Department of Biochemistry, Faculty of Science, Ege University, Bornova, 35100 Izmir, Turkey.
Department of Medical Microbiology, Faculty of Medicine, Ege University, Bornova, 35100 Izmir, Turkey.
ACS Sens. 2021 Aug 27;6(8):2988-2997. doi: 10.1021/acssensors.1c00854. Epub 2021 Jul 16.
The global pandemic of COVID-19 continues to be an important threat, especially with the fast transmission rate observed after the discovery of novel mutations. In this perspective, prompt diagnosis requires massive economical and human resources to mitigate the disease. The current study proposes a rational design of a colorimetric lateral flow immunoassay (LFA) based on the repurposing of human samples to produce COVID-19-specific antigens and antibodies in combination with a novel dye-loaded polymersome for naked-eye detection. A group of 121 human samples (61 serums and 60 nasal swabs) were obtained and analyzed by RT-PCR and ELISA. Pooled samples were used to purify antibodies using affinity chromatography, while antigens were purified magnetic nanoparticles-based affinity. The purified proteins were confirmed for their specificity to COVID-19 commercial LFA, ELISA, and electrochemical tests in addition to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polymersomes were prepared using methoxy polyethylene glycol--polycaprolactone (mPEG--PCL) diblock copolymers and loaded with a Coomassie Blue dye. The polymersomes were then functionalized with the purified antibodies and applied for the preparation of two types of LFA (antigen test and antibody test). Overall, the proposed diagnostic tests demonstrated 93 and 92.2% sensitivity for antigen and antibody tests, respectively. The repeatability (92-94%) and reproducibility (96-98%) of the tests highlight the potential of the proposed LFA. The LFA test was also analyzed for stability, and after 4 weeks, 91-97% correct diagnosis was observed. The current LFA platform is a valuable assay that has great economical and analytical potential for widespread applications.
新型冠状病毒肺炎（COVID-19）全球大流行仍构成重大威胁，尤其是在发现新型突变后观察到的快速传播率。在这种情况下，及时诊断需要大量的经济和人力资源来减轻疾病。本研究提出了一种基于重新利用人体样本的比色横向流动免疫分析（LFA）的合理设计，用于产生 COVID-19 特异性抗原和抗体，并结合新型染料负载聚合物囊泡进行肉眼检测。收集了 121 个人体样本（61 份血清和 60 份鼻拭子），并通过 RT-PCR 和 ELISA 进行了分析。使用亲和层析从混合样本中纯化抗体，而抗原则通过基于磁性纳米颗粒的亲和技术进行纯化。使用商业 LFA、ELISA 和电化学测试以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析对纯化蛋白的 COVID-19 特异性进行了验证。使用甲氧基聚乙二醇-聚己内酯（mPEG-PCL）嵌段共聚物制备聚合物囊泡，并加载考马斯亮蓝染料。然后将聚合物囊泡用纯化的抗体进行功能化，并用于制备两种类型的 LFA（抗原测试和抗体测试）。总体而言，所提出的诊断测试对抗原测试和抗体测试的灵敏度分别为 93%和 92.2%。测试的重复性（92-94%）和再现性（96-98%）突出了所提出的 LFA 的潜力。还对 LFA 测试的稳定性进行了分析，4 周后，观察到 91-97%的正确诊断。当前的 LFA 平台是一种有价值的分析方法，具有广泛应用的巨大经济和分析潜力。
