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Colloids and Surfaces A: Physicochem. Eng. Aspects 380 (2011) 29–34

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

journa l homepage: www.e lsev ier .com/ locate /co lsur fa

olvothermal fabrication of adsorptive polymer monolith with large nanoporesowards biomolecules immobilization

hu Weia, Yong-Lai Zhangd, Hong Dinga, Jiayin Liuc, Jing Suna, Yinyan Hea, Zhengqiang Li c,eng-Shou Xiaob,∗

State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, Jilin University, Changchun 130012, ChinaDepartment of Chemistry, Zhejiang University, Hangzhou 310028, ChinaKey Lab for Molecular Enzymology and Engineering, Jilin University, Changchun 130023, ChinaState Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012, China

r t i c l e i n f o

rticle history:eceived 4 October 2010eceived in revised form 24 January 2011ccepted 28 January 2011

a b s t r a c t

Reported in this paper is solvothermal fabrication of nanoporous polymer monolith for biomoleculesadsorption. General functional groups such as carboxyl and amido could be easily introduced into thenanoporous polymers by in situ polymerization of divinylbenzene and the monomers of methacrylicacid or acrylamide. The synthesized polymers exhibit high BET surface areas (105–499 m2/g), large pore

vailable online 15 March 2011

eywords:anoporous polymerarge nanoporesolvothermal synthesis

volumes (0.41–1.22 cm3/g), and large pore size (10–100 nm), which were quite suitable for biomoleculesadsorption. As representative tests, myoglobin was chosen as a target adsorbate, and all these nanoporouspolymers show much higher adsorptive capacities than conventional materials such as commercial resin(XAD-4) and activated carbon. Our nanoporous polymer monolith would be potentially important forenzyme immobilization.

iomolecular adsorptiondsorption rate and capacity

. Introduction

Enzymes, biological catalysts with high selectivities and activ-ties, have been paid much attention due to their extensivepplications in the fields of biochemical synthesis, biosensors andrugs [1–5]. However, most of native enzymes only exhibit highatalytic activities and selectivities under normal environments6,7]. Organic solvents, reagents, mechanical treatments, extremeemperatures or pH values would induce denaturation of enzymes,hus losing the activities. In addition, the recovery and reusagef enzymes are also disadvantages for their practical applica-ions. To overcome these problems, researchers have developedeveral strategies, in which immobilization of enzymes on solidupports is considered as one of the most effective approaches8–17].

To date, various supports have been used for enzyme immo-ilization such as silica gels [18–21], mesoporous silica materials

22–29], and porous resins [30,31]. Silica gels are porous and ther-

ally stable, however, due to their uncontrollable pore sizes andelatively low surface areas, the immobilized enzymes usuallyhow lower activities than the free enzymes. Mesoporous silicas

∗ Corresponding author. Tel.: +86 431 85168590; fax: +86 431 85168624.E-mail address: fsxiao@mail.jlu.edu.cn (F.-S. Xiao).

927-7757/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2011.01.059

© 2011 Elsevier B.V. All rights reserved.

with controllable ordered mesopores and large surface areas arepromising materials in biomolecular immobilization, but the rel-atively small pore sizes for large enzymes are still challenging.Porous resins with organic framework are kinds of suitable carri-ers for enzymes due to their oleophilic frameworks. However, theiradsorptive capacity is still not very high due to their low BET surfaceareas and unsuitable pore sizes. In addition, the hydrophobic-ity of organic supports significantly debases their performance inaqueous solutions. Therefore, it is urgent for developing polymer-based nanoporous materials with large surface areas, suitablepore sizes, and controllable wettability for biomolecular adsorp-tion.

Recently, we have reported a novel and facile synthesis ofnanoporous polymers with a large surface area and superhy-drophobicity, giving excellent adsorptive property for organiccompounds [32–35]. However, the superhydrophobicity of thiskind of nanoporous polymers strongly hinders the adsorption ofenzymes in aqueous solution. Additionally, the enzyme immobi-lization normally requires the presence of large mesopores, whichare favorable for the adsorption and transfer of bulky enzymes.

Herein, we have demonstrated successful syntheses of nanoporouspolymers with carboxyl/amido as functional groups for surfacewettability tuning. These materials have abundant nanopores andhigh BET surface areas, and therefore, show high adsorptive rateand capacity for myoglobin.

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. Experimental

.1. Materials

Divinylbenzene (DVB) and methacrylic acid (MA) were pur-hased from Tianjin–Guangfu Chemical Company. AcrylamideAM), azobisisobutyronitrile (AIBN), and ethyl acetate were pur-hased from Beijing Chemical Company. Amberlite XAD-4 wasurchased from Fluka. All chemicals were used as received withoutny further purification.

.2. Samples preparation

In a typical synthesis of nanoporous copolymer of divinylben-ene with methacrylic acid, divinylbenzene (DVB) and methacryliccid (MA) monomers (3 g, mass ratio: mDVB/mMA = 5, 4, 3, 2) wereissolved in 15 mL ethyl acetate, then 0.05 g azobisisobutyronitrileAIBN) was added. After stirring for 4 h at room temperature, theolution was transferred into a Teflon autoclave and then treatedt 100 ◦C for 24 h. The system was cooled to room temperaturend a solid monolith was obtained after the evaporation of ethylcetate solvent. The as-synthesized samples were designated asDVB-COOH-x, where x stands for the mass ratio of DVB to MA.

In a typical synthesis of nanoporous copolymer of divinylben-ene with acrylamide, divinylbenzene (DVB) and acrylamide (AM)onomers (3 g, mass ratio: mDVB/mAM = 5, 4, 3, 2) were dissolved

n 15 mL ethyl acetate, then 0.05 g azobisisobutyronitrile (AIBN)as added. After stirring for 4 h at room temperature, the solu-

ion was transferred into a Teflon autoclave and then treated at00 ◦C for 24 h. The system was cooled to room temperature and aolid monolith was obtained after the evaporation of ethyl acetateolvent. The as-synthesized samples were designated as PDVB-ONH2-y, where y stands for the mass ratio of DVB to AM.

Pure poly divinylbenzene (PDVB) was polymerized in the sameay without addition of MA or AM.

.3. Characterization

Nitrogen isotherms were measured with a Micromeritics Tristar

000 analyzer at −196 ◦C. The samples were pretreated by out-assing for 10 h at 150 ◦C. Transmission electron micrograph (TEM)xperiments were performed on a Philips CM 200 LaB6 operat-ng at an accelerating voltage of 200 kV. Thermogravimetric (TG)urves were carried out on a Netzsch STA 449C at a heating rate of

ig. 1. (a) Nitrogen adsorption/desorption isotherms and pore size distribution (inset)sotherms and pore size distribution (inset) of PDVB-CONH2-y (y = 5, 4, 3, 2) samples. Eac

chem. Eng. Aspects 380 (2011) 29–34

20 ◦C/min from room temperature to 800 ◦C under flowing air. FT-IRspectra were performed on an IFS 66V/S (Bruker) IR spectrometerin the range of 400–2000 cm−1, and the samples were measureduntil the final pressure was 0.004 mbar.

2.4. Enzyme adsorption

The experiments for adsorption of myoglobin on thenanoporous polymers were conducted by mixing 15 mL of0.5 mg/mL protein solution (potassium phosphate as buffer at pH7.0 and 8.0) with 90 mg of nanoporous polymers under stirringat 25 ◦C in a vessel covered to prevent evaporation. Sampleswere obtained periodically for immediate analysis. The amountsadsorbed were estimated from the differences in enzyme con-centration before and after adsorption measured by UV–visspectroscopy at 280 nm (Shimadzu UV1700).

To further evaluate the capacity of the nanoporous polymers formyoglobin, 40 mg of nanoporous polymer was suspended in 5 mLof 15 mM buffer solution (potassium phosphate as buffer at pH 7.0)with different myoglobin concentrations (from 0.1 to 3.0 mg/mL).The resulting mixture was stirred wildly at 25 ◦C in a vessel cov-ered to prevent evaporation for 72 h, which was confirmed to belong enough to reach the adsorption equilibrium. The mixture wasthen filtered through a membrane of 0.22 �m pore diameter andthe supernatant was analyzed by UV–vis spectroscopy at 280 nmto determine the amount adsorbed of enzyme according to thefollowing equation:

q = V0(C0 − C)W

(1)

where q is the equilibrium adsorbed amount in the materials, C0 andC are the myoglobin concentrations at initial and equilibrium solu-tions, respectively, V0 is the volume of the initial enzyme solution,W is weight of the adsorbent [27–29].

3. Results and discussion

3.1. N2 adsorption

Fig. 1 shows nitrogen isotherms and pore size distribution ofPDVB-COOH-x and PDVB-CONH2-y samples with different PMA(polymethacrylic acid) and PAM (polyacrylamide) contents (x,y = 5–2). All samples behave as typical H4 hysteresis loops at highrelative pressure (P/P0 at 0.8–1.0), but they show quite differ-

of PDVB-COOH-x (x = 5, 4, 3, 2) samples and (b) nitrogen adsorption/desorptionh isotherm has been offset by 100 cm3/g along the vertical axis for clarity.

S. Wei et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 380 (2011) 29–34 31

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ig. 2. TEM images of (a) PDVB-COOH-5, (b) PDVB-COOH-4, (c) PDVB-COOH-3, (d)DVB-CONH2-5 sample.

nt nitrogen uptakes. These results indicate that these samplesave nanoporous structures whereas their BET surface areas arebviously different. For example, PDVB-COOH-2 sample exhibitssmall hysteresis loop and relatively low nitrogen uptake, givingBET surface area of 304 m2/g and a pore volume of 0.41 cm3/g

Table 1). Compared with PDVB-COOH-2, larger hysteresis loops

nd higher nitrogen uptakes could be observed in the isothermsf PDVB-COOH-3, PDVB-COOH-4 samples. The two samples haveET surface areas of 415 and 473 m2/g and pore volumes of 0.69nd 0.73 cm3/g (Table 1), respectively. Furthermore, PDVB-COOH-5ample exhibits the highest nitrogen uptake. It has a BET sur-

COOH-2, (e) PDVB-CONH2-2, and (f) PDVB-CONH2-5 samples. Inset of (f): photo of

face area of 499 m2/g and a pore volume of 1.10 cm3/g (Table 1).Obviously, the surface areas increase with the content of PDVB inthe samples, which is reasonably related to that of the linear andloose PMA or PAM segments formed under solvothermal condition,would fill in the as-formed pores by PDVB in the presence of ethylacetate solvent. The textural parameters for various samples are

presented in Table 1.

Furthermore, it is worth noting that PDVB-COOH-x and PDVB-CONH2-y samples synthesized in the presence of ethyl acetatesolvent have pore sizes centered at 35–55 nm (inset of Fig. 1),which are much larger than those (3.8–3.9 nm) of nanoporous PDVB

32 S. Wei et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 380 (2011) 29–34

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ig. 3. FT-IR spectra of pure PDVB, PDVB-COOH-2, and PDVB-CONH2-2 samples.

nd PDVB-COOH-x synthesized in the presence of tetrahydrofuranTHF) [32,33]. These results indicate that the ethyl acetate solventlays an important role for the formation of very large nanopores.ossibly, during the polymerization the phase separation betweenhe polymers and ethyl acetate solvent is much stronger than thatetween the polymers and THF solvent, and similar phenomena arelso reported in the literature [36].

.2. TEM images

Fig. 2 shows TEM images of PDVB-COOH-x and PDVB-CONH2-(x, y = 5–2) samples, giving abundant disordered nanopores at

0–100 nm, which is in good agreement with the results derived

ig. 5. Shapes of the water droplets on the tablet of (a) PDVB-COOH-5 (CA at 136◦), (b) PDCA at 120◦) samples.

Fig. 4. TG curves of PDVB-COOH-2, PDVB-COOH-5, PDVB-CONH2-2, and PDVB-CONH2-5 samples.

from nitrogen isotherms. As an example, the photograph of PDVB-CONH2-5 shows a perfect solid monolith (inset in Fig. 2f), which ischaracteristic of nanoporous PDVB materials.

3.3. FT-IR spectra

Fig. 3 shows FT-IR spectra of pure PDVB, PDVB-COOH-2, and

PDVB-CONH2-2 samples. Notably, there is no obvious peak inthe region of 1600–1750 cm−1 for PDVB sample. However, PDVB-COOH-2 exhibits a clear peak at 1702 cm−1 associated withcharacteristic of C O stretching frequency in the carboxylic group.PDVB-CONH2-2 gives a strong peak at 1670 cm−1 assigned to C O

VB-CONH2-5 (CA at 132◦), (c) PDVB-COOH-2 (CA at 121◦), and (d) PDVB-CONH2-2

hysicochem. Eng. Aspects 380 (2011) 29–34 33

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both sample surface area and sample wettability are adjusted.These PDVB-COOH-x and PDVB-CONH2-y samples exhibit goodadsorption property for bulky biomolecules of myoglobin in aque-ous solution.

Table 1Textural parameters of carboxyl/amido functionalized nanoporous polymers, XAD-4, and activated carbon.

Sample Mass ratio ofDVB/MA (AM)

SBET (m2/g) VP (cm3/g) dP (nm)

PDVB-COOH-5 5 499 1.10 53.0PDVB-COOH-4 4 473 0.73 50.5PDVB-COOH-3 3 415 0.69 48.5PDVB-COOH-2 2 304 0.41 51.5PDVB-CONH2-5 5 405 1.13 34.8PDVB-CONH2-4 4 352 1.22 33.3

S. Wei et al. / Colloids and Surfaces A: P

tretching frequency in the amido group. The presence of carboxylicnd amido groups in the samples is reasonably attributed to the usef MA and AM in the synthesis of PDVB-COOH-2 and PDVB-CONH2-, respectively.

.4. TG curves

Fig. 4 shows TG curves of PDVB-COOH-2, PDVB-COOH-5, PDVB-ONH2-2, and PDVB-CONH2-5 samples. Weight loss at about 200 ◦C∼2%) is due to desorption of the adsorbates, while weight loss inhe temperature range from 280 to 600 ◦C is related to the decom-osition of the polymers. Notably, these samples are thermallytable below 200 ◦C, indicating their good thermal stabilities. Whenhe temperature is over 200 ◦C, a significant reduction of weightoss occurs for the samples. However, PDVB-CONH2 samples are

ore stable than PDVB-COOH samples. For the same PDVB-COOHamples, the sample with more oxygen content has lower decom-osition temperature.

.5. Wettability

The wettability is an important factor for the adsorption ofnzyme on the samples in aqueous solutions. For example, super-ydrophobic samples are difficult to contact with water because a

arge amount of enzymes usually dissolved in water. Fig. 5 showsettability of PDVB-COOH-5, PDVB-COOH-2, PDVB-CONH2-5, and

DVB-CONH2-2 samples. Notably, pure PDVB shows a high staticontact angle (CA) of about 155◦ (Appendix A Supplementary Fig.1), indicating its superhydrophobic property [37–40]. However,hen the monomers of MA and AM are introduced, PDVB-CONH2-5

nd PDVB-COOH-5 samples give the angles at 132–136◦. A fur-her increase of MA and AM monomers results in the formation ofDVB-COOH-2 and PDVB-CONH2-2 samples with lower angles at20–121◦. These results suggest that the sample wettability coulde adjusted by the content of MA and AM monomers in the startingixture.

.6. Enzyme adsorption

As a typical example for enzyme adsorption, Fig. 6 showsdsorption performance of myoglobin on large nanoporous poly-ers, commercial resin and activated carbon at pH values of 7.0

nd 8.0. XAD-4 (BET surface area at 750 m2/g and pore size at12.9 nm), a commercial resin mainly consisted of polystyrenend polydivinylbenzene, exhibits low adsorptive capacity (∼5% ofotal in the buffer, pH = 7.0, Fig. 5a), which is possibly related tots relatively small pore size. Activated carbon has a large surfacerea (BET surface area ∼940 m2/g) and hierarchical pores (mainly2 nm, 10–100 nm) [32], still giving low adsorption at first 24 h∼5% of total in the buffer, pH = 7.0, Fig. 5a). This phenomenon

ight be assigned to the disability for adsorption of bulky myo-lobin (∼3.5 nm) in micropores which contribute most of its BETurface area. Interestingly, both MA and AM functionalized PDVBshow very high adsorptive rate and capacity. For example, whendsorption time reaches to 8 h, PDVB-COOH-5 and PDVB-CONH2-5how the capacity at 95 and 98% of total myoglobin in the bufferpH = 7.0, Fig. 5a). Comparing with the adsorption on activated car-on, it is suggested that the large nanopores in PDVBs play a criticalole for the adsorption of bulky myoglobin enzymes. Furthermore,t is observed that PDVB-COOH-5 and PDVB-CONH2-5 have fasterdsorption rate than PDVB-COOH-2 and PDVB-CONH2-2 (pH = 7.0,

ig. 5a), which are reasonably attributed to their distinguishableET surface area (Table 1). When pH value in the buffer is 8.0, PDVB-OOH-x and PDVB-CONH2-y samples still show higher adsorptionate and capacity (Fig. 5b) than XAD-4 and activated carbon. There-ore, by introducing functionalized groups of MA or AM in PDVBs,

Fig. 6. Adsorption capacity at pH of (a) 7.0 and (b) 8.0 for myoglobin on PDVB-COOH-2, PDVB-COOH-5, PDVB-CONH2-2, PDVB-CONH2-5, XAD-4, and activated carbonsamples.

PDVB-CONH2-3 3 225 0.75 34.0PDVB-CONH2-2 2 105 1.19 52.2XAD-4 – 750 0.99 12.9Activated carbon – 940 0.49 <2; 10–100

SBET: BET surface area; VP: total pore volume; dP: average pore size distribution.

34 S. Wei et al. / Colloids and Surfaces A: Physico

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[39] X. Zhang, F. Shi, J. Niu, Y.G. Jiang, Z.Q. Wang, Superhydrophobic surfaces: from

ig. 7. Adsorption isotherms of myoglobin on PDVB-COOH-5, PDVB-CONH2-5, XAD-, and activated carbon samples at pH = 7.0. The solid lines are fitting results of theangmuir equation.

Furthermore, Fig. 7 shows the isotherms of myoglobin on PDVB-OOH-5, PDVB-CONH2-5, activated carbon, and commercial resinAD-4 at 25 ◦C. All isotherms show a sharp initial rise and theneach maximum adsorption amounts (plateau) at about 0.5 mg/mLquilibrium concentrations, indicating that these isotherms areangmuir-type. However, both PDVB-CONH2-5 and PDVB-COOH-5ad much higher adsorption capacity (133.75 and 121.25 mg myo-lobin/g) than activated carbon (68.8 mg myoglobin/g) and XAD-47.5 mg myoglobin/g). These results confirm that the PDVBs arexcellent adsorbents for enzyme adsorption of myoglobin.

. Conclusion

Nanoporous polymers with adjustable contents of car-oxyl/amido groups (PDVB-COOH-x and PDVB-CONH2-y) and largeanopores have been successfully synthesized by a facile solvother-al route. These polymers exhibit excellent property for enzyme

dsorption of myoglobin, which may therefore be potentially sig-ificant in enzyme immobilization.

cknowledgements

This work was supported by State Basic Research Project ofhina (2009CB623507) and National Natural Science Foundationf China (20973079).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.colsurfa.2011.01.059.

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