Design of white-light SPR apparatus and study of binding kinetics of indolicidin analogues and DNA.
Translated title
Design af white-light SPR apparat og studie af binding kinetics af indolicidin analoger og DNA.
Author
Francek, Per
Term
4. term
Education
Publication year
2011
Pages
28
Abstract
This thesis addresses the need for new antimicrobial strategies by focusing on indolicidin and its less cytotoxic analogues IL4 and IL8. The aims are to design and implement a white-light Surface Plasmon Resonance (SPR) apparatus and to use SPR to investigate the binding kinetics between these peptides and DNA. The work outlines the physical principles of SPR (including excitation via ATR and the requirement for p-polarized light) and describes robust strategies for immobilizing DNA on gold chips via thiol/mercaptohexanol monolayers or streptavidin–biotin capture. To mitigate mass transport effects, it includes the design of serial and parallel flow chambers and calibration of tubing length, and applies the Langmuir model to extract association and dissociation rate constants and equilibrium constants from equilibrium and kinetic measurements. The excerpt covers protocols and assay setups for IL4, IL8, and indolicidin binding and the development of the white-light SPR instrumentation; however, specific quantitative outcomes are not included here. Overall, the thesis establishes an experimental framework for comparing the DNA-binding kinetics of indolicidin and its analogues.
Dette speciale adresserer behovet for nye antimikrobielle strategier ved at fokusere på indolicidin og de mindre cytotoksiske analoger IL4 og IL8. Formålet er dels at designe og implementere et hvidlys-baseret Surface Plasmon Resonance (SPR) apparat og dels at anvende SPR til at undersøge bindingskinetikken mellem disse peptider og DNA. Arbejdet gennemgår de fysiske principper for SPR (herunder excitation via ATR og krav om p-polariseret lys), og beskriver robuste strategier for immobilisering af DNA på guldchips enten via thiol/mercaptohexanol-monolag eller via streptavidin–biotin-fangst. For at reducere masseoverførselseffekter indgår design af gennemstrømningsceller (serie og parallel) samt kalibrering af slangelængde, og data behandles med Langmuir-modellen til at udlede associerings- og dissociationshastigheder samt ligevægtskonstanter fra ligevægts- og kinetikmålinger. Uddraget omfatter protokoller og forsøgsopsætninger for IL4-, IL8- og indolicidin-binding samt udviklingen af hvidlys-SPR-instrumentationen; specifikke kvantitative resultater fremgår dog ikke af dette uddrag. Samlet etablerer specialet en eksperimentel ramme for sammenligning af DNA-bindingskinetik for indolicidin og dets analoger.
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