Two commercially available ultraviolet (UV) curable molds (PUA, acryl), one thermal curable mold (PDMS), and one self-made UV curable mold (Teflon) were used. A PUA mold (MINS-311RM; Minuta Technology, Osan, South Korea), an acryl mold (OrmoStamp; Micro Resist Technology GmbH, Berlin, Germany), PDMS (SYLGARD 184 Silicon Elastomer Kit; Dow Corning, Midland, MI, USA), Teflon (Fluolink MD 700; Solvay, Brussels, Belgium; and 1-hydroxycyclohexyl phenyl ketone; Sigma-Aldrich, St. Louis, MO, USA), polyethylene terephthalate (PET) (SH71S; SKC, Seoul, South Korea) were used. In the cell culture process, Dulbecco’s modified Eagle’s Medium (DMEM; Welgene, Gyeongsan, South Korea), fetal bovine serum (Welgene), penicillin-streptomycin (Welgene), glutaraldehyde solution (Sigma-Aldrich), ethanol (Daejung, Busan, South Korea), phosphate-buffered saline (Sigma-Aldrich), and osmium tetroxide solution (Sigma-Aldrich) were used.

The flexible Nafion micro-patterned mold was fabricated by a silicon master (linewidth, 800 nm; space, 800 nm; depth, 600 nm; line pattern). Four different molds were also fabricated based on PDMS, PUA, acryl, and Teflon. The PUA, acryl, and Teflon molds were exposed under a 365-nm UV lamp for 90 s at 40 mJ/s for curing. To create the PDMS mold, a PDMS solution mixed with an elastomer and curing agent (10:1) was dropped onto the PUA mold and incubated for 2 hours at 80°C.

After that, 3 substrates (Si wafer, glass wafer, PET film) were exposed to O₂ plasma for 3 minutes at 15 sccm (120 mTorr, 50 W, LF-plasmaster 100; JNE, Korea) to make a hydrophilic surface. Then, a thin film of Nafion was deposited by spin coating (Spin 300 A; Midas System, Daejeon, South Korea) with 5% Nafion (Nafion perfluorinated resin solution; Sigma-Aldrich) solution as the substrate. The micro-pattern was fabricated using a mold by thermal nanoimprinting (T-NIL; Midas System) for 10 minutes at 105°C and 10 bar.

The sensors were first sterilized by immersion in 70% ethanol (Daejung), followed by UV light exposure for 20 minutes. Then, MDA-MB-231 (Korean Cell Line Bank; Seoul, South Korea) and MCF-7 (Korean Cell Line Bank) were seeded on each sample in a six-well plate (SPL life Science, Pocheon, Korea) and incubated at 37℃ and 5% CO₂ in DMEM supplemented with 10% (v/v) fetal bovine serum, and 1% (v/v) penicillin-streptomycin.

Mechanically flexible, biocompatible sheets can be used as platforms for applications of biosensors and tissue scaffolds [29]. A micro-pattern can give an opportunity to guide cells or control the cell morphology. In this work, micro-fabrication of Nafion films was performed using various molds and substrates and patterned by photolithographic techniques. Nafion is suitable for use in cell adhesion because it provides a solid support, expressing a high surface charge density and good water wettability. To make a micro-pattern Nafion film, we chose a Nafion solution, not a Nafion membrane, because the Nafion solution has advantages for making patterns on diverse substrates and electrodes. As previously stated, we used different substrates to optimize the micro-pattern, so we chose the Nafion solution. First, a soft mold was fabricated to make a micro-pattern Nafion film, and we used PDMS, PUA, Teflon, and acryl molds to optimize the micro-pattern of the Nafion film. A soft mold was fabricated using a silicon master, and the micro-pattern was fabricated by thermal lithography using these molds as a base (Fig. 1A). To demonstrate the structural integrity and scalability at the microscale, SEM images were taken. The original dimensions of the pattern were 800 nm linewidth, 800 nm space, and 600 nm height. As shown in Fig. 1B, each mold had diverse dimensions; of particular note, the PDMS mold had reduced linewidth, height, and space. Furthermore, some pattern destruction was observed in the Teflon mold. Of these options, acryl showed the best performance and accurate dimensions (800 nm linewidth, 790 nm space, 590 nm height).

The films were also observed by optical microscopy to visualize large areas (Fig. 2A). In this process, Nafion films were not removed from the base substrate when using a PUA mold. The Nafion films still remained on the base substrate. We suggest that the adhesion force between Nafion and the second substrate was insufficient to remove the Nafion from the base (first) substrate. Some destruction was also observed on the Nafion films when using the Teflon mold. The PDMS and acryl molds showed the best performance on optical microscopy. SEM and AFM images were observed to optimize the mold (Fig. 2B and 2C). Acryl showed a clear and accurate line pattern on the SEM images. PDMS and Teflon showed an accurate linewidth and space, but the height was different from that of the acryl mold. AFM showed the height differences between the PDMS and acryl molds; the acryl mold had an accurate height, whereas the PDMS mold had a height of 150 nm.

Optical and AFM images of the films on the different substrates are shown in Fig. 3. An advantage of using a Nafion solution, not a Nafion membrane, is that Nafion could be deposited on various substrates. Nafion was deposited on the silicon wafer, the glass wafer, and the PET flexible film using the acryl mold. Due to the optical transparency of the entire structure, Nafion films were applied in optics for fabrication. The lines had a high structural fidelity and resolution, demonstrating the accuracy of this fabrication process to form micro-patterns over large areas [6]. AFM images are shown in Fig. 3B, with patterns showing a linewidth of around 800 nm and a space of 800 nm. However, the silicon wafer had a height of about 400 nm, while the glass wafer and PET film had a height of 100 nm. Thus, the silicon wafer showed the best performance as a substrate to make micro-patterned Nafion films.

As previously mentioned, the Nafion micro-patterns obtained by the molding process were able to control the cell-adhesive regions, unlike previously reported cell patterning techniques using stamping, enzymes, and other proteins. Fig. 4 shows the cells on films coated with MCF-7 cells (Fig. 4A and 4B) and MDA-MB-231 cells (Fig. 4C and 4D). The cells clearly showed cell adhesion patterns aligning with the designed organization. Most interestingly, although the morphology of cell adhesion of MDF-7 and MDA-MB-231 was different, both MDF-7 and MDA-MB-231 cells migrated from the micro-patterns. These data suggest that the Nafion coating with a micro-pattern organization played a crucial role through its ability to guide and enhance cell growth and adhesion according to a specifically designed order. The cells were guided by the surface morphology and the stiffness differences between the pattern and the substrate. These Nafion films can therefore be used as cell culture sheets for cell-based platforms, where the micro-pattern can be used to guide and align the cells.