EA3798 Détection et Approches Thérapeutiques Nanotechnologiques dans les Mécanismes Biologiques de Défense, Université de Reims Champagne-Ardenne, 51100 Reims, France.
Anal Biochem. 2011 Sep 15;416(2):180-5. doi: 10.1016/j.ab.2011.05.018. Epub 2011 May 20.
Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs' advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs' amines with the sulfhydryl groups issued from the reduced Abs' disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD-Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy-light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD-Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals.
半导体量子点（QDs）被证明是独特的荧光标记物，为高通量检测和诊断提供了极好的可能性。为了充分发挥 QDs 在亮度、光稳定性、大斯托克斯位移和尺寸可调荧光发射方面的优势，它们应该在生物流体中保持稳定，并与靶特异性捕获分子标记。理想的基于 QD 的纳米探针的直径不应超过 15nm，并且应该在其表面上包含多个均匀定向的高活性亲和分子，例如抗体（Abs）。通过将 QD 的胺与从还原 Abs 的二硫键发出的巯基交联，将 QD 与 Abs 直接偶联是常见的技术。然而，该过程通常会产生偶联物，其中 QD 表面上功能活性 Abs 的数量并不总是符合预期，并且通常较低。在这里，我们开发了一种具有优化的 Ab 还原、亲和纯化和 QD-Ab 偶联关键步骤的先进程序。我们成功地还原了 Abs，使得还原反应产生了高度功能性的、部分切割的 75kDa 重轻 Ab 片段。这些 Ab 片段的亲和纯化，随后与 QD 标记，生成的 QD-Ab 偶联物与根据标准程序产生的偶联物相比，具有大大改善的功能。所开发的方法可以扩展到与任何类型的 Ab 与不同的半导体、贵金属或磁性纳米晶体的偶联。
