A Simplified and Robust Activation Procedure of Glass Surfaces for Printing Proteins and Subcellular Micropatterning Experiments

Abstract

Depositing biomolecule micropatterns on solid substrates via microcontact printing (µCP) usually requires complex chemical substrate modifications to initially create reactive surface groups. Here, we present a simplified activation procedure for untreated solid substrates based on a commercial polymer metal ion coating (AnteoBind^TM Biosensor reagent) that allows for direct µCP and the strong attachment of proteins via avidity binding. In proof-of-concept experiments, we identified the optimum working concentrations of the surface coating, characterized the specificity of protein binding and demonstrated the suitability of this approach by subcellular micropatterning experiments in living cells. Altogether, this method represents a significant enhancement and simplification of existing µCP procedures and further increases the accessibility of protein micropatterning for cell biological research questions.

Keywords: fluorescence microscopy; live cell analysis; micro-contact printing; micropatterning.

Figure 1

Schematic workflow of simplified large-area protein micropatterning on activated glass substrates. In short, untreated glass substrates are activated by coating with AnteoBind^TM reagent (A). In parallel, a large-area PDMS stamp is incubated with BSA (or BSA-Cy5) solution for surface passivation (B). The stamp is subsequently placed onto the substrate, resulting in a transfer of the micron-scale BSA grid for surface passivation (C,D). After the stripping of the stamp, the patterned glass substrate is bonded with a 96-well plastic casting. Next, streptavidin and biotinylated antibodies are sequentially incubated (E). In a final step, cells are seeded onto the antibody-patterned surfaces for fluorescence microscopy analysis (F). Exemplary TIRF images of BSA-Cy5 printed surfaces with FITC-labeled antibody patterns (G). AFM image of micron-scale BSA grid and respective line profile. Scale bar = 3 µm (H).

Figure 2

Characterization of AnteoBind^TM coating for protein micropatterning via µCP. (A) Glass substrates were precoated with indicated concentrations of AnteoBind^TM reagent (diluted in ddH₂O) prior µCP with BSA-inked (5 mg/mL) PDMS stamps. BSA-patterned substrates were further modified with Cy5-labeled streptavidin (50 µg/mL). Binding of STA-Cy5 to uncoated glass surfaces was used as control. (B) Glass substrates were precoated with AnteoBind^TM reagent (1:20 in ddH₂O) prior µCP with BSA-inked (indicated BSA concentrations) PDMS stamps. BSA-patterned substrates were further modified with Cy5-labeled strep(tavidin (50 µg/mL). Binding of STA-Cy5 to coated but unpatterned surfaces was used as control. (C) Representative line profiles of STA-Cy5 fluorescence signal at indicated AnteoBind^TM dilution as shown in A. (D) Representative line profiles of STA-Cy5 fluorescence signal at indicated BSA concentration as shown in B.

Figure 3

Applicability for subcellular micropatterning experiments. (A) Schematic presentation of the subcellular micropatterning assay. Cells are transiently cotransfected with fluorescently labeled bait (and prey) molecules and grown on antibody-patterned surfaces. Upon specific antibody–bait interactions, bait proteins are rearranged in the plasma membrane according to the surface pattern. (B) Representative TIRF microscopy images of GFP-ErbB2-expressing cells grown on BSA-Cy5-patterned surfaces consisting of: no antibodies (top), unspecific anti-HA antibodies (middle) and specific anti-GFP antibodies (bottom). (C) Box plots show quantitation of GFP contrast of cells grown under conditions as in (B) (n = 18 cells; **** p < 0.0001 for comparison with GFP-Ab). (D) Representative TIRF microscopy images of cells coexpressing GFP-ErbB2 and RFP-Lact-C2 grown on anti-GFP antibody-patterned surfaces.

Figure 4

Evaluation of antibody specificity. (A) Representative TIRF microscopy images of GFP-ErbB2 expressing cells grown on solely anti-GFP antibody-patterned surfaces (left) and with an additional STA layer in between (right). (B) Box plots show quantitation of GFP contrast of cells grown under indicated conditions (n > 29 cells; **** p < 0.0001 for comparison of the two different conditions). (C) Representative TIRF microscopy images of GFP-ErbB2-expressing cells grown on surfaces bearing different concentrations of anti-GFP antibody. (D) Dot plot shows quantitation of antibody concentration-dependent mean GFP contrast (n = 20 cells per concentration).