Micro protein patterning using a lift-off process with fluorocarbon thin film

Sensors and Actuators B 99 (2004) 623–632

Micro protein patterning using a lift-off process with fluorocarbon thin film Sang-Ho Lee a , Chang-Soo Lee b , Dong-Sik Shin b , Byung-Gee Kim b , Yoon-Sik Lee b , Yong-Kweon Kim a,∗ a

b

School of Electrical Engineering and Computer Science (ENG 420-007), Seoul National University, Seoul 151-742, South Korea School of Chemical Engineering, Seoul National University, Seoul 151-742, South Korea

Received 2 April 2003; received in revised form 18 November 2003; accepted 24 November 2003

Abstract This paper discusses micro protein patterning using fluorocarbon (FC) thin films patterned by a lift-off process to prevent the nonspecific binding of protein. 3M’s FluoradTM and Asahi Glass’s CYTOPTM were evaluated in FC film patterning using the lift-off process. Surface characterization of patterned FC films was performed for various static and dynamic contact angles, surface energy, roughness, surface morphology, and fluorescence intensity after each step of the lift-off process and surface modification for protein patterning. The CYTOPTM films were found to have better biochemical resistance than the FluoradTM films for surface characterization. The CYTOPTM films preserved the surface properties, demonstrating a narrow variation of static and dynamic contact angles, contact angle hysteresis, and surface roughness in repeated surface modifications for the protein patterning over 17 h. In a nonspecific binding test of BSA, it was found that the nonspecific binding on the CYTOPTM film appeared considerably above 2 ␮g/ml from the surface characterization. We fabricated 100 ␮m diameter circular silicon nitride (SiN) patterns with the CYTOPTM film background. FITC-labeled BSA was patterned on the silicon nitride surface after chemical oxidation for Si–OH group activation, silanization, and aldehyde treatment. © 2004 Elsevier B.V. All rights reserved. Keywords: Fluorocarbon film; Lift-off process; Contact angle; Protein patterning; Nonspecific binding

1. Introduction It is a desirable and useful technique to immobilize biomolecules in specific locations with micron size, in creating lab on a chip (LOC). Recently, research on biopatterning has concentrated on selective immobilization of proteins with prevention of nonspecific binding at undesired locations on a chip [1–3]. Several techniques for creating micron-sized arrays of proteins have been reported, including conventional photolithography [1,4,5], photochemistry [1], micro-contact printing [2], patterning using fluorocarbon (FC) film backgrounds [6,7], laminar flow patterning using polydimethylsiloxane (PDMS) microchannels [3], polymer-based dry lift-off [8] and ink-jet-based robotic printing [1].

∗ Corresponding author. Tel.: +82-2-880-7440; fax: +82-2-873-9953. E-mail addresses: [email protected] (S.-H. Lee), [email protected] (Y.-K. Kim).

0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2003.11.040

FC film patterning using a microfabrication process to achieve control of the cell or protein adsorption is a recent introduction. Because FC film has low surface energy, excellent chemical resistance, and thermal stability, it is an excellent candidate for use in the design of selective biomaterial adsorption [6,7]. At present, the widely studied commercial aqueous FC solutions are FluoradTM (3M, St. Paul, MN, USA), Teflon AF® (Du Pont Polymers, Wilmington, DE, USA) and CYTOPTM (Asahi Glass, Japan). In addition to the LOC applications, these FC solutions have diverse applications such as lubricant, protective coating for electronic components, low dielectric material in microelectronics, optical film, anti-reflection coating, and as an anti-sticking layer in microelectromechanical systems [7,9,10]. FC film patterning methods generally utilize spin-coating and O2 plasma-based dry etching. After spin-coating the FC solution, the FC films are patterned by O2 plasma-based etching using a photoresist etching mask [5,7,9]. Photoresist has demonstrated poor adhesion to the FC film surface.

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Therefore, to improve adhesion between the photoresist and the FC film, a thin aluminum layer must be deposited on the FC film surface by thermal evaporation or sputtering, as an adhesion promotion layer. It is also important to ensure that the resultant FC film surface is free of any aluminum related species, as they may provide more active sites causing nonspecific binding of protein [9]. Andersson et al. [11] reported the suitability of C4 F8 film patches as hydrophobic valves in microfluidic biochemical applications. The C4 F8 film was deposited on photoresist opening patterns in an inductively coupled plasma (ICP) etcher, and patterned by a lift-off process. The lift-off process is a very effective method in FC patterning as it can preserve the inherent FC film properties. The plasma polymerization using an ICP etcher demands expensive vacuum equipment and source material. In addition, the plasma chemistry in the film growth could lack reproducibility compared with FC solution spin-coating. The FC solution spin-coating method guarantees reproducible Teflon-like film properties and does not require any surface pretreatment, such as silanization in self-assembled monolayer (SAM) coatings. Hydrophobic SAM coatings generally need silanization and depend on surface coverage of the silanol groups [1,2]. This study introduces an FC film patterning method using a lift-off process and a spin-coating method to obtain a reproducible hydrophobic surface. 3M’s FluoradTM and Asahi glass’s CYTOPTM were evaluated for FC film patterning. Silicon nitride (SiN) has a wide range of applications, including as an insulator in microelectroncis, an optical waveguide in optoelectronics, a cantilever, and a membrane in micro and nanoelectromechanical systems (MEMS and NEMS) [12–16]. Chemical, optical, and mechanical properties of SiN can be controlled by changing the stoichiometry by varying the process conditions, such as in a low-pressure chemical vapor deposition (LPCVD) and a plasma-enhanced vapor deposition (PECVD) [12–14]. SiN has been evaluated as a substrate for biosensing in protein covalent binding and cell detection as it can be used for a wide range of biosensing applications on the LOC using MEMS and NEMS technologies [16–20]. This study also introduces a micro protein patterning on the SiN surface with a non-polar FC film background using a fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA). Contact angle analysis, an atomic force microscope (AFM) analysis, and fluorescence intensity measurement were carried out to investigate changes of the surface properties of the FC films in the lift-off process and after repeated biochemical processing.

2. Experiment 2.1. Materials Bare silicon wafers (1 0 0, p-type, LG Siltron, Korea) and SiN-deposited silicon wafers were prepared as substrates. A silicon wafer was used to characterize the FC film

surface during the lift-off process and biochemical resistance test. It is possible to detect delicate surface changes of FC films on a silicon substrate, minimizing the topological change of the substrate because of the silicon wafer’s extremely flat surface. Silicon surfaces, as received from the supplier, had a root mean square roughness of 2–3 Å. SiN deposited on a silicon wafer was used for protein patterning. The SiN was deposited on the bare silicon wafer using an STS 310PC plasma-enhanced chemical vapor deposition reactor with a gas mixture of SiH4 , N2 , and NH3 . The deposited SiN had a refractive index of 2.2–2.5, which was close to the measured refractive index of intrinsic silicon-rich nitride in other research results [12–14]. The wafers were diced into substrates of 10 mm × 19 mm. Two commercial FC solutions were selected for FC film spin-coating. One is a mixture of FC-722 and FC-40 (FluoradTM , 3M, MN, USA), and the other is a mixture of CTL-809M and ST-Solv 180 (CYTOPTM , Asahi glass, Japan). Fluorescein isothiocyanate-labeled BSA, streptavidin, 3-aminopropyl triethoxysilane (␥-APS), Tween 20, and glutaraldehyde 25% solution (Grade II) were obtained from Sigma (St. Louis, MO, USA). The BSA was obtained from AGROS Chemicals (NJ, USA). Other chemicals of analytical or research grade were used. Deionized (D.I.) water (H2 O, polar, 72.8 dyn/cm), formamide (HCONH2 , polar, 58 dyn/cm), and diiodomethane (CH2 I2 , non-polar, 50.8 dyn/cm) were used as probe liquids for the contact angle analysis. 2.2. Analytical instruments The refractive index of the deposited SiN was measured using a Gaertner L116 B ellipsometer. Contact angle measurement is widely used, including for adhesion, surface treatment, and polymer film modification. Although contact angles are macroscopically observable consequences of surface interactions, contact angle analysis is a simple and sensitive method for measuring surface changes at a monolayer level [21–23]. A Krüss G10 contact angle analyzer was used to investigate the wettability of FC films. Contact angles of D.I. water (H2 O), formamide (HCONH2 ), and diiodomethane (CH2 I2 ) were measured on the FC films to estimate wettability and surface energy. Contact angle measurements were taken at least five times at different locations on the surface. The average values were used in contact angle analysis. Measurements showed a standard deviation of less than 2◦ . To observe the surface morphology and surface roughness by atomic force microscopy, we used PSIA Auto probe M5 in noncontact mode, and Digital Instruments Nanoscope III in tapping mode. Auto probe M5 was used for surface characterization of the surface modified FC film surfaces. The Nanoscope III was used to observe the FC film surfaces after protein adsorption, as it displays topology and surface roughness more reliably than the Auto probe M5 does.

S.-H. Lee et al. / Sensors and Actuators B 99 (2004) 623–632

These two AFM instruments showed similar data acquisition performance on the same sample (sonicated CYTOPTM FC film) when comparing average roughness (Ra ) and root mean square roughness (Rrms ). The Auto Probe M5 measured 6.33(Ra )/8.23 (Rrms ) and the Nanoscope III measured 6.04 (Ra )/7.92(Rrms ). All fluorescence images were acquired with a BioRad MRC1024 laser-scanning confocal fluorescence microscopy. FITC was excited at 488 nm using a krypton/argon laser. Micro patterned substrates were viewed with a 10× magnifying objective lens. Because the fluorescence has an emission peak in the frequency corresponding to the color green, only the green component of the image was analyzed. Photoshop v. 6.0 (graphic program) was used to calculate fluorescence intensity on the acquired images. 2.3. Fluorocarbon film patterning method Fig. 1(a–c) shows the process flow for the FC film patterning by the lift-off process. To define circular patterns with the FC film background, 3 ␮m thick positive photoresist (PR) (AZ 4330, Clariant Co., USA) was spin-coated on silicon wafers and SiN-deposited silicon wafers, and patterned by a photolithography (Fig. 1(a)). The FC thin film was then spin-coated on the wafers with the photoresist patterns at 1500 rpm (Fig. 1(b)). After hard baking at 110 ◦ C for 10 m, unnecessary FC film and photoresist patterns were

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stripped in acetone, then rinsed in methanol and D.I. water. The removal was performed in an ultrasonic bath with water as the contact liquid (Fig. 1(c)). The advantage of using the lift-off process for FC film patterning is its simplicity compared with O2 plasma dry etching. O2 plasma dry etching-assisted FC patterning is complicated and requires expensive vacuum processes such as aluminum (Al) thermal evaporation and O2 plasma etching. Al-patterning also requires preparation of phosphoric acid (H3 PO4 )-based wet chemicals [6,7]. FC film patterning using the lift-off process does not require any microfabrication except photolithography. Other processes are performed using general laboratory equipment, such as a convection oven and an ultrasonic bath. The FC film can retain its inherent surface properties during patterning because there is no chemical exposure except to general organic solvents, such as acetone and methanol during the fabrication processes. Our experiments demonstrated that the performance of FC film patterning using the lift-off process depended on the mixing ratio of the FC solutions. The solutes (FC-722, CTL-809M) should be diluted with a solvent (FC-40, CT-Solv 180) to a greater degree than the company-recommended recipe. The final mixing ratios were 1–5 for FC-722 and FC-40, and 1–10 for CTL-809M and ST-Solv 180.

2.4. Surface modification processes for protein patterning

Fig. 1. Process flow of fluoropolymer patterning and protein immobilization: (a) photoresist patterning; (b) fluorocarbon solution spin-coating and baking; (c) lift-off patterning using sonication; (d) chemical oxidation and aminosilanization; (e) glutaraldehyde-adsorption; and (f) FITC-labeled BSA binding.

Fig. 1(d–f) shows the protein patterning processes using the FC film patterned SiN substrate. First, the substrates were rinsed in acetone for 10 m, in ethanol for 15 m, and in D.I. water for 15 m to remove the residual photoresist chemicals. They were then carefully blow-dried under a nitrogen stream. To activate the silanol (Si–OH) groups for aminosilane binding on the SiN surface by chemical oxidation, the substrates were soaked in a mixture of CH3 COOH and H2 O2 for 3 h; this was followed by D.I. water rinsing and N2 -drying [24–26]. The water contact angle on the SiN surface changed from 72.1◦ before chemical oxidation to 0◦ after hydrophilization by chemical oxidation. Second, to introduce amine functional groups on the Si–OH groups activated on the SiN surface, the activated SiN substrates were incubated with a 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 D.I. water to remove unbound silane compounds, the substrates were dried under a nitrogen stream (Fig. 1(d)). Third, the amino-silanized substrates were reacted with 10% glutaraldehyde in 1 mM PBS buffer (pH 7.5) at 30 ◦ C for 1 h (Fig. 1(e)). After several rinses with D.I. water, FITC-labeled BSA in 5 mM PBST buffer (phosphate buffer, 0.5% Tween, pH 7.0) was applied to the substrates at room temperature for 1 h (Fig. 1(f)). The substrates were stored at 4 ◦ C until they were used.

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3. Results and discussion 3.1. Surface characterization of fluorocarbon film before and after the lift-off process The lift-off process, when applied to FC film patterning, can cause mechanical damage to the FC film surface during the sonication process. Further investigation is needed to determine whether the FC films maintain their surface properties both before and after the lift-off process using sonication. The FC films were patterned on a silicon wafer and the surface characterized by contact angle analysis and AFM analysis. 3.1.1. Static contact angles and surface energy Table 1 shows the resultant static contact angles of three probe liquids (D.I. water, HCONH2 , CH2 I2 ) on the FC films. The contact angles of liquids on the FC films indicated higher values than those of solid bulk Teflon sheet used as a reference sample for comparative study. The FluoradTM FC film before the lift-off process demonstrated the highest contact angle of surface responses to the probe liquids. Surface energy of a solid surface can be measured by indirect methods using contact angle measurements. Good et al. [21] developed a methodology for determining surface energy (γ s ), called a three-liquid procedure. Using this procedure, we can calculate the surface energy of a non-polar interaction and of an acid–base interaction between liquids and a solid surface. The calculated surface energies of the FC films were in the range of 8–13 dyn/cm lower than that of solid bulk Teflon. The calculated surface energies of FC films were also lower than the surface energies of surfaces containing –CF3 H or –CF2 – [22]. The lower surface energies suggest that the prepared FC thin films have considerable non-polar surface characteristics compared with the Teflon surface containing the –CF2 – chemical structure. The surface energy of the FluoradTM film increased slightly after the lift-off process and the static contact angles decreased by 7–12◦ . However, after the lift-off process, the CYTOPTM

Table 1 Static contact angles of FC films on a silicon substrate and the calculated surface energies Fluorocarbon film surfaces on silicon

Static contact angle (◦ ) θD.I. water

Teflon sheet (reference)

θHCONH2

Surface energy θCH2 I2

γ s (dyn/cm)

108.25

86.77

80.02

18.06

119.51 107.67

105.26 98.67

101.22 94.62

8.72 11.59

113.43 112.30

99.36 96.95

91.06 90.83

12.54 12.96

FluoradTM Before lift-off After lift-off (sonication) CYTOPTM Before lift-off After lift-off

Table 2 Surface roughness, dynamic contact angles of FC films on a silicon substrate, and hysteresis before and after the lift-off process Fluorocarbon film surface on silicon

AFM analysis Ra /Rrms (Å)

Dynamic contact angles θ advancing θ receding

Hysteresis H

FluoradTM Before lift-off After lift-off

3.39/4.42 14.4/18.1

122.91 112.23

82.42 77.66

40.49 34.57

CYTOPTM Before lift-off After lift-off

5.76/7.66 6.04/7.92

119.01 117.24

103.54 103.45

15.47 13.79

film maintained static contact angles and surface energy close to the initial value (before lift-off). 3.1.2. Surface roughness and dynamic contact angles In the AFM roughness analysis (Table 2), the FluoradTM film showed a fivefold increase after the lift-off process in root mean square roughness (Rrms ) and average roughness (Ra ). However, the CYTOPTM film showed a small variation of −1 Å in Rrms and Ra after patterning by the lift-off process. The increase in roughness of the FluoradTM film after sonication may imply that the FluoradTM film can be damaged by sonication, whereas the small variation in roughness of the CYTOPTM film implies that the CYTOPTM film resists sonication. For a more accurate interpretation of the FC film surface, a dynamic contact angle analysis was performed using a captive sessile drop method. The dynamic contact angles were divided into an advancing angle and a receding angle; contact angle hysteresis (H = θadvancing − θreceding ) is defined as the difference between the advancing angle (θ advancing ) and the receding contact angle (θ receding ). The contact angle hysteresis is a function of surface roughness, chemical heterogeneity, surface polarity, and molecular rearrangement of the surface during wetting and drying. A lower value of contact angle hysteresis indicates homogeneity of the surface [22,23]. The relationship between the heterogeneity and the contact angle hysteresis has been discussed by Johnson and Dettre, who modeled a heterogeneous surface and observed that the contact angle hysteresis varied with the percentage of low contact angle component surface coverage [21–23]. The contribution of the hydrophobic (non-polar) component is large for the advancing angle, whereas the contribution of the hydrophilic (polar) component is large for the receding angle [22,23]. As shown in Table 2, we found that the CYTOPTM film demonstrated smaller contact angle hysteresis than the FluoradTM film did, showing the highest contact angle and the lowest surface energy in the static contact angle analysis. In particular, after the lift-off process by sonication, the advancing and receding angles of the FluoradTM film showed a difference of 5–10◦ , whereas the advancing and receding angles had hardly changed on the CYTOPTM film.

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According to the interpretation of Johnson and Dettre’s model [21–23], the decrease of the advancing and receding contact angles can indicate that the percentage of hydrophilic components was increased by sonication damage in the case of the FluoradTM film. Given the results of the dynamic contact angle analysis and the surface roughness analysis, the CYTOPTM film can be considered to maintain its initial surface properties after the lift-off process, as the CYTOPTM film shows small contact angle hysteresis and small variation of surface roughness. 3.2. Biochemical resistance of FC films during surface modifications for protein patterning FC film surfaces should retain their novel surface properties after repeated various surface modifications while preventing protein nonspecific binding. The FC film was patterned on the silicon wafer and its surface was characterized by contact angle analysis, AFM analysis, and fluorescence intensity analysis. The FC film patterned SiN specimens were characterized at each step of surface modifications for protein patterning. In addition, nonspecific binding of protein on the FC films was investigated as a function of the FITC-labeled BSA concentration. 3M’s FluoradTM film was peeled off locally at the first chemical oxidation step for Si–O– group activation, using a mixture of H2 O2 and CH3 COOH. The FluoradTM film showed a large decrease of the D.I. water contact angle (from 110◦ to 70–80◦ ) and had a large contact angle hysteresis (greater than 60◦ ). The AFM analysis showed an obvious difference of surface morphology before and after chemical oxidation, as if the film surface were swollen. Near-circular hillocks appeared on the scanned area (Fig. 2(b)). However, the CYTOPTM film showed stable surface properties at the first chemical oxidation step. The static contact angle and dynamic contact angles maintained nearly constant values, showing a dismissible deviation within 2◦ (Table 4). Moreover, the surface morphology hardly changed after chemical oxidation in the AFM analysis (Fig. 2(c) and (d)). Therefore, we proceeded with the surface characterization only on the CYTOPTM film for the following surface modifications and protein adsorption. In the case of the CYTOPTM film, the static angles and dynamic angles (θ advancing and θ receding ) hardly changed, showing a variation within 2◦ before FITC-labeled BSA adsorption (Table 3). In addition, the contact angle hysteresis retained its initial values, close to the contact angle hysteresis as measured on the spin-coated film. However, AFM analysis showed a slight increase in surface roughness during surface modifications. Surface roughness of less than −0.1 ␮m does not usually contribute to contact angle hysteresis [27]. Recently, it was reported that there is no correlation between the nanometer scale surface roughness and the contact angle hysteresis regardless of whether the surface is polar or non-polar. In addition, the final conclusion in those reports was that the contact angle hysteresis

Fig. 2. AFM images of fluorocarbon film on a silicon substrate: (a) FluoradTM film before chemical oxidation (after sonication); (b) FluoradTM film after chemical oxidation; (c) CYTOPTM film before chemical oxidation (after sonication); and (d) CYTOPTM film after chemical oxidation (scan area: 3 ␮m × 3 ␮m, PSIA’s Auto Probe M5).

depends dominantly on the chemical heterogeneity or chemical interaction [28–30]. Therefore, we can conclude that there is no significant increase of chemical heterogeneity of the CYTOPTM film surface that can affect the contact angle hysteresis and static contact angles during the surface modifications. The small variations of the contact angle hysteresis suggests that CYTOPTM film can endure repeated surface modifications for 16 h (3 h chemical oxidation, 12 h aminosilanization, 1 h glutaraldehyde-treatment).

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Table 3 Surface roughness, dynamic contact angles, and static contact angles of CYTOPTM films on a silicon substrate at each step of surface modifications for protein patterning Surface modification (reaction time)

Sonication (4 m) Oxidation (3 h) Aminosilanization (12 h) Glutaraldehyde (1 h)

AFM analysis Ra /Rrms (Å)

6.04/7.92 7.80/12.3 10.9/15.0 10.8/14.1

Dynamic contact angle θ advancing

θ receding

115.39 114.84 114.91 114.03

101.90 103.83 103.42 101.67

To investigate the nonspecific binding of BSA protein on the CYTOPTM films, we examined the effect of various protein concentrations on the CYTOPTM films that had been surface modified for the protein patterning method. All specimens were soaked in BSA for 1 h. Table 4 shows the variation of contact angles and roughness on CYTOPTM films as a function of BSA concentration. As the protein concentration increased, advancing and receding angles decreased and the contact angle hysteresis was increased. The advancing and the receding angles decreased by 4–9◦ compared with their value after sonication. The receding angle was sensitive to surface degradation by the nonspecific binding of the BSA protein. Interestingly, the static angles of water maintained the initial value of 111◦ within ±1◦ We found that the static contact angles could hardly discriminate for surface degradation of the FC films. However, dynamic contact angle analysis implied the indication of nonspecific binding on the surface. AFM images in Fig. 3(a–d) show the remarkable surface topological change where the fine grains became enlarged; this effect increased as the BSA concentration increased on the CYTOPTM film. These phenomena were obvious at levels above 2 ␮g/ml. In a 10 ␮g/ml BSA adsorption, the surface roughness of CYTOPTM film increased three times in Ra and Rrms to its values of the sonication step. The analytical results in terms of dynamic contact angles and AFM prove that the increased BSA concentration can cause nonspecific binding of BSA on the CYTOPTM film surface. In the same manner, fluorescence intensity on the CYTOPTM film increased with the increased FITC-labeled BSA concentration. The correlation of the dynamic contact angle analysis, AFM analysis, fluorescence analysis, and the nonspecific binding appeared to be a serious problem at concentrations greater than 2 ␮g/ml of the BSA protein.

Hysteresis H

Static contact angle θD.I. water

13.49 11.01 11.49 12.36

112.19 111.63 112.98 109.29

These findings were gathered from the surface roughness, the topological change, and the fluorescence intensity increased considerably above 2 ␮g/ml; furthermore, the dynamic contact angles indicated a decreasing trend above 2 ␮g/ml, although the dynamic contact angles decreased out of the error range (>4◦ ) from 5 ␮g/ml. There are many factors involved in the nonspecific binding of proteins, including the protein’s three-dimensional structure, hydrogen bonding force, hydrophobic interaction, electrostatic force properties, and reactive functional groups on the protein surface. Because these factors also change with the buffer pH, isoelectric point of the protein, surface charge density, chemical property of the solid surface, and adsorption of low molecular chemicals [31], it is not easy to identify those specific factors playing major roles in nonspecific binding. One recent interesting finding is that elimination of the hydrogen bonding donor group, incorporation of the hydrogen bonding group, and maintenance of electrically neutral charge balance appear to be key structural elements in making protein resistant surfaces [32]. The CYTOPTM film, having low surface energy (−13 dyn/cm), can partially meet the requirements of no hydrogen bonding donor group, neutral charge balance, and high hydrophobicity to prevent nonspecific binding at less than 1 ␮g/ml protein concentration. Nonspecific binding may result from the increase in hydrophobic interactions between the hydrophobic thin film and hydrophobic groups of the protein on the CYTOPTM film. 3.3. Protein patterning on silicon nitride patterns with the CYTOPTM film background Circular SiN pattern arrays were fabricated using the lift-off process for the CYTOPTM film for protein patterning.

Table 4 Surface roughness, dynamic contact angles and static contact angles of CYTOPTM films on a silicon substrate as a function of FITC-labeled BSA concentration BSA adsorption concentration, 1 h reaction (␮g/ml)

AFM analysis Ra /Rrms (Å)

1 2 5 10

7.27/11.81 10.20/19.09 9.81/16.40 16.11/23.21

Dynamic contact angle θ advancing

θ receding

114.07 113.51 111.50 111.82

99.37 97.64 93.30 92.72

Hysteresis H

Static contact angle θD.I. water

14.70 15.87 18.20 19.10

110.87 111.47 112.12 111.82

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The diameter of the circular SiN pattern was 100 ␮m and its center-to-center pitch was 450 ␮m. The FITC-BSA proteins specifically immobilized on the SiN patterns were measured using a confocal microscope, and the variance of the fluorescence intensity from both the intra patterns in a single chip (same batch) and inter patterns (batch-to-batch trials; chip to chip) was calculated. To confirm whether a protein pattern resulted from the covalent binding or nonspecific adsorption, we examined the extent of adsorption of protein on the patterned surface as a control experiment for each modified surface. Fig. 4 shows that no significant fluorescence intensity was observed on the SiN patterns except for the aldehyde-modified SiN surface. This result suggests that the nonspecific FITC-BSA binding is negligible on the SiN patterns modified by chemical oxidation, aminosilanization, and the CYTOPTM film background. In other words, we conclude that the BSA specific immobilization is attributed to the covalent binding between FITC-BSA and glutaraldehyde.

Fluorescence Intensity (Arb. Unit)

Fig. 3. AFM images of CYTOPTM film surface on a silicon substrate as a function of the adsorption concentration of FITC-labeled BSA: (a) 1 ␮g/ml; (b) 2 ␮g/ml; (c) 5 ␮g/ml; and (d) 10 ␮g/ml (scan area: 3 ␮m × 3 ␮m, Digital Instruments’ Nanoscope III).

70 60 50

intensity on SiN TM intensity on SiN / intensity on CYTOP TM intensity on SiN / intensity on CYTOP

40 30 20 10 0

ct In ta

a ce s u rf

Oxi

d a ti

on am

ila in o s

n iz a

t io n

a ld e

h

tre yd e

a tm

ent

Fig. 4. Fluorescence intensity analysis of 100 ng/ml FITC-labeled BSA adsorbed on the SiN patterns (diameter: 100 ␮m, center-to-center pitch; 450 ␮m) after each surface modification for investigation of covalent binding of BSA protein.

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Fig. 5. Optical micrograph of the CYTOPTM fluorocarbon film patterned on an SiN substrate (diameter: 100 ␮m, center-to-center pitch; 450 ␮m): (a) and fluorescence intensity profile of FITI-labeled BSA immobilized on SiN patterns with the CYTOPTM film background; (b) at 1 ␮g/ml of FITC-BSA; and (c) at 2 ␮g/ml of FITC-BSA.

The FITC-labeled BSA (10 ng/ml–2 ␮g/ml) was covalently immobilized on the surface modified patterned SiN surface. Fig. 5(a) shows the patterning result of the CYTOPTM film on the SiN substrate. Fig. 5(b) and (c) show the fluorescence images of patterned FITC-BSA on the SiN patterns. When the concentration of FITC-labeled BSA varied from 10 to 600 ng/ml, the ratios of the fluorescence intensity on SiN to the fluorescence intensity on the CYTOPTM film background linearly increased from 2.27 ± 0.08 to 22.99 ± 2.32 (Fig. 6). The variations of the fluorescence

intensity on the same batch, as well as batch-to-batch, were approximately within 12%. These results were collected from the analysis of 342 circles per substrate from three separate batch experiments. As expected in the experiment on biochemical resistance, a significant reduction of fluorescence intensity on the CYTOPTM film background was observed for less than 1 ␮g/ml FITC-BSA concentration, suggesting that the CYTOPTM film efficiently prevents nonspecific protein binding. However, the slight increase of fluorescence intensity on the CYTOPTM film background,

TM

140

intensity on SiN TM intensity on SiN / intensity on CYTOP TM intensity on CYTOP 50

120

40

100 80

30

60 20 40 10

20 0

0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

intensity on SiN / intensity on CYTOP

Fluorescence Intensity (Arb. Unit)

S.-H. Lee et al. / Sensors and Actuators B 99 (2004) 623–632

FITC-BSA concentration (mg/mL) Fig. 6. Fluorescence intensity of FITC-labeled BSA immobilized on the micro SiN patterns as a function of the concentration of the FITC-labeled BSA.

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The receding angle was sensitive to surface degradation by the nonspecific binding of BSA protein. Given surface characterization results, the nonspecific binding appeared considerably higher at concentrations above 2 ␮g/ml. FITC-labeled BSA protein was patterned successfully on the SiN surface, preventing nonspecific binding on the background by the CYTOPTM film at less than 1 ␮g/ml BSA concentration. Circular SiN patterns with a 100 ␮m diameter and 450 ␮m center-to-center pitch were fabricated with the patterned CYTOPTM film background. The fluorescence intensity on the SiN patterns increased linearly within a range of 10 ng/ml–1 ␮g/ml without significant increase of fluorescence intensity on the CYTOPTM film background. The developed protein patterning method, using an FC thin film background, is expected to contribute to a protein chip study using protein patterns of several hundred micrometers at ng/ml concentration level.

Acknowledgements

indicating nonspecific protein binding, was observed at 2 ␮g/ml protein concentration, where the ratio of the fluorescence intensity on SiN to the fluorescence intensity on the CYTOPTM film background decreased to 11.75 ± 1.13.

This paper was supported by the Nano Bioelectronics and Systems Research Center of the Seoul National University, Korea. The authors thank Professor J.-G. Park’s group of Hanyang University for supporting the contact angle and AFM measurements.

4. Conclusions

References

The FC thin film patterning method using the lift-off process has been developed to control a background surface for the prevention of nonspecific binding. 3M’s FluoradTM and Asahi Glass’s CYTOPTM were evaluated comparatively by various surface characterization methods, including static contact angles, dynamic contact angles, surface energy, roughness, surface morphology, and fluorescence intensity. The prepared FC films have low surface energies (8–13 dyn/cm) proving a non-polar surface. We confirmed that the CYTOPTM film maintains the initial surface properties with little variation in the static and dynamic contact angles, surface roughness, and small contact angle hysteresis after the lift-off process. However, FluoradTM films showed large contact angle hysteresis and increased roughness. In addition, the static and dynamic angles decreased by 5–12◦ after the lift-off. The CYTOPTM film demonstrated considerably better biochemical resistance than the FluoradTM film. The CYTOPTM film had a dismissible variation of static and dynamic contact angles, contact angle hysteresis and roughness during the surface modifications for the protein patterning for 17 h (3 h chemical oxidation, 12 h aminosilanization, 1 h glutaraldehyde-treatment, 1 h 1 ␮g/ml BSA adsorption). However, after chemical oxidation, the surface of 3M’s FluoradTM film was degraded, showing an obviously swollen surface resembling a morphological change, and large contact angle hysteresis greater than 60◦ .

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