Immobilization of avidin on the functionalized carbon nanotubes

Synthetic Metals 156 (2006) 938–943

Immobilization of avidin on the functionalized carbon nanotubes Young-Soon Kim a , Joong-Hee Cho a , S.G. Ansari a , Hyung-Il Kim a , M.A. Dar a , Hyung-Kee Seo a , Gil-Sung Kim a , Dai-Soo Lee a , Gilson Khang b , Hyung-Shik Shin a,∗ b

a School of Chemical Engineering, Chonbuk National University, 664-14, Duckjingu, Jeonju 561-756, Republic of Korea Department of Polymer Nano Science and Technology, Chonbuk National University, 664-14, Duckjingu, Jeonju 561-756, Republic of Korea

Received 9 March 2006; received in revised form 12 June 2006; accepted 15 June 2006 Available online 25 July 2006

Abstract Immobilization of avidin was carried out by functionalizing the multi-wall carbon nanotubes (MWCNTs). Treatment with nitric acid and sulfuric acid mixture (1:3, optimized ratio) leads to the functionalization of nanotubes as observed from Fourier transform infrared absorption spectroscopy (FTIR) measurements. Avidin was coupled with the solution of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylamino propyl)-carbodiimide hydrochloride (EDC) and then immobilized on nanotubes. X-ray photoelectron spectroscopic (XPS) studies show a shift in the peak position of C 1s towards lower energy side and changes in the bands with treatment/immobilization. The shoulders and shift in photoelectron peak positions indicate the destruction of the graphite structure of the surface layer. SEM images showed that after functionalization, the nanotubes are seen with open ends, granular surface and are joined together. This indicates that after treatment, the CNTs reactivity increased at the ends as well as at the sidewalls. It is believed that the NHS often assist the carbodiimide coupling in the presence of EDC, reacts with the amine function to yield the amide bond. The carbodiimide catalyzes the formation of amide bonds between carboxylic acids and amines by activating carboxyl. The reaction of complex containing avidin can form a covalent bond with functionalized carbon nanotubes as observed from FTIR and XPS measurements. © 2006 Elsevier B.V. All rights reserved. Keywords: Carbon nanotube; Functionalization; Immobilization; Avidin

1. Introduction There has been intense interest on carbon nanotubes since their discovery by Iijima in 1991 because of their excellent mechanical, electrical and nonlinear optical properties [1]. These properties and their unique structure made them a potential candidate for nanoelectronics, sensors, electrochemical storage of energy, structural composites and high-tech electrical and optical actuator applications. For some of these applications, highly purified carbon nanotubes are necessary. Nevertheless, it is widely reported in the literature that the purification method leads to the structural changes and affect the CNT functionality/properties [2–8]. These structural changes can have important implications for their novelistic applications in addition to

∗

Corresponding author. Tel.: +82 63 270 2438; fax: +82 63 270 2306. E-mail address: [email protected] (H.-S. Shin).

0379-6779/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.synthmet.2006.06.003

the functionalization [9]. The literature review shows that the purification by acid washing creates an open end termini in the structure that are stabilized by COOH and OH groups left bonded to the nanotubes at the end termini and/or the sidewall defect sites [10–12]. The COOH can be coupled to various biochemical groups depending on the choice of the coupling chemicals and/or bio-materials. Bioelectronics is a rapidly progressing interdisciplinary research field that combines biotechnology, chemistry, microelectronics, physics and materials science. Avidin is a protein present in raw egg white, which binds biotin. Biotin (Vitamin B-6) is required for cell growth and for the production of fatty acids. Biotin also plays a central role in carbohydrate and protein metabolism and is essential for the proper utilization of the other B-complex vitamins. Avidin, when synthesized in the hen oviduct, is a glycoprotein of MW 68,000 Da which occupies about 0.05% (w/w) of the total protein content of the hen egg white. The ability of avidin to bind biotin (Vitamin H) with

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exceptionally high affinity (kd = 10−15 M) has been the basis for its exploitation as a molecular tool in biotechnological, biochemistry, collectively known as avidin–biotin technology [13]. The highly charged protein with pI ∼ 10.5 and the presence of carbohydrate can cause non-specific binding to extraneous material in certain applications, and these properties, can be of use in biochemical applications [14]. In addition to these, a wide range of macromolecules including proteins, polysaccharides and nucleic acids can be readily linked to biotin without serious affect on their biological, chemical or physical characteristics. Avidin can be covalently conjugated to enzymes using cross-linking or other conjugation reagents. These reagents couple through functional groups on each molecule. The incorporated part of a cross-linker acts as a spacer between the conjugated molecules. The method and reagent used to make avidin conjugates are important because both the enzymatic activity and biotin binding ability must be preserved in the conjugate. There arises a need to study the coupling of avidin to various chemical materials and study the complex structure resulting in novel biochemical applications. An attempt is made in this paper to investigate the formation of such complex with carbon nanotubes. In this study, we describe the complex formation of avidin with multi-wall carbon nanotubes (MWCNT). 2. Experimental The purified MWCNTs (99% pure) were purchased from ILJIN Nanotech Co., Ltd., Korea. The quality was further improved by refluxing with concentrated HNO3 for 1 h at room temperature. In order to assure that there are no residues of the HNO3 , the mixture was diluted with distilled water and decanted until a pH of above 7 was achieved. A filter system and vacuum pump was erected in which the nanotube mixture was washed and poured through. It was completely dried on a filter paper in the vacuum oven at 80 ◦ C for 48 h. For the functionalization of carbon nanotubes, 10 mg of purified carbon nanotubes was mixed with the solution of HNO3 (67% concentration) and H2 SO4 (98% concentration) in a 1:3 ratio and was sonicated for 3 h. The mixture was then filtered using a 0.2 ␮m pore sized hydrophilic filter (PVDF). In order to remove acids, the mixture was rinsed with the 1 M NaOH solution and washed three to four times with distilled water. These were then dried on the filter paper in a vacuum oven at 80 ◦ C for 48 h. For the formation of a complex containing avidin, functionalized carbon nanotube and avidin were mixed in a solution of N-hydroxysuccinimide (NHS, Acros organics Co.) and 1ethyl-3-(3-dimethylamino propyl)-carbodiimide hydrochloride (EDC, Tokyo Kasei Kogyo Co., Ltd.). EDC is a water soluble carbodiimide which couples proteins to other molecules in a single reaction mixture and NHS acts as cross-linkers for protein conjugation applications. For investigating the optical properties, the fluorescent avidin conjugated fluorescein isothiocyanate (FITC-labeled avidin, Sigma) was used. The pH was kept below the isoelectric point of the avidin.

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Fourier transform infrared absorption spectroscopic (FTIR) measurements were used to analyze the chemical bonding. FTIR spectra were recorded with a MAGNA-IR750 Fourier transform infrared instrument by KBr pellets in the frequency range 4000–500 cm−1 . Optical properties of the samples were determined using photoluminescence (PL) excited with 325 nm line He–Cd laser. The surface chemical bonding was analyzed by using X-ray photoelectron spectroscopy (XPS, Model—Kratos Amicus). The microstructures of samples were examined by the field emission scanning electron microscope (FE-SEM, JEOL JSM-6330F). 3. Results and discussion Fig. 1 shows the FTIR spectra of (a) MWCNTs, (b) MWCNTs treated with nitric acid and (c) MWCNTs functionalized with nitric acid and sulfuric acid in a 1:3 ratio. For FTIR measurement, the amount of CNTs added to the KBr must be strictly controlled as the black CNTs absorb infrared rays if the amount of is excessive. Therefore, only a weak C C peak (ca. 1632 cm−1 ) can be seen in Fig. 1(a). The FTIR spectra exhibit bands both at about 3394 and 1632 cm−1 in Fig. 1(b). Various bands were observed at about 3394, 2885, 1716, 1632, 1280, 1188 and 1064 cm−1 when functionalized (Fig. 1(c)). It is considered that the peak at 3394 corresponds to OH stretching and peaks at 2885, 1280 and 1188 cm−1 corresponds to CH stretching. The peak corresponding to the carboxylic acid group (C O, 1716 cm−1 ) could not be observed in the acquired FTIR spectra. This indicates that C O was present at the ends of the MWCNT, which helped in the fictionalization of carbon nanotubes. Fig. 2, FTIR spectrum of immobilized avidin over functionalized MWCNTs, shows the existence of vibration modes corresponding to C C bonds at 1632 cm−1 , as well as band related to the OH stretching at 3394 cm−1 . As can be observed from Fig. 3(a), CNTs did not show the fluorescent property. Therefore, we used FITC-labeled avidin which shows a peak at 2.2 eV photon energy. After functional-

Fig. 1. FTIR spectra of (a) MWCNT, (b) MWCNT treated with sulfuric acid and (c) MWCNT functionalized with nitric acid and sulfuric acid in a 1:3 ratio.

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Fig. 2. FTIR spectrum of immobilized avidin with fuctionalized MWCNT.

ization/immobilization, carbon nanotubes and avidin were seen mixed in the solution with NHS and EDC, and this resulted in mixed peaks in the PL spectra (Fig. 3(c)). For surface chemistry of carbonaceous material, additional characterization of evolution of the functional groups was performed by XPS studies of C 1s (Fig. 4(a)), O 1s (Fig. 4(b)) and N 1s (Fig. 4(c)) peaks of MWCNTs, functionalized and immobilized CNTs. Before acquiring the spectra, all the samples were sputtered in argon atmosphere for 50 s. The C 1s peak has been fitted to several symmetrical components according to the standard peak assignment procedures [14–17]. Fig. 4(a) shows the core level spectra of C 1s for CNTs, functionalized CNTs and immobilized CNTs. The changes in the bands with treatment are evident in these spectra. There is a clear shift of the main peak towards lower energy side, which indicates a chemical shift due to the functionalization and immobilization. In case of CNTs, the main peak assigned to the C 1s is at 284.55 eV. The shoulder of this peak is assigned to C O (287.25 eV). In case of functionalized CNTs, a shift in the C 1s peak (−0.695 eV), with three additional

Fig. 3. PL spectra of (a) MWCNT (b) avidin and (c) immobilized avidin with fuctionalized MWCNT.

peaks are observed. When immobilized with avidin, the symmetry of C 1s peak is deteriorated and two additional small peaks [C H (283.75 eV) and C O (284.55 eV)] with various shoulders, towards higher energy side (284.55–288.0 eV) are observed. This indicates the destruction of the graphite structure of the surface layer resulting from the treatment process. These features agree well with the observations from the FTIR spectra. The narrow scan O 1s spectra are presented in Fig. 4(b). The peak position for oxygen is centered at around 532.5 eV. In case of CNTs, a slight shift in peak position (by +0.35 eV) of O 1s is observed. This change in binding energy (∼0.35 eV) can be considered insignificant based on the energy resolution of the XPS instrument. The spectra in Fig. 4(b) show the changes in the peak position, symmetry and shift towards lower energy with the treatment process. These changes can be ascribed to the increase in surface area with functionalization and immobilization with avidin. This can also be associated with the stabilization of open end structures, created while functionalization, by the COOH and OH groups [15–19]. Satros et al. reported that EDC reacts with carboxyl group on the protein to produce an intermediate that reacts with a primary amine on another protein. This method is efficient for protein-to-protein conjugation because most proteins contain both primary amines and carboxyl groups; however, the possibility of self-polymerization exists due to the presence of both primary amines and carboxyl groups on most proteins. They further investigated that the NHS often assist the carbodiimide coupling in the presence of EDC, which includes the formation of the intermediate active ester (the product of condensation of carboxylic groups and NHS) that further reacts with the amine function to yield finally the amide bond. The carbodiimide catalyzes the formation of amide bonds between carboxylic acids and amines by activating the carboxyl. In this reaction, EDC converts the carboxylic acid into a reactive intermediate which is susceptible to attack by amines [Satros]. Therefore, the reaction of complex containing avidin can form a covalent bond with functionalized carbon nanotubes, as observed from FTIR and XPS measurements. The evidence of such reaction can be seen in Fig. 4(c), which shows the narrow scan N 1s spectra (un-fitted data) of MWCNTs, functionalized and immobilized CNTs. It is clearly evident from this measurement that when immobilized with avidin, an amide bond exists between the carboxylic acids and amines by activating carboxyl. Fig. 5 shows the scanning electron images of MWCNTs, functionalized MWCNTs and immobilized with avidin. MWCNTs’ diameter is in the range of 10–30 nm and the length of the CNTs is about few micrometer. Fig. 5(a) shows the images of the as purchased MWCNT where one can observe the open ends of the CNTs. With functionalization (Fig. 5(b)), the CNTs are seen with more open ends (bright spots on the image) with granular surface and joining tubes together. This indicates that after treatment, the CNTs became reactive at the ends as well as at the sidewalls. The immobilization with avidin can be seen in Fig. 5(c). The observation from XPS and FTIR studies can be supported from these images,

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Fig. 4. XPS narrow scan spectra of (a) C 1s, (b) O 1s and (c) N 1s for MWCNT, functionalized MWCNT, and immobilized avidin.

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Fig. 5. FESEM images of (a) MWCNT, (b) functionalized MWCNT and (c) immobilized avidin.

which clearly show the increase in reactivity and immobilization of CNT. 4. Conclusion Nitric acid and sulfuric acid mixture (1:3, optimized ratio) treatment leads to the functionalization of nanotubes as observed from Fourier transform infrared absorption spectroscopy (FTIR) measurements. This functionalization process leads to an important amount of functional groups such as carbonyl, carboxyl and hydroxyl are introduced by this treatment step. The XPS studies showed a shift in the C 1s peak position and changes in the bands with treatment. There is a clear shift of the main peak towards lower energy side indicating a chemical shift due to the functionalization and immobilization of CNT. The shoulders and shift in peak positions indicate the destruction of the graphite structure of the surface layer resulting from the treatment process. The O 1s XPS spectra clearly show the changes in the peak position, symmetry and shift towards lower energy with the treatment process. These changes can be ascribed to the increase in surface area with functionalization and influence of immobilization with avidin. This can also be associated with the stabilization of open end structures, created while functionalization, by the COOH and OH groups. SEM images showed that after functionaliza-

tion, the CNTs are seen with more open ends (bright spots on the image) with granular surface, and joining tubes together. This indicates that after treatment, the CNTs became reactive at the ends as well as at the sidewalls. The observation from XPS and FTIR studies can be supported from these images, which clearly show the increase in reactivity and immobilization. It is believed that the NHS often assist the carbodiimide coupling in the presence of EDC reacts with the amine function to yield the amide bond. The carbodiimide catalyzes the formation of amide bonds between carboxylic acids and amines by activating carboxyl. The reaction of complex containing avidin can form a covalent bond with functionalized carbon nanotubes as observed from the FTIR and XPS measurements. Acknowledgments This work was supported by the Korea Institute of Industrial Technology Evaluation & Planning (ITEP) through the Advanced Materials Research Center, the Korea Science and Engineering Foundation (research grant no. R01-2004-00010792-0). S.G. Ansari would like to acknowledge the financial support from Korean Research Foundation and the Korean Federation of Science and Technology Society Grant (Brain pool program).

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