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Archived at the Flinders Academic Commons: http://dspace.flinders.edu.au/dspace/ This is the publisher’s copyright version of this article. The original can be found at: http://dx.doi.org/DOI: 10.1117/12.759351 Clements, L., Khung, Y., Thissen, H.W., & Voelcker, N.H., “Preparation of 2-directional gradient surfaces for the analysis of cell-surface interactions”. Proceedings of SPIE, 6799, 67990W-1-67990W-9 (2007). Copyright 2007 Society of Photo-Optical Instrumentation Engineers. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited.

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Preparation of 2-directional gradient surfaces for the analysis of cell-surface interactions Lauren R. Clementsa,b,c*, Yit-Lung Khunga, Helmut Thissenb,c, Nicolas H. Voelckera,c School of Chemistry, Physics and Earth Sciences, Flinders University, GPO Box 2100, Bedford Park 5042 SA, Australia; b CSIRO Molecular and Health Technologies, Bayview Avenue, Clayton VIC 3168, Australia; c CSIRO Food Futures Flagship, 5 Julius Avenue, North Ryde NSW 2113, Australia a

ABSTRACT The ability to evaluate and control the cellular response to substrate materials is the key to a wide range of biomedical applications ranging from diagnostic tools to regenerative medicine. Gradient surfaces provide a simple and fast method for investigating optimal surface conditions for cellular responses such as attachment and growth. By using two orthogonal gradients on the same substrate, a large space of possible combinations can be screened simultaneously. Here, we have investigated the combination of a porous silicon (pSi) based topography gradient with a plasma polymer based thickness gradient. pSi was laterally anodised on a 1.5 x 2.5cm2 silicon surface using hydrofluoric acid to form a pore size gradient along a single direction. The resulting pSi was characterised by SEM and AFM and pore sizes ranging from macro to mesoporous were found along the surface. Plasma polymerisation was used to form a thickness gradient orthogonal to the porous silicon gradient. Here, allylamine was chosen as the monomer and a mask placed over the substrate was used to achieve the thickness gradient. The analysis of this chemistry based gradient was carried out using profilometry and XPS. It is expected that orthogonal gradient substrates will be used increasingly for the in vitro screening of materials used in biomedical applications. Keywords: Two-directional gradients, surface modification, porous silicon, plasma polymerisation

1. INTRODUCTION Understanding cell-material interactions is of fundamental importance for a variety of biomedical applications. Cells and living tissue often come into direct contact with synthetic substrate materials and a number of properties of these surfaces have previously been shown to have a direct influence on the cellular response1, 2. Many surface properties have been demonstrated to influence the efficiency of cell attachment and proliferation as well as the phenotype1, 2. Some of these properties include topography3, 4, elasticity5, surface chemistry6, hydrophobicity7, 8 and ligand density9. For this reason, much attention has been directed towards the surface modification of substrates, for example to enhance or minimise cellular attachment10, 11. For example, extracellular matrix proteins such as fibronectin and collagen have been used to promote cellular attachment9 while poly(ethylene oxide) and other polymers have been used to effectively reduce protein adsorption and subsequent cell attachment12. It has been shown previously that the cellular response can be directly influenced by topography alone. For example, ridges (2 µm wide and 3-5 µm high) have been formed on surfaces and 95% of cells were observed to grow along the axis of these ridges, regardless of any additional chemistry applied to the surface13. Commonly termed ‘contact guidance,’ this phenomenon has been used to direct cell growth. The interaction of cells with a given surface is not generic, in that different cell types favour different surface conditions. Screening for a favourable cellular response in a specific application can therefore be a time-consuming process as the combination of screening factors can be numerous. At the same time, sample sizes used in such experiments are often large (1 cm2 or more). For this reason, several high - throughput techniques have recently emerged that allow screening * Email: [email protected]; Phone: +61 8 8201 2168

BioMEMS and Nanotechnology III, edited by Dan V. Nicolau, Derek Abbott, Kourosh Kalantar-Zadeh, Tiziana Di Matteo, Sergey M. Bezrukov, Proc. of SPIE Vol. 6799, 67990W, (2007) · 0277-786X/07/$18 · doi: 10.1117/12.759351

Proc. of SPIE Vol. 6799 67990W-1


for optimal surface conditions in regard to cell attachment, proliferation or phenotype. High-throughput experiments based on microarrays in particular are gaining increasing popularity in biomedical research as they allow for several characteristics to be investigated at once. While microarray technologies allow for the simultaneous analysis of several properties at once, gradient surfaces can be used to optimise the cellular response to a small set of variables. Gradient surfaces can be described as surfaces on which at least one surface property changes in a controlled fashion. Thus, with the use of gradients, it is possible to effectively reduce sample size and analysis time in in vitro experiments. For example, recent studies3, 4 have shown that surface topography gradient surfaces can be used to screen for specific cellular morphology. Cell attachment was significantly reduced within a certain range of topography, while other topography regions stimulate the cells to assume specific morphologies. In this study, porous silicon (pSi) was chosen as the base substrate. pSi has been shown to be both biocompatible and biodegradable14 and hence has attracted much attention in the field of biomaterials15. Its high surface area has been exploited in drug delivery16 and biosensor applications17, 18, where high levels of drug loading were reported. The effectiveness of pSi as a scaffold for bone regeneration also paves the way for tissue engineering applications19. pSi can be easily tailored to produce various pore sizes, depths and porosities through judicious choice of the etching conditions. The influence of etching current density on pore size affords the opportunity to generate lateral pore size gradients on this material. A further enhancement of the usefulness of gradient substrates is the development of 2-directional gradients. Here, we have deposited a secondary chemical-based thickness gradient orthogonal to the direction of the tailored pSi gradient. Several research groups have already described thickness gradients incorporating two different polymers and subsequent cellular attachment and morphology20, 21. The motivation behind our studies was to develop 2-directional gradients as screening tools for cell surface interactions. Further studies will investigate the cellular response to combinations of surface topography and surface chemistry based on this screening tool.

2. EXPERIMENTAL 2.1 Preparation of porousity gradients Porous silicon (pSi) gradients were prepared using anodisation of p++ type silicon (0.0005-0.001 Ω.cm resistivity, orientated, Boron doped from Virginia Semiconductors). Etching was performed in a custom built rectangular etching cell using a rectangular platinum mesh counter electrode positioned at one side of the etching cell as shown in Figure 1. Hydrofluoric acid (HF) solutions were prepared using 49% aqueous HF and 100% ethanol. For surface gradient preparation a 1:1 HF/ethanol solution was used, applying a current density of 21.5mA/cm2 for 60 seconds. Following anodisation, samples were rinsed with ethanol, methanol, acetone and dichloromethane and subsequently dried with a gentle stream of nitrogen. Pt electrode

HF/EtOH solution

O-ring Si wafer Teflon Etching Cell

Back contact

Figure 1: Schematic of the etching cell used in this study.

2.2 Preparation of thickness gradients Plasma polymerisation experiments were conducted using a custom-built reactor as described elsewhere22. Briefly, the reactor consisted of 2 circular electrodes enclosed within a cylindrical chamber with a height of 35 cm and a diameter of 17 cm. Allylamine (Aldrich, 98% purity) was used as the monomer. In order to achieve a thickness gradient, a glass slide, tilted 12° to the substrate surface with the sides enclosed, was placed over the silicon wafer. The outer edge of the silicon wafer was protruding 10mm from the opening of the glass slide. Allylamine plasma polymers (ALAPP) were



deposited at a pressure of 0.2 mbar for 25 seconds with a frequency of 200 kHz and a power of 20 W. This procedure was repeated 4 times to achieve an adequate thickness gradient. 2.3 Preparation of 2-directional gradients A two-directional gradient was prepared by combining the porosity gradient and thickness gradient on the same substrate. In the first step, the pSi gradient was formed on the surface. The thickness gradient was subsequently deposited orthogonal to the porosity gradient. As the two gradients ran perpendicular to each other, every x/y position on the substrate had unique surface properties in regard to the pore size and coating thickness as shown schematically in Figure 2.

Figure 2: Schematic for the preparation of a 2-directional gradient whereby the second gradient is generated perpendicular to the first gradient.

2.4 X-ray photoelectron spectroscopy (XPS) X-ray photoelectron spectroscopy (XPS) analysis of the plasma polymer surface was performed an AXIS HSi spectrometer (Kratos Analytical Ltd.) fitted with a monochromatic Al Kα source. Elemental compositions were obtained from survey spectra collected at a pass energy of 320 eV. C1s high resolution spectra were obtained at a pass energy of 40 eV. Binding energies were determined using the aliphatic carbon peak at 285.0 eV as a reference. Vision 1.5 software (Kratos Analytical Ltd.) was used for peak fitting of high resolution spectra. 2.5 Scanning electron microscopy (SEM) Scanning electron microscopy of pSi samples was conducted on the Philips XL30 field emission scanning electron microscope. Samples to be analysed were coated with platinum to avoid charging of the substrate surface. An acceleration voltage of 10 kV was used for all experiments, with images captured at intervals of 1 mm along the gradient. All capture angles were at 90º relative to the substrate surface. 2.6 Profilometry In order to determine the thickness of the deposited plasma polymers, a polymer mask was applied to the flat silicon surface prior to polymer deposition according to a previously described method23. Briefly, poly(D,L-lactide) (PDLLA) was solvent cast onto the edge of a silicon wafer and allowed to dry for 10 minutes prior to plasma treatment. Following plasma polymer deposition, PDLLA masks were carefully removed using tweezers. A Dektak 6M stylus profilometer (Veeco) was then employed to determine the plasma polymer thickness along the gradient. A diamond tip with a radius of 12.5 µm was used with a scan speed of 0.1 mm/sec while applying 10 mg of force to the sample. The plot was levelled and the average step height values were obtained using the Detak 6M software. 2.7 AFM Atomic force microscopy (AFM) analysis of pSi surfaces was performed on a Nanoscope IV (Veeco, Santa Barbara, CA). Analysis of the thickness of the deposited plasma polymer was performed on a MPF 3D AFM (Asylum Research). NSC15 AlBr probes (Micromasch) were used for all air imaging with a tip resonance frequency in the range of 310-341 kHz. Tapping mode was utilised for all experiments, using scan rates of 0.8-1.2 Hz and a target amplitude between 0.651.15V. Images were plane-fitted and pore sizes were measured using the section analysis tool within the Nanoscope 5.30r3 software.



3. RESULTS AND DISCUSSION 3.1 Preparation of porous silicon gradients Porous silicon was generated at a current density of 21.5 mA/cm2 using the asymmetric electrode configuration shown in figure 1. A 1:1 HF/ethanol electrolyte solution was used in order to provide a lateral gradient spanning over a surface distance of approximately 3 cm. Under the anodisation regime used here, electropolishing can be observed in the region near the electrode. This confirms the current density chosen for the anodisation of the gradient is optimal and suggests that any further increase in current density will result in the collapse of the pore structure. Furthermore, the distribution in structural colour and visual characteristics were similar in comparison to previously published lateral gradients reported by Sailor et al.24 and Khung et al.4. The topography of the pSi surface was first characterised with atomic force microscopy (AFM). The pores were expected to be the largest in the region closest to the electrode as it would experience the largest electric field, while the smallest pores were expected to be obtained from regions furthest from the electrode. AFM images were taken at approximately 5 mm intervals and revealed that in the region closest to the electrode, the largest pores were observed with a pore size of 800 nm-1.2 µm while pores with a size of 300-500 nm were observed in the middle region (12 mm from the electrode). Further away from the electrode (24 mm from the electrode), a pore size of only 50-100 nm was observed as shown in Figure 3. However, the ability for AFM to extensively characterise the entire surface was limited by the scan size of the piezo scanner and the difficulty in determining the exact imaging position on the surface. For this reason, other imaging techniques were also employed.

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Figure 3: AFM analysis of etched p++ type porous silicon (current density 21.5mA/cm2, 60 seconds) with a) 2 mm, b) 12 mm and c) 24 mm from electrode. Scale bar represents 1 µm (a,b), 200nm (c). z-scale is 500nm (a,b), 100nm (c).

SEM was performed at 1 mm intervals along the lateral gradient shown in Figure 4a in order to further characterise the surface. As shown in Figure 4b-g, the average pore size decreased at increasing distances from the electrode, which corroborated the observations made by AFM. Pore width and depth were measured using the SEM analysis software. The relationship between the pore width and depth and the distance from the electrode is shown in Figure 5. The pore size rapidly decreased near the electrode for approx 6 mm before a plateau in pore size was observed. In the region closest to the electrode, pores ranging from 1.6 µm-900 nm were observed. At a distance of 2 mm from the electrode, pore sizes were greatly reduced to 1 µm-500 nm in width. 400-100 nm pores were observed 10 mm from the electrode. At distances of 15 mm and 19 mm, pore sizes of 250-100 nm and 200-80 nm were observed, respectively. Finally pore sizes ranging from 100-50 nm were observed at a distance of 25 mm from the electrode. A distribution of pore sizes was evident throughout regions on the gradient, which can be seen in particular in Figure 4c. Similarly, the pore depth decreased with increasing distances from the electrode. The non-linear nature of the gradient could be attributed to the non-linear decrease in current density across the substrate. Previous studies have observed similar results24, 25. Several HF/ethanol ratios were also investigated and it was found that increasing the concentration of HF led to a steeper decline in the pore size along the gradient.



c

Figure 4: Laterally etched p++ type silicon (current density of 21.5 mA/cm2 for 60 s) with a) photograph of the pSi gradient and b)-g) SEM images of gradient pSi along the length of the gradient: b) 0 mm, c) 2 mm, d) 10mm, e) 15 mm, f) 19 mm and g) 25 mm from the electrode. Scale bar represents: a) 5 mm, b)-d) 400 nm and e)-g) 100 nm. Images were captured 90º to the substrate surface.

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distance from electrode (nun) Figure 5: Pore characteristics at varying distances from the electrode as determined from SEM analysis.



3.2 Preparation of plasma polymer gradients Thickness gradients were prepared on flat silicon and pSi by plasma polymerisation using a tilted glass slide with the sides enclosed placed over the silicon substrate. While the approximate thickness of the layer deposited on the surface during the process can be monitored by optical inspection, the exact film thickness of the polymer was determined by profilometry. AFM has been used previously for similar studies however the method is time consuming as imaging must be performed at a precise position23. Control AFM experiments were performed in parallel on the deposited surfaces and the results from AFM and profilometry experiments were in good agreement with each other. Hence profilometry was chosen for the analysis of film thickness. PDLLA was used for masking during plasma polymerisation. This polymer is ideally suited for this purpose as it is soluble in many organic solvents and PDLLA coatings can be mechanically removed easily23. A typical section analysis profile of the plasma polymer at the border of a coated and uncoated (masked) region is shown in Figure 6a. The sharp boundary between the coated and uncoated regions indicates that little damage has occurred to the polymer film upon removal of the polymer mask. The Detak 6M software was used to create an average step height, from which a plot of film thickness vs distance was drawn (Figure 6b). The deposited ALLAPP gradient was found to range from a thickness of 1.1 µm at the opening of the glass slide to a thickness of 230 nm on the covered end. The thickness could be tuned easily by the number and duration of plasma polymer depositions performed on the substrate. In the experiments described here, four subsequent depositions were carried out. The length of the gradient on the substrate was found to be highly dependent on the protrusion of the silicon wafer with respect to the glass slide. The linearity of the gradient was optimal when the substrate was protruding from the glass slide by approximately 10 mm. This allowed for the gradient to extend over a length of 3 mm as shown in Figure 6b.

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Figure 6: Allylamine plasma polymer (ALLAP) film thickness as determined by profilometry studies with a) typical step height profile of ALAPP coating and b) film thickness at varying distances from the exposed end of the glass slide.

3.3 Preparation of orthogonal gradients Our research is focused on the preparation of orthogonal gradients which allows two key surface properties to be modified on a single substrate. Here, we have combined a topography gradient with a coating thickness gradient. The final appearance after incorporation of both a physical and chemical gradient is shown in Figure 7. It is important to note that upon the deposition of the chemical gradient, we observed a slight intensifying in structural colour of the pSi as shown in Figure 7c. This is due to the change in refractive index as a result of the deposition on the underlying pSi film.



c

b

a

+ Figure 7: Preparation of 2-directional gradient surfaces with a) pore size gradient on silicon, b) plasma polymer thickness gradient on silicon and c) orthogonal pore size and thickness gradients generated on silicon.

XPS was used to determine the surface chemistry of the deposited plasma polymer film. The ALAPP surface was analysed after being deposited orthogonally to the pore size gradient. XPS analysis of the 2-directional gradient showed a consistent elemental composition across the topography gradient (within the experimental error of the technique) as shown in Table 1. In addition, the complete attenuation of the Si signal indicates a thickness of more than 10 nm (the information depth of the XPS technique) over the entire wafer. These results indicate that the surface chemistry was not altered over the length of the topography gradient. It can therefore be assumed that when culture studies are implemented, any cellular response to the surface is related to the thickness of the coating and not to a difference in surface chemistry. Table 1: XPS data obtained on a sample representing orthogonal pSi and ALAPP thickness gradients. The elemental composition was obtained along the pore size gradient, starting from the electropolished region.

Distance (mm)

C (%)

O (%)

N (%)

0 5 10 15 20 25

73.0 73.5 73.6 74.0 72.9 73.9

14.9 14.1 14.1 14.3 14.8 14.3

12.1 12.4 12.3 11.7 12.3 11.8

4. CONCLUSIONS The application of 2-directional continuous gradient substrates is a promising approach towards the identification, control and optimisation of cell-biomaterial interactions. Here we have demonstrated the preparation of substrates representing two independent orthogonal gradients. A pSi gradient was prepared by anodisation of silicon in aqueous hydrofluoric acid with pore sizes ranging from 1.6 µm to 50 nm. In addition, an allylamine plasma polymer coating was deposited onto the porous substrate. This surface coating represented a thickness gradient. The application of 2D gradient substrates representing topography, chemical and biological signal gradients is a promising approach towards the identification, control and optimisation of cell-biomaterial interactions with a variety of potential applications, including applications in stem cell technologies. It is expected that in particular orthogonal gradient substrates will be used increasingly in a variety of biomedical applications.



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