Department of Chemical Engineering, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
ACS Appl Mater Interfaces. 2012 Oct 24;4(10):5125-33. doi: 10.1021/am301637q. Epub 2012 Oct 9.
A novel surface modification method was investigated. The surface of siliceous materials was modified using polystyrene, poly(acrylic acid), poly(N-isopropylacrylamide), and poly(p-acrylamidophenyl-α-mannoside) synthesized by reversible addition-fragmentation chain transfer polymerization. Thiol-terminated polymers were obtained by reduction of the thiocarbonate group using sodium borohydride. The polymers were immobilized on the surface via the thiol-ene click reaction, known as the Michael addition reaction. Immobilization of the polymers on the maleimidated surface was confirmed by X-ray photoelectron spectroscopy, infrared spectroscopy, and contact angle measurements. The polymer-immobilized surfaces were observed by atomic force microscopy, and the thickness of the polymer layers was determined by ellipsometry. The thickness of the polymer immobilized by the maleimide-thiol reaction was less than that formed by spin coating, except for polystyrene. Moreover, the polymer-immobilized surfaces were relatively smooth with a roughness of less than 1 nm. The amounts of amine, maleimide, and polymer immobilized on the surface were determined by quartz crystal microbalance measurements. The area occupied by the amine-containing silane coupling reagent was significantly less than the theoretical value, suggesting that a multilayer of the silane coupling reagent was formed on the surface. The polymer with low molecular weight had the tendency to efficiently immobilize on the maleimidated surface. When poly(p-acrylamidophenyl-α-mannoside)-immobilized surfaces were used as a platform for protein microarrays, strong interactions were detected with the mannose-binding lectin concanavalin A. The specificity of poly(p-acrylamidophenyl-α-mannoside)-immobilized surfaces for concanavalin A was compared with poly-l-lysine-coated surfaces. The poly-l-lysine-coated surfaces nonspecifically adsorbed both concanavalin A and bovine serum albumin, while the poly(p-acrylamidophenyl-α-mannoside)-immobilized surface preferentially adsorbed concanavalin A. Moreover, the poly(p-acrylamidophenyl-α-mannoside)-immobilized surface was applied to micropatterning with photolithography. When the micropattern was formed on the poly(p-acrylamidophenyl-α-mannoside)-spin-coated surface by irradiation with ultraviolet light, the pattern of the masking design was not observed on the surface adsorbed with fluorophore-labeled concanavalin A using a fluorescent microscope because of elution of poly(p-acrylamidophenyl-α-mannoside) from the surface. In contrast, fluorophore-labeled concanavalin A was only adsorbed on the shaded region of the poly(p-acrylamidophenyl-α-mannoside)-immobilized surface, resulting in a distinctive fluorescent pattern. The surface modification method using maleimidation and reversible addition-fragmentation chain transfer polymerization can be used for preparing platforms for microarrays and micropatterning of proteins.
研究了一种新的表面改性方法。通过可逆加成-断裂链转移聚合合成了聚苯乙烯、聚丙烯酸、聚（N-异丙基丙烯酰胺）和聚（p-丙烯酰胺基苯基-α-甘露糖苷），对硅质材料的表面进行了修饰。使用硼氢化钠还原硫代碳酸盐基团得到了巯基聚合物。通过硫醇-烯点击反应将聚合物固定在表面上，该反应也称为迈克尔加成反应。通过 X 射线光电子能谱、红外光谱和接触角测量证实了聚合物在马来酰亚胺化表面上的固定。通过原子力显微镜观察了聚合物固定化表面，并通过椭圆偏振法确定了聚合物层的厚度。通过马来酰亚胺-巯基反应固定的聚合物的厚度小于旋涂形成的聚合物的厚度，除了聚苯乙烯。此外，聚合物固定化表面相对光滑，粗糙度小于 1nm。通过石英晶体微天平测量确定了表面固定的胺、马来酰亚胺和聚合物的量。含胺硅烷偶联剂的面积明显小于理论值，表明表面形成了多层硅烷偶联剂。低分子量的聚合物有在马来酰亚胺化表面上高效固定的趋势。当聚（p-丙烯酰胺基苯基-α-甘露糖苷）固定化表面用作蛋白质微阵列的平台时，与甘露糖结合凝集素伴刀豆球蛋白 A 检测到强烈的相互作用。与聚-l-赖氨酸涂覆的表面相比，聚（p-丙烯酰胺基苯基-α-甘露糖苷）固定化表面对伴刀豆球蛋白 A 的特异性进行了比较。聚-l-赖氨酸涂覆的表面非特异性地吸附了伴刀豆球蛋白 A 和牛血清白蛋白，而聚（p-丙烯酰胺基苯基-α-甘露糖苷）固定化表面优先吸附伴刀豆球蛋白 A。此外，聚（p-丙烯酰胺基苯基-α-甘露糖苷）固定化表面应用于光刻微图案化。当通过紫外线照射在聚（p-丙烯酰胺基苯基-α-甘露糖苷）旋涂表面上形成微图案时，使用荧光显微镜观察到表面吸附的荧光标记的伴刀豆球蛋白 A 上没有掩模设计的图案，因为聚（p-丙烯酰胺基苯基-α-甘露糖苷）从表面洗脱。相比之下，荧光标记的伴刀豆球蛋白 A 仅吸附在聚（p-丙烯酰胺基苯基-α-甘露糖苷）固定化表面的阴影区域，形成独特的荧光图案。使用马来酰亚胺化和可逆加成-断裂链转移聚合的表面改性方法可用于制备蛋白质微阵列和微图案化的平台。
