In the experiment with low linker concentration, we observe most (80-99%) of our colloids became stuck after contact with the lipid bilayer. Follow up research needs to look for ways to increase repulsion between the surface and the colloid. We would like to propose two experiments to increase repulsion by increasing the steric stabilization.
Increasing Steric Stabilization: PEG on Colloids and Lipid Bilayer
In preliminary experiments we added biotinylated PEG5000 to the colloids before incubation in a 10:1 PEG2000:DNA Linker molar ratio to increase the steric stabilization resulting from the interaction between the PEG in bilayer and on the colloid. Preliminary results did not produce diffusing colloids (they all got stuck), but the influence of factors as sample age or nuclease contamination on this result cannot be ruled out. Further investigation is needed.
Repulsion can also be increased by adding PEG5000 instead of PEG2000 to the lipid bilayer. The length of a PEG molecule scales with the square root of its molecular weight [35]. We estimate the length of PEG5000 molecule to be shorter than the DNA linker therefore providing stronger repulsion than PEG2000.
Outlook: 3D Substrates
When 2D diffusion of colloids can be accomplished for all colloids on glass substrates the next step is to use ORMOCER substrates. Based on our conclusion of part I, we expect diffusion to be slower on these substrates as the diffusion coefficients of lipid bilayers are generally lower on ORMOCER. This is not a fundamental problem as studying diffusion is also possible at low diffusion coefficients.
When 2D diffusion of colloids on ORMOCER substrates is accomplished TPA printed structures can be coated with a lipid bilayer and the full sys- tem can be completed. In principle no major changes in the setup of the system are required. Density matching will be necessary to prevent the sed- imentation of colloids. 3D imaging may prove a challenge as bright field
will refract the light beams (act as a lens), impairing the imaging. Fast z-stack acquisition in resonant confocal micropcopy might be an option.
Sample List
Figure Filename (.nd2)
40a 20150820 sample 21C FRAP 003
40b 20150820 sample 21C with-linkers FRAP RED 001
40c 20150820 sample 21C with-linkers FRAP GREEN 003
40d 20150820 sample 21C with-beads movie 001 crop
40e 20150820 sample 21C with-beads movie 001 crop
41a 20150825 sample 25A1 with-beads movie 002
41b 20150825 sample 25A1 with-beads movie 002
42a 20150722 sample 18A with-linkers red FRAP 002
42b 20150722 sample 18A with-linkers red FRAP 002
5
Acknowledgements
First of all I would like to thank my direct supervisor Casper van der Wel for your guidance and support during my master research project. Your great knowledge of experimental tools, microscopy, analysis and theory propelled this research and made my stay in the group a very instructive period. I would like to thank my supervisor Daniela Kraft; on one part for your ent- housiasm and insight in the project, but equally important, for proving the framework in which this research was conducted: An enthusiastic and profes- sionally organized research group that fosters creativity and a positive work environment. I would like to thank Indrani Chakraborty for all your help with the DNA linkers and many borrowed samples from linkers to colloids and SUVs. I would like to thank all members of the Soft Matter Physics group for good cooperation and the great I time had in this group!
Finally I would like thank and acknowledge Sebastian Hasselmann from the Fraunhofer Institute in W¨urzburg for the ORMOCER samples and helpful communication.
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