Patterning of COC Polymers by Middle‐Energy Ion Beams for Selective Cell Adhesion in Microfluidic Devices

Microfluidic devices play a crucial role in advanced cell biology applications, including cell separations, cultivations, migration and interaction studies, diagnostic devices, and organ‐on‐chips. One of the frequent purposes of such devices is the ability to selectively address the attachment of cells at defined locations on the surface. This study explores the application of middle‐energy carbon, oxygen, and nitrogen ions to locally modify the surface of cyclic olefin copolymer (COC) thermoplastic material, allowing selective cell growth on patterned polymer surfaces. The investigation considers ion element type, ion beam energy, and ion irradiation fluence, analyzing their influence on the modification effect. Characterization of the modified surfaces involves various surface‐analytical methods such as contact angle, energy dispersive spectroscopy (SEM‐EDX), atomic force microscopy (AFM), x‐ray photoelectron spectroscopy (XPS), rutherford backscattering spectrometry (RBS), and elastic recoil detection analysis (ERDA). The study extends to practical aspects, with a representative cancer cell line, MCF‐7, grown on the patterned surface to evaluate the degree of selective attachment. Additionally, the stability of the irradiated patterns is tested under elevated temperatures beyond the glass transition temperature (Tg), demonstrating the compatibility of the approach with hot embossing technology. The findings underscore the potential of ion beam treatment for COC in cell‐biology‐related applications, offering insights into surface modification techniques for enhanced functionality in microfluidic devices.


Introduction
Microfluidic devices have emerged as a powerful tool in the field of cell biology, enabling precise manipulation and analysis of cells at the microscale. [1]These devices offer several advantages, including the ability to study cellular behavior in a controlled environment, precise control over small volumes of liquid, reduced sample volumes, and the potential for high-throughput analysis and automation. [2,3]These properties make them suitable for use in applications such as cell culturing, [4] cell sorting, [5] and single-cell analysis, [6] and can enable the investigation of cell behavior, [7] cell-cell interactions, [8] and cellular responses to external stimuli. [9]However, to utilize the full potential of microfluidic devices for cell culture applications, it is essential to optimize the surfaces within these devices to selectively promote cell adhesion.
The selection of suitable material plays an important role in the design of the microfluidic device, and several aspects need to be considered.The material must be usable for the available fabrication process, have to resist all chemicals that are used, resist the elevated temperatures, be compatible with biochemical and biophysical protocols, and most importantly for bioapplications, be biocompatible. [10][13] COP have several advantages over other materials, including resistance to chemicals like polar solvents, a low water absorption rate, and good electrical insulating properties. [14]They are also an excellent material for optical components, as they are highly transparent, especially in the UV part of the spectrum. [13][21] However, the inertness and low energy of the surface can also be detrimental to some biomedical applications, especially cell attachment.
To achieve selective cell adhesion within microfluidic devices, surface modifications of thermoplastic polymers play a crucial role. [22]Surface engineering techniques have been extensively explored to enhance the biocompatibility and functionalization of COP and COC.These methods involve modifying the surface chemistry to promote or inhibit cell adhesion selectively.Advantages of surface modifications include the ability to control cell attachment, [23] improved biocompatibility, [24,25] and tailored functionality for specific applications. [26][41] However, each method has its advantages and disadvantages, particularly when considering mass production in microfluidic device fabrication.For example, when plasma is used, the treated polymer tends to progressively change through aging and compensation processes. [42]Chemical and biofunctionalization may not resist the elevated temperatures often needed for bonding and final packaging of the device. [43]ne of the well-established approaches for surface modification of thermoplastic polymers is ion beam irradiation/implantation. [44]This technique involves the irradiation of the polymer surface with energetic ions to induce irreversible structural and chemical changes in the molecular structure of the material.These changes include molecular chain branching, cross-linking, and molecular degradation or scission. [45,46]Chain branching and cross-linking increase the molecular weight of the polymer; on the other hand, degradation or scission causes a reduction in the initial molecular weight. [47][50][51][52][53][54][55] The ions can be selected based on their energy and mass to achieve desired effects, such as etching, [56,57] cross-linking, [58] or surface functionalization and roughening. [53,59,60]oreover, ion irradiation of polymers with the micrometerscale beams offers an efficient technique for highly local and spatially accurate (order of micrometers) modification of chemical and functional properties.This technique allows for the selection of desired properties, including degree of reduction, modification of sp 2 /sp 3 ratio, carbon hybridization ratio, and dielectric properties (conductivity). [61]Generally, ion fluences used for polymer irradiation range between 10 9 and 10 14 cm −2 , higher fluences can lead to polymer destruction and significant carbonization. [62]Ion fluences exceeding 10 13 cm −2 cause overlapping of individual ion tracks, promoting the growth and aggregation of -bonded carbon clusters. [63]An increase in fluence above 5 × 10 13 cm −2 leads to the nucleation of nano-sized carbon-enriched clusters and the formation of a quasi-continuous carbon-embedded layer. [61]The conductivity of the irradiated polymers is then attributed to the hopping or tunneling of electrons between these conductive carbon islands. [64]iddle-energy (order of MeV) ion beam patterning offers several advantages for polymer modification in cell-based applications.It provides precise control over surface properties, such as wettability, roughness, and chemical composition, allowing for selective cell adhesion.][67][68] Ion beams in the MeV range are well-suited for ion beam writing and patterning.Furthermore, the process can be integrated into existing microfabrication workflows like hot embossing, enabling scalability for mass production. [69,70]his study focuses on the surface modifications of thermoplastic COC polymers, specifically the TOPAS brand, for achieving selective cell adhesion using middle-energy ion beam patterning.We explored the use of energetic ions of oxygen, nitrogen, and carbon with fluences of 3.75 × 10 12 , 3.75 × 10 13 , and 3.75 × 10 14 cm −2 at an energy of 600 keV.These ions were selected due to their ability to alter the material properties and enhance cell adhesion. [50,71,72]Modified materials were characterized by several methods, such as contact angle (CA) measurement, energy dispersive spectroscopy (SEM-EDX), atomic force microscopy (AFM), x-ray photoelectron spectroscopy (XPS), rutherford backscattering spectrometry (RBS), and elastic recoil detection analysis (ERDA).The patterned foils were then tested for selective cell adhesion to TOPAS polymers before and after subjecting them to temperatures 10 °C above their glass transition temperature.By investigating the surface modifications of thermoplastic polymers, this research contributes to the development of improved techniques for surface modifications potentially usable in bio-applications and microfluidics.

Results and Discussion
The primary goal of this study was to determine how ion beam irradiation can alter the surface properties of COC and if the technique can produce patterns with a positive impact on localized cell adhesion during cultivation experiments.Additionally, we investigated whether the induced cell adhesion effect can resist the exposure of COC to temperatures above T g .Carbon, nitrogen, and oxygen ions with ion fluences of 3.75 × 10 12 , 3.75 × 10 13 , and 3.75 × 10 14 cm −2 were selected to induce surface modifications on TOPAS 112 and 011 foils.The modified surface was characterized by several surface analytical techniques, and the biological assay was utilized to investigate the irradiated patterns and their ability to promote cell adhesion.

Wettability and Surface Energy
CA measurement is influenced by several factors, such as the chemical composition of the surface, droplet size, surface roughness, and image analysis.This makes direct comparisons between materials measured in different ways challenging.The contact angle of pristine COC also varies from 90°-109°depending on supplier and brand, as reported in various publications. [12,28,73,74]n our experiment, the CA of deionized water on pristine TOPAS 112 and 011 foils was 100 ± 2°and 101 ± 2°, respectively (Figure 1A).The measured angles are within the reported range of COC polymers. [12,28,73]In irradiated areas, there is a visible trend in the decrease of CA while the ion fluence increases.This effect is significant for fluences of 3.75 × 10 13 and 10 14 cm −2 .For the lowest fluence, the effect is significant only in the cases of TOPAS 011 modification and TOPAS 112 irradiated by nitrogen ions.Furthermore, it is evident that the change in ion type has minimal effect, and the trends are similar for all ions.
The OWKR method was used to calculate the surface energy.The SFE of pristine TOPAS 112 and 011 was found to be 14.3 ± 1.7 and 13.4 ± 1.3 mN m −1 , respectively (Figure 1B), which fall into the category of low-energy polymers that include polytetrafluoroethylene, polydimethylsiloxane, and polypropylene.These polymers have typical values of SFE in the range of 18-30 mN m −1 . [75]he lowest ion fluence had a significant impact on TOPAS 011 foils; this applies to all ion types.In the case of TOPAS 112, only nitrogen ions made significant changes to the foils.The ion treatment (fluences of 3.75 × 10 13 and 10 14 cm −2 ) by all ion types increased the surface energy, reaching up to 25 mN m −1 .The observed changes in SFE suggest that the middle-energy ions interact with both TOPAS materials, which results in the transformation of their hydrophobic properties to slightly hydrophilic.
The findings demonstrate that various ion modifications of TOPAS materials affect the CA and SFE, increasing the hydrophilicity of the surface, with a decrease of the contact angle down to 85°for higher fluences of ions.This decrease could be attributed to the increased presence of oxygen-containing groups on the surface caused by chain braking and oxidation. [45,74]

Surface Morphology
Material morphology plays an important role in cell attachment. [76]Therefore, we first documented the optical changes to the surface by optical microscopy.We also employed contact profilometry together with AFM to show changes in the topography of the modified surfaces.
At the higher fluences, there is a visible optical effect of the irradiation (Figure 2A), where the transparent material transforms and acquires a light yellow (fluence of 3.75 × 10 13 cm −2 ) and brown (fluence of 3.75 × 10 14 cm −2 ) tint.This color shift can be attributed to the generation of color-sensitive conjugated bonds, which subsequently leads to an increase in ion absorption. [77]he decrease in transmittance can be caused by the alteration of the chemical structure by chain scission and the generation of free radicals, potentially leading to the development of molecular cross-links, and in the case of higher fluences, even increased carbonization. [62,77]e also tried to determine if the ion modification affected the surface profile of polymers (Figure 2B).In the areas of the modification, we were able to measure polymer compaction of ≈200 nm in depth for both TOPAS foils and all ion types.However, this effect was possible to measure only on the foils irradiated with the fluence (3.75 × 10 14 cm −2 ).This effect is probably caused by material ablation, as melting would be more profound in the case of TOPAS 011 as it has a lower glass transition temperature.
Additionally, we measured the topography of the modified zones by AFM (Figure 3).It is apparent from the root mean square roughness, that with increased ion fluence, the measured roughness decreases in the majority of cases.The observed alterations in the morphology and reduced surface roughness of COC are likely attributed to the process of material ablation, wherein the original impurities present on the surface are eliminated through ion bombardment.An additional significant factor to consider is the structure of polymeric chains.Semicrystalline polymers like polyethylene (PE) exhibit increased roughness due to the higher rate of ablation for the amorphous portion of the material compared to the crystalline portion. [78]n the other hand, amorphous polymers, which include COC, undergo ablation at an even rate, resulting in no increase or, in some cases, even a decrease in roughness. [22,79]ased on the surface topography measurements, it can be concluded that the impact of surface roughness on cell adhesion enhancement will be low, as there is no structuring that would directly promote cell attachment.However, the decrease in roughness can contribute to the changes in SFE and wettability of the surface, indirectly influencing the cell interaction with the surface. [80]

Surface Chemical Composition
The backbone of the COC monomer contains no oxygen and, therefore, no chemical groups that would increase its hydrophilicity. [13]Changes in the chemical composition play a crucial role in cell interactions with the material. [81]Therefore, we first employed back-scattered electrons (BSE) to identify any changes in modified areas, together with EDX and XPS to determine the elemental composition in areas patterned by ion irradiation.
The BSE measurement (Figure 4A), where the highest fluence of 3.75 × 10 14 cm −2 is presented, shows clear material contrast between unmodified parts of the foil and irradiated zones (an array of 200 μm 4 × 4 squares).Overall, there seem to be no visible differences between TOPAS 112 and 011 material and different ions used for the irradiation.
To determine the chemical composition of the surface, we first employed the EDX method.We focused the analysis on carbon and oxygen, as no other elements were detected.Based on our analysis using EDX, it is evident that the oxygen concentration in both the pristine TOPAS 112 and 011 samples is minimal, as depicted in Figure 4B.The lowest ion fluence of 3.75 × 10 12 cm −2 significantly increases the oxygen content in TOPAS 112 material, and this effect is independent of the ion type.However, this is not true when compared to the TOPAS 011 modification, where the lowest fluence has no significant effect on the oxygen increase.The alteration in ion fluence to 3.75 × 10 13 and 10 14 cm −2 , leads to an increase in the oxygen content in both TOPAS foils, reaching up to 6%.Furthermore, it is worth noting that this impact remains consistent regardless of the specific type of ion employed for the modification (Figure 4B).
The minimal differences in carbon-oxygen ratio between fluences of 3.75 × 10 13 and 10 14 cm −2 together with the coloring of the patterned areas, might suggest increased carbonization of the material. [62,77]This effect could be responsible for the negligible difference between ion fluences, and therefore, a further increase in ion fluence will probably not affect the increase in oxygen content.We also did not detect any other elements, particularly nitrogen, in the spectra.This observation may indicate that there is a lack of chemical interaction between the nitrogen ions and the foils.Alternatively, it is possible that the detection limit of the EDX technique employed is insufficient to capture the presence of nitrogen ions or that the ions have penetrated deeper into the material, surpassing the detection range of the EDX instrument.
The elements that were detected and their respective concentrations, as determined by the XPS analysis, are presented in Table 1.The results exhibit a good correlation with the EDX measurements, indicating a consistently low oxygen content in the pristine TOPAS 112 and 011 foils.Furthermore, our findings revealed the absence of any atoms other than carbon and oxygen, suggesting that the 600 keV ions are not engaging in any direct chemical interactions with the surface.The increase in oxygen is approximately three times higher (up to 15%) compared to the increase observed in EDX measurement.The reason for this is the higher surface sensitivity of XPS, which can detect elements down to 10 nm, compared to EDX, which can scan depths of several hundred nm with a 10 kV acceleration voltage.Because of bond scissions and chain breakage, the surface probably undergoes increased oxidation.Consequently, the surrounding atmosphere's oxygen can diffuse into it more readily.It is evident that at higher ion fluences the effect of oxidation is more profound; this is especially true for oxygen ions.In the case of carbon and nitrogen ions, the differences between fluences of 3.75 × 10 13 and 10 14 are not so prominent.
In order to have a more accurate understanding of the impact of ion irradiation, the shape of the C 1s peak was evaluated.From a deconvolution analysis of the C 1s peaks and positions of each sub-peak, the concentration of each chemical component with C 1s and O 1s and the respective functional groups were calculated and summarized in Table S1 (Supporting Information).From the data, it is apparent that a pristine surface exhibit two XPS peaks (a C─C peak at 285 eV and a C─O peak at 286.5 eV), whereas three peaks (C─C, C─O, and a C═O peak at 285, 286.5, and 287.9 eV, respectively) were observed for surfaces modified by ion fluences 3.75 × 10 12 and 3.75 × 10 13 .Irradiating the surface with the highest ion fluence further increases the amount of C─O functionalities and introduces a peak corresponding to COOR at 289 eV.
To show how the elemental composition of the irradiated samples changes deeper into the material, we employed ion-beam spectroscopic methods (RBS and ERDA with 2.0, 2.5, and 3.04 MeV He ions) on samples irradiated by the highest ion fluence.The projected range of the 600 keV carbon, oxygen, and nitrogen ions in TOPAS is ≈1.6-1.7 μm, and the ratio of electronic to nuclear stopping is 6-8.
The RBS spectra of pristine and irradiated TOPAS 112 and 011 foils are shown in Figure 5A,B, respectively.The corresponding ERDA spectra can be seen in Figure 5C,D.The atomic concentrations of carbon, hydrogen, and oxygen and their ratios are presented in Table 2. Apart from the carbon and hydrogen, the non-modified TOPAS samples also show very low concentrations of oxygen (below 1%).After ion irradiation, the oxygen concentration increases for all used ions.The most significant increase is evident after irradiation using oxygen ions, where the oxygen concentration increases to 9.9% in the case of TOPAS 112 and  9.0% in the case of 011 TOPAS foils.If the carbon and nitrogen ions were used, the % ratio decreased to a value of 7.7 for all cases.On the other hand, the hydrogen concentration decreased after ion irradiation, and the most significant decrease is in the case of the TOPAS 011 sample irradiated using 600 keV oxygen ions, where the hydrogen concentration decreased from 49.4% to 32.7%.
The increase in oxygen concentration may be connected with post-irradiation oxidation of the damaged layer.The structure of both types of pristine TOPAS is mainly composed of C─C (3.4 eV) and C─H (4.3 eV) bonds, [82] while the energy of the energetic ions used (600 keV) is two orders of magnitude higher.The ion irradiation, in this case with predominant electronic stopping, leads to bond scission, and oxygen from the surrounding atmosphere can diffuse into the open structures of polymers and be captured in the defects. [63,83]The hydrogen release can be caused by the cleavage of macromolecular chains and the formation of free radicals, predominantly in the electronic stopping mode. [84]he hydrogen reduction can be attributed to the oxygen introduction to the structure of the polymer.The introduction of oxygen can be regarded as the main factor contributing to the increase in hydrophilicity of the irradiated samples. [82]ontrol of surface properties is very important in microfluidic devices because the increase in hydrophilicity of the surface helps to minimize the adsorption of certain analytes (like proteins) while also improving cell adhesion.The physicochemical characteristics of the polymer surface undergo alterations as a result of oxidation, degradation, and cross-linking during the treatment process. [30]The most typical example is the use of plasma mostly using argon and oxygen gases. [28,30,74,85]There also have been some studies on the nitrogen plasma. [86,87]The interaction between radicals, ions, and electrons generated during plasma treatment and the polymer results in the formation of a hydrophilic surface with increased surface energy, caused by the creation of C═O, ─OH, ─NH 2 , ─C≡N, ─CONH 2 , and COOH groups with nitrogen during the oxidation process. [30]Surfaces with nitrogen-containing groups are generally more favorable to cell attachment. [30,87,88]Electron beam treatment (EB) is also a viable option for surface modifications.Some studies have reported the influence of EB treatment parameters on polymer wettability, resulting in the formation of polar ─OH and C═O functional groups. [32,89,90]n contrast to other methods, the implantation of ions not only breaks the chemical chain of the polymer but also promotes their interaction with free radicals present on the chain, resulting in the formation of new chemical bonds and groups, including polar bonds and unsaturated bonds.
Nitrogen ion implantation can introduce nitrogen atoms into the COC matrix, which can chemically bond with carbon atoms in the polymer chain and form C─N, C═N, and C≡N bonds, thereby introducing polar groups into the polymer surface. [91]Oxygen ion implantation is effective in incorporating oxygen-containing functional groups (e.g., ─OH, C═O, C─O─C) onto the polymer surface. [72]This process can significantly enhance surface wettability and energy.The introduction of oxygen groups can also lead to oxidative degradation of the polymer chains, affecting the mechanical and thermal properties of the surface layer. [92]Carbon ion implantation into COCs could lead to the formation of a modified surface layer enriched with carbon. [93]This process may induce the cross-linking of polymer chains, enhancing mechanical properties such as hardness and wear resistance.Additionally, carbon implantation can lead to the formation of sp2-bonded carbon clusters, contributing to increased electrical conductivity and potentially altering optical properties. [94]ased on our findings, it is clear that ions with energies of 600 keV penetrate deeply into the material and do not interact with the surface layer.This is evident from the absence of nitrogen-containing groups in the EDX and XPS data.Therefore, the primary process that enhances wettability involves the breakdown of molecular chains and the introduction of free radicals, followed by the oxidation of the surface layer of the foils upon exposure to the atmosphere.

Cell Culture Studies
The interaction between cells and the material surface is a critical aspect of cell biology research. [81]In this study, we further investigated the effects of surface modifications by carbon, oxygen, and nitrogen ion irradiation of the surface of TOPAS 112 and 011 foils on cell behavior.These ions can influence various aspects of the cell's overall behavior and their interaction and adhesion to the surface.
After two days of cell incubation on the samples, most cells migrated to the modified pattern as shown, in Figure 6.Minimal variations were observed in cellular growth among substrates subjected to distinct ion modifications.Consequently, only substrates subjected to oxygen ion modification are depicted in Figure 6.Supporting data about substrates modified with nitrogen and carbon ions are presented in Figures S1 and S2 (Supporting Information).No significant differences were observed in cell confluence patterns when comparing fluences of 3.75 × 10 13 and 10 14 cm −2 however, noticeable variations were observed at a fluence of 3.75 × 10 12 cm −2 .At these conditions, cell confluence exhibited reduced robustness compared to higher ion fluences.There was also no difference in cell confluence between the TOPAS 112 and 011 foils.
For COC materials, there is no possibility to directly compare the modification effects of ion irradiation on cell behavior because, to our knowledge, there are no publications that would deal with this topic.However, there are several works examining improved cell adhesion by ion irradiation on other polymer materials such as polyurethanes, [66] polystyrene (PS), [68] polyethylene (PE), [95][96][97] polytetrafluoroethylene, [98] and biodegradable materials like polylactic acid. [99]Irradiating non-polar polyolefins (such as PE, PP, PS, and fluoropolymers) results in the formation of polar groups on the surface of the polymer. [96]This process improves the capacity of the polymer to be wetted, adhere to other materials, and interact with biological components.The irradiation causes macromolecular chain scission and cross-linking. [100]t higher ion fluences, the original polymer structure undergoes destruction, resulting in the formation of a highly carbonized layer that exhibits enhanced electrical conductivity. [94]It has been shown that exposing a polymer surface to high-energy ions results in a notable enhancement of cell adhesion and proliferation, which leads to improved biocompatibility of the polymer. [49,68,96]rom our data, we believe the increase in biocompatibility of the TOPAS material is caused mainly by the oxidation of the surface, and the increase in wettability is the main contributor to the enhanced cell attachment.

Aging and Thermal Changes
The important aspect of microfluidic devices being usable for bioor medical applications and being commercially viable is costeffectiveness.Therefore, the use of fabrication techniques that are suitable for mass production, such as injection molding or hot embossing, is necessary to keep the price of the final device low.However, techniques like hot embossing are often not compatible with standard modification methods due to the detrimental effect of increasing the temperature necessary for bonding the final device. [101]The often-used methods such as UV photooxidation, photografting, and the most commonly used plasma modification are prone to aging, [29,30,102] and thermal degradation. [103]lso, grafted biomolecules might not survive the increase in temperature.
Therefore, we first tried to establish how the modified surface changes over time.We measured the aging effect six months after irradiation (Figure S3, Supporting Information) and we observed no significant changes to the wettability or surface energy of the modified surfaces.This is in contrast to the other methods, mainly the plasma treatment, where the effects of the COC modification rapidly degrade during the first several days after the treatment. [29]o simulate changes to the surface during hot embossing or final packaging by thermal bonding, we subjected the modified foils to elevated temperatures of T g + 10 °C.We chose these con-ditions to take into account different embossing and bonding protocols, that are dependent on the specific hardware.To verify the stability of the modification after heating, we performed the CA measurement together with XPS.
From Figure 7, we can observe, that the heating of the foils beyond their glass transition temperature has no effect on the pristine surface, as the water CAs of both TOPAS foils are comparable to the unheated samples.The absence of differences between ion types persists; however, the effect of a decrease in wCA with increasing fluences seems to diminish.Also, overall, the values of wCAs decrease after the thermal treatment even further, but only in ion irradiated areas.
The XPS data in Table 3 also shows negligible changes to the pristine surface of both types of foils after heating.However, in the modified areas, the oxygen concentration increases by ≈1-2%.The rise in oxygen concentration can be attributed to the additional oxidation of the surface caused by surface heating, [103] which is accountable for the enhanced wettability.The key finding from these experiments is that the alteration remains intact and withstands the heat treatment that simulates the microfluidic device's final packaging.
Given the minimal disparity observed between ion modifications, the decision was made to exclusively proceed with oxygen ion modification for subsequent cell culture experiments, owing to its perceived potential for optimal attributes in cellular applications.As depicted in Figure 8, the persistent impact of substrate patterning was evident even after subjecting the substrates to a temperature of T g + 10 °C.Although this temperature induced some changes outside the pattern area, resulting in increased cell confluence outside of the pattern, the cellular growth retained its distinctive adherence to the modified pattern on both types of TOPAS foils.
In contrast to other methods of surface modification, mainly plasma and UV exposure, and also to a lesser degree electron beam treatment, [32] our approach does not suffer from the aging effect that leads to a rapid increase in contact angle and degradation of the modification. [85]This aging effect might be potentially further enhanced by elevated temperatures, even further diminishing the effect of modification.Another issue of plasmabased methods is the need for other techniques such as lithography and grafting of molecules [104] or soft-lithography [105] (where elastomeric stamp needs to be fabricated by other methods) for the creation of patterns on the surface.The advantage of our approach is the ability to use either a microstructured stencil mask for higher throughput patterning or direct ion beam writing to achieve higher resolutions, which is not possible with standard plasma treatment.
As shown in our work, the ion modification can resist the necessary increase in temperature needed for final packaging.This should allow the modified patterns to be enclosed within the microfluidic device and enable the possibility of having commercially viable microfluidic devices where controlled cellmaterial interactions in defined nano-or micro-patterns are necessary.
Table 3.The XPS measurement of TOPAS 112 and 011 foils irradiated by oxygen ions with a fluences of 3.75 × 10 12 , 3.75 × 10 13 , and 3.75 × 10 14 cm −2 after thermal treatment T g + 10 °C.The elemental composition was determined through the analysis of XPS spectra.

Conclusion
In conclusion, this study successfully modified the COC (TOPAS 112 and 011) foils through ion (carbon, nitrogen, and oxygen) irradiation, enhancing their surface properties to improve cell attachment.The ion-modified surfaces exhibited increased hydrophilicity and oxygen content, positively influencing cellmaterial interactions as shown by improved cell adhesion.Our approach also allows patterning through masks or direct writing without the need for additional methods such as lithography techniques.For use in hot embossing and thermal bonding processes, we verified the resilience of the modification to aging by heating the materials beyond the T g .These findings suggest the broad potential of our TOPAS modification method for various bio-applications and polymer microfluidics, where controlled cell-material interactions in defined nano-and micro-patterns are essential.Further research and exploration in this direction hold promise for advancing biomedical and biotechnological applications.

Experimental Section
Materials: The following TOPAS substrates were used for ion exposure and subsequent material and biological analysis: mcs-foil 112 with a thickness of 175 μm and T g of 142 °C (corresponds to TOPAS grade 6015 with norbornene content 52-57%); [13] mcs-foil 011 with a thickness of 140 μm and T g of 78 °C (corresponds to TOPAS grade 8007 with norbornene content 35-45%) [13] obtained from (ChipShop, Germany).
Ion Irradiation: The TOPAS 112 and 011 polymer foils were irradiated by 600 keV carbon, oxygen, and nitrogen ions in the implantation chamber installed on the 3 MV Tandetron MC 4130 at the Nuclear Physics Institute of CAS.The ion irradiation was accomplished at ion current densities in the range of 5-8 nA cm −2 and ion fluences of 3.75 × 10 12 , 3.75 × 10 13 , and 3.75 × 10 14 cm −2 .The projected range of the 600 keV carbon, oxygen, and nitrogen ions in TOPAS is ≈1.6-1.7 μm, and the ratio of electronic to nuclear stopping is 6-8.The implantation chamber was evacuated to a pressure of ≈6.9 × 10 −6 mbar.The two sets of samples were prepared in this way on both of the TOPAS 112 and 011 foils.One set was irradiated throughout the large surface area (used for CA, XPS, AFM, RBS, and ERDA measurement), and the second set of patterned samples was prepared through the stainless-steel stencil mask with the pattern of arrays composed of 4 × 4 square openings with a size of 200 × 200 μm (used for EDX, profilometry, and biological experiments).
Contact Angle: Changes in the wettability of the surface were determined by CA measurement.The Sessile Drop method was utilized on a Drop Shape Analyzer DSA30 (Krüss, Germany), and the measurement was carried at an ambient room temperature of 22-24 °C with instrument accuracy ± 0.1°.A 2 μL drop of deionized water or diiodomethane was deposited by a microneedle on the surface at a constant of 2 μL min using a syringe pump.sample was measured four times with a drop of water and diiodomethane after 10 s of stabilization.The static contact angle was calculated for both water and diiodomethane through the Advance software (sessile drop method) with the fitting method Ellipse (tangent-1) and an automatic baseline (if not possible, a manual baseline was used).All drops were documented with a camera image and saved.
Surface Free Energy: SFE was calculated in Advance software employing the Owens, Wendt, Rabel, and Kaelble (OWRK) method from the contact angles of two liquids: water as polar and diiodomethane (CH 2 I 2 ) as non-polar.[108] In total, eight values from each liquid were used to calculate the surface free energy.
Scanning Electron Microscopy: The surface morphology of the patterned TOPAS112 and 011 foils, shape, and material contrast of the irradiated zones were determined by SEM using a Hitachi SU-5000 (Hitachi, Japan).The applied acceleration voltage for SE and BSE observation was 10 kV, with a working distance set to ≈10 mm.Due to the non-conductivity of the samples, the low vacuum mode was used for the observation with a pressure set to 40 Pa.
Surface Contact Profilometry: SCP was used for the determination of the depth of modified zones.The measurement was conducted on surface profiler DektakXT (Brucker, Germany).
Atomic Force Microscopy: Surface topography was determined by AFM (AFM NTEGRA AURA, NT-MDT) The measurement was performed in tapping mode with the silicon high-resolution probe (ETALON series, ScanSens GmbH) with a resonant frequency of 77 kHz and force constant 3.5 N m −1 .The data were analyzed using Gwyddion software version 2.65. [109]ergy-Dispersive X-Ray Spectroscopy: EDX was used for the determination of the elemental concentration with a relative error of 10%.The measurement was carried out on a Hitachi SU-5000 (Hitachi, Japan) together with an EDAX Octane Elect Super analyzer and SDD detector (AMETEK EDAX, USA) in low vacuum mode set to 40 Pa.The applied acceleration voltage for EDX was 10 kV with a working distance of 10 mm.
X-Ray Photoelectron Spectroscopy: The XPS spectra were measured using the hemispheric analyzer Phoibos 100 (Specs, Germany) operated in FAT mode.The X-ray source was non-monochromatic (XR50), where an Al anode (line Al K) was used.The survey spectra were recorded at pass energy 40 eV (step 0.5 eV) and high-resolution spectra at pass energy 10 eV (step 0.1 eV), repeated five times to improve the signal-to-noise ratio.The spectra were referenced to the peak of aliphatic C-C bonds at 285 eV.
Software CasaXPS was used to evaluate the XPS spectra.Chemical composition was calculated from high-resolution spectra using default RSF factors from CasaXPS (RSF C = 1, RSF O = 2.93).Analyses of the C 1s peaks were performed according to. [30,110,111]Four bond types were considered: C─C, C─O, C═O, and COOR.Corresponding chemical shifts were +1.5 eV for C─O bonds, +2.9 eV for C═O bonds, and +4.0 eV for COOR.Higher shifts (e.g., 4.3 eV that would correspond to the COOH group) were not observed.The positions of the deconvolution components were fixed according to the above-mentioned chemical shifts; the FWHMs were not fixed but checked.
Rutherford Backscattering Spectrometry and Elastic Recoil Detection Analysis: RBS and ERDA were employed for the compositional study of the TOPAS foils before and after the ion irradiation with the highest ion fluence.The RBS spectra were collected using a beam of 2.0 and 3.04 MeV He ions.An Ultra-Ortec PIPS detector recorded the He ions backscattered at a laboratory scattering angle of 170°.The ERDA spectra were measured using 2.5 MeV He ions, with the primary beam coming at an angle of 75°w ith respect to the substrate surface normal and with hydrogen atoms recoiled at a scattering angle of 30°registered with the PIPS detector covered by a 12 μm Mylar foil.The typical ion current used during the RBS and ERDA analyses was ≈5 nA.To reduce the effects of the sample degradation during the RBS/ERDA analysis, several particular spectra were measured on different beam spots on the sample surface, and the final spectrum was obtained by summing the individual spectra.RBS/ERDA spectra were evaluated by SIMNRA code. [112,113]hermal Treatment of Foils: TOPAS 112 and 011 foils underwent thermal treatment to investigate how increased temperatures can alter the surface properties of the foils and affect the cell behavior in patterned areas.A stack of a 4-inch diameter silicon wafer, a modified TOPAS foil, a glass slide with an opening above the foil patterns (the opening was cut by a micro-abrasive lathe, Comco Inc., USA), and another 4-inch diameter silicon wafer were assembled.The stack was loaded into the SB6 Gen2 wafer bonder (SUSS MicroTec, Germany) chamber, and the automated recipe started, setting the top and bottom tool temperatures to 50 °C.The chamber was evacuated to 1000 mbar.The top tool compressed the inserted stack with 200 mbar pressure to achieve full contact between the top and bottom tools with the stack.Then, the temperature was raised to the target temperature through a full ramp.The TOPAS 112 foil's target temperature was set to 152 °C, and the TOPAS 011 foil's target temperature was set to 88 °C (10 °C above the glass transition temperature for both foils).The temperature ramp process lasted ≈5 min for both recipes.After the target temperature was achieved, the foils within the stack were subjected to the target temperature for 10 min.Then, the tools' pressure was released, and the chamber was purged.Both the top and bottom tools' heating was stopped, and they were cooled down to 55 °C by nitrogen flow.Finally, the processed stack was taken out of the chamber, disassembled, and the foil was ready for the cell cultivation experiments.
In Vitro Cell Culture: Human mammary gland adenocarcinoma MCF-7 (ATCC HTB-22TM adenocarcinoma cell line; ATCC) was employed as the model cell line in the investigation.The cell line was grown in high-glucose DMEM medium (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma-Aldrich), 0.1% (w/v) penicillin, and 0.1% (w/v) streptomycin (Sigma-Aldrich).The cell line was maintained in the culture flasks (surface area 25 cm 2 , maximum volume 10 mL) and cultured in the cell culture incubator (CelCulture CO 2 incubator, Esco Micro) at 37 °C in a humidified atmosphere (95% relative humidity), including 5% CO 2 .
After reaching a confluence of 40-60%, cells were harvested and used for experiments twice a week.Before subculturing the cells, the medium was removed, and the cells were rinsed with PBS buffer (5 mL) (Phosphate-Buffered Saline, Sigma-Aldrich).The cells were trypsinized with trypsin solution (0.5 mL) (Trypsin-EDTA, Sigma-Aldrich, diluted 10×) for 5 min.Cells were then suspended in the cell culture (2 mL) medium and loosened apart by repeated pipetting.The cells left in the flask were mixed with the fresh medium (5 mL) and left for incubation.The number of viable cells determined by trypan blue exclusion on a CellDrop FL (DeNovix, USA).
Foil Cell Cultivation: Foils irradiated by an ion beam were submerged for 30 s in 70% ethanol (PENTA, Czech Republic) to sterilize the surface, rinsed with MiliQ water, and dried with a stream of nitrogen gas.The substrates were then inserted into 6 well plates and submerged in cell culture media (2 mL), and to each foil, cell suspension (1 mL, 100 000 cells mL −1 ) was seeded on the surface of irradiated foils.Cell cultivation was done in the CelCulture CO 2 incubator (Esco Micro, Singapore) at 37 °C in a humidified atmosphere (95% relative humidity) including 5% CO 2 for 48 h.
Fluorescence Microscopy: Cells on foil were stained with Hoechst 33 342 (1 μg mL −1 ) (Thermo Fisher Scientific) for 30 min in the cell incubator.Afterward, the foils were rinsed by briefly submerging them into the PBS buffer.The surface of the irradiated foils with cells was documented using an inverted fluorescence microscope, the Olympus IX71 (Olympus, Japan), coupled with the CCD camera, the QImaging Retiga 2000R (QImaging), using the pE-4000 (CoolLED) as the excitation source.The cells were imaged with the objective Olympus UPLFLN 4× and UP-lanFLN 10×.For Hoechst-labeled cells, a 360-370 nm excitation filter, a 400 nm dichroic mirror, and a 420-460 nm emission filter (Chroma Technology) was used.

Figure 2 .
Figure 2. Surface characterization of TOPAS 112 (red-colored) and TOPAS 011 (blue-colored) foils modified by carbon, nitrogen, and oxygen ions with fluences of 3.75 × 10 12 , 3.75 × 10 13 , and 3.75 × 10 14 cm −2 .A) Images of color changes induced by ion irradiation from optical microscopy (no changes observed on foils modified by the lowest fluence).Scale bar = 2 mm.B) Contact profilometry measurement was used to determine the average depth (the effect of compaction) of areas modified by the highest fluence of 3.75 × 10 14 cm −2 of carbon, nitrogen, and oxygen ions.

Figure 4 .
Figure 4. Surface characterization of TOPAS 112 (red-colored) and 011 (blue-colored) foils modified by carbon, nitrogen, and oxygen ions with fluences of 3.75 × 10 12 , 3.75 × 10 13 , and 3.75 × 10 14 cm −2 .A) Images from scanning electron microscopy of the surface of both types of foils modified with the highest fluence of 3.75 × 10 14 cm −2 by carbon, nitrogen, and oxygen ions.The material contrast of modified zones is shown as a signal from backscattered electrons.Scale bar = 200 μm.B) The EDX measurement.The elemental composition was determined through the analysis of EDX spectra.The data are presented as mean ± SD (n = 12) and were analyzed using a 2-way ANOVA, comparing the pristine data set versus the modified material data sets.The symbol * represents a p-value ≤ 0.05, and *** represents p ≤ 0.001.Data with no symbols are considered statistically insignificant.

Figure 5 .
Figure 5.The spectra of ion beam analytical methods for elemental composition of TOPAS 112 and 011 foils modified by carbon, nitrogen, and oxygen ions with a fluence of 3.75 × 10 14 cm −2 .A) The RBS spectra of pristine and modified TOPAS 112 foils.B) The RBS spectra of pristine and modified TOPAS 011 foils.C) The ERDA spectra of pristine and modified TOPAS 112 foils.D) The ERDA spectra of pristine and modified TOPAS 011 foils.

Figure 6 .
Figure 6.Representative images of the MCF-7 cells after 48 h incubation on the surface of TOPAS 112 and 011 foils modified by oxygen ions (with fluences of 3.75 × 10 12 , 3.75 × 10 13 , and 3.75 × 10 14 cm −2 ) taken by fluorescence microscopy.Left, cells and modified areas in transmitted light; right, cell nuclei visualized by Hoechst 32 258 together with details of modified areas; cytoplasm visualized by CellTracker Orange.Scale bar = 100 μm.

Figure 7 .
Figure 7. Surface wettability characterization of TOPAS 112 (T g = 142 °C) and TOPAS 011 (T g = 78 °C) foils modified by carbon, nitrogen, and oxygen ions with fluences of 3.75 × 10 12 , 3.75 × 10 13 , and 3.75 × 10 14 cm −2 after thermal treatment T g + 10 °C.A) Water CA measurement.B) SFE values calculated by the OWRK method.The data are presented as mean ± SD (n = 4) and were analyzed using a 2-way ANOVA, comparing the pristine data set versus the modified material data sets.The symbol ** represents a p-value ≤ 0.01, and *** p ≤ 0.001.Data with no symbols are considered statistically insignificant.

Figure 8 .
Figure 8. Representative images of the MCF-7 cells after 48 h incubation on the surface of TOPAS 112 and 011 foils modified by oxygen ions (with fluences of 3.75 × 10 12 , 3.75 × 10 13 , and 3.75 × 10 14 cm −2 ) taken by fluorescence microscopy.Foils were heated before the cell seeding in the bonder at T g + 10 °C.Left, cells and modified areas in transmitted light; right, cell nuclei visualized by Hoechst 32 258.Scale bar = 100 μm.

Table 2 .
The results of the RBS and ERDA measurements.The elemental composition of TOPAS 112 and 011 foils irradiated by carbon, nitrogen, and oxygen ions with a fluence of 3.75 × 10 14 cm −2 .