Protein patterning utilizing region-specific control of wettability by surface modification under atmospheric pressure Donghee Lee, Min-Sung Kwon, Ji-Chul Hyun, Chang-Duk Jun, Euiheon Chung et al. Citation: Appl. Phys. Lett. 103, 123701 (2013); doi: 10.1063/1.4821438 View online: http://dx.doi.org/10.1063/1.4821438 View Table of Contents: http://apl.aip.org/resource/1/APPLAB/v103/i12 Published by the AIP Publishing LLC.
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APPLIED PHYSICS LETTERS 103, 123701 (2013)
Protein patterning utilizing region-specific control of wettability by surface modification under atmospheric pressure Donghee Lee,1,a) Min-Sung Kwon,2 Ji-Chul Hyun,3 Chang-Duk Jun,2 Euiheon Chung,1,3 and Sung Yang1,3,b)
1 Department of Medical System Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, South Korea 2 School of Life Science, GIST, Gwangju 500-712, South Korea 3 School of Mechatronics, GIST, Gwangju 500-712, South Korea
(Received 21 April 2013; accepted 30 August 2013; published online 18 September 2013) Wettability control can be crucial in improving the uniformity of selective protein immobilization in high-density microarrays. In this study, we propose an atmospheric-pressure plasma-enhanced chemical vapor deposition (AP-PECVD)-based method in conjunction with photolithography to implement region-specific control of wettability on Si substrate. The proposed PECVD method under atmospheric pressure condition would be a useful alternative of conventional reactive plasma-based treatments methods requiring vacuum condition for uniform protein patterning. Layers with dissimilar wettability and roughness prepared by AP-PECVD process using tetraethoxysilane (TEOS) or TEOS-O2 as precursors could realize uniform protein patterning in a C 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4821438] micrometer-scale. V Microarray technology has been established as a useful bioanalytical means for various applications including basic biological study, disease diagnostics, and drug development in pharmaceutical industry.1,2 DNA chips have been commercialized in the field of genomics.3 Furthermore, the applications of microarray technology have been expanded to proteomics with the aim of analyzing, profiling, and quantifying activities and interactions of protein molecules using protein chips/microarrays. Protein microarrays can be used to rapidly and reliably profile the expression levels of and the interactions between the protein molecules involved in cell signaling and disease progression or other biologically significant events.4,5 The most common method of fabricating protein microarrays in industry is based on attaching protein molecules to chemically modified substrates, to which solutions containing the protein molecules are delivered using high-precision printing robots.6 However, spot inhomogeneity, which can be identified by the “coffee-ring” effect, occur frequently and have been attributed to the hydrophilicity of the substrates that are commonly used to develop microarrays.7,8 Since the spot inhomogeneity significantly increase statistical variation in the data gathered from the arrays, suitable substrates for suppressing the coffee-ring effect have been developed recently, resulting in improved spot homogeneity in microarrays, with the spots now being as small as 10 lm for high-density microarrays.9–13 To minimize the coffee-ring effect, various methods have been used to make physicochemically active patterns on the substrates to limit protein immobilization within the desired areas by controlling the region-specific wettability of the patterns.14–16 Reactive plasma-based treatments can prepare microarray substrates with region-specific control of wettability, while a)
Current address: Korea Photonics Technology Institute (KOPTI), Gwangju 500-779, South Korea b) Author to whom correspondence should be addressed. Electronic mail:
[email protected]. 0003-6951/2013/103(12)/123701/5/$30.00
simultaneously allowing for high-density spot formation and uniform protein binding. However, for conventional plasmabased processing to fabricate microarray, low-pressure conditions are essential,12,13,17,18 requiring vacuum chamber and pumping system. Thus, it has limitations in processing time and substrate size. Atmospheric-pressure plasma enhanced chemical vapor deposition (AP-PECVD) has recently drawn significant attention since the PECVD process can be performed at an atmospheric pressure without requiring a vacuum chamber or a pumping system.19–21 Hence, there is no limit to the size of the substrate to be treated with APPECVD, while only small substrates can be treated with lowpressure plasma-enhanced chemical vapor deposition.22–25 Furthermore, AP-PECVD allows for parallel and in-line processing as well.26 On the other hand, nontoxic, non-explosive, and highly safe precursors are preferred when AP-PECVD is employed to perform reactive plasma treatments because during the process the reactor and chamber are not as controllable as the vacuum chambers for low-pressure PECVD. The latter can handle highly explosive or hazardous gases, which are either used during the treatment process or are reactive byproducts generated owing to the use of H2, CxFy, SF6, and various ethylene glycol methyl ethers.13,17,27 Here, we report the creation of uniform protein patterning using AP-PECVD for selective protein immobilization on 2D surface or 3D structure. Our approach is based on the difference in the adsorption of proteins on specific regions with different wettabilities, thus the region-specific control of wettability was utilized for protein patterning in micrometer scale, fabricated by applying AP-PECVD and lift-off process. Tetraethoxysilane (TEOS; Si-(O-(C2H5))4), a liquid organosilicon for generating silicon dioxide thin films, has been used in numerous applications.20 It is reported that either SiOxCyHz-like or SiO2-like layer can be deposited using TEOS or a mixture of TEOS and O2 as the precursor during the PECVD process.28–30 We demonstrated protein patterning on 2D surface or 3D structured substrates from few
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FIG. 1. SEM images and static contact angles of various AP-PECVD-treated Si surfaces (observed under 100 k magnification). (a) A single layer deposited via AP-PECVD using TEOSO2 (TEOS-O2/Si), (b) A single layer deposited via AP-PECVD using TEOS (TEOS/Si), (c) A double layer deposited via AP-PECVD using TEOS and TEOS-O2 sequentially (TEOS-O2/ TEOS/Si), (d) A double layer deposited via AP-PECVD using TEOS-O2 and TEOS (TEOS/TEOS-O2/Si). The contact angles can be seen from the images inset in top right corners of the SEM images.
micrometer scale to hundreds micrometer scale utilizing the region-specific control of wettability for both a proteinadherent area and a protein-repellent area. The selective immobilization of proteins onto the Si substrate with regionspecific control of wettability could be realized since the adsorption of proteins has dependence on the wettability of the surface.31 Furthermore, the bioactivities of the immobilized antibodies were confirmed by immunoreactions and the specific cell adhesion. Considering several benefits of atmospheric-pressure condition comparing to vacuum condition to fabricate micro-patterning, we believe that this proposed patterning method is a time-saving, cost-effective alternative for microarrays, biochips and biosensors where selective immobilization of biomolecules is essential. Four different surface types were investigated including two single layers deposited by AP-PECVD using either TEOS-O2 (TEOS-O2/Si) or TEOS (TEOS/Si) as the precursor; and two heterogeneous double layers sequentially deposited using TEOS-O2 and TEOS (TEOS/TEOS-O2/Si) or TEOS and TEOS-O2 (TEOS-O2/TEOS/Si), which were applied to implement the region-specific control of wettability for protein patterning in this study. Figure 1 shows surface morphologies of the AP-PECVD-deposited layers measured by scanning electron microscopy (SEM; S-4700, Hitachi, Japan) with each contact angle (Phoenix 300, SEO Co., Republic of Korea). SEM images showed that modified surfaces present dissimilar surface morphology. SEM images on TEOS-O2/Si surface (Fig. 1(a)) and TEOS/Si surface (Fig. 1(b)) showed that the TEOS/Si surface was relatively smooth, but the TEOS-O2/Si surface is considerably rough due to the presence of the nanoparticles with tens of nanometers in size. In case of heterogeneous double layers (TEOS-O2/ TEOS/Si and TEOS/ TEOS-O2/Si), all surface were rough since the heterogeneous double layers have contained the nanoparticles layers deposited by AP-PECVD process using TEOS-O2 as precursor in common. The SEM image of the surface of the TEOS/TEOSO2/Si (Fig. 1(d)) double layer showed it was made of a coating on a layer of nanoparticles whose diameters were larger (about 80 nm) than those of the nanoparticles (about 20 nm) in the TEOS-O2/Si (Fig. 1(a)). It can be inferred that the thin layer, deposited using TEOS, is conformally coated on the
SiOx-like nanoparticles layer deposited using TEOS-O2 layer in case of TEOS/TEOS-O2/Si. As next step, AFM analysis has been conducted to quantify the surface roughness of the surfaces prepared. Table I(a) showed the quantitative roughness values of the modified surfaces by AP-PECVD obtained by atomic force microscopy (AFM; XE-100, Park System, Republic of Korea) analysis, which are consistent with the ones obtained by SEM image analysis. All surfaces containing layer deposited using TEOS-O2 (TEOS-O2/Si, TEOS/TEOSO2/Si and TEOS-O2/ TEOS/Si) presented abrupt increase in the roughness values comparing with the untreated silicon surface (Si). On the contrary, TEOS deposition does not change the roughness of the layers prepared in advance, implying that the layer deposited using TEOS is conformally coated (TEOS/ Si and TEOS/TEOS-O2/Si). Depending on the precursor in CVD process, the chemical composition of a deposited layer is determined.32,33 To clarify the chemical composition ratio of the layers by APPECVD using TEOS and/or TEOS-O2, X-ray photoelectron spectroscopy (XPS; MultiLab 2000, Thermo Electron Corp., MA, US) analysis was conducted as shown in Table I(b). The chemical composition ratios of TEOS/Si and TEOS/TEOS-O2/Si were very similar regardless of the existence of the middle layer prepared using TEOS-O2 in case of TEOS/TEOS-O2/Si. It was observed that the low O/C TABLE I. Quantitative results of the surface characterization of the untreated silicon and various AP-PECVD-treated surfaces: (a) root mean square (rms) surface roughness values with standard deviation calculated from AFM analysis (Unit: nm), (b) chemical composition ratio (Unit: at. %) obtained from XPS analysis and relative composition ratio such as oxygen versus carbon ratio (O/C) or oxygen versus silicon ratio (O/Si). (b)
Si TEOS/Si TEOS-O2/Si TEOS-O2/TEOS/Si TEOS/TEOS-O2/Si
(a)
C
O
Si
O/C
O/Si
0.2 6 0.0 0.4 6 0.4 53.3 6 2.1 55.1 6 4.1 48.5 6 3.3
13.1% 53.5% 6.6% 9.8% 56.0%
28.9% 32.7% 65.5% 60.0% 30.7%
58.0% 13.8% 27.9% 30.2% 13.3%
2.21 0.61 9.92 6.12 0.55
0.50 2.37 2.35 1.99 2.31
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FIG. 2. Schematic diagram for protein patterning on Si substrate with a region-specific control of wettability by applying AP-PECVD using TEOS and TEOSO2. (a) Procedure for protein patterning on a 2D Si surface; (b) Procedure for protein patterning on a 3D Si structure.
ratios in the layers deposited using TEOS (0.61 in case of TEOS/Si and 0.55 in case of TEOS/TEOS-O2/Si) and the high O/C ratios in the layers deposited using TEOS-O2 (9.92 in case of TEOS-O2/Si and 6.12 in case of TEOS-O2/ TEOS/Si). Thus, it could be easily predicted that the layers with the higher O/C ratios will exhibit the more hydrophilic and vice versa.34 In other words, the cases having the TEOS-O2 treated layer as the top-most layer will exhibit the more hydrophilic than the others and are consistent with the contact angle analysis. Next, in order to see how the surface morphology and chemical composition effect on the wetting property, the contact angle has been measured. It can be seen from the contact angle images that the AP-PECVD process altered the wetting properties of Si substrate, turning their surfaces from hydrophilic to be superhydrophilic or less hydrophilic depending on the precursors used in the deposition. The TEOS-O2/Si and TEOS-O2/TEOS/Si surfaces exhibit superhydrophilicity (contact angle; 43 !4 lm) and shapes on 2D/3D Si
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substrates that can be realized depending on the mask and etching depth. To analyze the uniformity of the protein patterns formed, the fluorescence intensity from each fluorescence image was digitized using the ImageJ for quantitative analysis. The coefficient of variation (CV) of the fluorescent intensity was used to evaluate the inter- and intra-spot uniformities.13 The calculated “inter-spot” CV values of the mean fluorescent values obtained from the randomly selected 12 spots were as low as 3.9% for 2D surface and 4.0% for 3D surface and are superior compared to the one reported (14 21%) with coffee-ring effect.10 And the “intra-spot” CV values were 3.4 6.4% on 2D surface and 5.9 8.4% on 3D surface. It is believed that these low CV values for the inter- and intra-spots are comparable to the ones reported for the high density microarrays;10,13,39 the “inter-spot” CV of the mean values (3.6 8.8%) and the “intra-spot” CV values (3.1 9.6%). The detailed statistics could be found in the supplemental material (Tables SI and SII).42 As a final step of this study, the bioactivity of the immobilized proteins has been investigated to see how much the region-specific control of the wettability preserves the protein function. As well known, the bioactivity of the immobilized proteins is of critical importance in protein microarrays. Immobilized proteins usually adhere to the substrate surface in a random fashion, which reduces their bioactivities.40 Although the fluorescence signal was clearly observed only in the area, where the proteins are selective immobilized, that alone is insufficient to guarantee that bioactivities of the proteins are conserved. Thus, the biofunctionality of the immobilized proteins could be determined via the immunoreactions and the specific cell adhesion since the mouse anti-human antibody LFA-1 IgG (TS2/4) is specific to the leukocyte-function-associated antigen-1 (LFA-1) in Jurkat T cells. LFA-1 is a member of the integrin family and is important for leukocyte adhesion and migration.41 To examine the conservation of the bioactivity of the
FIG. 3. Fluorescent microscope images of the patterned fluorescent dye-labeled antibodies on wettability-patterned Si substrates with Cy3-labeled mouse antihuman antibody LFA-1 IgG (TS2/4-Cy3) and Rhodamine-labeled goat anti-rabbit IgG (Anti-rabbitIgG-TRITC). (a) Selective immobilization of proteins on 2D Si surface with region-specific control of wettability (Left: circular-spot array of TS2/4-Cy3 with 200 lm in diameter, Center: honeycomb pattern of TS2/ 4-Cy3 with 10 lm in line width, Right: square-spot array of Anti-rabbit IgG-TRITC with 4 lm in width and length), (b) Selective immobilization of proteins on 3D Si structure (Left: circular-spot array of TS2/4-Cy3 with 200 lm in diameter and 12 lm in height, Center: honeycomb pattern of TS2/4-Cy3 with line width of 10 lm and 12 lm in height, Right: circular-spot array of Anti-rabbit IgG-TRITC with 10 lm in diameter and 8 lm in height).
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immobilized proteins, we tested two immunoreactions simultaneously: (1) one is the antibody-antigen reaction of the fluorescein isothiocyanate (FITC)-labeled anti-mouse antibody IgG (secondary antibody for TS2/4) with the Fc-specificity for TS2/4 and (2) the other is the cell adhesion test with the cell-tracking dye (CMRA)-labeled Jurkat T cells. The fluorescence signal implied that the CMRA-labeled T cells were clearly confined to the TEOS-treated region on the wettability-patterned substrate, while also indicating that the immobilized antibody TS2/4 was functionally bioactive and had reacted with LFA-1 on the T cells (Fig. S3).42 Therefore, one can conclude, on the basis of the selective adhesion of T cells with immobilized TS2/4, that the antibodies immobilized on the wettability-patterned substrates remained bioactive. Utilizing the selective immobilization of T cells on substrate by applying TS2/4 antibodies, this patterning method can be utilized in protein patterning as well as cell patterning. In summary, selective protein immobilization was achieved with region-specific dissimilar wettability on Si substrate prepared by AP-PECVD and a conventional lift-off process. The surface properties of the four layers with single or double layers deposited were characterized by SEM, AFM, XPS, and goniometry analyses. The uniform selective immobilization of protein has been demonstrated on 2D surface or 3D structure. The results of binding assays showed that the fluorescent dye-labeled antibodies have been selectively immobilized specifically only on the TEOS-treated region of the substrate, while they were strongly prohibited from immobilization on the TEOS-O2-treated region. The biofunctionality of our patterning method was confirmed by adhesion test of Jurkat T-cells with the immobilized TS2/4. Our method would be a useful alternative for an efficient and cost-effective technique in various applications where selective immobilization of proteins is required. Authors would like to thank Dr. Daekyung Sung for invaluable discussions. This work was partially supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 20110028861), and a grant from the Institute of Medical System Engineering (iMSE) at the Gwangju Institute of Science and Technology (GIST), Republic of Korea. 1
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