Ultrafine Silk-Derived Nanofibrous Membranes ... - ACS Publications

Research Article pubs.acs.org/journal/ascecg

Ultrafine Silk-Derived Nanofibrous Membranes Exhibiting Effective Lysozyme Adsorption Shixiong Yi,†,‡ Fangyin Dai,† Yue Ma,‡ Tingsheng Yan,‡ Yang Si,*,‡ and Gang Sun*,‡ †

State Key Laboratory of Silkworm Genome Biology & College of Biotechnology, Southwest University, Chongqing, 400715, P.R. China, and ‡ Fiber and Polymer Science, University of California, Davis, California 95616 United States S Supporting Information *

ABSTRACT: Protein separation materials that are both selective and effective could have wide applications in fields of bioengineering and pharmaceutical industry. However, preparation of such materials has proven to be extremely challenging. Herein, we present a scalable methodology to prepare carboxyl group functionalized nanofibrous membranes (SFNM) by combining sustainable silk and electrospinning. The naturally abundant silk is thus reconstructed into nanofibrous membranes with tunable surface functions. The resultant SFNMs exhibit integrated properties of ultrathin fiber diameter (125 nm), larger surface area (14 m2 g−1), high porosity, superhydrophilicity, and negatively charged fiber surface, which can reversibly adsorb lysozyme with a robust capacity of 710 mg g−1 and high durability, matching well with the requirements for purifying protein solutions. The fabrication of such fascinating materials may provide new insights into the design and development of multifunctional separation membranes for various applications. KEYWORDS: Silk, Nanofibers, Membranes, Protein adsorption, Lysozyme

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area.11,12 One dimensional nanofibers provide ultrahigh surface area and, when they are fabricated into membranes, exhibit high porosity, good interconnectivity, and ease of surface modification with functional groups (carboxyl, hydroxyl, and mercapto groups).12−15 In previous studies, ethylene-vinyl alcohol copolymer, polyacrylonitrile, and poly(vinyl alcohol) nanofibrous membranes have been prepared by electrospinning technology for electric charge adsorption of lysozyme,16−18 which exhibited the good adsorption capacities. However, these polymers are derived mainly from nonrenewable fossil resources and are not biodegradable, the disposal of the waste separation materials might cause serious environmental concerns. From the viewpoint of sustainable applications, it is highly desirable to prepare robust and environmentally friendly protein separation nanofibrous membranes at a large scale. Nature, however, offers a remarkably simple alternative idea that has nothing to do with the fossil resources, yet again capitalizes on renewable biomass: instead of directly using synthetic polymers, biomasses, such as cellulose, lignin, alginate, and protein, are readily available, inexpensive, and sustainable resources that can be converted into nanofibers.19−22 Bombyx mori (silkworm) silk, as the forefront of advanced natural materials, combines promising features of relatively slow

INTRODUCTION The separation and purification of proteins have become one of important requisites in present pharmaceutical and biomedical industries.1−3 Lysozyme (N-acetylmuramide glycanhydrolase), as a very small enzyme (14.4 kDa), has been widely used in industrial applications, such as a food additive, cell disrupting agent, antibacterial agent, and anticancer agents.4−6 Because of its valuable properties, separation and purification of lysozyme have attracted great attention. Highly efficient, economical, and simple techniques have been further studied to get the highly purified lysozyme.7 The conventional separation and purification methods for lysozyme are ultrafiltration, precipitation, chromatography, and reverse micelles extraction.7,8 Although these separation methods are effective, they are usually complicated, time-consuming, and expensive. Generally, the production of lysozyme was mainly derived from the egg-white source. The most abundant proteins in egg-white are ovalbumin, ovotransferrin, ovomucoid, lysozyme, and ovomucin. Among these proteins, lysozyme exhibit the highest isoelectric point (pI) values (pI = 10.8), which is obviously higher than that of ovalbumin (pI = 4.5), ovotransferrin (pI = 6.1−6.6), ovomucoid (pI = 3.9−4.3), and ovomucin (pI = 4.5− 5.0).9,10 Therefore, the separation strategy based on electric charge adsorption could easily purify lysozyme from egg-white with relative selectivity. As the development of the nanoscience and nanotechnology, nanomaterials have been prepared and utilized as adsorbents because of their large active surface © 2017 American Chemical Society

Received: May 18, 2017 Revised: August 21, 2017 Published: August 24, 2017 8777

DOI: 10.1021/acssuschemeng.7b01580 ACS Sustainable Chem. Eng. 2017, 5, 8777−8784

Research Article

ACS Sustainable Chemistry & Engineering

Scheme 1. Schematic Illustration for (a) the Prepared Process of SFNM by Electrospinning Technology, (b) the Preparation of PMDA-SFNM and the Lysozyme Adsorption Process, and (c) the Graft Reactions between Silk and PMDA

pyromellitic dianhydride (PMDA), tetrahydrofuran (THF), disodium hydrogen phosphate (Na2HPO4), monosodium orthophosphate (NaH2PO4), coomassie brilliant blue G-250, sodium chloride (NaCl), sodium hydroxide (NaOH), and all other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA.) or Fisher Scientific (Pittsburgh, PA, USA). All reagents were used as received without any further purification. Double-distilled and deionized water were used throughout the study. Preparation of Silk Fibroin Nanofibrous Membranes (SFNM). The degummed silk was first dissolved in mixed solvent systems of CaCl2/CH3CH2OH/H2O (1/2/8 in molar ratios) at 70 °C for 6 h, which was used for further dialyzing to remove any small molecule impurities. After dialysis with cellulose tubular membrane (Sigma Co., 250-7u) in distilled water for 3 days, the silk fibroin solution was filtered and lyophilized to obtain regenerated silk fibroin sponges. Finally, to prepare the homogeneous spinning solution, the purified silk fibroin was dissolved in 98% formic acid with stirring for 3 h, the concentration of silk solution was 10 wt %. During the electrospinning process, a high electric potential was used to a droplet of silk fibroin solution at the tip (ID 0.495 mm) of a syringe needle, as shown in Scheme 1a. The electrospun nanofibers were collected on a target drum which was placed at a distance of 7 cm from the syringe tip. A voltage of 25 kV was applied to the collecting target by a high voltage power supply. Flow rate of the polymer solution was 1.0 mL h−1. Chemical Modification of SFNM. The silk fibroin is a natural polymer contains many amino acids with amino or hydroxyl groups at side chain, such as arginine (1.045%), serine (13.135%), and threonine (1.191%),26 as summarized in Table S1. These amino/hydroxyl groups at the side chain of silk could easily react with PMDA to form amide or ester bonds due to the high reactivity of anhydrides groups, as shown in Scheme 1b and c; thus, negatively charged carboxyl groups could be generated. The experimental details were performed as follows: A solution was prepared by adding PPA (1.0 wt %) and PMDA (5.0 wt %) in THF solution. The PMDA grafted SFNM (PMDA-SFNM) were obtained by immersing SFNM in the mixed solution at 80 °C for 1 h. Subsequently, the treated nanofibrous membranes were washed with water and dried. Finally, the PMDA-SFNM was collected. The PMDA

degradation, biocompatibility, ease of processing into various formats, excellent mechanical performance, and sufficient supply, etc.23−25 The protein has amino acids with functional groups at side chain, such as arginine, serine, and threonine.25,26 The immobilization of different functional molecules onto the surface of biomedical materials can be performed easily through covalent bonding with the functional groups.27,28 We thus expected that silk (regenerated silk fibroin, RSF) nanofibers can obtain promising protein absorption properties by being functionalized in a similar manner to that of common synthetic polymers. Herein, we present a robust strategy for creating carboxyl group functionalized silk fibroin nanofibrous membranes (SFNM) for effective lysozyme adsorption (seen in Scheme 1). The premise of this design is that naturally abundant and sustainable silk is reconstructed into nanofibrous membranes with tunable surface functions on a large scale. These SFNMs exhibited the integrated properties of ultrathin fiber diameter (125 nm), larger surface area, high porosity, superhydrophilicity, negative charged fiber surface, electric charge adsorption to lysozyme, and high adsorption capacity (710 mg g−1), all resulting from the synergistic effects of 3D nanofibrous architectures and well-functionlized, carboxyl group incorporated nanofibers.

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EXPERIMENTAL SECTION

Materials. Cocoons of Bombyx mori silkworm and silk fabrics were obtained from the Silkworm Gene Bank at Southwest University (Chongqing, China). The silkworm cocoons were degummed with boiling Na2CO3 aqueous solution (0.5 wt %) for 45 min to remove sericin. Lysozyme powder from chicken egg white was purchased from MP Biomedicals, LLC, Solon, OH, USA. Bromelain, papain, bovine serum albumin (BSA), ovalbumin, and pepsin were provided by Sangon Biotech Co., Ltd., China. Polyphosphoric acid (PPA), 8778



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grafted silk fabrics (PMDA-silk fabrics) were obtained by using the same grafting method. Adsorption Capacity Measurements. Lysozyme solutions at different concentrations were prepared by dissolving a certain amount of lysozyme powder in a phosphate buffer solution with different pH values. To measure the static adsorption performance, 0.5 g of PMDASFNM was immersed in the lysozyme solution (100 mL) for a certain time, subsequently the absorbance intensity change at 280 nm was detected using an ultraviolet−visible UV−vis spectrophotometer (Evolution 600, Thermo Scientific), and then the amount of proteins adsorption at equilibrium was calculated by using the equation

Q t = (C0 − C t)V0/m

Research Article

RESULTS AND DISCUSSION Nanofibrous Morphology and Structure. Adsorption performance of the nanofibrous membrane is usually determined by the following four decisive criteria: (1) large surface area and high tortuous porous structure, (2) stable physical structure and chemical properties, (3) hydrophilic surface and insolubility in water, and (4) rich and reactive functional groups on surfaces. First, the SEM images of SFNM and silk fabrics before and after grafting PMDA are shown in Figure 1a−d. No obvious change of the two silk fibers was

(1)

where Qt is the amount of proteins adsorbed at a given time (mg g−1), C0 is the initial concentrations of the proteins (mg mL−1), Ct is the concentrations of the proteins at a given time during adsorption process (mg mL−1), V0 is the volume of the solution (mL), and m is the mass of dry adsorbents (g). To study the dynamic adsorption performance of PMDA-SFNM, 5 layers of PMDA-SFNM were packed together and set in the middle of a plastic filter syringe column in a diameter of 15 mm. A 5 mg mL−1 of lysozyme solution was prepared at pH 6 and added into the column gradually, and the solution flowed through the membranes continually. The concentrations of lysozyme of the effluent solutions were instantaneously measured in every 2 mL until it reached the initial concentration values. All of the measurements were driven by a stable gravity pressure drop (750 Pa) and controlled at the same liquid level. To examine the selectivity of the PMDA-SFNM, the adsorption capacities for different proteins, such as lysozyme, bromelain, papain, BSA, ovalbumin, and pepsin, were measured and compared, respectively. In addition, the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to evaluate the adsorption capacity of the PMDA-SFNM for the protein mixture. The reusability of the PMDA-SFNM was evaluated. The adsorption and desorption experiments were carried out for 10 cycles. After adsorption of lysozyme, the PMDA-SFNM was immersed in 1.0 mol L−1 of NaCl solution until there was no lysozyme detected in the effluent. Afterward, the membranes were washed for 3 times and then dried in the oven. Finally, the regenerated membranes were repeatedly used for adsorption of lysozyme. The adsorption capacity was also evaluated. Lysozyme Separation from Fresh Chicken Egg-White. Prior to lysozyme separation, chicken egg-white from fresh chicken eggs was diluted 5 times in a phosphate buffer (PBS) (pH 6.0) with stirring for 3 h, then the solution was centrifuged (15 000 rpm) for 20 min to remove the sediment, the supernatant solution was used as the lysozyme resource. In a typical adsorption test, 50 mg of PMDASFNM was immersed in 50 mL of prepared chicken egg-white solution with shaking at room temperature for 2 h. After it was washed by PBS three times, the adsorbed lysozyme was eluted by using 1 mol L−1 of NaCl aqueous solution. The composition of residual chicken egg white and the eluted lysozyme was examined by SDS-PAGE (4−20% gradient gel from Bio-Rad with standard Coomassie blue staining protocols). Characterization. Surface morphologies of adsorbents before and after grafting PMDA were observed by using a field emission scanning electron microscope (FE-SEM, Philips XL30, USA). Fourier transform infrared spectra (FT-IR) were recorded by a Nicolet 6700 FTIR spectrometer (Thermo Electron Co., USA) in transmittance mode for all the samples in the range of 4000−400 cm−1, and the scan times and resolution were 64 and 4 cm−1, respectively. Dynamic water permeation capacity was measured and analyzed by using a Dino Capture software bundled with a Dino-Lite microscope (AM413T5 Dino-Lite Pro, AnMo Electronics Corporation, Hsinchu, Taiwan). The Brunauer−Emmet−Teller (BET) surface area was characterized using N2 adsorption−desorption isotherms with a surface area analyzer (ASAP 2020, micromeritics Co., USA). The selectivity capacity was analyzed by SDS-PAGE (4−20% gradient gel from Bio-Rad with standard Coomassie blue staining protocols).

Figure 1. SEM images of (a) silk fabrics, (b) PMDA-silk fabrics, (c) SFNM, and (d) PMDA-SFNM. (e) Diameter distribution of SFNM and (f) of PMDA-SFNM.

observed before and after grafting PMDA, indicating that the physical structure of the SFNM remained stable. The SEM images of PMDA-SFNM revealed a randomly oriented threedimensional nonwoven structure with about 125 nm diameters, which was 2 orders of magnitude smaller than that of PMDAsilk fabrics (about 11 μm). Moreover, clear adhesive points among nanofibers could be observed in the PMDA-SFNM samples, which could be attributed to the surficial swell of nanofibers during modification reaction in THF. These adhesive points could further provide the stable ability of the nanofibrous structure.13 In addition, the diameter distribution of SFNM and PMDA-SFNM are shown in Figure 1e and f, revealing that there was no obvious change in its diameter after the grafting treatment of SFNM. The intrinsic hydrophilicity of a surface could be improved by it being textured with multiple scales of roughness, and the unique nanofibrous architecture and ultrafine nanofibers effectively create functional silk surfaces with improved water wettability.29,30 Water contact and permeation measurements of the PMDA-SFNM and PMDA-silk fabrics are shown in Figure 2a and b. The PMDA-SFNM exhibits promising super8779


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Figure 2. Dynamic photographic measurements of water permeation on the surface of (a) PMDA-SFNM and (b) PMDA-silk fabrics. (c) FT-IR spectra of SFNM and PMDA-SFNM.

hydrophilicity with fast infiltration of water in 1 s, which is obvious superior to that of the conventional PMDA-silk fabrics (about 5 s). Moreover, the effective grafting of PMDA on surfaces of SFNM was also confirmed by the FTIR spectra in Figure 2c. The appearance of new peaks of CO and C−O−C (around 1720 and 1196 cm−1) indicated the successful grafting of COOH onto the SFNM. The incorporation of the negatively charged COOH groups provided possibility for the PMDASFNM to absorb the lysozyme. The fascinating hierarchical nanofibrous structure of asprepared membranes enabled us to deeply investigate their porous structure. The relevant N2 adsorption−desorption isotherms displayed in Figure 3a exhibit the isotherm of type IV with a H3 hysteresis loop, the narrow hysteresis loop was clearly visible over the relative pressure region, which indicated the typical mesoporous capillary condensation behaviors. And the hysteresis between adsorption and desorption isotherms reveal that the relevant mesopores are open, thus it cause the difference of capillary force for N2 adsorption and desorption.31,32 Brunauer−Emmett−Teller (BET) surfaces areas analysis indicated that the SFNM and PMDA-SFNM membranes possessed similar surface areas of 14.25 and 14.02 m2 g−1, respectively, significantly higher than that of commercial fibrous materials. Moreover, fractal analysis was performed to quantitatively evaluate the irregular porous structure by employing the modified Frenkel−Halsey−Hill (FHH) theory of multilayer gas adsorption. As can be seen from the Figure 3b, the relevant FHH plots reconstructed from N2 adsorption isotherms exhibited two distinct linear regions with different slopes. The slopes according to the high coverage regions were taken to ensure a reliable determination of fractal dimension (D).33,34 The resultant calculated D values of SFNM and PMDA-SFNM membranes were 2.65 and 2.59, respectively, confirming the typical surface fractal feature with irregular porous structure. The slight decrease of D values for PMDA-SFNM revealed the relatively lower structure irregularity, which could be attributed to partial adhesion among nanofibers, as demonstrated by FE-SEM observations.

Figure 3. (a) N2 adsorption−desorption isotherms of SFNM before and after grafting. (b) FHH plots of ln(V/Vmono) against ln(ln(p0/p)) reconstructed from the relevant N2 adsorption isotherms.

Optimization of the Adsorption Performance. PMDASFNM and PMDA-silk fabrics were used for lysozyme adsorption and their adsorption capacities were examined and compared. Figure 4a shows that the adsorption capacity of PMDA-SFNM and PMDA-silk fabrics were increased rapidly with the increasing adsorption time and reached equilibrium within 4 and 8 h, respectively. The adsorption capacity of the PMDA-SFNM reached 710 mg g−1, which is significantly superior to that of the previously reported separation nanofibrous membranes.16,17,35 This performance is also more than two times higher than that of the PMDA-silk fabrics (330 mg g−1), confirming the feature of smaller diameters and better hydrophilicity of PMDA-SFNM. The coomassie brilliant blue G-250 (with brown color) was used to dye the lysozyme in solution so as to display the concentration changes of the protein before and after adsorption using PMDA-SFNM, and the results are shown in Figure 4b. The lysozyme solution initially presented brilliant blue and the color changed to cyan after adsorption by PMDA-SFNM for the decrease in lysozyme concentration. Additionally, the adsorption capacity of lysozyme on PMDA-SFNM increased with increasing concentration of PMDA and finally attains equilibrium when PMDA contents reached 4 wt %. To achieve optimiztic performance, the effect of conditions, such as pH values and concentration of salt on adsorption of lysozyme, were intensively examined. The PMDA-SFNM membranes trapped proteins via electric charge adsorption. 8780


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Figure 4. (a) Kinetic adsorption performance of PMDA-silk fabrics and PMDA-SFNM as a function of time. (b) Adsorption performance of PMDASFNM at different PMDA concentrations. The insets are the optical images of (I) lysozyme aqueous solution, (II) Coomassie brilliant blue G-250, and (III and IV) before and after adsorbing lysozyme. (c and d) Effect of pH values and NaCl concentration on the adsorption capacity of PMDASFNM.

Thus, this electrostatic interactions could be broken by pH and ionic strength in solution. The pI value of lysozyme is about 10.8, which is positively charged below its pI value. As shown in Figure 4c, the PMDA-SFNM exhibited negative charge due to the surface modified carboxyl groups, which could facilely adsorb positively charged lysozyme in acidic and neutral conditions (pH < 7).36 However, with further increasing the pH to 10 (close to the pI values of lysozyme), the positive charge of lysozyme was rapidly decreased to neutral, causing the obvious decrease of adsorption capacity. Moreover, the process is also reversible, and desorption of the proteins by facilely using NaCl aqueous solution. It can be seen from Figure 4d that the adsorption capacity of PMDA-SFNM for lysozyme was decreased with increasing concentration of NaCl. This was attributed to the weakened electrostatic forces between lysozyme and carboxyl groups with increasing ionic strength, thereby resulting in the deceased adsorption capacity. Kinetic Study of the Adsorption Process. Adsorption equilibrium uptakes by the PMDA-SFNM under different initial lysozyme concentrations were measured and compared, respectively, and the results are shown in Figure 5a. The adsorption capacity gradually reached a plateau with increasing concentration of lysozyme, and the equilibrium value of 728 mg g−1 was obtained. Moreover, the adsorption data from Figure 5a were evaluated by means of two popular adsorption models, Langmuir and Freundlich equations. The applicability of the isotherm equation to describe the adsorption process was judged by the correlation coefficients (R2 values). For Langmuir isotherm, the plots of inverse equilibrium lysozyme adsorption capacity (1/Qe) versus equilibrium lysozyme concentration (1/ Ce) give a straight line and Qmax (the maximum adsorption capacity when the nanofibrous membranes is fully covered with

Figure 5. (a) Effect of the initial lysozyme concentration on the adsorption capacity of PMDA-SFNM. (b) Breakthrough curve of lysozyme solution through the PMDA-SFNM.

lysozyme), and kL (Langmuir adsorption constant) can be calculated from the intercept and slope of the plots.38 In the case of Freundlich isotherm, the plot of log Qe versus log Ce gives a straight line with slope of 1/n.38,39 Thus, Freundlich 8781


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ACS Sustainable Chemistry & Engineering constants KF and n were calculated. The results are shown in Table 1. The correlation coefficient (R2) for Langmuir was Table 1. Langmuir and Freundlich Model Parameters for Lysozyme Adsorption on PMDA-SFNM Langmuir

Freundlich

1/Qe = 0.012/Ce − 0.001

log Qe = 1.318 log Ce + l.938

Qmax (mg g−1)

KL (mL mg−1)

R2

KF

n

R2

769.2308

0.1048

0.9990

86.8560

0.7583

0.9860

0.9990, which was higher than that for Freundlich model (0.9860), indicating that the adsorption process followed the Langmuir monolayer adsorption rather than heterogeneous surface adsorption. Moreover, the theoretical maximum capacity calculated based on Langmuir model was 769 mg g−1, which was close to the experimental result. Dynamic lysozyme adsorption performance of PMDASFNM was examined, respectively, by measuring concentrations of lysozyme in effluent solution. Figure 5b shows that the effluent concentration of lysozyme increased gradually with increasing elution volume and reached the equilibrium value when the elution volume was approximately 20 mL. The dynamic binding capacity was calculated to be about 656 mg g−1, which was approximately 90% of the maximum static adsorption capacity. It is worth noting that this dynamic breakthrough experiment was carried out under the pressure of 1000 Pa, which was actually lower than that of the high driven pressure for other commercial adsorbents (higher than 105 Pa).40,41 It demonstrated that the PMDA-SFNM could greatly decrease the energy consumption and improve the reaction rate, which is very vital for their real applications. Selectivity and Reversibility. Selective adsorption ability for different types of proteins also plays a significant role in the practical applications of protein adsorbents. In this study, six proteins including lysozyme, bromelain, papain, BSA, ovalbumin, and pepsin were used for adsorption test by the PMDASFNM under the same conditions. The corresponding pI values of the proteins are 10.8, 9.5, 8.75, 4.8, 4.7, and 1, respectively.37 It is shown from Figure 6a that the PMDA-SFNM could only adsorb positively charged proteins including lysozyme, bromelain, and papain with capacities of 710, 157, and 379 mg g−1, respectively. The difference in adsorption capacities might be attributed to the distinctions in surface charges and molecule size among different types of proteins.37,42 In addition, the adsorption capacity toward different proteins were measured and analyzed by SDS-PAGE before and after adsorption. It was seen from Figure 6b that bands of positively charged proteins were reduced for the electrostatic attractive forces between proteins and PMDA-SFNM, indicating that the PMDA-SFNM exhibited the good adsorption capacity for positively charges proteins. To further prove the selective adsorption function, we have tested the lysozyme adsorption from fresh chicken egg-white, which is the most widely studied model to evaluate the performance of lysozyme affinity absorbents since egg-white is composed of a mixture of competitive proteins. Briefly, 50 mg of PMDA-SFNM was immersed in 50 mL of diluted egg-white, and it was shaken at room temperature for 2 h. After washing by PBS, the adsorbed lysozyme was eluted by NaCl solution. The results were analyzed by SDS-PAGE, as shown in Figure 7. As can be seen from lane 2, the fresh chicken egg white

Figure 6. (a) Adsorption capacity of PMDA-SFNM toward different proteins. (b) SDS-PAGE analysis for purification of a mixture of lysozyme, bromelain, papain, BSA, ovalbumin, and pepsin by staining coomassie blue: (1) the protein ladder, (2) the protein solution, and (3) the protein solution after adsorption by PMDA-SFNM.

Figure 7. SDS-PAGE analysis of protein solution from standard broad range ladder (lane 1), fresh chicken egg-white (lane 2), standard lysozyme (lane 3), chicken-egg white after adsorption (lane 4), and eluted protein solutions from absorbed PMDA-SFNM (lane 5).

exhibited the typical major protein components, ovotransferrin, ovalbumin, ovomucoid, and lysozyme with molecular weights at 76k, 45k, 28k, and 14k Dalton, respectively. After adsorption by PMDA-SFNM, the intensity of lysozyme pattern was obvious decreased (lane 3), indicating the adsorption of lysozyme. Moreover, the eluate from the PMDA-SFNM exhibited only one strong protein band (lane 4), revealing the high purity of eluted lysozyme. These promising results demonstrate the great potential of the as-prepared PMDA-SFNM for real lysozyme 8782


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ACS Sustainable Chemistry & Engineering *E-mail: [email protected].

separation applications since the egg-white is the major bio source for lysozyme extraction. To evaluate the economic plausibility, it is very important for us to discuss the reusability of the functional membranes. We performed the reversibility of PMDA-SFNM for 10 times. The influence of repetitive usage on adsorption of lysozyme shown in Figure 8, the stable adsorption capacity of PMDA-SFNM

ORCID

Yang Si: 0000-0001-7209-6206 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.

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ACKNOWLEDGMENTS This research was supported by National Natural Science Foundation of China (No. 21506173), Natural Sc ience Foundation of Chongqing (No. cstc2016jcyjA0210), China Postdoctoral Science Foundation (No. 2015M582503), Chongqing Postdoctoral Science Foundation (No. Xm2015122), and the USA National Institute of Food and Agriculture (USDA-NIFA Award 2015-68003-23411).

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Figure 8. Reuse capacity of PMDA-SFNM for 10 cycles. The insets are the SEM images of PMDA-SFNM after 10 cycles.

(1) Jun, J. I.; Kim, K. H.; Lau, L. F. The matricellular protein CCN1 mediates neutrophil efferocytosis in cutaneous wound healing. Nat. Commun. 2015, 6, 7386. (2) Li, Y.; Budamagunta, M. S.; Luo, J.; Xiao, W.; Voss, J. C.; Lam, K. S. Probing of the assembly structure and dynamics within nanoparticles during interaction with blood proteins. ACS Nano 2012, 6, 9485−9495. (3) Müller, K.; Faeh, C.; Diederich, F. Fluorine in pharmaceuticals: looking beyond intuition. Science 2007, 317, 1881−1886. (4) Kiristi, M.; Singh, V. V.; Esteban-Fernández de Avila, B.; Uygun, M.; Soto, F.; Aktaş Uygun, D.; Wang, J. Lysozyme-based antibacterial nanomotors. ACS Nano 2015, 9, 9252−9259. (5) Muriel-Galet, V.; Talbert, J. N.; Hernandez-Munoz, P.; Gavara, R.; Goddard, J. M. Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications. J. Agric. Food Chem. 2013, 61, 6720−6727. (6) Raheja, A.; Agarwal, A.; Muthuvijayan, V.; Chandra, T. S.; Natarajan, T. S. Studies on encapsulation of bovine serum albumin, lysozyme and insulin through coaxial electrospinning. J. Biomater. Tissue Eng. 2013, 3, 669−672. (7) Ercan, D.; Demirci, A. Recent advances for the production and recovery methods of lysozyme. Crit. Rev. Biotechnol. 2016, 36 (6), 1078−1088. (8) Stradner, A.; Sedgwick, H.; Cardinaux, F.; Poon, W. C.; Egelhaaf, S. U.; Schurtenberger, P. Equilibrium cluster formation in concentrated protein solutions and colloids. Nature 2004, 432, 492−495. (9) Abeyrathne, E.; Lee, H.; Ahn, D. Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agentsA review. Poult. Sci. 2013, 92, 3292−3299. (10) Campbell, L.; Raikos, V.; Euston, S. R. Modification of functional properties of egg-white proteins. Nahrung 2003, 47, 369− 376. (11) Li, Y.; Lin, T.-y.; Luo, Y.; Liu, Q.; Xiao, W.; Guo, W.; Lac, D.; Zhang, H.; Feng, C.; Wachsmann-Hogiu, S.; et al. A smart and versatile theranostic nanomedicine platform based on nanoporphyrin. Nat. Commun. 2014, 5, 4712. (12) Faivre, D. Multifunctional materials: Dry but flexible magnetic materials. Nat. Nanotechnol. 2010, 5, 562−563. (13) Si, Y.; Yu, J.; Tang, X.; Ge, J.; Ding, B. Ultralight nanofibreassembled cellular aerogels with superelasticity and multifunctionality. Nat. Commun. 2014, 5, 5802. (14) Si, Y.; Fu, Q.; Wang, X.; Zhu, J.; Yu, J.; Sun, G.; Ding, B. Superelastic and superhydrophobic nanofiber-assembled cellular aerogels for effective separation of oil/water emulsions. ACS Nano 2015, 9, 3791−3799.

remained after being reused for 10 cycles. The SEM image of the membranes used for 10 cycles was obtained (insert, in Figure 8), without obvious changes for its structure. Moreover, we have measured the released amount of the lysozyme after each recycling test, as shown in Figure S1. After each test, the released amounts of the lysozyme were about 630−660 mg g−1, indicating over 90% of the recycling efficiency. The obtained results suggested that the prepared PMDA-SFNM would greatly improve the practical application for the adsorption of lysozyme.

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CONCLUSIONS In summary, we have presented a sustainable and synergistic assembly strategy for the scalable fabrication of functionalized SFNM by a combination of electrospinning technique and chemical modification of silk proteins. For the first time, naturally abundant silk is reconstructed into nanofibrous membranes with tunable surface function on a large scale. With their ultrathin fiber diameter (125 nm), larger surface area, high porosity, superhydrophilicity, negatively charged fiber surface, the SFNM can selectively and reversibly adsorb lysozyme with robust capacity of 710 mg g−1 and high durability. We envision that such exceptional SFNM will open up numerous opportunities as the next generation of protein adsorbents for rapid, massive, and cost-effective separation and purification of proteins.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01580. Table of amino acids with amino and hydroxyl at side chain and results for the released amount of the lysozyme after each recycling test (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. 8783


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Ultrafine Silk-Derived Nanofibrous Membranes ... - ACS Publications

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