Preparation of Unsymmetrical Microspheres at the Interfaces

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Preparation of Unsymmetrical Microspheres at the Interfaces Keiji Fujimoto,* Kazumichi Nakahama, Miwako Shidara, and Haruma Kawaguchi Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan Received January 7, 1999. In Final Form: April 6, 1999 Many synthetic microspheres are spherical and symmetrical because of the thermodynamical limitations of the reaction systems. We developed modification techniques to prepare spherical microspheres with intrinsic unsymmetry, for example, a microsphere with both an anionic and a cationic part. The modification was performed at the liquid-solid interface. Reactive microspheres with p-nitrophenyl moieties were settled onto the IgG-preadsorbed substrate, and the reaction between the activated ester moieties and IgG molecules proceeded only at the interface upon attachment. After the reaction, the modified microspheres were detached from the substrate with ultrasonication. In another modification at the air-water interface, the dispersion of reactive microspheres in ethanol was first spread on a water surface to produce the monolayer of microspheres, and then hydrolysis at the water side of the monolayer was performed by adding a NaOH aqueous solution to the subphase. After this unsymmetrical hydrolysis, we carried out IgG immobilization with the remaining reactive moieties. In both interface methods, the immobilized IgG molecules were labeled with colloidal gold particles conjugated with anti-IgG antibodies. Transmission electron microscopy indicated that gold particles could be observed on only the immobilized side. Small microspheres were coupled to the reactive microsphere, and its unsymmetry was confirmed by scanning electron microscopy. Such unsymmetrical microspheres will be applicable to the electrical rheology, diagnosis, display technology, and creation of functional devices through the assembling of microspheres.

Introduction The unsymmetry of materials is divided into morphological and intrinsic aspects. The former means that the material is unsymmetrical in shape, and the latter, irrespective of its shape, occurs when elements composing the material are oriented in the mass to form a direction in the material itself. Many microspheres have been obtained by conventional polymerization techniques such as emulsion and suspension polymerizations. They are generally spherical and symmetrical because of the thermodynamical limitations of the reaction. Vanderhoff and co-workers found that nonspherical microspheres could be produced by a seeded polymerization technique which was attributed to phase separation.1 Some researchers have also succeeded in obtaining morphologically unsymmetrical microspheres, such as a raspberrylike surface and a dumbbell form.2,3 On the other hand, we can find intrinsic unsymmetry in fluorescent chemicals, proteins, and liquid crystals. Many globular proteins show dielectric properties and have large dipole moments.4 This is caused by the distribution of fixed charges on the surface of protein molecules. In this manner, molecules and their assembling materials are sometimes given specific properties by the unsymmetry of each molecule. Frechet and co-workers have prepared unsymmetrically functionalized dendritic molecules designed to possess large dipole moments.5 Takei and Shimizu also reported that latex spheres were chemically modified on only one side by gold * Telephone: +81-45-563-1141 ext. 3457. Fax: +81-45-562-7625. E-mail: [email protected]. (1) Shue, H. R.; EL-Aasser, M. S.; Vanderhoff, J. W. J. Polym. Sci., Part A 1990, 28, 653-667. (2) Okubo, M.; Kanaida, K.; Matsumoto, T. Colloid Polym. Sci. 1987, 265, 876-881. (3) Skjeltorp, A. T.; Ugelstad, J.; Ellingsen, T. J. Colloid Interface Sci. 1986, 113, 577-582. (4) Takahashi, S.; Sano, Y. Biopolymers 1993, 33, 59-68.

evaporation and chemisorption.6 These are some instances of intrinsic unsymmetry, but here, we describe a simple new method for introducing intrinsic unsymmetry into microspheres at the liquid-solid or air-liquid interfaces. Microspheres with intrinsic unsymmetry that are expected to be fabricated with our techniques are shown in Figure 1. A part of one hemisphere is anionic and a part of the other hemisphere is cationic. Only a part of the microsphere is swellable or shrinkable, these microspheres have hairy polymers similar to flagella. Such unsymmetries will lead to anisotropic behavior and show unique functions as a particle. Each unsymmetrical microsphere will be utilized as a “microsphere block”. By assembling these blocks in a one-, two-, or three-dimensional form, we will be able to create functional devices. Experimental Section Materials. Methacrylic acid (MAc), glycidyl methacrylate (GMA), styrene (St), and divinylbenzene (DVB) were purified by distillation under reduced pressure to remove their inhibitors. Azobisamidinopropane dihydrochloride (V-50) was used without further purification. p-Nitrophenyl acrylate (NPA) was synthesized by adding acryloyl chloride to an ethyl acetate solution of p-nitrophenol (HONP) and recrystallized from hexane according to a method described previously.7 Methylenebisacrylamide (MBAAm) and azobis(isobutyronitrile) (AIBN) were used without further purification. Human immunoglobulin G (IgG), 4-phenylspyro[furan-2(3H),1′-futalan]-3,3′-dione (fluorescamine), and monolaurate (Tween-20), purchased from Sigma Chemical Co., were used without further purification. Gold colloid (15 nm in diameter) conjugated with anti-human IgG was purchased from Biocell Co. Other chemicals were purchased from Wako Pure Chemicals Co. (5) Wooley, K. L.; Hawker, C. J.; Frechet, J. M. J. J. Am. Chem. Soc. 1993, 115, 11496-11505. (6) Takei, H.; Shimizu, N. Langmuir 1997, 13, 1865-1868. (7) Kashiwabara, M.; Fujimoto, K.; Kawaguchi, H. Colloid Polym. Sci. 1995, 273, 339-345.

10.1021/la990023v CCC: $18.00 © 1999 American Chemical Society Published on Web 05/26/1999

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Figure 1. Illustration of unsymmetrical microspheres and their assembly. Table 1. Characterization of Reactive Microspheres mol % of crosslinker code

15

20

25

MAc/NPA/MBAA (mol in feed) conversion (%) remaining NPA (%) diam of dried microsphere (µm)a diam of microsphere in PBS (µm)b sector’s angle (°)c

10:10:3.5 26.2 24.3 1.69 5.91 165 ( 38

10:10:5 42.8 31.3 168 4.55 121 ( 50

10:10:6.7 55.5 36.9 1.52 4.06 100 ( 35

Figure 2. Schematic procedure of preparation of unsymmetrical microspheres by the liquid-solid interface method. (a) Adsorption of IgG was carried out by pouring the protein solution into the polystyrene dish. (b) The dish was rinsed with PBS. (c) The dispersion of reactive microspheres was placed on the IgG-preadsorbed dish. (d) Microspheres were allowed to react with IgG molecules. (e) Hydrolysis was carried out by adding a NaOH aqueous solution. (f) Modified microspheres were detached from the dish by ultrasonication.

Preparation of Reactive Microspheres. Two types of reactive microspheres were prepared in this work. Large reactive microspheres (MN microspheres) were prepared as follows. MAc, NPA, and MBAAm were dissolved in ethanol, and precipitation polymerization was initiated by adding an AIBN solution at 60 °C according to a method described previously.7 The polymerization recipe is tabulated in Table 1. Small microspheres (SG microspheres) were prepared by soap-free emulsion polymerization. GMA, St, and DVB were copolymerized in a soap-free aqueous medium using V-50 according to a method described previously.8 To obtain small microspheres having amino groups (SGN microspheres), ammonia was allowed to react with the epoxy groups of the SG microspheres. Preparation of Unsymmetrical Microspheres. LiquidSolid Interface Method. Surface modification was carried out at the liquid-solid interface as shown in Figure 2. The concentration of human IgG was adjusted to 400 ppm with the phosphatebuffered saline (PBS, pH 7.4) for adsorption. Protein adsorption was carried out at 4 °C by pouring the protein solution into the polystyrene dish. After the adsorption was allowed to proceed for 24 h, the dish was rinsed with PBS. The concentration of the dispersion of MN microspheres was adjusted to 2 mg/mL with PBS. The dispersion (1 mL) was placed on the IgG-preadsorbed

dish and allowed to stand for 24 h at 4 °C to carry out the reaction between the activated ester moieties of MN microspheres and the amino or the thiol groups of IgG molecules. After being washed with PBS three times to remove unreacted microspheres, hydrolysis was carried out by adding a NaOH aqueous solution of 0.1 mol/L to the MN microsphere-settling dish. To detach modified microspheres from the dish, the dish was immersed in a vessel-type ultrasonic cleaner (VS-70R, Iuchi Co., Ltd.), and ultrasonication was carried out at 46 kHz and 65 W for 10 s. The amount of IgG’s immobilized onto MN microspheres was determined by a fluorometric assay with fluorescamine as described by Stocks et al.9 To obtain the calibration curve, symmetrical (conventional) immobilization of IgG molecules onto MN microspheres was carried out as follows. MN microspheres (2 mg) were dispersed in 1 mL of PBS containing 200 ppm human IgG and allowed to stand for 5 h at 4 °C. Then, modified microspheres were centrifuged and dispersed in PBS three times, respectively, to remove unreacted IgG’s. Additionally, 2 mg of MN microspheres was added into a 0.1 mol/L NaOH aqueous solution and allowed to stand at 4 °C for 30 min to obtain completely hydrolyzed MN microspheres. The mixtures (0.4 mg) of human IgG-immobilized MN microspheres and completely hydrolyzed MN microspheres were prepared at different mixing ratios. Fluorescamine (0.3 mg/mL) in acetone (0.5 mL) was added to 1.5 mL of each mixture in the phosphate-buffered solution (pH 8.0) while they were vortexed. After 3 min of incubation, the fluorescence intensity at 500 nm was measured upon excitation at 400 nm. Then, the intensity for unsymmetrical microspheres of 0.4 mg was measured to estimate the amount of immobilized human IgG and the modified area using the calibration curve. Air-Liquid Interface Method. At the air-water interface, introduction of unsymmetry into the microspheres was performed as shown in Figure 3. MN microspheres (4 mg) were dispersed

(8) Inomata, Y.; Kawaguchi, H.; Hiramoto, M.; Wada, T.; Handa, H. Anal. Biochem. 1992, 206, 109-114.

(9) Stocks, S. J.; Jones, A. J. M.; Ramey, C. W.; Brooks, D. E. Anal. Biochem. 1986, 154, 232-234.

a Measured by transmission electron microscopy. b Measured by optical microscopy (reactive ester were completely hydrolyzed). c Mean ( standard deviation.

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Figure 3. Schematic procedure of preparation of unsymmetrical microspheres by the air-liquid interface method. The microsphere monolayer was formed at the air-water interface. (a) To hydrolyze the surface region dipping to the water phase, a NaOH aqueous solution was poured to the subphase. After neutralization with excess hydrochloric acid, the resulting microspheres were centrifuged and redispersed in PBS. IgG molecules were allowed to react with the remaining reactive moieties on microspheres. (b) The latex of SGN microspheres was injected into the subphase to allow them to react with the underside of the monolayer. in ethanol of 0.1 mL. The dispersion was spread on a water surface with a pipet to produce a monolayer of microspheres in a Teflon trough filled with distilled water. The spreading area was 175 cm2. Compression of the microspheres was carried out at 5 mm/ min by an L-B film deposition apparatus (NL-LB240N-MWC, Nippon Laser & Electronics Lab.), and the surface pressure was measured using a Wilhelmy plate. Hydrolysis of reactive moieties of microspheres at the water side was performed for 5 min by adding the 0.01 mol/L NaOH aqueous solution to the subphase. Then, excess hydrochloric acid was added to neutralize the medium. After the resulting microspheres were centrifuged and redispersed in PBS, IgG molecules were allowed to react with the remaining reactive moieties on microspheres in the same manner as the symmetrical immobilization described in the liquid-solid interface method. The amount of immobilized human IgG was detected with the above-mentioned fluorescamine method. Introduction of SGN microspheres to the underside of the monolayer was carried out to coat an MN microsphere with SGN microspheres on only one side. As described above, a monolayer of MN microspheres was formed at the air-water interface. Instead of a NaOH aqueous solution, the latex of SGN microspheres was added to the subphase to a final concentration of 0.2 mg/mL. After allowing the introduction to proceed for 2 h, the latex in the subphase was diluted with water and the modified monolayer was transferred to a glass plate (120 mm in width, 70 mm in length, and 1 mm in depth). Air-Liquid and Liquid-Solid Interface Method. SGN microspheres were coupled to MN microspheres in another method. The schematic illustration of this method is shown in Figure 4. The monolayer of MN microspheres was first produced by spreading them on a water surface. After the monolayer was unsymmetrically hydrolyzed for 5 min by adding a 0.01 mol/L NaOH aqueous solution to the subphase, it was transferred to the glass plate. The obtained plate was placed at the bottom of a 500 mL beaker, which was filled with water of 400 mL. The dispersion of SGN microspheres was poured to the beaker to a final concentration of 0.2 mg/mL and allowed to stand at room

Fujimoto et al.

Figure 4. Schematic procedure of preparation of unsymmetrical microspheres by the air-liquid/liquid-solid interface method. The monolayer was unsymmetrically hydrolyzed with a NaOH aqueous solution, and it was transferred to the glass plate. The dispersion of SGN microspheres was poured and allowed to stand at room temperature for 2 h. Then, modified microspheres were detached from the glass plate by ultrasonication. temperature for 2 h. Then, the glass plate was taken out of the beaker and rinsed with water. Finally, modified microspheres were detached from the glass plate by ultrasonication at 46 kHz and 65 W for 10 s. Observation of Unsymmetrically Modified Microsphere Surfaces. The obtained microspheres were incubated with colloidal gold particles conjugated with anti-human IgG antibodies (15 nm in diameter) to label the immobilized human IgG molecules at 4 °C for 24 h in the dark. To remove the unbound gold particles, microspheres were recovered by centrifuging (14 000 rpm, 30 min) and washing twice with PBS, then with the phosphate-buffered solution containing 1 M NaCl, and with PBS containing Tween-20, respectively. Microspheres labeled with gold particles were embedded in an epoxy resin and thinsectioned. Their sections of approximately 100 nm were observed with the transmission electron microscope (TEM). On the other hand, MN microspheres covered with SG microspheres were observed with the scanning electron microscope (SEM).

Results Modification at the Liquid-Solid Interface. We intended to perform the unsymmetrical imprinting at the interfaces (liquid-solid and air-liquid) to introduce the unsymmetry to the microsphere. Reactive microspheres were prepared for introduction of various chemicals into their surface layer. The obtained MN microsphere is a monodisperse hydrogel with reactive moieties which are attributed to NPA units. Table 1 also gives the amount of remaining NPA after polymerization and the microsphere diameter. The diameters of each dried microsphere were almost the same size. After swelling in a phosphatebuffered solution, the diameters increased in the order of decreasing cross-linking density. Because ester moieties of NPA were appreciably decomposed during polymerization, only 31.3% of the charged NPA was obtained after polymerization. This is because an ionized carboxylate group reacts with the neighboring reactive ester group along the polymer chain to form an acid anhydride, and

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Figure 6. Pressure-area phase diagram for microspheres spread on the water surface. The amount of microspheres spread on the water was 6 (O), 8 mg (4), and 12 mg (0), respectively.

Figure 5. TEM micrographs of thin-sectioned microspheres: (a) symmetrically modified, (b) completely hydrolyzed, and (c) unsymmetrically modified (prepared by the liquid-solid interface method). The modified regions were visualized by gold particles (15 nm in diameter) conjugated with anti-IgG antibodies, that were bound to IgG molecules introduced onto the microsphere.

simultaneously, an ethanolysis reaction proceeds in ethanol.10,11 The IgG molecule was selected to visualize the modified region by labeling it with anti-IgG antibodycarrying gold particles. Because desorption of IgG adsorbed onto the PS substrate is likely to occur, we washed the IgG-preadsorbed PS substrate with PBS until IgG’s were no longer detectable from the rinsing solution, therefore avoiding the reaction of IgG’s with the unfavorable region of the microsphere surface. The optical microscopic observation revealed that MN microspheres were settled and closely packed on the IgG-preadsorbed dish and were not detached even by rinsing strongly. Brief ultrasonication was used to detach the microsphere-IgG complex from the substrate. TEM photographs of symmetrically modified MN microspheres showed gold particles binding over the microsphere surface in a symmetrical form (Figure 5a), and virtually no gold particles could be observed on the completely hydrolyzed microspheres (Figure 5b). In the case of the microspheres modified with the liquid-solid method, the gold particles could be observed at the limited region in an unsymmetrical form (Figure 5c), indicating that the intrinsic unsymmetry could be successfully imprinted onto the microsphere. In the same method, three microspheres having different crosslinking densities were prepared to change the unsymmetrically immobilized area. As described above, we could alter the swelling ratio of these microspheres by changing the concentration of the cross-linker. To estimate the unsymmetrically modified area, we measured the arc labeled with gold particles from TEM views of thinsectioned microspheres. The angle of the sector composed of each arc was tabulated in Table 1. The modified area was larger than we expected, and it decreased as the crosslinking density increased. Modification at the Air-Liquid Interface. The other preparation was carried out at the air-water interface. Optical microscopy revealed that MN microspheres were spread at the air-water interface and that a monolayer “island” composed of the microspheres was formed on the water surface. When the NaOH aqueous solution of 0.01 mol L-1 was added to the subphase, the microspheres swelled and spread on the water surface, resulting in the lateral packing of microspheres. On the (10) Gaetjens, E.; Morawetz, H. J. Am.. Chem. Soc. 1961, 83, 17381742. (11) Morawetz, H.; Zimmering, P. E. J. Phys. Chem. 1954, 58, 753756.

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Table 2. Packing Area of Monomicrosphere Layer amt of microsphere (mg) 6 8 12 a

packing area (cm2) exptl ideal 58 78 120

58.9 78.6 117.9

packing area per microsphere (10-8 cm2/microsphere) exptl ideala 2.41 2.43 2.49

2.46 2.46 2.46

Calculated from hexagonal packing.

Figure 8. SEM views of MN microspheres (large circles) binding SGN microspheres (small circles): (a) air-liquid interface method (pH 6.5, 2 h, latex concentration 0.20 g L-1) and (b) air-liquid/liquid-solid interface method (pH 6.5, 2 h, latex concentration 0.20 g L-1).

Figure 7. TEM micrographs of thin-sectioned microspheres prepared by the air-liquid interface method.

other hand, there was no significant change when distilled water was added. These results indicate that hydrolysis occurs rapidly after the addition of the NaOH aqueous solution. In fact, we could also find that the amount of esters rapidly decreased by adding the NaOH aqueous solution and that the decomposition of esters depended on the concentration of added NaOH aqueous solution (data not shown). Figure 6 shows the curves of the surface pressure-area isotherm on the distilled water. The packing area was very proportional to the number of

spreading microspheres. When the region of spreading microspheres was gradually reduced at a constant speed, the pressure for each microsphere suddenly increased at a certain area, indicating that the microspheres were closely packed in the same manner as the solid LB membrane.12 From the comparison between the experimental packing area and the calculated one, we found that the microsphere monolayer was arranged almost in a hexagonal packing form by exerting the surface pressure to the layer of spreading microspheres (Table 2). The microspheres were hydrolyzed for 5 min by the NaOH aqueous solution of 0.01 mol/L, after the monolayer was compressed into a hexagonal form. After hydrolyzed microspheres were recovered, centrifuged, and resus(12) Horvolgyi, Z.; Nemeth, S.; Fendler, J. H. Langmuir 1996, 12, 997-1004.

Preparation of Unsymmetrical Microspheres

pended in PBS, the remaining reactive sites were allowed to react with IgG’s. Unlike the results of the liquid-solid method, unsymmetrical IgG immobilization could not be so completely achieved with the microsphere. TEM photographs of microspheres labeled with gold particles are shown in Figure 7. Most of IgG molecules seemed to be unsymmetrically immobilized onto the microsphere surface, although it was observed that several IgG’s were scattered. However, when the monolayer was not fully compressed, only symmetrical microspheres were produced. It is presently thought that the imprinting of the unsymmetry in the air-liquid method is less efficient than that in the liquid-solid method. Modification at Air-Liquid and Liquid-Solid Interface Method. We intended to prepare a microsphere with a large dipole moment. MN microspheres have a negative charge derived from carboxyl groups, and SGN microspheres are positive because of the amino groups of their surface. At first, we carried out the unsymmetrical binding of SGN to MN microspheres by the air-liquid method. The SEM photograph of the obtained microspheres is shown in Figure 8a. It was observed that a few SGN particles were assembled and bound to the region, probably in the air-liquid interface where the reaction took place. An attempt to prepare such microspheres was also made by the air-liquid/liquid-solid interface method. Added SGN microspheres were deposited on a monolayer of MN that was transferred to the glass plate. After the binding reaction, only the limited surface region of MN microspheres was covered with some SGN microspheres as shown in Figure 8b. The number of bound SGN microspheres increased with increases in both pH (3.5-6.5) and the concentration of the added SGN latex (data not shown). Discussion We successfully obtained intrinsicly unsymmetrical microspheres by our modification techniques at the interfaces. In the liquid-solid interface method, as shown in Table 1, there is a tendency for the modified area to become larger with decreasing cross-linking density. This is probably because the microsphere’s softness leads to the enlargement of the area where the microsphere can bind to the substrate. In a similar way, we will be able to control the modified area with the softness of microspheres or substrates. We attempted to carry out unsymmetrical modification using a water-soluble substrate such as poly(vinyl alcohol). We found that the microspheres were covered with the polymers of the substrate and it was too difficult to remove them. Contrary to our expectation, the unsymmetrical imprinting by the air-liquid interface method was less efficient than that by the liquid-solid method. This is probably because the rotation of microspheres was not adequately suppressed by the compression

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of the microsphere monolayer. This also suggests that we must consider that the small molecules such as IgG can diffuse to the hydrolyzed region by capillary attraction. Further improvement is needed for unsymmetrical imprinting by the air-liquid interface method. On the other hand, the liquid-solid interface method requires that adhesion between the microsphere and the substrate is strong enough to interrupt the rotational motion of the microspheres. Therefore, we developed the air-liquid/ liquid-solid interface method for the unsymmetrical introduction of various materials. For these, because the lateral packing of microspheres is strong enough maintain the monolayer even after modifications at the air-liquid interface, the imprinted unsymmetry can be completely transferred to the solid surface. Variation of the unsymmetry to be introduced provides a variety of interesting properties and applications. As shown in Figure 1, if a part of one hemisphere is anionic and a part of the other hemisphere is cationic, unsymmetrical microspheres have large dipole moments. Such microspheres can be oriented in an electrical field, and their suspensions will be able to function as an electrorheological fluid and therefore be applicable to the switching device. They may also be utilized as components such as liquid crystals in display technology. If one hemisphere is hydrophilic and the other is hydrophobic, the microsphere may be oriented along the water-oil interface, stabilizing the droplets as a surfactant.13 Suspension of microspheres form a variety of ordered structures at an interface in response to an electric field.14 The lattice structure, orientation, and size of the colloid crystals can be tailored by template-directed colloidal crystallization.15 With the use of unsymmetrical microspheres, we will be able to produce one-, two-, and threedimensional structures applicable to functional devices in the processing of materials. Whitesides et al. described an approach for the three-dimensional self-assembly of millimeter-scale components.16 Further applications, such as self-assembly, will be expected if we can strictly control the region to be modified. If the interactions among microspheres are reversible, the assembling structures might exhibit a similar reversibility. We hope that our methodology will be able to provide the creation of a unsymmetrical microsphere and that many devices will be designed, synthesized, and applied to a wide variety of fields. LA990023V (13) Velev, O. D.; Furusawa, K.; Nagayama, K. Langmuir 1996, 12, 2374-2384. (14) Yeh, S.-R.; Seul, M.; Shraiman, B. I. Nature 1997, 386, 57-59. (15) van Blaaderen, A.; Ruel, R.; Wiltzius, P. Nature 1997, 385, 321324. (16) Terfort, A.; Bowden, N.; Whitesides, G. M. Nature 1997, 386, 162-164.