Materials Science and Engineering C 38 (2014) 46–54
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Multi-scale carbon micro/nanofibers-based adsorbents for protein immobilization Shiv Singh a, Abhinav Singh a, Vaibhav Sushil Singh Bais b, Balaji Prakash b, Nishith Verma a,c,⁎ a b c
Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India Department of Biological Science and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, India Center for Environmental Science and Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India
a r t i c l e
i n f o
Article history: Received 10 November 2013 Received in revised form 26 December 2013 Accepted 22 January 2014 Available online 31 January 2014 Keywords: Activated carbon fiber Carbon nanofiber Adsorption Immobilization Bovine serum albumin Glucose oxidase
a b s t r a c t In the present study, different proteins, namely, bovine serum albumin (BSA), glucose oxidase (GOx) and the laboratory purified YqeH were immobilized in the phenolic resin precursor-based multi-scale web of activated carbon microfibers (ACFs) and carbon nanofibers (CNFs). These biomolecules are characteristically different from each other, having different structure, number of parent amino acid molecules and isoelectric point. CNF was grown on ACF substrate by chemical vapor deposition, using Ni nanoparticles (Nps) as the catalyst. The ultrasonication of the CNFs was carried out in acidic medium to remove Ni Nps from the tip of the CNFs to provide additional active sites for adsorption. The prepared material was directly used as an adsorbent for proteins, without requiring any additional treatment. Several analytical techniques were used to characterize the prepared materials, including scanning electron microscopy, Fourier transform infrared spectroscopy, BET surface area, poresize distribution, and UV–vis spectroscopy. The adsorption capacities of prepared ACFs/CNFs in this study were determined to be approximately 191, 39 and 70 mg/g for BSA, GOx and YqeH, respectively, revealing that the carbon micro-nanofibers forming synthesized multi-scale web are efficient materials for the immobilization of protein molecules. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bioactive substances including proteins are considered as potential substitutes of traditional chemical compounds for a variety of applications, ranging from the compact sensors to catalysts. The superiority of these bioactive substances over conventional chemicals is because of their ability of carrying out complex chemical reactions with high selectivity [1]. In spite of the promising advantages, their widespread applications are, however, limited by the factors such as instability, relative high cost of synthesis, and also, high cost involved in separating the substances from the reactor effluents. Immobilization of proteins is an effective strategy to counter some of the aforementioned hurdles. Immobilization provides stability to their structure, thus enabling their applications under different conditions of temperatures, pH and organic solvents that are bio-active. In such context, the choice of immobilization technique is important. Currently, different strategies are used for the immobilization of biomolecules, for example, cross-linking, entrapment and adsorption. Materials produced using cross-linking and entrapment often have low reproducibility,
⁎ Corresponding author at: Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India. Tel.: +91 512 2596352x7704; fax: +91 512 2590104. E-mail address: [email protected] (N. Verma). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.042
high pore diffusion resistance and non-uniform deposition of solute molecules. On the contrary, adsorption is considered to be a simple, economical and efficient method for the immobilization of biomolecules [2]. Furthermore, adsorption does not alter the structure of biomolecules, and therefore, the activities of the biomolecules are retained to a large extent following adsorption [3,4]. There are several materials used as the substrate to immobilize proteins by adsorption. The common examples are silica, alumina, glass, methyl methacrylate, catalytic filamentous carbon and activated carbon [5–14]. In general, a substrate should possess high surface area and should be non-toxic, mechanically strong and amenable to surface functionalization. In particular, the adsorption capacity of the material for the biomolecules should be large. Recently, multi-scale web of activated carbon microfibers (ACFs) and carbon nanofibers (CNFs) has been extensively used for different environmental applications, for examples, as adsorbents and antibacterial agents [15–18]. The salient advantage of ACFs/CNFs is that it can be directly used in an end-application, without requiring any postsynthesis step. In accordance with their variety of applications, CNFs are grown on ACF substrate by chemical vapor deposition (CVD), using different metal nanoparticles (Nps) as catalyst, for example, nickel (Ni), copper (Cu) and silver (Ag). The prepared materials have large surface area [15–18]. Furthermore, the prepared CNFs are less cytotoxic in comparison to conventional adsorbents such as zeolite, granular activated carbon and carbon nanotubes (CNTs) [19].
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In the present study, the multi-scale web of ACFs/CNFs was used to immobilize different proteins: bovine serum albumin (BSA), glucose oxidase (GOx) and a laboratory purified YqeH. BSA is similar to human serum albumin, the most abundant protein found in blood plasma. It is used as a clinical reagent for ELISA testing, and also, helps in binding of hydrophobic steroids [20]. GOx is used as a biosensor to determine the free-glucose in human blood [3]. Along with these commercially available proteins, adsorption on YqeH protein, synthesized in our laboratory and expressed using cloned enzyme, has also been studied. YqeH has been chosen owing to the facts that its biochemical role and catalytic machinery are well characterized. YqeH is a GTPase and plays an important role in the assembly of small ribosomal (30S) subunit in bacteria [21]. Considering its role in ribosome maturation, which is an essential process, the enzyme is conserved across all kingdoms of life. Recently, its catalytic machinery has been deciphered and shown to employ K+ ion for GTP hydrolysis [22]. In this study, the multi-scale web of ACFs/CNFs was grown by CVD using benzene (C6H6) as a carbon source and Ni metal Nps as the CVD catalyst. Following ultra-sonication, the Nps were removed from the tip of the CNFs to offer increased adsorption sites for proteins. The prepared ACF/CNF substrate was characterized for its physico-chemical properties using various analytical techniques, including scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, Brunauer–Emmet–Teller (BET) surface area and poresize distribution (PSD). The adsorption tests for different proteins were performed under batch conditions at different temperatures (5, 10 and 15 °C) to determine the equilibrium loading on the prepared substrate. 2. Experimental 2.1. Materials Ni(NO3)2·6H2O (purity N 97%), sodium dodecyl sulfate (SDS, purity N 99%), C 6 H 6 , acetone and other reagents used for preparing buffers including NaCl, KCl, Na 2 HPO 4 , KH 2 PO 4 , MgCl 2 , 2-mercaptoethanol, glycerol, sodium acetate and glacial acetic acid were procured from Merck, India. The phenolic resin precursor based ACFs were purchased from Gun Ei Chemical Industry Co. Ltd., Japan. BSA, GOx and Bradford reagent were purchased from SRL (India), Calbiochem (Germany) and Sigma-Aldrich (Germany), respectively. Nitrogen and hydrogen gases (purity N 99.999%) were purchased from
47
Sigma Gases, India. Milli-Q water was used for preparing the required solutions in the experiment. 2.2. Pretreatment of ACFs and synthesis of ACFs/CNFs The as-received ACFs were pretreated by 5 ml of 0.03 M HNO3in 1 l of Milli-Q water at 80 °C for 1 h to remove any impurities from the ACF surface. Next, the ACF samples were dried at room temperature (30 ± 5 °C) for 6 h, followed by hot air-oven and vacuum drying at 120 and 200 °C, respectively for 12 h each. The dried ACF samples were impregnated with 0.4 M Ni (NO3)2·6H2O salts dispersed in acetone solution, using the wet incipient method described in previous studies [16,23]. The anionic SDS (0.3% w/w) surfactant was used in the impregnation solution to prevent the agglomeration of the salt crystals in the solution and facilitate uniform dispersion of the crystals on the ACFs. After impregnation, samples were dried in the static air for 6 h and in the oven for 12 h at 120 °C. Next, calcinations, reduction and CVD were sequentially carried out on the impregnated ACFs. Fig. 1 illustrates the schematic representation of the experimental set-up used for calcinations, reduction and CVD in the present study. The details of the experimental set-up have been described in the previous study [18]. The impregnated samples were calcined at 400 °C in N2 atmosphere for 4 h, followed by reduction at 550 °C in H2 atmosphere (flow rate = 200 sccm) for 2 h. The calcinations and reduction temperatures were determined by the temperature programmed reduction. After calcination and reduction, Ni(NO3)2 particles dispersed in ACFs were converted to Ni Nps. These Ni Nps acted as the catalyst for the tipgrowth of CNFs during the CVD. The CVD was performed at 800 °C for 2 h, using liquid C6H6 as the carbon source. The temperature of the liquid C6H6 was maintained at 8 °C using Freon unit bubbled with N2. C6H6 vapors were carried by N2 gas (100 sccm) to the quartz reactor where it was decomposed by Ni Nps dispersed in the ACFs to affect the CNF growth. After CVD, the reactor was gradually cooled to the room temperature under N2 flow. Next, the prepared material (ACFs/CNFs) was ultra-sonicated in 0.05 M HNO3 solution for 5 min to remove Ni Nps from the tip of the prepared CNFs. 2.3. Purification of YqeH The recombinant plasmid for YqeH was freshly transformed into Escherichia coli BL21 (DE3) CODON PLUS cells. Fresh transformant was
Fig. 1. Schematic of vertical tubular reactor used for calcinations, reduction and CVD.
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grown in Luria Broth containing 100 μg/ml ampicillin at 37 °C with constant aeration. Exponentially growing culture (A600 of ~ 0.6) was induced with 0.1 mM IPTG and further grown for 12–14 h at 18 °C. Cells were harvested and lysed by sonication in lysis buffer (phosphate buffer containing 5% glycerol, 1 mM β-mercaptoethanol and protease inhibitor HIS cocktail (Sigma)). The cell lysate was clarified at 15,000 g for 45 min and supernatant was loaded onto a pre-equilibrated Ni-NTA column (His-Trap FF GE Healthcare). The column was washed with equilibration buffer of 50 mM Tris–Cl pH 8, 500 mM NaCl, 5 mM MgCl2, 3 mM β-mercaptoethanol, and 10% glycerol. His-tagged proteins were eluted using a 0 to 500 mM linear gradient of imidazole in equilibration buffer. At the final step of purification, all eluted fractions (obtained in the previous step) containing His-tagged proteins were pooled and loaded onto 26/60 Superdex200 High Load (HL) gel filtration column (GE Healthcare) and then pre-equilibrated with buffer (50 mM Tris–Cl pH 8, 500 mM NaCl, 5 mM MgCl2, 3 mM β-Mercaptoethanol, 10% glycerol) to achieve the requisite homogeneity in protein preparation. 2.4. Adsorption of proteins on ACFs/CNFs The sonicated ACFs/CNFs were the templates for the adsorption study of biomolecules in this study. Different buffer solutions were used as the dispersing medium for different proteins: 10 mM phosphate buffer of pH 7 for BSA [20,24], 100 mM of acetate buffer of pH 4.2 for GOx [3], and 50 mM Tris–Cl pH 8, 500 mM NaCl, 5 mM MgCl2, 3 mM β-mercaptoethanol, and 10% glycerol for YqeH. Different test solutions of BSA (200–2000 ppm), GOx (200–1400 ppm) and YqeH (300–1400 ppm), each of 1.0 cm3 volume, along with their respective buffers were transferred to 15.0 cm3 borosilicate culture tubes. Next, the sonicated ACFs/CNFs (0.01 g) were added to the test- and buffer solution containing tubes and kept in a shaking water bath (100 rpm) for 24 h at three different temperatures of 5, 10 and 15 °C to immobilize the proteins. The loading of the biomolecules on the ACFs/CNFs was determined by measuring the concentrations of the solute in the buffer solution before and after adsorption. Therefore, the ACF/CNF absorbents were repeatedly (8–10 times) rinsed with the same buffer solution after adsorption to remove the loosely bound proteins from the ACF/ CNF surface, back into the solution. The concentrations of the solutions were determined by the Bradford assay method, using UV–vis spectroscopy. Briefly, a 3 ml-volume of the commercially available Bradford
reagent was mixed with 0.1 ml of the solutions and incubated at 25 °C for 10 min. The reagent color changed from brown to dark blue. The equilibrium loadings were calculated from the difference between initial and final concentrations of solutions, using the following equation: Q ¼ ðC i −C e ÞV=w
ð1Þ
here, Q, Ci, Ce, V and w were the calculated equilibrium loading, initial concentration of biomolecules in the buffer, final concentration of biomolecule after adsorption, volume of the test solution and weight of adsorbent, respectively. Fig. 2 describes the schematic representation of the steps involved in the material synthesis and immobilization, including impregnation, calcinations, reduction, CVD, ultra-sonication and batch study for the adsorption of proteins. 3. Surface characterization The prepared ACFs/CNFs were characterized using several analytical techniques, including SEM (Supra 40 VP, Zeiss), FTIR (Tensor 27 Bruker), BET surface area, total pore volume (VTotal) and PSD (Autosorb-1C Quantachrome) and UV–vis spectroscopy (Cary 100 Bio, Varian). SEM was used to investigate the surface morphology of the substrate ACFs and ACFs/CNFs. Surface functional groups were identified using FTIR in wave number range of 400–4000 cm−1 at attenuated total reflectance mode. The scan rate was 100 over a resolution of 4 cm−1. The Raman spectroscopy analysis was carried out to determine the relative amount of graphitic carbon present in ACFs and ACFs/CNFs, using a confocal Raman instrument (Model: Alpha, Make: Witec, Germany with k = 543 nm). The data were collected in the range of 200–4000 cm−1 at room temperature in air at a resolution of 2.0 cm−1. BET surface area (SBET), VTotal and PSD were calculated at − 196 °C. Microporous and mesoporous volumes were determined by density functional theory (DFT) and the Barrett–Joyner–Halenda (BJH) method, respectively. 4. Result and discussion 4.1. Surface morphology Fig. 3 shows the SEM images of the surface morphology of ACFs, ACFs/CNFs and sonicated ACFs/CNFs. Uniformly distributed pores and
Fig. 2. Schematic of preparation of Ni-ACFs/CNFs and immobilization of different biomolecules on ACFs/CNFs.
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smooth surface of ACFs are clearly evident in Fig. 3(a–a1). Fig. 3(b–b1) shows the SEM images of ACFs/CNFs and confirms the uniform and dense CNF-growth. Shiny particles may be observed at the tip of CNFs, which confirmed the tip-growth mechanism of the CNFs on the ACF substrate. Fig. 3(c–c1) presents the SEM images of the sonicated ACF/ CNF sample. It may be observed that post-ultra-sonication, most of the Ni Nps were removed from the tip of the CNFs. Fig. 4 shows the SEM images of the ACF/CNF sample after the immobilization of the proteins, namely, BSA, GOx and YqeH, at low and high magnifications. Comparing the SEM images (5 KX) shown in Figs. 3c and 4(a–c) for pre- and post-adsorbed samples, respectively, a distinct change may be observed in the surface morphology of ACFs/CNFs. In Fig. 4(a–c), a thin layer of material (adsorbed protein) is clearly observed over the surface of ACFs/CNFs. 4.2. FTIR analysis Fig. 5 shows the FTIR spectrum of ACF/CNF sample. The analysis was performed to identify different functional groups present on the surface of ACFs/CNFs. As observed from the spectrum,
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peaks were observed at 650, 1575, 1690 and 3650 cm− 1, attributed to C`C, N\O, C_O and O\H stretch, respectively. C`C, C_O and O\H stretches were intrinsically present on the ACF surface. The N\O stretch appeared because of HNO 3 used for the pretreatment of ACF. As also observed from the figure, the N\O stretch disappeared at high temperatures, because of the high temperature processing of the material, viz. calcinations, reduction and CVD. The other stretches remained intact on the ACF/CNF surface. Few additional common peaks were identified in the post-proteinimmobilized ACF/CNF samples which confirm the adsorption of proteins on the surface of ACFs/CNFs. The peaks observed at 1265, 1485, 1610, 1716 and 3400 cm− 1 were attributed to N\H (3°), N\H (2°), N\H (1°), C_O and N\H/O\H stretch, respectively. N\H stretches originated from peptide bonds (\CO\NH\) of different amino acids present in the BSA, GOx and YqeH, whereas C_O stretch originated either from \COOH group of the protein-side free chain or from the buffer used during the immobilization. Multiple peaks were also observed between 3600 and 3800 cm−1and were assigned for the O\H stretch of hydroxyl group [18,25–27].
Fig. 3. SEM images of (a–a1) ACFs, (b–b1) Ni-ACFs/CNFs and (c–c1) sonicated ACFs/CNFs.
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Fig. 4. SEM images of (a–a1) BSA-ACFs/CNFs, (b–b1) GOx-ACFs/CNFs and (c–c1) YqeH-ACFs/CNFs.
4.3. Raman spectra Fig. 6 describes the Raman spectrum of ACF and ACF/CNF samples. As observed from the figure, there are two peaks for both samples. The first peak observed at 1340.4 cm−1 (D-band) corresponds to the disordered phase of carbon, whereas the second peak observed at 1570.4 cm−1 (Gband) corresponds to the graphitic phase of the material. The ID/IG peak ratio of the ACF was determined to be 1.22. The ratio decreased to 1.04 in the ACF/CNF sample. Considering that ID/IG ratio reflects the magnitude of the disordered phase relative to the graphitic phase present in the material, it was concluded that the graphitic phase was higher in the ACF/CNF than in the ACF substrate. In other words, graphitic phase in ACFs increased following the growth of CNFs on ACFs.
4.4. Surface area analysis Fig. 7 describes the N2 adsorption isotherms for the prepared materials: substrate ACFs and ACFs/CNFs with and without sonication. SBET, VTotal and PSD were calculated from the data plotted in Fig. 7. It may be observed that the adsorption isotherm of ACFs is of Type I and
those for ACFs/CNFs and sonicated ACFs/CNFs are of Type II in accordance with the BET classifications. The Type I isotherms are exhibited mostly by microporous materials, with the isotherm curve significantly increasing up to a certain level and thereafter, continuing in the horizontal plateau. The adsorption curve of Type II isotherm monotonically increases with increasing relative pressures (P/P0), as observed in Fig. 7 also, which is the unique characteristic of mesoporous adsorbents. Table 1 summarizes SBET, VTotal and PSD of the prepared materials. As observed, SBET and microporous contents of ACFs were 1187 m2/g and ~ 88%, respectively. On the other hand ACFs/CNFs had comparatively smaller SBET (371 m2/g) and VTotal (0.40 cm3/g). ACFs/CNFs were mostly mesoporous (~51%). Following sonication, SBET (~450 m2/g) and VTotal (0.54 cm3/g) increased, as observed from the data shown in the table for the sonicated ACF/CNF sample. This increase is attributed to the removal of the Ni Nps from the tip of the CNFs, exposing the internal surface area also to N2 adsorbate molecules. 4.5. Adsorption As mentioned earlier in the text, the adsorption capacity of the prepared ACFs/CNFs for different proteins was determined at three
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Fig. 5. FTIR spectra of ACF, ACF/CNF and biomolecules-adsorbed ACF/CNF samples.
different temperatures (5, 10 and 15 °C). In the present tests, adsorption of the proteins was found to be exothermic (later shown in the paper), with decrease in the adsorption capacity with increasing temperatures. Therefore, the adsorption tests were performed at relatively lower temperatures (5, 10 and 15 °C). Moreover, although BSA was found to be stable at room temperature (~ 25 °C), we observed that GOx and the lab purified YqeH were denatured in 5–6 h only, when the tests were performed at 25 °C. Therefore, 37 °C was not considered for the tests and the highest temperature considered in the study was 15 °C. The adsorption tests were performed at different pHs also to determine the optimum pH for the maximum equilibrium loading of the solutes. The significance of performing tests at different pHs for different proteins is explained later in the text. After determining the equilibrium loading (Q) of the biomolecules at different concentrations (Ce), the adsorption data were fitted with Langmuir and Freundlich isotherms and the
corresponding thermodynamic parameters were extracted. The linearized forms of Langmuir and Freundlich equations may be expressed as follows: Ce ¼ Q
Ce Qm
þ
1 KlQ m
ð2Þ
ln Q ¼ lnK f þ n ln C e
ð3Þ
where, Ce, Q, and Qm are equilibrium concentrations, equilibrium loading, and the maximum saturation capacity of the adsorbent, respectively. Kl is the Langmuir adsorption constant; Kf and n are the Freundlich parameters. Thermodynamic parameters for the adsorption of different biomolecules were calculated at different temperatures (278, 283 and 288 K) using the following equations: 0
ΔG ¼ −RT lnK
ð4Þ
450
Volume (cc/g)
400 350
ACF Sonicated ACF/CNF ACF/CNF
300 250 200 150 100 0.0
0.2
0.4
0.6
0.8
1.0
Relative Pressure (P/Po) Fig. 6. Raman spectrum of ACF and ACF/CNF samples.
Fig. 7. Nitrogen adsorption isotherms of ACF, ACF/CNF and sonicated ACF/CNF samples.
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Table 1 SBET, VTotal and PSD of prepared materials. SBET (m2/g)
Sample
ACFs Ni-ACFs/CNFs Sonicated-ACFs/CNFs
K¼
C i −C e Ce
0
0
ΔG ¼ ΔH −TΔS
lnK ¼ −
1187 371 450
VTotal (cm3/g)
0.61 0.40 0.54
PSD (%) Micro
Meso
Macro
88.20 35.82 21.96
3.88 51.24 57.93
7.83 12.93 20.11
ð5Þ
0
ΔH 0 ΔS0 þ RT R
ð6Þ
ð7Þ
where, K, ΔG0, ΔH0 and ΔS0 are the equilibrium constant, Gibbs free energy change, change in enthalpy and entropy change, respectively. Fig. 8 describes the equilibrium isotherms of different proteins on the ACF substrate and the sonicated ACFs/CNFs at different temperatures. As observed from Fig. 8a, equilibrium loading of BSA on ACFs increased with increasing BSA concentrations and the maximum adsorption capacity was determined to be ~ 166 mg/g corresponding to ~1500 ppm concentration at 5 °C. The capacity decreased with increasing temperatures, suggesting the adsorption of BSA to be exothermic, as shown in Table 2 (ΔH0 = 64.8 kJ/mol) [28–31]. Further, not much significant difference was observed between the solute loadings at 10 and 15 °C. Fig. 8b describes the adsorption isotherm of BSA on sonicated ACF/CNF sample. As observed from the figure, the equilibrium loadings of the protein molecules are larger on the sonicated materials than the parent materials, with the maximum adsorption capacity of ~192 mg/g observed for the sonicated ACFs/CNFs at 5 °C (ΔH0 = 73.8 kJ/mol). Herein, it is mentioned that the ultra-sonication of the prepared ACFs/ CNFs resulted in dislodging of the Ni-Nps from the tips of the CNFs,
which opened up the pores. Thus, the interior surface of the CNFs was also exposed to the biomolecules for adsorption, in particular that of mesopores. Increase in the surface area of the sonicated ACFs/CNFs following the sonication was confirmed by the SBET analysis. As observed from Table 1, the SBET of the sonicated ACFs/CNFs was ~ 20% larger than that of ACFs/CNFs. Further, the multi-scale web had relatively larger mesopore contents. Therefore, the larger size of BSA (8.4 nm × 8.4 nm × 4.8 nm) inhibited adsorption in micropores [32,33]. This may also be one of the reasons for the lesser loading of BSA observed on ACFs than on ACFs/CNFs. Table 2 shows the parameters calculated by fitting of the isotherm data with Freundlich and Langmuir equations. The regression coefficients (R2) were found to be 0.994 and 0.991 corresponding to the Freundlich and Langmuir models, respectively, indicating that the adsorption of BSA followed the Freundlich isotherm. Fig. 8c describes the adsorption of GOx on ACFs at 5 °C. The tests were performed at pH = 4.2. The maximum Qe was determined to be ~21 mg/g at the liquid phase concentration of 1200 ppm. No significant loadings of GOx on ACFs were observed at 10 and 15 °C. As shown in Table 2, R2 was calculated to be higher (0.995) and closer to 1, if the data were fitted with the Langmuir equation, suggesting that the monolayer coverage of GOx proteins occurred on ACFs. Fig. 8d shows the adsorption isotherms of GOx on ACFs/CNFs at different temperatures. As observed from the figure, the maximum adsorption loading was obtained to be ~39 mg/g at 5 °C. Relatively less adsorption occurred at 10 and 15 °C, suggesting that lower temperature was favorable for the GOx adsorption and the GOx-adsorption was exothermic (ΔH0 = 73. kJ/mol). As observed from Table 1, the adsorption isotherm followed the Langmuir model (R2 = 0.999) and Qm was determined to be ~59 mg/g. Fig. 8e describes the equilibrium loadings of the laboratory synthesized YqeH proteins [19] on ACFs/CNFs at different temperatures. The adsorption tests were performed at pH = 8. The maximum Qe of YqeH on the multi-scale web was determined to be ~70 mg/g at 5 °C. Relatively lesser adsorption occurred at high temperatures, suggesting the adsorption to be exothermic (ΔH0 = 56.2 kJ/mol) in this case also. The adsorption of YqeH on ACFs/CNFs also followed the Freundlich model at three temperatures. The values of R2 were calculated to be relatively higher for the Freundlich model, compared to the Langmuir
Fig. 8. Adsorption isotherm of (a) BSA on ACFs, (b) BSA on ACFs/CNFs, (c) GOx on ACFs, (d) GOx on ACFs/CNFs, and (e) YqeH on CNFs at different temperatures.
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Table 2 Freundlich and Langmuir isotherm constants, correlation coefficients and thermodynamic parameters for the adsorption of different biomolecules at different temperatures. Temp (°C)
BSA-ACFs
BSA-ACFs/CNFs
GOx-ACFs GOx-ACFs/CNFs
YqeH-ACFs/CNFs
5 10 15 5 10 15 5 5 10 15 5 10 15
Freundlich model
Langmuir model
Thermodynamic parameter
Kf (l/mg)1/n
1/n
R2
Qm (mg/g)
Kl (l/mg)
R2
K
lnK
ΔH (kJ/mol)
0.740 0.665 3.047 0.632 1.888 0.950 3.197 0.868 0.593 0.232 0.448 0.388 0.138
0.765 0.722 0.490 0.805 0.602 0.689 0.275 0.545 0.565 0.670 0.778 0.705 0.789
0.994 0.994 0.978 0.998 0.999 0.999 0.916 0.987 0.999 0.992 0.989 0.995 0.977
321.915 240.437 141.159 417.501 249.626 232.740 25.303 58.941 53.810 46.885 194.746 98.913 68.017
0.001 0.001 0.002 0.001 0.001 0.001 0.005 0.002 0.001 0.001 0.001 0.001 0.001
0.941 0.956 0.953 0.973 0.973 0.949 0.995 0.999 0.999 0.917 0.944 0.988 0.915
0.6664 0.2797 0.2531 1.0258 0.3498 0.3408 – 0.9429 0.3635 0.3201 0.8083 0.7708 0.3462
−0.4058 −1.2739 −1.3738 0.0255 −1.0503 −1.0765 – −0.0588 −1.0119 −1.1390 −0.2128 −0.2603 −1.0609
−64.8
model. It is also observed from Table 2 that K and Qe calculated for different biomolecules used in this study decreased with increasing temperatures, confirming the adsorption to be exothermic. Also, ΔH0 and K values were found to be comparable (within the same order of magnitude) to the reported literature data for the adsorption of proteins on different substrates [34,35]. Based on the adsorption data presented in this study for three different proteins on the ACF/CNF multi-scale web, a plausible mechanism for adsorption is proposed herein (Fig. 8). The adsorption of BSA was carried out at pH 7 in the PBS buffer, which is above the isoelectric point (IP) 4.7 of the BSA molecules. Thus, the proteins were negatively charged under the experimental conditions. Considering the negatively charged surface of ACFs and ACFs/CNFs, attributed to the presence of different oxygenated surface functional groups, the adsorption by the electrostatic attraction is not considered. Such surface characteristics (negatively charged functional groups) of ACFs/CNFs have been corroborated by the FTIR and Zeta potential measurements [18,19]. The most prominent mechanism for the adsorption of BSA on the substrate is the hydrophobic interaction. Further, BSA has multiple adsorption sites via several amino acids such as cysteine, tyrosine and tryptophan. Therefore, the physico-chemical adsorption of BSA because of hydrogen bonding and electro-active amino acids is also likely to occur, although at a relatively smaller magnitude [24,36]. With regard to GOx, it was found that the enzyme retained its maximum activity between pH 4 and 5 and was denatured or deactivated
−73.8
−72.3
−56.2
beyond this range. Similar observations were made in other studies with regard to the stability of GOx [37,38]. Interestingly, the IP of GOx is approximately 4.2. Therefore, at the experimental pH = 4.2, the ACF/CNF surface was amphoteric. It follows that the adsorption of GOx on ACFs/CNFs occurs primarily by the hydrophobic interaction. It is also mentioned that the CNF surface contained large amounts of O\H surface functional groups, as evident from the FTIR spectra. Therefore, the adsorption of GOx on ACFs/CNFs is also ascribed to the binding between the N\H or C_O groups of amino acids contained in GOx and the O\H groups present on the surface of the adsorbent. There is also a likelihood of the electrostatic attraction between the functional groups of GOx and the ACF/CNF surface, considering that the latter is neutrally charged at pH = 4.2. The IP of YqeH protein was measured to be 6.03. However, the adsorption tests were carried out at pH 8 because this protein was found to be the most active at pH 8 and was denatured at pH less than 6 and above 9. Under the experimental conditions, the protein molecule was negatively charged. Therefore, the hydrophobic interaction with the adsorbent surface was considered to be the major contributor to the adsorption of YqeH protein on ACFs/CNFs, similar to the characteristics of the BSA adsorption. Fig. 9 depicts the various mechanisms of adsorption proposed for different proteins. A comparison of the equilibrium loading obtained for different proteins on the multi-scale web of ACFs/CNFs was made with that obtained on the other substrates discussed in the literature. Table 3 presents the
Fig. 9. Proposed adsorption mechanism of BSA (1, 2 and 4), GOx (1, 3 and 4) and YqeH (1and 4) on ACFs/CNFs.
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S. Singh et al. / Materials Science and Engineering C 38 (2014) 46–54
Table 3 A comparison of the immobilization of proteins on ACFs/CNFs with different substrates described in the literatures. References
Material used for immobilization
Biomolecules
Ce (mg/l)
Loading of biomolecules on substrate (mg/g)
Present study
ACFs/CNFs
[5] [6] [9] [10] [11] [13] [14] [26]
Aldehyde functional ReSynTM microspheres Polyvinyl alcohol-coated glass powder surfaces Mesoporous molecular sieve silicate Mesoporous silica Catalytic filamentous carbons p(HEMA-co-MMA) copolymer Mesostructured silica materials Hydroxyapatite (HA) nanoparticles
BSA GOX YqeH BSA BSA BSA BSA BSA BSA GOx BSA
~1400 ~1200 ~800 – 1400 1400 1400 1400 1400 1400 –
192 39 70 292 22 40–380 10–320 8–75 20 12 28
comparative data. It may be observed that the solute loading on ACFs/ CNFs is relatively larger in most cases and comparable in some cases. 5. Conclusions The multi-scale web of ACFs/CNFs prepared in this study has considerably large BET surface area and pore volume, potentially suitable to be applied as an adsorbent. The adsorption tests exhibited significant and uniform adsorption of proteins on the ACF/CNF surface. The adsorption capacities of the prepared adsorbent for BSA, GOx and YqeH were experimentally determined to be approximately 192, 39 and 70 mg/g, respectively. The micro-mesoporous ACFs/CNFs follow Type I and II adsorption isotherms. The adsorption data for different proteins were fitted by Langmuir or Freundlich model, signifying mono- or multilayer coverage of the biomolecules. Further, the adsorption was exothermic, as the equilibrium solute loading decreased with increasing temperatures. The adsorption of different biomolecules on ACFs/CNFs under the experimental conditions is attributed to single- or a combination of physico-chemical, hydrophobic and electrostatic mechanisms. The method to prepare ACFs/CNFs is simple, and the prepared adsorbent in this study may be used for separating and purifying pharmaceutical effluents. Acknowledgments The authors acknowledge the support from DBT (New Delhi, India) for providing a research grant. The authors are also thankful to Gun Ei Chemical Industry Co. Ltd., Japan for supplying ACFs. References [1] D.A.R. Mahmoud, W.A. Helmy, J. Appl. Sci. Res. 12 (2009) 2466–2476. [2] F.R.R. Teles, L.P. Fonseca, Mater. Sci. Eng. C 28 (2008) 1530–1543. [3] A.R. Hood, N. Saurakhiya, D. Deva, A. Sharma, N. Verma, Mater. Sci. Eng. C 33 (2013) 4313–4322. [4] M.D. Saikia, N.N. Dutta, React. Funct. Polym. 68 (2008) 33–38. [5] B.V. Twala, B.T. Sewell, J. Jordaan, Enzyme Microb. Technol. 50 (2012) 331–336. [6] A.K. Bajpai, J. Appl. Polym. Sci. 78 (2000) 933–940. [7] J.F. Cabrita, L.M. Abrantes, A.S. Viana, Electrochim. Acta 50 (2005) 2117–2124. [8] C.X. He, J.H. Liu, L.Y. Xie, Q.L. Zhang, C.H. Li, D.Y. Gui, G.Z. Zhang, C. Wu, Langmuir 25 (2009) 13456–13460.
[9] S. Kumar, V.S. Chauhan, P. Nahar, Microchem. J. 89 (2008) 148–152. [10] S.M.L. dos Santos, K.A.B. Nogueira, M. de Souza Gama, J.D.F. Lima, I.J. da Silva Júnior, D.C.S. de Azevedo, Microporous Mesoporous Mater. 180 (2013) 284–292. [11] G.A. Kovalenko, E.V. Kuznetsova, Y.I. Mogilnykh, I.S. Andreeva, D.G. Kuvshinov, N.A. Rudina, Carbon 39 (2001) 1033–1043. [12] F.B.O. Daoud, S. Kaddour, T. Sadoun, Colloids Surf. B 75 (2010) 93–99. [13] A. Denizli, H. Yavuz, Y. Arıca, Colloids Surf. A 174 (2000) 307–317. [14] J.L. Blin, C. Carteret, R. Bleta, M.J. Stébé, Colloids Surf. A 357 (2010) 128–135. [15] M. Bikshapathi, S. Singh, B. Bhaduri, G.N. Mathur, A. Sharma, N. Verma, Colloids Surf. A 399 (2012) 46–55. [16] A. Chakraborty, D. Deva, A. Sharma, N. Verma, J. Colloid Interface Sci. 359 (2011) 228–239. [17] G.B. Zheng, K. Kouda, H. Sano, Y. Uchiyama, Y.-F. Shi, H.-J. Quan, Carbon 42 (2004) 635–640. [18] S. Singh, M. Ashfaq, R.K. Singh, H.C. Joshi, A. Srivastava, A. Sharma, N. Verma, New Biotechnol. 30 (2013) 656–665. [19] M. Ashfaq, S. Singh, A. Sharma, N. Verma, Ind. Eng. Chem. Res. 52 (2013) 4672–4682. [20] P.K. Yadav, F. McKavanagh, P.D. Maguire, P. Lemoine, Appl. Surf. Sci. 258 (2011) 361–369. [21] B. Anand, P. Surana, S. Bhogaraju, S. Pahari, B. Prakash, Biochem. Biophys. Res. Commun. 386 (2009) 602–606. [22] B. Anand, P. Surana, B. Prakash, PLoS One 5 (2010) e9944. [23] R.M. Singhal, A. Sharma, N. Verma, Ind. Eng. Chem. Res. 47 (2008) 3700–3707. [24] B.B. Prasad, A. Prasad, M.P. Tiwari, Biosens. Bioelectron. 39 (2013) 236–243. [25] V. Helenius, J. Korppi-Tommola, S. Kotila, J. Nieminen, R. Lohikoski, J. Timonen, Chem. Phys. Lett. 280 (1997) 325–332. [26] J. Liu, X. Zhang, H. Pang, B. Liu, Q. Zou, J. Chen, Biosens. Bioelectron. 31 (2012) 170–175. [27] G. Bayramoğlu, A.Ü. Metin, B. Altıntas, M.Y. Arıca, Bioresour. Technol. 101 (2010) 6881–6887. [28] T. Zhang, Y.M. Zhou, M. He, Y.X. Zhu, X.H. Bu, Y.J. Wang, Chem. Eng. J. 219 (2013) 278–285. [29] M. Matsukata, M. Nishino, J.P. Gong, Y. Osada, Y. Sakurai, T. Okano, Colloids Surf. B 13 (1999) 203–211. [30] T.S. Anirudhan, S.R. Rejeena, A.R. Tharun, Colloids Surf. B 93 (2012) 49–58. [31] J. Han, P. Silcock, A.J. McQuillan, P. Bremer, Colloids Surf. A 349 (2009) 207–213. [32] R. Gadiou, E.A. dos Santos, M. Vijayaraj, K. Anselme, J. Dentzer, G.A. Soares, C. Vix-Guterl, Colloids Surf. B 73 (2009) 168–174. [33] S.K. Swain, D. Sarkar, Appl. Surf. Sci. 286 (2013) 99–103. [34] Q. Wang, P. Liu, X. Zhou, X. Zhang, T. Fang, P. Liu, X. Min, X. Li, J. Photochem. Photobiol. C 230 (2012) 23–30. [35] X.-L. Han, F.-F. Tian, Y.-S. Ge, F.-L. Jiang, L. Lai, D.-W. Li, Q.-L. Yu, J. Wang, C. Lin, Y. Liu, J. Photochem. Photobiol. B 109 (2012) 1–11. [36] J. Kim, R.J. Desch, S.W. Thiel, V.V. Guliants, N.G. Pinto, Microporous Mesoporous Mater. 170 (2013) 95–104. [37] Q. Liu, A.M. Rauth, X.Y. Wu, Int. J. Pharm. 339 (2007) 148–156. [38] Z. Wen, B. Niemeyer, J. Chromatogr. B 857 (2007) 149–157.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Multi-scale carbon micro/nanofibers-based adsorbents for protein immobilization Shiv Singh a, Abhinav Singh a, Vaibhav Sushil Singh Bais b, Balaji Prakash b, Nishith Verma a,c,⁎ a b c
Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India Department of Biological Science and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, India Center for Environmental Science and Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India
a r t i c l e
i n f o
Article history: Received 10 November 2013 Received in revised form 26 December 2013 Accepted 22 January 2014 Available online 31 January 2014 Keywords: Activated carbon fiber Carbon nanofiber Adsorption Immobilization Bovine serum albumin Glucose oxidase
a b s t r a c t In the present study, different proteins, namely, bovine serum albumin (BSA), glucose oxidase (GOx) and the laboratory purified YqeH were immobilized in the phenolic resin precursor-based multi-scale web of activated carbon microfibers (ACFs) and carbon nanofibers (CNFs). These biomolecules are characteristically different from each other, having different structure, number of parent amino acid molecules and isoelectric point. CNF was grown on ACF substrate by chemical vapor deposition, using Ni nanoparticles (Nps) as the catalyst. The ultrasonication of the CNFs was carried out in acidic medium to remove Ni Nps from the tip of the CNFs to provide additional active sites for adsorption. The prepared material was directly used as an adsorbent for proteins, without requiring any additional treatment. Several analytical techniques were used to characterize the prepared materials, including scanning electron microscopy, Fourier transform infrared spectroscopy, BET surface area, poresize distribution, and UV–vis spectroscopy. The adsorption capacities of prepared ACFs/CNFs in this study were determined to be approximately 191, 39 and 70 mg/g for BSA, GOx and YqeH, respectively, revealing that the carbon micro-nanofibers forming synthesized multi-scale web are efficient materials for the immobilization of protein molecules. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bioactive substances including proteins are considered as potential substitutes of traditional chemical compounds for a variety of applications, ranging from the compact sensors to catalysts. The superiority of these bioactive substances over conventional chemicals is because of their ability of carrying out complex chemical reactions with high selectivity [1]. In spite of the promising advantages, their widespread applications are, however, limited by the factors such as instability, relative high cost of synthesis, and also, high cost involved in separating the substances from the reactor effluents. Immobilization of proteins is an effective strategy to counter some of the aforementioned hurdles. Immobilization provides stability to their structure, thus enabling their applications under different conditions of temperatures, pH and organic solvents that are bio-active. In such context, the choice of immobilization technique is important. Currently, different strategies are used for the immobilization of biomolecules, for example, cross-linking, entrapment and adsorption. Materials produced using cross-linking and entrapment often have low reproducibility,
⁎ Corresponding author at: Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India. Tel.: +91 512 2596352x7704; fax: +91 512 2590104. E-mail address: [email protected] (N. Verma). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.042
high pore diffusion resistance and non-uniform deposition of solute molecules. On the contrary, adsorption is considered to be a simple, economical and efficient method for the immobilization of biomolecules [2]. Furthermore, adsorption does not alter the structure of biomolecules, and therefore, the activities of the biomolecules are retained to a large extent following adsorption [3,4]. There are several materials used as the substrate to immobilize proteins by adsorption. The common examples are silica, alumina, glass, methyl methacrylate, catalytic filamentous carbon and activated carbon [5–14]. In general, a substrate should possess high surface area and should be non-toxic, mechanically strong and amenable to surface functionalization. In particular, the adsorption capacity of the material for the biomolecules should be large. Recently, multi-scale web of activated carbon microfibers (ACFs) and carbon nanofibers (CNFs) has been extensively used for different environmental applications, for examples, as adsorbents and antibacterial agents [15–18]. The salient advantage of ACFs/CNFs is that it can be directly used in an end-application, without requiring any postsynthesis step. In accordance with their variety of applications, CNFs are grown on ACF substrate by chemical vapor deposition (CVD), using different metal nanoparticles (Nps) as catalyst, for example, nickel (Ni), copper (Cu) and silver (Ag). The prepared materials have large surface area [15–18]. Furthermore, the prepared CNFs are less cytotoxic in comparison to conventional adsorbents such as zeolite, granular activated carbon and carbon nanotubes (CNTs) [19].
S. Singh et al. / Materials Science and Engineering C 38 (2014) 46–54
In the present study, the multi-scale web of ACFs/CNFs was used to immobilize different proteins: bovine serum albumin (BSA), glucose oxidase (GOx) and a laboratory purified YqeH. BSA is similar to human serum albumin, the most abundant protein found in blood plasma. It is used as a clinical reagent for ELISA testing, and also, helps in binding of hydrophobic steroids [20]. GOx is used as a biosensor to determine the free-glucose in human blood [3]. Along with these commercially available proteins, adsorption on YqeH protein, synthesized in our laboratory and expressed using cloned enzyme, has also been studied. YqeH has been chosen owing to the facts that its biochemical role and catalytic machinery are well characterized. YqeH is a GTPase and plays an important role in the assembly of small ribosomal (30S) subunit in bacteria [21]. Considering its role in ribosome maturation, which is an essential process, the enzyme is conserved across all kingdoms of life. Recently, its catalytic machinery has been deciphered and shown to employ K+ ion for GTP hydrolysis [22]. In this study, the multi-scale web of ACFs/CNFs was grown by CVD using benzene (C6H6) as a carbon source and Ni metal Nps as the CVD catalyst. Following ultra-sonication, the Nps were removed from the tip of the CNFs to offer increased adsorption sites for proteins. The prepared ACF/CNF substrate was characterized for its physico-chemical properties using various analytical techniques, including scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, Brunauer–Emmet–Teller (BET) surface area and poresize distribution (PSD). The adsorption tests for different proteins were performed under batch conditions at different temperatures (5, 10 and 15 °C) to determine the equilibrium loading on the prepared substrate. 2. Experimental 2.1. Materials Ni(NO3)2·6H2O (purity N 97%), sodium dodecyl sulfate (SDS, purity N 99%), C 6 H 6 , acetone and other reagents used for preparing buffers including NaCl, KCl, Na 2 HPO 4 , KH 2 PO 4 , MgCl 2 , 2-mercaptoethanol, glycerol, sodium acetate and glacial acetic acid were procured from Merck, India. The phenolic resin precursor based ACFs were purchased from Gun Ei Chemical Industry Co. Ltd., Japan. BSA, GOx and Bradford reagent were purchased from SRL (India), Calbiochem (Germany) and Sigma-Aldrich (Germany), respectively. Nitrogen and hydrogen gases (purity N 99.999%) were purchased from
47
Sigma Gases, India. Milli-Q water was used for preparing the required solutions in the experiment. 2.2. Pretreatment of ACFs and synthesis of ACFs/CNFs The as-received ACFs were pretreated by 5 ml of 0.03 M HNO3in 1 l of Milli-Q water at 80 °C for 1 h to remove any impurities from the ACF surface. Next, the ACF samples were dried at room temperature (30 ± 5 °C) for 6 h, followed by hot air-oven and vacuum drying at 120 and 200 °C, respectively for 12 h each. The dried ACF samples were impregnated with 0.4 M Ni (NO3)2·6H2O salts dispersed in acetone solution, using the wet incipient method described in previous studies [16,23]. The anionic SDS (0.3% w/w) surfactant was used in the impregnation solution to prevent the agglomeration of the salt crystals in the solution and facilitate uniform dispersion of the crystals on the ACFs. After impregnation, samples were dried in the static air for 6 h and in the oven for 12 h at 120 °C. Next, calcinations, reduction and CVD were sequentially carried out on the impregnated ACFs. Fig. 1 illustrates the schematic representation of the experimental set-up used for calcinations, reduction and CVD in the present study. The details of the experimental set-up have been described in the previous study [18]. The impregnated samples were calcined at 400 °C in N2 atmosphere for 4 h, followed by reduction at 550 °C in H2 atmosphere (flow rate = 200 sccm) for 2 h. The calcinations and reduction temperatures were determined by the temperature programmed reduction. After calcination and reduction, Ni(NO3)2 particles dispersed in ACFs were converted to Ni Nps. These Ni Nps acted as the catalyst for the tipgrowth of CNFs during the CVD. The CVD was performed at 800 °C for 2 h, using liquid C6H6 as the carbon source. The temperature of the liquid C6H6 was maintained at 8 °C using Freon unit bubbled with N2. C6H6 vapors were carried by N2 gas (100 sccm) to the quartz reactor where it was decomposed by Ni Nps dispersed in the ACFs to affect the CNF growth. After CVD, the reactor was gradually cooled to the room temperature under N2 flow. Next, the prepared material (ACFs/CNFs) was ultra-sonicated in 0.05 M HNO3 solution for 5 min to remove Ni Nps from the tip of the prepared CNFs. 2.3. Purification of YqeH The recombinant plasmid for YqeH was freshly transformed into Escherichia coli BL21 (DE3) CODON PLUS cells. Fresh transformant was
Fig. 1. Schematic of vertical tubular reactor used for calcinations, reduction and CVD.
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grown in Luria Broth containing 100 μg/ml ampicillin at 37 °C with constant aeration. Exponentially growing culture (A600 of ~ 0.6) was induced with 0.1 mM IPTG and further grown for 12–14 h at 18 °C. Cells were harvested and lysed by sonication in lysis buffer (phosphate buffer containing 5% glycerol, 1 mM β-mercaptoethanol and protease inhibitor HIS cocktail (Sigma)). The cell lysate was clarified at 15,000 g for 45 min and supernatant was loaded onto a pre-equilibrated Ni-NTA column (His-Trap FF GE Healthcare). The column was washed with equilibration buffer of 50 mM Tris–Cl pH 8, 500 mM NaCl, 5 mM MgCl2, 3 mM β-mercaptoethanol, and 10% glycerol. His-tagged proteins were eluted using a 0 to 500 mM linear gradient of imidazole in equilibration buffer. At the final step of purification, all eluted fractions (obtained in the previous step) containing His-tagged proteins were pooled and loaded onto 26/60 Superdex200 High Load (HL) gel filtration column (GE Healthcare) and then pre-equilibrated with buffer (50 mM Tris–Cl pH 8, 500 mM NaCl, 5 mM MgCl2, 3 mM β-Mercaptoethanol, 10% glycerol) to achieve the requisite homogeneity in protein preparation. 2.4. Adsorption of proteins on ACFs/CNFs The sonicated ACFs/CNFs were the templates for the adsorption study of biomolecules in this study. Different buffer solutions were used as the dispersing medium for different proteins: 10 mM phosphate buffer of pH 7 for BSA [20,24], 100 mM of acetate buffer of pH 4.2 for GOx [3], and 50 mM Tris–Cl pH 8, 500 mM NaCl, 5 mM MgCl2, 3 mM β-mercaptoethanol, and 10% glycerol for YqeH. Different test solutions of BSA (200–2000 ppm), GOx (200–1400 ppm) and YqeH (300–1400 ppm), each of 1.0 cm3 volume, along with their respective buffers were transferred to 15.0 cm3 borosilicate culture tubes. Next, the sonicated ACFs/CNFs (0.01 g) were added to the test- and buffer solution containing tubes and kept in a shaking water bath (100 rpm) for 24 h at three different temperatures of 5, 10 and 15 °C to immobilize the proteins. The loading of the biomolecules on the ACFs/CNFs was determined by measuring the concentrations of the solute in the buffer solution before and after adsorption. Therefore, the ACF/CNF absorbents were repeatedly (8–10 times) rinsed with the same buffer solution after adsorption to remove the loosely bound proteins from the ACF/ CNF surface, back into the solution. The concentrations of the solutions were determined by the Bradford assay method, using UV–vis spectroscopy. Briefly, a 3 ml-volume of the commercially available Bradford
reagent was mixed with 0.1 ml of the solutions and incubated at 25 °C for 10 min. The reagent color changed from brown to dark blue. The equilibrium loadings were calculated from the difference between initial and final concentrations of solutions, using the following equation: Q ¼ ðC i −C e ÞV=w
ð1Þ
here, Q, Ci, Ce, V and w were the calculated equilibrium loading, initial concentration of biomolecules in the buffer, final concentration of biomolecule after adsorption, volume of the test solution and weight of adsorbent, respectively. Fig. 2 describes the schematic representation of the steps involved in the material synthesis and immobilization, including impregnation, calcinations, reduction, CVD, ultra-sonication and batch study for the adsorption of proteins. 3. Surface characterization The prepared ACFs/CNFs were characterized using several analytical techniques, including SEM (Supra 40 VP, Zeiss), FTIR (Tensor 27 Bruker), BET surface area, total pore volume (VTotal) and PSD (Autosorb-1C Quantachrome) and UV–vis spectroscopy (Cary 100 Bio, Varian). SEM was used to investigate the surface morphology of the substrate ACFs and ACFs/CNFs. Surface functional groups were identified using FTIR in wave number range of 400–4000 cm−1 at attenuated total reflectance mode. The scan rate was 100 over a resolution of 4 cm−1. The Raman spectroscopy analysis was carried out to determine the relative amount of graphitic carbon present in ACFs and ACFs/CNFs, using a confocal Raman instrument (Model: Alpha, Make: Witec, Germany with k = 543 nm). The data were collected in the range of 200–4000 cm−1 at room temperature in air at a resolution of 2.0 cm−1. BET surface area (SBET), VTotal and PSD were calculated at − 196 °C. Microporous and mesoporous volumes were determined by density functional theory (DFT) and the Barrett–Joyner–Halenda (BJH) method, respectively. 4. Result and discussion 4.1. Surface morphology Fig. 3 shows the SEM images of the surface morphology of ACFs, ACFs/CNFs and sonicated ACFs/CNFs. Uniformly distributed pores and
Fig. 2. Schematic of preparation of Ni-ACFs/CNFs and immobilization of different biomolecules on ACFs/CNFs.
S. Singh et al. / Materials Science and Engineering C 38 (2014) 46–54
smooth surface of ACFs are clearly evident in Fig. 3(a–a1). Fig. 3(b–b1) shows the SEM images of ACFs/CNFs and confirms the uniform and dense CNF-growth. Shiny particles may be observed at the tip of CNFs, which confirmed the tip-growth mechanism of the CNFs on the ACF substrate. Fig. 3(c–c1) presents the SEM images of the sonicated ACF/ CNF sample. It may be observed that post-ultra-sonication, most of the Ni Nps were removed from the tip of the CNFs. Fig. 4 shows the SEM images of the ACF/CNF sample after the immobilization of the proteins, namely, BSA, GOx and YqeH, at low and high magnifications. Comparing the SEM images (5 KX) shown in Figs. 3c and 4(a–c) for pre- and post-adsorbed samples, respectively, a distinct change may be observed in the surface morphology of ACFs/CNFs. In Fig. 4(a–c), a thin layer of material (adsorbed protein) is clearly observed over the surface of ACFs/CNFs. 4.2. FTIR analysis Fig. 5 shows the FTIR spectrum of ACF/CNF sample. The analysis was performed to identify different functional groups present on the surface of ACFs/CNFs. As observed from the spectrum,
49
peaks were observed at 650, 1575, 1690 and 3650 cm− 1, attributed to C`C, N\O, C_O and O\H stretch, respectively. C`C, C_O and O\H stretches were intrinsically present on the ACF surface. The N\O stretch appeared because of HNO 3 used for the pretreatment of ACF. As also observed from the figure, the N\O stretch disappeared at high temperatures, because of the high temperature processing of the material, viz. calcinations, reduction and CVD. The other stretches remained intact on the ACF/CNF surface. Few additional common peaks were identified in the post-proteinimmobilized ACF/CNF samples which confirm the adsorption of proteins on the surface of ACFs/CNFs. The peaks observed at 1265, 1485, 1610, 1716 and 3400 cm− 1 were attributed to N\H (3°), N\H (2°), N\H (1°), C_O and N\H/O\H stretch, respectively. N\H stretches originated from peptide bonds (\CO\NH\) of different amino acids present in the BSA, GOx and YqeH, whereas C_O stretch originated either from \COOH group of the protein-side free chain or from the buffer used during the immobilization. Multiple peaks were also observed between 3600 and 3800 cm−1and were assigned for the O\H stretch of hydroxyl group [18,25–27].
Fig. 3. SEM images of (a–a1) ACFs, (b–b1) Ni-ACFs/CNFs and (c–c1) sonicated ACFs/CNFs.
50
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Fig. 4. SEM images of (a–a1) BSA-ACFs/CNFs, (b–b1) GOx-ACFs/CNFs and (c–c1) YqeH-ACFs/CNFs.
4.3. Raman spectra Fig. 6 describes the Raman spectrum of ACF and ACF/CNF samples. As observed from the figure, there are two peaks for both samples. The first peak observed at 1340.4 cm−1 (D-band) corresponds to the disordered phase of carbon, whereas the second peak observed at 1570.4 cm−1 (Gband) corresponds to the graphitic phase of the material. The ID/IG peak ratio of the ACF was determined to be 1.22. The ratio decreased to 1.04 in the ACF/CNF sample. Considering that ID/IG ratio reflects the magnitude of the disordered phase relative to the graphitic phase present in the material, it was concluded that the graphitic phase was higher in the ACF/CNF than in the ACF substrate. In other words, graphitic phase in ACFs increased following the growth of CNFs on ACFs.
4.4. Surface area analysis Fig. 7 describes the N2 adsorption isotherms for the prepared materials: substrate ACFs and ACFs/CNFs with and without sonication. SBET, VTotal and PSD were calculated from the data plotted in Fig. 7. It may be observed that the adsorption isotherm of ACFs is of Type I and
those for ACFs/CNFs and sonicated ACFs/CNFs are of Type II in accordance with the BET classifications. The Type I isotherms are exhibited mostly by microporous materials, with the isotherm curve significantly increasing up to a certain level and thereafter, continuing in the horizontal plateau. The adsorption curve of Type II isotherm monotonically increases with increasing relative pressures (P/P0), as observed in Fig. 7 also, which is the unique characteristic of mesoporous adsorbents. Table 1 summarizes SBET, VTotal and PSD of the prepared materials. As observed, SBET and microporous contents of ACFs were 1187 m2/g and ~ 88%, respectively. On the other hand ACFs/CNFs had comparatively smaller SBET (371 m2/g) and VTotal (0.40 cm3/g). ACFs/CNFs were mostly mesoporous (~51%). Following sonication, SBET (~450 m2/g) and VTotal (0.54 cm3/g) increased, as observed from the data shown in the table for the sonicated ACF/CNF sample. This increase is attributed to the removal of the Ni Nps from the tip of the CNFs, exposing the internal surface area also to N2 adsorbate molecules. 4.5. Adsorption As mentioned earlier in the text, the adsorption capacity of the prepared ACFs/CNFs for different proteins was determined at three
S. Singh et al. / Materials Science and Engineering C 38 (2014) 46–54
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Fig. 5. FTIR spectra of ACF, ACF/CNF and biomolecules-adsorbed ACF/CNF samples.
different temperatures (5, 10 and 15 °C). In the present tests, adsorption of the proteins was found to be exothermic (later shown in the paper), with decrease in the adsorption capacity with increasing temperatures. Therefore, the adsorption tests were performed at relatively lower temperatures (5, 10 and 15 °C). Moreover, although BSA was found to be stable at room temperature (~ 25 °C), we observed that GOx and the lab purified YqeH were denatured in 5–6 h only, when the tests were performed at 25 °C. Therefore, 37 °C was not considered for the tests and the highest temperature considered in the study was 15 °C. The adsorption tests were performed at different pHs also to determine the optimum pH for the maximum equilibrium loading of the solutes. The significance of performing tests at different pHs for different proteins is explained later in the text. After determining the equilibrium loading (Q) of the biomolecules at different concentrations (Ce), the adsorption data were fitted with Langmuir and Freundlich isotherms and the
corresponding thermodynamic parameters were extracted. The linearized forms of Langmuir and Freundlich equations may be expressed as follows: Ce ¼ Q
Ce Qm
þ
1 KlQ m
ð2Þ
ln Q ¼ lnK f þ n ln C e
ð3Þ
where, Ce, Q, and Qm are equilibrium concentrations, equilibrium loading, and the maximum saturation capacity of the adsorbent, respectively. Kl is the Langmuir adsorption constant; Kf and n are the Freundlich parameters. Thermodynamic parameters for the adsorption of different biomolecules were calculated at different temperatures (278, 283 and 288 K) using the following equations: 0
ΔG ¼ −RT lnK
ð4Þ
450
Volume (cc/g)
400 350
ACF Sonicated ACF/CNF ACF/CNF
300 250 200 150 100 0.0
0.2
0.4
0.6
0.8
1.0
Relative Pressure (P/Po) Fig. 6. Raman spectrum of ACF and ACF/CNF samples.
Fig. 7. Nitrogen adsorption isotherms of ACF, ACF/CNF and sonicated ACF/CNF samples.
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S. Singh et al. / Materials Science and Engineering C 38 (2014) 46–54
Table 1 SBET, VTotal and PSD of prepared materials. SBET (m2/g)
Sample
ACFs Ni-ACFs/CNFs Sonicated-ACFs/CNFs
K¼
C i −C e Ce
0
0
ΔG ¼ ΔH −TΔS
lnK ¼ −
1187 371 450
VTotal (cm3/g)
0.61 0.40 0.54
PSD (%) Micro
Meso
Macro
88.20 35.82 21.96
3.88 51.24 57.93
7.83 12.93 20.11
ð5Þ
0
ΔH 0 ΔS0 þ RT R
ð6Þ
ð7Þ
where, K, ΔG0, ΔH0 and ΔS0 are the equilibrium constant, Gibbs free energy change, change in enthalpy and entropy change, respectively. Fig. 8 describes the equilibrium isotherms of different proteins on the ACF substrate and the sonicated ACFs/CNFs at different temperatures. As observed from Fig. 8a, equilibrium loading of BSA on ACFs increased with increasing BSA concentrations and the maximum adsorption capacity was determined to be ~ 166 mg/g corresponding to ~1500 ppm concentration at 5 °C. The capacity decreased with increasing temperatures, suggesting the adsorption of BSA to be exothermic, as shown in Table 2 (ΔH0 = 64.8 kJ/mol) [28–31]. Further, not much significant difference was observed between the solute loadings at 10 and 15 °C. Fig. 8b describes the adsorption isotherm of BSA on sonicated ACF/CNF sample. As observed from the figure, the equilibrium loadings of the protein molecules are larger on the sonicated materials than the parent materials, with the maximum adsorption capacity of ~192 mg/g observed for the sonicated ACFs/CNFs at 5 °C (ΔH0 = 73.8 kJ/mol). Herein, it is mentioned that the ultra-sonication of the prepared ACFs/ CNFs resulted in dislodging of the Ni-Nps from the tips of the CNFs,
which opened up the pores. Thus, the interior surface of the CNFs was also exposed to the biomolecules for adsorption, in particular that of mesopores. Increase in the surface area of the sonicated ACFs/CNFs following the sonication was confirmed by the SBET analysis. As observed from Table 1, the SBET of the sonicated ACFs/CNFs was ~ 20% larger than that of ACFs/CNFs. Further, the multi-scale web had relatively larger mesopore contents. Therefore, the larger size of BSA (8.4 nm × 8.4 nm × 4.8 nm) inhibited adsorption in micropores [32,33]. This may also be one of the reasons for the lesser loading of BSA observed on ACFs than on ACFs/CNFs. Table 2 shows the parameters calculated by fitting of the isotherm data with Freundlich and Langmuir equations. The regression coefficients (R2) were found to be 0.994 and 0.991 corresponding to the Freundlich and Langmuir models, respectively, indicating that the adsorption of BSA followed the Freundlich isotherm. Fig. 8c describes the adsorption of GOx on ACFs at 5 °C. The tests were performed at pH = 4.2. The maximum Qe was determined to be ~21 mg/g at the liquid phase concentration of 1200 ppm. No significant loadings of GOx on ACFs were observed at 10 and 15 °C. As shown in Table 2, R2 was calculated to be higher (0.995) and closer to 1, if the data were fitted with the Langmuir equation, suggesting that the monolayer coverage of GOx proteins occurred on ACFs. Fig. 8d shows the adsorption isotherms of GOx on ACFs/CNFs at different temperatures. As observed from the figure, the maximum adsorption loading was obtained to be ~39 mg/g at 5 °C. Relatively less adsorption occurred at 10 and 15 °C, suggesting that lower temperature was favorable for the GOx adsorption and the GOx-adsorption was exothermic (ΔH0 = 73. kJ/mol). As observed from Table 1, the adsorption isotherm followed the Langmuir model (R2 = 0.999) and Qm was determined to be ~59 mg/g. Fig. 8e describes the equilibrium loadings of the laboratory synthesized YqeH proteins [19] on ACFs/CNFs at different temperatures. The adsorption tests were performed at pH = 8. The maximum Qe of YqeH on the multi-scale web was determined to be ~70 mg/g at 5 °C. Relatively lesser adsorption occurred at high temperatures, suggesting the adsorption to be exothermic (ΔH0 = 56.2 kJ/mol) in this case also. The adsorption of YqeH on ACFs/CNFs also followed the Freundlich model at three temperatures. The values of R2 were calculated to be relatively higher for the Freundlich model, compared to the Langmuir
Fig. 8. Adsorption isotherm of (a) BSA on ACFs, (b) BSA on ACFs/CNFs, (c) GOx on ACFs, (d) GOx on ACFs/CNFs, and (e) YqeH on CNFs at different temperatures.
S. Singh et al. / Materials Science and Engineering C 38 (2014) 46–54
53
Table 2 Freundlich and Langmuir isotherm constants, correlation coefficients and thermodynamic parameters for the adsorption of different biomolecules at different temperatures. Temp (°C)
BSA-ACFs
BSA-ACFs/CNFs
GOx-ACFs GOx-ACFs/CNFs
YqeH-ACFs/CNFs
5 10 15 5 10 15 5 5 10 15 5 10 15
Freundlich model
Langmuir model
Thermodynamic parameter
Kf (l/mg)1/n
1/n
R2
Qm (mg/g)
Kl (l/mg)
R2
K
lnK
ΔH (kJ/mol)
0.740 0.665 3.047 0.632 1.888 0.950 3.197 0.868 0.593 0.232 0.448 0.388 0.138
0.765 0.722 0.490 0.805 0.602 0.689 0.275 0.545 0.565 0.670 0.778 0.705 0.789
0.994 0.994 0.978 0.998 0.999 0.999 0.916 0.987 0.999 0.992 0.989 0.995 0.977
321.915 240.437 141.159 417.501 249.626 232.740 25.303 58.941 53.810 46.885 194.746 98.913 68.017
0.001 0.001 0.002 0.001 0.001 0.001 0.005 0.002 0.001 0.001 0.001 0.001 0.001
0.941 0.956 0.953 0.973 0.973 0.949 0.995 0.999 0.999 0.917 0.944 0.988 0.915
0.6664 0.2797 0.2531 1.0258 0.3498 0.3408 – 0.9429 0.3635 0.3201 0.8083 0.7708 0.3462
−0.4058 −1.2739 −1.3738 0.0255 −1.0503 −1.0765 – −0.0588 −1.0119 −1.1390 −0.2128 −0.2603 −1.0609
−64.8
model. It is also observed from Table 2 that K and Qe calculated for different biomolecules used in this study decreased with increasing temperatures, confirming the adsorption to be exothermic. Also, ΔH0 and K values were found to be comparable (within the same order of magnitude) to the reported literature data for the adsorption of proteins on different substrates [34,35]. Based on the adsorption data presented in this study for three different proteins on the ACF/CNF multi-scale web, a plausible mechanism for adsorption is proposed herein (Fig. 8). The adsorption of BSA was carried out at pH 7 in the PBS buffer, which is above the isoelectric point (IP) 4.7 of the BSA molecules. Thus, the proteins were negatively charged under the experimental conditions. Considering the negatively charged surface of ACFs and ACFs/CNFs, attributed to the presence of different oxygenated surface functional groups, the adsorption by the electrostatic attraction is not considered. Such surface characteristics (negatively charged functional groups) of ACFs/CNFs have been corroborated by the FTIR and Zeta potential measurements [18,19]. The most prominent mechanism for the adsorption of BSA on the substrate is the hydrophobic interaction. Further, BSA has multiple adsorption sites via several amino acids such as cysteine, tyrosine and tryptophan. Therefore, the physico-chemical adsorption of BSA because of hydrogen bonding and electro-active amino acids is also likely to occur, although at a relatively smaller magnitude [24,36]. With regard to GOx, it was found that the enzyme retained its maximum activity between pH 4 and 5 and was denatured or deactivated
−73.8
−72.3
−56.2
beyond this range. Similar observations were made in other studies with regard to the stability of GOx [37,38]. Interestingly, the IP of GOx is approximately 4.2. Therefore, at the experimental pH = 4.2, the ACF/CNF surface was amphoteric. It follows that the adsorption of GOx on ACFs/CNFs occurs primarily by the hydrophobic interaction. It is also mentioned that the CNF surface contained large amounts of O\H surface functional groups, as evident from the FTIR spectra. Therefore, the adsorption of GOx on ACFs/CNFs is also ascribed to the binding between the N\H or C_O groups of amino acids contained in GOx and the O\H groups present on the surface of the adsorbent. There is also a likelihood of the electrostatic attraction between the functional groups of GOx and the ACF/CNF surface, considering that the latter is neutrally charged at pH = 4.2. The IP of YqeH protein was measured to be 6.03. However, the adsorption tests were carried out at pH 8 because this protein was found to be the most active at pH 8 and was denatured at pH less than 6 and above 9. Under the experimental conditions, the protein molecule was negatively charged. Therefore, the hydrophobic interaction with the adsorbent surface was considered to be the major contributor to the adsorption of YqeH protein on ACFs/CNFs, similar to the characteristics of the BSA adsorption. Fig. 9 depicts the various mechanisms of adsorption proposed for different proteins. A comparison of the equilibrium loading obtained for different proteins on the multi-scale web of ACFs/CNFs was made with that obtained on the other substrates discussed in the literature. Table 3 presents the
Fig. 9. Proposed adsorption mechanism of BSA (1, 2 and 4), GOx (1, 3 and 4) and YqeH (1and 4) on ACFs/CNFs.
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S. Singh et al. / Materials Science and Engineering C 38 (2014) 46–54
Table 3 A comparison of the immobilization of proteins on ACFs/CNFs with different substrates described in the literatures. References
Material used for immobilization
Biomolecules
Ce (mg/l)
Loading of biomolecules on substrate (mg/g)
Present study
ACFs/CNFs
[5] [6] [9] [10] [11] [13] [14] [26]
Aldehyde functional ReSynTM microspheres Polyvinyl alcohol-coated glass powder surfaces Mesoporous molecular sieve silicate Mesoporous silica Catalytic filamentous carbons p(HEMA-co-MMA) copolymer Mesostructured silica materials Hydroxyapatite (HA) nanoparticles
BSA GOX YqeH BSA BSA BSA BSA BSA BSA GOx BSA
~1400 ~1200 ~800 – 1400 1400 1400 1400 1400 1400 –
192 39 70 292 22 40–380 10–320 8–75 20 12 28
comparative data. It may be observed that the solute loading on ACFs/ CNFs is relatively larger in most cases and comparable in some cases. 5. Conclusions The multi-scale web of ACFs/CNFs prepared in this study has considerably large BET surface area and pore volume, potentially suitable to be applied as an adsorbent. The adsorption tests exhibited significant and uniform adsorption of proteins on the ACF/CNF surface. The adsorption capacities of the prepared adsorbent for BSA, GOx and YqeH were experimentally determined to be approximately 192, 39 and 70 mg/g, respectively. The micro-mesoporous ACFs/CNFs follow Type I and II adsorption isotherms. The adsorption data for different proteins were fitted by Langmuir or Freundlich model, signifying mono- or multilayer coverage of the biomolecules. Further, the adsorption was exothermic, as the equilibrium solute loading decreased with increasing temperatures. The adsorption of different biomolecules on ACFs/CNFs under the experimental conditions is attributed to single- or a combination of physico-chemical, hydrophobic and electrostatic mechanisms. The method to prepare ACFs/CNFs is simple, and the prepared adsorbent in this study may be used for separating and purifying pharmaceutical effluents. Acknowledgments The authors acknowledge the support from DBT (New Delhi, India) for providing a research grant. The authors are also thankful to Gun Ei Chemical Industry Co. Ltd., Japan for supplying ACFs. References [1] D.A.R. Mahmoud, W.A. Helmy, J. Appl. Sci. Res. 12 (2009) 2466–2476. [2] F.R.R. Teles, L.P. Fonseca, Mater. Sci. Eng. C 28 (2008) 1530–1543. [3] A.R. Hood, N. Saurakhiya, D. Deva, A. Sharma, N. Verma, Mater. Sci. Eng. C 33 (2013) 4313–4322. [4] M.D. Saikia, N.N. Dutta, React. Funct. Polym. 68 (2008) 33–38. [5] B.V. Twala, B.T. Sewell, J. Jordaan, Enzyme Microb. Technol. 50 (2012) 331–336. [6] A.K. Bajpai, J. Appl. Polym. Sci. 78 (2000) 933–940. [7] J.F. Cabrita, L.M. Abrantes, A.S. Viana, Electrochim. Acta 50 (2005) 2117–2124. [8] C.X. He, J.H. Liu, L.Y. Xie, Q.L. Zhang, C.H. Li, D.Y. Gui, G.Z. Zhang, C. Wu, Langmuir 25 (2009) 13456–13460.
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