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Browsing Cranfield Health by Author "Altintas, Zeynep"
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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 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.Item Open Access 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 analysisItem Open Access 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