Article pubs.acs.org/Langmuir
Nanoporous Gyroid-Structured Epoxy from Block Copolymer Templates for High Protein Adsorbability Xin-Bo Wang,‡,† Tze-Chung Lin,† Han-Yu Hsueh,† Shih-Chieh Lin,† Xiao-Dong He,§ and Rong-Ming Ho*,† ‡
School of Materials Science and Engineering, Harbin Institute of Technology at Weihai, Weihai, Shandong 264209, China Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan § Center for Composite Materials and Structures, Harbin Institute of Technology, Harbin 150080, China †
S Supporting Information *
ABSTRACT: Nanoporous epoxy with gyroid texture is fabricated by using a nanoporous polymer with gyroid-forming nanochannels as a template for polymerization of epoxy. The nanoporous polymer template is obtained from the self-assembly of degradable block copolymer, polystyrene-b-poly(L-lactide) (PS−PLLA), followed by hydrolysis of PLLA blocks. Templated polymerization can be conducted under ambient conditions to create well-defined, bicontinuous epoxy networks in a PS matrix. By taking advantage of multistep curing of epoxy, well-ordered robust nanoporous epoxy can be obtained after removal of PS template, giving robust porous materials. The through-hole nanoporous epoxy in the film state can be used as a coated layer to enhance the adsorbability for both lysozyme and bovine serum albumin.
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tion.15,16 One feasible method to fulfill the requirements for the fabrication of well-defined through-hole nanoporous epoxy is to acquire it through templated synthesis using self-assembled materials as templates. In recent decades, block copolymers (BCPs) have received intensive attention because of their ability to self-assemble into various ordered nanostructures such as sphere, cylinder, gyroid, and lamellae.17,18 Moreover, the nanostructures and the microdomain sizes of BCPs can be easily regulated by simply changing volume fraction and molecular weight. Among microphase-separated phases from the self-assembly of BCPs, gyroid is one of the most appealing morphologies because of its unique texture, composed of cocontinuous, interpenetrated networks in a matrix.19,20 BCPs containing chemically degradable blocks are versatile precursors to fabricate welldefined nanoporous materials.21,22 There are different methods to etch the degradable block of BCPs such as ozonolysis,23 UV degradation,24 and reactive ion etching.25,26 Also, polylactidecontaining BCPs, such as polystyrene-b-poly(D,L-lactide) (PS− PLA)27 and polystyrene-b-poly(L-lactide) (PS−PLLA),28 have been used to fabricate nanoporous polymer materials by hydrolysis at which polylactides can be hydrolytically degraded in base aqueous solution. With the use of gyroid-forming degradable BCPs, well-ordered nanoporous materials possessing interconnected nanochannels with through-hole property can be easily fabricated through selective etching of the minor
INTRODUCTION High-performance polymers, such as epoxy, provide a wide range of applications in novel devices. Cured epoxy exhibits good chemical resistance, outstanding adhesion, high electrical insulation and thermal stability, in addition to excellent mechanical properties and has been extensively used as a matrix in anticorrosive paints, microelectronic packaging, composites, and construction.1 One promising approach for the use of epoxy in practical applications is to create welldefined nanopores within the polymer matrix. Porous materials have scientific and technological interests because of their wide applications such as catalyst carriers,2,3 separators,4−6 sensors,3,7,8 photonics,9,10 absorbers,11 and reactors,12 yet many porous materials have micrometer-sized pores, which limit their further applications in nanotechnologies. Therefore, porous materials with nanometer-sized pores, in particular, with precise design and tailoring, are highly demanded to give desired functionalities and properties at nanoscale (e.g., high porosity and large specific surface area). With the accessibility for the control of uniform pore size and architecture (in particular, continuous and interpenetrating nanochannels with throughhole property), important attention has been paid to the adsorption capability of nanoporous epoxy because of the feasibility for the functionalization of the nanopore with numerous polar groups, such as hydroxyl, amine, and ether linkage.13,14 As a result, with the combination of the advantage of polar and biocompatible properties, it is highly appealing to use nanoporous epoxy in the applications of biosupporters for tissue attachment and bioscaffolds for protein immobiliza© XXXX American Chemical Society
Received: May 8, 2016
A
DOI: 10.1021/acs.langmuir.6b01765 Langmuir XXXX, XXX, XXX−XXX
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Figure 1. Schematic illustration of fabricating nanoporous gyroid-structured epoxy from a BCP template: (a) gyroid-forming PS−PLLA, (b) nanoporous gyroid PS template after hydrolysis of PLLA blocks, (c) PS/epoxy nanohybrids from templated synthesis of epoxy via thermal curing, and (d) nanoporous gyroid-structured epoxy after removal of PS templates.
template to fabricate a mesoporous silica KIT-6/epoxy polymer composites, which can be served as low thermal expansion material.35 Taylor and coworkers prepared a gyroid-structured epoxy/block copolymer composites using a triblock copolymer in which the triblock copolymer is the gyroid skeleton and epoxy is a matrix.36 Nevertheless, it is still a challenge to fabricate nanoporous epoxy with well-defined nanostructured textures. Herein, we aim to demonstrate a new method for the fabrication of well-ordered nanoporous gyroid polymer such as epoxy from BCP templates. In contrast with the nanostructured epoxy as described above, the approach demonstrated in this study provides the feasibility to fabricate nanoporous epoxy with ordered gyroid textures and controllable pore sizes from a hard-template method instead of a soft-template method.22 For hard-template method it is straightforward because it is much easier for the control of templated morphologies and also for the fabrication of ordered nanomaterials with orientation control in large area. Figure 1 schematically illustrates the approach used in this study. Figure 1a shows the texture of a gyroid phase with cocontinuous PLLA networks in a PS matrix from the self-assembly of PS−PLLA after solution casting. PLLA networks are selectively hydrolyzed to give a nanoporous gyroid PS as a template for polymerization of epoxy (Figure 1b). Epoxy and hardener are driven into the nanoporous PS through capillary force. Subsequently, gyroid-structured PS/ epoxy nanohybrids can be formed (Figure 1c) through templated polymerization of epoxy. After the removal of the PS template, nanoporous gyroid-structured epoxy can be successfully fabricated (Figure 1d), giving nanoporous epoxy with high porosity due to the gyroid-forming texture and thus large specific surface area, as demonstrated by the feasibility of using such a nanoporous materials with through-hole character for high protein adsorbability.
phase in the self-assembled gyroid phase, giving high porosity and large specific surface area. Consequently, the well-ordered nanoporous polymer can be used as a template (nanoreactor) for templated syntheses, such as sol−gel reaction, electroplating, electroless plating, and atomic layer deposition, to fabricate a variety of nanohybrids.22 After the removal of the polymer template, various well-defined nanoporous materials, such as nanoporous metal oxides and nanoporous metals, can be formed. For the fabrication of well-defined through-hole nanoporous epoxy, one straightforward approach is to create epoxycontaining BCPs with gyroid phase and degradable components as constituted block, yet it requires BCPs from precise synthesis (i.e., living polymerization) to create the BCPs with narrow molecular weight distribution for self-assembly to give the well-ordered cocontinuous texture. For the synthesis of epoxy, controlled polymerization with living character is unfeasible. It is noted that the self-assembly of BCPs has been developed for fabrication of nanostructured epoxy.29−31 Hillmyer and coworkers employed amphiphilic BCPs containing epoxy-miscible block to form well-ordered amphiphilic BCP/epoxy nanocomposites.32 Lodge and coworkers prepared a three-dimensionally bicontinuous epoxy network by using a solid nanoporous template derived from a polymeric bicontinuous microemulsion (BμE), giving two interpenetrating, three-dimensionally continuous networks with extensive interfacial area and periodicity on the order of 100 nm. Furthermore, by replicating the inverse structure of such a BμEderived nanoporous materials through nanocasting, porous epoxy can be obtained after crystallization or vitrification of one component and selective extraction of another.33 Thomas and coworkers fabricated periodic bicontinuous porous epoxy using interference lithography for the fabrication of porous templates, which revealed enhanced energy dissipation performance.34 Yamauchi and coworkers used gyroid-structured silica as a B
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Langmuir Table 1. Characterization of PS−PLLA Synthesized
a
code
Mn,PS (g/mol)a
ĐM,PSa
Mn,PLLA (g/mol)b
ĐM a
Mn,total
f PLLAv
phase
d spacing (nm)c
BCP-L BCP-M BCP-H
10 200 32 400 45 000
1.14 1.16 1.15
7000 23 200 29 400
1.15 1.24 1.17
17 200 55 00 74 400
0.35 0.37 0.35
gyroid gyroid gyroid
16.9 46.9 50.7
Data were calculated according to GPC.
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b1
H-NMR results cSAXS results. conducted at 37 °C for 2 h under gentle shaking. The suspension was then centrifuged and washed with PBS five times. The supernatant was collected together, filtered through a membrane with the pore diameter 0.45 μm, and then diluted to 500 mL with PBS buffer solution. Subsequently, the supernatant was analyzed by ultraviolet absorbance at 280 nm to determine the adsorption amount of protein. The adsorption test was performed three times and each value of final adsorption amount was averaged. The following formula was adopted to determine the adsorption amount of protein (A, mg/g) based on epoxy
EXPERIMENTAL SECTION
Synthesis Procedures. The detailed synthetic routes are shown in the Supporting Information (SI). H-Spectrum of Nuclear Magnetic Resonance (1H NMR). All 1 H NMR spectra were record on a Varian INOVA 500 NMR spectrometer. The 1H NMR samples were prepared in deuterated chloroform with concentration 1 wt %. Gel Permeation Chromatography. Polymers were welldissolved in tetrahydrofuran (THF) at a concentration of 0.2 wt % and passed through a 0.45 μm Teflon syringe filter to remove possible particles. The gel permeation chromatography (GPC) is equipped with three columns (PL-gel 5 μ, 30 cm mixed-C, Polymer Laboratories) at 35 °C with a flow rate of 1 mL/min as mobile phase. The elution time was detected by Waters 2414 Refractive Index Detector. The molecular weight and polydispersity were calculated by the Water Breeze software. Calibration was performed using narrow distribution polystyrene standards (Polymer Laboratories). Small-Angle X-ray Scattering. Small-angle X-ray scattering (SAXS) experiments were conducted at the synchrotron X-ray beamline X27C at the National Synchrotron Radiation Research Center (NSRRC) in city of Hsinchu, Taiwan. The wavelength of the X-ray beam was 0.155 nm. A MAR CCD X-ray detector (MAR USA) was used to collect the 2D SAXS patterns. 1D linear profile was obtained by integration of the 2D pattern. The scattering angle of the SAXS patter was calibrated using silver behenate, with the first-order scattering vector q* (q* = 4πλ−1 sin θ, where 2θ is the scattering angle) being 1.076 nm−1. Transmission Electron Microscopy. Bright-field transmission electron microscopy (TEM) images were obtained using the mass− thickness contrast with a JEOL JEM-2100 LaB6 transmission electron microscope (at an accelerating voltage of 200 kV). Microtoming was carried out at room temperature by using a Leica Ultramicrotome to acquire microsections with thickness of ∼50 nm; then, the microsections were stained by exposing to the vapor of a 4% aqueous RuO4 solution for 1 h. The RuO4 can react with the double bonds of benzene ring in PS blocks, rendering the microphase-separated domains dark under TEM due to the mass−thickness contrast. Field-Emission Scanning Electron Microscopy. Field-emission scanning electron microscopy (FESEM) observations were performed on a JEOL JSM-6700F using accelerating voltages of 1.5 to 3 keV. Before observations, the samples were dried at 60 °C in vacuum overnight and then sputter-coated with 2 to 3 nm of platinum to avoid the charge effect. The platinum coating thickness was estimated from a calculated deposition rate and experimental deposition time. Specific Surface Area and Pore Size Analyzer. Nitrogen sorption isotherms were conducted on a BET-201A sorptometer from Porous Materials at −196 °C. The porous epoxy powders were degassed for 12 h at 60 °C and then for 24 h at room temperature prior to investigation. Specific surface areas were calculated using the multipoint Brunauer−Emmett−Teller (BET) method. Pore size distributions were determined by Barrett−Joyner−Halenda (BJH) method using nitrogen on carbon at −196 °C with the cylindrical pore model. Protein Adsorption Test. A recognized method was adopted for protein adsorption analysis, and the test procedure was adjusted appropriately on the basis of the examined system studied here.37 The proteins were dissolved in phosphate-buffered saline (PBS, 0.01 mol/ L, pH 7.4) to give a final concentration of 20 mg/mL. 40 mg epoxy slices (∼1 mm thick) were placed in a 6 mL centrifuge tube, to which 2 mL of prepared protein solution was added. Adsorption was
A=
(C0 V0 − Cr Vr) m
(1)
where C0 is the initial protein concentration in PBS buffer solution (mg/mL), V0 is initial volume of PBS buffer solution with protein (mL), Cr is the residual concentration of protein in PBS buffer solution (mg/mL), Vr is the residual volume of PBS buffer solution with protein (mL), and m is the mass of epoxy (g). Raman Microspectrometer. The depth profiling of protein adsorption was determined by confocal Raman microscopy. The chemical composition depth profile of the epoxy/protein bulk was characterized using a Raman microspectrometer, which combines a Renishaw Raman spectroscope and an inverted Leica DMIRBE microscopy. The 532 nm laser was used as an excitation source and was focused through a 40× objective to ∼1 μm light spot on the sample surface. Scattered light from the sample surface was collected through the same objective. Raleigh scattering light was cut off by a holographic notch filter. Raman light was passed through an entrance slit with a 65 μm opening and a 1200 L/mm diffraction grating and then measured by a CCD camera. For the distribution depth profile of the protein within the epoxy/protein bulk, Raman spectra were acquired from the surface of the focal planes at different depths.
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RESULTS AND DISCUSSION Synthesis of Block Copolymers. PS−PLLA block copolymers were synthesized successively by atom transfer radical polymerization (ATRP) and ring-opening polymerization (ROP) using a double-headed initiator. Synthetic details (Scheme S1) were described in our previously published results, except the catalyst of ROP was changed into AlEt3.38 The number-average and weight-average molecular weight as well as the molecular weight distribution (ĐM) of PS block were determined by GPC. The ĐM of PS−PLLA was determined by GPC, and the numbers of L-lactide repeating units versus styrene repeating units were determined by 1H NMR analysis to calculate the molecular weight synthesized. On the basis of the molecular weight synthesized, the volume fraction of PLLA ( f PLLAv) was calculated by assuming the densities of PS and PLLA are 1.02 and 1.251 g/cm3, respectively. The characterizations of synthesized PS−PLLA were summarized in Table 1. Three PS−PLLA BCPs with different molecular weights were synthesized and denoted as BCP-L, BCP-M, and BCP-H in accordance with their corresponding molecular weights at which L, M, and H stand for low, intermediate, and high molecular weights, respectively. Self-Assembly of PS−PLLA. Bulk samples of PS−PLLA BCPs were prepared by solution casting from dichloromethane (CH2Cl2) solution (10 wt % of PS−PLLA) at room C
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The [110] projection images suggest the formation of gyroid phase for all samples examined. The interdomain spacings of (211)G plane can be approximately determined as 16, 45, and 49 nm from the TEM images for the BCP-L, BCP-M, and BCP-H, respectively. Figure 3a shows 1D SAXS profile with diffraction peaks at the relative q values of √6:√8:√14:√16:√26:√32:√38:√50, as marked by the triangle arrows, suggesting the formation of gyroid phase (Figure 2a−c). According to the primary reflection, the interdomain spacings of (211)G plane can be found to be increased with the molecular weight of the BCPs and determined as 16.9, 46.9, and 50.7 nm for the BCP-L, BCPM, and BCP-H, respectively; the results are consistent to the measurements from TEM micrographs. Fabrication of Gyroid-Forming PS Template. The PLLA blocks of the thermally treated PS−PLLA bulk samples were removed by hydrolysis using a 0.5 M basic solution that was prepared by dissolving 2 g of sodium hydroxide in a 40/60 (by volume) solution of methanol/water. After 3 days of hydrolysis at room temperature, the hydrolyzed samples were rinsed using a mixture of deionized water and methanol and then used as templates for the following polymerization of epoxy. After hydrolysis in a mild aqueous base solution, the PLLA blocks of the PS−PLLA can be removed completely. The diffraction peaks at the relative q values remain unchanged as compared with those of corresponding BCPs (as represented in Figure 3b for BCPM), reflecting the successful preservation of the self-assembled texture after hydrolysis. As a result, the interdomain spacing of (211)G plane of the nanoporous PS template can be determined as 46.9 nm from the primary reflection, which is equivalent to that of intrinsic BCP from self-assembly. Pore-Filling Nanoporous PS Template. For a successful pore-filling process in templated synthesis, appropriate solvents are required to enhance the wetting tendency of the reactants into the PS template through capillary force. Considering the hydrophobicity of PS inner wall, amphiphilic substances, such as short chain alcohols, are usually used. It is noted that there is a competition between polymerization of epoxy from curing and its pore-filling process into the PS templates. If the polymerization occurs before pore-filling process, blocking of nanopores will terminate the following templated synthesis.
temperature in a glass bottle well sealed by aluminum foil with punch holes for vapor releasing. After standing for 2 weeks, the bulk samples were dried in a vacuum oven at 65 °C for 3 days. The dried samples were first heated to the maximum annealing temperature, Tmax = 180 °C, for 5 min to eliminate the crystalline residues of the PLLA block that were formed during the preparation procedure. After thermal treatment, the samples were quenched to ambient condition for SAXS experiments and then sectioned by ultramicrotome for TEM observation. Figure 2a−c shows the TEM images of PS−PLLA BCPs with different molecular weights at which the PS microdomains
Figure 2. TEM images of PS−PLLA BCPs with different molecular weights: (a) BCP-L, (b) BCP-M, (c) BCP-H, and (d) TEM micrograph of PS/epoxy nanohybrids from (b) after hydrolysis followed by templated synthesis. Panels a−c show results from RuO4 staining, whereas panel d is from OsO4 staining.
appear dark, whereas the PLLA microdomains are bright because of the mass−thickness contrast from RuO4 staining.
Figure 3. 1D SAXS profiles of (a) BCP-L, BCP-M and BCP-H, (b) BCP-M, nanoporous PS template from BCP-M and PS/epoxy nanohybrids fabricated from templated polymerization, and (c) porous epoxy (acquired from BCP-M) with multistep thermal curing (solid) and single-step thermal step (dashed). D
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Langmuir Note that through the curing reaction of the epoxy resins at room temperature the blocking problem for pore-filling process can be alleviated by lowering the reaction rate due to the low reaction temperature. A mixture of Bisphenol A type epoxy (DER 331, Dow Chemical) and triethylene tetramine (TETA) (DEH 24, Dow Chemical) dissolved in methanol (epoxy/ TETA/methanol = 1.33 g:0.17 g:1 mL) to form a transparent precursor solution was prepared first. To acquire complete pore-filling process, the nanoporous PS templates prepared was immersed into the methanol solution with the mixture for several hours at room temperature. Subsequently, precuring polymerization at 60 °C below the glass-transition temperature of the PS template was carried out for fabrication of PS/epoxy (low cross-linking density) to preserve the templated morphology. A multistep postcuring will be carried out to give robust network after removal of the PS matrix (see below for details). Fabrication of PS/Epoxy Nanohybrids. The PS template (∼1 cm2 in area and 1 mm in thickness) was immersed in the precursor solution with stirring at room temperature for several hours. During this stage, epoxy and its hardener were taken into the nanoporous PS template by virtue of capillary force. It is at the end of this stage that epoxy appears in the rubber state due to low cross-linking density, which makes PS template able to be separated from its ambient rubber-like epoxy. Subsequently, the epoxy pore-filled PS was acquired, then heated to 60 °C for 12 h followed by 150 °C for 0.5 h. Figure 2d shows the TEM image of PS/epoxy nanohybrids. Note that OsO4 can be used to stain the C−O−C bond in the cured epoxy, giving significant mass−thickness contrast for TEM observations. As a result, in contrast with the TEM image of BCP-M (Figure 2b), inverse mass−thickness contrast can be observed in Figure 2d. The epoxy microdomains appear dark, whereas the PS microdomains are bright by using OsO4 for staining. Consequently, the [110] projection image from the epoxy microdomains suggests the formation of gyroid-forming epoxy networks in the PS matrix. The diameter of the gyroid-structured epoxy is ∼26 nm as determined from Figure 2d, nearly equal to the diameter of PLLA microdomain in Figure 2b. The 1D SAXS results (Figure 3b) further confirm the observed gyroid phase in which gyroid characteristic peaks at the relative q value of √6 and √8 can be clearly identified. In contrast with the nanoporous PS template and PS−PLLA, two major peaks at the relative q value of √6 and √8 can still be clearly identified for the PS/epoxy nanohybrids, even though the characteristic peaks of gyroid at high relative q value region are diminished due to the low electron-density contrast for X-ray scattering between epoxy and PS, reflecting that the polymerization of epoxy can be successfully carried out within the nanochannels of the PS template. Notably, the interdomain spacing of (211)G plane of the PS/epoxy nanohybrids is ∼46 nm, as determined from the primary reflection of 1D SAXS, indicating a slightly shrinkage from the dimension of PS template after the templated polymerization. We speculate that the change is attributed to the intrinsic behavior from the curing of epoxy at which shrinkage should be expected due to the cross-linking reaction of epoxy resins. To further confirm the formation of the PS/ epoxy nanohybrids, we applied Fourier transform infrared spectroscopy (FTIR) to identify the chemical structure of PS/ epoxy nanohybrids. Scheme 1 shows the epoxy and amine hardener used in this study and the suggested chemical reactions of epoxy curing. It
Scheme 1. Curing of Epoxy Resin: (a) Epoxy and Amine Hardener Used in This Study and (b) Curing Mechanism of Epoxy39
is known that amines are efficient hardeners for epoxy to perform curing reaction. Here bisphenol A type of epoxy resin and its hardener were used for the curing of epoxy within the nanoporous PS template (Scheme 1a). When epoxy and amine are mixed in appropriate proportions, epoxy group and amine group react easily even at room temperature. Eventually, crosslinked macromolecules structure forms after complete curing reaction (Scheme 1b). Note that there is ether bond along the backbone of epoxy and large number of hydroxyl at the side chains; after complete curing reaction, there will be the formation of tertiary amine. A model study for the corresponding reactions as illustrated was carried out; as shown in Figures 4i,ii, the epoxy group at 915 cm−1 will disappear after thermal treatment at 150 °C for 30 min, indicating the occurrence of a complete curing reaction with high-degree cross-linking density after the thermal treatment.40 By contrast, for the PS template (Figure 4iii), an out-of-plane vibration of the benzene ring at 906 cm−1 can be found and distinguished from the neighboring peak of the epoxy group at 915 cm−1. As a result, it is feasible to use the characteristic peak at 915 cm−1 as an indicator to trace the degree of cross-linking density of the epoxy from curing. Moreover, the characteristic peak of C−N stretching vibration of the epoxy can also be clearly identified at 1249 cm−1.41 On the basis of the spectroscopic results, it is reasonable to suggest that a complete curing of the epoxy resins can be successfully achieved through appropriate thermal treatments. E
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enhance the solvent resistance of the gyroid-forming epoxy networks. A post cure is to provide additional heat for the development of the epoxy to reach its full physical characteristics. With the evolution of curing and thus hardening, the ability for the molecules of resin to react with hardener for cross-linking gradually becomes difficult. Although internal stress can be eliminated by increasing curing time at lower temperature (i.e., precuring), the optimal curing conditions of epoxy for performance (e.g., Tg, hardness and mechanical strength) is difficult to achieve. As a result, a postcuring treatment at higher temperature is usually conducted after precuring at lower temperature.42 High-temperature curing enables fast reaction of chemical cross-linking and gives higher cross-linking density, but it will cause higher internal stress due to fast reaction. One way to solve the dilemma is to carry out a multistep curing. In this case, the temperature is gradually raised to the final setting temperature for curing to provide a good balance for acquiring higher degree of curing and less building of internal stress.43 At first, direct heating from room temperature to 150 °C was conducted, but it will cause the disordering of the forming morphology after thermal treatment, as shown in Figure 5a and further evidenced by SAXS results (Figure 3c, dashed). This is attributed to the fast heating as mentioned above, which results in high internal stress, leading to the deformation of epoxy skeleton while using styrene to dissolve the PS matrix. Consequently, a multistep curing was used by heating the sample to 60 °C for 12 h, followed by 150 °C for 30 min. As evidenced in Figure 4v, after the thermal treatment, the characteristic peak at 915 cm−1 will completely disappear and only the characteristic peak at 906 cm−1 remains, suggesting that the cross-linking density should be high enough (similar to the results from the model system) to provide the required mechanical strength for the following solvent treatment to remove the PS template. As shown in Figure 4vi, neither characteristic peak of epoxy group at 915 cm−1 nor neither characteristic peak of PS at 906 cm−1 can be recognized once epoxy resins are completely reacted, followed by removal of the PS template using styrene as solvent. Therefore, nanoporous robust epoxy networks with gyroid nanostructure can be obtained (Figure 5b), and, most importantly, the ordered structure can be preserved as evidenced by the significant reflection results (Figure 3c, solid) in contrast with the disorder-like scattering (Figure 3c, dashed). In Figure 3c, the SAXS result was acquired from the nanoporous epoxy (Figure 5b) at which the removal of PS template is to carry out by using
Figure 4. FTIR spectra of (i) uncured epoxy, (ii) cured epoxy obtained on the basis of a model study (heated for 30 min at 150 °C), (iii) PS template, (iv) PS/epoxy nanohybrids with thermal treatment at 60 °C for 12 h, (v) PS/epoxy nanohybrids with thermal treatment at 60 °C for 12 h, followed by at 150 °C for 30 min, and (vi) nanoporous epoxy after removal of PS matrix from (v).
Fabrication of Nanoporous Gyroid-Structured Epoxy. For the fabrication of nanoporous gyroid-structured epoxy, it is necessary to completely remove the PS template. Nevertheless, the texture of the nanoporous gyroid-structured epoxy is strongly dependent on the cross-linking degree of epoxy resin from postcuring. As found, it is difficult to remain the gyroidforming epoxy networks after removal of the PS matrix by using UV irradiation because of the simultaneous degeneration of epoxy resin. Therefore, removal of the PS template was carried out by using selective solvent; however, the forming gyroidstructured epoxy will be destroyed after the removal of the PS template by the use of selective solvents due to the swelling of the epoxy. Styrene monomer was thus used as solvent for the removal of PS to alleviate the deterioration of templated morphology, but the problem remains. We speculate that the underlying reason for solvent swelling is attributed to the low degree of cross-linking resulting from a low reaction temperature. Obviously, further thermal treatment at elevated temperature will be required. As evidenced in Figure 4iv, in addition to the peak at 906 cm−1 attributed to the PS template, the intensity of 915 cm−1 (shoulder peak) can still be found, indicating that the degree of curing is indeed not high enough to sustain the gyroid texture from styrene treatment. Accordingly, to alleviate the swelling problem, postcuring at higher temperature was conducted to
Figure 5. FESEM micrographs of porous epoxy by (a) single-step curing and (b) multistep curing. F
DOI: 10.1021/acs.langmuir.6b01765 Langmuir XXXX, XXX, XXX−XXX
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Figure 6. (a) N2 adsorption−desorption isotherms of nanoporous PS template from BCP-M; (b) corresponding pore size distribution of panel a; (c) N2 adsorption−desorption isotherms of nanoporous epoxy using PS template from BCP-M; and (d) corresponding pore size distribution of panel c. The specific surface area and the average pore size of the PS template from BCP-M are 173 m2 g−1 and 25 nm, respectively; those of nanoporous epoxy using the PS template are 411 m2 g−1 and 16 nm. The average pore size is calculated using the BJH method.
styrene to dissolve the PS matrix. Note that the PS template was prepared by the self-assembly of PS−PLLA, followed by hydrolysis; the formation of multiple grain boundary is inevitable. As a result, it is highly possibly to cause the decrease on the long-range order, while the styrene may prefer to diffuse to the grain boundaries and possibly swell the PS domains to cause the reduction in long-range order. This is exactly what we observed at which the primary reflections remain but the high q reflections gradually diminish with the dissolution time of using styrene for the purpose. Combined with the morphological observations (Figure 2d) and the scattering result (Figure 3b) as well as the FTIR characterization, the curing of epoxy after pore-filling into nanoporous gyroid PS templates can be successfully achieved to give well-ordered PS/epoxy nanohybrids. As shown in Figure 5, in contrast with the distorted gyroid (that is a cocontinuous texture but lack of ordered texture) from single-step curing, well-defined gyroid nanostructure with 3-fold symmetry texture and ordered structure can be formed from the thermal treatment with multistep curing. After multistep curing, the epoxy pore-filled PS was immersed in flowing styrene for 2 weeks at room temperature and nanoporous epoxy (∼0.75 cm2 in area and 0.8 mm in thickness) with high cross-linking density was obtained. The morphological observations are consistent with the scattering results (Figure 3c) in which there are no SAXS peaks after
single-step (dashed) curing for the nanoporous epoxy fabricated. Because of the bicontinuous networks with wellordered texture, the nanoporous gyroid-structured epoxy can be free-standing after the removal of the template, resulting in high porosity and large specific surface area. Characterization of Nanoporous Gyroid-Structured Epoxy. N2 adsorption−desorption isotherms were carried out to characterize the nanoporous epoxy fabricated. Figure 6a shows a typical type-IV isotherm (IUPAC classification). The curve exhibits a significant hysteresis loopin the high relative pressure region from 0.85 to 1 (type-IV isotherm), indicating uniform pore-size distribution in the nanoporous PS template. The hysteresis loop is close to a type H1 in which both the adsorption−desorption branches are nearly vertical, indicating that the nanoporous PS template possesses ordered texture with uniform mesopores.44 The average pore size was determined to be ∼25 nm (Figure 6b). In Figure 6c, the curves in the relative pressure between 0.85 and 1.0 show an H1 hysteresis loop, and both the adsorption and desorption curves are close to vertical, indicating that the nanoporous epoxy possesses ordered mesopores. There is no closure for the curves in the relative pressure region between 0.2 and 0.8. We speculate that this might be attributed to the dissolved nitrogen that swells the epoxy matrix, resulting in a lack of closure of the isotherm at low relative pressures. In general, G
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Article
Langmuir
interaction between protein and charged surface that plays a major role in the adsorption behavior of protein. With numerous polar groups, epoxy is believed to be a suitable adsorbent. To demonstrate the advantage of using well-ordered nanoporous gyroid-structured epoxy fabricated, two model proteins, lysozyme and bovine serum albumin (BSA), are selected for protein adsorbability study. Lysozyme contains a large number of −NH2 groups whose pI is 11; it is a small ellipsoid-shaped protein with molecular mass of 14.7 kDa and molecular size of 3 nm × 3 nm × 4.5 nm.47 BSA is a carboxylic acid-rich protein with the pI = 4.7; it is a large ellipsoid-shaped protein with molecular mass of 67 kDa and molecular size of 4 nm × 4 nm × 14 nm.48 These two proteins appear to be adsorbed into the channels of nanoporous epoxy, and lysozyme should be adsorbed much easier than BSA due to its smaller dimension from the consideration of size effect; note that the average pore size of the nanoporous epoxy is ∼16 nm. Most importantly, the nanoporous gyroid texture will be a good carrier for the adsorption of the protein than other textures due to its through-hole property. Moreover, for practical applications, it is feasible to fine-tune the pore size of the nanoporous gyroid-structured epoxy by changing the molecular weight of the BCP. As is known, there are many functional groups in the cured epoxy such as tertiary amine, hydroxyl, and ether bond. In the case of the nitrogen atom and oxygen atom, the long pair electrons in these groups give cured epoxy the tendency to associate with protons due to the interactions between its functional groups and protein. With the large specific surface area (411 m2 g−1) and through-hole property, the nanoporous gyroid-structured epoxy is expected to give the high capability for protein adsorbability. On the basis of the adsorption measurements, for nonporous epoxy, the adsorption capabilities of lysozyme and BSA were determined as approximately 0.29 and 0.16 mg m−2 , respectively. According to the specific surface area value of 411 m2 g−1, it is straightforward to expect the adsorbabilities of the nanoporous epoxy for lysozyme and BSA should be 119 and 65 mg g−1, respectively. Consistently, the adsorbabilities of the nanoporous epoxy for lysozyme and BSA were determined to be 116 and 57 mg g−1, respectively, indicating that the nanoporous gyroid-structured epoxy fabricated is indeed a through-hole texture and possesses the intrinsic adsorption capability like the nonporous one because the chemical structures of the nonporous and nanoporous epoxy should be the same. The variations in adsorbability between nonporous and nanoporous epoxy might be attributed to the slight effect of dimension on pore filling. Accordingly, the measured adsorbability of the nanoporous gyroid-structured epoxy reflects the feasibility of using such a nanoporous texture for practical uses as protein adsorbent and carrier. It is also noted that there are two factors to determine the protein adsorption using the nanoporous epoxy: the size of the pore and the charge of protein. Because BSA has larger size than lysozyme, it is, in principle, much easier for lysozyme to pore fill the channels of the nanoporous gyroid-structured epoxy (with pore size of ∼16 nm) than that for BSA. Namely, the dimension of the pore is the necessary condition to be considered for the adsorption of the proteins onto the inner wall of the nanoporous epoxy. Once satisfying the size criteria, the charge of the protein will be considered to fulfill the conditions for the adsorption. Note that proteins can exhibit their best activities for adsorption only under physiological conditions, that is, about neutral pH value conditions as we
nonclosure of the physisorption isotherms is referred to structural rearrangements due to the softness of the materials. Note that usually the swelling results in a certain degree of mesopores collapse;45 however, the nanoporous epoxy fabricated is a highly cross-linked polymer and should possess a high structural stability. We speculate that the open isotherm is attributed to the difficulty for the removal of the dissolved N2 during depressurization. In contrast with the cross-linked epoxy, the PS template should have high flexibility to alleviate this effect. As shown in Figure 6d, there are two populations for the average pore sizes; one is ∼10 nm and the other is ∼25 nm, which is consistent with the FESEM results. The small population of the mesopores with an average pore size of 10 nm is attributed to the existence of randomly distributed mesopores resulting from the deformation from dissolved nitrogen and network shifting after removal of the template that also cause the broadening of the pore size distribution. To further characterize the fabricated well-order nanoporous gyroid-structured epoxy, we carried out systematic analysis with respect to the pore size of the nanoporous epoxy. The specific surface areas of PS templates from BCPs used in this study can be calculated by the following eq 2
S=
C ρD
(2)
where S represents the specific surface area, C is a dimensionless constant (for gyroid, C = 5.8), ρ represents the solid bulk density, and D is the ligament size.46 For PS templates from BCP-L, BCP-M, and BCP-H, D values are calculated as approximately 11, 29, and 32 nm from Figure 2, respectively. As ρ = 1.02 g cm−3 for bulk PS, the calculated specific surface areas using eq 2 for the PS templates from BCP-L, BCP-M, and BCP-H are Scalc‑L = 516 m2 g−1, Scalc‑M = 196 m2 g−1, and Scalc‑H = 177 m2 g−1, respectively. The calculated Scalc‑M value 196 m2 g−1 and the experimental SBET‑M value 173 m2 g−1 are comparable, having ∼13% deviation acquired from BET result (Figure 6a). This analysis indicates the validity of using eq 2 for the calculation of the S values. Accordingly, the Scalc values of the PS templates can also be used to determine their measured values approximately. As a result, templates with specific surface areas from 177 to 516 m2 g−1 can be obtained by regulating the corresponding BCPs. For nanoporous epoxy using PS template from BCP-M with ρ = 1.15 g cm−3, C = 5.8, and D = 26 nm acquired from Figure 2d, the calculated specific surface area of the nanoporous epoxy (Scalc‑E) is 194 m2 g−1 using eq 2. In contrast with the value from BET (411 m2 g−1), there is a large discrepancy between the calculated and experimental ones. This might be attributed to the dissolved nitrogen that swells the epoxy matrix, resulting in different pore size, as shown in Figure 6d. Therefore, the smaller pores may contribute significantly to the increase in the specific surface area of the nanoporous epoxy fabricated. On the basis of the specific surface area analysis of PS templates, we can infer that the specific surface area of the nanoporous gyroid-structured epoxy from this study can be much larger than 411 m2 g−1. Nanoporous Epoxy for Protein Adsorption. Proteins are the condensation products of amino acids and contain different functional groups. They usually show a net charge under normal physiological conditions. At a pH value, the net charge is zero on the surface of each protein, that is, the isoelectric point of the protein (pI). It is the electrostatic H
DOI: 10.1021/acs.langmuir.6b01765 Langmuir XXXX, XXX, XXX−XXX
Langmuir
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CONCLUSIONS A well-defined nanoporous gyroid-structured epoxy monolith was fabricated from block copolymer templates. Uniform pore size on nanoscale can be adjusted by changing the molecular weight of degradable block copolymer for self-assembly, followed by hydrolysis, giving the nanoporous polymers with controlled pore sizes. The well-defined nanoporous polymers can be used as templates for the fabrication of nanoporous gyroid-structured epoxy through templated polymerization. The obtained nanoporous gyroid-structured epoxy displays a large specific surface area (as large as 411 m2 g−1) and thus gives an adsorption capacity of 116 mg g−1 to lysozyme. Accordingly, this fabrication method of nanoporous gyroidstructured epoxy, as demonstrated, suggests a promising approach to prepare other polymeric materials with nanoporous gyroid structure for practical uses.
examined here. At the adsorption test condition (pH 7.4 which is between pI values of lysozyme and BSA), the net charge of lysozyme should be positive, while that of BSA is negative, which results in the better adsorbability of lysozyme than BSA due to the redundant lone -pair electrons of the nanoporous epoxy. We speculate that the enhanced adsorption of the lysozyme within the nanoporous epoxy is attributed to the combined actions of size and charge effects. Both of these two effects are indispensable, but the charge effect might be conquered. With the through-hole property of gyroid texture and the corresponding large specific surface area, it is reasonable to enhance the adsorbability of porous epoxy as observed. Because of the possibility of degeneration at strong base and acid condition for lysozyme and BSA, it is impractical to examine the adsorption of the lysozyme under the conditions with pH >11 (pI value of lysozyme) and that of the BSA with pH
