Mohammad A Wahab1*, Farzana Darain1#, Azharul Karim2 and Jorge N Beltramini1*
1NANOMAC (ARC Nanomaterials Centre) AIBN, UQ, 1#Australian Institute of Bioengineering and Nanotechnology (AIBN), The University of Queensland, St Lucia, Brisbane, QLD 4072, Queensland, Australia 2Chemistry, Physics and Mechanical Engineering, Science and Engineering Faculty, Queensland University of Technology, 2 George Street, QLD 4001 Australia
Received: 25 May, 2015; Accepted: 31 July, 2015; Published: 05 August, 2015
Mohammad A Wahab, NANOMAC (ARC Nanomaterials Centre) AIBN, UQ, Australian Institute of Bioengineering and Nanotechnology (AIBN), The University of Queensland, St Lucia, Brisbane, QLD 4072, Queensland, Australia, Tel: +6133463817; Fax: +61 7 3346 397; E-mail:
Wahab MA, Darain F, Karim A, Beltramini JN (2016) Nano-Confined Synthesis of Fullerene Mesoporous Carbon (C60-FMC) with Bimodal Pores: XRD, TEM, Structural Properties, NMR, and Protein Immobilization. Int J Nanomater Nanotechnol Nanomed 2(1): 001-008. DOI: 10.17352/2455-3492.000006
© 2015 Wahab MA. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Nanoconfined synthesized crystalline fullerene mesoporous carbon (C60-FMC) with bimodal pore architectures of 4.95 nm and 10-15 nm pore sizes characterized by XRD, TEM, nitrogen adsorption/desorption isotherm and solid-state NMR, and the material was used for protein immobilization. The solid-state 13C NMR spectrum of C60-FMC along with XRD, BET and TEM confirms the formation of fullerene mesoporous carbon structure C60-FMC. The immobilization of albumin (from bovine serum, BSA) protein biomolecule in a buffer solution at pH 4.7 was used to determine the adsorption properties of the C60-FMC material and its structural changes investigated by FT-IR. We demonstrated that the C60-FMC with high surface area and pore volumes have excellent adsorption capacity towards BSA protein molecule. Protein adsorption experiments clearly showed that the C60-FMC with bimodal pore architectures (4.95 nm and 10-15 nm) are suitable material to be used for protein adsorption.
The immobilization of biomolecules onto porous solid supports has attracted much attention due to its scientific, technological importance and application in many areas, such as bio catalysis, separation and transport, immobilization, advanced materials (electrode materials), adsorption science, and nanobiotechnology [1-9]. Meanwhile, different porous solid materials (silica, inorganic oxides, carbon etc.) have been considered as support for the adsorption of biomolecules [1-3]. For example, much progress on the adsorption of biomolecules onto nanoporous silica materials has been reported but the use of porous silica supports usually deteriorate the native characteristics of a protein molecule and even leads to the loss of its coherent structure due to the electrostatic interactions between the –OH groups of silica and the surface charge on the amino acid residues on the surface of the proteins [1-10]. It was also reported that the structure of the porous silica adsorbent in aqueous media is not stable due to the hydrolysis of their siloxane bridges (≡Si-O-Si≡) although the efficiency of biochemical devices largely depends on the stability of solid supports/adsorbents and solid support-protein interactions [1-8,11]. Therefore, new porous adsorbents structures need to be found that can be effectively used in the development of nanobiomedical devices. In this context, recently thermo-mechanically robust porous carbon materials that are very suitable for bio-applications have received much attention due to their unique characteristics, namely: a large specific surface area and high pore volume with pore sizes distribution that can be tuned over a wide pore range of 2 nm to > 50 nm . At the same time, several examples of enzyme immobilization and biomolecules or drugs adsorption on mesoporous carbons have been reported in the literatures [13-20]. It is also observed that mesoporous carbon materials exhibited better performance for these applications than that of counterpart silica material. Then, instead of using a conventional carbon source as mentioned above [1-4,12-20] it will be necessary to find a new carbon source to create a new mesoporous carbon solid framework that will improve the adsorption of biomolecules. As such, Fullerenes (C60) as carbon source consists of nanoscopic building block with surface tailorable that can give unique functionalities to the mesoporous carbon framework [21-23]. Previously, fullerene C60 has been partly used along with porous silica for extending their surface functionalities [24-32] but C60 has not been reported to be used as sole carbon source for preparing fullerene mesoporous carbon that can be useful for protein immobilization.
With this concept in mind, this paper, successfully demonstrate the use of fullerene C60 as carbon source to prepare a new class of fullerene mesoporous carbon (C60-FMC) with bimodal pore architectures via pore filling method and its properties for protein immobilization.
Tetraethylorthosilicate (TEOS), Pluronic surfactant P123 (EO20PPO70EO20), Fullerene C60, Trimethylbenzene (TMB) and Albumin from Bovine serum (BSA) from Sigma-Aldrich were used as received without further purification.
Synthesis of mesoporous SBA-15 silica nano-hard template
In order to prepare mesoporous SBA-15 template, first 2 g of Pluronic surfactant P123 (EO20PPO70EO20) was stirred in 60 ml of 2M HCl at 38 oC for 2 h for making a homogenous solution. Then 4.2 g of TEOS precursor was added to the above surfactant containing acidic solution. The mixture was stirred for only 6-8 min and then left the solution to stand for 24 h at 38 oC [29,30]. The mixture was subsequently transferred into an autoclave for another 24 h at 100 oC. The as-synthesized SBA-15 silica was collected by filtration, dried and then calcined at 550 oC for 6 h in air to remove surfactant.
Nano-confined synthesis of fullerene mesoporous carbon (C60-FMC)
A pore fill nanocasting method was used for the synthesis of high surface area fullerene mesoporous carbon (C60-FMC) by using mesoporous SBA15 silica as a hard template [12,30]. A targeted amount of fullerene C60 was added into trimethylbenzene in the appropriate proportion and then sonicated for few hours. Afterwards, the sonicated solution was thoroughly mixed with mesoporous SBA-15 silica template. Then the fullerene impregnated SBA-15 composite was heated at 100 oC for 6 h and later temperature was increased to 160 oC holding for another 6 h for polymerization. Then the polymerized fullerene C60/silica nanocomposite material was transferred into an aluminum quartz boat that was placed at the middle of a tube furnace and carbonized at 850 oC under nitrogen atmosphere for 6 h. After heat-treatment, the mesoporous fullerene/silica nanocomposite was washed thoroughly with 5 wt% HF to remove silica template. As a result of this step a highly porous fullerene mesoporous carbon was created. Mesoporous SBA-15 silica template-free C60-FMC samples were used for all subsequent characterization and application.
Adsorption study of BSA to fullerene mesoporous carbon (C60-FMC)
Prior to the adsorption studies, the fullerene mesoporous carbon materials were dried overnight at 120 oC. Then BSA was dissolved in 10 mM acetate buffer, pH 4.7 to prepare stock solution. 10 mg of the support samples were suspended in BSA solution and mixture was continuously shaken at 500 rpm at room temperature. The adsorption kinetics of BSA was recorded at various pre-determined time periods. Suspensions were separated by centrifugation at 10000 g for 3 min at room temperature and then 20 µL of supernatant were taken out for measurement of the protein concentration using NanoDrop® ND-1000 spectrophotometer at 280 nm. The amount of immobilized BSA protein was calculated by subtracting the amount of BSA in the supernatant liquid after adsorption from the amount of protein present before adding the samples. Finally, the BSA-immobilized C60-FMC supports were washed three times with acetate buffer solution then separated by centrifugation and characterized using attenuated total reflection IR. A leaching test was carried out using BSA immobilized mesoporous materials suspended in 10 mM acetate buffer, pH 4.7 which was continuously shaken at 500 rpm at room temperature for 24 h. After centrifuging at 10000 g for 3 min, the amount of protein leakage was investigated using NanoDrop® ND-1000 spectrophotometer at 280 nm. The isoelectric point for BSA is at pH 4.7. Therefore, the protein stock solution for BSA was prepared in acetate buffer solution with a pH equal to its isoelecric point since proteins tended to bind well at or around their isoelectric point. In addition, at the isoelectric point, protein involved in hydrophobic interactions with the three-dimensional mesopore structure.
Mesoporous SBA15 silica nano-hard template and fullerene mesoporous carbon (C60-FMC) materials were comprehensively characterized by using X-ray diffraction (XRD, Bruker D8 Advanced X-ray diffractometer with Ni-filtered CuKa radiation at a voltage of 40 mV and a current of 30 mA) to confirm the formation of fullerene mesoporous carbon (C60-FMC) structure, whereas transmission electron microscope (TEM) was used to determine the pore structure of C60-FMC. TEM (TEM, FEI Tecnai 20, 200kV) imaging was carried out on a F20 microscope with an accelerating voltage 200 kV. The dispersed particles in ethanol were undergone for Ultra sonication for 10 min for ensuring good dispersion and homogeneity of particles in ethanol. The solution was then settled on carbon coated Cu grids for TEM imaging. X-ray diffraction (XRD) patterns were also recorded on a Rigaku Miniflex diffractometer (Japan) with Cu Kα radiation (λ=0.154 nm) at a scanning rate of 2 degree/min in the 2θ range from 10 to 80°. The BET surface areas and textural properties were obtained using an automated adsorption analyzer (Quadrasorb SI, Quantachrome, USA). The N2-adsorption–desorption isotherms were measured at 77 K on a nitrogen-adsorption apparatus after degassing the samples at 180 OC for 6 h. The Brunauer–Emmett–Teller (BET) surface areas were determined at a relative pressure (P/Po) of 0.3005. The pore-size distribution (PSD) was obtained by using the Barrett-Joyner-Halenda (BJH) model from the desorption branch (31,32). Then the attenuated total reflection FT-IR spectra of BSA-immobilized mesoporous supports were obtained using a Thermo Nicolet 5700 spectrometer. The measurement was based on 32 scans with a resolution of 4 cm-1. The samples were scanned in the spectral range of 1800–1200 cm-1. Solid-state NMR was performed on the Avance III spectrometer (Bruker), operating at 75.468 MHz for 13C. The samples were placed in the 4 mm zirconium rotor and rotated at magic angle with 5 kHz frequency. The spectra were recorded using SP-hpdec technique (single pulse with high power proton decoupling). The parameters included 42ms acquisition time with sweep width of 50 kHz; 2K data points were collected. High-power decoupling utilized tppm15 scheme at 65.7 kHz proton power. Between 200 and 7000 scans were collected. The recycle times were from 10s to 60s, according to the sample’s T1.
Results and Discussion
Preparation and structural features of mesoporous SBA15 nano-hard template
In order to prepare fullerene mesoporous carbon, meosoporous SBA-15 silica template needs to show a very ordered structure with large pore size that allows the impregnation of fullerene C60 inside the SBA-15 silica pores. As shown in Figure 1, the silica based SBA-15 has three well-resolved peaks at 2° values between 0.75 and 5°, indicating the formation of hexagonal mesoporous SBA15 silica material, which is also supported by the TEM images in Figure 2. The fact that the type IV N2 adsorption-desorption isotherms confirm the mesoporous structure of the SBA-15 silica with capillary condensation step at relative pressure of 0.618 to 0.81, corresponding to the existence of mesopores with narrow pore size distribution as shown in Figure 3 (inset) [11,33-36]. The specific surface area (SBET), total pore volume, and BJH pore size of the highly ordered mesoporous SBA15 silicate were 719 m2/g, 1.12 cm3/g and 9.15 nm, respectively. The textural properties are in good consistency with previous results on the highly ordered mesoporous SBA-15 silicate.