It has recently been shown that elevated C-reactive protein (CRP) levels are associated with a blunted systemic endothelial vasodilator function, indicative of a systemic inflammatory response. It has also been recognized that inflammation may contribute to all stages of the atherosclerotic process. Several prospective studies have shown that the level of CRP is a strong predictor of future myocardial dysfunction, stroke, peripheral arterial disease, and vascular death among individuals without known cardiovascular disease. CRP is also found in association with the senile plaques and neurofibrillary tangles of Alzheimer disease. Hence, determination of the blood serum levels of CRP is of a great clinical importance. / This thesis discusses the results on the development of two electrochemical immunosensors: (i) the avidin (model) immunosensor, and (ii) the CRP immunosensor. The suitability of using a range of electrochemical techniques in probing antibody-antigen interactions was also investigated. / It was shown that a gold working electrode surface could be successfully modified by covalent binding of NHS-biotin to a self-assembled-monolayer of cystamine dihydrochloride, resulting in the construction of the avidin immunosensor. Avidin could then bind to this immunosensor (biotinated electrode) and detected using the electrochemical techniques of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) and chronoamperometry (CA). It was also shown that polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) can be used to qualitatively and quantitatively characterize avidin bound to the immunosensor. The results demonstrated that all experimental techniques used were suitable in characterizing with high sensitivity the avidin-biotin interaction, and the amount of avidin in solution. The avidin calibration curves showed high linearity in concentrations ranging from 10-10 to 5 x 10-7 M. AFM imaging confirmed that avidin forms clusters upon binding to the immunosensor, with the cluster size ranging between 28 and 33 nm. The avidin-biotin binding constant was determined to be 3.09 x 1012 M-1 . It was also determined that the avidin-biotin equilibrium is reached in ca. 20 minutes. / The CRP immunosensor was then designed on the basis of the avidin immunosensor architecture. Using a range of electrochemical techniques and PM-IRRAS, it was demonstrated that a gold electrode surface could be functionalized by CRP antibodies, covalently attached to the surface through a duplex cystamine/glutaraldehyde layer. This architecture represents the CRP immunosensor. It was then shown that CRP antigen specifically binds to the CRP immunosensor (i.e. the immobilized CRP antibody). This interaction could be characterized with high sensitivity using the electrochemical techniques of CV, EIS, DPV and CA. The CRP concentration range investigated was 10-14 to 10-8 M. A linear calibration plot was obtained. The CRP antibody-antigen binding constant was determined to be 3+/-1 x 108 M-1. The corresponding binding equilibrium is reached in ca. 10 minutes. The results show that the developed CRP immunosensor is a good candidate for further research towards developing a commercial CRP immunosensor.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.101138 |
| Date | January 2006 |
| Creators | Hennessey, Hooman. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Chemical Engineering.) |
| Rights | © Hooman Hennessey, 2006 |
| Relation | alephsysno: 002614793, proquestno: AAIMR32717, Theses scanned by UMI/ProQuest. |
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