Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería, 8, 33006, Oviedo, Spain; Scholarship By CNPq, Brazil.
Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería, 8, 33006, Oviedo, Spain.
Talanta. 2020 Jan 1;206:120228. doi: 10.1016/j.talanta.2019.120228. Epub 2019 Aug 5.
The asymmetric flow field-flow fractionation (AF4) coupled on-line with elemental (inductively coupled plasma-mass spectrometry, ICP-MS) and molecular (fluorescence and UV) detection has been investigated as a powerful tool for the characterization of bioinorganic nano-conjugates. In this study, we described methods for the characterization of biotin-antibody complexes bioconjugated with streptavidin quantum dots (QDs-SA-b-Ab). Operating parameters of AF4 separation technique were optimized and two procedures are proposed using a channel thickness of 350 μm and 500 μm. The use of a 500 μm spacer allowed to achieve an efficient AF4 separation of the QDs-SA-b-Ab complexes from the excess of individual species used in the bioconjugation that was required for a proper characterization of the bioconjugates. Optimization of the AF4 allowed a separation resolution good enough to isolate the QDs-SA-b-Ab bioconjugates from the free excess of b-Ab and QD-SA. The efficiency of the bioconjugation process could be then calculated, obtaining a value of 86% for a 1 QDs-SA: 5 b-Ab bioconjugation ratio. In addition, sample recovery around 90% was achieved.
不对称流场流分离（AF4）与元素（电感耦合等离子体质谱，ICP-MS）和分子（荧光和 UV）检测在线联用，已被研究作为表征生物无机纳米缀合物的有力工具。在本研究中，我们描述了用于表征生物素-抗体复合物与链霉亲和素量子点（QDs-SA-b-Ab）生物缀合的方法。优化了 AF4 分离技术的操作参数，并提出了两种使用通道厚度为 350 μm 和 500 μm 的程序。使用 500 μm 的间隔物可以有效地将 QDs-SA-b-Ab 复合物从生物缀合中所需的过量单个物种中分离出来，这对于正确表征生物缀合物是必要的。AF4 的优化允许分离分辨率足够好，足以将 QDs-SA-b-Ab 生物缀合物与游离过量的 b-Ab 和 QD-SA 分离。然后可以计算生物缀合过程的效率，对于 1 QDs-SA：5 b-Ab 的生物缀合比，获得 86%的值。此外，实现了约 90%的样品回收率。
