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Functionalized anodic aluminum oxide (AAO) membranes for affinity protein separation
Journal of Membrane Science 325 (2008) 801–808
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Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
Functionalized anodic aluminum oxide (AAO) membranes for affinity protein separation Wei Shi a,∗ , Yuqing Shen a , Dongtao Ge a,∗ , Maoqiang Xue b , Huihui Cao a , Sanqing Huang a , Jixiao Wang c , Guoliang Zhang c , Fengbao Zhang c a Department of Biomaterials/Biomedical Engineering Research Center, College of Materials, Xiamen University, Xiamen 361005, China b Department of Preclinical Medicine, Medical College, Xiamen University, Xiamen 361005, China c School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, China
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Article history: Received 11 June 2008 Received in revised form 1 September 2008 Accepted 3 September 2008 Available online 10 September 2008 Keywords: Affinity membrane AAO Chitosan Adsorption Uniformity membrane
a b s t r a c t An ideal affinity membrane should own well uniformities. However, most existing microporous membranes used as affinity matrices generally have wide pore size distribution and some thickness variation. In this paper, chitosan (CS)–anodic aluminum oxide (AAO) composite membrane with excellent uniformities, such as narrow pore size and porosity distribution, as well as uniform membrane thickness, was fabricated, for the first time. Cu2+ -attached affinity membrane was obtained by immobilizing Cu2+ on the CS–AAO membrane. The contents of CS and Cu2+ of affinity membranes were ∼49.7 and 27.15 mg/g membrane, respectively. The Cu2+ -attached affinity membranes were used to recover a model protein, hemoglobin, from hemoglobin–phosphate solution (batch manner) and from the hemolysate (dynamic manner). The protein adsorption indicated that the adsorption capacity of hemoglobin was ∼17.5 mg/g membrane, and the adsorption isotherm fitted the Freundlich model well. Elution of protein showed desorption ratio was up to 91.2% using 0.5 M imidazole aqueous solution as the desorption agent. The adsorption capacities of all the tested affinity membranes did not significantly change during the repeated adsorption–desorption operations. The result of dynamic experiment showed Cu2+ -attached affinity membranes can well purify the hemoglobin from the red cell lysate. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The rapid development of biotechnology and biomedicine requires more reliable and efficient separation techniques for the isolation and purification of biomolecules such as proteins, nucleic acids and hormones [1]. Affinity chromatography is ideal for achieving the very high selectivity needed for the purification of proteins and pharmaceuticals. As a new technology in affinity separation, membrane affinity chromatography possesses shorter diffusion time than those obtained in conventional affinity chromatography, because the interactions between molecules and active sites on the membrane occur in convective through pores, rather than in stagnant fluid inside the pores of an adsorbent particle [2,3]. For this reason, affinity membrane chromatography has the potential to maintain high efficiencies both at high flow rates and for the use of large biomolecules with small diffusivities [4,5].
∗ Corresponding authors. Tel.: +86 592 2185299; fax: +86 592 2185299. E-mail addresses: [email protected] (W. Shi), [email protected] (D. Ge). 0376-7388/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2008.09.003
One of the most important factors in membrane affinity chromatography is the identification of suitable membranes [6]. To date, most membranes for affinity separation were made from organic materials. In general, organic materials can be roughly divided into two categories, namely, natural and synthetic polymers [7,8]. The natural macromolecular materials, such as agarose, dextrin, and chitosan (CS) are good affinity matrices because they are compatible with the usual ligands, but are often difficult to process as membranes [9,10]. In contrast, synthetic polymers such as nylon [11,12], polysulfone [13,14] and glycidyl methacrylate [9] are apt to be manufactured into membranes, however, are less suitable for the immobilization of the ligand due to their low compatibility and low concentration of actively functional groups in their structure. Coating of these membranes with natural macromolecules is an alternative for depressing non-specific binding of proteins and heightening capacities for affinity ligands [6,12]. Based on our and other theoretical analysis [15–18], besides enough reactive groups and good compatibility, an ideal affinity membrane should own well uniformities, such as narrow pore size and porosity distribution, as well as uniform membrane thickness. Most polymeric microporous membranes used as affinity matri-
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Fig. 1. Reaction scheme for preparation of the CS–AAO composite membrane. (A) AAO membrane was activated by GPTMS; (B) CS was coupled on the activated membranes.
ces were manufactured by phase-inversion method. Membranes produced from this method generally have wide pore size distribution and some thickness variation. These nonuniformites may severely degrade the performance of affinity membranes [15,17,18]. Compared with organic membranes, porous alumina membrane (AAO membrane) as a typical inorganic membrane possesses superuniform structure, such as straight and parallel ordered pore channels, homogeneous pore size distribution, as well as uniform membrane thickness [19]. Moreover, AAO membrane owns better mechanical strength, reusability and resistance to microbial degradation, heat and chemical attack [20]. Therefore, AAO membrane is an interesting candidate for the affinity chromatography applications. However, few publications reported the use of AAO membrane as affinity matrix. Recently, Sun et al. [21] used atom transfer radical polymerization to grow polymer brushes in AAO membrane to modify this inorganic matrix for affinity separation protein. Although membrane pores were not clogged, the pore size of modified AAO membrane was reduced evidently; as a result, the mass transfer resistance should be increased significantly. In this paper, CS–AAO composite membrane that holds the ideal pore structure of AAO membrane was fabricated and used as affinity matrix, for the first time. AAO membrane was activated with 3-glycidoxypropyltrimethoxysilane (GPTMS), and then CS was coupled on the activated membrane to increase reactive sites. Cu2+ as ligand was immobilized onto the CS–AAO composite membrane. The applicability of Cu2+ -chelated CS–AAO composite membrane for affinity purification hemoglobin was presented and discussed in detail. 2. Experimental 2.1. Chemicals and apparatus AAO membranes (60 m thickness, 0.2 m pore size and 47 mm diameter) were purchased from Whatman. GPTMS was obtained from Danyang Organosilane Material Chemical Company (China). Chitosan (CS, average molecular weight was about 3,00,000 and the degree of deacetylation was over 90%) was purchased from Zhejiang Aoxing Biotechnology Company (China). Fresh bovine blood was a gift from the Huangjinxiang Company (China). Bovine hemoglobin (Hb) and Tris-(hydroxymethyl)aminomethane were obtained from Sigma. Ninhydrin H2 O, imidazole and Coomassie brilliant blue R250 were the products of Sinopharm Chemical Reagent Company (China). Sodium dodecyl sulfate (SDS), glycine and EDTA disodium salt were obtained from BBI. Prestained protein ladder (11–170 kDa) was purchased from Fermentas. All other chemicals were of analytical grade and used without further purification. All solutions were prepared using deionized Milli-Q water (Millipore). The concentrations of Hb, Cu2+ , and CS were determined using a UV-752 spectrophotometer (Shanghai Instrument Co. Ltd., China).
Refrigerated Centrifuge (Beckman Avanti J-25, USA) was used for the preparation of hemolysate. The membrane cartridge (donated amicably from the Dalian Chemical and Physical Institute, China) was used to load the membrane stack. A peristaltic pump (Model BT-100, Shanghai, China) was used for the feeding of hemolysate solutions. The morphologies of the membranes were visualized with the field-emission scanning electron microscope (FE-SEM) (LEO, Germany). 2.2. Preparation of affinity membrane 2.2.1. Activation of AAO membrane Ten AAO membrane disks were immersed in a solution of 5 ml GPTMS–30 ml ethanol–2 ml sodium acetate buffer solution (50 mM, pH 5.0). The polytetrafluoroethylene device containing the above solution was vacuumized for 5 min to remove air from the pores of the membranes. The membranes remained in this solution for another 15 min under ambient pressure before being rinsed with ethanol. The membranes were then cured by heating in vacuum at 100 ◦ C for 1 h. 2.2.2. Preparation of CS–AAO composite membrane CS was dissolved in 5 wt% acetic acid aqueous solution to form 2 wt% CS solution. The solution was stirred at room temperature for 24 h, and then was filtered to remove any indissolvable solids and impurities. The activated membranes were shaken in 10 ml prepared CS solution for 1 h at room temperature. The membrane disc was placed onto a sintered glass filter holder (Autoscience) and the remaining CS solution was sucked slowly through the membrane disc by reducing pressure until no further drop was formed at the filtrate side. This was followed by drying the wetted membrane in an oven at 85 ◦ C for 45 min. Non-covalently bound CS was removed by washing the membranes with 1 vol.% acetic acid solution and deionized water. Reaction scheme for the preparation of CS-coated AAO membrane is shown in Fig. 1. The amount of the CS coupled onto the AAO membranes was determined by the ninhydrin method [6]. Briefly, the ninhydrin reagent was prepared by dissolving 70 mg SnCl2 and 500 mg ninhydrin into a mixture of 15 ml DMSO and 2 ml of 2 M sodium acetate. 40 mg dry CS–AAO composite membrane pieces were added to a mixture of 1.0 ml of pure water and 2.0 ml ninhydrin reagent. The mixture was incubated in a boiling water bath for 30 min and then cooled to room temperature. A n-propanol solution (7.0 ml, 50% (v/v) in pure water) was added to the mixture. The optical density of the resulting solution was determined at 570 nm wavelength against a reference which was obtained from the AAO membrane. The standard curve was created using 1–6 mg solid CS.
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Fig. 2. FE-SEM photos of two frontal surfaces of AAO membrane and CS–AAO composite membrane. (A and B) Original alumina membrane; (C and D) CS–AAO composite membranes.
2.2.3. Immobilization of Cu2+ CS is a well-known adsorbent for metal ions, such as Cu2+ , 2+ Ni , Cr2+ , Zn2+ and Hg2+ , because the amine group (–NH2 ) and the hydroxyl group (–OH) can serve as coordinate sites for metals. The porous CS–AAO composite membranes were kept in a solution of CuSO4 (0.05 M) and shaken overnight at room temperature [22]. After rinsing with distilled water and drying, Cu2+ -attached affinity membranes were obtained. The amount of Cu2+ immobilized on membranes was calculated from the difference in pre- and post-reaction concentration of Cu2+ . Cu2+ ions were analyzed in the supernatant as described below. A 9 ml EDTA solution (0.05 M) was added to 1 ml of solution containing Cu2+ and the absorbance of Cu–EDTA complex was measured directly by UV–vis spectrophotometry at 740 nm [23]. 2.3. Cu2+ ions leakage from the affinity membrane The effects of metal ion leakage and resulted toxicity during adsorption and elution are also significant issues to be evaluated [24,25]. The leakage of Cu2+ ions from the affinity membrane was determined in the media of 10 mM phosphates buffer solution (pH 6.8 and 7.8) and 0.5 M imidazole aqueous solution. Membranes were incubated in above media with shaking at room temperature for 10 days. The media were renewed every day, and the amount of the Cu2+ release into the medium was measured cumulatively by UV–vis spectrophotometry at 740 nm.
were mixed with 5 ml of various concentrations of Hb solution (40–450 mg/l). The mixture was shaken at 25 ◦ C for 8 h, which proved to be a sufficient period to reach adsorption equilibrium. The amount of Hb adsorbed was determined by the following equation: Q =
(C0 − Ct )V m
(1)
where Q is the amount of Hb adsorbed on unit mass of the membranes (mg/g); C0 and Ct are the initial and final concentrations of the Hb solution, respectively (mg/l); V is the volume of the Hb solution (l); and m is the mass of the membranes (g). The concentration of the Hb solution was detected by UV–vis spectrophotometry at 405 nm. 2.5. Desorption and regeneration Hb desorption experiments were performed in a 0.5 M imidazole aqueous solution. Hb adsorbed membranes were placed in the desorption medium and shaken for 5 h at room temperature, followed by the procedure of washing with large volume of distilled water and phosphate buffer (pH 6.8). The final Hb concentration within the desorption medium was determined by spectrophotometry. The desorption ratio was calculated from the amount of Hb adsorbed on the membranes and the amount of Hb desorbed. Hb adsorption–desorption operation was done five times using the same adsorbent for checking the reusability of the membranes. 2.6. Dynamic adsorption of Hb from hemolysate
2.4. Batch adsorption experiments The Cu2+ -attached affinity membranes were tested for the adsorption of Hb in 10 mM phosphate buffer (pH 6.8) by batch experiment. Approximately 40 mg of affinity membranes
2.6.1. Preparation of hemolysate Hemolysate was prepared as described by Stocker-Majda et al. [26]. Briefly, fresh bovine blood (ca. 500 ml) was centrifuged at 2000 rpm/min and 4 ◦ C for 10 min, afterwards the plasma was
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removed by aspiration. The remaining red blood cells (ca. 250 ml) were washed five times with three volumes of 0.9% NaCl. The cells were lysed by osmotic shock at 4 ◦ C. Hemolysate after cell lysis was pretreated by centrifugation (2000 rpm/min, 4 ◦ C, 30 min) to remove cell debris. The resultant hemolysate was adjusted with 10 mM phosphate buffer, pH 6.8, to a Hb concentration of 200 mg/l.
2.6.2. Dynamic adsorption experiment The dynamic experiments were carried out in the cartridge to investigate the breakthrough performance. Ten sheets of affinity membrane with a diameter of 47 mm were set into a stack. The stack was first equilibrated with the equilibrium buffer. Then hemolysate was impelled by peristaltic pump with two different flow rate (i.e., 2 and 4 ml/min) to flow through the membrane stack. The concentration of Hb in the feed was kept at 200 mg/l. The amounts of Hb through the membrane cartridge were measured in succession with a spectrophotometer. After adsorption the stack was washed with 0.5 M imidazole aqueous solution until A405 was <0.02.
2.6.3. SDS-PAGE electrophoresis The purity of Hb was assayed by sodium dodecylsulfate– polyacrylamide gel electrophoresis (SDS-PAGE) using 15% separating gel and 5% stacking gel. Gels were stained with 0.25% (w/v) Coomassie brilliant R250 in methanol–acetic acid–water (4.5:1:4.5, v/v/v) and destained in methanol–acetic acid–water (4.5:1:4.5, v/v/v). Electrophoresis was run for 1.5 h with a voltage of 120 V. Bovine hemoglobin (Sigma) was used as a standard.
3. Results and discussion 3.1. Characteristics of the membranes The morphologies of AAO membrane and CS–AAO composite membrane were observed by FE-SEM and were illustrated in Fig. 2. The AAO membrane is composed of a high-purity alumina matrix that is manufactured electrochemically. This unique material has a precise, non-deformable honeycomb pore structure (Fig. 2A and B) with no lateral cross over between individual pores. After coating CS onto the AAO membrane, the pore size of the membrane has not apparently changed (Fig. 2C and D). Moreover, the CS–AAO composite membrane also exhibited homogeneous surfaces. The precise pore structure and narrow pore size distribution of the CS–AAO composite membrane can significantly reduce diffusion resistance, possess high flow rates, and ensure a high level of affinity adsorption performance in purification of bioproducts. The CS content of CS–AAO membrane was ∼49.7 mg/g membrane. Since there are plentiful reactive groups (–NH2 and –OH) on the CS molecule, the compatibility with the usual ligands was greatly enhanced by coating CS on the AAO membrane. The energydispersive X-ray spectroscopy (EDS) data (Fig. 3) showed there was Cu element on the affinity membranes, which verified that Cu2+ was immobilized on the CS–AAO composite membrane. The amount of Cu2+ immobilized on affinity membrane can be determined spectrophotometrically at 740 nm, and the Cu2+ content was ∼27.15 mg/g membrane. The leakage of Cu2+ in the media showed that there was nearly no Cu2+ release in the 10 mM phosphates buffer solution (pH 6.8 and 7.8) and 0.5 M imidazole aqueous solution. This result suggested that Cu2+ ions were strongly chelated on the CS–AAO composite membrane. Some of the characteristics of the Cu2+ -attached affinity membrane were listed in Table 1.
Fig. 3. EDS image of Cu2+ -attached affinity membranes.
Table 1 The characteristics of Cu2+ -attached affinity membrane Items
Property
Membrane material Average pore size of the membrane (m) Thickness of membrane (m) CS density (mg/g membrane) Cu2+ content (mg/g membrane)
AAO, CS and Cu2+ 0.2 60 49.7 27.15
3.2. Adsorption rate The adsorption rate curves of Hb are exemplified in Fig. 4. There was relatively faster adsorption rates observed at the beginning of the adsorption process, and then the adsorption equilibrium was achieved gradually for about 6.5 h. This figure also shows the nonspecific and specific adsorption of Hb onto the AAO, CS–AAO and Cu2+ -attached membranes. Although Whatman Company alleged that AAO membrane (Anodisc® ) exhibited low protein binding, AAO membrane owns high non-specific adsorption capacity for Hb, ∼11.8 mg/g membrane. The AAO membrane is composed of a high-purity alumina matrix that is manufactured electrochemically. During its preparation, the oxide layer that was derived by the anodizing technique would be coated on aluminium substrates. When the AAO membrane is directly in contact with the
Fig. 4. Adsorption rates of Hb on the membranes. Cu2+ content of affinity membrane: 27.15 mg/g membrane; Hb concentration: 200 mg/l; temperature: 25 ◦ C; medium: phosphate buffer (pH 6.8, 10 mM); Hb solution volume: 5 ml; membrane weight: 0.040 g.
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2+
Fig. 5. Freundlich adsorption isotherm of Hb on Cu -attached affinity membrane. Cu2+ content of affinity membrane: 27.15 mg/g membrane; temperature: 25 ◦ C; medium: phosphate buffer (pH 6.8, 10 mM); Hb solution volume: 5 ml; membrane weight: 0.040 g; adsorption time: 8 h.
protein solution, the protein molecules are apt to be adsorbed on the oxide layer of the AAO membrane [27]. After coating CS onto the AAO membrane, the non-specific Hb adsorption of the membrane reduced significantly, and the amount was about 1.95 mg/g membrane only. While much higher specific adsorption capacity, up to 17.5 mg/g membrane was obtained after ligand (Cu2+ ) immobilization. 3.3. Analysis of the adsorption mechanism Freundlich adsorption isotherms were applied for the description of the adsorption mechanism for Hb on Cu2+ -attached membrane. The isotherms can be described as follows: q = KC 1/n
(2)
where q is the adsorption capacity of Hb on unit mass of the affinity membrane at equilibrium (mg/g membrane); C is the equilibrium concentration of Hb (mg/l); n and K are the physical constants of the Freundlich adsorption isotherm. Eq. (2) can be transformed as follows: log q = log K +
1 log C n
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Fig. 6. Effects of temperature on the Hb adsorption capacities of Cu2+ -attached affinity membrane. Cu2+ content of affinity membrane: 27.15 mg/g membrane; Hb concentration: 200 mg/l; medium: phosphate buffer (pH 6.8, 10 mM); Hb solution volume: 5 ml; membrane weight: 0.040 g; adsorption time: 8 h.
0.125 and 1, respectively. This result further demonstrated that the affinity CS–AAO membrane owns excellent uniformities. 3.4. Effect of temperature The effect of temperature was studied under 4, 25 and 37 ◦ C. As shown in Fig. 6, the adsorption capacity increased with increasing temperature. The hypothesis for these phenomena is that the adsorption was endothermic, and higher temperature was favored for the adsorption. The result was similar to other related experiments [28]. The Hb adsorption was ∼17.9 mg/g membrane, which was obtained at 37 ◦ C. However, the effect of temperature on Hb adsorption is not notable. For example, the adsorption capacity of Hb increases by ∼2.29%, when the temperature changes from 25 to 37 ◦ C. 3.5. Effects of ionic strength The effect of the ionic strength on Hb adsorption was presented in Fig. 7. As seen in the figure, the adsorption capacity decreased with increasing the concentration of NaCl in the binding buffer (10 mM, phosphate, pH 6.8). When the added concentration of NaCl
(3)
Although the Freundlich equation was proposed originally as an empirical equation, it can be derived, however, from a sound theoretical footing. It is obtained by assuming that all surfaces are not really smooth at a microscopic level. The use of the Freundlich isotherm to characterize affinity adsorption has a number of advantages [28]. First, the Freundlich isotherm is based on a heterogeneous exponentially decaying distribution, which fits well to the tailing portion of the heterogeneous distribution of affinity membrane. In addition, the logarithmic form of the Freundlich isotherm is easily applied as it can be transformed into a linear function (Eq. (3)). This analysis is a useful diagnostic for identifying sources of error, as deviations from linearity are visually evident. Finally, the slope of the straight-line fit yields n, which is a measure of the heterogeneity of a system. A more homogeneous system will have an n value approaching unity. Fig. 5 shows the linear relationship of the Freundlich isotherm for the adsorption of Hb with Cu2+ -attached affinity membrane. The correlation coefficient (r) of the isotherm was 1, indicating that the data fit the model very well. The values of K and n for our adsorption system were found from the straight-line plot of log q versus log C by linear regression, and were
Fig. 7. Effect of NaCl concentration on the Hb adsorption capacities of Cu2+ -attached membrane. Cu2+ content of affinity membrane: 27.15 mg/g membrane; Hb concentration: 200 mg/l; temperature: 25 ◦ C; medium: phosphate buffer (pH 6.8, 10 mM); Hb solution volume: 5 ml; membrane weight: 0.040 g; adsorption time: 8 h.
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Fig. 8. Effect of pH on Hb adsorption capacities on Cu2+ -attached affinity membrane. Cu2+ content of affinity membrane: 27.15 mg/g membrane; Hb concentration: 200 mg/l; temperature: 25 ◦ C; Hb solution volume: 5 ml; membrane weight: 0.040 g; adsorption time: 8 h.
changes from 0 to 0.8 M, the adsorption capacity of Hb was ∼18.42% decrease. The electrostatic interaction between protein molecules and metal-chelated affinity system could be reduced in the presence of salt because the surface charges of protein are screened [29]. An increase in ionic strength also causes a change in the interaction mechanism due to more contributions from hydrophobic and protein–protein interactions [30]. More ions may also be attached to Hb molecules at high ionic strengths. This causes further stabilization of the protein molecules (higher solubility), which may lead to lower adsorption of Hb on the affinity membrane. In addition, the protein is expected to become more flexible with increasing electrolyte concentration because the intra-electrostatic interactions become less important [31,32]. For this reason, the protein molecule may lose the ability to expose its histidine residue to the affinity membranes, which is the dominant adsorption site in protein binding with an attached metal ion. 3.6. Effects of pH The principle of immobilized metal ion chelating affinity chromatography (IMAC) suggested that the adsorption of protein to the adsorbent was governed by the coordination of metals with electron-donor ligands exposed on the surface of the proteins [33,34]. The medium pH is very important parameter which determined the adsorption capacity of the affinity membrane to the target protein in the biological fluids. The adsorption medium pH affects the net charge on the immobilized metal ions as well as on the protein molecule in the adsorption medium. In this study, the effect of pH of the equilibration buffer on the adsorption capacity of Hb on Cu2+ -attached affinity membrane was investigated and the obtained results were shown in Fig. 8. It indicated the pH dependence of the amount of Hb adsorbed on the Cu2+ -attached affinity membranes. In all the cases investigated, the maximum adsorption of Hb was observed at pH 6.8. Significantly lower adsorption capacities were obtained with all affinity membranes in more acidic and in more alkaline pH regions. It has been shown that proteins have no net charge at their isoelectric points (pI), and therefore the maximum adsorption from aqueous solutions is usually observed at their pI (pI of Hb: 6.8) [35,36]. When pH < pI, the protonation effect on the amino acid residue (histidine) will be increased and hence their coordination ability to immobilized metal ions will be reduced [37,38]. These could explain that the adsorption capacity decreased from pH 6.8 to 4.8. At an alkaline pH, coordination with
Fig. 9. Breakthrough curves at different feed-rates. Separation chromatogram of Hb from hemolysate on a stack of 10 membranes with 47 mm diameter.
amino and hydroxyl groups took place and resulted in less effective adsorption of Hb [39]. 3.7. Regeneration of the membranes Regeneration is a crucial step in all affinity chromatography techniques. It was thus necessary to evaluate the regeneration efficiency of the affinity adsorbents after each cycle. Histidine units bind to chelated divalent metal ions in the unprotonated state, and then the adsorbed proteins can be eluted by either lowering the pH or introducing a high concentration of free imidazole [40]. Desorption of Hb from the affinity membranes was carried out in batch system. For all the tested Cu2+ -attached affinity membranes up to 91.2% of the adsorbed Hb was desorbed by using 0.5 M imidazole aqueous solution. In order to show the reusability of the Cu2+ -attached affinity membranes, the adsorption–elution cycle was repeated five times using the same affinity membranes. There was no remarkable reduce in the adsorption capacity of the membranes. The Hb adsorption capacity decreased only 6% after five cycles. Moreover, no obvious changes of the morphology of the affinity membranes were found in the recycling process. These results demonstrated the stability of the present CS–AAO membranes as a metal-chelated affinity adsorbent. 3.8. Dynamic separation of Hb from hemolysate Hb is found in red blood cells and responsible for oxygen transfer from the lung to the body tissues as well as the return transport of carbon dioxide and protons. To investigate the selectivity and dynamic behavior of Cu2+ -attached affinity membrane, red cell lysate (hemolysate) was loaded onto the affinity stack of 10 membranes with 47 mm diameter in a single-pass mode at constant flow rate. Fig. 9 presents the ratio between permeate and feed concentrations C/C0 as function of the permeate time. As shown in this figure, higher velocity of the hemolysate makes out a rapid breakthrough and the lower one shows a laggard penetration. It also shows a high rate for Hb adsorption in dynamic experiments. Because the mass transfer in dynamic experiment is convective mode, the resistance is greatly reduced. As a result, the operation for Hb adsorption could be speeded up. The key performance criteria for affinity membrane processes are breakthrough curve sharpness at the adsorption stage [41]. In fact, not only the feed rate of solute and ligand content but also the nonuniformites in membrane thickness and pore size may have significant effects on the breakthrough curve sharpness [15]. The uniform affinity membrane can enhance solute recovery
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branes could be described with Freundlich adsorption isotherms. The adsorbed Hb on the affinity membranes was desorbed up to 91.2% with 0.5 M imidazole aqueous solution as the desorption agent. It was possible to reuse these metal-chelated affinity membranes without significant reduction in the protein adsorption capacities. The result of dynamic adsorption showed Cu2+ -attached affinity membranes have been successfully used for the separation and purification of Hb from the red cell lysate. These features make the CS–AAO composite membranes very good candidate for use in metal-chelated affinity separation of proteins and would be effective in processing large volume of biological fluid containing a target protein. It is important to emphasize that many ligands can be easily immobilized on the CS–AAO composite membrane by activating the –NH2 and/or –OH groups of the CS, therefore we expect for a wide range of affinity systems could be considered applying this strategy. Acknowledgments We are extremely grateful to the National Nature Science Foundation of China (No. 30500127) and the Natural Science Foundation of Fujian Province (No. C0510005) for supporting this research.
Fig. 10. SDS-PAGE assay of Hb obtained from hemolysate. From left to right: lane A, marker; lane B, purified hemoglobin after affinity chromatography; lane C, hemolysate; lane D, standard bovine hemoglobin (Sigma).
efficiency and ligand utilization efficiency [15]. Due to the excellent uniformites of affinity CS–AAO membranes, both breakthrough curves under two feed rates are sharper, indicating a high affinity purification performance. Using the data presented in Fig. 9 one can calculate the so-called dynamic capacity, i.e., the uptake of the membrane before the exiting feed concentration reaches 10% of the inlet value. The dynamic capacities for the flow rates of 2 and 4 ml/min are 12.8 and 11.6 mg/g membrane, 27% and 34% smaller than the corresponding static adsorption capacity, respectively, because some adsorptive capacity remains unused mainly due to slow sorption kinetics and resistance from mass transport rate processes. Full adsorption capacities can only be achieved if the flow rate through the affinity membrane is slow enough to allow each Hb molecules to diffuse to the adsorptive site and to rearrange and/or unfold their structure to its most favorable state before the interstitial volume continues to move through the membrane. After adsorption the stack was eluted with 0.5 M imidazole aqueous solution, the quality of the purified Hb was assayed by SDS-PAGE electrophoresis. Fig. 10 gives the electrophoresis of Hb before and after purification by membrane chromatography. The purified Hb showed single band in SDS-PAGE, indicating the well selectivity of the prepared affinity membrane for Hb. 4. Conclusions A novel organic–inorganic composite membrane with excellent uniformities, such as narrow pore size and porosity distribution, as well as uniform membrane thickness, was prepared by using AAO membrane as supported matrix. AAO membrane was activated with GPTMS, followed by coupling CS to enhance the amount of active groups of the AAO membrane. The content of CS on the CS–AAO composite membrane was ∼49.7 mg/g membrane. The prepared CS–AAO membrane integrated virtues of organic and inorganic membrane, including plentiful active groups and well uniformities. After chelating with Cu2+ the affinity membranes were used for adsorption of Hb. The adsorption capacity of Hb was ∼17.5 mg/g membrane, and the adsorption behavior of Hb on affinity mem-
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Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
Functionalized anodic aluminum oxide (AAO) membranes for affinity protein separation Wei Shi a,∗ , Yuqing Shen a , Dongtao Ge a,∗ , Maoqiang Xue b , Huihui Cao a , Sanqing Huang a , Jixiao Wang c , Guoliang Zhang c , Fengbao Zhang c a Department of Biomaterials/Biomedical Engineering Research Center, College of Materials, Xiamen University, Xiamen 361005, China b Department of Preclinical Medicine, Medical College, Xiamen University, Xiamen 361005, China c School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, China
a r t i c l e
i n f o
Article history: Received 11 June 2008 Received in revised form 1 September 2008 Accepted 3 September 2008 Available online 10 September 2008 Keywords: Affinity membrane AAO Chitosan Adsorption Uniformity membrane
a b s t r a c t An ideal affinity membrane should own well uniformities. However, most existing microporous membranes used as affinity matrices generally have wide pore size distribution and some thickness variation. In this paper, chitosan (CS)–anodic aluminum oxide (AAO) composite membrane with excellent uniformities, such as narrow pore size and porosity distribution, as well as uniform membrane thickness, was fabricated, for the first time. Cu2+ -attached affinity membrane was obtained by immobilizing Cu2+ on the CS–AAO membrane. The contents of CS and Cu2+ of affinity membranes were ∼49.7 and 27.15 mg/g membrane, respectively. The Cu2+ -attached affinity membranes were used to recover a model protein, hemoglobin, from hemoglobin–phosphate solution (batch manner) and from the hemolysate (dynamic manner). The protein adsorption indicated that the adsorption capacity of hemoglobin was ∼17.5 mg/g membrane, and the adsorption isotherm fitted the Freundlich model well. Elution of protein showed desorption ratio was up to 91.2% using 0.5 M imidazole aqueous solution as the desorption agent. The adsorption capacities of all the tested affinity membranes did not significantly change during the repeated adsorption–desorption operations. The result of dynamic experiment showed Cu2+ -attached affinity membranes can well purify the hemoglobin from the red cell lysate. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The rapid development of biotechnology and biomedicine requires more reliable and efficient separation techniques for the isolation and purification of biomolecules such as proteins, nucleic acids and hormones [1]. Affinity chromatography is ideal for achieving the very high selectivity needed for the purification of proteins and pharmaceuticals. As a new technology in affinity separation, membrane affinity chromatography possesses shorter diffusion time than those obtained in conventional affinity chromatography, because the interactions between molecules and active sites on the membrane occur in convective through pores, rather than in stagnant fluid inside the pores of an adsorbent particle [2,3]. For this reason, affinity membrane chromatography has the potential to maintain high efficiencies both at high flow rates and for the use of large biomolecules with small diffusivities [4,5].
∗ Corresponding authors. Tel.: +86 592 2185299; fax: +86 592 2185299. E-mail addresses: [email protected] (W. Shi), [email protected] (D. Ge). 0376-7388/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2008.09.003
One of the most important factors in membrane affinity chromatography is the identification of suitable membranes [6]. To date, most membranes for affinity separation were made from organic materials. In general, organic materials can be roughly divided into two categories, namely, natural and synthetic polymers [7,8]. The natural macromolecular materials, such as agarose, dextrin, and chitosan (CS) are good affinity matrices because they are compatible with the usual ligands, but are often difficult to process as membranes [9,10]. In contrast, synthetic polymers such as nylon [11,12], polysulfone [13,14] and glycidyl methacrylate [9] are apt to be manufactured into membranes, however, are less suitable for the immobilization of the ligand due to their low compatibility and low concentration of actively functional groups in their structure. Coating of these membranes with natural macromolecules is an alternative for depressing non-specific binding of proteins and heightening capacities for affinity ligands [6,12]. Based on our and other theoretical analysis [15–18], besides enough reactive groups and good compatibility, an ideal affinity membrane should own well uniformities, such as narrow pore size and porosity distribution, as well as uniform membrane thickness. Most polymeric microporous membranes used as affinity matri-
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Fig. 1. Reaction scheme for preparation of the CS–AAO composite membrane. (A) AAO membrane was activated by GPTMS; (B) CS was coupled on the activated membranes.
ces were manufactured by phase-inversion method. Membranes produced from this method generally have wide pore size distribution and some thickness variation. These nonuniformites may severely degrade the performance of affinity membranes [15,17,18]. Compared with organic membranes, porous alumina membrane (AAO membrane) as a typical inorganic membrane possesses superuniform structure, such as straight and parallel ordered pore channels, homogeneous pore size distribution, as well as uniform membrane thickness [19]. Moreover, AAO membrane owns better mechanical strength, reusability and resistance to microbial degradation, heat and chemical attack [20]. Therefore, AAO membrane is an interesting candidate for the affinity chromatography applications. However, few publications reported the use of AAO membrane as affinity matrix. Recently, Sun et al. [21] used atom transfer radical polymerization to grow polymer brushes in AAO membrane to modify this inorganic matrix for affinity separation protein. Although membrane pores were not clogged, the pore size of modified AAO membrane was reduced evidently; as a result, the mass transfer resistance should be increased significantly. In this paper, CS–AAO composite membrane that holds the ideal pore structure of AAO membrane was fabricated and used as affinity matrix, for the first time. AAO membrane was activated with 3-glycidoxypropyltrimethoxysilane (GPTMS), and then CS was coupled on the activated membrane to increase reactive sites. Cu2+ as ligand was immobilized onto the CS–AAO composite membrane. The applicability of Cu2+ -chelated CS–AAO composite membrane for affinity purification hemoglobin was presented and discussed in detail. 2. Experimental 2.1. Chemicals and apparatus AAO membranes (60 m thickness, 0.2 m pore size and 47 mm diameter) were purchased from Whatman. GPTMS was obtained from Danyang Organosilane Material Chemical Company (China). Chitosan (CS, average molecular weight was about 3,00,000 and the degree of deacetylation was over 90%) was purchased from Zhejiang Aoxing Biotechnology Company (China). Fresh bovine blood was a gift from the Huangjinxiang Company (China). Bovine hemoglobin (Hb) and Tris-(hydroxymethyl)aminomethane were obtained from Sigma. Ninhydrin H2 O, imidazole and Coomassie brilliant blue R250 were the products of Sinopharm Chemical Reagent Company (China). Sodium dodecyl sulfate (SDS), glycine and EDTA disodium salt were obtained from BBI. Prestained protein ladder (11–170 kDa) was purchased from Fermentas. All other chemicals were of analytical grade and used without further purification. All solutions were prepared using deionized Milli-Q water (Millipore). The concentrations of Hb, Cu2+ , and CS were determined using a UV-752 spectrophotometer (Shanghai Instrument Co. Ltd., China).
Refrigerated Centrifuge (Beckman Avanti J-25, USA) was used for the preparation of hemolysate. The membrane cartridge (donated amicably from the Dalian Chemical and Physical Institute, China) was used to load the membrane stack. A peristaltic pump (Model BT-100, Shanghai, China) was used for the feeding of hemolysate solutions. The morphologies of the membranes were visualized with the field-emission scanning electron microscope (FE-SEM) (LEO, Germany). 2.2. Preparation of affinity membrane 2.2.1. Activation of AAO membrane Ten AAO membrane disks were immersed in a solution of 5 ml GPTMS–30 ml ethanol–2 ml sodium acetate buffer solution (50 mM, pH 5.0). The polytetrafluoroethylene device containing the above solution was vacuumized for 5 min to remove air from the pores of the membranes. The membranes remained in this solution for another 15 min under ambient pressure before being rinsed with ethanol. The membranes were then cured by heating in vacuum at 100 ◦ C for 1 h. 2.2.2. Preparation of CS–AAO composite membrane CS was dissolved in 5 wt% acetic acid aqueous solution to form 2 wt% CS solution. The solution was stirred at room temperature for 24 h, and then was filtered to remove any indissolvable solids and impurities. The activated membranes were shaken in 10 ml prepared CS solution for 1 h at room temperature. The membrane disc was placed onto a sintered glass filter holder (Autoscience) and the remaining CS solution was sucked slowly through the membrane disc by reducing pressure until no further drop was formed at the filtrate side. This was followed by drying the wetted membrane in an oven at 85 ◦ C for 45 min. Non-covalently bound CS was removed by washing the membranes with 1 vol.% acetic acid solution and deionized water. Reaction scheme for the preparation of CS-coated AAO membrane is shown in Fig. 1. The amount of the CS coupled onto the AAO membranes was determined by the ninhydrin method [6]. Briefly, the ninhydrin reagent was prepared by dissolving 70 mg SnCl2 and 500 mg ninhydrin into a mixture of 15 ml DMSO and 2 ml of 2 M sodium acetate. 40 mg dry CS–AAO composite membrane pieces were added to a mixture of 1.0 ml of pure water and 2.0 ml ninhydrin reagent. The mixture was incubated in a boiling water bath for 30 min and then cooled to room temperature. A n-propanol solution (7.0 ml, 50% (v/v) in pure water) was added to the mixture. The optical density of the resulting solution was determined at 570 nm wavelength against a reference which was obtained from the AAO membrane. The standard curve was created using 1–6 mg solid CS.
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Fig. 2. FE-SEM photos of two frontal surfaces of AAO membrane and CS–AAO composite membrane. (A and B) Original alumina membrane; (C and D) CS–AAO composite membranes.
2.2.3. Immobilization of Cu2+ CS is a well-known adsorbent for metal ions, such as Cu2+ , 2+ Ni , Cr2+ , Zn2+ and Hg2+ , because the amine group (–NH2 ) and the hydroxyl group (–OH) can serve as coordinate sites for metals. The porous CS–AAO composite membranes were kept in a solution of CuSO4 (0.05 M) and shaken overnight at room temperature [22]. After rinsing with distilled water and drying, Cu2+ -attached affinity membranes were obtained. The amount of Cu2+ immobilized on membranes was calculated from the difference in pre- and post-reaction concentration of Cu2+ . Cu2+ ions were analyzed in the supernatant as described below. A 9 ml EDTA solution (0.05 M) was added to 1 ml of solution containing Cu2+ and the absorbance of Cu–EDTA complex was measured directly by UV–vis spectrophotometry at 740 nm [23]. 2.3. Cu2+ ions leakage from the affinity membrane The effects of metal ion leakage and resulted toxicity during adsorption and elution are also significant issues to be evaluated [24,25]. The leakage of Cu2+ ions from the affinity membrane was determined in the media of 10 mM phosphates buffer solution (pH 6.8 and 7.8) and 0.5 M imidazole aqueous solution. Membranes were incubated in above media with shaking at room temperature for 10 days. The media were renewed every day, and the amount of the Cu2+ release into the medium was measured cumulatively by UV–vis spectrophotometry at 740 nm.
were mixed with 5 ml of various concentrations of Hb solution (40–450 mg/l). The mixture was shaken at 25 ◦ C for 8 h, which proved to be a sufficient period to reach adsorption equilibrium. The amount of Hb adsorbed was determined by the following equation: Q =
(C0 − Ct )V m
(1)
where Q is the amount of Hb adsorbed on unit mass of the membranes (mg/g); C0 and Ct are the initial and final concentrations of the Hb solution, respectively (mg/l); V is the volume of the Hb solution (l); and m is the mass of the membranes (g). The concentration of the Hb solution was detected by UV–vis spectrophotometry at 405 nm. 2.5. Desorption and regeneration Hb desorption experiments were performed in a 0.5 M imidazole aqueous solution. Hb adsorbed membranes were placed in the desorption medium and shaken for 5 h at room temperature, followed by the procedure of washing with large volume of distilled water and phosphate buffer (pH 6.8). The final Hb concentration within the desorption medium was determined by spectrophotometry. The desorption ratio was calculated from the amount of Hb adsorbed on the membranes and the amount of Hb desorbed. Hb adsorption–desorption operation was done five times using the same adsorbent for checking the reusability of the membranes. 2.6. Dynamic adsorption of Hb from hemolysate
2.4. Batch adsorption experiments The Cu2+ -attached affinity membranes were tested for the adsorption of Hb in 10 mM phosphate buffer (pH 6.8) by batch experiment. Approximately 40 mg of affinity membranes
2.6.1. Preparation of hemolysate Hemolysate was prepared as described by Stocker-Majda et al. [26]. Briefly, fresh bovine blood (ca. 500 ml) was centrifuged at 2000 rpm/min and 4 ◦ C for 10 min, afterwards the plasma was
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removed by aspiration. The remaining red blood cells (ca. 250 ml) were washed five times with three volumes of 0.9% NaCl. The cells were lysed by osmotic shock at 4 ◦ C. Hemolysate after cell lysis was pretreated by centrifugation (2000 rpm/min, 4 ◦ C, 30 min) to remove cell debris. The resultant hemolysate was adjusted with 10 mM phosphate buffer, pH 6.8, to a Hb concentration of 200 mg/l.
2.6.2. Dynamic adsorption experiment The dynamic experiments were carried out in the cartridge to investigate the breakthrough performance. Ten sheets of affinity membrane with a diameter of 47 mm were set into a stack. The stack was first equilibrated with the equilibrium buffer. Then hemolysate was impelled by peristaltic pump with two different flow rate (i.e., 2 and 4 ml/min) to flow through the membrane stack. The concentration of Hb in the feed was kept at 200 mg/l. The amounts of Hb through the membrane cartridge were measured in succession with a spectrophotometer. After adsorption the stack was washed with 0.5 M imidazole aqueous solution until A405 was <0.02.
2.6.3. SDS-PAGE electrophoresis The purity of Hb was assayed by sodium dodecylsulfate– polyacrylamide gel electrophoresis (SDS-PAGE) using 15% separating gel and 5% stacking gel. Gels were stained with 0.25% (w/v) Coomassie brilliant R250 in methanol–acetic acid–water (4.5:1:4.5, v/v/v) and destained in methanol–acetic acid–water (4.5:1:4.5, v/v/v). Electrophoresis was run for 1.5 h with a voltage of 120 V. Bovine hemoglobin (Sigma) was used as a standard.
3. Results and discussion 3.1. Characteristics of the membranes The morphologies of AAO membrane and CS–AAO composite membrane were observed by FE-SEM and were illustrated in Fig. 2. The AAO membrane is composed of a high-purity alumina matrix that is manufactured electrochemically. This unique material has a precise, non-deformable honeycomb pore structure (Fig. 2A and B) with no lateral cross over between individual pores. After coating CS onto the AAO membrane, the pore size of the membrane has not apparently changed (Fig. 2C and D). Moreover, the CS–AAO composite membrane also exhibited homogeneous surfaces. The precise pore structure and narrow pore size distribution of the CS–AAO composite membrane can significantly reduce diffusion resistance, possess high flow rates, and ensure a high level of affinity adsorption performance in purification of bioproducts. The CS content of CS–AAO membrane was ∼49.7 mg/g membrane. Since there are plentiful reactive groups (–NH2 and –OH) on the CS molecule, the compatibility with the usual ligands was greatly enhanced by coating CS on the AAO membrane. The energydispersive X-ray spectroscopy (EDS) data (Fig. 3) showed there was Cu element on the affinity membranes, which verified that Cu2+ was immobilized on the CS–AAO composite membrane. The amount of Cu2+ immobilized on affinity membrane can be determined spectrophotometrically at 740 nm, and the Cu2+ content was ∼27.15 mg/g membrane. The leakage of Cu2+ in the media showed that there was nearly no Cu2+ release in the 10 mM phosphates buffer solution (pH 6.8 and 7.8) and 0.5 M imidazole aqueous solution. This result suggested that Cu2+ ions were strongly chelated on the CS–AAO composite membrane. Some of the characteristics of the Cu2+ -attached affinity membrane were listed in Table 1.
Fig. 3. EDS image of Cu2+ -attached affinity membranes.
Table 1 The characteristics of Cu2+ -attached affinity membrane Items
Property
Membrane material Average pore size of the membrane (m) Thickness of membrane (m) CS density (mg/g membrane) Cu2+ content (mg/g membrane)
AAO, CS and Cu2+ 0.2 60 49.7 27.15
3.2. Adsorption rate The adsorption rate curves of Hb are exemplified in Fig. 4. There was relatively faster adsorption rates observed at the beginning of the adsorption process, and then the adsorption equilibrium was achieved gradually for about 6.5 h. This figure also shows the nonspecific and specific adsorption of Hb onto the AAO, CS–AAO and Cu2+ -attached membranes. Although Whatman Company alleged that AAO membrane (Anodisc® ) exhibited low protein binding, AAO membrane owns high non-specific adsorption capacity for Hb, ∼11.8 mg/g membrane. The AAO membrane is composed of a high-purity alumina matrix that is manufactured electrochemically. During its preparation, the oxide layer that was derived by the anodizing technique would be coated on aluminium substrates. When the AAO membrane is directly in contact with the
Fig. 4. Adsorption rates of Hb on the membranes. Cu2+ content of affinity membrane: 27.15 mg/g membrane; Hb concentration: 200 mg/l; temperature: 25 ◦ C; medium: phosphate buffer (pH 6.8, 10 mM); Hb solution volume: 5 ml; membrane weight: 0.040 g.
W. Shi et al. / Journal of Membrane Science 325 (2008) 801–808
2+
Fig. 5. Freundlich adsorption isotherm of Hb on Cu -attached affinity membrane. Cu2+ content of affinity membrane: 27.15 mg/g membrane; temperature: 25 ◦ C; medium: phosphate buffer (pH 6.8, 10 mM); Hb solution volume: 5 ml; membrane weight: 0.040 g; adsorption time: 8 h.
protein solution, the protein molecules are apt to be adsorbed on the oxide layer of the AAO membrane [27]. After coating CS onto the AAO membrane, the non-specific Hb adsorption of the membrane reduced significantly, and the amount was about 1.95 mg/g membrane only. While much higher specific adsorption capacity, up to 17.5 mg/g membrane was obtained after ligand (Cu2+ ) immobilization. 3.3. Analysis of the adsorption mechanism Freundlich adsorption isotherms were applied for the description of the adsorption mechanism for Hb on Cu2+ -attached membrane. The isotherms can be described as follows: q = KC 1/n
(2)
where q is the adsorption capacity of Hb on unit mass of the affinity membrane at equilibrium (mg/g membrane); C is the equilibrium concentration of Hb (mg/l); n and K are the physical constants of the Freundlich adsorption isotherm. Eq. (2) can be transformed as follows: log q = log K +
1 log C n
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Fig. 6. Effects of temperature on the Hb adsorption capacities of Cu2+ -attached affinity membrane. Cu2+ content of affinity membrane: 27.15 mg/g membrane; Hb concentration: 200 mg/l; medium: phosphate buffer (pH 6.8, 10 mM); Hb solution volume: 5 ml; membrane weight: 0.040 g; adsorption time: 8 h.
0.125 and 1, respectively. This result further demonstrated that the affinity CS–AAO membrane owns excellent uniformities. 3.4. Effect of temperature The effect of temperature was studied under 4, 25 and 37 ◦ C. As shown in Fig. 6, the adsorption capacity increased with increasing temperature. The hypothesis for these phenomena is that the adsorption was endothermic, and higher temperature was favored for the adsorption. The result was similar to other related experiments [28]. The Hb adsorption was ∼17.9 mg/g membrane, which was obtained at 37 ◦ C. However, the effect of temperature on Hb adsorption is not notable. For example, the adsorption capacity of Hb increases by ∼2.29%, when the temperature changes from 25 to 37 ◦ C. 3.5. Effects of ionic strength The effect of the ionic strength on Hb adsorption was presented in Fig. 7. As seen in the figure, the adsorption capacity decreased with increasing the concentration of NaCl in the binding buffer (10 mM, phosphate, pH 6.8). When the added concentration of NaCl
(3)
Although the Freundlich equation was proposed originally as an empirical equation, it can be derived, however, from a sound theoretical footing. It is obtained by assuming that all surfaces are not really smooth at a microscopic level. The use of the Freundlich isotherm to characterize affinity adsorption has a number of advantages [28]. First, the Freundlich isotherm is based on a heterogeneous exponentially decaying distribution, which fits well to the tailing portion of the heterogeneous distribution of affinity membrane. In addition, the logarithmic form of the Freundlich isotherm is easily applied as it can be transformed into a linear function (Eq. (3)). This analysis is a useful diagnostic for identifying sources of error, as deviations from linearity are visually evident. Finally, the slope of the straight-line fit yields n, which is a measure of the heterogeneity of a system. A more homogeneous system will have an n value approaching unity. Fig. 5 shows the linear relationship of the Freundlich isotherm for the adsorption of Hb with Cu2+ -attached affinity membrane. The correlation coefficient (r) of the isotherm was 1, indicating that the data fit the model very well. The values of K and n for our adsorption system were found from the straight-line plot of log q versus log C by linear regression, and were
Fig. 7. Effect of NaCl concentration on the Hb adsorption capacities of Cu2+ -attached membrane. Cu2+ content of affinity membrane: 27.15 mg/g membrane; Hb concentration: 200 mg/l; temperature: 25 ◦ C; medium: phosphate buffer (pH 6.8, 10 mM); Hb solution volume: 5 ml; membrane weight: 0.040 g; adsorption time: 8 h.
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Fig. 8. Effect of pH on Hb adsorption capacities on Cu2+ -attached affinity membrane. Cu2+ content of affinity membrane: 27.15 mg/g membrane; Hb concentration: 200 mg/l; temperature: 25 ◦ C; Hb solution volume: 5 ml; membrane weight: 0.040 g; adsorption time: 8 h.
changes from 0 to 0.8 M, the adsorption capacity of Hb was ∼18.42% decrease. The electrostatic interaction between protein molecules and metal-chelated affinity system could be reduced in the presence of salt because the surface charges of protein are screened [29]. An increase in ionic strength also causes a change in the interaction mechanism due to more contributions from hydrophobic and protein–protein interactions [30]. More ions may also be attached to Hb molecules at high ionic strengths. This causes further stabilization of the protein molecules (higher solubility), which may lead to lower adsorption of Hb on the affinity membrane. In addition, the protein is expected to become more flexible with increasing electrolyte concentration because the intra-electrostatic interactions become less important [31,32]. For this reason, the protein molecule may lose the ability to expose its histidine residue to the affinity membranes, which is the dominant adsorption site in protein binding with an attached metal ion. 3.6. Effects of pH The principle of immobilized metal ion chelating affinity chromatography (IMAC) suggested that the adsorption of protein to the adsorbent was governed by the coordination of metals with electron-donor ligands exposed on the surface of the proteins [33,34]. The medium pH is very important parameter which determined the adsorption capacity of the affinity membrane to the target protein in the biological fluids. The adsorption medium pH affects the net charge on the immobilized metal ions as well as on the protein molecule in the adsorption medium. In this study, the effect of pH of the equilibration buffer on the adsorption capacity of Hb on Cu2+ -attached affinity membrane was investigated and the obtained results were shown in Fig. 8. It indicated the pH dependence of the amount of Hb adsorbed on the Cu2+ -attached affinity membranes. In all the cases investigated, the maximum adsorption of Hb was observed at pH 6.8. Significantly lower adsorption capacities were obtained with all affinity membranes in more acidic and in more alkaline pH regions. It has been shown that proteins have no net charge at their isoelectric points (pI), and therefore the maximum adsorption from aqueous solutions is usually observed at their pI (pI of Hb: 6.8) [35,36]. When pH < pI, the protonation effect on the amino acid residue (histidine) will be increased and hence their coordination ability to immobilized metal ions will be reduced [37,38]. These could explain that the adsorption capacity decreased from pH 6.8 to 4.8. At an alkaline pH, coordination with
Fig. 9. Breakthrough curves at different feed-rates. Separation chromatogram of Hb from hemolysate on a stack of 10 membranes with 47 mm diameter.
amino and hydroxyl groups took place and resulted in less effective adsorption of Hb [39]. 3.7. Regeneration of the membranes Regeneration is a crucial step in all affinity chromatography techniques. It was thus necessary to evaluate the regeneration efficiency of the affinity adsorbents after each cycle. Histidine units bind to chelated divalent metal ions in the unprotonated state, and then the adsorbed proteins can be eluted by either lowering the pH or introducing a high concentration of free imidazole [40]. Desorption of Hb from the affinity membranes was carried out in batch system. For all the tested Cu2+ -attached affinity membranes up to 91.2% of the adsorbed Hb was desorbed by using 0.5 M imidazole aqueous solution. In order to show the reusability of the Cu2+ -attached affinity membranes, the adsorption–elution cycle was repeated five times using the same affinity membranes. There was no remarkable reduce in the adsorption capacity of the membranes. The Hb adsorption capacity decreased only 6% after five cycles. Moreover, no obvious changes of the morphology of the affinity membranes were found in the recycling process. These results demonstrated the stability of the present CS–AAO membranes as a metal-chelated affinity adsorbent. 3.8. Dynamic separation of Hb from hemolysate Hb is found in red blood cells and responsible for oxygen transfer from the lung to the body tissues as well as the return transport of carbon dioxide and protons. To investigate the selectivity and dynamic behavior of Cu2+ -attached affinity membrane, red cell lysate (hemolysate) was loaded onto the affinity stack of 10 membranes with 47 mm diameter in a single-pass mode at constant flow rate. Fig. 9 presents the ratio between permeate and feed concentrations C/C0 as function of the permeate time. As shown in this figure, higher velocity of the hemolysate makes out a rapid breakthrough and the lower one shows a laggard penetration. It also shows a high rate for Hb adsorption in dynamic experiments. Because the mass transfer in dynamic experiment is convective mode, the resistance is greatly reduced. As a result, the operation for Hb adsorption could be speeded up. The key performance criteria for affinity membrane processes are breakthrough curve sharpness at the adsorption stage [41]. In fact, not only the feed rate of solute and ligand content but also the nonuniformites in membrane thickness and pore size may have significant effects on the breakthrough curve sharpness [15]. The uniform affinity membrane can enhance solute recovery
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branes could be described with Freundlich adsorption isotherms. The adsorbed Hb on the affinity membranes was desorbed up to 91.2% with 0.5 M imidazole aqueous solution as the desorption agent. It was possible to reuse these metal-chelated affinity membranes without significant reduction in the protein adsorption capacities. The result of dynamic adsorption showed Cu2+ -attached affinity membranes have been successfully used for the separation and purification of Hb from the red cell lysate. These features make the CS–AAO composite membranes very good candidate for use in metal-chelated affinity separation of proteins and would be effective in processing large volume of biological fluid containing a target protein. It is important to emphasize that many ligands can be easily immobilized on the CS–AAO composite membrane by activating the –NH2 and/or –OH groups of the CS, therefore we expect for a wide range of affinity systems could be considered applying this strategy. Acknowledgments We are extremely grateful to the National Nature Science Foundation of China (No. 30500127) and the Natural Science Foundation of Fujian Province (No. C0510005) for supporting this research.
Fig. 10. SDS-PAGE assay of Hb obtained from hemolysate. From left to right: lane A, marker; lane B, purified hemoglobin after affinity chromatography; lane C, hemolysate; lane D, standard bovine hemoglobin (Sigma).
efficiency and ligand utilization efficiency [15]. Due to the excellent uniformites of affinity CS–AAO membranes, both breakthrough curves under two feed rates are sharper, indicating a high affinity purification performance. Using the data presented in Fig. 9 one can calculate the so-called dynamic capacity, i.e., the uptake of the membrane before the exiting feed concentration reaches 10% of the inlet value. The dynamic capacities for the flow rates of 2 and 4 ml/min are 12.8 and 11.6 mg/g membrane, 27% and 34% smaller than the corresponding static adsorption capacity, respectively, because some adsorptive capacity remains unused mainly due to slow sorption kinetics and resistance from mass transport rate processes. Full adsorption capacities can only be achieved if the flow rate through the affinity membrane is slow enough to allow each Hb molecules to diffuse to the adsorptive site and to rearrange and/or unfold their structure to its most favorable state before the interstitial volume continues to move through the membrane. After adsorption the stack was eluted with 0.5 M imidazole aqueous solution, the quality of the purified Hb was assayed by SDS-PAGE electrophoresis. Fig. 10 gives the electrophoresis of Hb before and after purification by membrane chromatography. The purified Hb showed single band in SDS-PAGE, indicating the well selectivity of the prepared affinity membrane for Hb. 4. Conclusions A novel organic–inorganic composite membrane with excellent uniformities, such as narrow pore size and porosity distribution, as well as uniform membrane thickness, was prepared by using AAO membrane as supported matrix. AAO membrane was activated with GPTMS, followed by coupling CS to enhance the amount of active groups of the AAO membrane. The content of CS on the CS–AAO composite membrane was ∼49.7 mg/g membrane. The prepared CS–AAO membrane integrated virtues of organic and inorganic membrane, including plentiful active groups and well uniformities. After chelating with Cu2+ the affinity membranes were used for adsorption of Hb. The adsorption capacity of Hb was ∼17.5 mg/g membrane, and the adsorption behavior of Hb on affinity mem-
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