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Protein patterning on silicon-based surface using background hydrophobic thin film
Biosensors and Bioelectronics 18 (2003) 437 /444 www.elsevier.com/locate/bios
Protein patterning on silicon-based surface using background hydrophobic thin film Chang-Soo Lee a,b, Sang-Ho Lee c, Sung-Soo Park a,b, Yong-Kweon Kim c, ByungGee Kim a,b,* a School of Chemical Engineering, Seoul National University, Kwanak-Ku, Seoul 151-742, South Korea Institute of Molecular Biology and Genetics, Seoul National University, Kwanak-Ku, Seoul 151-742, South Korea c School of Electrical Engineering and Computer Science, Seoul National University, Kwanak-Ku, Seoul 151-742, South Korea b
Received 13 December 2001; accepted 31 July 2002
Abstract A new and convenient protein patterning method on silicon-based surface was developed for protein array by spin coating of hydrophobic thin film (CYTOPTM). Photolithographic lift-off process was used to display two-dimensional patterns of spatially hydrophilic region. The background hydrophobic thin film was used to suppress nonspecific protein binding, and the hydrophilic target protein binding region was chemically modified to introduce aldehyde group after removal of the photoresist layer. The difference in surface energy between the hydrophilic pattern and background hydrophobic film would induce easier covalent binding of proteins onto defined hydrophilic areas having physical and chemical constraints. Below 1 mg/ml of total protein concentration, the CYTOPTM hydrophobic film effectively suppressed nonspecific binding of the protein. During the process of protein patterning, inherent property of the hydrophobic thin film was not changed judging from static and dynamic contact angle survey. Quantitative analysis of the protein binding was demonstrated by streptavidin /biotin system. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Protein patterning; Nonspecific binding; CYTOPTM; Contact angle
1. Introduction The patterning of biomolecules on solid surfaces, while preventing nonspecific binding of unwanted areas and species, is of keen interest to the recent development for specific protein/protein interactions, immobilization of biomolecules or cells, biosensors, and biochips. In the development of diagnostic kits, biohybrid material, biomaterial devices, protein sensor, and protein chip, nonspecific binding can often be the bottleneck to obtain reliable sensitivity and reproducibility, and to lower positive false error (Blawas and Reichert, 1998; Schneider et al., 2000). Other desirable methods should meet several requirements such as minimum number of
* Corresponding author. Address: Lab of Molecular Biotechnology and Biomaterials, School of Chemical Engineering, Seoul National University, Kwanak-Gu, Seoul 151-742, South Korea. Tel.: /82-2880-6774; fax: /82-2-874-1206 E-mail address: [email protected] (B.-G. Kim).
processing steps, minimal nonspecific binding in the backgrounds, and low production cost for a single-use. For patterning of proteins onto chip substrate, many researchers used both physical adsorption and covalent binding (Williams and Blanch, 1994; Schena et al., 1995; Belosludtsev et al., 2001). Until now, a couple of protein patterning methods were reported and these methods can be classified into four categories; photochemical method, microcontact printing (mCP), microfluid network (mFN) and spotting or spraying. Although photochemical method is well-established technique and can be used repeatedly on the same surface with different proteins to create a multiple protein patterns, UV light (265 /275 nm) is detrimental to protein activity with arylazide, benzophenone, and diazirine chemistries for protein patterning on derivatized surfaces during the irradiation step. However, alternative approach with NO2 or perfluoro-substitution on arylazide ring can make these compounds to be activated at higher wavelength, eliminating the need for the light in the
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range of 265 /275 nm that is more likely to damage proteins (Blawas and Reichert, 1998; Nicolau and Cross, 2000). As an alternative to the photochemical method, Whitesides et al. introduced mCP method for patterning self-assembled monolayers (SAMs) of alkanethiols onto gold surfaces (Jackman et al., 1995; Kane et al., 1999). The mCP is simple, flexible, and well adapted for research laboratories not equipped with photolithographic tools and clean room facilities. But the success of transferring biomolecules from stamp to chip surface depends upon the times taken for steps of both drying the stamp and printing proteins. For example, after 1 min in the dry state at 55% ambient humidity, the protein transferring efficiency was decreased substantially. Because the mechanism of transferring biomolecules was mainly relied on patterned SAM on the immobilizing surfaces, the density of printed proteins is relatively low (Bernard et al., 2000). In mFN method, high aspect ratio of polydimethylsiloxane (PDMS) capillary channels is required for obtaining stable biomolecules patterning. But deeper capillaries are prone to collapse either spontaneously or during processing by inherent structural instability of the PDMS elastomer, and shallow capillaries often tend to be blocked by dust particles or confer poor mass transfer of other proteins in sample solution (Delamarche et al., 1997). Finally, direct spotting or spraying methods are generally used for making various microarray. Multiple arrays of biomolecules were deposited by using a robotic controlled spotter or electrospray deposition (ESD). These methods are adaptable for mass production of microarray but its facility is very expensive for laboratory research. In spotting, there are several variation factors such as robot’s dwell time on the slide, solution viscosity, humidity, temperature, and contaminants. Therefore, many researchers prefer to use an enclosure and air filtration system (Zubritsky, 2000). In ESD method, a critical problem is how to conserve biomolecules functional activity on electrospray and on subsequent impact of the charged electrospray products with substrate surface (Morozov and Morozova, 1999). Without such expensive machines, these methods have some limits on control of quantity of deposited biomolecules, reproducibility, low interaction efficiencies, and high false error rates. Furthermore, although the spotting method can reduce protein activity loss during the immobilization or patterning, the adsorbed proteins are easily washed or removed when the chip is to be used as a unit for lab on a chip (LOC). Therefore, there are other needs to develop efficient protein immobilization techniques for orderly patterning proteins on microfabricated surfaces for protein chips (Kane et al., 1999). To overcome the above problems, we have devised a protein patterning method using hydrophobic thin film
to reduce nonspecific binding on the background and to make various patterns of protein at micron scale from aqueous solution. Even though previous investigation had examined the usage of hydrophobic teflon film for neural cell patterning (Makohliso et al., 1998) and surface modification for MALDI-TOF substrates (AnchorchipTM from Bruker Daltonics) to enhance detection sensitivity of protein and peptides samples by concentrating the micro sample, no attempts were made to use hydrophobic thin film at micron level scale for protein patterning. In our approach, fluorocarbon based hydrophobic film that reduces nonspecific binding and is resistant to harsh chemical and physical treatment is used. Attractions of this method are easy patterning of proteins on various surfaces with flexible pattern shape or various pattern size, and becoming a simple tool for quantitative analysis, so that protein chip made using this method can be easily used for LOC applications. To our knowledge, it is the first trial for the protein patterning on solid surface using hydrophobic thin film.
2. Materials and methods 2.1. Materials Silicon nitride (Si3N4) layer was grown on silicon substrate using plasma enhanced chemical vapor deposition method. Bare silicon (Si), silicon oxide (SiO2, orientation [100], p-type), and silicon nitride (Si3N4) wafers were diced into 1 by 1 cm. FITC (fluorescein isothiocyanate) labeled BSA (Bovine Serum Albumin), streptavidin, g-APS (3-amiopropyl triethoxysilane), Tween 20, glutaraldehyde 25% solution (Grade II) were obtained from Sigma (St. Louis, MO). CYTOPTM is a mixture of cyclized perfluoro polymer (CPFP) and its solvents such as CTL-809M and ST-Solv 180, which were purchased from Asahi glass Co (Tokyo, Japan). There are some variations in its properties depending on its concentration, quality and solvent. CYTOP of code CTL-809M was selected for spin coating on the silicon wafer in our experiment. BSA was obtained from AGROS Chemicals (NJ, USA). Sulfo-NHS-LC-LCBiotin was obtained from Pierce (Pierce, IL). The procedures of biotinylation were performed according to the recommended protocols from Pierce. Other chemicals of analytical or research grade were used. 2.2. Microfabrication of well-defined features using liftoff process All of the microfabrication was performed in the clean room (The Inter-university Semiconductor Research Center, Seoul National University). Silicon based surfaces were precleaned in the mixture of H2SO4 and H2O2 (4:1) and completely rinsed with deionized water.
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we have obtained highly ordered micro patterns on silicon based surfaces with hydrophobic thin film background.
2.3. Protein patterning
Fig. 1. Schematic illustration of the microfabrication process of CYTOPTM on substrates. At first, photoresist patterns were made by conventional photolithography. CYTOPTM polymer was spun on the substrate. The CYTOPTM film coated wafer was then baked in a convectional oven at 110 8C for 10 min. The baked thin film was stripped from the photoresist features by acetone solvent. (a) Thick PR (photoresist) spin coating and patterning. (b) CYTOPTM film spin coating and baking in a convectional oven. (c) PR stripping for remove CYTOPTM attached to PR. (d) Chemical modification with aminosilane and aldehyde linkage. (e) Protein loading from aqueous buffer.
Microfabrication involves several steps as follows (Fig. 1): firstly, in order to use a lift-off process, 3 mm-thick photoresist (AZ 4330) was spun on the substrates and patterned by the photolithography. Second, subsequent hydrophobic thin film (CYTOPTM) was spin-coated on the substrates at a speed of 2000 rpm. Third, the hydrophobic thin film attached to the patterned photoresist was selectively removed by acetone solution. Specimens were sequentially rinsed by methanol and deionized water in the ultrasonic bath for 2 min. Then
The substrate is subsequently rinsed in acetone for 10 min, in ethanol for 15 min and in deionized water for 15 min to remove residual photoresist chemicals. It is carefully dried under blowing nitrogen stream until the next step. Microfabrication and protein patterning method involves several steps (Fig. 1). Firstly, for silicon oxidation step, piranha solution is used (Raman Suri and Mishra, 1996; Weiping et al., 1999) but H2SO4 in the solution is replaced with CH3COOH to preserve the hydrophobic thin film safely. After substrates are soaked with the modified piranha solution for 3 h, the substrates are carefully dried on the convectional oven at 110 8C for 3 h. Secondly, to introduce amine functional group on the surface with 3-amiopropyl triethoxysilane, the above oxidized substrate is incubated with 10% solution of 3-aminopropyl triethoxysilane in absolute ethanol at room temperature for 12 h. Following several rinses of the substrates with 95% ethanol and deionized water to remove unbound silane compounds, the substrates were dried under nitrogen stream. Thirdly, amino-silanized substrate is reacted with 10% glutaraldehyde in 1 mM PBS buffer (pH 7.5) at 30 8C for 1 h. After several rinses with deionized water, protein solution in 5 mM PBST buffer (phosphate buffer, 0.5% Tween, pH 7.0) was applied to the substrate at room temperature for 1 h and stored at 4 8C until use. In streptavidin /biotin system, FITC / biotin /BSA solution was reacted with streptavidin patterned chip for 1 h. The amount of immobilized proteins on the desired areas (100 mm diameter, pitch; 450 mm distance, chip; 1 by 1 cm) was measured with commercial FITC labeled BSA and variances of the fluorescence intensity from both intra patterns and inter patterns (batch to batch trials) were calculated.
2.4. Contact angle Prior to the measurement of contact angle analysis, the substrates were rinsed with deionized water and dried under nitrogen stream. Contact angles of the substrates were determined after each reaction step within 24 h. Kru¨ss G10 contact angle analyzer (Kru¨ss GmbH, Hamburg, Germany) was used to investigate wettability and surface tension of hydrophobic thin film. All the measurements were carried out at room temperature and ambient humidity. Each reported value was the average of contact angles measured for five times.
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2.5. Fluorescence Analysis All fluorescence image was acquired with laser-scanning confocal fluorescence microscopy (BioRad MRC 1024, Bio-Rad Laboratories Inc., California, USA). FITC was excited at 488 nm using a krypton /argon laser. Micro patterned substrates were viewed with 10 / objective. Because the fluorescence has an emission peak in a frequency corresponding to green color, only the green component of the image was analyzed.
3. Results and discussion 3.1. Protein patterning FITC labeled BSA (10 /2 mg/ml) was immobilized onto the patterned circles (100 mm diameter) on plasma enhanced chemical vapor deposited silicon nitride surface. Spatially high ordered two dimensional protein patterns were observed with high signal-to-noise ratio (pattern intensity/background intensity). By varying the concentration of FITC labeled BSA from 10 to 600 ng/ ml, the signal to noise ratio linearly increased in the range from 2.279/0.08 to 22.999/2.32 (Fig. 2). In the case of control experiments for the extent of adsorption
Fig. 2. Fluorescence analysis of model protein patterning with FITC labeled BSA on the surface; Correlation between the dosing protein concentration and fluorescence intensity. FITC labeled BSA was well immobilized onto chemically modified hydrophilic surface of 100 mm (diameter) circles; Signal (j), Noise (m), Signal/Noise ("), respectively. In the inset graph, the adsorption experiment on each modified surface was carried out with 200 ng/ml of FITC /BSA. Black, white and hatched bars represent signal, noise, and signal/noise ratio. Number 1, 2, 3, and 4 mean intact surface, oxidation, aminosilanization, and aldehyde surface, respectively.
of protein to each modified surface, no significant fluorescence intensity is observed on the protein patterns except aldehyde surface and the CYTOPTM film background (inset graph of Fig. 2), suggesting that nonspecific protein binding is negligible on the modified surface, and low nonspecific binding of FITC /BSA is found on the CYTOPTM background. This result indicates that protein patterning is attributed to covalent binding. The variations of the fluorescence signal intensity among intra and inter patterns were approximately within 12% (Fig. 2). These results were collected from the analysis of 342 circles per one substrate from 3 separated batch experiments. Significant reduction in background signals was observed below 1 mg/ml FITC / BSA concentration, suggesting that the hydrophobic thin film (CYTOPTM) efficiently prevents nonspecific protein binding. However, slight increase in background signals indicating nonspecific protein binding was observed at 2 mg/ml protein concentration, where S/N ratio of 11.759/1.13 was observed (Fig. 3). The higher fluorescence intensity of background might result from the increase in hydrophobic interactions between hydrophobic thin film and hydrophobic groups of the protein. The experiments approve that this patterning method is quite efficient in preventing nonspecific protein binding onto hydrophobic thin film below 1 mg/ml of protein concentration. However, there are many factors involved in the nonspecific binding of proteins such as protein three-dimensional structure, hydrophobicity, hydrogen bonding force, hydrophobic interaction, electrostatic force properties, and reactive functional groups on protein surface. Since these factors also change according to buffer pH, isoelectric point of protein, surface charge density, chemical property of solid surface, adsorption of low molecular chemicals, etc. (Ortega-Vinuesa et al., 1998), it is not easy to identify specific factors playing major roles for the nonspecific binding. One recent interesting finding is that elimination of hydrogen bonding donor group, incorporation of hydrogen bonding group and keeping electrically neutral charge balance appear to be key structural elements in making protein resistant surfaces (Chapman et al., 2000). According to the result, the CYTOPTM (amorphous cyclized perfluoro polymer) coated surface is such a kinds partially fulfilling requirements of having no hydrogen bonding donor group, neutral charge balance, and high hydrophobicity. 3.2. Wetting property during protein patterning CYTOPTM has similar material properties to those of Teflon, and is only solubilized by other fluorocarbon compounds (Matsumoto, et al., 1998). Because coating a hydrophobic thin film is an effective method to prevent stiction in micromachining technology, CYTOPTM is considered as an attractive material for circuit
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Fig. 3. Micropatterns displayed on CYTOPTM coated surface, and the line fluorescence intensity profile of the micropatterns from its image; Upper and lower images show the results at 1 mg/ml of FITC /BSA and at 2 mg/ml of FITC /BSA, respectively.
protection of integrated sensors. The fabricated silicone nitride surface has hydrophilic and hydrophobic regions made from CYTOPTM film coating and lift-off process, respectively. If our observation is correct, chemical reaction for the protein patterning only occurs in the hydrophilic region, and hydrophobic property of the CYTOPTM film would be critical to evaluate the nonspecific protein binding. Therefore, static contact angle was measured to examine the hydrophobicity of micropatterned CYTOPTM film surface. As shown in Fig. 4, the substrates coated with CYTOPTM shows larger static contact angles, indicating that the coating gives more hydrophobic surface than the bare silicon oxide and PE silicon nitride substrates. While the water contact angle of bare substrates substantially increased during each protein patterning step, CYTOPTM coated and micropatterned substrate retained its inherent contact angle. The micropatterned surfaces approximately show 111.89/1.28 of average water contact angle, which is almost identical to the reported value from the producer (Asahi Glass Co.). Regardless of different substrates, the contact angle changes during the protein patterning were negligible, suggesting that inherent wettability of the hydrophobic thin film coated substrates was not changed during chemical pretreatment and subsequent protein covalent binding.
Fig. 4. Comparison of the static water contact angles on either bare substrate or CYTOPTM patterned substrate surface after each patterning step (black bar; after chemical oxidation, white bar; aminosilanization, gray bar; glutaraldehyde treatment, hatched bar; protein patterning, respectively).
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The precise comparison between the wetting properties of substrates, however, is somewhat difficult because of the fact that the static contact angle based on Young’s Equation is limited by heterogeneous and rough surfaces (Lam et al., 2001). Therefore, measurement of static contact angle cannot alone provide detailed interpretation of surface energetics. To further analyze the average microscopic property of the surface of the coated substrates, advancing and receding contact angles */dynamic contact angle */were measured by captive sessile drop (1 ml) method. As can be seen in Table 1, the difference between advancing and receding angle is from 8.42 to 16.358. In general, advancing contact angle (ua) is larger than receding contact angle (ur), and their difference is known as contact angle hysteresis (DH). The contact angle hysteresis (DH ) is a function of surface roughness, surface polarity, chemical heterogeneity, molecular rearrangement of the surface during the wetting and dewetting process (Lam et al., 2001). It is known that the advancing angle (ua) is more sensitive to the hydrophobic part such as CF2 or CF3 groups and the receding angle (ur) is more sensitive to hydrophilic part. Therefore, a decrease in the surface heterogeneity corresponds to a decrease in the hysteresis. While the hysteresis of bulk Teflon is approximately 308, the prepared CYTOPTM thin films show 8.42 /16.358 of small contact angle hysteresis, suggesting the surface is very homogeneous so that its homogenous property can become an efficient physical and chemical barrier to the protein adsorption. 3.3. Quantitative analysis with streptavidin /biotin system It is crucial for protein chip to have ability for quantitative analysis of target molecules on the chip surface. To evaluate the performance of the quantitative analysis, 1 mg/ml of unlabeled streptavidin protein solution was used for protein patterning, and then different concentrations of FITC /BSA /biotin solution were incubated with patterned streptavidin surface to quantify binding of FITC /BSA /biotin concentrations.
Fig. 5. The quantitative analysis with streptavidin /biotin system; firstly, streptavidin (1 mg/ml, PBST) was patterned onto PE Si3N4 surface and then free aldehyde was blocked by ethanolamine buffer for 30 min. The patterned protein activity was measured using from 1 to 500 ng/ml FITC /BSA /biotin solution; Signal (j), Noise (m), Signal/ Noise ratio ("), respectively. In the inset graph, the same experiment was carried out with 100 ng/ml of FITC /BSA /biotin and FITC / BSA, respectively. Black, white and hatched bars represent signal, noise, and signal/noise ratio, respectively.
As streptavidin and biotin interaction has high binding constant, this assay is generally the most powerful method in molecular biology, immunoassay, diagnostics, and biosensor. The feasibility of streptavidin and biotin reaction system is first criteria to the fundamental study in biosensor, protein chip, and other miniaturized analytical device (Busse et al., 2002). Fig. 5 shows the magnitude of the fluorescence intensity obtained by the analysis of 342 patterned circles per one substrate from three separated batch experiments. The fluorescence signal intensity linearly increases with the increase in the concentration of the FITC /BSA /biotin up to 100 ng/ml. The limit of detection of this assay was 1 ng/ml,
Table 1 The dynamic water contact angles on CYTOPTM patterned silicon oxide and PE Si3N4 surface
Oxidation Aminosilanization Glutaraldehyde Protein
SiO2 Advancing
Receding
DH
PE Si3N4 Advancing
Receding
DH
113.9791.43 115.7390.88 115.1890.25 114.7590.47
101.5591.65 105.9291.69 98.8293.3 102.6695.75
12.4290.51 9.8290.81 16.3593.05 12.0995.28
112.6493.87 112.7493.41 114.0090.85 113.1091.12
104.0092.69 104.3295.09 101.6791.39 104.1895.30
8.6491.77 8.4291.68 12.3390.73 9.6894.17
The values were measured by 5 times after the substrates were dried under the nitrogen chamber. Generally, the static contact angles are smaller than the advancing contact angle.
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corresponding to 15.63 pmol/l of FITC /BSA /biotin in considering the signal to noise ratio(i.e. S/N 2.349/0.23). The level of signal to noise ratio( /2.0) indicates reliable range of detection in chip based assay (Lee et al., 2000). In general, the cutoff value for serodiagnosis is normally around 10 ng/ml. Therefore this method shows reasonable sensitivity for practical diagnoses (Sato et al., 2001). However, compared to the FITC /BSA patterning in Fig. 2, there is 35% reduction in fluorescence intensity at the saturation point, which might result from the loss of streptavidin functional activity in the covalent binding onto silicon surface. In the case of control experiments such as no dosage and FITC /BSA (i.e. 100 ng/ml) in the inset graph of Fig. 5, no significant fluorescence intensity is observed on the features of protein pattern and the CYTOPTM film background, suggesting that there is no nonspecific protein /protein interaction between streptavidin and FITC /BSA under this condition, and low nonspecific binding of FITC /BSA is found on the CYTOPTM background. These results indirectly display the sensitivity and effectiveness of this method for quantitative analysis of interesting targets.
4. Conclusions In this paper, to reduce protein nonspecific adsorption on the background on silicon based surfaces, feasibility of a new protein patterning method was examined using hydrophobic thin film (CYTOPTM). This method has several attractive points: (1) By using different surface energy, protein patterning is easily obtained from aqueous solution. (2) Even if this paper only shows silicon based substrates, this method can be applied to various substrates including glass, gold, and polymer. (3) This method can be regio-selectively used to pattern biomolecules or cells without spotting or dispensing machine and have flexibility in pattern shape and size (4) Reproducibility and efficiency of biomolecules patterning can be improved by increasing dwell time with chemically pretreated hydrophilic regions. (5) This method can be interfaced with robotic spotting machine, piezoelectric, electrospray, or inkjet dispenser system to immobilize many different biomolecules on defined patterns. (6) This method is applicable to quantitative analysis for target molecules by either changing the size of the patterns or the concentration of patterning proteins. (7) The simple, reliable fabrication method is desirable to mass production. Furthermore, CYTOPTM film can become a physical and chemical barrier for reducing nonspecific protein binding. Significant improvement in controlling nonspecific protein binding from this method can be applied to other systems requiring distinct protein surfaces such as protein biosensors, protein chip, cell chip and LOC.
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Acknowledgements This work was supported by the Nano Bioelectronics & Systems Research Center. The authors thank to Dr Hyung-Soo Kim (Institute of Microbiology, Seoul National University) for helpful discussion and analysis of confocal microscopy.
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Protein patterning on silicon-based surface using background hydrophobic thin film Chang-Soo Lee a,b, Sang-Ho Lee c, Sung-Soo Park a,b, Yong-Kweon Kim c, ByungGee Kim a,b,* a School of Chemical Engineering, Seoul National University, Kwanak-Ku, Seoul 151-742, South Korea Institute of Molecular Biology and Genetics, Seoul National University, Kwanak-Ku, Seoul 151-742, South Korea c School of Electrical Engineering and Computer Science, Seoul National University, Kwanak-Ku, Seoul 151-742, South Korea b
Received 13 December 2001; accepted 31 July 2002
Abstract A new and convenient protein patterning method on silicon-based surface was developed for protein array by spin coating of hydrophobic thin film (CYTOPTM). Photolithographic lift-off process was used to display two-dimensional patterns of spatially hydrophilic region. The background hydrophobic thin film was used to suppress nonspecific protein binding, and the hydrophilic target protein binding region was chemically modified to introduce aldehyde group after removal of the photoresist layer. The difference in surface energy between the hydrophilic pattern and background hydrophobic film would induce easier covalent binding of proteins onto defined hydrophilic areas having physical and chemical constraints. Below 1 mg/ml of total protein concentration, the CYTOPTM hydrophobic film effectively suppressed nonspecific binding of the protein. During the process of protein patterning, inherent property of the hydrophobic thin film was not changed judging from static and dynamic contact angle survey. Quantitative analysis of the protein binding was demonstrated by streptavidin /biotin system. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Protein patterning; Nonspecific binding; CYTOPTM; Contact angle
1. Introduction The patterning of biomolecules on solid surfaces, while preventing nonspecific binding of unwanted areas and species, is of keen interest to the recent development for specific protein/protein interactions, immobilization of biomolecules or cells, biosensors, and biochips. In the development of diagnostic kits, biohybrid material, biomaterial devices, protein sensor, and protein chip, nonspecific binding can often be the bottleneck to obtain reliable sensitivity and reproducibility, and to lower positive false error (Blawas and Reichert, 1998; Schneider et al., 2000). Other desirable methods should meet several requirements such as minimum number of
* Corresponding author. Address: Lab of Molecular Biotechnology and Biomaterials, School of Chemical Engineering, Seoul National University, Kwanak-Gu, Seoul 151-742, South Korea. Tel.: /82-2880-6774; fax: /82-2-874-1206 E-mail address: [email protected] (B.-G. Kim).
processing steps, minimal nonspecific binding in the backgrounds, and low production cost for a single-use. For patterning of proteins onto chip substrate, many researchers used both physical adsorption and covalent binding (Williams and Blanch, 1994; Schena et al., 1995; Belosludtsev et al., 2001). Until now, a couple of protein patterning methods were reported and these methods can be classified into four categories; photochemical method, microcontact printing (mCP), microfluid network (mFN) and spotting or spraying. Although photochemical method is well-established technique and can be used repeatedly on the same surface with different proteins to create a multiple protein patterns, UV light (265 /275 nm) is detrimental to protein activity with arylazide, benzophenone, and diazirine chemistries for protein patterning on derivatized surfaces during the irradiation step. However, alternative approach with NO2 or perfluoro-substitution on arylazide ring can make these compounds to be activated at higher wavelength, eliminating the need for the light in the
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range of 265 /275 nm that is more likely to damage proteins (Blawas and Reichert, 1998; Nicolau and Cross, 2000). As an alternative to the photochemical method, Whitesides et al. introduced mCP method for patterning self-assembled monolayers (SAMs) of alkanethiols onto gold surfaces (Jackman et al., 1995; Kane et al., 1999). The mCP is simple, flexible, and well adapted for research laboratories not equipped with photolithographic tools and clean room facilities. But the success of transferring biomolecules from stamp to chip surface depends upon the times taken for steps of both drying the stamp and printing proteins. For example, after 1 min in the dry state at 55% ambient humidity, the protein transferring efficiency was decreased substantially. Because the mechanism of transferring biomolecules was mainly relied on patterned SAM on the immobilizing surfaces, the density of printed proteins is relatively low (Bernard et al., 2000). In mFN method, high aspect ratio of polydimethylsiloxane (PDMS) capillary channels is required for obtaining stable biomolecules patterning. But deeper capillaries are prone to collapse either spontaneously or during processing by inherent structural instability of the PDMS elastomer, and shallow capillaries often tend to be blocked by dust particles or confer poor mass transfer of other proteins in sample solution (Delamarche et al., 1997). Finally, direct spotting or spraying methods are generally used for making various microarray. Multiple arrays of biomolecules were deposited by using a robotic controlled spotter or electrospray deposition (ESD). These methods are adaptable for mass production of microarray but its facility is very expensive for laboratory research. In spotting, there are several variation factors such as robot’s dwell time on the slide, solution viscosity, humidity, temperature, and contaminants. Therefore, many researchers prefer to use an enclosure and air filtration system (Zubritsky, 2000). In ESD method, a critical problem is how to conserve biomolecules functional activity on electrospray and on subsequent impact of the charged electrospray products with substrate surface (Morozov and Morozova, 1999). Without such expensive machines, these methods have some limits on control of quantity of deposited biomolecules, reproducibility, low interaction efficiencies, and high false error rates. Furthermore, although the spotting method can reduce protein activity loss during the immobilization or patterning, the adsorbed proteins are easily washed or removed when the chip is to be used as a unit for lab on a chip (LOC). Therefore, there are other needs to develop efficient protein immobilization techniques for orderly patterning proteins on microfabricated surfaces for protein chips (Kane et al., 1999). To overcome the above problems, we have devised a protein patterning method using hydrophobic thin film
to reduce nonspecific binding on the background and to make various patterns of protein at micron scale from aqueous solution. Even though previous investigation had examined the usage of hydrophobic teflon film for neural cell patterning (Makohliso et al., 1998) and surface modification for MALDI-TOF substrates (AnchorchipTM from Bruker Daltonics) to enhance detection sensitivity of protein and peptides samples by concentrating the micro sample, no attempts were made to use hydrophobic thin film at micron level scale for protein patterning. In our approach, fluorocarbon based hydrophobic film that reduces nonspecific binding and is resistant to harsh chemical and physical treatment is used. Attractions of this method are easy patterning of proteins on various surfaces with flexible pattern shape or various pattern size, and becoming a simple tool for quantitative analysis, so that protein chip made using this method can be easily used for LOC applications. To our knowledge, it is the first trial for the protein patterning on solid surface using hydrophobic thin film.
2. Materials and methods 2.1. Materials Silicon nitride (Si3N4) layer was grown on silicon substrate using plasma enhanced chemical vapor deposition method. Bare silicon (Si), silicon oxide (SiO2, orientation [100], p-type), and silicon nitride (Si3N4) wafers were diced into 1 by 1 cm. FITC (fluorescein isothiocyanate) labeled BSA (Bovine Serum Albumin), streptavidin, g-APS (3-amiopropyl triethoxysilane), Tween 20, glutaraldehyde 25% solution (Grade II) were obtained from Sigma (St. Louis, MO). CYTOPTM is a mixture of cyclized perfluoro polymer (CPFP) and its solvents such as CTL-809M and ST-Solv 180, which were purchased from Asahi glass Co (Tokyo, Japan). There are some variations in its properties depending on its concentration, quality and solvent. CYTOP of code CTL-809M was selected for spin coating on the silicon wafer in our experiment. BSA was obtained from AGROS Chemicals (NJ, USA). Sulfo-NHS-LC-LCBiotin was obtained from Pierce (Pierce, IL). The procedures of biotinylation were performed according to the recommended protocols from Pierce. Other chemicals of analytical or research grade were used. 2.2. Microfabrication of well-defined features using liftoff process All of the microfabrication was performed in the clean room (The Inter-university Semiconductor Research Center, Seoul National University). Silicon based surfaces were precleaned in the mixture of H2SO4 and H2O2 (4:1) and completely rinsed with deionized water.
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we have obtained highly ordered micro patterns on silicon based surfaces with hydrophobic thin film background.
2.3. Protein patterning
Fig. 1. Schematic illustration of the microfabrication process of CYTOPTM on substrates. At first, photoresist patterns were made by conventional photolithography. CYTOPTM polymer was spun on the substrate. The CYTOPTM film coated wafer was then baked in a convectional oven at 110 8C for 10 min. The baked thin film was stripped from the photoresist features by acetone solvent. (a) Thick PR (photoresist) spin coating and patterning. (b) CYTOPTM film spin coating and baking in a convectional oven. (c) PR stripping for remove CYTOPTM attached to PR. (d) Chemical modification with aminosilane and aldehyde linkage. (e) Protein loading from aqueous buffer.
Microfabrication involves several steps as follows (Fig. 1): firstly, in order to use a lift-off process, 3 mm-thick photoresist (AZ 4330) was spun on the substrates and patterned by the photolithography. Second, subsequent hydrophobic thin film (CYTOPTM) was spin-coated on the substrates at a speed of 2000 rpm. Third, the hydrophobic thin film attached to the patterned photoresist was selectively removed by acetone solution. Specimens were sequentially rinsed by methanol and deionized water in the ultrasonic bath for 2 min. Then
The substrate is subsequently rinsed in acetone for 10 min, in ethanol for 15 min and in deionized water for 15 min to remove residual photoresist chemicals. It is carefully dried under blowing nitrogen stream until the next step. Microfabrication and protein patterning method involves several steps (Fig. 1). Firstly, for silicon oxidation step, piranha solution is used (Raman Suri and Mishra, 1996; Weiping et al., 1999) but H2SO4 in the solution is replaced with CH3COOH to preserve the hydrophobic thin film safely. After substrates are soaked with the modified piranha solution for 3 h, the substrates are carefully dried on the convectional oven at 110 8C for 3 h. Secondly, to introduce amine functional group on the surface with 3-amiopropyl triethoxysilane, the above oxidized substrate is incubated with 10% solution of 3-aminopropyl triethoxysilane in absolute ethanol at room temperature for 12 h. Following several rinses of the substrates with 95% ethanol and deionized water to remove unbound silane compounds, the substrates were dried under nitrogen stream. Thirdly, amino-silanized substrate is reacted with 10% glutaraldehyde in 1 mM PBS buffer (pH 7.5) at 30 8C for 1 h. After several rinses with deionized water, protein solution in 5 mM PBST buffer (phosphate buffer, 0.5% Tween, pH 7.0) was applied to the substrate at room temperature for 1 h and stored at 4 8C until use. In streptavidin /biotin system, FITC / biotin /BSA solution was reacted with streptavidin patterned chip for 1 h. The amount of immobilized proteins on the desired areas (100 mm diameter, pitch; 450 mm distance, chip; 1 by 1 cm) was measured with commercial FITC labeled BSA and variances of the fluorescence intensity from both intra patterns and inter patterns (batch to batch trials) were calculated.
2.4. Contact angle Prior to the measurement of contact angle analysis, the substrates were rinsed with deionized water and dried under nitrogen stream. Contact angles of the substrates were determined after each reaction step within 24 h. Kru¨ss G10 contact angle analyzer (Kru¨ss GmbH, Hamburg, Germany) was used to investigate wettability and surface tension of hydrophobic thin film. All the measurements were carried out at room temperature and ambient humidity. Each reported value was the average of contact angles measured for five times.
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2.5. Fluorescence Analysis All fluorescence image was acquired with laser-scanning confocal fluorescence microscopy (BioRad MRC 1024, Bio-Rad Laboratories Inc., California, USA). FITC was excited at 488 nm using a krypton /argon laser. Micro patterned substrates were viewed with 10 / objective. Because the fluorescence has an emission peak in a frequency corresponding to green color, only the green component of the image was analyzed.
3. Results and discussion 3.1. Protein patterning FITC labeled BSA (10 /2 mg/ml) was immobilized onto the patterned circles (100 mm diameter) on plasma enhanced chemical vapor deposited silicon nitride surface. Spatially high ordered two dimensional protein patterns were observed with high signal-to-noise ratio (pattern intensity/background intensity). By varying the concentration of FITC labeled BSA from 10 to 600 ng/ ml, the signal to noise ratio linearly increased in the range from 2.279/0.08 to 22.999/2.32 (Fig. 2). In the case of control experiments for the extent of adsorption
Fig. 2. Fluorescence analysis of model protein patterning with FITC labeled BSA on the surface; Correlation between the dosing protein concentration and fluorescence intensity. FITC labeled BSA was well immobilized onto chemically modified hydrophilic surface of 100 mm (diameter) circles; Signal (j), Noise (m), Signal/Noise ("), respectively. In the inset graph, the adsorption experiment on each modified surface was carried out with 200 ng/ml of FITC /BSA. Black, white and hatched bars represent signal, noise, and signal/noise ratio. Number 1, 2, 3, and 4 mean intact surface, oxidation, aminosilanization, and aldehyde surface, respectively.
of protein to each modified surface, no significant fluorescence intensity is observed on the protein patterns except aldehyde surface and the CYTOPTM film background (inset graph of Fig. 2), suggesting that nonspecific protein binding is negligible on the modified surface, and low nonspecific binding of FITC /BSA is found on the CYTOPTM background. This result indicates that protein patterning is attributed to covalent binding. The variations of the fluorescence signal intensity among intra and inter patterns were approximately within 12% (Fig. 2). These results were collected from the analysis of 342 circles per one substrate from 3 separated batch experiments. Significant reduction in background signals was observed below 1 mg/ml FITC / BSA concentration, suggesting that the hydrophobic thin film (CYTOPTM) efficiently prevents nonspecific protein binding. However, slight increase in background signals indicating nonspecific protein binding was observed at 2 mg/ml protein concentration, where S/N ratio of 11.759/1.13 was observed (Fig. 3). The higher fluorescence intensity of background might result from the increase in hydrophobic interactions between hydrophobic thin film and hydrophobic groups of the protein. The experiments approve that this patterning method is quite efficient in preventing nonspecific protein binding onto hydrophobic thin film below 1 mg/ml of protein concentration. However, there are many factors involved in the nonspecific binding of proteins such as protein three-dimensional structure, hydrophobicity, hydrogen bonding force, hydrophobic interaction, electrostatic force properties, and reactive functional groups on protein surface. Since these factors also change according to buffer pH, isoelectric point of protein, surface charge density, chemical property of solid surface, adsorption of low molecular chemicals, etc. (Ortega-Vinuesa et al., 1998), it is not easy to identify specific factors playing major roles for the nonspecific binding. One recent interesting finding is that elimination of hydrogen bonding donor group, incorporation of hydrogen bonding group and keeping electrically neutral charge balance appear to be key structural elements in making protein resistant surfaces (Chapman et al., 2000). According to the result, the CYTOPTM (amorphous cyclized perfluoro polymer) coated surface is such a kinds partially fulfilling requirements of having no hydrogen bonding donor group, neutral charge balance, and high hydrophobicity. 3.2. Wetting property during protein patterning CYTOPTM has similar material properties to those of Teflon, and is only solubilized by other fluorocarbon compounds (Matsumoto, et al., 1998). Because coating a hydrophobic thin film is an effective method to prevent stiction in micromachining technology, CYTOPTM is considered as an attractive material for circuit
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Fig. 3. Micropatterns displayed on CYTOPTM coated surface, and the line fluorescence intensity profile of the micropatterns from its image; Upper and lower images show the results at 1 mg/ml of FITC /BSA and at 2 mg/ml of FITC /BSA, respectively.
protection of integrated sensors. The fabricated silicone nitride surface has hydrophilic and hydrophobic regions made from CYTOPTM film coating and lift-off process, respectively. If our observation is correct, chemical reaction for the protein patterning only occurs in the hydrophilic region, and hydrophobic property of the CYTOPTM film would be critical to evaluate the nonspecific protein binding. Therefore, static contact angle was measured to examine the hydrophobicity of micropatterned CYTOPTM film surface. As shown in Fig. 4, the substrates coated with CYTOPTM shows larger static contact angles, indicating that the coating gives more hydrophobic surface than the bare silicon oxide and PE silicon nitride substrates. While the water contact angle of bare substrates substantially increased during each protein patterning step, CYTOPTM coated and micropatterned substrate retained its inherent contact angle. The micropatterned surfaces approximately show 111.89/1.28 of average water contact angle, which is almost identical to the reported value from the producer (Asahi Glass Co.). Regardless of different substrates, the contact angle changes during the protein patterning were negligible, suggesting that inherent wettability of the hydrophobic thin film coated substrates was not changed during chemical pretreatment and subsequent protein covalent binding.
Fig. 4. Comparison of the static water contact angles on either bare substrate or CYTOPTM patterned substrate surface after each patterning step (black bar; after chemical oxidation, white bar; aminosilanization, gray bar; glutaraldehyde treatment, hatched bar; protein patterning, respectively).
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The precise comparison between the wetting properties of substrates, however, is somewhat difficult because of the fact that the static contact angle based on Young’s Equation is limited by heterogeneous and rough surfaces (Lam et al., 2001). Therefore, measurement of static contact angle cannot alone provide detailed interpretation of surface energetics. To further analyze the average microscopic property of the surface of the coated substrates, advancing and receding contact angles */dynamic contact angle */were measured by captive sessile drop (1 ml) method. As can be seen in Table 1, the difference between advancing and receding angle is from 8.42 to 16.358. In general, advancing contact angle (ua) is larger than receding contact angle (ur), and their difference is known as contact angle hysteresis (DH). The contact angle hysteresis (DH ) is a function of surface roughness, surface polarity, chemical heterogeneity, molecular rearrangement of the surface during the wetting and dewetting process (Lam et al., 2001). It is known that the advancing angle (ua) is more sensitive to the hydrophobic part such as CF2 or CF3 groups and the receding angle (ur) is more sensitive to hydrophilic part. Therefore, a decrease in the surface heterogeneity corresponds to a decrease in the hysteresis. While the hysteresis of bulk Teflon is approximately 308, the prepared CYTOPTM thin films show 8.42 /16.358 of small contact angle hysteresis, suggesting the surface is very homogeneous so that its homogenous property can become an efficient physical and chemical barrier to the protein adsorption. 3.3. Quantitative analysis with streptavidin /biotin system It is crucial for protein chip to have ability for quantitative analysis of target molecules on the chip surface. To evaluate the performance of the quantitative analysis, 1 mg/ml of unlabeled streptavidin protein solution was used for protein patterning, and then different concentrations of FITC /BSA /biotin solution were incubated with patterned streptavidin surface to quantify binding of FITC /BSA /biotin concentrations.
Fig. 5. The quantitative analysis with streptavidin /biotin system; firstly, streptavidin (1 mg/ml, PBST) was patterned onto PE Si3N4 surface and then free aldehyde was blocked by ethanolamine buffer for 30 min. The patterned protein activity was measured using from 1 to 500 ng/ml FITC /BSA /biotin solution; Signal (j), Noise (m), Signal/ Noise ratio ("), respectively. In the inset graph, the same experiment was carried out with 100 ng/ml of FITC /BSA /biotin and FITC / BSA, respectively. Black, white and hatched bars represent signal, noise, and signal/noise ratio, respectively.
As streptavidin and biotin interaction has high binding constant, this assay is generally the most powerful method in molecular biology, immunoassay, diagnostics, and biosensor. The feasibility of streptavidin and biotin reaction system is first criteria to the fundamental study in biosensor, protein chip, and other miniaturized analytical device (Busse et al., 2002). Fig. 5 shows the magnitude of the fluorescence intensity obtained by the analysis of 342 patterned circles per one substrate from three separated batch experiments. The fluorescence signal intensity linearly increases with the increase in the concentration of the FITC /BSA /biotin up to 100 ng/ml. The limit of detection of this assay was 1 ng/ml,
Table 1 The dynamic water contact angles on CYTOPTM patterned silicon oxide and PE Si3N4 surface
Oxidation Aminosilanization Glutaraldehyde Protein
SiO2 Advancing
Receding
DH
PE Si3N4 Advancing
Receding
DH
113.9791.43 115.7390.88 115.1890.25 114.7590.47
101.5591.65 105.9291.69 98.8293.3 102.6695.75
12.4290.51 9.8290.81 16.3593.05 12.0995.28
112.6493.87 112.7493.41 114.0090.85 113.1091.12
104.0092.69 104.3295.09 101.6791.39 104.1895.30
8.6491.77 8.4291.68 12.3390.73 9.6894.17
The values were measured by 5 times after the substrates were dried under the nitrogen chamber. Generally, the static contact angles are smaller than the advancing contact angle.
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corresponding to 15.63 pmol/l of FITC /BSA /biotin in considering the signal to noise ratio(i.e. S/N 2.349/0.23). The level of signal to noise ratio( /2.0) indicates reliable range of detection in chip based assay (Lee et al., 2000). In general, the cutoff value for serodiagnosis is normally around 10 ng/ml. Therefore this method shows reasonable sensitivity for practical diagnoses (Sato et al., 2001). However, compared to the FITC /BSA patterning in Fig. 2, there is 35% reduction in fluorescence intensity at the saturation point, which might result from the loss of streptavidin functional activity in the covalent binding onto silicon surface. In the case of control experiments such as no dosage and FITC /BSA (i.e. 100 ng/ml) in the inset graph of Fig. 5, no significant fluorescence intensity is observed on the features of protein pattern and the CYTOPTM film background, suggesting that there is no nonspecific protein /protein interaction between streptavidin and FITC /BSA under this condition, and low nonspecific binding of FITC /BSA is found on the CYTOPTM background. These results indirectly display the sensitivity and effectiveness of this method for quantitative analysis of interesting targets.
4. Conclusions In this paper, to reduce protein nonspecific adsorption on the background on silicon based surfaces, feasibility of a new protein patterning method was examined using hydrophobic thin film (CYTOPTM). This method has several attractive points: (1) By using different surface energy, protein patterning is easily obtained from aqueous solution. (2) Even if this paper only shows silicon based substrates, this method can be applied to various substrates including glass, gold, and polymer. (3) This method can be regio-selectively used to pattern biomolecules or cells without spotting or dispensing machine and have flexibility in pattern shape and size (4) Reproducibility and efficiency of biomolecules patterning can be improved by increasing dwell time with chemically pretreated hydrophilic regions. (5) This method can be interfaced with robotic spotting machine, piezoelectric, electrospray, or inkjet dispenser system to immobilize many different biomolecules on defined patterns. (6) This method is applicable to quantitative analysis for target molecules by either changing the size of the patterns or the concentration of patterning proteins. (7) The simple, reliable fabrication method is desirable to mass production. Furthermore, CYTOPTM film can become a physical and chemical barrier for reducing nonspecific protein binding. Significant improvement in controlling nonspecific protein binding from this method can be applied to other systems requiring distinct protein surfaces such as protein biosensors, protein chip, cell chip and LOC.
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Acknowledgements This work was supported by the Nano Bioelectronics & Systems Research Center. The authors thank to Dr Hyung-Soo Kim (Institute of Microbiology, Seoul National University) for helpful discussion and analysis of confocal microscopy.
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