Development of a micro fluorescence detector for m-TAS based on surface plasmon field enhancement

本研究的目的是开发一种基于表面等离子体增强激发的高性能荧光检测方法，用于集成到微量全分析系统(μ-TAS)中。我们采用了纳米印迹法制备的PMMA纳米锥阵列(直径250 nm；高度750 nm；间距250 nm；宏观尺寸5×5 mm^2)作为衬底。分别用单宁酸和硝酸银对其进行表面处理。将德州红固定在表面后，利用荧光显微镜比较有无纳米锥阵列在远场照射下相同区域的荧光强度，来评价纳米锥阵列的荧光增强性能。镀银纳米锥阵列的荧光强度大约是平面的50倍。这一结果表明，镀银纳米锥阵列的远场照射产生了局域等离子体，而强烈增强的电磁场有效地激发了固定在表面的荧光团。然后，我们演示了这种激励方法的实际应用。将抗-Ig G固定在镀银纳米锥阵列上，表面分别覆盖有PDMS微流体通道。将荧光标记的免疫球蛋白溶液和牛血清白蛋白溶液分别引入两个通道，洗涤后进行荧光显微成像。两个通道之间的荧光强度的明显差异表明，镀银纳米锥作为等离子体增强的底物可用于荧光生物分析。

The aim of this study is development of a high-performance fluorescence detection method based on surface plasmon enhanced excitation for integration into a micro total analysis system (μ-TAS). We employed a PMMA nanocone array (diameter, 250nm ; height, 750 nm ; pitch, 250 nm ; macroscopic dimension, 5×5mm^2) prepared by nanoimprinting as a substrate. The surface was treated with tannic acid and silver nitrate successively. After immobilization of Texas-Red on the surface, fluorescence enhancement property of the nanocone array was estimated by comparison between the fluorescence intensity of the same areas in the presence and absence of nanocone array upon far-field illumination using a fluorescence microscope. The silver-coated nanocone array showed the fluorescence intensity approximately 50 times larger than that of the flat surface. This result suggests that far-field illumination of the silver-coated nanocone array induces local plasmons and the strongly enhanced electromagnetic field effectively excites the fluorophores immobilized on the surface. Then we have demonstrated a practical application of this excitation method. Anti-IgG was immobilized on a silver coated nanocone arrays and the two sections of the surface were covered with PDMS microfluid channels, respectively. Fluorescence labeled IgG solution and BSA solution were independently introduced into the two channels and then, after careful washing, fluorescence microscopic imaging was carried out. The clear difference in the fluorescence intensity between the two channels demonstrates that the silver-coated nanocone is useful for fluorescence bioassays as a plasmon-enhanced substrate.