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No description available.

Angiotensinogen is a blood protein that plays a critical role in the regulation of blood pressure in the body. This protein exists in two forms, oxidised and reduced, determined by the presence or absence of a disulfide bridge between Cys 18 and Cys 138. The ratio of oxidised to reduced angiotensinogen is 60:40 in the blood of healthy individuals - an equilibrium that is disrupted in women who develop pre-eclampsia, leading to a higher proportion of oxidised angiotensinogen in the blood. Pre-eclampsia, one of the leading causes of premature births, is a severe and potentially fatal pregnancy condition characterised by the sudden onset of symptoms such as high blood pressure and proteinuria typically during the third trimester. This condition is responsible for an estimated 550,000 deaths globally each year, and with no available treatment or cure other than early delivery of the child, there is a desperate need for a reliable and predictive diagnostic test for this condition. Can we use angiotensinogen as a biomarker for the early diagnosis of pre-eclampsia? Being able to distinguish between reduced and oxidised angiotensinogen and determine the relative amounts of each in blood samples would be of a huge diagnostic value for this condition. This thesis outlines the expression and purification of recombinant human angiotensinogen in Escherichia coli, and the development of an antibody-based SPR assay for angiotensinogen that was subsequently used to probe whether reduced and oxidised angiotensinogen can be distinguished experimentally. The assay developed was sensitive and reproducible, and demonstrated that the reduced and oxidised forms can be distinguished experimentally. The antibody bound the two forms with differential affinity, due to differences in both the association and dissociation rates of the two forms with the monoclonal antibody. Finally, in an attempt to further elucidate the differences between the two redox states of angiotensinogen, molecular dynamic simulations were carried out on angiotensinogen in the presence or absence of the disulfide bond between Cys 18 and Cys 138. These simulations revealed some quite striking differences in the dynamics between the two forms. Reduced angiotensinogen was found to be more dynamic in regions critical for binding to renin, providing a possible explanation for the reported differential affinity that renin displays for the two forms.1 Thus, reduced and oxidised angiotensinogen show some quite distinct differences and can be distinguished in an SPR-based assay, highlighting their potential for use as a biomarker in a diagnostic bioassay.

Surface plasmon waves are TM polarized charge density waves that propagate at the interface of two media with real dielectric constants of opposite sign (i.e. liquid dielectric and certain metals). Surface plasmon resonance (SPR) sensors use these waves to detect refractive index changes adjacent to the metal layer. Refractive index changes arise from the binding of an analyte (e.g. a target molecule, protein, or bacterium) to the functionalized metal layer or from interfering effects such as changes in solution index. Standard, single channel SPR sensors cannot differentiate these two effects as their design allows only one mode to be coupled. This novel self-referencing technique employs two surface plasmon modes to simultaneously measure surface binding and solution refractive index. Dual surface plasmon modes are achieved by matching the refractive indices on either side of the metal film. The two modes generated - symmetric, long-range surface plasmon (LRSP) and anti-symmetric, short-range surface plasmon (SRSP) - have different field profiles and hence assist in differentiating solution refractive index changes from surface layer formation. Amorphous Teflon, with a refractive index close to water, is chosen as the buffer layer and gold is chosen as the metal layer. Magnesium fluoride, with a higher index than Teflon, is used as the buffer layer when using ethanol as the base solution. The sensor operation was optimized through simulations to yield higher sensitivity, lower reflectivity and resonances within the spectrometers range. Optimization results showed good performance over a wide range for Teflon, MgF2 and gold thicknesses which helped in the fabrication of the sensor. Demonstration of self-referencing operation was done through two different sets of experiments: (1) formation of an alkanethiol self-assembled monolayer on gold in the presence of ethanol and methanol solutions having different refractive indices and (2) streptavidin-biotin binding with solutions of different NaCl concentration and thus different refractive indices. In both these experiments, the resonance wavelengths were accurately predicted, reflectivity varied by 10-15% and sensitivity by 25% from that of the simulated values.

Surface plasmon resonance (SPR) sensors are advantageous to other techniques of sensing chemical binding, offering quantitative, real-time, label-free results. Previous work has demonstrated the effectiveness of using dual-mode SPR sensors to differentiate between surface and background effects, making the sensors more robust to dynamic environments. This work demonstrates a technique that improves upon a previously optimized planar film dual-mode SPR sensor’s LOD by introducing a periodic array of subwavelength nano-gaps throughout the plasmon supporting material. First, general figures of merit for a sensor having an arbitrary number of modes are studied. Next, the mode effective index dispersion and magnetic field profiles of the two strongly bound modes found using a gap width of 20nm are analyzed. Qualitative analysis of the results demonstrates how such a design can enable better LODs in terms of each figure of merit. By optimizing a nano-gap enhanced sensor containing 20nm gaps, it is quantitatively demonstrated that the resulting modes improve upon almost every figure of merit, especially with respect to the orthogonality and magnitude of the sensitivity vectors, resulting in LODs approximately a factor of five less than the optimal planar design.

Surface plasmons are bound TM polarized electromagnetic waves that propagate along the interface of two materials with real dielectric constants of opposite signs. Surface plasmon resonance (SPR) sensors make use of the surface plasmon waves to detect refractive index changes occurring near this interface. For sensing purposes, this interface typically consists of a metal layer, usually gold or silver, and a liquid dielectric. SPR sensors usually measure the shift in resonance wavelength or resonance angle due to index changes adjacent to the metal layer. However this restricts the limit of detection (LOD), as the regions of low slope (intensity vs. wavelength or angle) in the SPR curve contain little information about the resonance. This work presents the technique of tunable laser interrogation of SPR sensors. A semiconductor laser with a typical lasing wavelength of 650nm was used. A 45nm gold layer sputtered on a BK7 glass substrate served as the sensor. The laser wavelength is tuned to always operate in the region of highest slope by using a custom-designed LabVIEW program. It is shown that the sensitivity is maximized and LOD is minimized by operating around the region of high slope on the SPR curve.

Protein-protein and protein-molecule interactions are complicated phenomena due to the tendency of proteins to change shape and function in response to their environment. Protein aggregation whether onto surfaces or in solution, can pose numerous problems in industry. Surface plasmon resonance (SPR) devices and quartz crystal microbalances (QCM) are two real-time, label free methods that can be used to detect the interactions between molecules on surfaces. These devices often employ self-assembled monolayers (SAMs) to produce specific surfaces for studying protein-protein interactions. The objective of this work was to develop methodologies utilizing SPR to better understand protein-protein and protein-molecule interactions with possible applications in the food and separation industrial sectors. A very well characterized whey protein, β-lactoglobulin (BLG), is used in numerous applications in the food industry. BLG can undergo different types of self-aggregation due changes in external environment factors such as buffer strength, pH or temperature. In this work, a hydrophilic SAM was developed and used to study the interaction and non-specific adsorption of BLG and palmitic acid (PA), a molecule which is known to bind to BLG. It was found that PA tended to reduce BLG conformational changes once on the surface, resulting in a decrease in its surface adhesion. Fluorescent excitation emission matrices (EEM’s) using a novel fluorescence probe technique were utilized to detect protein on the surface as well as conformational changes on the surface of the sensor, although the extent these changes could not be quantified. Another whey protein, α-lactoglobulin (AL), was utilized as a surrogate protein to study the adsorption of colloidal/particulate and protein matter (CPP) extracted from filtration studies of river water. A large fraction of natural organic matter (NOM), the major foulant in membrane based water filtration, is CPP and protein. Understanding the interactions between these components is essential in abating NOM membrane fouling. Several SPR methods were investigated in order to verify the interactions. A mixture of AL and CPP particles in solution prevented the non-specific adsorption of AL to the SAM surface. This change in association was then detected through SPR. Fluorescent EEM’s of the sensor surface verified that CPP and AL bound to the surface. This finding has fundamental significance in the interpretation of NOM-based membrane fouling. To better understand the mechanisms behind non-specific adsorption, a mechanistic mathematical model was developed to describe the adsorption of BLGs onto the hydrophilic SAM. The resulting model performed well in terms of predicting adsorption based on SPR data. The model incorporated the monomer-dimer equilibrium of BLG in solution, highlighting the impact of protein aggregation on non-specific adsorption mechanisms. For future studies, improvement in fluorescent FOP surface scan methodology would help identify different protein/molecules and conformations on the surface.

This research investigates an extension of the glancing angle deposition (GLAD) technique and a biosensing application of films produced by GLAD. The extension to GLAD, called phi-sweep (PS), improves column isolation compared to films grown by traditional GLAD (TG) as well as modifies the column tilt angle, , of the slanted columns according to tan(_{PS}) = tan(_{TG}) cos(), where is the sweep angle. The biosensing application makes use of localized surface plasmon resonance in noble metal GLAD films functionalized with rabbit immunoglobulin G (rIgG) to detect binding of anti-rabbit immunoglobulin G (anti-rIgG) to the films' surface. The extinction peak red-shifts a distance dependent on the concentration of anti-rIgG solution in a manner described by the Langmuir isotherm with a saturation value, _{max}, of 29.4 0.7 nm and a surface confined thermodynamic binding constant, K, of (2.7 0.3)10 M. / Microsystems and Nanodevices

Surface immobilization of probe molecules in surface based assays is a key area of research in the continued development of immunoassay microarrays. Interest continues to grow in microarray based immunoassays given their potential as a high throughput technique for immunodiagnostics. Therefore, it is important to thoroughly study and understand the implications of interfacial chemistry and immobilization conditions on the performance of the assay. This thesis presents a body of work that examines the impact of probe density, interfacial chemistry, and enhancement factors for arrays read with surface plasmon resonance (SPR) imaging. An array of structurally similar Salmonella disaccharides was immobilized at varying densities and the interface formed was thoroughly investigated to determine the properties of the interface. The arrays were then used with SPR imaging to evaluate the binding of an antibody specific for one disaccharide of the three stereoisomers on the array. A dilute disaccharide surface was found to provide optimal antibody binding. Higher densities result in steric hindrance of antibody binding by not allowing the disaccharide to insert into the antibody binding pocket. The role of interfacial chemistry in antibody attachment was studied to determine optimum conditions. The study examined physical adsorption, covalent attachment, and affinity capture. It was found that covalent attachment provided the most stable attachment and resulted in the lowest levels of antigen detection. Both the physical adsorption and affinity capture provided larger antigen binding capacity and therefore more sensitive antigen detection. The covalent attachment was chosen to evaluate an enhanced assay with the incorporation of gold nanoparticles. These particles provided detection limits that were an order of magnitude improved over those excluding the nanoparticles. A novel surface chemistry for antibody immobilization in SPR imaging studies was evaluated. This involved the electrochemical driven formation of mono- to multilayers of diazonium benzoic acid films. The studies showed the ability to control the thickness of the films formed and also the ability of the antibody chips to capture antigen from solution.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 6 uppsatser.