Chinese Science Bulletin © 2009
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Preparation and characterization of 3-(triethoxysilyl) propyl isocyanate self-assembled monolayer on surface of chip XIE Yao, GENG LiNa, QU Feng, LUO AiQin, QU Feng & DENG YuLin† School of Life Science and Technology, Beijing Institute of Technology, Beijing 100081, China
Monolayer of 3-(triethoxysilyl) propyl isocyanate was prepared on the slide by self-assembled technique. X-ray photoelectron spectroscopy (XPS) was employed to analyze the elementary composition of the film. Contact angle of distilled water was measured to characterize the surface state. It was shown that 3-(triethoxysilyl) propyl isocyanate had been successfully assembled on the slide. The increase of contact angle to 80° demonstrated that the hydrophobicity of the surface of chip was increased significantly. Moreover, further self-assembly of bovine serum albumin (BSA) on 3-(triethoxysilyl) propyl isocyanate was also carried out with the advantages such as simple and convenient preparation. Therefore, the potential of broader applications in the modification of micro-channel in the μ-TAS system, the immobilization of protein or peptide and the surface modification of materials are all expectative. 3-(triethoxysilyl) propyl isocyanate, self-assembled monolayers, characterization, covalent bonding
Self-assembly (SA), which was developed in the late 1980s, was used for functional material assembling at ― molecule level[1 3]. The organics and inorganics compounds were usually induced to form the monolayer or multilayer nano-film. So far, these techniques have been used in the research of many fields, such as material science, life science, and machinery. Compared with corresponding macroscopic material, the properties of the self-assembled monolayers (SAMs) could be developed significantly, and even showed some new performances. Furthermore, self-assembly techniques were broadly used in the construction of medical material by ― layer-by-layer self-assembled technique[4 7], self-asse― mbly biosensor[8 10] and self-assembly biomolecule de― vices[11 13] in life science. With the development of self-assembly method, functional self-assembled materials attracted more and more attention. 3-(triethoxysilyl) propyl isocyanate was one of the most broadly used double functional groups reagents, which contains functional groups of — NCO and www.scichina.com | csb.scichina.com | www.springerlink.com
—OCH2CH3. Due to the high reactivity of these functional groups, 3-(triethoxysilyl) propyl isocyanate was often used as the crosslinking reagent in the synthesis reactions. Lately, its further applications in the synthesis of new compounds and assembling hybrid materials ― were also reported[14 19]. In recent years, the studies on the separation of biomolecules by “labs-on-chips” or micro total analysis systems (μ-TAS) were developed deeply. In all kinds of chips, the glass chip was broadly used in protein or peptide analysis because of its light transmittance, rapid heat-dispersing and easy surface modification[20]. However, due to the electrostatic ironic interaction between protein and silanol groups of the glass, the adsorption of protein on the surface of glass could not be avoided, not matter what kind of work could be finished on chips. Received March 19, 2008; accepted July 23, 2008 doi: 10.1007/s11434-009-0059-9 † Corresponding author (email:
[email protected]) Supported by the National Natural Science Foundation of China (Grant No. 20435020, 20275005 and 20705005)
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follows: (1) The glass slides were first immerged in the cleanser solution overnight. After this step, the slides were rinsed in turn by running water, distilled water and de-ionized water. (2) The cleaned slides were treated in ethanol, acetone and de-ionized water by ultrasonic washing for 10 min to remove the surface organic impurities. (3) The glass substrates used for assembling were treated for 30 min in piranha solution (H2SO4:H2O2 = 7/3 (V/V)) at 90℃. Then the substrates were carefully rinsed with de-ionized water and dried. (4) The dried glass slides were then immerged in the solution prepared with NH3·H2O:H2O2:H2O=1:1:5 (V/V) for 24 h. After that, the substrates were dried in N2 after washing by de-ionized water at room temperature.
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Therefore, just the modified chips were used in the protein separation. Usually, the modification coating methods contain dynamic coating and static coating[21,22]. Furthermore, the self-assembly technology was also successfully used in the chip surface modification. The applications in such as protein separation and enrichment, and biosensor were reported lately. The epoxy polymer with high hydrophilicity was assembled on the surface of PDMS chip by Wu et al.[23]. As the excellent performance in the inhibition of electroosmosis flow (EOF), the assembly film was well used in the separation of protein, peptide and DNA fragment. Moreover, Desai’s group[24] reported the studies of layer-by-layer biomimetic threedimensional structures in microfluidics. Yu’s group[25] used self-assembling aptamer in biosensors to realize a low detection of lysozyme with the advantages being alternative, sensitive, and versatile. Along with the advantages mentioned above, self-assembly technique holds great advantages in direct chip surface modification, in which it is quite difficult to realize complex chemical reactions. Monomolecular film of 3-(triethoxysilyl) propyl isocyanate was prepared on the slide by self-assembled technique in our study, and the assembling of bovine serum albumin (BSA) on this monomolecular film was also studied. The X-ray photoelectron spectroscopy (XPS) was employed to investigate the elementary composition of the film. Moreover, the contact angle of distilled water was used to characterize the surface state.
1.3 SAMs preparation (1) 3-(triethoxysilyl) propyl isocyanate SAMs. The pretreated glass substrates were placed in the double functional group reagent (a mixture of 5 mL methylbenzene, 0.1 mL 3-(triethoxysilyl) propyl isocyanate and 0.1 mL triethylamine) for 12 h. Lifted from the solution, the chip substrates were rinsed with methylbenzene, acetone and de-ionized water, respectively. Moreover, they were dried with a nitrogen flush at room temperature and finally put into a vacuum drying oven at 120℃ for 30 min. The surface assembling reaction was showed below (Figure 1).
1 Experimental 3-(triethoxysilyl) propyl isocyanate (99%, Fluka, USA) was used without further treatment. Methylbenzene, methanol, acetone, ethanol, hydrogen peroxide and other reagents purchased from Beijing Chemical Reagent Company were all of analytical grade. Bovine serum albumin was purchased from Sigma-Aldrich (USA). Microfluidic chip slides (SG2506, 63 mm×63 mm×1.65 mm) used as the substrates were bought from Shaoguang Chromic Slide Co. Ltd. (Changsha, China). De-ionized water was prepared by a Milli-Q system (Millipore, Bedford, MA, USA). 1.2 Pretreatment of the glass chip The glass chip used for assembling was cut into square (0. 5 cm×0. 5 cm), and the pretreatment protocol was as
Figure 1 Schematic of the preparation of 3-(triethoxysilyl) propyl isocyanate SAMs.
(2) BSA SAMs. The 3-(triethoxysilyl) propyl isocyanate substrates were dipped into the BSA solution (1 mg/mL) for 60 min at room temperature. At last, the substrates were cleaned with de-ionized water to remove the unreacted BSA and dried in N2.
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1.1 Chemicals
1.4 Characterization of the SAMs In this study, XPS analysis was conducted on a PHI-5300 XPS system, using Mg Kα irradiation at a pass energy of 29.35 eV and the degree of vacuum was below 1.33×10−6 Pa. The binding energy of contaminated carbon (C(1s): 284.6 eV) was used as reference. The static contact angles of distilled water on the film surface were measured in ambient air at room temperature using a JY-82 contact angle measuring system. The values reported were the averaged values of at least 5 measurements for every sample.
2 Results and discussion 2.1 XPS analysis of the SAMs XPS is a highly diagnostic tool for the assessment of the chemical state of elements. The XPS spectra of 3-(triethoxysilyl) propyl isocyanate film are showed in Figure 2. As expected, the bare substrate did not show
Figure 2 740
XPS spectra of 3-(triethoxysilyl) propyl isocyanate film.
any noticeable N(1s) signal, whereas the coated substrate showed a significant N(1s) band. The N(1s) peak (Figure 2(a)) located at Eb = 399.0 eV was attributed to the nitrogen in the “—NCO” group and the C(1s) peak (Figure 2(b)) located at Eb = 287.7 eV belonged to the carbon also in the “—NCO” group. These results demonstrated that the 3-(triethoxysilyl) propyl isocyanate was successfully assembled on the surface of the glass. At the same time, in atomic content quantification based on the XPS, the atomic contents of C found in the 3-(triethoxysilyl) propyl isocyanate film increased to a certain extent. As showed in Figure 2(b), the area of the carbon element was 5390, while the contaminated carbon was 3331. Therefore, it was strengthened that the 3-(triethoxysilyl) propyl isocyanate self-assembly was realized based on the area changes of the carbon element. The XPS spectra of BSA film are showed in Figure 3. After further assembling of the protein, the N(1s) char-
Figure 3
XPS spectra of BSA film.
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2.2 Contact angles measurement of the SAMs Wettability is one of the most important performances of the solid material surface. As the contact angle was extremely sensitive to the surface state, the structure and the composition changes of the material surface could be investigated by the measurement of contact angle[26,27]. The water contact angle values of the substrate surface were studied here and the results are showed in Table 1. It was found that the substrate gave a contact angle of 31º without any treatment, and the water contact angle was dramatically reduced to 5º after the pretreatment. This indicated that the substrate was fit for assembling with high hydrophilicity after the activation treatment. The 3-(triethoxysilyl) propyl isocyanate substrate showed a contact angle of 80º after assembling. By contrast, the BSA film obtained on the 3-(triethoxysilyl) propyl isocyanate-treated substrate showed a lower contact angle of 52° with the decrease in hydrophobicity. Therefore, the dramatical changes of the contact angle clearly demonstrated the success of each step of self-assembly. The BSA had been immobilized on the substrate surface by bonding with the first monolayer, 3-(triethoxysilyl) propyl isocyanate. Table 1 Contact angle result obtained for both the bare and the treated substrates Treatment taken for substrates Without any treatment
Contact angle (º) 31
After pre-treatment
5
3-(triethoxysilyl) propyl isocyanate SAMs
80
BSA SAMs
52
3 Conclusions Monomolecular film of 3-(triethoxysilyl) propyl isocy1
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anate was prepared on the chip substrate surface based on the self-assembled technique. The bovine serum albumin (BSA) was further assembled on this functional monomolecular film in our studies. Surface contact angle measurement and XPS were used for surface characterization. All the results demonstrated that the 3-(triethoxysilyl) propyl isocyanate film could be further used in the BSA assembling. Being functional reagent, the —NCO and —OCH2CH3 groups of 3-(triethoxysilyl) propyl isocyanate could react with many other groups due to their high reactivity. Therefore, 3-(triethoxysilyl) propyl isocyanate was usually used as the crosslinking reagent in the film assembling to obtain the ideal compound materials. Usually, the end group of the silane was firstly hydrolyzed and then the polycondensation with the surface was taken to form the covalent bond in the self-assembly processes. After the hydrolysis and the polycondensation reaction occurred in the silane, the self-assembled monolayers could be covalently linked by the stable siloxane group. Compared with the double functional reagent of 3-Aminopropyltrimethoxysilane (3-APS) in the application of protein assembling, the reactive exposed —NCO groups on the surface of the 3-(triethoxysilyl) propyl isocyanate film could react with protein directly without aldehyde group modification. Thus obviously, the whole processes of biomolecular assembling were simplified and the unstable factors brought by multi-reaction could be reduced. Compared with the conventional chemical modification, self-assembly techniques have the advantages such as simple operation, easy to control, mild reaction condition and the ability to rebuild. Taking the modification of the chip channel for example, the solvent evaporation problem under high temperature condition could be avoided well. Three dimensional structures could be established in a simpler and more effective manner by using self-assembly technique. Therefore, the potential of broader applications in molecular device, bio-chip, biosensor and the μ-TAS system are all expectative.
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acteristic peak (Figure 3(a)) located at Eb = 400.0 eV appeared. It was attributed to the nitrogen formed in the acid amide linkage after the hydrolysis of —NCO group and the nitrogen in the acid amide linkage existing in the protein. Meanwhile, the C(1s) peak (Figure 3(b)) at Eb = 286.3 eV attributed to the carbon in the —COOH group in BSA could also be found. All of these results verified that BSA was successfully immobilized on the 3-(triethoxysilyl) propyl isocyanate film.
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