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Sci China Ser B-Chem | Jul. 2009 | vol. 52 | no. 7 | 969-974

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Honeycomb-patterned films of polystyrene/ poly(ethylene glycol): Preparation, surface aggregation and protein adsorption

WAN LingShu, KE BeiBei, LI XiaoKai, MENG XiangLin, ZHANG LuYao & XU ZhiKang†

Key Laboratory of Macromolecular Synthesis and Functionalization of Ministry of Education, Institute of Polymer Science and State Key Laboratory of Chemical Engineering, Zhejiang University, Hangzhou 310027, China

Highly ordered honeycomb-patterned polystyrene (PS)/poly(ethylene glycol) (PEG) films were prepared by a water-assisted method using an improved setup, which facilitated the formation of films with higher regularity, better reproducibility, and larger area of honeycomb structures. Surface aggregation of hydrophilic PEG and adsorption of bovine serum albumin (BSA) on the honeycomb-patterned films were investigated. Field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) were used to observe the surface morphologies of the films before and after being rinsed with water. As confirmed by the FESEM images and the AFM phase images, PEG was enriched in the pores and could be gradually removed by water. The adsorption of fluorescence-labeled BSA on the films was studied in visual form using laser scanning confocal microscopy. Results clearly demonstrated that the protein-resistant PEG was selectively enriched in the pores. This water-assisted method may be a la-tent tool to prepare honeycomb-patterned biofunctional surfaces.

honeycomb-patterned film, water-assisted method, polystyrene, poly(ethylene glycol), surface aggregation

1 Introduction

Honeycomb-patterned films with highly ordered pores have many potential applications. For example, the films can be used for the assembly of nanoparticles[1－3], tem-plates[4,5], optical materials[6－9], biointerfaces for tissue engineering[10－16], molecularly imprinted membranes[17], biomimetic surfaces[12,18], and superhydrophobic sur-faces[19]. In 1994, Francois et al.[20] discovered that hon-eycomb-patterned films could be formed from the solu-tion of star-shaped polystyrene (PS) dissolved in carbon disulphide under a flow of moist gas. This process is very convenient and is called breath figure method, water-assisted method, or water drop templating method[21－25].

Early reports claimed that very specific polymers such as star-shaped polymers were required for the fab-rication of honeycomb-patterned films[26,27]. Thereafter,

it was found that linear polymers with polar end groups or amphiphilic polymers could form films with high re- gularity[26－28]. The addition of surfactants or hydrophilic additives could also improve the film formation. Fur-thermore, it was reported that honeycomb-patterned film could be prepared from a linear PS without any polar end groups[29]. Nevertheless, polymers with high seg-ment density (e.g., star polymers) or polar moieties, and the addition of surfactants or hydrophilic additives can stabilize water drops to prevent their coalescence, thus a wide range of casting conditions can be tolerated[26,27].

Received September 22, 2008; accepted November 5, 2008; published online Janu-ary 22, 2009 doi: 10.1007/s11426-009-0007-1 †Corresponding author (email: xuzk@zju.edu.cn) Supported by the National Natural Science Foundation of China (Grant No. 50803053), the National Natural Science Foundation of China for Distinguished Young Scholars (Grant No. 50625309), the National Postdoctoral Science Founda-tion of China (Grant Nos. 20070421172 & 20081466) and the National Undergradu-ate Innovative Test Program

970 WAN LingShu et al. Sci China Ser B-Chem | Jul. 2009 | vol. 52 | no. 7 | 969-974

It has been proposed that a system with hydrophilic blocks or additives can result in film pores enriched with the hydrophilic moieties[26]. A series of amphiphilic block copolymers have been used for the fabrication of honeycomb-patterned films[18,30－33]. The surface aggre-gation of hydrophilic blocks was confirmed through X-ray photoelectron spectroscopy and water contact an-gle measurements. Cui et al.[34] studied the water- induced reversible switch of surface morphology for PS/poly(2-vinlypyridine) system. Recently, Kim et al.[35] prepared honeycomb-patterned films from a blend of PS and poly(ethylene glycol) (PEG). They elucidated the distribution of PEG on the film surface by measuring the composition of droplets using gel permeation chroma-tography. Direct visual evidence for the distribution of PEG should be further given. On the other hand, PEG is a well-known protein-resistant polymer. It is interesting to investigate the protein adsorption behavior on the honeycomb-patterned film surface. Therefore, in this work, we describe an improved instrumental setup for film formation by the water-assisted method, and try to elucidate the surface aggregation of hydrophilic PEG and its protein resistance using field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and laser scanning confocal microscopy (LSCM).

2 Experimental

2.1 Materials

Polystyrene (PS, MW = 235k, MWD = 2.89) was pro-vided by Zhenjiang Chiemei Chemicals. Poly(ethylene glycol) (PEG, MW = 400) was purchased from Sino-pharm Chemical Reagent and was used as received. Poly(ethylene terephthalate) (PET) film was kindly pro-vided by Hangzhou Tape Factory and was cleaned using acetone for 0.5 h before use. Rhodamine-labeled bo-vine serum albumin (RBITC-BSA, bioreagent) was purchased from Beijing Biosynthesis Biotechnology Co., Ltd. Water used in all related experiments was deionized and ultrafiltrated to 18 MΩ with an ELGA LabWater system.

2.2 Preparation of honeycomb-patterned films

PS and PEG were blended at a weight ratio of 70/30 and dissolved in toluene with 5% (mass fraction) initial polymer concentration. The solution was cast by a mi- croinjector onto a PET film placed on an aluminum dish.

The volume of solution injected was about 100 microli-ters in the normal case. The carrier nitrogen gas was bubbled through distilled water and sent with water va-por onto the solution surface. The gas flow was con-trolled by a needle valve and was measured with a flow meter. The polymer solution started to evaporate and water vapor was condensed onto the solution surface simultaneously. After complete evaporation of the sol-vent, a honeycomb-patterned film was formed. The rela-tive humidity and the temperature in the incubator were measured by a hygro-thermograph (DT-321S, CEM Corporation, HongKong).

2.3 Characterization

A field emission scanning electron microscope (FESEM, Sirion-100, FEI, USA) was applied to observing the surface morphology of films after being sputtered with gold using ion sputter JFC-1100. Height and phase im-ages were recorded simultaneously by an atomic force microscope (AFM, SEIKO SPI3800N) under tapping conditions. Distribution of the adsorbed RBITC-BSA on the film surfaces was evaluated by a laser scanning con-focal microscope (LSCM). The procedure for protein adsorption is as follows: RBITC-BSA was dissolved in phosphate buffer solution (PBS, pH 7.36, 0.01 mol/L) resulting in a protein concentration of 0.03 mg/mL. Films were incubated in the solution for 12 h, washed using the PBS, and observed using LSCM. LSCM was performed on a Leica TCS SP5 confocal setup mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Wetzlar, Germany) and was operated under the Leica Application Suite Advanced Fluores-cence (LAS AF) program. High-resolution image for the sample was taken using a 63× NA 1.4 lambda blue oil objective.

3 Results and discussion

It has been mentioned in the introduction section that for polymers without specific structures, polar blocks, or polar end groups, the film formation process is much less robust and thus the casting conditions need to be optimized[26]. Molecular weights and architectures of polymers are significant in determining the quality of the formed films. Casting conditions including polymer concentration, solvent properties, temperature, and rela-tive humidity are also important. Generally, ordered structures can be formed only under a high relative hu-

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midity, which can enhance the condensation of water vapor.

In practice, we found that the shape of the outlet af-fected both the stability and the relative humidity of the gas flow, thus affecting the quality of films. We com-pared three types of outlets, which were termed funnel (Figure 1(a)), funnel with filter (Figure 1(b)) and in-verted-wineglass-shaped funnel (Figure 1(c)). The rela-tive humidity at the bottom of the outlets was measured (Figure 2). It can be seen that when a common funnel was used, the relative humidity was very stable at a moderate level. The relative humidity lowered and be-came less stable when the common funnel was added with a filter. With the improved inverted-wineglass- shaped funnel, the gas flow was not only very stable but also had the highest relative humidity. Actually, the im-proved setup facilitated the formation of films with

Figure 1 Schematic representation for the experimental setup. (a) A funnel, (b) a funnel with a filter, and (c) an inverted-wineglass-shaped funnel.

Figure 2 Relative humidity of the gas flow for the three types of outlets. Curves (a), (b) and (c) correspond to the outlets shown in Figure 1, re-spectively. Flow rate is 2 L/min, temperature is 26.5℃, and the environ-mental relative humidity is ~60%.

higher regularity, better reproducibility, and larger area of honeycomb structures.

Figure 3 shows the FESEM images of PS/PEG films before and after being rinsed with water. As can be seen from Figure 3, ordered honeycomb-patterned PS/ PEG films were successfully prepared. For Kim et al.[35] only observed the films at low magnification, detailed information in the pores was not given. In fact, at high magnification, it could be clearly observed that the morphology in the pores was different from the outer surface of the film (Figure 3(e)). It seems that there was something deposited into the pores during the film formation process. However, in our case, FESEM is not sensitive to the surface chemistry. Considering that PS is water insoluble whereas PEG is water soluble, the films were therefore rinsed with water and the change of surface morphologies was examined using FESEM. As shown in Figure 3(f), after being rinsed with water, the matter enriched in the pores was fully removed. This result indicated that hydrophilic PEG was enriched in the pores, which is consistent with that reported by Cui et al.[34].

AFM was also used to evaluate the surface aggrega-tion of PEG. Figure 4 shows AFM height and phase im-ages of the films rinsed with water for different time. AFM height images only provide information of sample surface topography, whereas phase images reveal a high sensitivity to fine surface details[36]. For example, phase images can provide a fairly detailed view of the chemi-cally heterogeneous surface that has harder and softer components, generating different contrast regions that are darker or lighter[37]. As shown in the upper row of Figure 4, the films show similar surface topography. However, it can be seen from the lower row of the said Figure that the phase image contrast changed with rins-ing time. For the nascent film (Figure 4(a)), the lighter region was separated round, representing the pores en-riched with PEG, whereas the darker region was PS-enriched phase. After being rinsed with water for 2 h, the pore region became partially darker indicating that a part of PEG was removed. By increasing the time to 24 h, the entire region became darker (Figure 4(c)). It is clear that these AFM phase images reflected the distri-bution of PEG.

PEG is a well-known polymer that is highly pro-tein-resistant. Therefore, if PEG is indeed enriched in the pores, the pores should be protein-resistant differing from the outer surface of the film enriched with hydro-

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Figure 3 FESEM images of PS/PEG (70/30, w/w) films cast from a 5 wt% toluene solution under 2 L/min N2 flow before (a, c, e) and after (b, d, f) being rinsed with water for 24 h. The magnification of (a, b), (c, d), and (e, f) is 2000×, 10000× and 20000×, respectively.

Figure 4 AFM height (upper row) and phase (lower row) images of PS/PEG (70/30, w/w) films cast from a 5 wt% toluene solution under 2 L/min N2 flow before (a) and after being rinsed with water for 2 h (b), and 24 h (c).

phobic PS. The adsorption of RBITC-BSA on the films before and after being rinsed with water was preliminar-ily explored. Figure 5 shows the LSCM images of films that did not adsorb proteins (control samples) and those

of films adsorbed with RBITC-BSA. For the film was enriched with PEG in the pores, BSA was mainly ad-sorbed on the outer surface, that is, the PS-enriched re- gion. Fluorescence emission could only be observed

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Figure 5 Laser scanning confocal microscopy images of PS/PEG (70/30, w/w) films cast from a 5 wt% toluene solution under 2 L/min N2 flow. (a) Nas-cent film; (b) film rinsed with water for 24 h; (c) RBITC-labeled BSA adsorbed film; and (d) RBITC-labeled BSA adsorbed film rinsed with water for 24 h. from the outer surface region. However, for the sample rinsed with water, fluorescence emission could be ob-served from the outer surface as well as from a part of the pores. This result clearly demonstrated that the wa-ter-assisted method could result in ordered honey-comb-patterned surfaces enriched with hydrophilic moieties in the pores. Therefore, this method may be a latent tool for preparing patterned biofunctional surface.

4 Conclusions

PS/PEG films with ordered honeycomb patterns were

prepared by water-assisted method. Using an inverted- wineglass-shaped funnel as the outlet of moist gas in-creased its relative humidity and stability, thus improv-ing the quality and reproducibility of the films. FESEM and AFM phase images confirmed the surface aggregation of hydrophilic PEG, which was enriched in the pores instead of the outer surface of the film. The adsorption of fluorescence-labeled BSA also proved the enrichment of PEG in the pores. Our future work will focus on the preparation and biofunctions of honey-comb-patterned films with enriched functional moieties.

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