MeV ion beam lithography of biocompatible halogenated Parylenes using aperture masks

Nuclear Instruments and Methods in Physics Research B 354 (2015) 34–36

Contents lists available at ScienceDirect

Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb

MeV ion beam lithography of biocompatible halogenated Parylenes using aperture masks Harry J. Whitlow a, Rattanaporn Norarat a,b,c,⇑, Marta Roccio d, Patrick Jeanneret a, Edouard Guibert a, Maxime Bergamin a, Gianni Fiorucci a, Alexandra Homsy a, Edith Laux a, Herbert Keppner a, Pascal Senn d,e a

University of Applied Sciences (HES-SO), Haute Ecole Arc Ingénierie, Eplatures-Gris 17, CH-2300 La Chaux-de-Fonds, Switzerland Faculty of Science and Agriculture, Rajamangala University of Technology Lanna, Chiang Rai, 57120 Chiang Rai, Thailand Department of Physics, University of Jyväskylä, P.O. Box 35, Jyväskylä FI-40014, Finland d Inner Ear Research Laboratory, Univ. Department of Otorhinolaryngology, Head and Neck Surgery, Inselspital, CH-3010 Bern, Switzerland e Department of Otolaryngology, Head and Neck Surgery, Hopitaux Universitaires de Genève, CH-1211 Geneva, Switzerland b c

a r t i c l e

i n f o

Article history: Received 18 July 2014 Received in revised form 19 September 2014 Accepted 21 October 2014 Available online 21 November 2014 Keywords: MeV ion beam lithography Parylene-C Parylene-F Biocompatibility Murine spiral ganglion cells

a b s t r a c t Parylenes are poly(p-xylylene) polymers that are widely used as moisture barriers and in biomedicine because of their good biocompatibility. We have investigated MeV ion beam lithography using 16O+ ions for writing defined patterns in Parylene-C, which is evaluated as a coating material for the Cochlear Implant (CI) electrode array, a neuroprosthesis to treat some forms of deafness. Parylene-C and -F on silicon and glass substrates as well as 50 lm thick PTFE were irradiated to different fluences (1  1013 1  1016 1 MeV 16O+ ions cm 2) through aperture masks under high vacuum and a low pressure (<10 3 mbar) oxygen atmosphere. Biocompatibility of the irradiated and unirradiated surfaces was tested by cell-counting to determine the proliferation of murine spiral ganglion cells. The results reveal that an oxygen ion beam can be used to pattern Parylene-C and -F without using a liquid solvent developer in a similar manner to PTFE but with a 25 smaller removal rate. Biocompatibility tests showed no difference in cell adhesion between irradiated and unirradiated areas or ion fluence dependence. Coating the Parylene surface with an adhesion-promoting protein mixture had a much greater effect on cell proliferation. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Parylenes are a class of polymers that can be deposited as thin films with very high conformity on a variety of substrates from the vapour phase using the Gorham process [1]. The precursors are [2,2]paracyclophane dimer derivatives [2] where NH2, halogens or organic groups such as esters etc. may be substituted on the aliphatic groups or benzene rings. The different possible substitutions give rise to a wide range of Parylenes with different properties. Parlylene-C is deposited from the precursor (pseudoortho-dichloro[2,2]paracyclophane). Of particular interest for surgical applications are the barrier properties of Parylene-C [3,4] and its high biocompatibility. Lithographic patterning of polytetrafluorethylene (PTFE), a linear chain fluoropolymer, has been reported using a MeV 16O+ beam [5,6] or other ion beams where the exposure takes place in a O2 rich atmosphere [7] and in vacuum [8]. Here we report ⇑ Corresponding author at: Faculty of Science and Agriculture, Rajamangala University of Technology Lanna, Chiang Rai, 57120 Chiang Rai, Thailand. E-mail address: [email protected] (R. Norarat). http://dx.doi.org/10.1016/j.nimb.2014.10.024 0168-583X/Ó 2014 Elsevier B.V. All rights reserved.

investigations into lithographic pattering of Parylene-C and -F and the biocompatibility after 1 MeV 16O+ irradiation. 2. Experimental Samples of 5.8 lm thick Parylene-C were deposited on (100) Si substrates and standard soda-lime glass microscope slides from precursor using a COMELEC CVD reactor [9]. The samples of Parylene-F on (100) Si substrates were purchased from Comelec [9] (100) Si substrates with deposited Parylene films were used to measure the 16O ion-induced removal. This was done to facilitate measurement of the thickness of material removed using a stylus profilometer from the step height corresponding to the mask edge. A 50 lm PTFE sheet was also used in the study as a reference material because its properties under oxygen ion irradiation were previously studied [5,6]. Masks (Fig. 1) were fabricated by fs-laser machining in 50 lm thick Mo sheet. Mo was chosen because it has a small grain size which resulted in straighter edges than in Cu or stainless steel sheets. Masks with single isolated lines down to 15 lm in width with edge roughness of a few lm could be

35

H.J. Whitlow et al. / Nuclear Instruments and Methods in Physics Research B 354 (2015) 34–36

Fig. 1. Photograph of the laser-cut Mo mask. The circular regions are connected by 30 lm wide lines. The dashed line denotes the region on the irradiated samples that was covered with adhesion promoting proteins (see text).

fabricated with this technique. The masks were clamped in contact with the Parylene coated substrates and 50 lm PTFE sheet. Irradiation was carried out at the 1.7 MV Tandetron in La Chauxde-Fonds in the ion irradiation end-station using magnetically rastered and neutral trapped 16O+ beams. Unless otherwise specified, 1 MeV 16O+ beams were used for the irradiation. The ion beam 0.35–0.84 lA was focused to a roughly circular spot of 0.5 cm2 area. To obtain a uniform irradiation fluence the scanning amplitude was adjusted to be about 1.5 cm larger than the aperture that defines the irradiated area on the target holder onto which the mask and sample were clamped. The fluence end-point was measured by integrating the secondary electron suppressed current from the target holder. Tests using the discolouration of paper (cellulose) targets showed no evidence of non-uniformity in the beam fluence. Normally the irradiation was carried out at the base pressure of the turbo-pumped irradiation chamber (2.2  10 6 mbar). In order to test the effect of oxygen ambient, O2 gas (99.999% pure) was fed into the irradiation chamber using a needle valve and the pumping speed of the chamber turbo-pump was reduced by throttling. Biocompatibility measurements were carried out using crossshaped mask with five 2 mm dia. open circles at the centre and the end of the arms (Fig. 1). The protocol is given in Table 1. 3. Results and discussions Fig. 2 presents the thickness of the film removed by the ion irradiation. The rate of removal for Parylene-C and -F is about 25 times smaller than for PTFE. The data also revealed that up to a fluence of 1014 16O+ ions cm 2 Parylene-F exhibited a swelling of the surface, which was not observed in the case of Parylene-C or PTFE. This swelling corresponds to a negative amount of removed material and hence is not represented in Fig. 2. Presumably, this is due to internal pressure build up from gas evolution [10] and possibly oxygen retention. The reason why this is only

Fig. 2. Thickness removed with 1 MeV

16

O+ion fluence.

observed in Parylene-F is not clear. The average effective atomic sputter yields derived from the densities [11] and repeating unit structure and the straight line slopes were similar for Parylene-C and -F (2600 and 2900 atoms per ion, respectively). PTFE on the other hand had an average atomic sputter yield of 6.9  104 and the fluence dependence of the sputtering yield exhibited a saturation behaviour; falling to 3.6  104 for the highest fluencies. It was observed that irradiation of Parylene-C and PTFE in an O2-rich ambient (data not shown), at a pressure of 1:0  10 4 mbar above the chamber base pressure, did not enhance the thickness removed per ion. Increasing the O2 pressure to 5  10 4 mbar lead to a reduction in thickness removed per ion. This may be attributed to neutralisation in collisions with gas atoms. Tests of the energy dependence of the removal rate for Parylene-C and PTFE showed this increased by a factor 2 for both materials when the 16O+ ion energy was increased from 0.6 to 2 MeV (data not shown). Taken together with the absence of an enhancement in removal rate in an O2-rich atmosphere, indicates that the energy deposited by the oxygen ions is most probably the removal-rate limiting factor. Biocompatibility was assayed by culturing cells isolated from murine spiral ganglia on irradiated samples. Cell adhesion and proliferation on irradiated versus unirradiated areas were compared. The biocompatibility tests were performed following the protocol in Table 1 using spiral ganglion cells because the ultimate application of this technology is the modification of the cochlear implant prosthesis electrode surface to allow growth and docking of spiral ganglion neurons on them [12–15]. Irradiated and unirradiated areas were selectively coated with adhesion molecules (poly-Llysine and laminin as shown in Fig. 1 and Table 1. In Fig. 3 shows the confluence (areal density) of SG-cells on poly-L-lysine and laminin coated and uncoated regions of Parylene-C for different ion fluences. Considering first the zero fluence data in Fig. 3, this shows

Table 1 SG-cell biocompatibility testing protocol.  Spiral ganglion cells were obtained by excising the temporal bones and dissection in sterile conditions in Hanks solution at 4 °C of the cochlea from three C57 black 6 animals that were sacrificed by approved procedure at day 7 without using chemical agents  After removal of the organ of corti the spiral ganglia were isolated and dissociated by incubation at room temperature in Acumax [16] for 20 min  The explants were mechanically triturated using p1000 pipet tips in neurobasal medium with B27 supplement [17]  Subsequently, the explant cells were centrifuged at 130 g for 5 min and resuspended in neurobasal medium and passed through a 100 lm strainer  The Parylene-C samples were sterilised in UVC light and coated by placing a drop of poly-L-Lysine 0.01% solution [18] and laminin 50 lg/ml [19] mix and incubating for 15 min at 37 °C followed by washing two times in medium. The drop was placed so the attachment promoting protein mix partly covered the irradiated and unirradiated areas of Parylene-C (Fig. 1)  The cells were seeded on the Parylene-C coated substrates and incubated at 37 °C in 5% CO2: air environment for 7 days  The cytoskeleton of the cells was stained with Phallodin [20] and the cell nuclei using DAPI (green and red channels respectively in Fig. 4)  Microscope images were taken and cell counting carried out by selecting the colour channel corresponding to the nuclei and counting these using the cell counting image recognition software in ImageJ [21]

36

H.J. Whitlow et al. / Nuclear Instruments and Methods in Physics Research B 354 (2015) 34–36

 At low fluences Parylene-F exhibits a swelling of the surface that was not observed for Parylene-C or PTFE.  Irradiation in a pressure of 1:0  10 4 mbar O2 did not enhance the removal rate for Parylene-C or PTFE.  The removal of Parylene-C and PTFE per ion was observed to increase for increasing 16O+ ion energy.  No significant differences in adhesion and proliferation of SG cells was observed between irradiated and unirradiated areas. Ethics statement

Fig. 3. SG cell proliferation on protein (laminin/PLL) adhesion layer coated and uncoated Parylene-C for different ion fluences. Note: the zero fluence data is the mean of all the controls.

The animal procedures were approved by the Animal Research Ethics Committee of the Canton Bern, Switzerland (License nr. BE117/12). Acknowledgements The work was carried out under the auspices of the EU-FP7 NANOCI project (No. 281056). RN was supported by the Academy of Finland Center of Excellence in Nuclear and Accelerator Based Physics (Ref. 251353), the Magnus Erhnrooth Foundation and Rajamangala University of Technology Lanna. Dr. Orapin Chienthavorn is thanked for helpful advice. References

Fig. 4. Optical micrograph of SG cell proliferation on protein adhesion layer coated Parylene-C (the circular region defines the irradiated area). The image has been manipulated by expanding the contrast to enhance visibility and by colourmapping the fluorescence from Phallodin green and DAPI red.

that the poly-L-lysine (PLL) and laminin coating enhances the SGcell proliferation with more 95% confidence. It can also be seen from this figure that for the uncoated Parylene-C, there is statistically no evidence that the ion irradiation either promotes or inhibits the confluence of SG-cells. Likewise, no evidence is seen in Figs. 3 and 4 that 16O+ irradiation degrades the enhancement of cell adhesion by PLL and laminin coating applied after the irradiation. The implication is that the irradiated surface does not degrade cell proliferation either directly by creating a chemical insult on the surface on which SG cells grow, or by degrading the adsorption of adhesion proteins (laminin/PLL) on the surface. The implication is that irradiation of Parylene-C surfaces do not affect cell adhesion or proliferation, or protein (laminin/PLL) adsorption to the substrate. The weak green contrast in the irradiated region of Fig. 4 might be due to autofluorescence or conjugation of the Phallodin stain to the surface. 4. Conclusions  MeV 16O+ bombardment can be used for lithographically patterning Parylene-C and -F in a similar way to that reported for PTFE [5,6] using a broad beam in a parallel exposure process in combination with a stencil mask.  The removal rate for Parylene-C and -F was about 25 times smaller than for PTFE.

[1] W.F. Gorham, A new, general synthetic method for the preparation of linear poly-p-xylylenes, J. Polym. Sci. A-1 4 (1966) 3027–3039, http://dx.doi.org/ 10.1002/pol.1966.150041209. [2] H.J. Reich, D.J. Cram, Macro rings. XXXVII. Multiple electrophilic substitution reactions of [2.2]paracyclophanes and interconversions of polysubstituted derivatives, J. Am. Chem. Soc. 91 (13) (1969) 3537. [3] A. Hogg, T. Aellen, S. Uhl, B. Graf, H. Keppner, Y. Tardy, J. Burger, J. Micromech. Microeng. 23 (2013) 075001. [4] A. Hogg, S. Uhl, F. Feuvrier, Y. Girardet, B. Graf, T. Aellen, H. Keppner, Y. Tardy, J. Burger, Protective multilayer packaging for long-term implantable medical devices, Surf. Coatings Technol., Available online 15 March 2014, ISSN 02578972, http://dx.doi.org/10.1016/j.surfcoat.2014.02.070. [5] S. Brun, G. Guibert, C. Meunier, E. Guibert, H. Keppner, S. Mikhailov, Rapid and direct micro-machining/patterning of polymer materials by oxygen MeV ion beam irradiation through masks, Nucl. Instr. Meth. B 269 (2011) 2422–2426. [6] S. Brun, V. Savu, S. Schintke, E. Guibert, H. Keppner, J. Brugger, H.J. Whitlow, Application of stencil masks for ion beam lithographic patterning, Nucl. Instr. Meth. B 306 (2013) 292. [7] G.W. Grime, C.J. Sofield, I. Gomez-Morilla, R. Gwilliam, M.D. Ynsa, O. Enguita, Rapid direct micromachining of PTFE in an oxygen rich atmosphere, Nucl. Instr. Meth. B 231 (2005) 378–385. [8] A. Kitamura(Ogawa), T. Satoh, M. Koka, T. Kobayashi, T. Kamiya, Fabrication of micro-prominences on PTFE surface using proton beam writing, Nucl. Instr. Meth. B 306 (2013) 288–291. [9] Comelec-SA, La Chaux-de-Fonds, Switzerland. [10] A. Chapiro, Chemical modification of irradiated polymers, Nucl. Instr. Meth. B 32 (1988) 111. [11] http://www.comelec.ch/en/parylenetableaux.php. [12] F. Edin, W. Liu, H. Li, F. Atturo, P.U. Magnusson, H. Rask-Andersen, 3-D gel culture and time lapse video microscopy of the human vestibular nerve, Acta Otolaryngol. 134 (12) (2014) 1211–1218, http://dx.doi.org/10.3109/00016489. 2014.946536. [13] G.O. Donoghue, Cochlear implants, N. Engl. J. Med. 369 (2013) 1190. [14] G.M. Clark, The multichannel cochlear implant for severe-to-profound hearing loss, Nat. Med. 19 (2013) 1236, http://dx.doi.org/10.1038/nm.3340. [15] Y. Brand, P. Senn, M. Kompis, N. Dillier, J.J. Allum, Cochlear implantation in children and adults in Switzerland, Swiss Med. Wkly. 144 (2014) 1, http:// dx.doi.org/10.4414/smw.2014.13909. [16] http://www.millipore.com//item/scr006. [17] http://www.lifetechnologies.com/order/catalog/product/21103049. [18] http://www.sigmaaldrich.com/catalog/product/sigma/p8920. [19] https://www.lifetechnologies.com/order/catalog/product/23017015, ICID = search-product. [20] Alexa Fluor 488 Phalloidin, . [21] http://imagej.nih.gov/ij/.