An approach for liposome immobilization using sterically stabilized micelles (SSMs) as a precursor for bio-layer interferometry-based interaction studies.
Non-fluidic bio-layer interferometry (BLI) has rapidly become a standard tool for monitoring almost all biomolecular interactions in a label-free, real-time and high-throughput manner. High-efficiency screening methods which measure the kinetics of liposomes with a variety of compounds require the immobilization of liposomes. In this work, a method is described for immobilizing liposomes for interaction studies, based on the biophysical principles of this biosensor platform. The immobilization approach includes the loading of DSPE-PEG(2000)-biotin containing sterically stabilized micelles (SSMs) which are restructured in a buffer change step, resulting in an accessible substrate for liposome immobilization. Liposomes in a concentration of 5mM of varying composition and fluidity were immobilized on the sensor surface by inserting the hydrophobic residues of the former loaded SSMs. This proof of principle was carried out using Cytochrome C as a membrane-interacting model protein. The binding of Cytochrome C to the immobilized liposomes was demonstrated, and the derived kinetic and affinity constants were similar to values given in the literature. In order to obtain a detailed understanding of this surface, and to show the integrity of the liposomes, confocal fluorescence microscopy was used. Images of immobilized liposomes containing calcein in the aqueous core indicated intact vesicles. A combination of this simple liposome immobilization approach, the possibility of automation on BLI systems with high throughput within an acceptable timescale and excellent reproducibility makes this assay suitable for basic research as well as for industrial and regulatory applications.
Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria. Electronic address: Jakob.Wallner@boku.ac.at.
Jakob
J
Wallner
Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria.
Gabriele
G
Lhota
Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria.
Markus
M
Schosserer
Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria.
Karola
K
Vorauer-Uhl
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D000327:Adsorption; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D002308:Cardiolipins / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry*; D029721:Drosophila Proteins / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays; D057927:Hydrophobic and Hydrophilic Interactions; D007368:Interferometry / Q000379:methods*; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D008823:Micelles; D008856:Microscopy, Fluorescence; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D054645:Protein Phosphatase 1 / Q000737:chemistry; D015203:Reproducibility of Results
C519576:1,1',2,2'-tetraoleoylcardiolipin; C519184:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol 2000); D002308:Cardiolipins; D029721:Drosophila Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D008823:Micelles; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010715:Phosphatidylglycerols; D015060:1,2-Dipalmitoylphosphatidylcholine; C081581:colfosceril palmitate; D011092:Polyethylene Glycols; D001710:Biotin; D045304:Cytochromes c; C585050:PPP1R15 protein, Drosophila; D054645:Protein Phosphatase 1; C017251:1,2-oleoylphosphatidylcholine; C007740:fluorexon; C030345:1,2-dipalmitoylphosphatidylglycerol
Bio-layer interferometry; Biosensor; Liposomes; Octet; Protein/membrane interactions; Sterically stabilized micelles