Bovine Serum Albumin Adsorption on Gluteraldehyde Cross-Linked

Article pubs.acs.org/jced

Bovine Serum Albumin Adsorption on Gluteraldehyde Cross-Linked Chitosan Hydrogels Subrata Mondal,† Cunben Li,‡ and Kean Wang*,† †

Department of Chemical Engineering, The Petroleum Institute, P. O. Box 2533, Abu Dhabi, United Arab Emirates School of Life Science & Chemical Technology, Ngee Ann Polytechnic, Singapore 599489

‡

ABSTRACT: Chitosan hydrogels were cross-linked with glutaraldehyde and examined for the adsorption of a model protein (bovine serum albumin, BSA) at different pH and cross-linking ratios. It was observed that the cross-linking significantly improved the chemical and structural stability of the hydrogel beads. The adsorption equilibrium was described by the Langmuir isotherm. The optimal adsorption capacity was identified at the cross-linking ratio of 0.2 and a pH of 5.5. The cross-linked chitosan hydrogels as well as BSA adsorbed chitosan hydrogels were characterized, and based on that, the adsorption mechanisms were discussed.

1. INTRODUCTION The protein separation is important for food, pharmaceutical, and environmental industries. Many proteins are negatively charged while chitosan (CS) is positively charged in the mild acidic and near neutral pH conditions, which makes CS and its derivatives excellent adsorbents for these applications. Besides, CS adsorbents possess such advantages as low cost, biodegradability, nontoxicity, and versatility, etc.1−5 Because of its low mechanical strength and poor stability in acidic medium, native CS is often surface modified for a specific application. For example, Xu et al. treated the CS with (NH4)2SO4 solution to preserve amine groups for adsorbing a dye at basic conditions.6 Li et al. grafted cyclodextrin onto CS matrix for the specific separation of milk whey.7 Wang et al. prepared magnetic carboxymethyl CS nanoparticles and achieved a capacity of ∼150 mg·g−1 for adsorbing a model protein (bovine serum albumin, or BSA).8 Zhang et al. fabricated genipin-cross-linked hydrophobic CS microspheres and stearic acid modified CS beads, which achieved BSA adsorption capacities of ∼124.0 mg·g−1 and 131.0 mg·g−1, respectively.9 Yoshida and Kataoka reported the BSA adsorption capacity of ∼165 g·L−1 on a cross-linked wet CS resin.10 However, cross-linking also reduces the adsorption capacity of CS, mainly due to the consumption of the functional groups (NH2/OH) by the cross-linking agent (Figure 1a). The knowledge of this trade-off is critical for developing a CS-based adsorbent. In this study, CS hydrogel beads were prepared by phase inversion method, cross-linked with a popular crosslinker (gluteraldehyde, or GLA) at different mass ratios, structurally characterized, and afterward tested for BSA adsorption at various conditions.

bovine serum albumin (MW, 66.4 kDa; pI, 4.7) and gluteraldehyde were obtained from Fisher Chemicals. All other chemicals were of laboratory reagent grade and used without further purification. 2.2. Preparing CS Hydrogel Beads. CS hydrogel beads were prepared via the phase inversion method.11 Briefly, 2.0 g of CS flakes was dissolved into 100 mL of 1% (v/v) acetic acid solution. The mixture was stirred overnight, filtered, and pumped through a syringe needle (27G) dropwise into 1 M NaOH solution under stirring. The spherical gel beads were formed with a diameter ∼ 2 mm. The hydrogel beads were filtered out and rinsed repeatedly with tap water until neutral pH was achieved. The beads were further washed with ethanol and deionized (DI) water. To prepare GLA cross-linked beads (denoted as CSX), about 0.4 g of freeze-dried CS beads were added to a flask with 50 mL of DI water and a specific amount of GLA (Table 1). The flask was shaken for 6 h at 50 °C in an incubator. The CSX were filtered and washed with excessive DI water to remove the residues. 2.3. Adsorption Experiments. BSA adsorption was conducted in batch mode to measure adsorption equilibrium on CS/CSX beads. Each experiment was done in triplicate under the same conditions. About 0.2 g of sample adsorbent (W) was added into a flask containing BSA solution with predetermined volume (V), concentration (C0), and pH value. The pH was adjusted by phosphate and acetate buffers. The flasks were shaken at 200 rpm under ambient conditions for 24 h. The supernatants were then extracted and analyzed by UV−vis for the equilibrium concentration (Ce). The adsorbed phase

2. EXPERIMENTAL SECTION 2.1. Materials. Chitosan flakes (90% DD) were acquired from Bioline Co. Ltd., Banglamung, Thailand. A model protein,

Received: March 20, 2015 Accepted: July 13, 2015

© XXXX American Chemical Society

A

DOI: 10.1021/acs.jced.5b00264 J. Chem. Eng. Data XXXX, XXX, XXX−XXX

Journal of Chemical & Engineering Data

Article

Figure 1. (a) Structure of GLA cross-linked CS beads. (b) ATR-FTIR spectra of CS and CSX beads. (c) Thermogravimetric analysis TGA/DTA curves of CS and CSX beads. B



Article

concentration (Qe) was calculated using the following equation: Qe = (C0 − Ce)V/W. Two isotherm models were used to fit the equilibrium data, i.e.: (1) Langmuir equation for monolayer adsorption, qe = KLCe/(1 + αLCe), where KL and αL are the isotherm parameters while KL/αL gives the monolayer saturation capacity (Qm); (2) Freundlich equation for heterogeneous systems, qe = KFCe1/n, where n is the heterogeneity parameter and KF is roughly the adsorption capacity. 2.4. Characterization. The FTIR spectra were measured on a Vertex 70 (wavenumber range, 400−4000 cm−1; resolution,

Table 1. Amounts of GLA Added at the Different CrossLinking Ratios amount of cross-linking agent required

amount of native chitosan beads

amount of DI water used

cross-linking ratio

g

mL

mL

0.2 0.5 0.7 1.0

6 6 6 6

50 50 50 50

0.07233 0.18088 0.25316 0.36166

Figure 2. SEM micrographs of CS beads: (a) the clean surface, (b) the surface after BSA adsorption, and (c) the cross-section after adsorption.

Figure 3. Internal and external surface morphologies of CSX beads. C



Article

at 1653 cm−1 appeared on CSX. The peaks at 1588 and 1371 cm−1 correspond to the bending vibration of N−H (amide II) and the stretching vibration of C−N (amide III), respectively. The peak at 1247 cm−1, which corresponds to (C−N) functionality, disappeared after cross-linking probably as a result of the bonding interaction of the GLA and (C−N) groups.3 All of these changes signified the successful cross-linking of CS with GLA. Figure 1c shows the TGA results of CS (solid line) and CSX (dashed line). We can see that both samples showed weight loss at approximately three stages. The first stage of weight loss (at ∼100 °C) is mainly due to the physically adsorbed volatile chemicals and water. The second stage (at ∼225 °C) is due to the degradation of native CS,2 which includes dehydration of saccharide rings, depolymerization, and the decomposition of acetylated and deacetylated units. The third stage of degradation (>300 °C) is due to the further destruction of pyranose rings. In the first stage, CSX is seen to have more weight loss due to the desorption of physically adsorbed water. CSX contains less free amine and hydroxyl groups and presents a more robust porous structure which is less susceptible to swelling. As a result, a large fraction of water molecules are physically adsorbed and easy to remove around the boiling point of water. Hence, an earlier weight loss is observed on CSX. In comparison, native CS adsorbed more water molecules via hydrogen bonding.12 In the second stage, where the weight loss is mainly due to the degradation of crystalline domain, the two samples are close to each other but CS showed a more drastic change than CSX. In the third stage, CSX presents a remarkably better thermal stability until ∼600 °C; this is attributed to the cross-linking between CS and GLA. These results further confirmed that the cross-linking can significantly enhance the stabilities of CS. Figure 2 shows the SEM images of CS before and after BSA adsorption (at pH 5.5 and after 48 h). It was observed that (1) freeze-drying did not cause the collapse of the porous structure

Figure 4. BSA isotherms on CS at different pH values and the fittings of Langmuir (solid line) and Freundich (dashed line) equations.

Table 2. BSA Isotherm Parameters on CS Freundlich model

Langmuir model pH value Qm/(mg·(g of wet beads)−1) 6.0 6.5 6.9 8.0

134.4 44.3 30.2 39.7

R2

KF

n

R2

0.9768 0.9843 0.9797 0.9643

40.6 46.1 11.5 14.0

1.78 2.09 1.74 1.97

0.9757 0.9127 0.9856 0.8909

4 cm−1) at ATR-FTIR mode. Thermogravimetry analysis (TGA) experiments were conducted with 15 mg of sample in N2 stream using a heating rate of 10 °C/min from 25 to 800 °C. The samples were coated with platinum prior to the scanning electron microscope (SEM) analysis.

3. RESULTS AND DISCUSSION 3.1. Characterization of CS/CSX Beads. Figure 1b compared IR characteristic peaks of CS and CSX. We can see that the peak at 1588 cm−1 disappeared on CS while the new peak

Figure 5. BSA isotherms on CSX at different pH and the fittings of the Langmuir model. D



Article

Table 3. BSA Isotherm Data on CSX cross-linked ratio 0.20 0.50 0.70 1.00

0.20 5.00 0.70 1.00

0.20 0.50 0.70 1.00

0.20 0.50 0.70 1.00

concentration Ce/ppm Qe/(mg·g−1) Ce/ppm Qe/(mg·g−1) Ce/ppm Qe/(mg·g−1) Ce/ppm Qe/(mg·g−1)

19.14 1.17 23.42 0.31 22.00 0.60 23.43 0.31

32.00 3.60 39.14 2.17 43.43 1.31 46.29 0.74

pH 4.00 93.43 6.31 102.00 4.60 82.00 3.60 112.00 2.60

Ce/ppm Qe/(mg·g−1) Ce/ppm Qe/(mg·g−1) Ce/ppm Qe/(mg·g−1) Ce/ppm Qe/(mg·g−1)

12.00 2.60 12.00 2.60 13.43 2.31 14.86 2.03

13.43 7.31 14.86 7.03 17.71 6.46 20.57 5.89

36.29 12.74 34.85 13.03 39.14 12.17 46.29 10.74


6.28 1.49 3.43 4.31 3.43 4.31 12.00 2.60

9.14 6.34 7.71 8.45 7.71 8.46 19.14 6.17

13.43 14.63 20.57 15.88 23.43 15.31 36.29 12.74


14.86 2.03 19.14 1.17 17.71 1.46 23.43 0.31

17.71 6.46 23.42 5.31 22.00 5.60 27.71 4.46

54.86 14.03 60.57 12.89 40.57 11.86 76.29 9.74

196.29 10.74 200.57 9.90 103.42 4.31 222.00 5.6 pH 5.00 39.14 17.17 43.43 16.31 50.57 14.89 54.86 14.03 pH 5.50 34.86 26.05 32.00 18.60 36.29 17.74 49.14 15.17 pH 6.00 132.00 23.60 137.71 22.46 57.71 13.46 153.43 19.31

413.43 17.31 423.43 15.31 210.57 7.90 447.71 10.45

637.71 22.46 654.85 19.03 437.71 12.45 687.71 12.46

679.14 24.17 706.29 18.75 669.14 16.17 734.85 13.03

873.43 25.31 899.14 20.17 913.43 17.31 930.57 13.90

114.86 27.03 114.86 27.03 122.00 25.60 136.29 22.74

277.71 44.46 294.86 41.03 309.14 38.17 334.86 33.03

472.00 55.60 500.57 49.89 522.00 45.60 550.57 39.89

704.86 59.03 734.85 53.03 757.71 48.46 794.86 41.03

42.00 33.20 93.43 31.31 94.85 31.03 133.43 23.31

112.00 55.20 250.57 49.89 274.86 45.03 314.86 37.03

312.00 75.20 473.43 55.31 494.86 51.03 536.29 42.74

783.43 86.63 686.29 62.74 710.57 57.86 780.57 43.89

314.86 37.03 319.14 36.17 137.71 22.46 362.00 27.60

532.00 43.60 537.71 42.46 316.29 36.74 572.00 35.60

577.71 44.46 589.14 42.17 550.57 39.89 620.57 35.89

743.43 51.31 762.00 47.60 766.29 46.74 814.86 37.03

the fittings by Langmuir (solid line) and Freundich (dashed line) isotherm equations, respectively. The fitting results are listed in Table 2. It is seen that Langmuir isotherm fits the data better in general, confirming the monolayer adsorption of BSA on the homogeneous surface of the CS.13 In comparison, the derived parameter (n) of the Frendluich isotherm fluctuated from 1.7 to 2.1 without a clear trend, which suggests that the CS surface is not heterogeneous. Figure 5 shows the BSA isotherm (symbols) on CSX with different cross-linking ratios and at various pH values. The isotherm data were fitted to Langmuir isotherm (lines) and listed in Table 3. We can see that, compared with Figure 4, BSA capacity was decreased significantly with the increasing crosslinking ratio, due to the occupation of amine groups by the cross-linker. Moreover, the lower porosity of the exterior surface of CSX deters BSA molecules from diffusing into the internal porous networks. The pKa of CS is around 6.5. Therefore, more amine groups will be protonated when pH is below this value, which offers more binding sites for BSA. In addition, BSA has an isoelectric point (pI) of 4.7 and is adsorbed mainly via the electrostatic interactions when pH > 4.7.14 Therefore, 4.7 < pH < 6.0 is favorable for BSA adsorption on CS. However, the native CS

of CS (Figure 2a), (2) the external surface became smoother after adsorption (Figure 2b), and (3) the cross-section was largely porous with interconnected pores, unaffected by the adsorbed phase (Figure 2c). The surface characteristics of the hydrogel beads were investigated previously by measuring the standard N2 adsorption isotherm (at 77K) on the freeze-dried hydrogel samples.6,7 It was revealed that the BET surface area ranges from ∼35 m2·g−1 on the cross-linked beads7 to ∼53 m2·/g−1 on the native beads.6 The average pore size is ∼1.3 nm, and the porosity is ∼75% in water.6 Because chitosan matrix swells significantly in aqueous solution, these properties are for general information only. Figure 3 shows the SEM images of the cross-section (panels a−c) and external surface (panels d−f) of CSX with different cross-linking ratios. It can be seen that, in general, (1) the external surface is smoother/less porous than the internal surface and (2) as the mass ratio of GLA increases, both surfaces become smoother/less porous. These results are in line with the expectations that more cross-linking reactions occurred on the external surface of adsorbents. 3.2. BSA Adsorption. Adsorption Equilibrium. Figure 4 shows BSA isotherm data (symbols) on native CS at different pH values from 6.0 to 8.0. Also shown in the same figure are E



Article

beads will be swollen and disintegrated at pH < 5.5. The GLA cross-linking offers chemical stability but with the sacrifice of adsorption capacity. Therefore, an optimal cross-linking ratio exists and should be identified for BSA adsorption on CS. Figure 6 shows the 3D plot of BSA capacity vs pH and crosslinking ratio. The maximum capacity (96.4 mg·g−1) is found at

There are some notable differences between IR spectra of CS and BSA-CS; such as a new shoulder peak appeared for BSA-CS at 1644 cm−1, and N−H bending vibration of the secondary amide shifted to the lower wavenumber from 1587 to 1566 cm−1 (with the increased peak intensity). An increase in absorbance at 1644 cm−1 may indicate that carboxyl groups of CS were also involved in the adsorption process. In addition, the OH and N−H stretching at 3367 cm−1 has been sharpened and changed which may be attributed to the deformation of O−H and N−H bands due to the interaction between the functional groups of chitosan and BSA molecules.16 All of these changes evidenced that both amine and hydroxyl groups were involved in BSA adsorption.5

4. CONCLUSIONS The GLA-cross-linked chitosan hydrogel beads present good chemical stability and structural stability in acidic conditions and reached an optimal BSA capacity of ∼96 mg·/−1g at a mass cross-linking ratio of 0.2 and a pH of 5.5. The BSA adsorption was found to be monolayer and chemical adsorption in nature, which can be well described by the Langmuir isotherm.

■

Figure 6. BSA adsorption capacity with respect to pH and crosslinking ratio.

AUTHOR INFORMATION

Corresponding Author

pH ∼ 5.5 and a cross-linking ratio of 0.2. Compared with CS, this capacity is lower than that at pH of 6.0 (134 mg·g−1), but is much higher than that at pH of 6.5 (44.3 mg·g−1). In addition to the chemical stability, the robust porous structure of CSX can accommodate more physically adsorbed BSA molecules, which is in favor of BSA adsorption at low pH. When pH is further reduced to

Bovine Serum Albumin Adsorption on Gluteraldehyde Cross-Linked

Recommend Documents