Guangdong Provincial Key Laboratory of Food Quality and Safety/College of Food Science, South China Agricultural University, Guangzhou, 510642, China.
Department of Applied Physics, The Hong Kong Polytechnic University, Hung Hom, 999077, Kowloon, Hong Kong.
Biosens Bioelectron. 2022 Jun 1;205:114089. doi: 10.1016/j.bios.2022.114089. Epub 2022 Feb 16.
Rapid and quantitative detection of paraquat is crucial because of its high toxicity. Here, we developed an ultrasensitive time-resolved fluorescence immunochromatographic assay (TRFICA) strip based on our synthesized variable domain of heavy chain antibody (VHH, also called Nanobody) for paraquat detection. Briefly, the specific immunogen selected from six designed antigens was employed to immunize alpaca, and a high-efficiency capacity of 1.6 × 10 pfu mL phage display nanobody library was established for biopanning against paraquat. The selected nanobody exhibited high sensitivity (limit of detection (LOD) was 0.0090 ng mL and IC was 0.0588 ng mL in buffer) and stability to high temperatures and denaturants. The molecular docking results indicated that the π-π, cation-π, and hydrogen bond interactions between paraquat and the pocket-like structures of complementarity-determining regions (CDRs) in VHH played a critical role in the antibody-paraquat recognition, competition, and affinity processes. The constructed TRFICA recognized paraquat through a quantitative analysis using the strip reader, and showed no cross-reactivity with other herbicides, and a semi-quantitative analysis using the naked eye. Notably, the potential practical applications of the TRFICA evaluated by performing a quantitative analysis of paraquat in food samples (vegetables, fruits, and grain products) and biological samples (blood and urine) showed a recovery rate range between 76.7% and 133.3% with inter-assay coefficient variation lower than 18.5%. The nanobody from phage display libraries was effective for small molecule recognition and detection, and it is a vital tool for immunoassay.
百草枯毒性高，因此快速定量检测百草枯至关重要。本研究基于合成的重链抗体可变区（VHH，也称为纳米抗体），开发了一种超灵敏时间分辨荧光免疫层析检测（TRFICA）试剂盒，用于百草枯检测。简而言之，用从六个设计抗原中选择的特异性免疫原对羊驼进行免疫，建立了高效容量的 1.6×10pfu mL噬菌体展示纳米抗体文库，用于针对百草枯的生物淘选。选择的纳米抗体表现出高灵敏度（在缓冲液中，检测限（LOD）为 0.0090ng/mL，IC 为 0.0588ng/mL）和对高温和变性剂的稳定性。分子对接结果表明，百草枯与 VHH 互补决定区（CDRs）中的口袋样结构之间的π-π、阳离子-π 和氢键相互作用，对抗体-百草枯识别、竞争和亲和力过程起着关键作用。所构建的 TRFICA 通过使用条带读取器进行定量分析来识别百草枯，并且与其他除草剂没有交叉反应，也可以通过肉眼进行半定量分析。值得注意的是，通过对食品样品（蔬菜、水果和谷物产品）和生物样品（血液和尿液）中的百草枯进行定量分析，评估了 TRFICA 的潜在实际应用，其回收率范围在 76.7%至 133.3%之间，批内变异系数低于 18.5%。噬菌体展示文库中的纳米抗体可有效识别小分子并进行检测，是免疫分析的重要工具。
