Most of lateral flow immunoassay (LFIA) devices rely on gold nanoparticles (GNP) labeled antibodies or other biospecific proteins, to achieve reagent-less color-based detection. GNP size, GNP-protein conjugation level and its stability are crucial points for the development of precise and accurate methods. In addition, the purification of the GNP-protein conjugates from unreacted protein and GNP, is necessary for adequate analytical performance of the assay.

To assist the synthesis and production process of GNP and their protein conjugates, we use for the first time a non-destructive, particle separation-multi-detection approach based on miniaturized flow field flow fractionation (HF5). A separation method was developed to baseline size-separate GNP, GNP-protein, protein and GNP including BSA used as a surface coater in less than 30 minutes.

Freshly synthesized GNP were first characterized and then conjugated with two different model antibodies: a mouse immunoglobulin (IgG) and a fluorescein-labeled mouse immunoglobulin (FITC-IgG).

The IgG-GNP complexes were fractionated using the HF5 apparatus, able to separate IgG-GNP from free proteins by their hydrodynamic size, allowing purification of the conjugation product. Both IgG-GNPs and GNPs were characterized according to their size by the MALS detector, and according to their Surface Plasmon Resonance and spectrum by UV-Vis detection, improving the results obtained via batch characterization.

This simple non-invasive approach is very useful for the LFIA development and optimization: the use of HF5-mutidetection offers a unique tool for this purpose facilitating the industrialization of the process and the relate optimization and standardization.

大部分侧向免疫分析（LFIA）设备依靠金纳米颗粒（GNP）标记的抗体或其他生物特异性蛋白质，以实现无试剂的基于颜色的检测。GNP大小，GNP蛋白质结合水平及其稳定性是开发精确方法的关键。此外，从未反应的蛋白质和GNP纯化GNP-蛋白质偶联物对于测定的足够分析性能是必要的。

为了协助GNP及其蛋白结合物的合成和生产过程，我们首次使用了基于微型流场流分馏（HF5）的无损粒子分离-多重检测方法。开发了一种分离方法，可在不到30分钟的时间内将基线大小分离的GNP，GNP蛋白，蛋白质和GNP（包括BSA）用作表面涂层剂。

首先对新鲜合成的GNP进行表征，然后与两种不同的模型抗体偶联：小鼠免疫球蛋白（IgG）和荧光素标记的小鼠免疫球蛋白（FITC-IgG）。

IgG-GNP复合物使用HF5设备分级分离，能够通过其流体动力学大小从游离蛋白中分离IgG-GNP，从而纯化结合产物。IgG-GNP和GNP均通过MALS检测器根据其大小进行表征，并通过UV-Vis检测根据其表面等离子体共振和光谱进行表征，从而改善了通过批处理表征获得的结果。

这种简单的非侵入性方法对于LFIA的开发和优化非常有用：HF5-mutidetection为此目的提供了一个独特的工具，可促进过程的工业化以及相关的优化和标准化。