Water-Soluble Biomass Resist Materials Based on Polyglucuronic Acid for Eco-Friendly Photolithography

: This study presents the development of photolithography employing biomass-based resist materials derived from polyglucuronic acid. Traditional resist materials require coating and developing procedures involving organic solvents, whereas our approach enables the use of water-based spin-coating and developing processes. The water-soluble biomass resist material, derived from polyglucuronic acid, exhibited exceptional photosensitivity at an exposure wavelength of 365 nm and a dose of approximately 90 mJ/cm 2 . We successfully patterned the microstructures, creating 3 µ m lines and 6 µ m holes. This organic solvent-free coating process underscores its applicability in scenarios such as in the microfabrication on plastic substrates with limited organic solvent tolerance and surface-patterning biomaterials containing cells and culture components.


Introduction
Nanotechnology, operating at an atomic and molecular scale, represents the fourth industrial revolution, profoundly influencing contemporary society [1].In the realm of nanotechnology, microfabrication plays a pivotal role not only in manufacturing integrated circuits for the semiconductor industry but also in various domains, such as electronics, micro electromechanical systems (MEMS) devices, microfluidics, cell culture, and regenerative medicine in tissue engineering [2][3][4].Focusing on applications in the biological domain, micro-and nanofabrication techniques hold immense potential for tailoring biomaterials with desired characteristics [5,6].For instance, Otsuka et al. employed optical patterning to create a substrate surface featuring intricate three-dimensional structures, incorporating both cell-adhesive and non-adhesive regions for cultivating hepatocyte spheroids [7].Spheroids exhibit not only morphological but also functional parallels to native tissues and organs [8,9], making them an ideal platform for in vitro drug screening [10].Furthermore, stem cells cultured in two dimensions often encounter challenges in controlling differentiation pathways, resulting in a suboptimal differentiation efficiency [11].The transition from two-dimensional to three-dimensional culture, facilitated by microfabrication technology, holds great promise in advancing transplantation medicine, cell-based therapies, and regenerative medicine [12][13][14][15][16].
Additionally, microfabricating and coating processes are gaining increasing attention in the realm of biological applications.For instance, Fukuda et al. applied fine hole patterns of hyaluronic acid to a glass substrate through capillary force lithography, enabling localized cell co-culture [17].Li et al. created fine line patterns of gelatin gel to produce patterned biomaterials for skin regeneration [18].These methods employ contact-based approaches where a replica mold of dimethylpolysiloxane (PDMS) comes into direct contact with the biomaterial to transfer microstructures.However, such contact methods were restricted by the risk of deformation, pattern breakage, and pattern detachment from the substrate Coatings 2023, 13, 2038 2 of 12 due to adhesive and frictional forces at the interface during the release process [19][20][21][22][23].Moreover, indirect lithographic processes tend to be intricate [24] and offer room for enhanced throughput, which can be achieved through direct patterning of biomaterials at the micro-and nanoscales [25].Photolithography, another microfabrication technique, obviated the need for high-precision master molds and boasts the advantage of non-contact transfer of complex geometries, mitigating sample contamination through physical contact.Nevertheless, the presence of toxic chemicals in solvents, developers, and strippers used in conventional photolithography hinders biological or chemical functionalization of the target material [26].Given the contemporary focus on environmental concerns [27,28] and cytotoxicity, there is a pressing need for processes devoid of organic solvents.
For example, Park et al. developed a silk-based water-soluble resist material and conducted positive photolithography using 193 nm argon fluoride (ArF) exposure [29].Furthermore, Zhu et al. developed a water-soluble resist material with methacrylate groups grafted onto wool keratin (WK) through UV exposure, including 5 min for prebaking and 30 min for developing [30].In addition, our previous study revealed that UV exposure photolithography of amylopectin-based water-soluble resist materials enabled the processing of structures that were approximately 8 µm [31].
The aim of this study was to develop negative water-soluble biomass resist materials derived from polyglucuronic acid that were eco-and biologically friendly, devoid of organic solvents, and to apply the substrate that can be corroded and contaminated by organic solvents in biomaterial applications.Polyglucuronic acid has many carboxyl groups that are more reactive than hydroxyl groups and is characterized by its ability to dissolve in water at high concentrations, despite its large molecular weight.Figure 1 shows a comparison between photolithography of existing water-insoluble resist materials and water-soluble biomass resist materials on polymethylmethacrylate (PMMA) wafers.In contrast to conventional spin-coating and developing processes that employ organic solvents, we endeavor to establish water-soluble biomass resist materials on biocompatible PMMA wafers devoid of pattern defects, owing to the spin-coating processes using water and developing processes using water.Additionally, we explored the potential cell affinity of water-soluble biomass resist materials by assessing their Young's modulus and surface zeta potential.We discussed the cell affinity of water-soluble biomass resist materials because biomaterials should possess chemical and physical properties that either promote or inhibit interactions with specific cellular substrates, depending on the intended application [32,33].

Synthesis of Water-Soluble Biomass Resist Materials
Figure 2 shows the chemical synthesis of sugar acrylate polymer derivatives based on polyglucuronic acid, serving as the foundation for our water-soluble biomass resist

Synthesis of Water-Soluble Biomass Resist Materials
Figure 2 shows the chemical synthesis of sugar acrylate polymer derivatives based on polyglucuronic acid, serving as the foundation for our water-soluble biomass resist materials utilized in the aforementioned photolithography.The structure of linear sugar polymer derivatives, 2-isocyanatoethyl acrylate, and linear sugar acrylate polymer derivatives are also depicted in Figure 2. The alpha-linked disaccharides within the linear sugar acrylate polymer derivatives conferred water solubility to these eco-friendly biomass materials.The synthesis involved in the reaction of 2-isocyanatoethyl acrylate with hydroxy groups is present in linear sugar polymer derivatives, resulting in sugar acrylate polymer derivatives endowed with photosensitive acrylic and hydroxy groups.These derivatives were then blended with 2-isocyanatoethyl acrylate and methyl ethyl ketone.The solution comprised 18% sugar polymer derivatives and 2-isocyanatoethyl acrylate.To optimize the concentration of photosensitive groups and water-developing properties, the photosensitive acrylic and hydroxyl groups were mixed in three sugar acrylate polymer derivatives at molar ratios of approximately 32:68, 26:74, and 20:80, respectively.

Synthesis of Water-Soluble Biomass Resist Materials
Figure 2 shows the chemical synthesis of sugar acrylate polymer derivatives based on polyglucuronic acid, serving as the foundation for our water-soluble biomass resist materials utilized in the aforementioned photolithography.The structure of linear sugar polymer derivatives, 2-isocyanatoethyl acrylate, and linear sugar acrylate polymer derivatives are also depicted in Figure 2. The alpha-linked disaccharides within the linear sugar acrylate polymer derivatives conferred water solubility to these eco-friendly biomass materials.The synthesis involved in the reaction of 2-isocyanatoethyl acrylate with hydroxy groups is present in linear sugar polymer derivatives, resulting in sugar acrylate polymer derivatives endowed with photosensitive acrylic and hydroxy groups.These derivatives were then blended with 2-isocyanatoethyl acrylate and methyl ethyl ketone.The solution comprised 18% sugar polymer derivatives and 2-isocyanatoethyl acrylate.To optimize the concentration of photosensitive groups and water-developing properties, the photosensitive acrylic and hydroxyl groups were mixed in three sugar acrylate polymer derivatives at molar ratios of approximately 32:68, 26:74, and 20:80, respectively.Triethylamine (Tokyo Chem., Tokyo, Japan), at a concentration of 7.5%, was introduced to the solution, and the reactor temperature was subsequently raised at a rate of 5 °C per minute until reaching 61 °C under a nitrogen atmosphere.The solution was stirred at approximately 60 °C for 6.5 h under a nitrogen atmosphere.These chemical reactions Triethylamine (Tokyo Chem., Tokyo, Japan), at a concentration of 7.5%, was introduced to the solution, and the reactor temperature was subsequently raised at a rate of 5 • C per minute until reaching 61 • C under a nitrogen atmosphere.The solution was stirred at approximately 60 • C for 6.5 h under a nitrogen atmosphere.These chemical reactions were corroborated through attenuated total reflectance Fourier transform infrared spectroscopy, as documented in our prior investigation [34].The three resulting sugar acrylate polymer derivatives were subjected to gel permeation chromatography to ascertain their average molecular weights, yielding values of approximately 30,000; 29,000; and 29,000 for the three derivatives expected with photosensitive acrylic and hydroxy groups at molar ratios of approximately 32:68, 26:74, and 20:80, respectively.Subsequently, these sugar acrylate polymer derivatives were purified and filtered through nanoporous polytetrafluoroethylene membrane filters featuring an average pore size below 250 nm.
To facilitate water-based spin-coating and developing processes on PMMA wafers, a process prone to contamination and damage when employing organic solvents in resist solutions, we adjusted the concentrations of the constituents in the water-soluble biomass resist materials (including sugar acrylate polymer derivatives based on polyglucuronic acid, crosslinker, photo-radical initiators, surfactant, and water) to 12.2%, 1.2%, 0.4%, 0.15%, and 86%, respectively, by weight.The viscosity of the water-soluble biomass resist materials, with photosensitive acrylic and hydroxy groups at molar ratios of approximately 32:68, 26:74, and 20:80, was measured at 26 • C and falls within the range of 2.3-2.4 cP as determined using a rheometer (Visco Analyzer VAR100, Reologica, Lund, Sweden).

Exposure Sensitivity Measurement
The exposure sensitivity of water-soluble biomass resist materials was evaluated as follows: Three water-soluble biomass resist films, each featuring varying blends of photosensitive acrylic and hydroxyl groups (32:68, 26:74, 20:80), were assessed.Water-based spin-coating and developing processes were used in a photoresist coating and developing system CLEAN TRACK ACT (Tokyo Electron, Tokyo, Japan).The water-soluble biomass resist materials were spun at 2000 rpm for 60 s following spin coating at 500 rpm for 5 s on the PMMA wafer (Merck, Darmstadt, Germany).The wafer was pre-wetted at 1000 rpm for 10 s with pure water to enhance the thickness uniformity and surface smoothness and to mitigate coating defects.Following spin coating, the wafers were baked at 95 • C for 120 s.The photolithography processes were conducted using a system featuring an ASML PAS5500 i-line Stepper (ASML, Veldhoven, The Netherlands) operating at a light wavelength of 365 nm and a numerical aperture of 0.48 in the range of 16 to 149 mJ/cm 2 .Following treatment under the development conditions at 24 • C for 90 s using pure water, the water-soluble biomass resist material was dried, and the thickness was measured using an ellipsometer GES5E (SOPRALAB, Courbevoie, France).

Measurement of Film Thickness Uniformity
We assessed the uniformity of the resist film thickness on a 200 mm PMMA wafer, as well as the spin-coated properties of the water-soluble biomass resist material with sugar acrylate polymer derivatives.The water-soluble biomass resist material was spincoated onto a PMMA wafer under the above conditions.Subsequently, the water-soluble biomass resist materials were baked at 80 • C for 90 s, and the thickness was measured using the ellipsometer.

Water-Based Spin-Coating and Developing Processes in Eco-Friendly Photolithography
Eco-friendly photolithography to microfabricate the water-soluble biomass resist material was evaluated as follows.The water-soluble biomass resist materials, featuring a photosensitive acrylic group with a hydroxyl group ratio of 26:74, were spin-coated on PMMA wafers using the aforementioned photoresist coating and developing system CLEAN TRACK ACT-8 and baked at 95 • C for 120 s.The photolithography was performed on a PMMA wafer at a UV dose of 90 mJ/cm 2 .The water-soluble biomass resist material was developed using pure water and then dried.
The resist patterns of the water-soluble biomass resist material were observed using a cold field emission scanning electron microscope (SEM) (Regulus8100, Hitachi High-Tech, Tokyo, Japan).

Surface Zeta Potential Measurement
Utilizing a zeta potential and particle size analyzer (ELSZ-2000 series, Otsuka Electronics, Osaka, Japan), we measured the surface zeta potentials of flat solid surfaces featuring the existing water-insoluble resist film as a reference and the water-soluble biomass resist film.The constituents of the existing water-insoluble resist material, including epichlorohydrin and phenol-formaldehyde novolac etch resist (DOW, Midland, MI, USA), photo initiator, and surfactant, were adjusted to 94%, 5%, and 1%, respectively, by weight.As a prelude to the surface zeta potential measurement, a 10 mM NaCl solution was filtered through a 0.1 µm syringe filter and subsequently diluted by a factor of 200 to create a monitor particle stock solution.The existing reference water-insoluble resist material and the water-soluble biomass resist material, featuring a photosensitive acrylic group to hydroxyl group ratio of 26:74, were each affixed to a flat measuring cell, which served Coatings 2023, 13, 2038 5 of 12 as a cell holder.Subsequently, the monitor particle dispersion solution was introduced into the cell for measurement.Surface zeta potentials are determined using the electroosmotic plots, and can be calculated by applying the Mori and Okamoto equation and the Smoluchowski equation.

Evaluation of Film Thickness Uniformity
Figure 4 shows the spin-coated film thickness uniformity of the water-soluble b mass resist material with a photosensitive acrylic group to hydroxyl group ratio of 26 applied to a 200 mm PMMA wafer.The maximum, minimum, and average film thickn uniformity of the spin-coated water-soluble biomass resist material on the PMMA wa were 999 nm, 983 nm, and 988 nm, respectively, yielding a variance of ±8 nm.The dev tion from the median value of 991 nm was 8.0%.
Comparatively, the film thickness uniformity of previous biomass resist materi was examined.The maximum and minimum film thickness uniformity of the cellulo based biomass resist material [34] were 100.8 nm and 100.4 nm, respectively, yielding variance of ±0.2 nm on Si wafer.The deviation from the median value of 100.6 nm w 1.9%.This improvement was attributed to the use of organic solvents in the spin-coati processes, which enhanced thickness uniformity.The maximum and minimum film thi ness uniformity of the natural polysaccharide(pullulan)-based resist material [35] w 255.2 nm and 250.3 nm, respectively, yielding a variance of ±2.45 nm on the Si wafer.T deviation from the median value of 252.8 nm was 9.7%.It is more difficult to spin co PMMA wafers evenly than Si wafers, owing to their larger surface uniformity.The wat Irrespective of the ratio of photosensitive acrylic and hydroxyl groups, resist film thickness exhibited a minimal change up to an irradiation dose of 16 mJ/cm 2 , followed by a steep increase between 16 and 46 mJ/cm 2 .Notably, beyond 96 mJ/cm 2 , film thickness remained constant even with continued exposure.This suggested that nearly all chemical reaction sites have reacted at an exposure dose of 96 mJ/cm 2 .The material with 32 mol% photosensitive acrylic groups manifests as the most sensitive, followed by the 26 mol% and 20 mol% materials.This trend aligned with the experiential expectation that samples with a higher content of photosensitive acrylic groups were more likely to react.
The UV exposure sensitivity of water-soluble biomass resist films with sugar acrylate polymer derivatives ranged from 16 mJ/cm 2 to 96 mJ/cm 2 for all three content ratios (32 mol%, 26 mol%, and 20 mol% of photosensitive acrylic groups).

Evaluation of Film Thickness Uniformity
Figure 4 shows the spin-coated film thickness uniformity of the water-soluble biomass resist material with a photosensitive acrylic group to hydroxyl group ratio of 26:74 applied to a 200 mm PMMA wafer.The maximum, minimum, and average film thickness uniformity of the spin-coated water-soluble biomass resist material on the PMMA wafer were 999 nm, 983 nm, and 988 nm, respectively, yielding a variance of ±8 nm.The deviation from the median value of 991 nm was 8.0%.On the other hand, from the results of the 1.5 µm line patterns, several pattern collapses were observed in the water-soluble biomass resist material with an aspect ratio of 0.66 for a film thickness of approximately 1 µm.These results indicated that 3 µm line patterns with an aspect ratio of 0.33 can be fabricated without pattern collapse, but a structure with an aspect ratio higher than 0.33 required improvement by means of an underlying film that improves the adhesion between the resist material and the substrate.It was clearly seen that the selection of the coating solvent and developer affected the resolution capability of the water-soluble biomass resist material.In contrast to conventional chemical processes that rely on toxic and costly developer solutions, such as tetramethyl ammonium hydroxide, aqueous alkaline, and organic solvents, our water-based approach was considered to be cost-effective and eco-friendly.

Fine Processing Evaluation
A comparison of these results with those obtained in a prior photolithography process of water-soluble resist materials was carried out.
In this study, a resolution of less than half was achieved by optimizing the molecular weight and hydroxyl group concentration of sugar acrylate polymer derivatives based on polyglucuronic acid compared with our prior amylopectin-based water-soluble resist materials.
Although our study demonstrated a resolution 2 µm larger than that of Park et al., it should be noted that our process employed UV exposure (365 nm wavelength), offering a more cost-effective and versatile alternative to the expensive ArF excimer lasers.The choice of the technique should be based on the desired resolution.Comparatively, the film thickness uniformity of previous biomass resist materials was examined.The maximum and minimum film thickness uniformity of the cellulosebased biomass resist material [34] were 100.8 nm and 100.4 nm, respectively, yielding a variance of ±0.2 nm on Si wafer.The deviation from the median value of 100.6 nm was 1.9%.This improvement was attributed to the use of organic solvents in the spincoating processes, which enhanced thickness uniformity.The maximum and minimum film thickness uniformity of the natural polysaccharide(pullulan)-based resist material [35] were 255.2 nm and 250.3 nm, respectively, yielding a variance of ±2.45 nm on the Si wafer.The deviation from the median value of 252.8 nm was 9.7%.It is more difficult to spin coat PMMA wafers evenly than Si wafers, owing to their larger surface uniformity.The watersoluble biomass resist material, when spin coated on PMMA wafers for 65 s, demonstrated a film thickness uniformity comparable to that of the prior natural polysaccharide(pullulan)based resist materials, promising prospects for biomaterial applications.Wettability is also expected to be improved by removing traces of contamination on the PMMA wafer using plasma treatment or other methods [36].We plan to improve the uniformity of film thickness.In terms of throughput, the rapid 90 s developing time of the water-soluble biomass resist material derived from the sugar acrylate polymer derivatives based on polyglucuronic acid is inferred to represent a substantial advantage compared with WK water-soluble resist materials.Our future experimental plans involve the development of highly functional water-soluble biomass resist materials through the exploration of material compositions to further refine patterns and enhance sensitivity.

Mechanical Properties of Films Results
Figure 6 shows the results of Young's modulus measurements for water-soluble biomass resist films in comparison to the reference materials.The water-soluble biomass resist films exhibited a notably modest value of 0.95 GPa and superior pliancy when juxtaposed with polymethyl methacrylate (PMMA), a ubiquitous thermoplastic.It was noteworthy that the Young's modulus of the cellulose-based water-soluble biomass resist films [34] from our antecedent research reached 1.6 GPa, signifying a noticeable enhancement in film flexibility.In comparison to conventional resist materials utilizing organic solvents, the pliability of the water-soluble biomass resist films surpassed that of photoresist UV-6 (4.2 GPa) by approximately a factor of 4.4 and photoresist SU-8 (3.3 GPa) by about 3.5 times.
Given the paramount significance of cellular and substrate stiffness in the domain of biomaterials, which can profoundly influence in vitro cellular behavior [37], the precise tuning of material stiffness is of utmost importance and necessitates careful consideration.On the other hand, the mechanical strength results suggest that water-soluble biomass resist films offer a favorable level of flexibility in comparison to traditional resist materials, thus rendering them amenable to potential applications in biomaterials, particularly through spin coating on curved surfaces, capitalizing on their inherent flexibility.On the other hand, from the results of the 1.5 µm line patterns, several pattern collapses were observed in the water-soluble biomass resist material with an aspect ratio of 0.66 for a film thickness of approximately 1 µm.These results indicated that 3 µm line patterns with an aspect ratio of 0.33 can be fabricated without pattern collapse, but a structure with an aspect ratio higher than 0.33 required improvement by means of an underlying film that improves the adhesion between the resist material and the substrate.It was clearly seen that the selection of the coating solvent and developer affected the resolution capability of the water-soluble biomass resist material.In contrast to conventional chemical processes that rely on toxic and costly developer solutions, such as tetramethyl ammonium hydroxide, aqueous alkaline, and organic solvents, our water-based approach was considered to be cost-effective and eco-friendly.
A comparison of these results with those obtained in a prior photolithography process of water-soluble resist materials was carried out.
In this study, a resolution of less than half was achieved by optimizing the molecular weight and hydroxyl group concentration of sugar acrylate polymer derivatives based on polyglucuronic acid compared with our prior amylopectin-based water-soluble resist materials.
Although our study demonstrated a resolution 2 µm larger than that of Park et al., it should be noted that our process employed UV exposure (365 nm wavelength), offering a more cost-effective and versatile alternative to the expensive ArF excimer lasers.The choice of the technique should be based on the desired resolution.
In terms of throughput, the rapid 90 s developing time of the water-soluble biomass resist material derived from the sugar acrylate polymer derivatives based on polyglucuronic acid is inferred to represent a substantial advantage compared with WK water-soluble resist materials.Our future experimental plans involve the development of highly functional water-soluble biomass resist materials through the exploration of material compositions to further refine patterns and enhance sensitivity.

Mechanical Properties of Films Results
Figure 6 shows the results of Young's modulus measurements for water-soluble biomass resist films in comparison to the reference materials.The water-soluble biomass resist films exhibited a notably modest value of 0.95 GPa and superior pliancy when juxtaposed with polymethyl methacrylate (PMMA), a ubiquitous thermoplastic.It was noteworthy that the Young's modulus of the cellulose-based water-soluble biomass resist films [34] from our antecedent research reached 1.6 GPa, signifying a noticeable enhancement in film flexibility.In comparison to conventional resist materials utilizing organic solvents, the pliability of the water-soluble biomass resist films surpassed that of photoresist UV-6 (4.2 GPa) by approximately a factor of 4.

Surface Zeta Potential Measurement Results
The results of surface zeta potential measurements for flat films of the existing waterinsoluble resist material and water-soluble biomass resist material are depicted in Figure 7.The average surface zeta potential values were found to be −40.3mV for the reference water-insoluble resist film and −25.5 mV for the water-soluble biomass resist film.
Notably, cell surfaces feature large, negatively charged domains [38,39].It was considered that water-soluble biomass resist films with smaller absolute surface zeta potential values had a greater potential for cellular affinity than the existing water-insoluble resist films with larger absolute surface zeta potential values.
The interplay between biomaterials and cell substrates was considered to be influenced by multiple factors, including surface hydrophilicity, hydrophobicity [40], and surface zeta potential.We plan to continue detailed studies on the cell adhesion properties of water-soluble biomass resist films by investigating ways to improve the interaction between the biomaterial and the cell substrate by adding cell adhesive ligands to the designed biomaterials and other treatments.Given the paramount significance of cellular and substrate stiffness in the domain of biomaterials, which can profoundly influence in vitro cellular behavior [37], the precise tuning of material stiffness is of utmost importance and necessitates careful consideration.On the other hand, the mechanical strength results suggest that water-soluble biomass resist films offer a favorable level of flexibility in comparison to traditional resist materials, thus rendering them amenable to potential applications in biomaterials, particularly through spin coating on curved surfaces, capitalizing on their inherent flexibility.

Surface Zeta Potential Measurement Results
The results of surface zeta potential measurements for flat films of the existing waterinsoluble resist material and water-soluble biomass resist material are depicted in Figure 7.The average surface zeta potential values were found to be −40.3mV for the reference water-insoluble resist film and −25.5 mV for the water-soluble biomass resist film.

Conclusions
The development of water-soluble biomass resist materials, derived from sugar acrylate polymer derivatives originating from polyglucuronic acid, has introduced a promising avenue in photolithographic resist materials.These water-soluble biomass resist materials exhibit exceptional photosensitivity at a 365 nm exposure wavelength, requiring an approximate dosage of 90 mJ/cm 2 to fabricate 3 µm lines and 6 µm holes in sugar microstructures via water-based spin-coating and developing processes.Utilizing sugar as a photoresist material promotes eco-friendly photolithography by eliminating the need for organic solvents.Furthermore, Young's modulus measurements reveal that water-soluble biomass resist films possess superior flexibility compared to conventional resist materials, such as cellulose-based resists.The improved flexibility of the obtained film held promise for spin coating not only on flat wafers that were easily corroded by organic solvents, which was difficult to do with existing resists, but also on curved substrates.Moreover, the surface zeta potential measurements suggested that water-soluble biomass resist materials facilitated cell adhesion more effectively than the existing water-insoluble resist materials.This development expands the horizons of photoresists for advanced applications.Funding: This collaborative endeavor-uniting industry, academia, and government-received financial support through various sources, including the Japan Society for the Promotion of Science Bilateral Joint Research Projects (no.120229937) in conjunction with Belgium and the Toyama Notably, cell surfaces feature large, negatively charged domains [38,39].It was considered that water-soluble biomass resist films with smaller absolute surface zeta potential values had a greater potential for cellular affinity than the existing water-insoluble resist films with larger absolute surface zeta potential values.
The interplay between biomaterials and cell substrates was considered to be influenced by multiple factors, including surface hydrophilicity, hydrophobicity [40], and surface zeta potential.We plan to continue detailed studies on the cell adhesion properties of watersoluble biomass resist films by investigating ways to improve the interaction between the biomaterial and the cell substrate by adding cell adhesive ligands to the designed biomaterials and other treatments.

Conclusions
The development of water-soluble biomass resist materials, derived from sugar acrylate polymer derivatives originating from polyglucuronic acid, has introduced a promising avenue in photolithographic resist materials.These water-soluble biomass resist materials exhibit exceptional photosensitivity at a 365 nm exposure wavelength, requiring an approximate dosage of 90 mJ/cm 2 to fabricate 3 µm lines and 6 µm holes in sugar microstructures via water-based spin-coating and developing processes.Utilizing sugar as a photoresist material promotes eco-friendly photolithography by eliminating the need for organic solvents.Furthermore, Young's modulus measurements reveal that water-soluble biomass resist films possess superior flexibility compared to conventional resist materials, such as cellulose-based resists.The improved flexibility of the obtained film held promise for spin coating not only on flat wafers that were easily corroded by organic solvents, which was difficult to do with existing resists, but also on curved substrates.Moreover, the surface zeta potential measurements suggested that water-soluble biomass resist materials facilitated cell adhesion more effectively than the existing water-insoluble resist materials.This development expands the horizons of photoresists for advanced applications.

Coatings 2023 , 12 Figure 1 .
Figure 1.Comparison of existing water-insoluble resist material with organic solvents and watersoluble biomass resist material applied on PMMA wafer.

Figure 1 .
Figure 1.Comparison of existing water-insoluble resist material with organic solvents and watersoluble biomass resist material applied on PMMA wafer.

Figure 1 .
Figure 1.Comparison of existing water-insoluble resist material with organic solvents and watersoluble biomass resist material applied on PMMA wafer.

3 .Figure 3 .
Figure3illustrates the dependence of the UV exposure dose on the thickness of watersoluble biomass negative-type resist films containing sugar acrylate polymer derivatives.This measurement assessed their sensitivity to UV exposure with subsequent water-based developing processes.These measurements were standardized using an exposure dose of 149 mJ/cm 2 .Coatings 2023, 13, x FOR PEER REVIEW 6 of

Figure 3 .
Figure 3. Dependence of UV exposure dose on insoluble thickness of water-soluble biomass resist films.

Figure 4 .
Figure 4. Film thickness uniformity of water-soluble biomass resist film on 200 mm PMMA wafer.

Figure 5
Figure 5 shows photolithographic SEM images of the water-soluble biomass resist material with a photosensitive acrylic to hydroxyl group ratio of 26:74.Parts (a) and (b) show 3 µm and 1.5 µm line patterns; parts (c) and (d) show 9 µm and 6 µm hole patterns.The obtained water-soluble biomass resist material demonstrated the formation of 3 µm line patterns and 9 µm and 6 µm hole patterns on the PMMA wafers with contamination or damage from the organic coating solvent and developer in resist materials.On the other hand, from the results of the 1.5 µm line patterns, several pattern collapses were observed in the water-soluble biomass resist material with an aspect ratio of 0.66 for a film thickness of approximately 1 µm.These results indicated that 3 µm line patterns with an aspect ratio of 0.33 can be fabricated without pattern collapse, but a structure with an aspect ratio higher than 0.33 required improvement by means of an underlying film that improves the adhesion between the resist material and the substrate.It was clearly seen that the selection of the coating solvent and developer affected the resolution capability of the water-soluble biomass resist material.In contrast to conventional chemical processes that rely on toxic and costly developer solutions, such as tetramethyl ammonium hydroxide, aqueous alkaline, and organic solvents, our water-based approach was considered to be cost-effective and eco-friendly.A comparison of these results with those obtained in a prior photolithography process of water-soluble resist materials was carried out.In this study, a resolution of less than half was achieved by optimizing the molecular weight and hydroxyl group concentration of sugar acrylate polymer derivatives based on polyglucuronic acid compared with our prior amylopectin-based water-soluble resist materials.Although our study demonstrated a resolution 2 µm larger than that of Park et al., it should be noted that our process employed UV exposure (365 nm wavelength), offering a more cost-effective and versatile alternative to the expensive ArF excimer lasers.The choice of the technique should be based on the desired resolution.

Figure 4 .
Figure 4. Film thickness uniformity of water-soluble biomass resist film on 200 mm PMMA wafer.

Figure 5
Figure 5 shows photolithographic SEM images of the water-soluble biomass resist material with a photosensitive acrylic to hydroxyl group ratio of 26:74.Parts (a) and (b) show 3 µm and 1.5 µm line patterns; parts (c) and (d) show 9 µm and 6 µm hole patterns.The obtained water-soluble biomass resist material demonstrated the formation of 3 µm line patterns and 9 µm and 6 µm hole patterns on the PMMA wafers with contamination or damage from the organic coating solvent and developer in resist materials.

12 Figure 6 .
Figure6shows the results of Young's modulus measurements for water-soluble biomass resist films in comparison to the reference materials.The water-soluble biomass resist films exhibited a notably modest value of 0.95 GPa and superior pliancy when juxtaposed with polymethyl methacrylate (PMMA), a ubiquitous thermoplastic.It was noteworthy that the Young's modulus of the cellulose-based water-soluble biomass resist films[34] from our antecedent research reached 1.6 GPa, signifying a noticeable enhancement in film flexibility.In comparison to conventional resist materials utilizing organic solvents, the pliability of the water-soluble biomass resist films surpassed that of photoresist UV-6 (4.2 GPa) by approximately a factor of 4.4 and photoresist SU-8 (3.3 GPa) by about 3.5 times.Coatings 2023, 13, x FOR PEER REVIEW 9 of 12

Figure 7 .
Figure 7. Surface zeta potential of flat films of existing water-insoluble resist material and watersoluble biomass resist material.

Figure 7 .
Figure 7. Surface zeta potential of flat films of existing water-insoluble resist material and watersoluble biomass resist material.