Woolf M Shane, Dignan Leah M, Karas Scott M, Lewis Hannah M, Kim Sabrina N, Geise Geoffrey M, DeMers Haley L, Hau Derrick, Gates-Hollingsworth Marcellene A, AuCoin David P, Landers James P
Department of Chemistry, University of Virginia, USA.
Department of Chemical Engineering, University of Virginia, USA.
Anal Methods. 2023 Apr 13;15(15):1870-1880. doi: 10.1039/d3ay00073g.
We report clear proof-of-principle for centrifugally-driven, multiplexed, paper-based orthogonal flow sandwich-style immunocapture (cOFI) and colorimetric detection of Zaire Ebola virus-like particles. Capture antibodies are immobilized onto nanoporous nitrocellulose membranes that are then laminated into polymeric microfluidic discs to yield ready-to-use analytical devices. Fluid flow is controlled solely by rotational speed, obviating the need for complex pneumatic pumping systems, and providing more precise flow control than with the capillary-driven flow used in traditional lateral flow immunoassays (LFIs). Samples containing the antigen of interest and gold nanoparticle-labeled detection antibodies are pumped centrifugally through the embedded, prefunctionalized membrane where they are subsequently captured to generate a positive, colorimetric signal. When compared to the equivalent LFI counterparts, this cOFI approach generated immunochromatographic colorimetric responses that are objectively darker (saturation), more intense (grayscale), and less variable regarding total area of the color response. We also describe an image analysis approach that enables access to rich color data and area statistics without the need for a commercial 'strip reader' or custom-written image analysis algorithms. Instead, our analytical method exploits inexpensive equipment (, smart phone, flatbed scanner, ) and freely available software (Fiji distribution of ImageJ) to permit characterization of immunochromatographic responses that includes multiple color metrics, offering insights beyond typical grayscale analysis. The findings reported here stand as clear proof-of-principle for the feasibility of disc-based, centrifugally driven orthogonal flow through a membrane with immunocapture (cOFI) and colorimetric readout of a sandwich-type immunoassay in less than 15 minutes. Once fully developed, this cOFI platform could render a faster, more accurate diagnosis, while processing multiple samples simul-taneously.
我们报告了用于扎伊尔埃博拉病毒样颗粒的离心驱动、多重、基于纸的正交流夹心式免疫捕获（cOFI）和比色检测的明确原理证明。捕获抗体固定在纳米多孔硝酸纤维素膜上，然后将其层压到聚合物微流控盘中，以生产即用型分析装置。流体流动仅由转速控制，无需复杂的气动泵系统，并且比传统侧向流免疫分析（LFI）中使用的毛细管驱动流提供更精确的流量控制。含有感兴趣抗原和金纳米颗粒标记检测抗体的样品通过嵌入式预功能化膜进行离心泵送，随后在膜上被捕获以产生阳性比色信号。与等效的LFI对应物相比，这种cOFI方法产生的免疫色谱比色反应在客观上更暗（饱和度）、更强烈（灰度），并且颜色反应总面积的变化更小。我们还描述了一种图像分析方法，无需商业“试纸读取器”或定制编写的图像分析算法即可获取丰富的颜色数据和面积统计信息。相反，我们的分析方法利用廉价设备（智能手机、平板扫描仪）和免费软件（ImageJ的Fiji版本）来表征免疫色谱反应，该反应包括多个颜色指标，提供了超越典型灰度分析的见解。此处报告的研究结果明确证明了基于盘的离心驱动通过膜进行免疫捕获（cOFI）和在不到15分钟内对比色夹心型免疫分析进行比色读数的可行性。一旦完全开发出来，这个cOFI平台可以实现更快、更准确的诊断，同时处理多个样品。
