ibtisam E.
    A DNA-based assay for the detection of one-point mutation in TP53 gene, responsible for lung cancer, was developed using a surface plasmon resonance (SPR) and a quartz crystal microbalance (QCM) biosensor systems. Amine coupling was employed for the immobilization of NeutrAvidin on thiol-derivatized surface to capture the biotinylated target sequence. Two targets sequences and one control DNA sequence were investigated including, a fully complementary (30 mer), one-point mutation and a non-complimentary DNA using hybridization with a detection probe strand (27 mer). The most appropriate surface coating was also examined for both sensor platforms with hybridization and single nucleotide polymorphism (SNP) detection efficiency were then compare. A 0.03-2 μM concentration range of detection probe was detected using the SPR and QCM sensors on wild and mutant type target surfaces. The linear regression analysis was performed for both sensors resulting in a R 2 value for the SPR assay of 0.985 and 0.993 for perfect and mismatch reaction and of 0.978 and 0.976 for the QCM assay, respectively. The obtained results demonstrate that the used approach represents a very promising future method for the detection of one-point mutation in genetic-based health problem with highly sensitive, specific, and real-time analysis
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    Surface plasmon pesonance immunosensor for the detection of campylobacter jejuni
    (MDPI, 2017-05-16) Masdor, Noor Azlina; Altintas, Zeynep; Tothill, Ibtisam E.
    Campylobacteriosis is an internationally important foodborne disease caused by Campylobacter jejuni. The bacterium is prevalent in chicken meat and it is estimated that as much as 90% of chicken meat on the market may be contaminated with the bacterium. The current gold standard for the detection of C. jejuni is the culturing method, which takes at least 48 h to confirm the presence of the bacterium. Hence, the aim of this work was to investigate the development of a Surface Plasmon Resonance (SPR) sensor platform for C. jejuni detection. Bacterial strains were cultivated in-house and used in the development of the sensor. SPR sensor chips were first functionalized with polyclonal antibodies raised against C. jejuni using covalent attachment. The gold chips were then applied for the direct detection of C. jejuni. The assay conditions were then optimized and the sensor used for C. jejuni detection, achieving a detection limit of 8 × 106 CFU·mL−1. The sensitivity of the assay was further enhanced to 4 × 104 CFU·mL−1 through the deployment of a sandwich assay format using the same polyclonal antibody. The LOD obtained in the sandwich assay was higher than that achieved using commercial enzyme-linked immunosorbent assay (ELISA) (106–107 CFU·mL−1). This indicate that the SPR-based sandwich sensor method has an excellent potential to replace ELISA tests for C. jejuni detection. Specificity studies performed with Gram-positive and Gram-negative bacteria, demonstrated the high specific of the sensor for C. jejuni.
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    Surface plasmon resonance based immunosensor for the detection of the cancer biomarker carcinoembryonic antigen
    (Elsevier Science B.V., Amsterdam., 2011-10-30T00:00:00Z) Altintas, Zeynep; Uludag, Yildiz; Gurbuz, Yasar; Tothill, Ibtisam E.
    An immunoassay in optimised conditions with a highly sensitive surface plasmon resonance (SPR) based biosensor was developed for the detection of the cancer biomarker carcinoembryonic antigen (CEA). Different formats of the immunoassay were initially investigated on the surface of the gold sensor chip. A self- assembled monolayer (SAM) was formed on the gold chip using 11- mercaptoundecanoic acid (MUDA), before the immobilisation of the antibodies was conducted. The assay was then formed in a direct capture and a sandwich assay. In order to increase the sensor signal the CEA antigen was incubated with the detection/capture antibody before it was injected to the sensor chip surface and the results were recorded in real-time using the Biacore 3000 instrument. A detection limit of 3ngml-1 CEA was obtained with a dynamic detection range from 3ngml-1 to 400ngml-1 with correlation coefficients of 1.00 and 0.99 for the sandwich and rabbit anti-mouse (RAM) capture assay. Kinetic data analysis was performed for the standard capture test and subsequently for the developed assays and Rmax showed an increase from 215 RU for the standard capture test to 428 RU for the RAM-capture assay and 734 RU for the sandwich assay, respectively. The developed SPR immunosensor using the sandwich assay format showed high sensitivity and reproducibility for CEA detection which makes it a promising procedure for cancer biomarker analysis
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    Ultrasensitive detection of endotoxins using computationally designed nanoMIPs
    (Elsevier, 2016-06-21) Altintas, Zeynep; Abdin, Mohammed J.; Tothill, Alexander M.; Karim, Kal; Tothill, Ibtisam E.
    Novel molecularly imprinted polymer nanoparticles (nanoMIPs) were designed for endotoxin from Escherichia coli 0111:B4, using computational modeling. The screening process based on binding energy between endotoxin and each monomer was performed with 21 commonly used monomers, resulting in the selection of itaconic acid, methacrylic acid and acrylamide as functional monomers due to their strong binding interaction with the endotoxin template. The nanoMIPs were successfully synthesized with functional groups on the outer surface to aid in the immobilization onto sensor surface. The solid phase photopolymerization approach used for the synthesis of nanoMIPs ranging from 200 to 235 nm in diameter. The limit of detection and KD were significantly improved when endotoxin samples were prepared using a novel triethylamine method. This improved the efficiency of gold nanoparticle functionalization by targeting the subunits of the endotoxin. Compared to the vancomycin MIP control, the endotoxin MIPs displayed outstanding affinity and selectivity towards the endotoxin with KD values in the range of 4.4–5.3 × 10−10 M, with limits of detection of 0.44 ± 0.02 ng mL−1 as determined by surface plasmon resonance (SPR) sensor when itaconic acid was used as the functional monomer. The MIP surface can be regenerated >30 times without significant loss of binding activity making this approach highly cost effective for expensive analyte templates. The combination of molecular modeling and solid phase synthesis enabled the successful synthesis of nanoMIPs capable of recognition and ultrasensitive detection of endotoxins using the highly sensitive SPR biosensor with triethylamine method.