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

我们提出并制造了一种新型的垂直液流操作的化学反应器，以满足生物化学的广泛应用。为了快速、高产率地进行大量试剂间的反应，利用NewSuBARU公司LIGA光束线BL2的同步辐射，利用深X射线光刻技术，在200 PM厚的PMMA薄片上制作了直径为40 PM的2100个圆柱形通孔的滤光片。我们尝试将自制的立式反应器应用于夹心酶联免疫吸附试验(EL ISA)中，利用紫外光吸收光谱分析小鼠免疫球蛋白(Ig G)和壬基酚。我们成功地获得了小鼠免疫球蛋白的校正曲线，发现在总分析周期的45min内可以定量检测到浓度小于100 ng/ml的小鼠免疫球蛋白，这大约是常规微量滴定板法所需时间的三分之一。我们研究了将所提出的垂直微反应器用于竞争性酶联免疫吸附试验(ELISA)的可能性，该分析方法能够在一系列垂直流控操作中高灵敏地检测内分泌干扰物壬基酚。在用0.3 dg/ml的抗NP抗体处理的滤膜上，即使在0.01 ng/m1的游离NP浓度下，我们仍然观察到结合的抑制(B/B_0=90%)。在此条件下，获得了NP在0.01~10 ng/ml范围内的校正曲线，该灵敏度比用96孔微孔板和相同的抗NP抗体建立的常规ELISA法的灵敏度(5~100 ng/ml)提高了两个数量级。

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.