Wang Yu, Zhang Bingbing, Zhang Ruohui, Ding Dang, Ma Wantong, Wang Wenrui, Liu Ziye, Zhu Yanzhi, Wang Xin, Zhi Dejuan, Wang Dongsheng
Joint Drug Development and Innovation Centre for Neurological Disorders of Lanzhou University-China National Biotec Group-Lanzhou Biotechnology Development Co., School of Pharmacy, Lanzhou University, Lanzhou, Gansu, 730000, PR China; MOE Frontiers Science Center for Rare Isotopes, Lanzhou University, Lanzhou, Gansu, 730000, PR China.
Joint Drug Development and Innovation Centre for Neurological Disorders of Lanzhou University-China National Biotec Group-Lanzhou Biotechnology Development Co., School of Pharmacy, Lanzhou University, Lanzhou, Gansu, 730000, PR China.
Anal Chim Acta. 2025 Feb 1;1337:343546. doi: 10.1016/j.aca.2024.343546. Epub 2024 Dec 21.
Botulinum neurotoxin type A (BoNT/A) is the most potent and prevalent neurotoxin known to cause botulism, and is also widely used in medical and cosmetic applications. The detection of BoNT/A is of great significance for botulism diagnosis and drug potency determination. Currently, the mouse bioassay (MBA) has long been the gold standard method but has disadvantages of ethical concerns, long testing duration, and high costs. CFP/YFP-based FRET sensors for detecting BoNT/A biological activity require complicated experimental control due to crosstalk, as well as inconvenient operation, intricate data analysis and UV-induced phototoxicity.
In present work, a FRET-based EGFP-SNAP25(141-206)-DsRED molecular sensor was developed to analyze the endopeptidase activity of BoNT/A, and methodological validation was performed. The results showed that the sensor has a high sensitivity of 4 U, with a linear range from 7.8 U to 125 U, a recovery rate of 96.8 % to 122.7 %, and a precision within 20 %. Furthermore, a cellular sensor for FRET-based BoNT/A biological activity detection was successfully constructed with a Neuro-2a cell line stably expressing the EGFP-SNAP25-tDimer2 variant, enabling the monitoring of the entire process of BoNT/A action. The cell-based assay exhibited a sensitivity of up to 100 pM of BoNT/A with a linear range from 3.125 nM to 50 nM and a recovery rate of 86.3 % to 117.2 %.
The FRET-based molecular and cellular sensors provided a convenient and rapid method for measuring BoNT/A biological activity, making them suitable for assessing BoNT/A raw material biological activities in clinical and environmental samples, as well as batches during pharmaceutical production processes.
A型肉毒杆菌神经毒素（BoNT/A）是已知导致肉毒中毒的最有效且最常见的神经毒素，同时也广泛应用于医学和美容领域。BoNT/A的检测对于肉毒中毒诊断和药物效价测定具有重要意义。目前，小鼠生物测定法（MBA）长期以来一直是金标准方法，但存在伦理问题、检测时间长和成本高的缺点。用于检测BoNT/A生物活性的基于CFP/YFP的FRET传感器由于串扰需要复杂的实验控制，操作不便，数据分析复杂且存在紫外线诱导的光毒性。
在本研究中，开发了一种基于FRET的EGFP-SNAP25(141-206)-DsRED分子传感器来分析BoNT/A的内肽酶活性，并进行了方法学验证。结果表明，该传感器具有4 U的高灵敏度，线性范围为7.8 U至125 U，回收率为96.8%至122.7%，精密度在20%以内。此外，成功构建了一种基于FRET的用于检测BoNT/A生物活性的细胞传感器，该细胞传感器使用稳定表达EGFP-SNAP25-tDimer2变体的Neuro-2a细胞系，能够监测BoNT/A作用的全过程。基于细胞的测定法对BoNT/A的灵敏度高达100 pM，线性范围为3.125 nM至50 nM，回收率为86.3%至117.2%。
基于FRET的分子和细胞传感器为测量BoNT/A生物活性提供了一种方便快捷的方法，使其适用于评估临床和环境样品中BoNT/A原料的生物活性以及药物生产过程中的批次。
