Microcontact Printing of Biomolecules on Various Polymeric Substrates: Limitations and Applicability for Fluorescence Microscopy and Subcellular Micropatterning Assays

Abstract

Polymeric materials play an emerging role in biosensing interfaces. Within this regard, polymers can serve as a superior surface for binding and printing of biomolecules. In this study, we characterized 11 different polymer foils [cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polymethylmethacrylate (PMMA), DI-Acetate, Lumirror 4001, Melinex 506, Melinex ST 504, polyamide 6, polyethersulfone, polyether ether ketone, and polyimide] to test for the applicability for surface functionalization, biomolecule micropatterning, and fluorescence microscopy approaches. Pristine polymer foils were characterized via UV-vis spectroscopy. Functional groups were introduced by plasma activation and epoxysilane-coating. Polymer modification was evaluated by water contact angle measurement and X-ray photoelectron spectroscopy. Protein micropatterns were fabricated using microcontact printing. Functionalized substrates were characterized via fluorescence contrast measurements using epifluorescence and total internal reflection fluorescence microscopy. Results showed that all polymer substrates could be chemically modified with epoxide functional groups, as indicated by reduced water contact angles compared to untreated surfaces. However, transmission and refractive index measurements revealed differences in important optical parameters, which was further proved by fluorescence contrast measurements of printed biomolecules. COC, COP, and PMMA were identified as the most promising alternatives to commonly used glass coverslips, which also showed superior applicability in subcellular micropatterning experiments.

Figure 1

Schematic workflow of the preparation of BSA micropatterns by μCP on functionalized polymer substrates. In short, polymer foils are activated by air-plasma oxidation (A), followed by the introduction of epoxide functional groups (B). Next, a PDMS stamp with a feature size of 3 μm is incubated with the biomolecule solution (e.g., BSA or BSA-Cy5 for surface passivation) (C). After a washing step (D), the stamp is placed upside-down on the functionalized polymer substrate for biomolecule transfer (E). After stripping of the stamp, the patterned substrate is bonded with a 384-well plastic casting for stabilization of the flexible foil. Fluorescence micrograph shows representative TIRF microscopy image of a BSA-Cy5 patterned polymer substrate. Scale bar: 10 μm.

Figure 2

Transmission (black) and refractive index measurements (red) of different polymeric materials and epoxy-coated glass coverslips for substrate comparison.

Figure 3

Characterization of the fluorescence pattern contrast of printed BSA-Cy5 on various substrates. (A) Schematic overview of Cy5-labeled BSA printed to the substrate surface. (B) Quantitation of the BSA-Cy5 fluorescence contrast. (C) Representative epifluorescence microscopy images of indicated BSA-Cy5 printed polymer foils. Scale bar: 30 μm. Contrast values are presented as mean ± standard deviation. n = 7; ****p < 0.0001 for comparison of fluorescence contrast with glass substrate; ns, no significant difference.

Figure 4

Characterization of the fluorescence pattern contrast of incubated streptavidin-Cy5 (STA-Cy5) on various BSA-patterned substrates. (A) Schematic overview of STA-Cy5 bound to the activated areas of the substrate surface. (B) Quantitation of the STA-Cy5 fluorescence contrast. (C) Representative TIRF microscopy images of indicated STA-Cy patterned polymer foils. Scale bar: 10 μm. Contrast values are presented as mean ± standard deviation. n = 7; ****p < 0.0001, and ***p < 0.001 for comparison of fluorescence contrast with glass substrate; ns, no significant difference.

Figure 5

Characterization of the fluorescence pattern contrast of incubated FITC-Ab on various BSA-patterned substrates further functionalized with STA. (A) Schematic overview of micropatterned biotinylated mouse IgG detected with FITC-labeled anti-mouse IgG antibody. (B) Quantitation of FITC-Ab fluorescence contrast. (C) Representative TIRF microscopy images of indicated FITC-Ab patterned polymer foils. Scale bar: 10 μm. Contrast values are presented as mean ± standard deviation. n = 7; ****p < 0.0001, and ***p < 0.001 for comparison of fluorescence contrast with glass substrate; ns, no significant difference; ns: no significant difference.

Figure 6

Applicability of selected polymer substrates for subcellular micropatterning experiments. (A) Hela cells transiently expressing the recently published GFP-fused bait-PAR-Grb2 (30) were grown on anti-HA antibody patterned polymer COC and PMMA substrates. Representative TIRF microscopy images bait-PAR-Grb2 expressing cells (green, left), BSA-Cy5 printed grid (red, middle), and the respective brightfield image of the adhered cells (gray, right). Scale bar: 30 μm. (B) Quantitation of fluorescence contrast. Data represented mean ± standard deviation of 25 analyzed cells measured in two different experiments.