Sequential injection analysis for the investigation of biomolecular interactions

Abstract

This thesis presents the application of Flow and Sequential Injection technologies to problems of biomolecular interaction analysis. Both biochemical and biological systems are addressed.The biochemical application involved the development of a Sequential Injection-based Biosensor (SIB) for the automated investigation of biomolecular binding phenomena. Based on Flow Injection Renewable Surface (FIRS) technology, this system enables renewal of the sensing surface for individual analyses, thus rendering regeneration of the surface unnecessary. Here, the renewable surface format is combined with the lab-on-valve flowcell design and fiber optic-based UV/vis detection for the first time to produce a flexible, multi-purpose biosensor. Additionally, precise fluidic control allows the generation of a "square impulse" flow profile which facilitates analysis of biomolecular interaction data using the first order rate equation. The SIB system presented here is used for the determination of relative affinities for a panel of monoclonal antibodies for an immobilized ligant. The commercially available BIAcore instrument, which uses surface plasmon resonance to evaluate biomolecular interactions, represents the standard in biosensor instrumentation. Therefore, comparison is made between results from BIAcore and those obtained with the SIB system. Results confirm that SIB yields comparable affinity ranking data to the BIAcore instrument.Cellular calcium response dynamics examined by Flow Injection Microscopy (FIM) in a plated-cell format constitute the biological system of interest. An epifluorescent microscope provides detection of the cellular response to stimulant by means of the calcium indicator, fura-2. Application of the stimulant is controlled via a Flow Injection system. Main features of this work are the precise fluidic control and fine temporal response engendered in the plated-cell FIM system. Because stimulant is applied in a brief, well defined pulse, temporal components to cellular response can be examined. Further, the plated-cell format allows individual cells, or sets of cells, to be targeted for observation. Repetitive stimulation of the targeted cells controls for biological variation as well as facilitating the study of response desensitization. The development of a quick, accurate automated method for the determination of dose-response relationships using this system is presented. In addition, the capacitative influx of calcium from the extracellular medium is identified and characterized.