Research on micro environmental analysis using stacked miniaturized 3D chemical system

提出并制造了一种具有垂直流体流动操作的新型化学反应器，具有广泛的生化应用前景。为了实现大批量试剂之间的快速、高产反应，利用NewSUBARU的LIGA光束线BL2同步辐射，采用深x射线光刻技术，在200pm厚度的PMMA薄片上制备了2100个直径为40pm的圆柱通孔滤光片。我们尝试将自制的垂直反应器应用于夹心式酶联免疫吸附试验（ELISA），利用紫外吸收光谱分析小鼠免疫球蛋白（IgG）和壬基酚。我们成功获得小鼠IgG的校准曲线，发现小鼠IgG浓度小于100 ng/ml，在总分析周期45 min内即可定量检测，约为常规方法使用微量滴度板所需周期的三分之一。我们研究了使用所提出的垂直微反应器进行竞争性酶联免疫吸附试验（ELISA）的分析方法的可能性，该方法能够在一系列垂直流体操作中高灵敏度地检测内分泌干扰物壬基酚。在用0.3 Dg/ml抗NP抗体预处理的滤网中，即使在0.01ng/m1的游离NP浓度下，我们仍然观察到对结合的抑制作用（B/B_0=90%）。在此条件下，得到了NP在0.01 ~ 10ng/ml范围内的标定曲线。该灵敏度比使用96孔微滴板和同一抗np抗体的普通ELISA检测灵敏度（5-100 ng/ml）提高2个数量级。

We proposed and fabricated a new chemical reactor with a vertical fluid flow operation for the wide biochemical applications. For rapid and high yield reaction between reagents with large quantity, the filters, which possess 2,100-cylindrical through-bores with 40pm diameter in 200pm-thickness PMMA sheets, were fabricated by deep X-ray lithography using synchrotron-radiation from LIGA beamline BL2 of NewSUBARU. We tried the application of the fabricated vertical reactor to an sandwich enzyme-linked immunosorbent assay (ELISA) for the analysis of mouse immunoglobulin (IgG) and nonylphenol using ultra violet (UV) absorption spectroscopy. We succeeded to obtain calibration curve of Mouse IgG and found that the mouse IgG less than 100 ng/ml concentration was quantitatively detected within 45 min of total analytical period, which was about one third of the period required for the conventional method using micro titer plate. We investigated the possibility of the analysis methods using the proposed vertical micro reactor for competitive enzyme-linked immunosorbent assay (ELISA), which enable high sensitive detection of an endocrine disrupter nonylphenol in a series of vertical fluidic operation. In the assay using filters pretreated with 0.3 Dg/ml of anti-NP-antibody, even at 0.01ng/m1 of free NP, we still observed the inhibition of the binding (B/B_0=90%). Under this condition, calibration curve of NP was obtained at the range of 0.01-10ng/ml. This sensitivity was two orders higher than the sensitivity (5-100 ng/ml) obtained by ordinary ELISA using 96-wells-micro-titer plate and the same anti-NP-antibody.