Browsing by Author "Uludag, Yildiz"

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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 an affinity sensor for prostate cancer diagnosis
    (2010-11) Uludag, Yildiz; Tothill, Ibtisam E.
    Prostate carcinoma is a fatal malignancy and is a major cause of death in men in the population aged 55 and over. Early diagnosis of prostate cancer is very important for successful treatment of the disease. The increase in prostate specific antigen (PSA) levels in serum above the normal limits is the primary indication of prostate malignancy; therefore, PSA is used as a biomarker for the diagnosis and prognosis of prostate cancer. To date PSA testing still dominate as the best biomarker for prostate cancer detection even though it is not prostate specific. This project aims to develop a rapid, sensitive and specific biosensor to detect prostate cancer biomarkers in order to reduce the number of people who undergo invasive examinations for diagnosis. As part of the work, alternative surface chemistry techniques were investigated to optimise the sensors immobilisation capacity, such as the use of dendrimer monolayers and surface grafting polymers. It was found that dendrimer monolayers can be used to increase the sensor surface capacity by 1.5 times for surface plasmon resonance (SPR) sensor chips but not for quartz crystal microbalance (QCM) chips. A novel polymer (UVlink) surface was also fabricated and successfully used for biomolecule immobilisation for SPR and QCM chips. The QCMA-1 prototype biosensor was applied to develop PSA detection assays. Initially an immunoassay was constructed and optimised on the QCM sensor chip. A new buffer was then formulated to eliminate 98% of non-specific binding due to human sera proteins and this enabled the PSA detection assay to be performed in 75% human serum. Since QCMA-1 instrument is a prototype, to confirm the results the PSA assay was repeated using a commercial biosensor, Biacore 3000. This newly developed method showed a limit of detection of 0.29 ng mL"1 (in 75 % serum) with a linear dynamic detection range up to 150 ng mL"1 with Au nanoparticles employed for sensitivity enhancement using both instruments. With the achieved detection limit, it is possible to use the developed QCM and SPR assays for cancer detection in patient samples. PSA detection binding data fitted to 1:1 Langmuir binding model and KD calculated as 9.46 x IQ"10 M for the assay performed with Biacore 3000 instrument and using the QCMA-1 biosensor 5.56 x 10"10 M. These results were comparable and show that the affinity between PSA and PSA-capture antibody is in the region of 10'10 M and that QCMA-1 assay results are compatible with Biacore 3000 assay results. In the study incorporation of fPSA, cPSA and PSMA immunoassays to the prostate cancer detection test was also considered. However, the commercial available antibodies were formed to have low affinity for these markers. Patient samples collected from Bedford Hospital NHS Trust and control samples collected from Cranfleld University staff were tested and it was found that the developed QCMA-1 test clearly discriminates the PSA level of patients from the controls. A second biosensor platform based on label-free interdigitated capacitive sensing principle was also employed in the study. Interdigitated electrode (IDE) arrays were fabricated with conventional microelectronics-micromachining technologies on silicon wafers. Later IDE arrays were utilised for the detection of PSA using an LCR meter. This new prototype enabled capacitance reading of individual capacitors in turn and also enabled the detection to be performed in buffer solution. Antigen binding assays were developed to capture the PSA molecule in buffer solutions achieving a detection limit of 15.6 ng mL"1 with a linear dynamic detection range of 15.6 - 250 ng mL"1 PSA
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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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    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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    Synthesis of controlled polymeric cross-linked coatings via iniferter polymerisation in the presence of tetraethyl thiuram disulphide chain terminator
    (Elsevier Science B.V., Amsterdam., 2010-05-15T00:00:00Z) Bossi, Alessandra; Whitcombe, Michael J.; Uludag, Yildiz; Fowler, S.; Chianella, Iva; Subrahmanyam, S.; Sanchez, I.; Piletsky, Sergey A.
    A “grafting from” approach has been used for controlled deposition of cross- linked polymers by living radical polymerisation. Borosilicate glass was modified with N,N-diethylaminodithiocarbamoylpropyl(trimethoxy)silane, in order to confine the iniferter reactive groups solely at its surface, then placed in solution with monomers and cross-linker. The polymerisation was initiated by UV irradiation. Formation of the cross-linked polymers was studied in terms of time course of the reaction, type of monomers incorporated and influence of oxygen. Grafted surfaces were characterised by AFM, FT-IR, ellipsometry and contact angle measurements. The ability to control the grafted layer improved dramatically when the chain terminator agent, N,N-N′,N′-tetraethyl thiuram disulphide (TED) was added. Upon irradiation TED increases the concentration of passive capping radicals and decreases the possibility of recombination of active macro-radicals, thus prolonging their lifetime. In the absence of TED the thickness of produced coatings was below 10 nm. TED added at different concentrations assisted in the formation of grafted layers of 10–130 nm thickness. Iniferter chemistry in the presence of TED can be used for growing nanometre-scale polymer layers on solid supports. It constitutes a robust general platform for controlled grafting and offer a general solution to address the needs of surface derivatisation in sensors t