Journal of Biomimetics, Biomaterials and Biomedical Engineering ...

Journal of Biomimetics, Biomaterials and Biomedical Engineering ISSN: 2296-9845, Vol. 26, pp 66-72 doi:10.4028/www.scientific.net/JBBBE.26.66 © 2016 Trans Tech Publications, Switzerland

Submitted: 2015-11-16 Revised: 2016-01-14 Accepted: 2016-01-14

PDMS Surface Modification Using Biomachining Method for Biomedical Application Yudan Whulanza1, a *, Hanif Nadhif1, b , Jos Istiyanto1, c, Sugeng Supriadi1, d and Boy Bachtiar2,e 1

Department of Mechanical Engineering, Faculty of Engineering, Universitas Indonesia, Indonesia 2

Department of Oral Biology, Faculty of Dentistry, Universitas Indonesia, Indonesia

a

[email protected], [email protected], [email protected], [email protected], e [email protected]

Keywords: Lab-on-Chip, PDMS modification, biomachining, groovy surface

Abstract. Engineering a cell-friendly material in a form of lab-on-chip is the main goal of this study. The chip was made of polydimethyl siloxane (PDMS) with a surface modification to realize a groovy structure on its surface. This groovy surface was naturally and randomly designed using biomachining process. This measure was aimed to improve the cell attachment on the PDMS surface that always known as hydrophobic surface. The biomachined surface of mold and also products were characterized as surface roughness and wettability. The result shows that the biomachining process were able to be characterized in three classes of roughness on the surface of PDMS. Introduction In this decade, study cell by using microfluidics technology is massively applied [1]. Previously, the common method is using petri dish which able to analyze and manipulate cells [2]. However, the dish has lack of environmental dynamic control. Lately, bioreactor is widely used to carry out the observation of cell. Although bioreactor allows dynamic control of the soluble environment, they often lack control of the cell-contact environment [3]. Microfluidic device itself gives unique environment to study small population of cell or even single-cell with ultra-small media volume [4]. Andersson et al also predicted that the use microfluidics use for quantitative cell analysis by bio imaging and bioinformatics method (known as cellomics) will be increased [5]. One reason is the cell target being studied is very suitable with common dimension of microfluidic device (10-200 μm). Moreover, rapid heat and mass transfer in microfluidic system is evident. Hence, it can be said that microfluidic system potentially to be used as a new cell analysis method [6]. Poly dimethylsiloxane (PDMS) has known to be a strong candidate for material used as invitro cell growth study. It has several benefit such as its low cost, ease to fabricate, transparent, oxygen permeable and biocompatible [7]. However, specific effort needs to be employed to enhance the capability of PDMS to be used in cell study. This study requires the use of aqueous or polar solvent which is not a favor for PDMS material because of its hydrophobic nature [8]. Many surface modifications have been performed such as by ultraviolet grafting [9], ozone treatment [10], microjet patterning [11], poly(ethylene glycol) photografting [12], radical polymerization [13]. Briefly, Wong and Ho [14] distinguished the PDMS modification in three categories which are surface activation, physical modification and chemical modification. This research aimed to modify surface of PDMS through natural and random mold design by biomachining process. Biomachining process utilized feroxidans bacteria and etched metal surface to result groovy structure in micro scale [15]. Then, the mold replicated the roughness into the surface of PDMS. Ultimately, a microenvironment was realized in a form of microfluidic system for cell study.

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Journal of Biomimetics, Biomaterials and Biomedical Engineering Vol. 26

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Material and Methods Copper material was prepared into a rectangular block with area of 1x1 cm2 and 5 mm thickness. The surface was cleaned and polished to give a shiny surface of specimen. This block of copper was ready to be used as main part of mold. Later, the metal component shall be surface modified using biomachining process according to Istiyanto [15]. Briefly, the metal specimen was dipped into a flask with solution that contain Acidithiobaccilus ferroxidans bacteria. The solution was kept at 37oC with continuous aeration of oxygen. The machining process was controlled by duration of metal dipping in the flask. Three dipping cases were employed which were 24 hours, 12 hours and without dipping as control parameter. Finally, the specimen was taken out, cleaned and ready to be used as mold. Polydimethyl siloxane (Sylgard 184 Dow Corning) was soaked on the mold that already went through biomachining process. Then, the mold was put inside a vacuum chamber for 20 minutes to eliminate air bubbles occurred in the mold. Later, the mold was placed on hot plate surface at 130oC and kept for 5 minutes. Ultimately, the PDMS membrane was carefully ripped from the metal mold and ready to be characterized. The surface roughness of metal mold and PDMS product were measured using Accretech Surfcom 2900SD3. Five samples were collected for each machining scenario. Additionally, nine points of measurement were taken for each specimen. Wettability of PDMS is an important property to be involved in characterization of material in medical application. This property was indicated by its contact angle of PDMS film surface. The contact angle was measured on 5 specimens of PDMS films. A fifty microliter of distillated water was dropped on PDMS surface and analyzed by Cosview microscopic camera. The acquired image from Cosview enable us to measure the contact angle of the distillated water. A cell viability test was also conducted to assure that the PDMS material is able to host and biocompatible with Candida albicans. It is important because the ultimate product of this material, lab-on-chip, was aimed to study the growth of Candida albicans. Initially, Candida albicans were incubated on the PDMS material for 24 hours and coated by Fetal Bovine Serum (FBS) from Gibco Thermo Fisher Scientific. Finally, in vitro toxicology assay kit, the yellow tetrazolium MTT from ATCC 30-1010K was applied in an ELISA kit reader from Thermo Fisher Scientific. Images of PDMS surface were also acquired to conform the attachment of Candida albicans on the PDMS material. A Scanning Electron Microscope from FEI Inspect F50 was used for each specimen with magnification of 500X, 1000X and 2000X. This step was directly taken after the incubation of Candida albicans was completed. Results and Discussions PDMS surface characterization consists three parameters which are geometric acquisition (qualitative), roughness measurement and wettability measurement. Firstly, the surface of metal mold was image analyzed to indicate the result of biomachining process using SEM. Later on, the PDMS products are also analyzed to show the molding process. Figure 1 shown the surface analysis images obtained by SEM in three different cases of biomachining process. In each case, three magnifications were acquired and depicted in horizontal way (figure 1). Briefly, it can be seen that the three cases of biomachining process also results three types of surface roughness. Note that the last case was the PDMS surface without biomachining process involved.

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Figure 1. SEM images of metal mold surface a) case of 24 hours of biomachining process; b) case of 12 hours biomachining process; and c) no biomachining process involved. The number 1, 2 and 3 indicates the magnification of 500X, 1000X and 2000X, respectively. It can be distinguished qualitatively that the type 1 has the roughest surface among the other types. On the other hand, type 3 of roughness resulted in the smoothest surface of PDMS. Note that figure 1a and 1b shows that type 1 and 2 has random pattern. Unmodified surface shows a relatively smooth surface as depicted in figure 1c. Intuitively, the biomachining process in case 1 has the longest duration which provides feroxidans bacteria to consume more metal mold rather than other cases. Therefore, the number of groovy structure are higher compare to other biomachining cases. Figure 2 shown the surface analysis results obtained by SEM of PDMS surface after molding step with corresponded metal mold. It confirms that the PDMS surface can also be distinguished in three types of roughness. Similarly, type 1 has the roughest surface and unmodified surface has the smoothest surface. Compare to that of figure 1, the roughness of PDMS in figure 2 shows a lower roughness of surface. This phenomenon caused by flattening of groovy structure during molding process.


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Figure 2. SEM images of PDMS surface after mold process a) case of using mold from 24 hours biomachining process; b) case of using mold from 12 hours biomachining process; and c) case of using unmodified surface mold. The number 1, 2 and 3 indicates the magnification of 500X, 1000X and 2000X, respectively. Figure 3 gives the result of roughness measurement for the metal mold and PDMS surface as mold product. The results are distinguished in three type as previously stated which are: type 1, type 2 and unmodified. In metal mold case, the roughness of type of 1, 2 and 3 are 2.020 ± 0.177 μm, 1.508 ± 0.197 μm, and 0.089 ± 0.014 μm, respectively. Additionally, the PDMS surfaces that produced have a roughness of 1.403 ± 0.039 μm, 1.137 ± 0.083 μm, and 0.160 ± 0.038 μm for type 1, 2 and 3 respectively.

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Figure 3. Surface roughness of metal mold and PDMS surface as mold product Note that the type 3 or unmodified metal mold and PDMS have the lowest roughness which indicate a smooth surface as depicted in figure 1 previously. The biomachining process confirms that a groovy structure were resulted significantly as depicted in roughness modification in type 1 and type 2. The measurement shows that this process increased 10 fold times of roughness in metal mold. However, during the molding into PDMS, the roughness get flattened become 8 fold times compare to that without modification. Figure 4 shown the result of contact angle measurement for all types of PDMS surface. It is aimed to measure the degree of hydrophobicity of PDMS. The roughness modification is required to physically change the PDMS surface to be more accommodate in handling aqueous medium. The measurement shows a slight increase of contact angle. This increasing represents a better contact with aqueous medium that carries cell in ultimate application. The contact angle of type 1 has an average of 90o which compare to 80o of unmodified PDMS material.

Figure 4. Contact angle of three types of PDMS surface resulted from the experiment.


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Ultimately, a viability cell test are conducted on PDMS surface to confirm the compatibility of candida albicans on the PDMS material. All types of PDMS specimens were seeded with candica albicans for 24 hours in aqueous environment. The viability is resulted from the measurement of vital sign of albicans through ELISA assay. Here, two scenarios of cell seeding on the PDMS surface are employed. First scenario is involving fetal bovine serum (FBS) coating on the PDMS surface before the cell seeding. On the other hand, there is no FBS coating on PDMS surface during the second scenario.

Figure 5. Result of cell viability based on PDMS substrate Figure 5 indicates cell viability of rough PDMS (type 1 and 2) compared to that the PDMS without surface modification. As the base calculation of cell viability, the ELISA result of candida albicans on the surface of multiwell is applied. It can be indicated that unmodified surface has a lower viability compare to the base result. It also shows that biomachined PDMS has significantly increasing viability for the scenario involving FBS addition. It also shows that the cell has a range of 27.2 % - 40.6 % more alive compare to the control well. Note that the unmodified PDMS has the same value of control well (100%). This indicates that the unmodified PDMS has a similar performance with control well in hosting the cell. This result also applies in the case without the involvement of serum during the viability test.

Figure 6. SEM imaging of C. albicans on PDMS surface from a) case 1 of biomachining process; b) case 2 of biomachining process; and c) case of no biomachining process involved. Figure 6 depicts images of PDMS seeded with C. albicans after viability test with 1,000X magnification. The roughness of PDMS can be indicated uneven shape of surface that showed in 6a and 6b. Qualitatively, it can be seen that C. albicans are able to live in the PDMS surface under all circumstances. However, the density of C. albicans on the area from case 1 has shown to have higher population.

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Conclusion These results indicates that roughness modification affect the PDMS in hosting the C.albicans as long as fetal bovine serum is provided. PDMS surface from 24 hours of biomachining process has a 5% higher viability compare to the 12 hours biomachining. It suggest that cell has a preference to live in microenvironment that enable the cell to attach to a groovier surface. This groovier structure is realized by a surface modification via a longer biomachining process. Acknowledgement This research is funded by Ministry of Research Technology and Higher Education of Indonesia in year 2014-2015 under scheme of International Collaboration and Publication grant. References [1] G. M. Whitesides, The origins and the future of microfluidics, Nature, 442 (2006) 368-373. [2] S. Zhang, Beyond the Petri dish, Nat. Biotechnol., 22 (2004) 151-152. [3] D. D.Carlo, L. P. Lee, Dynamic single-cell analysis for quantitative biology, Anal. Chem., 78 (2006) 7918-7925. [4] M. C. Kim, Z. Wang, R. H. Lam, T. Thorsen, Building a better cell trap: Applying Lagrangian modeling to the design of microfluidic devices for cell biology, J. Appl. Phys., 103 (2008) 044701. [5] H. Andersson, A. van den Berg, Microfluidic devices for cellomics: a review, Sensor Actuat. BChem., 92 (2003) 315-325. [6] M.Nikkhah, F. Edalat, , S. Manoucheri, A. Khademhosseini, Engineering microscale topographies to control the cell–substrate interface, Biomaterials, 33 (2012) 5230-5246. [7] J. Zhou, , A. V. Ellis, N. H. Voelcker, Recent developments in PDMS surface modification for microfluidic devices, Electrophoresis, 31 (2010) 2-16. [8] H. Makamba, J. H. Kim, K. Lim, N. Park, J H. Hahn, Surface modification of poly (dimethylsiloxane) microchannels, Electrophoresis, 24 (2003) 3607-3619. [9] S. Hu, X. Ren, M. Bachman, C. E. Sims, G. P.Li, N. Allbritton, N., Surface modification of poly (dimethylsiloxane) microfluidic devices by ultraviolet polymer grafting, Anal. Chem., 74 (2002) 4117-4123. [10] K. Efimenko, W. E. Wallace, J., Genzer, Surface modification of Sylgard-184 poly (dimethyl siloxane) networks by ultraviolet and ultraviolet/ozone treatment, J. Colloid Interf. Sci., 254 (2002) 306-315. [11] H. M. Tan, H. Fukuda, T. Akagi, T. Ichiki, T.. Surface modification of poly (dimethylsiloxane) for controlling biological cells’ adhesion using a scanning radical microjet, Thin Solid Films, 515 (2007) 5172-5178. [12] S. Sugiura, J. I. Edahiro, K. Sumaru, T. Kanamori, Surface modification of polydimethylsiloxane with photo-grafted poly (ethylene glycol) for micropatterned protein adsorption and cell adhesion, Colloid Surface B, 63 (2008) 301-305. [13] Y. Wu, Y. Huang, H. Ma, A facile method for permanent and functional surface modification of poly (dimethylsiloxane), J. Am. Chem. Soc., 129 (2007) 7226-7227. [14] I. Wong, C. M. Ho, Surface molecular property modifications for poly (dimethylsiloxane) (PDMS) based microfluidic devices, Microfluid. Nanofluid., 7 (2009) 291-306. [15]. J. Istiyanto, A. S. Saragih, T. J. Ko, Metal based micro-feature fabrication using biomachining process, Microelectron. Eng., 98 (2012) 561-565.