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Scalable Fabrication of Electrospun Nanofibrous Membranes Functionalized with Citric Acid for High-Performance Protein Adsorption Qiuxia Fu, Xueqin Wang, Yang Si, Lifang Liu, Jianyong Yu, and Bin Ding ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03107 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 28, 2016

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ACS Applied Materials & Interfaces

Scalable Fabrication of Electrospun Nanofibrous Membranes Functionalized with Citric Acid for High-Performance Protein Adsorption Qiuxia Fu†, Xueqin Wang‡, Yang Si‡, Lifang Liu†, Jianyong Yu§, and Bin Ding*,†,‡,§

†

Key Laboratory of Textile Science & Technology, Ministry of Education, College of

Textiles, Donghua University, Shanghai 201620, China ‡

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials,

College of Materials Science and Engineering, Donghua University, Shanghai 201620, China §

Nanofibers Research Center, Modern Textile Institute, Donghua University,

Shanghai 200051, China

* Corresponding author: Prof. Bin Ding (E-mail: [email protected])

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ACS Paragon Plus Environment


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ABSTRACT Fabricating protein adsorbents with high adsorption capacity and appreciable throughput is extremely important and highly desired for the separation and purification of protein products in the biomedical and pharmaceutical industries, yet still remains a great challenge. Herein, we demonstrate the synthesis of a novel protein adsorbent by in-situ functionalizing eletrospun ethylene-vinyl alcohol (EVOH) nanofibrous membranes (NFM) with critic acid (CCA). Taking advantage of the merits of large specific surface area, highly tortuous open-porous structure, abundant active carboxyl groups introduced by CCA, superior chemical stability and robust mechanical strength; the obtained CCA grafted EVOH NFM (EVOH-CCA NFM) present an excellent integrated protein (take lysozyme as the model protein) adsorption performance with a high capacity of 284 mg g-1, short equilibrium time of 6 h, ease of elution and good reusability. Meanwhile, the adsorption performance of EVOH-CCA NFM can be optimized by regulating buffer pH, ionic strength and initial concentration of protein solutions. More importantly, a dynamic binding efficiency of 250 mg g-1 can be achieved driven solely by the gravity of protein solution, which matches well with the demands of the high yield and energy conservation in the actual protein purification process. Furthermore, the resultant EVOH-CCA NFM also possess unique selectivity for positively charged proteins which was confirmed by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Significantly, the successful synthesis of such intriguing and economic EVOH-CCA NFM may provide a promising candidate for the next generation of protein adsorbents for rapid, massive and cost-effective separation and purification of proteins. KEYWORDS: electrospinning, ethylene-vinyl alcohol nanofibers, citric acid, surface modification, protein adsorption and purification 2


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1. INTRODUCTION Pharmaceutical protein products as one of the most important biological products, play extremely critical roles in fields of disease diagnosis, treatment and prevention, immunotherapy, as well as life sciences research.1-5 In general, high purity is rigorously stipulated by almost all the application fields because of its great influences on the pesticide effect and safety of the protein products. Meanwhile, the cost of the protein purification process nearly reaches up to over half of the total cost, therefore, biotechnological and pharmaceutical industries have devoted a great deal of efforts to develop downstream separation and purification methods as well as materials to obtain highly purified protein products efficiently, rapidly and cost-effectively.6,

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Conventionally, packed column chromatography by micro porous resin beads or gel microspheres have been applied most widely in the proteins separation and purification process owing to their high precision.8,

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However, their serious

limitations such as high pressure drops, high energy and buffer solution consumption, and low flow rates, which were caused by the dead-end inner porous structure of resin beads and the compaction of packed bed under a high driven pressure, thereby leading to a relatively low throughput and difficulties in efficiently scaling up production.10, 11 Alternatively, fibrous protein adsorbents with functional groups dispersed on the fiber surface present great potentials to address the shortcomings of the packed beds of resin beads due to their low pressure drop, high flow rate and low transmission resistance of protein, which are coming from their highly tortuous and interconnected open-porous structure, and easily accessible adsorption functional groups. For instance, Li et al. cross-linked the collagen fibers with glutaraldehyde for the separation of bovine hemoglobin and lysozyme with the capacities of about 40 and 120 mg g-1, respectively.12 Moreover, commercial cellulose membranes protein 3



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adsorbents (Sartobind C and S) with lysozyme adsorption capacity of about 51 mg g-1 also have been produced.13 These methods have successfully realized the application of various fibrous materials in the field of protein adsorption and separation, and the obtained adsorbents also showed great potential to decrease pressure drops in the manipulation. However, they still face sorts of challenges involving limited binding capacity and unfavorable rapid breakthrough resulted by their relatively small surface area, low porosity and insufficient adsorption functional groups.14-16 Nowadays, taking nanofibrous membranes (NFM) as matrix to replace the traditional ones for further coupling adsorption functional groups has been considered as one of the most promising approaches to solve these existing problems, yet need to be further explored. Since the discovery of electrospun nanofibers in 1745, they have been deemed to be extremely versatile platforms for various applications in the fields of adsorption, air-filtration, oil-water separation, electricity production and tissue engineering.17-22 These wide applications of nanofibrous memberans are due to their unique properties including large specific surface area (SSA), tunable tortuous open-porous structures, scalable synthesis from various materials and ease of functionalization.23-24 Meanwhile, these fantastic features also make them show great potential to be one of the

most perspective

candidates for the fabrication of

high-performance

chromatographic materials. Therefore, a large number of scientific researches have been conducted to develop forefront electrospun nanofibrous protein adsorbents based on different driving forces involving targeted affinity, dye-ligand affinity, electrostatic interaction, hydrophobic affinity, and so on.11 For example, Ma and co-workers modified and immobilized the polyethersulfone (PSU) NFM with protein A/G, the obtained materials could specifically adsorb protein IgG with capacity of 11.42 mg 4


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g-1;25 Zhang et al. modified hybrid chitosan/nylon-6 NFM with reactive dye of Cibacron Blue F3GA through a relatively simple process, and the resultant adsorbents could capture papain with capacity of about 70 mg g-1;26 Schneiderman et al. functionalized carbon NFM with cation ion-exchange groups by using the mixture of concentrated sulfuric and nitric acids, and the resulting carbon-based adsorbents possessed good acid and alkali resistance as well as relatively high adsorption capacity of 200 mg g-1 towards lysozyme.27 In addition, Lan et al. synthetized the cellulose diacetate nitrate by using concentrated nitric acids and then directly fabricated the NFM for the selective binding of negatively charged proteins of bovine serum albumin (BSA).28 These methods have fabricated different kinds of electrospun nanofibers chromatographic membranes and the performance of protein adsorbents have been improved in a certain extent, but they still subjected to several drawbacks such as complicated producing process, high cost of affinity and reactive dye ligands, relatively poor mechanical properties and low protein binding capacity which seriously limited their practical utilization. Accordingly, producing cost-effective nanofibrous protein adsorbents with excellent performance by simple approach under mild condition is still a big challenge to be resolved. In this contribution, we demonstrate the design and facile fabrication of intriguing and economic critic acid (CCA) grafted ethylene-vinyl alcohol (EVOH) NFM (EVOH-CCA NFM) under mild condition for high effective protein adsorption and purification, as shown in Scheme 1. To the best of our knowledge, this is the first time reporting the functionalization of electrospun EVOH NFM with CCA to fabricate chromatographic membranes for protein adsorption and separation. On the one hand, EVOH NFM are employed as substrates owing to their good hydrophilicity, excellent water insolubility and chemical stability, which endow the adsorbents with low 5



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non-specific protein adsorption. Meanwhile, the abundant active hydroxyl groups of EVOH could also be used for further derivatization. On the other hand, as an edible and environmentally friendly chemical substance, CCA performs well as a grafting agent, because it not only can introduce a large amount of adsorption groups (carboxyl groups) to the nanofibrous matrix, its relatively long carbon chains can also act as a spacer arms which can decrease the steric hindrance between the adsorption groups and adsorbed proteins, thereby can greatly improve the availability of active adsorption sites and conjugation between protein molecules.29 Benefiting from the large SSA, highly tortuous open-porous structure, abundant active hydroxyl groups of EVOH NFM as well as the numerous carboxyl groups of CCA, the resultant EVOH-CCA NFM exhibit integrated properties of high degree of adsorption functional groups, high open-pore tortuosity, and robust mechanical strength. Such fantastic morphology and chemical structure endow the obtained EVOH-CCA NFM with high efficient protein adsorption performance including large adsorption capacity, short equilibrium time, excellent dynamic adsorption property, favorable reusability and selectivity, signifying their great potential to be really used in the mass production of highly purified protein products. We anticipate that this work could offer a new approach for the development of chromatographic materials to satisfy the numerous requirements of actual protein adsorption and purification involving high productivity, low cost, energy consumption, and easy to scale up.

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Scheme 1. Schematic illustration of the fabrication process of EVOH-CCA NFM and their selective protein adsorption performance as well as regeneration process. 2. EXPERIMENTAL SECTION 2.1. Materials. EVOH (27 mol % ethylene content) was produced by Kuraray Co., Ltd., Japan. CCA, polyphosphoric acid (PPA), isopropyl alcohol (IPA), disodium hydrogen phosphate (Na2HPO4), monosodium orthophosphate (NaH2PO4), coomassie brilliant blue G250, sodium chloride (NaCl), lithium chloride (LiCl), potassium chloride (KCl), magnesium chloride (MgCl2), sodium hydroxide (NaOH), and phosphoric acid (H3PO4) were purchased from Shanghai Aladdin Reagent Co., Ltd., China. Lysozyme, BSA, bromelain, papain, pepsin as well as ovalbumin were supplied by Sangon Biotech Co., Ltd., China. Ultrapure water (resistivity higher than 18.2 MΩ) was supplied by utilizing a Heal-Force system. All the reagents were used without further refinement. 2.2. Preparation of EVOH NFM. The EVOH solution (8 wt %) was prepared by dissolving certain weight of EVOH in the mixture solvent of IPA and ultrapure water (7/3, v/v) with vigorous stirring in a water bath at 60 oC for 8 h.30 Following, the as-prepared EVOH solution was transferred to 10 mL plastic syringes capped with 7



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tubular metal needles. The electrospinning process was implemented by utilizing a DXES-1 spinning equipment supplied with an applied voltage of 30 kV, a controllable propulsion velocity of 4 mL h-1 and a receiving distance of 23 cm from spinneret to collector. During the entire spinning process, the temperature and relative humidity were sustained at 23 ± 2 oC and 45 ± 5%. The resultant EVOH NFM were collected on the grounded roller covered with paper and rotated at speed of 100 rpm, and then dried by using a vacuum oven at 40 oC for 12 h. 2.3. Fabrication of EVOH-CCA NFM. The modified processes for fabricating EVOH-CCA NFM were demonstrated as follows. Typically, the modification solutions with different CCA contents (0, 1, 2, 3, 4, 5 and 6 wt %) were first prepared via dissolving certain amount of CCA in ultrapure water with vigorous stirring, and 10 wt % PPA (as to the contents of CCA) act as catalyst was synchronously added into these solutions. Following, the as-prepared EVOH NFM were immersed in the modification solutions for 30 min with a fixed bath ratio (50/1, w/w). Subsequently, these membranes were taken out and transferred into an electric blast drying oven at 100 oC for 1 h to realize the esterification reaction between CCA and EVOH NFM. Finally, the EVOH-CCA NFM modified with different contents of CCA were obtained. 2.4. Static Protein Adsorption Performance Measurements. Based on the electrostatic interaction between negatively charged carboxyl groups of the NFM and the positively charged residue of proteins, lysozyme with isoelectric point (pI) of 10.8 and molecular weight (MW) of 14 kDa was selected as model protein to evaluate the protein adsorption performance of EVOH-CCA NFM. Typically, protein solutions in different concentrations were prepared by dispersing certain amount of protein in phosphate buffer, and the buffer solutions were prepared by mixing 0.2 M Na2HPO4 8


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and 0.2 M NaH2PO4 solutions according pH value of the buffer. The static protein adsorption capacities were measured as follows. Normally, 100 mg of dry EVOH-CCA NFM were immersed in 30 mL lysozyme solution, and then the mixed solution was shaken for a designated time at room temperature. Subsequently, the membranes were taken out and washed with buffer solution to remove the non-specifically adsorbed lysozyme. Finally, all the solutions were collected to measure the amount of residual lysozyme. The concentration of protein after adsorption was determined via detecting the absorbance intensity changes at 280 nm of the solutions by using an UV-vis spectrophotometer. The amount of adsorbed proteins were calculated based on the following formula: qt = V0 (C0 − Ct ) / m

where qt is the adsorption capacity at a given time, V0 is the protein solution’s volume,

C0 is the initial protein concentration, Ct is the protein concentration after adsorption for a given time, and m is the dry mass of the used EVOH-CCA NFM. 2.5. Dynamic Protein Adsorption Performance Measurements. To investigate the dynamic adsorption performance of EVOH-CCA NFM, 5 layers of NFM with accumulation thickness of about 0.4 mm were packed together and fixed in the middle of a homemade plastic filter syringe column with a diameter of 15 mm. Generally, lysozyme solution (1 mg mL-1, pH = 6) was poured into the column continuously and the solution flowed through the membranes slowly. Lysozyme concentration of the effluent solutions were immediately measured every 2 mL until it arrived the initial value. All of the operations were driven by a stable pressure drop of gravity (about 750 Pa) which was controlled by keeping a same liquid level. 2.6. Reusability Measurements. To evaluate the reusability of EVOH-CCA NFM, the adsorption-desorption experiments were conducted for 10 cycles. Generally, the 9



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membranes after saturation adsorption of lysozyme were treated with 1 M NaCl solution until non lysozyme could been detected in the eluent. After that, these membranes were washed with ultra-pure water for three times to remove NaCl and then dried in an air-circulating oven. Following, the regenerated membranes were repeatedly used in several cycle experiments to clarify the long-term usability. 2.7. Selectivity Measurements. To illustrate the selective adsorption properties of EVOH-CCA NFM, the adsorption ability towards different types of separate proteins and mixed proteins were tested. Herein, besides lysozyme, papain (pI of 8.75, MW of 23.4 kDa) and bromelain (pI of 9.5, MW of 33 kDa) were also selected as the model proteins which are positively charged. Meanwhile, BSA (pI of 4.8, MW of 67 kDa), ovalbumin (pI of 4.7, MW of 45 kDa), and pepsin (pI of 1, MW of 35 kDa) were selected as the negatively charged model proteins. In addition, the adsorption ability of EVOH-CCA NFM towards proteins mixture was analyzed by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 2.8. Apparatus and Characterization. The morphologies and energy-dispersive X-ray spectroscopy (EDX) of the relevant samples were observed by using a scanning electron microscopy (SEM, Vega 3, Tescan Ltd., Czech). The chemical structures of relevant samples were examined by employing a fourier transform infrared (FT-IR) spectrometer (Nicolet 8700, Thermo Nicolet Co., USA). The tensile strength of the samples were measured by using a tensile tester (XQ-1C, Shanghai New Fiber Instrument Co., Ltd., China). The N2 adsorption-desorption isotherms and BET surface area were characterized by an ASAP 2020 analyzer (Micromeritics Co., USA). The spectra of protein solutions were recorded by a UV-vis fiber-optic spectrometer (PG 2000pro, Idea Optics Technology Ltd., China). The pH value of buffer solutions were measured via utilizing a pH meter (PHS-3C, Shanghai Precision Scientific 10


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Instrument Co., Ltd., China). The dynamic water contact angles (WCA) (3 µL) were collected with a Kino SL200B contact angle measuring device. 3. RESULTS AND DISCUSSION 3.1. Morphologies and Structure. Based on the requirements of high-performance protein adsorbents, we designed and fabricated the nanofibrous protein adsorbents according to the following four principles: (1) the absorbents should be hydrophilic to prevent the non-specific protein adsorption, while should not be superhydrophilic to avoid the resistance to protein adsorption; (2) the absorbents should have large SSA and highly tortuous open-porous structure to provide numerous and easily accessible active adsorption sites; (3) the physical structure and chemical properties should keep stable during the fabricating and utilizing process; (4) abundant functional adsorption groups should be grafted on the substrates under convenient and mild conditions, and the adsorption groups should to be nontoxic.31 The first three principles were satisfied by taking EVOH NFM as substrates attributing to the physicochemical characteristics of EVOH and the unique features of NFM. To meet the last requirement, we combined the eletrospun EVOH NFM with surface modification by using CCA, because as a kind of poly-carboxylic acid, CCA not only can react with EVOH under mild conditions, but also can introduce more carboxyl groups on the surface of nanofibers in comparison with maleic anhydride used in our previous works.32, 33 The representative SEM image of EVOH NFM indicated a two dimensional, nonwoven-like structure piled up by randomly deposited nanofibers with an average diameter of 545 nm, as presented in Figure 1a. Notably, the compact bonding structure among fibers were formed along with the slight increase of average fiber diameter to 562 nm (Figure 1b) after modification, which was due to the graft polymerization of CCA and the impregnation of solution into fibers during the 11



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modification process. Moreover, the water wetting properties of the EVOH NFM were simultaneously improved attributing to the introduction of substantial carboxyl groups, as demonstrated in Figure 1c. It was clear that the EVOH-CCA NFM presented a smaller initial WCA of 120° and shorter completely infiltration time of 25 s than that of the pristine EVOH NFM (134°, 150 s). The effective grafting of carboxyl groups on surfaces of nanofibers was also confirmed by the analysis of FT-IR spectral (Figure 1d), the relatively decreased intensity of characteristic peak of -OH (around 3304 cm-1) and the appearance of new peaks of C=O and C-O-C (around 1720 and 1196 cm-1) illustrated the successful grafting of CCA on EVOH NFM.34 Benefiting from the formation of adhesion structure among fibers, the tensile properties of the NFM drastically enhanced from 3.76 to 8.68 MPa, and a relatively high breaking elongation of 91.2% (Figure S1) was obtained. The favorable mechanical properties can endow the EVOH-CCA NFM with long service life and good endurance towards high driven pressure.

Figure 1. SEM images of (a) EVOH NFM and (b) EVOH-CCA NFM. (c) Photographs of dynamic measurements of water permeation on the surface of EVOH and EVOH-CCA NFM. (d) FT-IR spectra of the relevant samples. 12


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To illustrate influences of the functionalization process on the hierarchical porous structure of the NFM, porous structure analysis was carried out via the N2 adsorption method. As showed in Figure 2a, the resultant N2 adsorption-desorption isotherm curves indicated typical IV type characteristic with H3 hysteresis loops, revealing the typical mesoporous structure in the pristine and modified EVOH membranes.35 And the BET surface area of EVOH and EVOH-CCA NFM were 3.33 and 2.52 m2 g-1, respectively. We attributed the slight reduction of the SSA to the formation of adhesion structure between nanofibers and the increase of fiber diameters within a narrow range, however, the SSA was still significantly larger than of flat films and commercial regenerated cellulose adsorbents.36, 37 In addition, fractal analysis was also performed to quantitatively analyze the porous structure by employing the modified Frenkel-Halsey-Hill (FHH) equation.38 As shown in Figure 2b, the FHH plots derived from the relevant N2 adsorption isotherms presented two obvious linear regions with different slopes, and the slope at high coverage regions around monolayer coverage was used to calculate the corresponding fractal dimension (D). The relevant D value of EVOH and EVOH-CCA NFM were 2.64 and 2.66, respectively, illustrating the typical surface fractal structure with irregular tortuous porous structure which could be recognized from the SEM images. Such fantastic irregular tortuous porous structure can provide substantial interconnected porous channels, thereby can significantly improve the mass transfer rate of proteins, the availability of adsorption ligands and operational flow rate.

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Figure 2. (a) N2 adsorption-desorption isotherms of EVOH and EVOH-CCA NFM, (b) FHH plots of ln(V/Vmono) against ln(ln(p0/p)) reconstructed from the relevant N2 adsorption isotherms. 3.2. Optimizing Protein Adsorption Performance of EVOH-CAA NFM. As the protein adsorption ability of EVOH-CAA NFM is based on the electrostatic forces between ionized carboxyl groups on the NFM and the positively charged residues on proteins, therefore, increasing the amount of carboxyl groups on membranes can effectively improve the protein adsorption performance of membranes. As for our research works, the amount of carboxyl groups grafted on EVOH NFM was depended on the content of CCA in the modification solutions, therefore it was necessary for us to optimize the adsorption performance by regulating the concentration of CCA (from 1 to 6 wt %). As illustrated in the resultant FT-IR spectra (Figure S2), the intensity of new peak at 1720 and 1196 cm-1 dramatically enhanced and the intensity at 3304 cm-1 14


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gradually weakened with the increase of CCA concentrations, demonstrating that increasing number of CCA molecules were grafted on EVOH NFM. However, the intensity of these characteristic peaks barely changed when the concentration of CCA reached and exceeded 4 wt %, suggesting that 4 wt % of CCA was enough to saturate the amount of carboxyl groups on the membranes. The changes of lysozyme adsorption capacity matched well with the above results. As displayed in Figure 3a, the adsorption capacities gradually increased with the increase of CCA contents, and the maximum static capacity of 284 mg g-1 was obtained when the content of CCA reached 4 wt %. The static adsorption capacity of EVOH-CCA NFM was about 4 times higher that of commercial Sartobind C and Sartobind S cation-exchange membranes adsorbents (about 51 mg g-1 towards lysozyme);13 meanwhile, it was nearly 1.5 times of the reported data (200 mg g-1),27, 32, 33

indicating that large amount of easily accessible carboxyl groups were dispersed on

the EVOH-CCA NFM. However, further increasing the CCA contents would not lead to an improvement of adsorption capacity, illustrating the adsorption process have reached saturation due to the limited carboxyl groups on the membranes. In addition, these membranes after saturation adsorption were dyed with coomassie brilliant blue to visually display the influence of the usage amount of CCA on the adsorption performance of the resultant EVOH-CCA NFM.39 As we can see from the insets of Figure 3, the pristine EVOH NFM presented whitey-brown due to its disability to adsorb protein, whereas the color changed to brilliant blue and gradually become darker with the increase of CCA contents, indicating that an increasing amount of lysozyme were adsorbed on membranes; finally, the color barely changed after the CCA content reached and exceeded 4 wt %, further demonstrating the saturation of adsorption. Moreover, the photograph of SDS-PAGE (Figure S3) showed that the 15



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band of lysozyme nearly disappeared after adsorption by EVOH-CCA NFM, further confirming the extraordinary high adsorption performance towards lysozyme.40,

41

Furthermore, EDX spectrum (Figure 3b) of the sample after adsorption equilibrium also indicated the effective adsorption of lysozyme on the EVOH-CCA NFM. Owing to the maximum lysozyme adsorption capacity, EVOH-CCA NFM modified with 4 wt % CCA were used to conduct the following experiments.

Figure 3. (a) Adsorption capacities of EVOH-CCA NFM modified with various contents of CCA within adsorption time of 12 h. The insets are the corresponding photographs of membranes colored by coomassie brilliant blue after saturation adsorption of lysozyme. (b) EDX pattern of EVOH-CCA NFM (take the NFM modified with 4 wt % of CCA as an example) after saturation adsorption of lysozyme. 3.3. The Kinetic Study of Adsorption. Fast kinetic adsorption performance is one of most crucial factors for protein adsorbents because it directly affects the actual 16


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efficiency of the adsorbents. Therefore, kinetic adsorption performance of EVOH-CCA NFM was studied by detecting the amount of adsorbed lysozyme in varying adsorption time (0, 1, 2, 4, 6, 8, 12, and 24 h). As presented in Figure 4, it was apparent that the adsorption capacity increased sharply at the beginning, then became gentle and finally reached equilibrium within 6 h, meanwhile, a saturation capacity of 284 mg g-1 was obtained. Furthermore, to investigate the type of adsorption force between

EVOH-CCA

NFM

and

lysozyme,

pseudo-first-order

and

pseudo-second-order kinetic models were employed to analyze the experimental data:42, 43

1   Pseudo-first-order model: qt = qe 1 − k1t   e  Pseudo-second-order model: qt =

k 2 qe 2t 1 + k 2 qet

Where qt is the adsorption amount at given contact time, qe is the calculated saturation capacity, k1 and k2 are relevant adsorption rate constants of the two models, t is the given contact time. The relevant fitted curves based on these two kinetics models were displayed in Figure 4, and the corresponding kinetic parameters were listed in Table 1. Notably, the kinetics adsorption process was followed well with the pseudo-first-order kinetic model attributing to the higher correlation coefficients (R2) of 0.99393. In other words, the adsorption mode between lysozyme and EVOH-CCA NFM was physisorption process. This result was consistent well with our design that the adsorption processes were driven by the physical electrostatic interactions between lysozyme and the EVOH-CCA NFM. Furthermore, benefiting from the numerous adsorption groups which were dispersed on the surface of NFM, a faster adsorption kinetic performance was obtained compared with other nanofibrous adsorbents previously reported.10, 34, 44 Obviously, the fast adsorption equilibrium rate 17



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will endow the EVOH-CCA NFM with a great potential to manufacture highly effective chromatographic columns for practical separation and purification of proteins.

Figure 4. The changes of lysozyme adsorption capacity with the elongation of adsorption time and relevant fitted kinetic adsorption curve based on the pseudo-first-order (solid line) and pseudo-second-order (dotted curve) model. Table 1. Kinetic parameters of lysozyme adsorption on the EVOH-CCA NFM pseudo-first-order kinetic model

pseudo-second-order kinetic model

qe (mg g-1)

k1 (min-1)

R2

qe (mg g-1)

k2 (min-1)

R2

295.84

0.43

0.99393

356.18

1.3

0.97131

3.4. Effects of pH and Ionic Strength. Adsorption capacity at varying pH values (4, 5, 6, 6.5, 7, 7.5, 8, 9, and 10) were measured to illustrate the influences of buffer pH on the adsorption performance of the EVOH-CCA NFM. The pH value of buffer solutions were regulated by utilizing 0.1 M NaOH and 0.1 M H3PO4 solution. As exhibited in Figure 5a, the membranes remained a relatively stable capacity of about 286 mg g-1 at the pH values ranging from 4 to 6, and then the capacity decreased sharply with the increase of buffer pH values from 6 to 10. This phenomenon might be explained by the fact that the buffer pH values have great effects on the electrostatic potential of both lysozyme and EVOH-CCA NFM. As the pI of lysozyme 18


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is approximately 10.8, thus the positive charges of lysozyme will increase with the decreasing buffer pH from 10 to 4.45 Conversely, the negative charges of the EVOH-CCA NFM barely changed because the carbonyl groups were existed as the ionic form in this pH range.46 Consequently, the electrostatic forces between lysozyme and carboxyl groups gradually enhanced with the decreasing of pH value, thereby leading to the increase of adsorption capacity. After adsorption capacity reached saturation, further enhancing the electrostatic interaction will not improve the adsorption capacity, resulting in the negligibly changes of the adsorption capacity at pH range from 4 to 6. In consideration of that neutral buffer is mild for proteins and more convenient for operation, pH value of 6 was applied to carry out the following tests. Besides buffer pH, ionic strength is another important factor which has great effects on the adsorption performance. Therefore, adsorption capacity under various ionic strengths were experimentally evaluated. Generally, the ionic strengths of buffer solution were regulated by adding NaCl with various concentration (from 0 to 1.0 M). Figure 5b demonstrated that the adsorption capacity barely changed when the concentration of NaCl solutions were lower than 0.1 M, whereas the capacity decreased rapidly with increasing NaCl concentrations from 0.1 to 1.0 M, the result was matched well with the previously reported studies.47, 48 This phenomenon may attributed to the fact that the electrostatic force between lysozyme and carboxyl groups were screened by the salt ions, in that case, the electrostatic interaction weakened with the increasing ionic strength, thereby resulting in the reduction of adsorption capacity. Moreover, Li+, Na+ and K+ ions with same charges but different ionic radius were selected as model ions to investigate the effects of ionic radius on the adsorption performance. As we can see from Figure 5c, the lysozyme adsorption 19



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capacities slightly decreased with the increase of the ionic radius. It might because of that the ions with same charges but larger radius could provide stronger shielding effects between lysozyme and carboxyl groups on the NFM, thereby resulting the decrease of the adsorption capacity. Furthermore, Mg2+ was selected as the model ion to study the influences of charges on the adsorption capacity by comparing with Na+ due to their similar ionic radius. As presented in Figure 5c, the adsorption capacity of EVOH-CCA NFM under the shielding effect of Mg2+ was lower than that of monovalent Na+, suggesting that ions with higher charges may have greater influences on the adsorption performance of the EVOH-CCA NFM.

20


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Figure 5. Effects of (a) pH value, (b) ionic strength, and (c) ionic radius and charges on the absorption capacity of EVOH-CCA NFM at the ion concentration of 0.2 M. 3.5. Adsorption Isotherm. To explore the effect of initial protein concentration on the performance of the EVOH-CCA NFM, adsorption equilibrium uptakes under varying initial lysozyme concentrations (from 0 to 1.2 mg mL-1) were tested (pH = 6 and at room temperature), and the results were presented in Figure 6a. Significantly, the adsorption amount almost linearly increased with the augment of lysozyme 21



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concentrations ranging from 0.1 to 1 mg mL-1 with a high linear coefficient of 0.99966 (Figure 6b). Further increasing lysozyme concentration, the adsorption capacity reached a plateau rapidly and the equilibrium adsorption capacity of 285mg g-1 was obtained. The results were reasonable by considering that the electrostatic interaction will strengthen with the increasing of concentration of lysozyme solution within the low concentration range under the theory of metastable equilibrium adsorption.49 Furthermore, to quantitatively analyze the adsorption process of lysozyme on EVOH-CCA NFM, two classical isotherm models (Langmuir and Freundlich model) were employed to fit the experimental data.50 The form of Langmuir and Freundlich isotherms were described by the following equations:43, 51 Langmuir model:

1 1 1 = + qe qmax K a qmax Ce

Freundlich model: lg qe =

lg Ce + lg K F n

where qmax is the maximum adsorption capacity, qe is the lysozyme adsorption amount at different initial concentration, Ce is the equilibrium lysozyme concentration, Ka is the Langmuir isotherm constant, KF is the Freundlich constant depicting adsorption capacity, 1/n is a constant indicating adsorption intensity. The relevant fitted curves were displayed in Figure S4, and the calculated isotherm parameters were listed in Table 2. It was evident that the R2 value calculated from Langmuir model (0.99936) was higher than that of Freundlich model (0.9936), indicating that the adsorption process obeyed Langmuir monolayer adsorption rather than heterogeneous surface adsorption.52 The explanation for this phenomenon might be related to the electrostatic attraction between carboxyl groups and lysozyme, because the carboxyl groups were unable to adsorb lysozyme after they were occupied by lysozyme. 22


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Moreover, the theoretical maximum capacity calculated based on Langmuir model was 296 mg g-1 which was similar with the experimental result, further confirming the high availability, easy accessibility and homogeneity of carboxyl groups on the EVOH-CCA NFM.

Figure 6. (a) Effect of the initial lysozyme concentration on the adsorption capacity of EVOH-CCA NFM and (b) fitted curve between capacity and initial concentration ranging from 0 to 1 mg mL-1.

Table 2. Calculated parameters of Langmuir and Freundlich isotherm models fitted to the experimental data in Figure 6. Langmuir model

Freundlich model

qmax (mg g-1)

Ka (mL mg-1)

R2

KF

1/n

R2

294.985

0.0507

0.99936

278.715

0.26661

0.9936

23



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Dynamic breakthrough analysis combined kinetic adsorption capacity with static equilibrium binding capacity is crucial for the practical evaluation of the application performance of the chromatographic columns, because it directly affects the productivity, time and energy consumption of the actual production processes.33, 53 Therefore, breakthrough curve measurement was performed to evaluate the dynamic adsorption capacity of the EVOH-CCA NFM. Figure 7 revealed a typical dynamic breakthrough curve, it was evident that the concentrations of the outlet gradually increased and then reached the initial value when the total elution volume was 22 mL, and the calculated dynamic binding capacity was 250 mg g-1 which was approximately 88% of the maximum static adsorption capacity and nearly 2 times of the reported data.27 More importantly, as the dynamic breakthrough experiment was conducted solely under the driven pressure of gravity (750 Pa) which was apparently lower than the high driven pressure (larger than 105 Pa) used by commercial fibrous adsorbents, demonstrating that the EVOH-CCA NFM can greatly improve the treatment rate and reduce the energy consumption, which is extremely important for their real applications.

Figure 7. Breakthrough curve of lysozyme solution through the EVOH-CCA NFM (5 layers with a total thickness of about 0.4 mm). Ideal protein adsorbents should possess such dynamic adsorption capacity which is 24


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90% of their equilibrium adsorption uptake when protein concentration of the effluent reach 10% of the initial value.54 However, the EVOH-CCA NFM showed a dynamic absorptive capacity of 131 mg g-1 (46% of the saturation capacity) when the concentration of effluent reached 0.1 mg mL-1. Such an unfavorable dynamic adsorption capacity might be ascribe to the inhomogeneous pore sizes of EVOH-CCA NFM, thereby resulting in a broadened protein mass transfer rate in the NFM. In addition, the thin stacking thickness (about 0.4 mm) of the used membranes might make the protein solution rapidly pass through the membranes and lead to short contact time between lysozyme and carboxyl groups, therefore preventing the full adsorption of lysozyme on the NFM. Fortunately, numerous efforts can be devoted to address the problem of fast breakthrough, such as systematically optimize the thickness of packed membranes, the flow rate of protein solution, and the distribution of the tortuous pore size.26 Consequently, the EVOH-CCA NFM could be used as an intriguing tool for the rapid and effective purification of proteins in the actual production process.

3.7. Selectivity of EVOH-CCA NFM. Selective adsorption ability towards different types of proteins also plays a significant role in the practical applications of protein adsorbents. As illustrated in Figure 8a, it was obvious that the EVOH-CCA NFM could only adsorb positively charged proteins including lysozyme, bromelain, and papain with capacities of 284, 58, and 150 mg g-1, respectively. The difference in adsorption capacities might attribute to the distinctions in surface charges and molecule size among different types of positively charges proteins. Generally, proteins with higher surface charges and smaller sizes are more easily adsorbed by adsorbents. The direct evidences of the selective adsorption toward positively charged proteins were revealed by FT-IR spectra of the EVOH-CCA NFM after adsorption of 25



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different proteins (Figure S5). As can be seen, new characteristic peaks (around 1655 and 1540 cm-1) of amide groups of proteins could be observed on the membranes after adsorption of positively charged proteins (lysozyme, bromelain, papain),55 while no characteristic peaks of amide groups could be observed on the NFM after adsorption of negatively charged proteins (BSA, ovalbumin, pepsin), confirming the conjectures that different charged proteins can be separated by the membranes. Moreover, the selectivity of EVOH-CCA NFM towards a protein mixture was also studied, as presented in Figure 8b. It was evident that the band color of lysozyme, bromelain and papain became lighter after they adsorbed by the membranes. What is worthy is that the fading degree of lysozyme band color was more obvious than that of bromelain and papain, illustrating that larger amount of lysozyme were adsorbed by the NFM. This result can be accepted by considering that the strength of the electrostatic attractions between proteins and the membranes adsorbents were affected by the pI of proteins, the buffer pH and the molecule size of proteins. Generally, the higher pI and the smaller molecule weight, the larger adsorption capacity. Contrarily, the bands of BSA, ovalbumin, and pepsin almost had no change due to their negative charges at the buffer pH of 6, which was same with surface charges of the carboxyl groups on the membranes. Consequently, the EVOH-CCA NFM could be used to realize the selective adsorption of different kinds of proteins by regulating and controlling the buffer pH based on the pI of the proteins.

26


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Figure 8. (a) The adsorptive capacities towards different types of separate proteins. (b) SDS-PAGE analysis of purification of the mixture of lysozyme, bromelain, papain, BSA, ovalbumin, and pepsin: M, protein maker; lane 1, original protein mixture; lane 2, protein mixture after adsorbed by EVOH-CCA NFM.

3.8. Reusability of EVOH-CCA NFM. Taking economic plausibility into consideration, it is extremely important for us to investigate the reusability of the materials. As displayed in Figure 9, the adsorption capacities of EVOH-CCA NFM kept relatively stable values of approximately same with the initial capacity within 10 cycles, indicating that the NFM possess a fantastic reusability. Such excellent reversibility can attribute to the stable physical and chemical structures of the EVOH-CCA NFM. The evidences was obtained from the SEM images (Figure S6) of the membranes after the first and tenth cycles, it was clear that no significant changes 27



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could be observed after it has been circularly used, indicating the stable structure and functionality of the EVOH-CCA NFM. We envision that the good cycling performance of the EVOH-CCA NFM can greatly meet the requirement of long serve life in their practical application.

Figure 9. Reversibility of the adsorption capacity over 10 cycles. 3.9. Comparative Study of Adsorption Performance. To further demonstrate the importance of nanofibrous structure on the performance of protein adsorbents, we compared the adsorption capacities of the obtained EVOH-CCA NFM with corresponding EVOH-CCA flat films, as exhibited in Figure 10. It was clear that UV-vis absorbance intensity at the wavelength of 280 nm of the lysozyme solution after adsorbed by NFM was obviously lower than that of flat films, and the calculated adsorption capacity of NFM (290 mg g-1) was approximately two times higher than that of flat films (99 mg g-1). It was because the feature of nanofibrous morphology which could endow the NFM with higher SSA and lager porosity than that of the flat films, therefore the NFM could graft more active adsorption groups for adsorbing proteins.56 In addition, electrospun NFM of EVOH and CCA (EVOH/CCA blend NFM) with different CCA contents (2, 3, and 4 wt %) were fabricated and their lysozyme adsorption performance were measured to have comparison with the EVOH-CCA NFM. As showed in Figure S7h, the lysozyme adsorption capacity of 28


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EVOH/CCA blend NFM within 12 h increased from 93 to 252 mg g-1 with the increase of CCA contents (from 2 to 3 wt %). However, further increasing CCA content to 4 wt %, the adsorption capacity decreased rapidly attribute to the flat film structure of the membranes after heat treatment. Therefore, the maximum lysozyme adsorption capacity of EVOH/CCA blend NFM (252 mg g-1) was slightly lower than that of the EVOH-CCA NFM (Figure 10b). This phenomenon might attribute the larger diameter of the EVOH/CCA blend NFM (larger than 859 nm) in comparison with the EVOH-CCA NFM prepared by submerging in acid solution, and the flat film structure of the blend membranes after heat treatment when the CCA contents reached 4 wt % (Figure S7a to g). More significantly, EVOH-CCA NFM also can be easily scaled up by using a large size pristine EVOH NFM as original materials, for example, a large-scale (65 × 60 cm) EVOH-CCA NFM (insert of Figure 10b) was obtained by modifying the large size EVOH NFM which was fabricated by a 10 needle multi-jets electrospinning equipment. It is highly possible to realize the massive industrial production by further enlarging the spinning equipment.57 Consequently, the EVOH-CCA NFM could be considered as a promising candidate for the synthesis of high-performance membranes chromatographic columns for the produce of protein products.

29



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Figure 10. (a) Adsorption spectrum of initial lysozyme solution as well as the solution after adsorption by flat films, EVOH-CCA NFM and electrospun EVOH/CCA blend NFM, (b) the relevant adsorption capacity. The inset is a photograph of large-scale (65 × 60 cm) EVOH-CCA NFM.

4. CONCLUSION In

conclusion,

we

have

described

a

novel,

effective,

facile

and

environmentally-friendly approach for fabricating highly carbonylated nanofibrous protein adsorbents by the organic combination of electrospun EVOH NFM and surface modification with CCA. Taking advantages of the unique integrated characteristics of the EVOH NFM and CCA, the resultant EVOH-CCA NFM exhibited an exceptionally excellent adsorption capacity of 284 mg g-1 towards lysozyme, relatively short equilibrium time of 6 h, and high mechanical strength of 8.68 MPa with large elongation of 91.2%. Furthermore, the obtained adsorbents also 30


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possessed a relatively high dynamic adsorption capacity of 250 mg g-1 solely driven by gravity. More importantly, the EVOH-CCA NFM presented an excellent reversibility, good selectivity performance, easy to enlarge and reserve, which can meet well with the requirements of massive scale separation and purification of the protein products. With the facility and cost-effectiveness of the synthesis process as well as the excellent performance of the resultant EVOH-CCA NFM, we envision that such fantastic nanofibrous protein adsorbents will provide a new kind of functional materials for the fast, effective, low-cost production of various highly purified protein products.

ASSOCIATED CONTENT Supporting Information. Stress–strain curves of the relevant pristine EVOH NFM and EVOH-CCA NFM (Figure S1). FT-IR spectra of EVOH-CCA NFM modified with various concentrations of CCA (Figure S2). SDS-PAGE analysis of purification of the separate lysozyme and BSA solution after adsorption by EVOH-CCA NFM (Figure S3). The fitted curves of Langmuir and Freundlich isotherm models for lysozyme adsorption on EVOH-CCA NFM (Figure S4). FT-IR spectra of the EVOH-CCA NFM after adsorption of different kinds of proteins (Figure S5). SEM images of EVOH-CCA NFM after the first and 10 times cycles (Figure S6). Morphologies and photographs of the blend EVOH/CCA NFM and comparison of the adsorption capacities between the EVOH-CCA NFM with blend EVOH/CCA NFM (Figure S7). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] 31



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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work is supported by the National Natural Science Foundation of China (No. 51322304), the Fundamental Research Funds for the Central Universities, the “DHU Distinguished Young Professor Program”, and the 111 Project (B07024).

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Biocompatible

Three-Dimensional

Scaffolds

of

Cellulose

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Microfiber/Gelatin

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Scalable Fabrication of Electrospun Nanofibrous ... - ACS Publications

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