SpyTag/SpyCatcher-based Self-Assembly Multivalent Anti-idiotypic Nanobody Mediated Environmentally Friendly Lateral Flow Immunoassay for Mycotoxin Zearalenone

IF 3.7 1区化学 Q1 CHEMISTRY, ANALYTICAL

Sensors and Actuators B: Chemical Pub Date : 2025-09-29 DOI:10.1016/j.snb.2025.138885

Yuanzhen Guo, Chong Cai, Xinyu Xu, Xiao Wu, Minwen Sun, Tailong Wei, Yujie Shi, Yubin Liu, Zhui Tu, Yanping Li, Qinghua He

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引用次数: 0

Abstract

Anti-idiotypic nanobodies (AIdnbs) are attractive as biosynthetic surrogate antigens due to their batch consistency, high tolerance and eco-friendly production. However, monomeric AIdnbs suffer from low immobilization efficiency and weak signal response on nitrocellulose filter (NC) membranes in lateral flow immunoassay (LFIA). To address this, we developed a self-assembly multivalent AIdnb system using the zearalenone (ZEN)-specific AIdnb Z6 as a model. We fused SpyTag or SpyCatcher to Z6 via flexible (GS) or semi-rigid (EK) linkers, then assembled bivalent Z6 through SpyTag/SpyCatcher and heptavalent Z6 via SpyTagged IMX313 scaffolds. EK-linked Z6-fusions consistently exhibited superior activity compared with GS-linked fusions due to the superior conformation for antibody binding of EK-linked variants. Adsorption kinetics and LFIA detection demonstrated the superior NC membranes’ immobilization efficiency and analytical performance of multivalent Z6 to monovalent Z6. The EK-linked bivalent Z6 demonstrated optimal performance in time-resolved microspheres-based LFIA, achieving a detection limit of 32.44 pg/mL for ZEN in 15 minutes, which is 4.7-fold lower than monovalent formats, along with a 4.5-fold broader detection range. Notably, this detection sensitivity surpassed conventional chemically synthesized antigens, achieving a 2.5-fold improvement. Furthermore, Maize, wheat and oat were tested as real samples, detection limits were 1.31–1.94 µg/kg for the bivalent Z6-based LFIA, and 1.62–2.12 µg/kg for heptavalent Z6-based LFIA. This study establishes a generalizable strategy for engineering high-performance multivalent biosynthetic antigens, enabling sensitive, eco-friendly LFIA for small-molecule detection.

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基于SpyTag/ spycatcher的自组装多价抗独特型纳米体介导的霉菌毒素玉米赤霉烯酮环境友好侧流免疫测定

抗独特型纳米体（AIdnbs）因其批量一致性、高耐受性和环保生产而成为生物合成的替代抗原。然而，在侧流免疫分析（LFIA）中，单体aidnb存在固定效率低、信号响应弱的问题。为了解决这个问题，我们开发了一种自组装多价AIdnb系统，使用玉米霉烯酮（ZEN）特异性AIdnb Z6作为模型。我们通过柔性（GS）或半刚性（EK）连接剂将SpyTag或SpyCatcher与Z6融合，然后通过SpyTag/SpyCatcher组装二价Z6，通过SpyTagged IMX313支架组装七价Z6。与gs连接的融合物相比，ek连接的z6融合物始终表现出更好的活性，这是由于ek连接的变体具有更好的抗体结合构象。吸附动力学和LFIA检测表明，NC膜具有较好的固定化效率和对多价Z6的分析性能。ek -联二价Z6在基于时间分辨微球的LFIA中表现出最佳性能，15分钟内ZEN的检出限为32.44 pg/mL，比单价格式低4.7倍，检测范围宽4.5倍。值得注意的是，这种检测灵敏度超过了传统的化学合成抗原，实现了2.5倍的提高。以玉米、小麦和燕麦为实际样品，二价z6基LFIA的检出限为1.31 ~ 1.94µg/kg，七价z6基LFIA的检出限为1.62 ~ 2.12µg/kg。本研究建立了一种可推广的策略来设计高性能的多价生物合成抗原，使敏感、环保的LFIA用于小分子检测。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Sensors and Actuators B: Chemical 工程技术-电化学

CiteScore

14.60

自引率

11.90%

发文量

1776

审稿时长

3.2 months

期刊介绍： Sensors & Actuators, B: Chemical is an international journal focused on the research and development of chemical transducers. It covers chemical sensors and biosensors, chemical actuators, and analytical microsystems. The journal is interdisciplinary, aiming to publish original works showcasing substantial advancements beyond the current state of the art in these fields, with practical applicability to solving meaningful analytical problems. Review articles are accepted by invitation from an Editor of the journal.