Low cost SU-8 lift-off process to fabricate a gold/glass microelectrodes array for culturing applications

Depósito de Investigación de la Universidad de Sevilla https://idus.us.es/ This is an Accepted Manuscript of an article published by Springer Science in Microsystem Technologies, Vol. 27 Issue 8 on August 2021, available at: https://doi.org/10.1007/s00542-020-05206-9 © 2022 Springer-Verlag GmbH. En idUS Licencia Creative Commons CC BY-NC-ND Low cost SU-8 lift-off process to fabricate a gold/glass microelectrodes array for culturing applications Miguel Cabello1*, Inmaculada Dom´ınguez2, Clara Mac´ıas1, Francisco Perdigones1, Carmen Aracil1, Jos´e M. Quero1 1Department of Electronic Engineering, University of Seville, Avda de los Descubrimientos sn, Seville, 41092, Spain. 2Department of Cell Biology, University of Seville, Av. de la Reina Mercedes, Seville, 41012, Spain. *Corresponding author(s). E-mail(s): mcabello[email protected]; Contributing authors: [email protected];[email protected]; p[email protected];[email protected];[email protected]; Abstract In this paper, a procedure to release SU-8 layers from glass substrates using typical undesirable effects is described. The proposed process can be used as lift off or stripping. The releasing method consists in taking advantage of the undesired thermal delamination, and the effect of UV overexposure on the delamination temperature, which are considered undesirable effects. This implies the use of SU-8 for both structural and lift off using the same facilities, without any additional photoresist, material, remover or developer. In this way, the fabrication of a glass/gold microelectrodes array (MEA) for cell culture applications is carried out as a proof of concept. The MEA is composed of gold tracks of 1.5 mm width and 8 electrodes with radii between 25 and 500 µm at its ends. The SU-8 photoresist is also used as an insulation layer releasing the pads and the electrodes. Finally, biological experiments with neuroblastoma cells SK-N-SH cultured on the MEA surface were carried out in order to validate and demonstrate the biocompatibility of both the MEA and the process. The fabrication and biological results were successful, showing a normal cell proliferation over the developed MEA. 1 1 Introduction The SU-8 photoresist is well known in MEMS applications, especially for rapid prototyping [1,2]. This material has been used for many applications in different fields. In this respect, SU-8 is one of the most important materials for polymer-based microfluidic circuits [3]. In addition, SU-8 has been used for fabricating sensors and actuators, such as microvalves [4,5], capacitive pressure sensors [6], accelerometers [7] and impulsion systems [8]. Moreover, SU-8 can also be used as an insulation layer [9,10], waveguides [11] and master molds for soft litography on glass [12], silicon [13] and printed circuit board (PCB) [14]. All these applications take advantage of the mechanical and chemical robustness of the SU-8, its transparency and biocompatibility. The majority of the devices fabricated with SU-8 requires a substrate where the deposited material remains. However, there are devices that require to be released from the substrate. The releasing of SU-8 from a substrate has been performed using different methods, for instance, a process based on the use of OmnicoatTM (Microchem Corp., MA, USA) which releases multilayer structures from silicon, but not glass [15] and using wet releasing [16]. The method reported on [17] consists in removing SU-8 after being an electroplating mold, using buckling-driven delamination assisted with a carbon dioxide snow jet . In addition, there are studies for releasing SU-8 structures using polystyrene as a sacrificial material [18], or even PDMS [19], as well as processes to strip the SU-8 [20]. The lift off process is widely used for planar microelectrodes array (MEA) fabrication [21]. This process is optimized for lift-off-resists (LOR) for example, AZ-5209E and AZ nLOF 2020 (Microchemicals GmbH). Although their objective are different, the lift off process and stripping are somehow related, that is, both of them imply to remove the SU-8 layers from the substrate. The typical fabrication process of the SU-8 includes several steps, that is, deposition, softbake, exposure, post exposure bake, development and optionally hardbake. The temperature, heating time, and UV dose have been studied to improve the understanding of the fabrication process, as well as the final results. All these studies are focused on avoiding undesirable effects, e.g., delamination, cracking or skin effect during the fabrication process [22–26]. In this paper, an alternative use of SU-8 as a lift off resist is proposed. The releasing method consists in taking advantage of the delamination, considered at first as an undesired effect, that can be used for lift off or stripping. This fact implies that the use of SU-8 is applied for both structural and lift off using the same facilities, without any additional photoresist, material, remover or developer. The target consists in introducing it as alternative method for working in abscense of LOR resists for any reason. The method is applied to the fabrication of a glass/gold MEA for cell culture, as a proof of concept. Microelectrode arrays are widely used for culture applications, such as neural networks studies [27], electrophysiology of prostate cancer cells [28] and electrostimulation of retinal explants [29]. Hence, the MEA is fabricated to validate the manufacturing process and then it is used for culturing neuroblastoma cells (SK-N-SH) to demonstrate the biocompatibility of the whole process. The rest of the paper is structured as follows. In Sect. 2, the proposed method is fully explained. The biocompatibility asssay is included in Sect. 3. The biological 2 experiments are described in Sect. 4. Finally, in Sect. 5, the main conclusions of this work are commented. 2 MEA fabrication process The aim of this method is to perform a lift-off process where the overexposure and overheating effects are used for promoting the delamination. The higher the applied temperature is, the larger the delamination. In addition, the higher the UV exposure dose, the lower the temperature at which the delamination starts and the larger the delamination [22]. The biocompatibility and transparency of the MEA materials are mandatory. For that reason, the substrate of this MEA is composed of glass, the SU-8 photoresist is used as an insulation layer, and the electrodes are made of gold. Figure 1shows the MEA design where the blue color represents the electrodes, tracks and pads which will be made of gold, and the glass is gray. Each electrode has a circular shape with different areas, ranging between 0.002 and 0.785 mm2, which means radii between 25 and 500 µm. Finally, the substrate has the standard dimensions of a glass slide, that is, 25 x 75 mm and 1 mm thick. Fig. 1 MEA design on glass substrate. The gray and blue colors represent the substrate and the gold, respectively Before manufacturing the MEA, the glass substrate is properly cleaned. In order to do so, the glass slide is rinsed using an ultrasonic cleaner and an isopropyl alcohol solution during 20 min. Then, the glass slide is rinsed on DI water and put once again inside the ultrasonic cleaner for 30 min. Finally, the glass slide is dried at room temperature for 15 min. Once the glass slide is completely dry, the fabrication process is carried out, as can be seen in Fig. 2. Figure 2A shows the glass substrate previuosly cleaned. Then, a SU-8 layer of 10 µm is spin coated at 1000 rpm (SMA - Spinner 600Pro, SMA SL), Fig. 2b. Then, the typical SU-8 fabrication process is carried out. The mask used in this step has the pattern of pads, tracks and electrodes. The result of this step can be seen in Fig. 2c. In this step, the substrate with the SU-8 is placed over a hot plate at 65 ◦C during 1 min and then at 95 ◦C for 5 min to perform the softbake. After that, the SU-8 is 3 Fig. 2 Step by step fabrication process of the MEA. a Glass slide; b Deposition of a SU-8 layer; c Photolithographic process on the SU- 8; d Sputtering of a gold layer on the substrate with SU-8; e Lift off process to promote the delamination and releasing of the SU-8 with gold; f Deposition of SU-8 as an insulation layer exposed to a UV-light source of 6.5 mW=cm2during 1 min and 40 s. Later on, the glass with the exposed SU-8 is placed over the hot plate at 65 ◦C for 1 min and then at 95 ◦C during 3 min for performing the post-exposure bake (PEB). Finally, after the SU-8 reaches the room temperature, it is immersed in the developer to remove the uncrosslinked photoresist. At this point, the glass slide is covered with cross-linked SU- 8, except those parts corresponding to the electrodes, tracks and pads. The next step consists in depositing gold on the glass slide with SU-8 (Fig. 2D). The gold deposition is performed by the sputtering technique (Sputter Coater Emitech K550X, Quorum Technologies). In this case, the gold layer has a thickness of 100 nm. After the gold sputtering, the SU-8 is released using the procedure described below. According to [22], to facilitate the delamination of the SU-8, a UV over-exposure is performed, in this case during 5 min, to reduce the delamination temperature. After that, the glass slide with SU-8 and gold is heated to temperatures over 120 ◦C for at least 15 min. After that time, the SU-8 layer can be easily removed using a jet of air, or manually, from the glass slide, remaining only the gold deposited directly on the substrate (Fig. 2e). Finally, a new SU-8 layer of about 5 µm thick is deposited over the glass slide with 4 gold, acting as an insulation layer. This final step is carried out following the same SU-8 fabrication process described on step C. The SU-8 insulation layer is covering the whole glass slide but releasing the pads and the electrodes, allowing the contact of the metal part with the outside (Fig. 2f). Figure 3shows a fabricated MEA on glass substrate. Figure 3a corresponds to an intermediate step of the fabrication process, particularly the step D, just before the lift off process, and, as can be seen, the SU-8 is starting to delaminate from the glass due to the high temperature. Figure 3b shows the final result of the MEA. Finally, Fig. 3c shows an enlarged image of one of the electrodes with SU-8 covering it, except a little circle of 100 µm of diameter which makes possible the contact between the electrode and any sample deposited on it. 3 Biocompatibility assay In order to assess the biocompatibility of the fabricated MEAs, they were used as cell substrates for culturing the neuroblastoma cell line SK-N-SH, which presents adherent growth. Neuroblastoma is a tumor formed by neuronal precursors of the sympathetic nervous system (SNS), derived from the neural crests. Neuroblastoma differentiated cells have larger nuclei and cytoplasm than those undifferentiated, which are normally round and small with scant cytoplasm [30]. The SK-N-SH cells were cultured in DMEM GlutaMAX (Dulbecco’s Modified Eagle Medium, Gibco) media supplemented with 10% fetal calf serum, penicillin (50 U/ml) and streptomycin (50 µg/ml), at 37 º C in a humidified 5% CO2 atmosphere. Cells were subcultured when 80–90% of confluence was reached. The duration of the experiment is 3 days. The MEA chips used in the experiments were made up of: (1) glass, (2) glass and gold, (3) glass, gold and SU- 8. Before using the MEAs as cell substrates, they were sterilized as follow. Firstly, they were immersed on ethanol 70% for 30 min. Then, the MEA chips were rinsed twice on sterile distilled water for 15 min each step to remove any rest of ethanol. Finally, they were exposed to UV light for 30 min and subsequently dried at 37 ◦C for at least 8 h, to avoid any rest of water to be absorbed by the substrate. The cell growth surface in each MEA was 10.20 cm2. For each experiment, 900,000 cells were seeded in each MEA. Cell growth was daily monitored using an inverted microscope, and images of the different cultures were taken. Cells growing on glass surface were considered the control cultures, and they showed around 90% of confluence 3 days after seeding. Conventional cell culture dishes for adherent cell lines were seeded in each experiment in order to control the normal cell growth rate. Dishes with no adherent surface were seeded to check that adequate substrate is necessary for correct adhesion and proliferation. 4 Experimental results and discussion The described MEAs have been used as substrates to assess cell adhesion and proliferation of neuroblastoma SK-N-SH cell line, with the aim of checking if there is any critical material or any step of the fabrication process which could cause lack of biocompatibility. For each experiment, cell growth was monitored during 3 days after cell 5 Fig. 3 Fabricated the MEA on glass substrate. a Before the lift off process; b Final result after the delamination of SU-8; c Enlarged image of the SU-8 with a hole of 100 µm of diameter to create the electrode seeding in the MEAs. Figure 4shows representative images of cell growth for days 1 and 3 for the different tested conditions, that is, glass, glass and gold, glass and gold and SU-8, and also for controls, polystyrene conventional treated surface and polystyrene hydrophobic surface. As can be seen, the cell proliferation and adhesion is similar on day 1 for the three devices (Fig. 4a, c e). The images taken on day 3 show more cells attached to the surface in all the three MEAs (Fig. 4b, d, f) reaching around the 90–95% confluence in all conditions. These results imply that proliferation and adhesion have occurred in a similar way in all the conditions. The presence of gold and SU-8 did not affect neither cell morphology nor growth rate as compared 6 with results obtained on glass alone. Regarding controls, the picture registered in conventional adherent dish shows the predictably cell growth in the third day (Fig. 4g). When cells were cultured in non adherent plastic substrates, they did not attach, and as expected, they died (Fig. 4h). According to the results, it is demonstrated that the MEA fabricated with the proposed method supports the whole process and allows the normal proliferation of SK-N-SH cells cultured on its surface. Fig. 4 Cell proliferation and growth on MEA chips: a Cells growth after 1 day of culture on glass substrate; b Cells growth after 3 days of culture on glass substrate. Cells have reached high confluence, filling the whole surface of the substrate; c Cells growth after 1 day of culture on glass substrate with gold; d Cells growth after 3 days of culture on glass substrate with gold. Gold patterned on glass does not have a negative effect on cell growth and high confluence is achieved; e Cells growth after 1 day of culture on glass substrate with SU-8 and gold; f Cells growth after 3 days of culture on glass substrate with SU- 8 and gold. SU-8 deposited on glass with gold does not have a negative effect on cells growth and high confluence is achieved; g Cell growth on adherent polystyrene conventional substrate after 3 days; h Cell death after 3 days from seeding on non adherent polystyrene surface 5 Conclusion In this paper, a low cost lift off process for SU-8 has been described, developed and validated. The process is based on using typical undesirable effects of the temperature and UV light dose over the SU-8 delamination. This process does not need additional sacrificial layers which are time consuming because of the spin coating step, the baking, and the UV exposure. In addition, the use of metals as a sacrificial layer makes the process more expensive. The proposed process does not need any auxiliary photoresist, additional developer or remover. Moreover, the unique used material is SU-8, its developer and the facilities to perform its typical photolithographic process. Although there are many LOR resists to this purpose, the proposed procedure is not intended to be a substitute method. The target consists in introducing it as alternative method for working in abscense of LOR resists for any reason. This lift off process is carried out to fabricate a transparent and biocompatible microelectrodes array for culturing applications, as a proof of concept. The materials of the MEA are glass as substrate, gold as electrodes and tracks and SU-8 as an insulation layer. The SU-8 is used for 7 both, the lift off process and the insulation layer. Therefore, In this paper, a low cost lift off process for SU-8 has been described, developed and validated. The process is based on using typical undesirable effects of the temperature and UV light dose over the SU-8 delamination. This process does not need additional sacrificial layers which are time consuming because of the spin coating step, the baking, and the UV exposure. In addition, the use of metals as a sacrificial layer makes the process more expensive. The proposed process does not need any auxiliary photoresist, additional developer or remover. Moreover, the unique used material is SU-8, its developer and the facilities to perform its typical photolithographic process. Although there are many LOR resists to this purpose, the proposed procedure is not intended to be a substitute method. The target consists in introducing it as alternative method for working in abscense of LOR resists for any reason. This lift off process is carried out to fabricate a transparent and biocompatible microelectrodes array for culturing applications, as a proof of concept. The materials of the MEA are glass as substrate, gold as electrodes and tracks and SU-8 as an insulation layer. The SU-8 is used for both, the lift off process and the insulation layer. Therefore, the whole process has been performed using only one photoresist. In order to validate the MEA biocompatibility and suitability for biological applications, neuroblastoma cells SK-N-SH has been cultured on the MEA, demonstrating a normal cell proliferation, which confirms the biocompatibility and viability of the proposed fabrication process. Acknowledgments. This work was supported by the Spanish Ministry of Economy and Competitiveness under Grant US-1265983. References [1] Lorenz, H., Despont, M., Fahrni, N., LaBianca, N., Renaud, P., Vettiger, P.: Su-8: a low-cost negative resist for mems. Journal of Micromechanics and Microengineering 7(3), 121 (1997) [2] Mukhopadhyay, S.: Microfluidic flow of aqueous ethanol and aqueous isopropyl alcohol in the su-8 based microchannel bends. J Aerospace Eng Technol 9(1), 23–26 (2019) [3] Narayan, S., Bae, K., Lehn, R., Yadav, S., Ott, M., Meckel, T., Stark, R.W.: Low-pressure bonding of monolithic su-8 microfluidic devices. 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