Post modification of injection molded polystyrene ...

Sensors and Actuators B 211 (2015) 187–197

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Short Communication

Post modification of injection molded polystyrene components using green solvents and flexible masks Si-Ying Wu, John P. Hulme ∗ Gachon Bio-Nano Institute, Gachon University, Seongnam-si 461-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 30 October 2014 Received in revised form 31 December 2014 Accepted 15 January 2015 Available online 28 January 2015 Keywords: Injection molding PDMS Diffractive Petri-dish PS polystyrene

a b s t r a c t In this paper two novel solvent based molding techniques were developed for the post modification of a selection of injection molded substrates. We will show that a variety of 2- and 3-D designs containing nano and micron size structures can be fabricated on flat, vertical and curved injection molded surfaces using a selection of green solvents in less than 48 h. The novel techniques used thin and thick film masters composed of polydimethylsiloxane and polyester in conjunction with a variety of pure and blended green solvents to modify injection molded polystyrene (PS). Moreover the said techniques allowed us to fabricate novel diffractive structures in injection molded polystyrene via multi-step soft imprint lithography (MSSIL). The new techniques were further validated by molding microfluidics components with integrated features ranging from 0.4 ␮m to 150 ␮m on dish in a single step. The effects of residual solvent on the viability and growth of E. coli O157 using grating cuvettes and petri-dishes were also measured and their effect on the hydrophobic recovery time of plasma treated injection molded polystyrene was recorded. We found the majority of the modified cuvettes did not exhibit any antioxidant or antibacterial properties and that the hydrophobic recovery time of oxidized polystyrene surfaces containing residual solvent did not significantly differ from plasma-treated pure polystyrene. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The petri-dish and cuvette are essential tools in the training of microbiological, analytical chemistry and regenerative medicine students. Current injection molding machines can fabricate a cuvette or dish in less than 6 s. Each device costs less than 10 cents, which is similar to micro-fluidic devices fabricated from paper [1–7]. Their high optical transparency and negligible absorption make them ideal candidates for fluorescent and absorbance based assays. Imprinting rather than the complete remolding of these substrates would permit the biologist to perform these modifications under normal laboratory conditions. Of the many fabrication processes reported previously, solvent embossing [8,9], solvent assisted molding [10], room temperature imprint lithography [11] and surface assisted micro-molding in capillaries (SAMIM) are potential techniques that can be used to modify short chain, high density

∗ Corresponding author. Tel.: +82 0317508550. E-mail address: [email protected] (J.P. Hulme). http://dx.doi.org/10.1016/j.snb.2015.01.067 0925-4005/© 2015 Elsevier B.V. All rights reserved.

polystyrene found in industrial grade plastics. The techniques often involved spin coating additional polymer solutions onto a rigid support and then imprinting at room temperature with a rigid silicon or flexible PDMS stamp. The primary solvents used in the preparation of the polymer films were toluene and tricholoroethylene (TCS). Yet the polystyrene (PS) used in injection molded components particularly petri-dishes and cuvettes is often low density, low grade material making it far less resistant to solvents such as acetone, chloroform and toluene thus preventing its use with previously reported techniques [8–11]. Over the past decade, a variety of rapid prototyping techniques have been reported [12,13]. Some of the quickest techniques produced a fully functional PDMS micro-fluidic device in less than an hour. However the height and geometry of these devices were constrained by the depth and resolution of the mask [13]. More recently [14] a new technique known as bench top remolding, was used to dissolve an entire petri-dish in gammabutyrolactone (GBL) over a period of 7 days producing structures as small as 3 ␮m and aspect ratios as high as 7. Yet the common petri-dish continues to be used in new and emerging areas of science such as 3-D fluidics and 3-D bio-printing [15–18]. Moreover their glass counter parts are frequently employed in the casting of

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ion selective polymer membranes necessary for clinical sensors. Their versatility, low cost and ease of handling means that they also employed as containers or support platforms for low volume microfluidic or miniaturize devices used in optical microscopy. Therefore a single device/platform that combines the advantages of the common petri-dish with those of microfluidics would appeal to both engineers and biologists alike. However such platforms would take days to fabricate using conventional injection molding techniques [19–26]. Another injection molded component that has remained unchanged for the last 40 years ago is the plastic cuvette. Like the common petri-dish it is primarily composed of low density polystyrene and designed for one shot applications. These components are often referred as “disposables” and are integral part every absorbance, fluorescent, scattering instrument on the planet. Modification or direct patterning of these components would allow the biologist to explore new types of optical and integrated fluidic devices in a much shorter time and at a reduced cost. In this paper we have demonstrated two new fabrication techniques permitting the internal and external surfaces of the common petri-dish and cuvette to be patterned using a selection of blended green solvents and a PDMS stamp. We will show that these simple techniques allowed us to create novel diffractive structures via multistep soft-imprint lithography on dish in less than 2 h. Such structures were then integrated with other micron features and transferred to a new petri-dish using the capillary solvent molding (CSM) method. 2. Experimental 2.1. Materials and reagents Designs were drawn using AutoCAD 2014 (Autodesk, Inc.) and printed on transparent plain polyester film at 16,000 dpi resolution. Aluminum coated holographic gratings (3600, 1800 and 1000 lines/mm), and polyester holographic grating films of the same dimensions were supplied by Edmund Scientific, Korea. A blaze grating (800 lines/mm) was also purchased from Horiba Japan. Injection molded blank substrates were obtained from Daihan Scientific Korea unless otherwise stated. A glass petri-dish was bought from A–Science Korea, polydimethylsiloxane (PDMS) was supplied in an easy to use format, Sylgard 184 Silicon Elastomer (Dow Corning, USA) Su-8 resist 2075 was purchased from Microchem, Korea. UV curable No. 81 was purchased from Norland Products, Inc. (New Brunswick, NJ). Bromocrescol purple, (−)-␤ & (+)-␣-pinene (B.P. 155 ◦ C), R-(+)-d-limonene (B.P. 176 ◦ C) acetone (B.P. 60 ◦ C) methanol (B.P. 56 ◦ C) and 2-propanol (82 ◦ C) were obtained from Aldrich Chemicals (Korea). E. coli O157 was supplied by Sungsoo Ann (Gachon University), broth and yeast extract supplied by Sigma–Aldrich. 2.2. Resist-free soft-imprinting process (imprinting diffraction gratings) PDMS copies of three diffractive structures (1000, 1800 and 3600 lines/mm) were fabricated by mixing PDMS pre-polymer with catalyst at a ratio of 10:1.2 mL of the mixture was then poured onto each of the gratings and cured for 24 h at room temperature. Solvent blends (v/v) were prepared by mixing different volumes of either pure (−)-␤-pinene or (+)-d-limonene with different volumes of acetone (Ace), isopropyl alcohol (IPA) or methanol (Meth). Once the size of the petri-dish to be modified was decided upon a known volume (300 ␮L for 90 mm or 150 ␮l for a 55 mm petri-dish) of a particular solvent blend was dispensed on the bottom. The dish was gently rotated by hand for a few seconds before a glass lid of similar dimensions was placed on top. The glass/polystyrene combo

was moved to a flat surface and allowed to rest for 5 min at room temperature. The lid was then removed and the plastic dish placed in a fume hood for 30 min until a hard clear glaze had formed. A PDMS grating (1 mm thick) was placed on top of the glaze and the two were heated on a hotplate at 88 ◦ C for a period of 2 h. The glaze/PDMS was cooled to room temperature and the PDMS grating removed. The imprinted glaze was place back on the hotplate and dried at 88 ◦ C for a further 12 h. A summary of the process can be seen in Fig. 1a. No additional force was applied to the PDMS. The pressure exerted on the glaze was determined by the weight of the PDMS (0.2 g) and the mold surface area (4 cm2 ). The corresponding pressure of 0.05 mbar is 6 orders smaller than typical pressures used in nano-imprint lithography (NIL) and 2 orders less than automated solvent assisted imprint lithography [11]. The process was repeated using PDMS gratings with periods of 1800 and 3600 lines/mm respectively. The process for MSSIL (qualitative example) was as follows: a PDMS holographic grating (1000 lines/mm, 180 nm in height) was placed on top of a fresh polystyrene glaze containing pure (−)-␤-pinene. The PDMS/glaze was left on hotplate at 60 ◦ C for 30 min and cooled to room temperature. The grating was removed from the glaze and rotated by 90◦ then placed back on top of the first imprint and returned to the oven (60 ◦ C) for a further 20 min. Finally the PDMS was taken off the glaze leaving the cross-haired pattern behind. Copies of the new master were then replicated in PDMS and used when required. For glaze thickness measurements, imprinted sample were placed in ultrasonic water (90 ◦ C) bath and sonicated for 5 h. Tali-step measurements of the partially stripped glaze were taken at different points across the surface of a 90 mm dish and a 2d map was constructed showing the difference in glaze thickness across the dish (see additional material). 2.3. Surface analysis For SEM and AFM analysis PDMS copies of all the polystyrene imprints/molds were fabricated as previously described. A small drop of UV curable Norland 81 adhesive was then added to the surface of each of the PDMS copies and clean glass substrates (5 mm × 5 mm) placed on top. The samples were heated in a 60 ◦ C oven for 2 min before being exposed to a UV source ( = 365 nm) for 10 min. The PDMS copies were removed and the polyurethane replicas examined with AFM in non-contact mode or coated with 50 nm of osmium for SEM analysis SEM (JEOL 6700F, Tokyo, Japan). All samples for AFM and SEM analysis were prepared in this manner unless otherwise stated. AFM imaging was performed in tapping mode with a multimode instrument (SPI 4000, Tokyo, Japan). Silicon tips (10 nm in diameter) were supplied by (Vecco, U.K. & Olympus, Japan). Unless otherwise stated, no magnified AFM images were used in the surface analysis of any of the samples. Contact angle measurements were carried out using a gonio-meter (Rame-Hart Instrument Co., USA). Light microscopy images were taken at 1000× magnification with an optical microscope (Nikon Ni-U, Japan), images were recorded with an infinity camera 2 (Lumenera, Japan) and analyzed using Infinity software. 2.4. Capillary solvent molding (CSM) with PE (thin film) and PU (thick) masters Cross-linked PDMS copies or replicas are known to experience severe swelling when washed or placed in direct contact with aromatic solvents [15]. Recent techniques have sought to address this issue by using Gamma-butyrolactone (GBL). GBL has a boiling point of 204 ◦ C is water soluble and compatible with PDMS. Unfortunately it is also a hallucinogen and its transportation is highly restricted outside the US. An alternative approach is not to use

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Fig. 1. Patterning of polystyrene lab-ware (petri-dish, 55 mm) using (a) the glaze method; (b) CSM method using a thin PE master; (c) CSM using a thick PU master.

PDMS but other materials such as polyester or polyurethane (cured Norland 81 optical adhesive). When cured, Norland 81 adhesive is significantly more resistant to solvent swelling than PDMS [16]. In addition polyester film is widely used in the fabrication of microfluidic devices and in the microelectronic industry [17]. Unlike the PDMS/polymer glaze method which combines the conformability of PDMS with the smooth surface of the polymer glaze, CSM utilizes the strong surface capillary forces between the wet polyester film/polyurethane master (cured Norland 81) and the polystyrene surface. The process for molding a polyester grating on dish is as follows: blank polystyrene dishes were placed on an 80 ◦ C hotplate for 1 h to remove excess water then left to cool to room temperature before use. During this time polyester grating film was immersed in a specific solvent blend to ensure complete wetting of the film. In total 7 different blends were used in this investigation. The film was removed from the solvent solution and placed on the surface of a blank dish. The dish/film was then heated on a 70 ◦ C hotplate for 1 h. It was found that for every 1 cm2 of the polyester film a minimum of 3 ␮L of solvent solution had to be present prior to molding. The dishes were subsequently cooled to room temperature and the polyester films removed. For the final step all samples were placed in a vacuum oven (90 mbar) at 25 ◦ C for 5 h. The temperature of the oven was increased by 10 ◦ C every 5h until it reached 85 ◦ C the samples were then taken out of the oven and allowed to cool to room temperature (RT). A summary of the process is shown in Fig. 1b. To test the validity of the molding process two controls C1 and C2 using 100% ␤-pinene and polyester grating film were fabricated in polystyrene. C1 involved molding polyester grating on a blank dish then leaving it to dry for 4 months at RT. The

conditions for C2 were the same, except after 4 months the sample was heated at 85 ◦ C for 5 h. Polyurethane replicas of C1 and C2 were then fabricated as previously described and analyzed with the AFM. The weight of the polyester film was 0.17 g and its surface area 17 cm2 . The corresponding pressure of 0.01 mbar is 6–7 orders smaller than typical pressures used in (NIL). However it is not only flat surfaces that can be modified with CSM. A selection of laser printed microfluidic designs were also molded on dish (see supplementary material, Fig. S1). The same technique permitted the sides of a blank-polystyrene petri-dish to be molded with polyester grating film, further highlighting the flexibility of this approach (see supplementary material, Fig. S2). For the next part of the experiment 2 integrated microfluidic masters [2 cm length (L) × 1 cm width (W) and 4 cm (L) ×1 cm (W)] with on-board coupling elements were fabricated using conventional photosensitive Su-8 negative resist [19]. A PDMS copy of the Su-8 master was then fabricated, from which several PU (Norland 81) replicas were made. The surface of the replicas was subjected to an O2 rich (80%) plasma for 5 min and then exposed to tri-decafluoro1,1,2,2tetrahydrooctyltricholosilane (CF3 (CF2 )5 CH2 CH2 SiCl3 ) vapour preventing adhesion between the surface of the replica and the polystyrene. The polyurethane replicas were briefly submersed for 2 min in a glass petri-dish containing 2 mL of (−)-␤-pinene. A 1 mL aliquot of (−)-␤-pinene was then added to a blank 55 mm polystyrene dish. The microfluidic copies were taken out of the ␤-P solution and placed face down in the PS dish and left for 5 min on a flat surface to degas. 800 ␮L of solvent was then removed, and the dish left to dry in a fume cupboard for 12 h. The PU/PS (replica/dish) was

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subsequently heated in an oven (85 ◦ C ± 2 ◦ C) for 10 h then left to cool at RT for 1 h. The replica was removed and the dish exposed to O2 plasma (Fermto Science, Korea) for 5 min. The hydrophilic PS devices were subsequently sealed with an oxidized PDMS top plate. A summary of the CSM process is illustrated in Fig. 1c, the process was the same for the 90 mm dish except 2 mL of solvent was added initially and then 1.4 mL were removed. 2.5. The effects of ˇ-pinene on the rate of hydrophobic recovery of PS gratings d-Limonene and ␤-pinene are known to be excellent O2 scavengers. Moreover (−)-␤-pinene has a very high biological oxygen demand value (B.O.D.) in (aq) solution and its vapor is known to react with ozone. Therefore it was necessary to measure the time it took for plasma treated polystyrene gratings to fully recover their hydrophobic nature. Moreover this phenomenon results in poor cell attachment, which negatively impacts the performance of PS devices for cell-based assays [14]. 5 petri-dishes were then molded with 100% (−)-␤-pinene and polyester grating film (steps 1–3, Fig. 1b) but this time they were dried (step 4) at RT and not in the oven as previously described. The effect of increasing drying time on the hydrophobic recovery of plasma treated polystyrene gratings was investigated by measuring the change in the contact angle of deionised water over a 4 day period and comparing them to blank plasma treated polystyrene (control). 2.6. The effects of ˇ-pinene and patterned PS surfaces on bio-film formation In this part of the investigation we measured the effects of ␤pinene and surface morphology on bio-film formation. It is well known that thin film plastics (cling film) doped with terpenes such as thymol or carvacol inhibit the formation of biofilms [20]. To the best of our knowledge no work has been conducted on the ability of nano-patterned doped PS to inhibit bio-film formation. To see the combinatorial effects of surface morphology and ␤-pinene on bio-film inhibition six different types of PS (petri-dish) surfaces were investigated. These were blank PS, plasma treated PS, PS doped with ␤-P, plasma treated PS doped with ␤-P, PS grating (1 ␮m pitch) molded with ␤-P, plasma treated PS grating molded with ␤-P. PS surfaces doped with ␤-P were fabricated using blank polyester sheet and ␤-pinene solution. All PS surfaces were dried at RT for 12 h (step 4, Fig. 1b) prior use. To each PS surface, 5 mL of LB broth containing 5 × 108 cfu/mL was then added. The dishes were placed on a hotplate at 37 ◦ C and incubated for 1 h. The bacterial solutions were discarded and each dish washed three times in deionized water then stained with crystal violet solution for 5 min. The PS surfaces were rinsed again in deionized water for 3 min then dried in a stream of nitrogen. The process was repeated for incubation times of 234,567 and 8 h. The average number of cells from five separate locations per dish was subsequently measured. Locations that exhibited >10% standard deviation between them were excluded. 2.7. The effects of solvent leaching on the growth of E. coli O157 In the next part of this investigation, we measured the effect of pure (−)-␤-pinene and (+)-␣-pinene and two additional blends Ace:IPA:D-Lim (1:1:1) and Ace:IPA:␤-P (1:1:2) on the growth of E. coli O157. Using a similar process shown in Fig. 1b the interior of twelve cuvettes (4 sides) were molded using polyester grating (1000 lines/mm) film and 4 different solvent solutions but this time the cuvettes were dried for 24 h at RT before testing.

Broth cultures were prepared by inoculating one single colony of E. coli O157 from an agar plate to a plastic tube containing 25 mL of sterile LB broth. These subcultures were diluted 1/100 with broth culture and 3 mL dispensed into each cuvette giving a starting absorbance (Ab) value of 0.04 units. Each cuvette was then placed in the incubator at 37 ◦ C and the Ab values measured every 2 h using a UV-21000 spectrophotometer (Cole Parmer, Korea). Each cuvette was weighed before and after the molding process and the percentage change weight calculated (see additional data). 2.8. Antioxidant activity 2,2-Diphenyl-1-picrylhydrazyl (DPPH)-scavenging assay was used to measure the antioxidant activity of a variety remolded cuvettes. DPPH is a cell-permeable, stable free radical that is commonly used to evaluate the ability of compounds to act as free radical scavengers or hydrogen donors and to measure the antioxidant activity of tissue extracts. The reaction of DPPH with an antioxidant or reducing compound produces the corresponding hydrazine DPPH2 , which can be followed by color change from purple (absorbance at 515–528 nm), to yellow [23]. For the DPPH assay, 0.1 mol/L DPPH stock solution was prepared in 70% ethanol solution. Approximately 3 mL of the stock solution were added to each modified cuvette. In total four new solvent solutions (pure ␤-P, IPA:D-Lim (5:1) blend, Ace:IPA:D-Lim (1:1:1) blend and ␣-P:Ace:IPA (1:1:1)) were used to mold the interior (4sides) of each cuvette using the CSM method previously described. After continuous incubation in the dark at 4 ◦ C for varying lengths of time 0.5, 1, 6, 12 and 24 h), the DPPH radical scavenging activity of each molded cuvette was assessed by measuring the absorbance at 517 nm against 70% aqueous ethanol/DPPH solution incubated in a blank polystyrene cuvette (positive control). For the negative control 4 ␮L of pure (−)-␤-pinene was added to 3 mL of a 70%/DPPH solution and incubated in the dark for 24 h at 4 ◦ C. The experiment was done in triplicate for each cuvette. The results were expressed as percentage decrease with respect to control values, and compared by one-way ANOVA and Turkey’s test. A difference was considered statistically significant if p ≤ 0.05. 3. Results and discussion 3.1. Evaluation of soft imprinting method It is well reported that PDMS swells in the presence of aromatic solvents. The swelling is dependent on the solvent concentration, thickness of the stamp and the imprinting temperature [8]. In addition, PDMS cannot be used in conventional hot-embossing or RTNIL because of its low modulus. Before the stamp is placed on top, no free solvent should be present otherwise the PDMS stamp will not conform to the surface of the polystyrene. Both larger and smaller dishes (90 and 55 mm) produced a uniform glaze. The surface roughness and flatness (Z) values for the glazed surfaces were on average 2–3 times smoother (AFM data provided in additional information) and 4–6 times flatter than the original injection molded surface thus eliminating the need for expensive electroformed or highly polished metal blanks prior the injection molded process. The fidelity of several polymer glazes was then tested using three types of PDMS gratings. Table 1 shows the average percent change in the aspect ratio (MAR) of 5 different types of glazes, imprinted with three different PDMS masters. The average percentage change for each of the imprinted glazes was calculated using Eq. (1), glazes formed using the methanol rich ternary Ace/Meth/D-Lim (1:2:1) blend exhibited the best fidelity with a MAR value of +2.3% when compared to the PDMS masters. Fidelity is dependent on several factors such as the

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Table 1 Imprinting efficiency using PDMS gratings with different glazed polystyrene surfaces formed from a selection of blended green solvents (n = 3). MAR is the average percentage change in the aspect ratio (AR) of the imprinted gratings compared to PDMS masters 1, 2 and 3. Gratings

PDMS Ace:Meth:D-Lim 1:2:1 Ace:Meth:␤-P 1:1:2 Ace:IPA:␤-P 1:1:2 Ace:IPA:␣-P 1:1:2 (+)-␤-Pinene (C)

Master 1

Master 2

MAR (%)

Height (nm)

Period (nm)

Height (nm)

Period (nm)

Height (nm)

1079 1022 1040 1067 1065 1031

180 169 160 142 125 107

550 537 543 550 541 546

174 177 165 100 110 80

279 253 277 273 270 267

81 76 62 55 51 45

solubility power of the blend [20], its miscibility [19], the boiling points of the solvents and the rate of solvent evaporation during the glazing process [20]. Out of all the blends used to form the glazes in Table 1 the solubility power (maximum amount of PS which will dissolve in a known volume of the blend) Ace/Meth/D-Lim (1:2:1) blend was the highest. The small increase in the MAR value suggests that the solvation (rate of dissolution of polystyrene in the blend) power of the Ace/Meth/D-Lim blend was a little high and a smaller fraction of acetone and limonene should have been used. Replacing d-limonene with an inferior solvent such as ␤-pinene is one way to do this. However the fidelity of the gratings produced from Ace/Meth/␤-P (1:1:2) glaze was considerably lower than glazes produced with the methanol rich blend Ace/Meth/D-Lim (1:2:1) exhibiting a MAR value of −11.5%. The decrease in aspect ratio was primarily due to a reduction in the height of the gratings and not the period. This trend continues as the values for the imprinted gratings fall from −11.5 to −31% and −34% when Ace/IPA/␤-P (1:1:2) and Ace/IPA/␣-P (1:1:2) blends were used. The MAR value of −44% occurred in glazes containing only pure (−)-␤-pinene. The general trend for the values in Table 1 implies that the fraction of Ace, Meth and IPA in all of the blends changes significantly during the glazing process (particularly at the glaze surface or the back of the solvent front) and that the resulting polystyrene glazes mostly contain low volatile bicyclic terpenes or d-limonene prior imprinting of the PDMS stamp. Therefore it is the rate of evaporation and changes in the molar fractions of the blended solvents during the glazing process that determine the MAR values shown in Table 1. MAR =

Master 3

Period (nm)

(ARI1 /ARM1 ) + (ARI2 /ARM2 ) + (ARI3 /ARM3 ) × 100 3−1

(1)

where ARI is the aspect ratio of imprinted or molded grating and ARM is the aspect ratio of polyester or PDMS masters. The aspect ratio is the ratio of the height of the grating to its period or cross section. Another problem is that of solvent pooling. Low volatile solvents such as ␤-pinene would inevitably pool on a planar surface leading large to variations in the thickness of the glaze across a short distant. The 2d map (see supplementary information, Fig. S3) shows that the thickness of an imprinted glaze ranges from 1.5 ␮m at the center (dish) to 1.76 ␮m (2 cm away from the center). At distances of 3 and 4 cm from the center the thickness of the glaze increases to 3.5 ␮m and 8 ␮m respectively. The small variation in glaze thickness is due to the bowed nature of the surface of the dish from the original injection molded process. This bowed feature essentially drains excess solvent causing it to pool around the side of the dish forming a ring like structure which in turn significantly improves the surface quality on and around the center of the dish by an order of magnitude (see AFM measurements, supplementary information, Fig. S4) These improvements in surface quality then allowed us to create novel diffractive features via MSSIL, previously only possible using high pressure imprint lithography and hard masks. In this case a PDMS grating with height of 180 nm and period of 1 ␮m

0 +2.3 −11.5 −31 −34 −44

was imprinted twice on a polystyrene/␣-P glaze. AFM imagery (see Fig. 2a and b) clearly shows the characteristic lattice structure consistent with conventional multistep nano-imprint lithography [21]. The displayed 2D imprinted features have a height of 60 (±3) nm. Features of larger aspect ratio can be realized by increasing the duration of the imprinting process, or by applying additional pressure. The effect of the presence of 1D and 2D periodic modulations on the diffraction of light is visible by eye, upon illuminating the gratings by a polychromatic source (Fig. 2c and d). Complex diffractive structures were also produced via MSSIL (5-step process) see supplementary information Figs. S5 and S6.

3.2. Evaluation of CSM method No reports to date have shown that either SAMIM or MIMIC are capable of molding integrated structures containing both submicron and sub mm features alone. In the next part of this investigation we combined the attributes of both techniques by investigating the role of solvent capillary pressure and the type and depth of structures that can be obtained using the CSM method. Like MSSIL, the CSM method allows new features to be directly molded into the supporting substrate unlike UV lithography. In the first part of this investigation we examined the molding efficiency of three polyester gratings using a selection of blended green solvents and disposable polystyrene dishes, the results are shown in Table 2. The MAR values in Table 2 were calculated using equation 1. On average the Ace:IPA:D-Lim (v/v) (1:4:1) blend produced the best gratings with a MAR value of −4.4% (see Fig. 3a and b). However when the fraction of IPA was reduced in blends (Ace:IPA:D-Lim) (1:2:1) and (Ace:IPA:␤-P) (1:1:1) the MAR values of the molded gratings fell to −11.2% and −30% respectively. This is due to a higher fraction of aromatic solvent and acetone in the blend and too short a drying time, resulting in moderate lateral (bulk) and vertical shrinkage. Unfortunately when binary blends containing no aromatic solvents such as Ace:IPA or Ace:Meth (see Fig. S7) were used, the polystyrene gratings demonstrated severe vertical and lateral shrinkage exhibiting MAR values of 72% and 45% respectively. Gratings molded with these blends displayed extensive surface cracking/splinters and clouding (1000s of lines, 100–200 nm in width and 2–20 mm long) observable 1–2 days after molding. Gratings molded using pure solvents ␣-P or ␤-P also exhibited MAR values ranging from −58% to −60%. This was due to significant amounts of solvent residing in the gratings promoting their dissolution at higher temperatures (85 ◦ C) [24]. To determine whether the amount of solvent was significant with regards to grating stability, at RT changes in the aspect ratios of 2 polystyrene control gratings C1 and C2 were measured once a week over a 4-month period. C1 showed a small change in the MAR value of −12% over the 4 months whereas the value for C2 was much greater at −61%. The difference in MAR values for C1 and C2 is due to difference in the solubility of PS in ␤-pinene at different

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Fig. 2. AFM and SEM analysis of the polymer glaze imprinting process. (a) First imprint on the glaze; (b) AFM cross section of the 2D grating (insert shows an SEM of the lattice structure); (c) photograph of the imprinted glaze and the diffraction pattern of the first imprint; (d) dish rotated (90◦ ) diffraction pattern of the 2nd imprint.

Table 2 Copying efficiency of the CSM method (for all AFM values n = 3) using ternary and binary blends and pure solvents. MAR is the average percentage change in the aspect ratios of the molded gratings compared to PE masters 1, 2 and 3. Gratings

PE original Ace:IPA:D.L 1:4:1 Ace:IPA:D.L 1:2:1 Ace:IPA:␤.P 1:1:1 Ace:IPA Ace:Meth (+)-␣-Pinene (−)-␤-Pinene (BP) (−)-␤-Pinene (BP) C1 (−)-␤-Pinene (BP) C2

Master 1

Master 2

Master 3

MAR (%)

Period (nm)

Height (nm)

Period (nm)

Height (nm)

Period (nm)

Height (nm)

1079 1053

176 180

550 547

180 170

280 267

100 83

0 −4.4

1039

176

535

154

263

70

−11.2

1009

130

525

114

255

57

−30.7

904 960 1039 1041 1060

260 212 70 70 172

460 460 540 546 540

230 200 65 56 155

200 200 284 276 280

135 120 47 45 75

+72.0 +45.4 −58 −60 −12

1046

73

546

53

271

41

−61

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Fig. 3. Atomic force microscopy images (AFM) of the molded polystyrene surfaces (petri-dish) using the CSM method. (a) Polyester thin film master with a periodicity of 1 ␮m; (b) polyurethane replica of a polystyrene grating molded with Ace:IPA:D-Lim (1:4:1) blend (MAR value −4.4%); (c) SEM images of the original blazed grating; (d) polystyrene copy of the blazed grating using an Ace:IPA:D-Lim (1:4:1) blend (magnified images can be supplied on demand if desired).

temperatures [20]. Additional optical structures (polystyrene blazed grating, 800 lines/mm) molded on dish using the CSM method are shown in Fig. 3c and d. Magnified images are available in the supplementary material if desired. In the next part of this paper we examined the extent to which CSM may be used to mold integrated features on dish. Although not previously stated one of our goals is to create an easy to handle microfluidic modular system (3–10 cm in diameter). For a microfluidic system to be modular it must be multifunctional and contain 2 or more different fluidic designs. Fig. 4 shows 2 different designs that were molded simultaneously on a 90 mm dish using the CSM method. The classic serpentine with integrated sub-micron features (horizontal image) can be seen as well as the standard Y mixer and cell culture chamber with on board coupling element. The average dimensions for the serpentine channel were 200 ␮m across by 150 ␮m deep; the arms of the Y micro-mixer were 60 ␮m across and 130 ␮m deep. SEM images of the main channel (micro mixer) are shown in Fig. 4b. The channel wall and the insert clearly show the sub-micron features patterned along the channel bottom. The Y mixer was then sealed and 5 ␮L of Bromocrescol purple was added. The characteristic flow dynamic can be seen as the solution fills the channel Fig. 4c. There are several advantages to this technique but the principal one is that many designs can be molded on dish in a

single step. Yet like SAMIM and MIMIC, CSM suffers from solvent reflow and trapping between the edge of the polyurethane master and polystyrene surface. Solvent reflow also depends on the type (low vapour pressure or high vapour pressure) and the volume of solvent used in the initial molding process. On the other hand the main concern with the resist free method is creating a glaze that expands and contracts uniformly during the imprinting procedure and the effect the solvent has on the porosity of the polystyrene. Yet the molding of microfluidic channels with integrated submicron structures in injection molded poly-methylmethacrylate PMMA and UV polymers and has been reported before [25,26]. These approaches required cumbersome and heavy machinery like, electro-polishers, roller embossers, injection molders, numerous fabrication steps and additional polymer layers. Moreover the cured polymers used in UV imprint lithography cannot be remolded unlike injection molded plastic [14].

3.3. The effects of ˇ-pinene on the rate of hydrophobic recovery of PS gratings We have shown that residual solvent can affect the fidelity of the imprinting/molding process but its effect on the hydrophobic recovery of plasma treated polystyrene was unknown.

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Fig. 4. Photographs showing different channel designs molded into 90 and 55 mm petri-dishes using CSM (thick PU masters) and soft imprinting methods (a) classic serpentine and cell culture design with on board diffractive components molded simultaneously into a 90 mm PS dish; (b) SEM picture of the bottom of the Y mixer (insert shows the diffractive structure along the base of the channel) fabricated using CSM; (c) a sealed channel filling with Bromocrescol purple.).

A potential disadvantage of CSM is that residual solvent trapped in the bulk polymer might accelerate the hydrophobic recovery of plasma treated PS gratings. Fig. 5a shows the effect of increasing drying time on the hydrophobic recovery of plasma PS gratings that had been molded using pure (−)-␤-pinene. PS gratings treated directly or dried at RT for 3 h prior to plasma treatment exhibited average rates of recovery (over the first 48 h) of 0.53◦ /h and 0.36◦ /h which are significantly faster than 0.21◦ /h exhibited by the control (blank plasma treated PS). Gratings dried for a minimum of 12 h and 24 h at RT before plasma treatment, exhibited rates of recovery 0.21◦ /h and 0.20◦ /h, suggesting the optimal drying time at RT was between 12 and 24 h after molding. Fig. 5b shows the change in the contact angle of the molded surface before and after plasma treatment. Before plasma treatment the contact angle of the PS grating is higher than the blank polystyrene. The small difference in the contact angle is due to the presence of solvent on the PS surface and not the low aspect (4 ␮m and clustered together, implying they are actively dividing but adhering to each other rather than the surface. After 8 h of incubation the number of cells found on all the surfaces was very similar (1200 cells/7000 ␮m2 ), thus flat hydrophobic (doped ␤-P or blank) PS surfaces do not significantly deter nor delay biofilm-formation.

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Fig. 5. (a) The effect of increasing drying time (step 4, Fig. 1b) on the contact angle of plasma treated patterned polystyrene petri-dishes (control is plasma treated blank PS); (b) changes in the contact angle of blank polystyrene and the polystyrene grating (PS) before and after plasma treatment (solvent drying time was 6 h for the polystyrene grating).

We then examined the effect of patterned PS surfaces on biofilm formation (insert Fig. 6b) as a function of incubation time. The number of bacteria found on plasma treated PS gratings increases in a bi-phasic manner similar to that seen on flat PS surfaces doped with ␤-P. However the number of bacteria found on untreated PS gratings barely increases over the 8 h period with a cell doubling time >240 min. Thus untreated PS gratings doped with ␤-P significantly delay the formation of the biofilm. The bacteria found on such surfaces (see image, Fig. 6b) are small,