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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

作者： Wu, Shaowen 1 ; Xu, Jintao 1 ; Chen, Wenxing 1 ; Wang, Fenghua 1 ; Tan, Xiaoliang 1 ; Zou, Xinlu 1 ; Zhou, Weijie 1 ; Huang, Wenjie 1 ; Zheng, Yixiong 2 ; Wang, Shihua 3 ; Yan, Shijuan 1 ;

作者机构： 1.Guangdong Acad Agr Sci, Agrobiol Gene Res Ctr, State Key Lab Swine & Poultry Breeding Ind, Guangdong Key Lab Crop Germplasm Resources Preserv, Guangzhou 510640, Peoples R China

2.Zhongkai Univ Agr & Engn, Guangzhou Key Lab Res & Dev Crop Germplasm Resourc, Guangzhou 510225, Peoples R China

3.Fujian Agr & Forestry Univ, Sch Life Sci, Key Lab Pathogen Fungi & Mycotoxins Fujian Prov, Fuzhou 350002, Peoples R China

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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