The production of carbon nanotubes using two-stage chemical vapor deposition and their potential use in protein purification

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Abstract

Carbon nanotubes (CNTs) were produced by gas phase two-stage chemical vapor deposition using ferrocene as a catalyst and acetylene (C2H2) and hydrogen (H2) as precursor gases. The CNTs were characterized using field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and by thermogravimetric analysis (TGA). Process parameters such as reaction temperature, reaction time and gas flow rates of C2H2 and H2 were optimized using Design-Expert software v.6.0.8. The statistical analysis reveals that the optimum conditions for the CNT production were reaction temperature of 850 °C, reaction time of 60 min and gases flow rates of 40 and 150 ml/min for C2H2 and H2, respectively. The optimum CNTs prepared were purified and functionalized using acid treatment. Both functionalized and nonfunctionalized CNTs were used as column chromatographic media for skim latex protein purification. Skim latex protein was used as the model protein for the recovery of useful proteins and enzymes. During purification, process parameters such as pH and ionic strength of the running buffer were optimized to enhance protein purification. Results revealed that CNTs behave efficiently as a hydrophobic interaction chromatographic (HIC) media.

Introduction

Carbon nanotubes (CNTs) have unique electrical and structural properties, including high aspect ratio, small tip radii of curvature, chemical inertness, high mechanical strength and high electrical conductivity. Since the miraculous discovery of carbon nanotubes by Iijima [1], various researchers have been carried out and have proved CNTs as one of the most promising nanostructured materials. The prospect of developing novel carbon-based nano-material has excited worldwide interest among researchers. CNTs continue to receive huge interest due to their extraordinary mechanical [2], [3], thermal [4] and electronic and magnetic [5], [6] properties. CNTs have wide industrial applications, which include flat panel field emission displays [7], nanoelectronic devices [8], chemical sensors [9], hydrogen storage [10], [11] and scanning probe tip [12]. Various methods to produce CNTs have been developed, including laser ablation [13], arc discharge [14] and chemical vapor deposition (CVD) [15].

The produced functionalized CNTs were purified by oxidation with nitric and sulfuric acids for the removal of impurities. It was reported that the purification of CNTs by acid washing creates open end termini in their structure which was stabilized by the carboxyl and hydroxyl groups [16]. Carboxylic group can also be introduced at the tube surface which can covalently bind proteins. This can be carried out via a two-step process of diimide-activated amidation between the carboxylic acid groups on the surface of CNTs and amine groups on proteins [17]. Carboxyl (–COOH) groups are readily derivatized by a variety of reactions [18] allowing immobilization of biomolecules such as proteins and enzymes despite the fact that the enzyme was negatively charged and produced strong electrostatic repulsions with the nanotubes [19], DNA [20], or metal nanoparticles [21] by multiuse processes. Davis et al. [22] reported immobilization of three proteins (Zn2Cd5-metalotionein, cytochrome c1, c3 and β-lactamase) on the carbon nanotubes showing that the adsorption was in the internal surface. Furthermore, glucose oxidase has been immobilized on aligned carbon nanotubes modified with a gold thin film [23], [24]. Gan et al. [25] have studied the immobilization of lactic dehydrogenase of mouse muscle. Recently, Yu et al. [26] have shown that myoglobin and horseradish peroxidase can be immobilized on CNTs using promoters in the media to favor the formation of amide bond between the terminal carboxylic and the lysine residues. Covalent immobilization of proteins or enzymes on the surface of CNTs, due to the best stability, accessibility and selectivity could be achieved through covalent bonding because of their capability to control the location of the bimolecule. The mechanism involved in this process is mainly adsorption of protein or enzyme on the surface of CNTs by covalent attachment. Chen et al. [27] reported an elegant method for protein immobilization by irreversibly attaching 1-pyrenebutanoic acid succinimidyl ester onto the sidewall of the SWNTs via π–π stacking of the pyrenees group. The active ester group of such a compound was used to covalently bind the lysine residue of protein pyrene derivatives which have been extensively studied as the basic non covalent functionalization of CNTs. Both functionalized and non functionalized CNTs are commonly used as a media for column chromatography. The CNT nano-sized dimensions provide large surface area, making it a suitable media for protein purification. In this work, the usage of CNTs in conjunction with protein separation creates many useful applications of CNTs

Skim latex serum is recovered from skim latex, the by-product of natural latex industries, which is usually considered as a waste and lavishly thrown away. It contains a dry rubber of 3–7% with very low impurities. Skim latex serum is the non rubber aqueous portion of latex resulting from acid coagulation or membrane filtration. The serum contains nitrogen, carbohydrates, proteins, lipids and trace metals. Some of these proteins are important enzymes which have great demand in pharmaceutical, food and detergent industries. Hence, there is a need to improve the efficiency and yield of protein separation [28]. CNTs have many great potential applications in the area of biotechnology and one of them is to use them as nano-support in the filtration of protein from skim latex serum.

In this study, CNTs were produced by two stage chemical vapor deposition (TS-CVD) using ferrocene (C10H10Fe) as a catalyst and acetylene (C2H2) and hydrogen (H2) as precursor gases. The preparation conditions were statistically optimized to produce the CNT yield in terms of mass and purity. The performance of the produced CNTs in the purification of protein from skim latex serum source was also investigated.

Section snippets

Gases

Three types of gases were used in the production of CNTs namely H2 (99.99% purity), C2H2 (99.9%) and Ar (99%). Ferrocene powder was used as a catalyst in the production of CNTs. Skim latex serum and its preparation for protein removal.

The skim latex sample was obtained from Rubber Research Institute Malaysia, Sungai Buloh, Selangor. The pH of the sample was first adjusted to 5 using acetic acid to promote the coagulation of small rubber particles. Centrifugation was then carried out at 10,000 

Statistical analysis of CNT production

Four level full factorial design was employed to attain all possible combinations of all the input factors with minimal number of experimental runs that could possibly optimize the response within the region of three-dimensional observation spaces. The results from the experiments were analyzed using analysis of variance (ANOVA) using DOE. The variables such as reaction temperature, reaction time, and gas flow rates were employed for the analysis in the design to obtain the CNT yield. The

Conclusions

CNTs have been successfully produced using two stage chemical vapor deposition method and the statistical analysis revealed that the optimum conditions for the best yield CNT production were reaction temperature of 850 °C, reaction time of 60 min and gas flow rates of 40 and 150 ml/min for C2H2 and H2, respectively. The TGA showed that the purity of CNT produced was 95%. FESEM and TEM analyses revealed that a uniformly dispersed vertical alignment of multiwall carbon nanotube (MWCNT) was observed.

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