Development of high-throughput analytical system for intracellular molecules with quantum dots

荧光半导体量子点（QDs）是一类新型的荧光探针，具有不同于传统荧光染料的光学性质。QD在可见波长处发射明亮的荧光，这取决于颗粒的大小，并且此外，这种明亮的荧光对光漂白具有抗性。在这个项目中，我们的目的是证明量子点是有用的量化细胞内的材料，并应用于细胞计数。首先，在免疫组织化学中用量子点代替有机荧光染料。用量子点标记的二抗检测到一些细胞内/核抗原，但有些抗原不能。这可能是由于两种荧光探针的理化性质不同造成的，我们尝试了不同的固定和染色程序，以找到最佳的免疫组化条件与量子点。例如，样品需要用Triton X-100处理并在柠檬酸钠缓冲液（pH 6）中微波暴露，以检测 ...更多信息 核内增殖细胞核抗原（PCNA）。我们计划确定各种抗原的最佳方法。我们验证了量子点是否结合到它们的目标化学计量以及经典的荧光染料。用量子点和Alexa 488同时免疫细胞化学标记PC-14细胞核内的PCNA，用激光扫描细胞仪检测细胞周期中细胞核内PCNA的表达水平。同时，用碘化丙啶（PI）染色测定细胞核DNA。QD和Alexa 488荧光的双变量DNA/PCNA含量细胞图是圆顶形的，并且彼此非常相似。结果表明，量子点在最佳条件下的免疫细胞化学结果与有机荧光染料的结果相当，量子点可作为一种有用的荧光染料应用于细胞计数。然而，我们并不总是成功地检测荧光强度的LSC或流式细胞仪，我们需要进一步检查的免疫细胞化学方法和光学条件的细胞仪。少

Fluorescent semiconductor quantum dots (QDs) are a novel class of fluorescent probe with optical properties that differ from those of conventional fluorochromes. QDs emit bright fluorescence at visible wavelengths, depending on size of the particle, and in addition this bright fluorescence is resistant to photobleaching. In this project, we aimed to demonstrate that QDs are useful to quantify intracellular materials and applied to cytometry. First, QDs were substituted for organic fluorochromes in immunohistochemistry. Some intracellular/nuclear antigens were detected by secondary antibodies labeled with QDs, but some antigens were not. This may be caused by the difference of physiochemical property between two types of fluoroprobes and we tried various fixations and dying procedure to find optimal condition for immunohistochemistry with QDs. For example, samples needed treatment with Triton X-100 and microwave exposure in sodium citrate buffer (pH6) for the purpose of the detection of … More intranuclear proliferating cell nuclear antigen (PCNA). We plan to determine the optimal methods for various antigens. We verified whether QDs bind to their targets stoichiometrically as well as classical fluorochromes. We labeled PCNA in nuclei of PC-14 cells immunocytochemically with QDs and Alexa 488 concomitantly and measured the level of intranuclear PCNA expression during cell cycle with laser scanning cytometry. At the same time, nuclear DNA stained with propidium iodide (PI) was measured. The bivariate DNA/PCNA content cytograms for QDs and Alexa 488 fluorescence were dome-shaped and very similar to each other. These indicated that immunocytochemistry with QDs under optimal condition provided the data equivalent to those with organic fluorescence dyes and QDs could be useful fluorochromes in cytometry. However, we didn't always succeed to detect fluorescence intensity by LSC or flow cytometer and we need further examination for immunocytochemical methods and optical condition in cytometry. Less