Figure 1.
Fabrication steps for the UV curable polymer stencil membrane.
A negative PDMS stamp is first fabricated using standard SU8 lithography. The stamp is then placed onto a flat substrate (e.g glass, PDMS, Petri dish). UV curable monomers are introduced by capillary flow into the gap between the stamp and the substrate. After UV curing the stamp is removed. The substrate is left for incubation with a dilute suspension of the protein of interest. Successive cycles of wash/passivation/incubation steps are then performed depending on the number of protein to be adsorbed on the patterns. After final washing the stencil is removed. Further incubation with an antifouling agent or another protein can then be performed. Notice that the proteins stay hydrated throughout the process.
Figure 2.
Reproducibility and accuracy tests for stencil patterning. A
: Circular patterns of BSA-Alexa555 (30 µm in diameter) on a glass substrate. The total covered area is 50,000 patterns. The inset is a close-up over a subset of 100 patterns. B: top: 1 µm wide lines patterned on a glass slide. Bottom: 15 µm triangle with vertex of 1.5 µm radius of curvature. Micron scale precision patterning can be achieved. C: Overlay of 400 intensity profiles of the patterns along their diameter. D: Intensity distribution of the patterns. A 7% standard deviation in protein coating is measured.
Figure 3.
Spreading assays and supported bilayers.
A: Spreading assay of macrophages on target antibodies. 30 µm patterns of BSA-Alexa647-mouse anti-BSA IgG and complementary coating with BSA-Alexa568. A brightfield image of macrophages spread onto the activating IgG area is overlaid. Post staining with secondary anti-mouse -Alexa 488 (green) indicates a specific coating of the anti BSA antibody on the pattern. B: Array of NBD-PC lipid bilayers made by stencil patterning. Top inset: close up of an individual patterned lipid bilayer. Notice the sharp boundaries with the rest of the BSA-Alexa555 passivated substrate. Bottom inset: the same pattern when the stencil is removed without BSA. The lipid bilayer radially spreads and typically dewets the glass in the centre. C: 10x10 arrays of E-cad patterns with adhering S180 cells. D: Overlay of the E-cad pattern (red stain with protein A-Alexa 647) and the endogeneous E-cadherin-GFP adhesion puncta at the glass/cell interface.
Figure 4.
A: BSA-Alexa555 round pattern on glass (30 µm diameter) surrounded by Fibrinogen-Alexa488. Notice the excellent complementarity of the coating of both proteins (Inset). B: Same protein but the top 3 rows of wells were incubated with 1 µg/ml BSA for only 20 minutes (see text for details) whereas the bottom rows were incubated with 10 µg/ml BSA to saturation. Right panel displays the average intensity of Alexa555 over a column showing that 20 minutes incubation with 1 µg/ml leads to a 20% saturation protein coating. Notice also the absence of cross contamination with the fibrinogen even in the low density zone due to the passivation step. C: Principles of multilayer stencilling to fabricate intertwined patterns of proteins. Stencil membrane are stacked and peeled one by one after each incubation/passivation step.1- first incubation on stacked layers,2- Rinse/Passivate, 3- Peel off the first layer,4- second incubation 5- Rinse/Passivate, 6- peel off last layer. D: 300 µm islands of 30 µm patterns of BSA-Alexa555 amidst patterns of BSA-Alexa488 obtained by multilayer stencilling.