Quantifizierung der Adsorption und Desorption von Proteinen durch Fluktuationsanalyse des Belegungsgrades auf nanoskopisch kleinen Sensoroberflächen
Final Report Abstract
Although the ultimate goal, to use equilibrium coverage fluctuations in order to extract the rate constants of adsorption and desorption in a single time trace, has not yet been achieved we have made considerable progress in assuming a position which, in principle, allows us to create such a device. Both prerequisites, a sensing device sensilive enough to pick up equilibrium coverage fluctuations and a reversible protein system, have been realized. In summary, experimental proof of concept for the construction of an optical biosensor using gold nanorods as sensing elements for monitoring lipid membrane mediated recognition events is presented. Plasmonic particles were immobilized on glass substrates and covered completely with lipid membranes such that the plasmon resonance shifts caused by protein induced changes in the dielectric properties at the membrane interface could be detected with single particle spectroscopy. A detailed analysis of the nanorods deposition on hydrophilic and amine-functionalized glass surfaces is performed in relation to various factors (such as the pH, ionic strength and concentration of the colloidal suspension, incubation time) in order to find the optimal conditions for obtaining a homogenous distribution of particles at the desired surface number density. The possibility of successfully draping lipid membranes over the gold particles immobilized on the glass substrate is demonstrated with complementary techniques of investigation such as phase imaging AFM, fluorescence microscopy (including FRAP) and single particle spectroscopy. The functionality and sensitivity of the proposed sensing platform is unequivocally certified by the resonance shifts of the plasmonic particles that were individually interrogated with dark field spectroscopy upon the adsorption of streptavidin to biotinylated lipid membranes. The specific interaction of annexin A1 and prothrombin with phospholipid bilayers containing acidi phospholipids has been scrutinized by means of scanning force and fluorescence microscopy, quartz crystal microbalance, ellipsometry, and modeled by dynamic Monte Carlo simulations. It was found that POPC/POPS bilayers exhibit phase separation in POPC- and POPS-enriched domains as a function of Ca2+ concentration. Annexin A1 interacts with POPC/POPS bilayers by forming irreversibly bound protein domains with monolayer thickness on POPS-enriched nanodomains, while the attachment of proteins to the POPC-enriched regions is fully reversible. A thorough kinetic analysis of the process reveals that both, the binding constant of annexin Al al the POPC-rich areas as well as the irreversible adsorption rate to the POPS-rich domains increases with calcium ion concentration. Based on the thermodynamic and kinetic data, a possible mechanism of the annexin Al membrane inieraction can be proposed. In conclusion, reversible protein-membrane systems can be provided that allow -in principleto study equilibrium coverage fluctuation. Combined with single particle sensing of adsorptin and desorption events a new sensor concept based on adsorption „noise" comes into reach.
Publications
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(2006) Adsorption and fluctuations of giant liposomes studied by electrochemical impedance measurements. Langmuir 22, 676-680
Sapper, A.; Reiss, B.; Janshoff, A.; Wegener, J.
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(2006) Partially reversible adsorption of annexin Al on POPC/POPS bilayers investigated by QCM, SFM and DMC-simulations. ChemBioChem 7, 106-115
Kastl, K.; Menke, M.; Lüthgens, E.; Faiß, S.; Gerke, V.; Janshoff, A.; Steinem, C.
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(2007) "Protein adsorption on TSM-resonators" Springer Ser. Chem. Sens. Biosens. 5,281-302
Steinem, C; Janshoff, A.
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(2008) Irreversible formation of protein domains on POPC/POPS solid supported membranes by annexin AI. Biochim. Biophys. Acta 1778, 1601-1610
Faiss, S.; Kastl, K.; Janshoff, A.; Steinem, C.
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(2008) Protein-membrane interaction probed by single plasmonic nanoparticles. Nano Lett. 8, 1724-1728
Baciu, C.; Becker, J.; Janshoff, A.; Soennichsen C.
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(2008) Protein-Membrane Interaction Probed by Single Plasmonic Nanoparticles. Plasmonics and Metamaterials, OSA Technical Digest (CD) (Optical Society of America, 2008), paper MWA5
Becker, J., Baciu, C., Janshoff, A., Sönnichsen, C.
