it would have been possible if you were using PS in your liposome's lipid composition. Even I faced the same problem because I use PC and CHOL.

Can you eaborate more?

I think I get some deposiion of lipids, but I am not sure that my protocole ensures a bilayer formation, I imagine more multilayers and liposomes...

Thanks for your help!

Hi. Did you ever check how your surface looks like, e.g. before how smooth it is and after whether you have a coverage (by afm)?! There are several publications where they deposited phosphocholine vesicles on silica measured by qcm. Check as possible authors Reimhult, Hook or Reviakine.

(for a start: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680641/ )

Actually you are right, you may get a bilayer, partly multilayers or even absorbed vesicles, that are just deformed and show a different curvature. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1303580/ )

You could try, based on electrostatic interactions, altering the DMPG content, modifying your surface chemistrt (oxidizing by piranha cleaning maybe) and/or adding a divalent ion such as CaCl2 (approx. 2mM )to your solution. Thus the divalent ion can bind to the surface oxygen and the lipidheadgroup.

Make sure that the transition temperature of the lipid is not creating problems. At 20 degree you have rather rigid vesicles that might not rupture/ bend that easily.

Hi Cornelis. Thanks a lot for our answer. Yes I have checked my surface and it looked like there was a big amount of lipids, not only one bilayer, but it looked quite flat. The thing is that I repeated the deposition with the similar lipids, and I have the feeling it does not deposit so well. I wanted to investigate how to control more this deposition phenomenon to make it more reliable, e.g. if there was surface treatmets that would improve deposition, or other tips or protocoles. I need to deposit the lipids on glass because the aim is to do fluorescence microscopy. So I will try the cleaning as you suggeste, and adding clacium (which I waws recommended already as well). Usally I make the deposition at temperature higher than the transition temperature. I do not know if there are minimum recommended incubation times before cleaning the excess liposomes, or recommended repetition of the liposomes incubation? Also the "washing" step is delicate. I was looking for a protocole that would enable to wash the excess lipids without distrubing or damaging the deposited liposomes on the surface (I deposit a drop of liposomes manually and wash manually as wel at the moment)

Thanks again

Anabel-Lise

Hi. Check the literature and the more recent papers, about kinetics of liposome deposition. The authors you have in my previous posts: Richter et al uses slbs all the time. I normally used a QCM chip only.... Once the lipids are in contac with the surface it is quite a rapid process. You can find the kinetics in any Qcm paper.... showing the typical bilayer formation. A couple of minutes is usually sufficient. The liquid was just running into my cells by gravitation. I guess if u place a droplet of lipid solution, let it sit for some minutes and then put buffer solution without lipids at the site to let it rinse slowly over the slide it should work. It definitely did not disturb my deposition. Do not pipette directly on the place where the lipids are because you do not want to apply to much stress.

I am not sure how you can ensure that you have only 1 single layer though.

Hope that helps.

Yes you are right, on an SPR chip it works very well but I use hydrophobic anchors and can check the signal rise and stability. It is more difficult to control outside, and simple deposition on glass; but you are right, it should work, though we can not know how many layers we have; but sometime it seems to work better than other and it is puzzling! Thanks for your time!

Dear Eric

Thanks a lot for sharing, these are indeed very interesting articles! Do you think it would also work with e.g. DMPC:DMPG 2:1 lipid mixtures? In this case, DMPG is maybe not so easily dissolved in isopropanol? I use to dissolve it in ChCl3:MetOH:H2O 60:32.5:7.5.

Thanks!

Anabel-Lise

Thanks Eric, I understand!

Slides Preparation 

-          Before each assay, microscopic glass slides are cleaned through ultrasonication in sodium dodecyl sulfate (SDS) solution (2 % w/w), (70 % v/v) ethanol and with MilliQ water (~18 µS), respectively. Then they are dried under stream of nitrogen. 

Chamber Mounting

-          A small chamber (Figure) is mounted on microscopic slide to reduce the volume of vesicles required for an effective bilayer formation (~ 30 μl). A vial cap is glued to the glass surface using nailpolish, and let in air dry for 10 min. 

-          The slides are exposured to UV-Ozone for 10 min to clean all of the possible organic – hydrocarbon contaminations and also make the surface hydrophilic.

Supported Lipid Bilayer (SLB) Formation

-          Liposome solution prepared according to a standard protocol is incubated into the chamber.

-          The surface is left for 1 h to allow for the formation of bilayers by vesicle fusion.

-          At the end of 1 h, the surface is rinsed with excess amount of filtered, degassed phosphate buffer to remove all non-adhered vesicles. To prevent the probable exposure of the SLB to air during washing, phosphate buffer rinsing is made by serial dilution within the chamber. So, the bilayer remained fully hydrated.

I found this link very useful and worked for me!

https://bitesizebio.com/28423/make-lipid-bilayers/

bests

Thanks! I actually found out a nice protocol

- Coverslips are first cleaned with H202 and ammonia (800mL H20, 160mL of H202 and 160mL of 30%ammonia) at 65C boilied for 20 min (and after drying, cooked 30 min at 65C).

- Vesicles are resuspended at 3mg/ml in 10mM Tris-base, 150mM NaCl, pH 7.4 and extruded to liposome

- Before deposition, liposome are diluted to 0.5mg/ml in fusion buffer

10mM PBS, 150mM NaCl, 10mM MgCl2, pH 7.5.

- Glass are cleaned with 20min oxygen-plasma cleaning and liposome immediately deposited and incubated.

- After buffer + water washes, the bilayer looks quite nice!

Dear Anabel, your protocol will surely work, but you should be able to get the same results with milder cleaning methods. I earlier used Piranha solution as well, but it's a very corrosive and dangerous way. you cold try this one:

- incubation for 20min at 65C in 2% Hellmanex II solution (in Milli Q water) (ideally put the beaker in a bath sonicator)

- rinse with lots of Milli Q

-blow dry with N2 gas

-plasma clean for 30s

then you can go on with addition of liposomes, SLBs form usually within 15min.

instead of plasma cleaning, you can also incubate the glass slides for 15min in 3M KOH.

using longer plasma cleaning increases the risk that the bilayer might seem ok, but with reduced lipid mobility. if the lipid mobility is 1-3 micron^2 /s, then all should be fine.

I see! What kind of coverslips are you using? some are very hydrophobic, and I am not sure that only hellmanex is enough to get them sufficiently hydrophilic? And are you talking about DOPC bilayers, or bit more complex mixture (lipids with charges for instance, or DPPC lipids?). I am wondering whether 30 s plasma cleaning for the glass would be sufficient, I mean it is a big difference to have aprotocolo with 30s versus 20 min!!?... but I'll check the lipid mobility in the conditions I explained indeed, I don't really understand why it would be lower with long plasma, is it because the plasma could cause defects in the glass? I was aware of this for PDMS, but not for glass? Thanks!!

I use #1.5 borosilicate from Menzel. it's true that glasses from some manufactures are not great for bilayer formation. I think, that the longer plasma cleaning creates too many surface charges or so, but that's just a guess. in fact, when looking with interferometric scattering microscopy at the bilayer formation, it seemed that the SUVs would not pop open on glass slides that were treated in the plasma cleaner at long times. it's not like with PDMS, where it creates actual defects/ cracks. it worked for me with DOPC, DPPC, POPC lipids. didn't try it yet with other lipids.

best regards

Darius