Scanning probe and optical tweezer investigations of biomolecular interactions

A complex array of intermolecular forces controls the interactions between and within biological molecules. The desire to empirically explore these fundamental forces has led to the development of several biophysical techniques. Of these, the atomic force microscope (AFM) and the optical tweezers have been employed throughout this thesis to monitor the intermolecular forces involved in biomolecular interactions. The AFM is a well-established force sensing technique capable of measuring biomolecular interactions at a single molecule level. However, its versatility has not been extrapolated to the investigation of a drug-enzyme complex. The energy landscape for the force induced dissociation of the DHFR-methotrexate complex was studied. Revealing an energy barrier to dissociation located ~0.3nm from the bound state. Unfortunately, the AFM has a limited range of accessible loading rates and in order to profile the complete energy landscape alternative force sensing instrumentation should be considered, for example the BFP and optical tweezers. Thus, this thesis outlines the development and construction of an optical trap capable of measuring intermolecular forces between biomolecules at the single molecule level. To demonstrate the force sensing abilities of the optical set up, proof of principle measurements were performed which investigate the interactions between proteins and polymer surfaces subjected to varying degrees of argon plasma treatment. Complementary data was gained from measurements performed independently by the AFM. Changes in polymer resistance to proteins as a response to changes in polymer surface chemistry were detected utilising both AFM and optical tweezers measurements. Finally, the AFM and optical tweezers were employed as ultrasensitive biosensors. Single molecule investigations of the antibody-antigen interaction between the cardiac troponin I marker and its complementary antibody, reveals the impact therapeutic concentrations of heparin have up on the association and dissociation of the complex. In the thesis the AFM and optical tweezers independently provide complementary data towards the understanding of biomolecular interactions

Scanning probe and optical tweezer investigations of biomolecular interactions

A complex array of intermolecular forces controls the interactions between and within biological molecules. The desire to empirically explore these fundamental forces has led to the development of several biophysical techniques. Of these, the atomic force microscope (AFM) and the optical tweezers have been employed throughout this thesis to monitor the intermolecular forces involved in biomolecular interactions. The AFM is a well-established force sensing technique capable of measuring biomolecular interactions at a single molecule level. However, its versatility has not been extrapolated to the investigation of a drug-enzyme complex. The energy landscape for the force induced dissociation of the DHFR-methotrexate complex was studied. Revealing an energy barrier to dissociation located ~0.3nm from the bound state. Unfortunately, the AFM has a limited range of accessible loading rates and in order to profile the complete energy landscape alternative force sensing instrumentation should be considered, for example the BFP and optical tweezers. Thus, this thesis outlines the development and construction of an optical trap capable of measuring intermolecular forces between biomolecules at the single molecule level. To demonstrate the force sensing abilities of the optical set up, proof of principle measurements were performed which investigate the interactions between proteins and polymer surfaces subjected to varying degrees of argon plasma treatment. Complementary data was gained from measurements performed independently by the AFM. Changes in polymer resistance to proteins as a response to changes in polymer surface chemistry were detected utilising both AFM and optical tweezers measurements. Finally, the AFM and optical tweezers were employed as ultrasensitive biosensors. Single molecule investigations of the antibody-antigen interaction between the cardiac troponin I marker and its complementary antibody, reveals the impact therapeutic concentrations of heparin have up on the association and dissociation of the complex. In the thesis the AFM and optical tweezers independently provide complementary data towards the understanding of biomolecular interactions

Surface-enhanced Raman scattering measurement from a lipid bilayer encapsulating a single decahedral nanoparticle mediated by an optical trap

We present a new technique for the study of model membranes on the length-scale of a single nanosized liposome. Silver decahedral nanoparticles have been encapsulated by a model unilamellar lipid bilayer creating nano-sized lipid vesicles. The metal core has two roles (i) increasing the polarizability of vesicles, enabling a single vesicle to be isolated and confined in an optical trap, and (ii) enhancing Raman scattering from the bilayer, via the high surface-plasmon field at the sharp vertices of the decahedral particles. Combined this has allowed us to measure a Raman fingerprint from a single vesicle of 50 nmdiameter, containing just ∼104 lipid molecules in a bilayer membrane over a surface area of <0.01 µm2, equivalent to a volume of approximately 1 zepto-litre. Raman scattering is a weak and inefficient process and previous studies have required either a substantially larger bilayer area in order to obtain a detectable signal, or the tagging of lipid molecules with a chromophore to provide an indirect probe of the bilayer. Our approach is fully label-free and bio-compatible and, in the future, it will enable much more localized studies of the heterogeneous structure of lipid bilayers and of membrane-bound components than is currently possible

Information Rx: Prescribing Good Consumerism and Responsible Citizenship

Recent medical informatics and sociological literature has painted the image of a new type of patient—one that is reflexive and informed, with highly specified information needs and perceptions, as well as highly developed skills and tactics for acquiring information. Patients have been re-named “reflexive consumers.” At the same time, literature about the questionable reliability of web-based information has suggested the need to create both user tools that have pre-selected information and special guidelines for individuals to use to check the individual characteristics of the information they encounter. In this article, we examine suggestions that individuals must be assisted in developing skills for “reflexive consumerism” and what these particular skills should be. Using two types of data (discursive data from websites and promotional items, and supplementary data from interviews and ethnographic observations carried out with those working to sustain these initiatives), we examine how users are directly addressed and discussed. We argue that these initiatives prescribe skills and practices that extend beyond finding and assessing information on the internet and demonstrate that they include ideals of consumerism and citizenship

Scanning probe and optical tweezer investigations of biomolecular interactions

A complex array of intermolecular forces controls the interactions between and within biological molecules. The desire to empirically explore these fundamental forces has led to the development of several biophysical techniques. Of these, the atomic force microscope (AFM) and the optical tweezers have been employed throughout this thesis to monitor the intermolecular forces involved in biomolecular interactions. The AFM is a well-established force sensing technique capable of measuring biomolecular interactions at a single molecule level. However, its versatility has not been extrapolated to the investigation of a drug-enzyme complex. The energy landscape for the force induced dissociation of the DHFR-methotrexate complex was studied. Revealing an energy barrier to dissociation located ~0.3nm from the bound state. Unfortunately, the AFM has a limited range of accessible loading rates and in order to profile the complete energy landscape alternative force sensing instrumentation should be considered, for example the BFP and optical tweezers. Thus, this thesis outlines the development and construction of an optical trap capable of measuring intermolecular forces between biomolecules at the single molecule level. To demonstrate the force sensing abilities of the optical set up, proof of principle measurements were performed which investigate the interactions between proteins and polymer surfaces subjected to varying degrees of argon plasma treatment. Complementary data was gained from measurements performed independently by the AFM. Changes in polymer resistance to proteins as a response to changes in polymer surface chemistry were detected utilising both AFM and optical tweezers measurements. Finally, the AFM and optical tweezers were employed as ultrasensitive biosensors. Single molecule investigations of the antibody-antigen interaction between the cardiac troponin I marker and its complementary antibody, reveals the impact therapeutic concentrations of heparin have up on the association and dissociation of the complex. In the thesis the AFM and optical tweezers independently provide complementary data towards the understanding of biomolecular interactions.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Chemical and spatial analysis of protein loaded PLGA microspheres for drug delivery applications

AbstractPolymer microspheres for controlled release of therapeutic protein from within an implantable scaffold were produced and analysed using complimentary techniques to probe the surface and bulk chemistry of the microspheres. Time of Flight – Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis revealed a thin discontinuous film of polyvinyl alcohol (PVA) surfactant (circa 4.5nm thick) at the surface which was readily removed under sputtering with C60. Atomic Force Microscopy (AFM) imaging of microspheres before and after sputtering confirmed that the PVA layer was removed after sputtering revealing poly(lactic-co-glycolic) acid(PLGA). Scanning electron microscopy showed the spheres to be smooth with some shallow and generally circular depressions, often with pores in their central region. The occurrence of the protein at the surface was limited to areas surrounding these surface pores. This surface protein distribution is believed to be related to a burst release of the protein on dissolution. Analysis of the bulk properties of the microspheres by confocal Raman mapping revealed the 3D distribution of the protein showing large voids within the pores. Protein was found to be adsorbed at the interface with the PLGA oil phase following deposition on evaporation of the solvent. Protein was also observed concentrated within pores measuring approximately 2μm across. The presence of protein in large voids and concentrated pores was further scrutinised by ToF-SIMS of sectioned microspheres. This paper demonstrates that important information for optimisation of such complex bioformulations, including an understanding of the release profile can be revealed by complementary surface and bulk analysis allowing optimisation of the therapeutic effect of such formulations