J ou rn al o f M at er ia ls R es ea rc h 2 02 3 w w w .m rs .o rg /jm r Vol.:(0123456789) DOI:10.1557/s43578-023-00909-x © The Author(s), under exclusive licence to The Materials Research Society 2023 Article Sub‑micron patterning of metal oxide surfaces via microcontact printing and microtransfer molding of amphiphilic molecules and antifouling application Josué Cordero‑Guerrero1, Gabriel Jiménez‑Thuel1, Sergio A. Paniagua2,3,a) 1 Present Address: Chemistry Graduate Program, Universidad de Costa Rica, San José, Costa Rica 2 Natural Sciences Department, Universidad de Costa Rica, West Campus, Alajuela, Costa Rica 3 Present Address: National Nanotechnology Laboratory (LANOTEC), CENAT-CONARE, San José, Costa Rica a) Address all correspondence to this author. e-mail: spaniagua@cenat.ac.cr; sergio.paniagua@ucr.ac.cr Received: 28 October 2022; accepted: 17 January 2023 Low cost, potentially scalable soft lithography approaches are employed here to generate nano and microscale line patterns of n‑octadecylphosphonic acid and n‑octadecyltriethoxysilane with thicknesses ranging from 2 up to 150 nm depending on conditions and substrates used. Elastomer stamps generated from commercial optical media (DVD‑R and CD‑R) are used to print the amphiphile n‑octadecylphosphonic acid (ODPA) on mica, Si(111), and aluminum AA6063, while n‑octadecyltriethoxysilane (OTES) is printed on AA6063. The thicker ODPA pattern generated via microtransfer molding is robust enough to act as resist for wet etch on aluminum alloy at its resolution limits, allowing a more permanent pattern directly on the substrate (resulting in a pitch of 1600 nm and crest full width half maximum of 820 nm). Upon surface modification with a non‑polar terminated monolayer (post‑patterning), the water contact angle is shown to increase relative to unpatterned hydrophobic control, resulting in significantly less bacteria adhesion. Introduction Research on soft lithography is on the rise due to its wide-rang- ing applications, low cost, scalability and nano- and microscale feature in areas up to 50  cm2 [1]. We focus here specifically on microcontact printing (µCP) and microtransfer molding (µTM, Fig. 1). In both cases, a liquid polydimethysiloxane (PDMS) elastomer is used to replicate a microstructure (at macroscopic scale) from a master. Once hardened, µCP is achieved by coating the stamp with the ink of interest and transferring the crest pat- tern to the substrate of interest, usually resulting in an ultra-thin coating [2]. In a typical µTM, a prepolymer solution is applied to the trenches of the PDMS stamp, which is transferred and heated to polymerize on the substrate of interest [3]. While µCP transfers the positive image of the stamp, µTM transfers the negative image of the stamp. Thus, a variety of materials, shapes and dimensions can be deposited using these strategies. Applications for such structures include wetting, tribology, and corrosion control, charge transfer, protein adsorption, and etch resist, among others [4]. In this study, we test the generation of nano and microstructures by soft lithography using amphiphilic molecules on mica, silicon, and an aluminum alloy, with the goal of testing the limits of these techniques for these kinds of molecules, with a special interest in modification of the observed water contact angles, which can lead to antifouling applications. The original interest for using soft lithography was in micro- electronics. Goetting, Deng & Whitesides [5] generated in octa- decylphosphonic acid (ODPA) patterns consisting of 6 µm wide lines separated by 4 µm and a height of 300 nm that could be reduced to monolayer with proper rinsing. The master for the PDMS stamp was created by convenstional photolithography. Chemical etching with an aqueous mixture of acids (nitric, ace- tic and phosphoric acid) known as “aluminum etchant type A” maintained the original pattern without loss of lateral resolution. Once the protective agent (phosphonic acid) was removed, con- ductive patterns separated from each other by insulating spaces were obtained. They hinted that the feature size limit may be 600 nm based on the edge roughness of 150 nm for their feature. More recently, Bao et al. [6] generated a superhydrophobic and abrasion-resistant surface through µCP. The template was a lotus leaf whose microtopography (a few micron tall and http://crossmark.crossref.org/dialog/?doi=10.1557/s43578-023-00909-x&domain=pdf http://orcid.org/0000-0002-5374-6258 J ou rn al o f M at er ia ls R es ea rc h 2 02 3 w w w .m rs .o rg /jm r © The Author(s), under exclusive licence to The Materials Research Society 2023 2 Article wide pillars) they replicated in PDMS. They coated the PDMS stamp with iron chloride (chemical etchant) and printed on a copper coating deposited on bamboo. The resulting mor- phology with stearic acid as modifying agent generated supe- rhydrophobicity (contact angle > 150°), and the observed hys- teresis (difference between advancing and receding contact angle) was evidence of a Cassie-Baxter type surface wetting. Bacterial interactions and bioflm formation at oil water interface have been studied on microscale-textured surfaces, with the aim of unraveling the mechanism of biofilm forma- tion in bioremediation processes [7]. Picoliter droplets of oil, consiting of 6 micron height, and varying length and pitch in the order of tens of microns were deposited by µTM. Research of this type has laid the foundations for developing new areas of study such as mechano-biology [8]. Cavallini et  al. [9] demonstrated nanoscale µTM with small molecule, as they patterned (in one step) 400 nm wide, 230 nm tall ridges with 20 nm protrusions using the common organic light emitter tris-(8-hydroxyquinoline)-aluminum (III) (AlQ3) on the native oxide of a Si wafer. Their employ- ment of volatile dichloromethane likely made it unnecessary to heat the print to remove solvent, but they stressed the need for proper pressure control. Tailoring the wetting behavior of surfaces is important for many applications [10], including customizing surfaces of biomedical interest for adhesion of repellency of a given type of cell, self -cleaning, antifogging, anti-icing, etc. In this study, we apply nano and microstructures generated by soft lithography to the modification of the observed water contact angles. A brief introduction to wetting on rough surfaces is presented below to explain how these structures can affect the observed contact angles. In the Wenzel-type wetting state, the droplet is completely resting on the surface area of a homoge- neous substrate. The contact angle of a liquid will depend on the roughness factor (r), which is defined as the surface area over the geometric area, calculated with Eq. 1, where θ is the intrinsic contact angle for the completely smooth surface and θ* the observed contact angle [11]: In the case of Cassie wetting, the liquid droplet rests on a heterogeneous surface and the cosine of the observed con- tact angle is an average of the contribution of each component accounted by their coverage factor (f) and their respective intrin- sic contact angles (Eq. 2). In addition, there is a special case called Cassie-Baxter in which the drop remains on the top of the solid and air is trapped between the microstructure, which sig- nificantly increases the hydrophobicity (Eq. 3) [12]. The param- eters involved in these equations (pattern, roughness factor and coverage) can be quantified by atomic force microscopy (AFM). Once the nanostructure is characterized, the static contact angle is measured and compared with the estimates of each equation, which will indicate what type of wetting the droplet displays on the substrate. Infection is one of the biggest problems associated with the use of biomedical devices such as intravascular and uri- nary catheters, heart valves, orthopedic implants [13]. The amount of antibiotic needed to eliminate a layer of biofilm on an implant is higher than that tolerated by humans and (1)cos(θ∗) = r · cos(θ) (2)cos(θ∗) = fi · cos(θi) + fj · cos(θj) (3)cos(θ∗) = fsolid · [1+ cos(θ)] −1 Figure 1: Soft lithography techniques used in this study: amphiphilic modifiers with low polarity 18-carbon chain (octadecylphosphonic acid—ODPA, or octadecyltriethoxysilane—OTES) with chemical affinity to metal oxides are shown. When ODPA is applied via µCP, a pattern with monolayer or few layers is printed. When ODPA or OTES are applied via µTM, a much thicker pattern is transferred, which serves as a protective resist for chemical etching. J ou rn al o f M at er ia ls R es ea rc h 2 02 3 w w w .m rs .o rg /jm r © The Author(s), under exclusive licence to The Materials Research Society 2023 3 Article requires the removal of the piece [14, 15]. Therefore, invest- ment in preventive or passive sterilization techniques should be considered. Nosocomial bacterial infection from surfaces that are not adequately disinfected or have developed bacte- rial resistance [16] could be avoided with new approaches. Bacterial resistance to antibiotics is increasing [17], and therefore, inhibitory measures of the initial interaction with surfaces are essential to reduce the probability of biofilm maturation [18]. On this matter, one of the approaches pur- sued in this project could be useful, as it proposes transfer by micromolding followed by chemical etching to obtain nano- topographies with high hydrophobicity and bacterial repel- lency on an aluminum alloy of industrial importance. Given that bacteria feed off nutrients through the solid-aqueous liquid interface, and most bacteria are hydrophilic [19], a repellent effect on water adsorption is expected to reduce not only the adhesion of bacteria but also their ease of nutrient acquisition. Previous studies have concluded that submicron topography has an effect on bacteria morphology, orientation and colonization rate [20]. We propose here an easy to fabri- cate test bed (using soft litho techniques) for continuation of this kind of research. Surfaces prepared via soft litho could be scaled for implementation once optimal dimensions are chosen, and with the appropriate etch chemistry could be used on other metallic substrates. Results and discussion Microcontact printing The internal plastic layer of a DVD-R displays 740 nm perio- dicity, and we measured crests´ full width at half maximum of 470 nm and pattern height of 140 nm via AFM [see Fig. S1(a)]. The PDMS stamp generated from such a template via replica molding results in the same pitch but slightly reduced heighs (ca. 125 nm) and width at half maximum (ca. 410 nm) as shown in Fig. S1(b). We used this stamp to transfer the original pattern but with molecular-scale pattern height to mica and silicon. Freshly cleaved atomically smooth mica displays a surface roughness (Sa) of less than 100 pm per 25 µm2 area; Fig. 2(a) shows the microcontact printing of an ODPA monolayer with, resulting in a pattern following the original pattern but with 6.7 ± 0.3 nm height. The contact angle reached 43 ± 5°, which indicates that the water droplet has significant interaction with the substrate (freshly cleaved mica showed complete wetting with water) compared to the substrate covered by ODPA via spread coating (78 ± 10° contact angle—the coverage is not full monolayer [21]); these results agree with a Cassie wetting regime. Si(111) wafers also have very low Sa (~ 200  pm per 25 µm2). Figure 2(b) shows the result when ODPA is printed with a DVD-R pattern (740 nm periodicity), which averaged 1.9 ± 0.4 nm height, with some nanoparticles present (likely due to work outside of a cleanroom). It is noteworthy that the Figure 2: AFM 5 × 5 µm images with microcontact-printed ODPA pattern with DVD-R topography on (a) mica (b) and Si(111). Cross sections corresponding to the white lines, with averaged heights, are shown below. J ou rn al o f M at er ia ls R es ea rc h 2 02 3 w w w .m rs .o rg /jm r Article © The Author(s), under exclusive licence to The Materials Research Society 2023 4 printed lines are more constant throughout their width. The 93 ± 5° contact angle is close to that expected from Wenzel wet- ting, with an ODPA monolayer oriented such that the non-polar termination is away from the surface. While microcontact printing of amphiphiles has been previ- ously reported on metals [1–3], patterning with an alkylphos- phonate at submicron feature width is reported for the first time on mica and silicon (to the best of our knowledge). This expands the possibilities in employing a similar lost-cost approach (per- haps using custom-made nanotemplates) for applied stud- ies in cell engineering research [22], biomedical devices [23], nanoscale control of work function in electronics [24], and even on nucleation and growth of nano/micro crystals [25]. Optical media pattern transfer by microtransfer molding and etching The nanostructures reproduced in PDMS maintain their mor- phology when heated below 100 °C, which broadens their use in techniques such as microtransfer molding. Traditionally µTM uses heat-curable agents for polymerization. The tech- nique applied in this study deposits multilayers of the non- thermocurable amphiphilic agent ODPA with heat applied (or OTES wihtout heat) along with pressure, and regenerates the original pattern with acceptable fidelity (Fig. 3). AA6063 aluminum alloy presents relatively high Sa (~ 70 nm in 25 µm2), and hence electropolishing is applied to smooth it, reaching a roughness of about one nanometer in a 25 µm2 area. The difference in appearance of AA6063 before and after elec- tropolishing is shown in Fig. 4. Even with this treatment, micro- contact printing of monolayers is hard to detect given that the height of a monolayer is on the order of magnitude of the surface roughness. Hence, for Al alloy we focused on creating thicker patterns, and in this way also influence the wetting via Cassie- Baxter regime should the pattern be sufficiently tall and robust. A CD-R plastic inner layer displays line pattern heights of 190 nm, with FWHM of 920 nm according to our AFM measurements, with a pitch of 1600 nm, while its replica- molded PDMS stamp has pattern heights of aproximately 180 nm with FWHM of 800 nm from our AFM measure- ments (Fig. S2). The result of the ODPA transfer from CD-R generated stamp via micromolding is shown in Fig. 5(a–c), and characterization after successive short etches with Etch- ant A (2 min × 3 cycles) is show in Fig. 5(d–f). Crests of up to 150 nm are withstanding the isotropic etch, although some defects are observed in the SEM and AFM [Fig. 5(d–e)]. The change in morphology after the etch is well emphasized by comparison of AFM images compare Fig. 5(b) with Fig. 5(e). We presume an anisotropic dry etch would result in sharper features. Although the µTM worked well with DVD-R ODPA Figure 3: Demonstration of µTM using commercial DVD of a movie. In (a) the original DVD template is shown, in (b) the stamp generated in PDMS from the DVD, and in (c) the micromolding transfer to electropolished Al AA6063, where the original grooves are reproduced with partial fidelity. Figure 4: Left: Photograph of AA6063 as is (a) and after electropolish (b). Center and Right: corresponding AFM images show the vast difference in roughness. J ou rn al o f M at er ia ls R es ea rc h 2 02 3 w w w .m rs .o rg /jm r Article © The Author(s), under exclusive licence to The Materials Research Society 2023 5 pattern printing to Al, its 740 nm pitch did not withstand the Etchant A wet etch under the conditions tested (images not shown), which is not surprising given the isotropic nature of a wet etch which leads to undercutting [26]. However, the 740 nm pitch DVD-R pattern printed with OTES via µTM can partially endure dilute phosphoric acid etch (Fig. 6); after a plasma OTES strip [Fig. 6(c)] the original 740 nm pitch is maintained but the crests have reduced width—AFM shows width at half maximum (FWHM) to be about 300 nm, while it is about 420 nm before etch [Fig. 6(a)]. At this feature sizes, we are at the limits of printing resolution (600 nm) and wet etch resolution (2 microns) mentioned by Goetting et al. [5] and Van Zant [26] respectively. The surface modification of CD-R patterned aluminum alloy with an ODPA monolayer does not change the morphol- ogy of the pattern already transferred to the aluminum. The contact angle for CD-R pattern transferred to electropolished aluminum and coated with ODPA is 110 ± 4°, while that of ODPA on electropolished Al (without pattern) is 95 ± 5°. This is due to a Cassie-Baxter-type behavior: when analyz- ing AFM data such as that in Fig. 5(c), representative of this structure in aluminum, a peak coverage fraction of 0.6 ± 0.2 is determined, so the air fraction corresponds to 0.4 ± 0.2 and the Cassie-Baxter equation predicts 117 ± 12°, a range that encompasses the observed value; the roughness factor is only 1.06, which results in a very small increase in the Figure 5: (a) SEM, (b) AFM image, and (c) AFM cross section through mid image of ODPA CD-R pattern on Al alloy (FWHM 850 ± 80 nm, height 150 ± 20 nm). (d) SEM, (e) AFM image and (f ) AFM cross section through mid image after Etchant A, resist strip and ODPA modification (FWHM 820 ± 80 nm, height 150 ± 30 nm). Insets in (a) and (d) are photos of substrates viewed at an angle, displaying light diffraction. Figure 6: AFM images of OTES DVD-R pattern on Al alloy: (a) as printed, FWHM 420 ± 40 nm, height 55 ± 6 nm, (b) after 3 h 1% phosphoric acid etch, FWHM 310 ± 50 nm, height 46 ± 2 nm, and (c) after resist strip with plasma, FWHM 300 ± 30 nm, height 33 ± 3 nm. Cross sections at mid image are shown below. J ou rn al o f M at er ia ls R es ea rc h 2 02 3 w w w .m rs .o rg /jm r Article © The Author(s), under exclusive licence to The Materials Research Society 2023 6 predicted contact angle according to Wenzel equation (Eq. 1). It should be possible to increase the air fraction by choosing higher pitch, taller crests, which should lead to an increase in observed contact angle provided geometric conditions still allow for the thermodynamic (meta)stability of Cassie-Baxter regime [27]. The main relevance of the findings in this section is perhaps the fact that the same elastomer used for micro- contact printing can be used for a modified micromolding transfer without the need of a prepolymer (or curing agent), but rather directly using amphiphilic molecules, such as alkanephosphonate and alkyltriethoxysilane. If the thickness is appropriate (as was especialy the case with the phospho- nate on aluminum alloy), transfer of the submicron pattern is feasible, although some fidelity loss is observed compared to original template, as well as pattern loss in some areas. We believe these are issues that can be minimized with additional process engineering as well as preparation within cleanroom environment. Bacterial repellency tests ODPA-modified etched samples such as that in Fig. 5(d), in addition to smooth hydrophilic (cleaned with Ar plasma) and smooth hydrophobic control with ODPA were subjected to bacterial repellence tests. Figure 7 plots the average number of bacteria counted on 63 × magnified fluorescence microscopy images versus the water contact angle of the substrates used. These bacteria remained adhered to the surface after half an hour of immersion in concentrated inoculum of E. coli, and were not washed away with the light rinse that was performed. The lowest bacteria count is that of the sample with CD-R pat- tern (8 ± 7), which has the highest contact angle (110 ± 4°). The difference with the hydrophobic control is significant both in the contact angle and in the number of bacteria according to t-student tests at 95% confidence, demonstrating that the hydro- phobicity increased by nano/microtopography is sufficient to cause less aqueous and bacterial adhesion, although it is not yet superhydrophobic. The contact angle correlates directly with bacterial adhesion: for the sample with CD-R pattern etched in Aluminum and coated with ODPA, the decrease in adhesion was 87% compared to the hydrophilic control and 72% compared to Figure 7: Fluorescence microscopy images (white scale bars are 10 microns; excitation at 365 nm and emission from 420 nm onwards) from controls (a-hydrophilic, b-hydrophobic with ODPA) and a sample (c) with CD-R etched pattern modified with ODPA (the pattern is magnified in the inset, scattering green light). The average adhered bacteria count (based on the images) versus contact angle with water (drops shown are 5 μL) is plotted in the bottom with their standard deviations as error bars (green-dyed bacteria are alive, while red-dyed bacteria are dead−most likely due to UV fluorescence source−making it easier to identify from green background). J ou rn al o f M at er ia ls R es ea rc h 2 02 3 w w w .m rs .o rg /jm r Article © The Author(s), under exclusive licence to The Materials Research Society 2023 7 the hydrophobic control. This result follows the reported hydro- philic character of E. coli that was determined by a variety of methods [28]. Examination of dried samples by SEM highlight how the E. coli bacteria interact with the transferred CD-R pattern. In some cases, bacteria is observed to rest on the trenches, which have an appropriate width to accommodate the bacteria longi- tudinally (Fig. 8). In future research, it would be interesting to determine if these structures promote or inhibit biofilm forma- tion after long periods, as well as study the proteomics of these and other bacteria which will interact differently depending on their morphology and the induced mechanical stress [29]. As has been shown [30], these interactions can disrupt efflux pumps in Gram-negative bacteria, making them less resistant to disinfectant processes. Conclusion We have shown here methods for the deposition of ODPA DVD-R-based patterns consisting of monolayers or a few lay- ers on silicon and mica by microcontact printing, as well as multilayered ODPA and OTES over 50 nm height on AA6063 aluminum alloy via non micromolding transfer. With carefully selected aluminum etching protocols, it was possible to transfer submicron patterns to the aluminum alloy by protection with these molecules: ODPA as resist in CD-R (1600 nm pitch) pat- tern with Etchant A treatment results in up to 150 nm tall etch transfer to the aluminum (with 820 nm crest FWHM), while OTES as resist and dilute phosphoric acid as etchant allowed DVD-R (740 nm pitch) pattern etch transfer (33 nm tall, 300 nm crest FWHM). Wenzel wetting was determined for monolayer DVD-R patterns on silicon and Cassie for few layers pattern on mica. Cassie-Baxter type wetting was determined for CD-R based patterns transferred and etched using ODPA-multilayers as resist. A significant increase in water contact angle is observed compared to the smooth monolayer of ODPA with its non-polar termination, which results in decreased attachment of bacteria after quick rinse, as determined via fluorescence microscopy. We believe that the low cost, potentially scalable soft litho patterning approaches demonstrated in this work could be considered for additional R&D for applications requiring micron and submi- cron patterns in chemistry, materials science, and microbiology that currently rely on more expensive machinery and materials. Experimental methods Stamp preparation and substrate pretreatments To prepare the templates, the nanotopographic plastic layer in CD-R (Maxell) or DVD-R (Egital) disks was exposed using scis- sors and tweezers, and the burnable dye was removed by rinsing with methanol for a few minutes. Replica molding was done with polydimethylsiloxane (PDMS, Sylgard 184 DOW) on areas typically less than half of total disk area following the provider instructions. Once cured, the stamps were cut as appropriate and characterized via AFM. 1 cm × 1 cm mica substrates (Microscopy Sciences) were cleaved using Scotch tape to expose an atomically smooth layer. 1 cm × 1 cm silicon(111) coupons (International Wafer Service) were cleaned with air plasma (Denton Desk V, 110 V, 3 mA, 1 min, 125 mTorr). Aluminum alloy AA6063 was donated by a local supplier (Extralum), and 2 cm × 6 cm pieces were soni- cated in acetone for 5 min prior to use. To planarize at the microscale, these were electropolished in 7% V/V perchloric acid dissolved in 95% ethanol for 2 min at 20 V at 0 °C with an Aluminum AA6063 counter electrode. 1 cm × 1 cm pieces were typically cut out of the electropolished sample for soft lithogra- phy procedures. Pattern generation by microcontact printing and smooth ODPA covered controls on mica and silicon Successful µCP is very dependent on the experimental condi- tions, the ink used, and the substrate [25]. For mica, the method that resulted in the best transfer was immersing the stamp in Figure 8: SEM images of bacteria (OD600 = 0.01) on the CD-R pattern transferred to aluminum alloy at low (a) and high (b) magnifications. J ou rn al o f M at er ia ls R es ea rc h 2 02 3 w w w .m rs .o rg /jm r Article © The Author(s), under exclusive licence to The Materials Research Society 2023 8 0.025 mM n-octadecylphosphonic acid (ODPA, Santa Cruz Biotechnology) dissolved in absolute ethanol. When the solvent dried, it was manually printed by microcontact for 30 s on the substrate of interest followed by annealing for 10 min at 70 °C. For Si (111), a similar procedure was followed, but a light pres- sure was applied during printing for 2 min followed by anneal- ing for 5 min at 160 °C. Hydrophobic smooth controls were also prepared. On mica, spread coating was done for 30 s with a Q-tip impregnated with 2.5 mM ODPA in ethanol, and the sample was then blown-dried and annealed at 100 °C for 5 min (similar procedure to that of Neves et al. [21]). On silicon, the ODPA was applied via spread coating and then annealed at 160 °C for 5 min (modified from Hanson et al. [31]). Pattern generation via microtransfer molding µTM proceeded in a similar way to µCP but with some changes for aluminum alloy: a higher concentration of ODPA was used (1.5 mM solution in ethanol) and the transfer was carried out on a hotplate at 70 °C for 10 min while applying a pressure of 220 g/cm2. Pattern transfer was evidenced by a visible diffraction pattern similar to that of the template used. The aluminum was chemically etched with three cycles of exposure to Aluminum Etchant Type A with Triton X-100 for 2 min with stirring (150 RPM), with intermediate rinses in water to remove microbub- bles and precipitates [32]. The remaining protective agent on the substrate was removed with Argon plasma (Denton Desk V, 3 mA, 1 min, 125 mTorr) and the substrate was finally turned hydrophobic by immersion in ODPA 10 mM in ethanol at 70 °C for 15 min with sonication in 5% trimethylamine in ethanol to remove any multilayers [33]. For printing with n-octadecyltri- ethoxysilane (OTES, Santa Cruz Biotechnology), a 12 mM solu- tion was prepared in hexane; a 3 s exposure to the stamp insured adsorption of the OTES without significant swelling by the hex- ane, which was left to dry for 1 min. Transfer of DVD-R pattern from stamp was achieved with 10 min of pressure (220 g/cm2) at room temperature. It must be noted that higher concentration of OTES resulted in improper transfer of pattern. Transfer etch was done with phosphoric acid (1%) at room temperature for 3 h, followed by rinses with water and 70% ethanol. Resist strip was done with Argon plasma. Preparation of AA6063 smooth controls Hydrophilic controls were prepared by Argon plasma cleaning (Denton Desk V, 7 mA, 1 min, 125 mTorr) electropolished alu- minum alloy. Hydrophobic controls were prepared by modifying unpatterned electropolished aluminum via immersion in ODPA as stated above. Characterization Atomic force microscopy (AFM) of smooth and patterned substrates was done with a MFP3D SA (Asylum Research) in intermittent contact mode with Nanosensors SSS-NCHR tips (42 N/m). Scanning electron microscopy (SEM) was done on a JSM- 6390LV (JEOL). In the latter case, the samples were cov- ered with a thin layer of gold (180 s in Denton Desk V). Static contact angles were measured with deionized water droplets in a Ramé-Hart 200 goniometer. Determination of E. coli adhesion under static conditions Escherichia coli (ATCC25922) inoculum was prepared in LB medium (Sigma) and incubated for 3.5 h at 37 °C. The medium was changed to water via centrifugation and wash cycles. Optical density (OD) was measured at 600 nm in a UV–VIS spectrophotometer (Shimadzu UV-1800) and the inoculum diluted to a value of OD600 = 0.5. Samples were immersed in this inoculum. After 30 min, they were washed with 2 mL of deionized water, and mounted on glass slides. Denovix Live/Dead Cell Drop Kit (5 µL propidium iodide and acridine orange) was used to stain the cells [34]. After cover- ing with coverslips, they were studied under an Axioscope A1 fluorescent microscope (Zeiss) at 63 × with two filters: (a) excitation 365 nm, beam splitter 395 nm and emission from 420 nm onwards, and (b) excitation 450–490 nm, beam split- ter 510 nm and emission from 515 nm onwards. A minimum of 11 images were acquired and the number of observed bac- teria was averaged. E. coli SEM imaging For characterization of bacteria on the ODPA-patterned alu- minum alloy, the aforementioned E. coli inoculum was diluted to OD600 = 0.01 and 10 μL dropcasted on the sample and left to dry in biosafety hood (without rinsing). Once dry, samples were gold-coated as mentioned above. Acknowledgments This work was supported by Office of Naval Research Grant N62909-20-1-2031, UCR Project 540-C0-125 (ascribed to the Programme of Natural and Health Sciences) and CeNAT 2020- 2021 and 2021-2022 scholarships. 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M. Castro, M. Vásquez, J. Cordero, M. Benavides, J. González, M.J. López, J. Vega, Y. Corrales, Surf Interfaces 30, 101881 (2022). https:// doi. org/ 10. 1016/j. surfin. 2022. 101881 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. https://doi.org/10.1016/j.surfin.2022.101881 Sub-micron patterning of metal oxide surfaces via microcontact printing and microtransfer molding of amphiphilic molecules and antifouling application Anchor 2 Introduction Results and discussion Microcontact printing Optical media pattern transfer by microtransfer molding and etching Bacterial repellency tests Conclusion Experimental methods Stamp preparation and substrate pretreatments Pattern generation by microcontact printing and smooth ODPA covered controls on mica and silicon Pattern generation via microtransfer molding Preparation of AA6063 smooth controls Characterization Determination of E. coli adhesion under static conditions E. coli SEM imaging Acknowledgments References