Protein nanoscaffold enables programmable nanobody-luciferase immunoassembly for sensitive and simultaneous detection of aflatoxin B1 and ochratoxin A

文献类型： 外文期刊

第一作者： Wu, Shaowen

作者： Wu, Shaowen;Xu, Jintao;Chen, Wenxing;Wang, Fenghua;Tan, Xiaoliang;Zou, Xinlu;Zhou, Weijie;Huang, Wenjie;Yan, Shijuan;Xu, Jintao;Zheng, Yixiong;Wang, Shihua

作者机构：

关键词： Immunoassay; Protein nanoscaffold; Nanobody-Luciferase conjugate; Mycotoxin; Simultaneous detection

期刊名称：JOURNAL OF HAZARDOUS MATERIALS （ 影响因子：13.6； 五年影响因子：12.7 ）

ISSN： 0304-3894

年卷期： 2024 年 462 卷

页码：

收录情况： SCI

摘要： Mycotoxins produced by fungi can contaminate various foods and pose significant health risks. Ensuring food safety demands rapid, highly sensitive analytical techniques. One-step Bioluminescent Enzyme Immunoassays (BLEIAs) employing nanobody-nanoluciferase fusion proteins have recently garnered attention for operational simplicity and heightened sensitivity. Nevertheless, fixed nanobody:nanoluciferase ratios in fusion proteins restrict the customization and sensitivity of traditional BLEIAs. In this study, we present a Scaffold Assembly -based BLEIA (SA-BLEIA) that overcomes these limitations through the programmable conjugation of nano -bodies and luciferases onto 60-meric protein nanoscaffolds using SpyTag/SpyCatcher linkages. These nano -scaffolds facilitate the adjustable coupling of anti-aflatoxin B1 and anti-ochratoxin A nanobodies with luciferases, optimizing nanobody/luciferase ratios and diversifying specificities. Compared to conventional methods, SA-BLEIA demonstrates considerably elevated sensitivity for detecting both toxins. The elevated local concentration of luciferase significantly amplifies bioluminescence intensity, permitting reduced substrate consumption and cost-effective detection. The usage of dual-nanobody conjugates facilitates the quantification orsimultaneous detection of both mycotoxins in a single test with shared reagents. The assay exhibits exceptional recovery rates in spiked cereal samples, strongly correlating with outcomes from commercial ELISA kits. Overall, this adaptable, highly sensitive, cost-effective, and multiplexed immunoassay underscores the potential of tunable scaffold assembly as a promising avenue for advancing bioanalytical diagnostic tools.

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