不对称流场的功能 - 在线与不同检测器联用的流分馏法用于对与生物分子偶联的水稳定量子点进行生物分子特征描述。

Capabilities of asymmetrical flow field - Flow fractionation on-line coupled to different detectors for characterization of water-stabilized quantum dots bioconjugated to biomolecules.

机构信息

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.

出版信息

Talanta. 2020 Jan 1;206:120228. doi: 10.1016/j.talanta.2019.120228. Epub 2019 Aug 5.

DOI:10.1016/j.talanta.2019.120228

PMID:31514892

Abstract

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.

摘要

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。