Department of Postharvest Science, Institute of Postharvest and Food Sciences, The Volcani Institute, Agricultural Research Organization, Bet Dagan 50250, Israel.
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Biosensors (Basel). 2022 Jan 25;12(2):63. doi: 10.3390/bios12020063.
Vertical flow immunoassays (VFIAs) are considered potential point-of-care testing (POCT) devices compared to lateral flow assays due to their ability to analyze a comparatively large sample volume and ease of multiplexing. However, VFIA devices are limited by low analytical sensitivity when coupled with a visual colorimetric signal. Herein, we carefully analyzed key parameters that accounted for the proper functionality of VFIA that can be modified to enhance the overall sensitivity of VFIA. In particular, we focused on improving the stability of conjugate pads impregnated with capture antibodies, maintaining a controlled flow rate to ensure higher analyte reactivity with capture antibodies, and enhancing the absorption efficiency. The results showed that air-drying of conjugate pads in the presence of 5% (/) lactose significantly improved the stability of antibodies during long-term storage. Integration of dissolvable polyvinyl alcohol (PVA) membrane of optimal concentration as a time-barrier film into the sensor delayed the flow of samples, thereby increasing the biorecognition interaction time between immunoreagents for the formation of immuno-complexes, which in turn led to higher sensitivity of the assay. Furthermore, the employment of an absorbent pad with higher water holding capacity significantly reduced the non-specific binding of immunocomplexes, thereby reducing the possibility of false-negative results.
垂直流免疫分析（VFIAs）相较于侧向流分析，因其能够分析较大样本量且易于多重检测而被认为是有潜力的即时检测（POCT）设备。然而，与可视化比色信号结合时，VFIAs 设备的分析灵敏度较低。在此，我们仔细分析了影响 VFIA 功能的关键参数，这些参数可进行修改以提高 VFIA 的整体灵敏度。具体而言，我们专注于改进带有捕获抗体的结合垫的稳定性，保持受控的流速以确保更高的分析物与捕获抗体的反应性，并提高吸收效率。结果表明，在存在 5%（/）乳糖的情况下，通过空气干燥使结合垫干燥可显著提高抗体在长期储存中的稳定性。将最佳浓度的可溶性聚乙烯醇（PVA）膜整合为时间屏障膜到传感器中，可延迟样品的流动，从而增加免疫试剂之间的生物识别相互作用时间，从而提高检测的灵敏度。此外，使用具有更高持水能力的吸收垫可显著减少免疫复合物的非特异性结合，从而降低假阴性结果的可能性。
