响应DNA修复的工程全细胞微生物传感器,用于对基因毒性杂质进行敏感和高通量查
Jie Li1, Haotian Ding1, Yuning Zhao1
1Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 211198, China.
Analytical chemistry
|August 17, 2023
概括
新的全细胞微生物传感器 (WCMS) 为检测基因毒性杂质 (GTIs) 提供了一种更安全,更敏感的方法. 这些工程系统改进了传统的测定方法,使药物和环境污染物的准确风险评估成为可能.
科学领域:
- 生物技术和生物传感技术
- 环境健康和毒理学
- 药品分析 药品分析
背景情况:
- 药品,食品和环境中的基因毒性杂质 (GTIs) 对人类健康构成重大风险.
- 目前的黄金标准方法,如艾姆斯检测有局限性,包括代谢活动不足,高GTI要求,以及敏感性和准确性不足.
- 对于评估GTIs的先进,敏感和高吞吐量方法有极大需求.
研究的目的:
- 开发新的全细胞微生物传感器 (WCMS) 系统,以灵敏,准确地检测遗传毒性杂质 (GTIs).
- 使用大肠杆菌和酵母来设计响应DNA修复的WCMS,以适应不同的GTI代谢途径.
- 与传统检测方法相比,要显著提高灵敏度并减少所需的GTI数量.
主要方法:
- 构建了两种对DNA修复反应的工程WCMS系统 (*Escherichia coli*-WCMS和酵母-WCMS) 使用特定的促进剂 (*ada*和*MAG1*,分别).
- 将DNA修复响应促进体与增强绿色光蛋白 (EGFP) 记者基因合在等离子体上,用于引入WCMS.
- 通过基因淘汰和突变对大肠杆菌和酵母菌株进行基因修饰,以最大限度地减少干扰并最大限度地保持GTI,从而提高灵敏度.
主要成果:
- 成功构建和验证了*E. coli*-WCMS和酵母-WCMS系统,能够根据DNA化和代谢酶要求检测GTIs.
- 实现了GTI检测极限的显著降低,甲基甲硫酸盐 (MMS) 的消费量降至0.46-8.53μg,氨酸的消费量降至0.068-2.65μg.
- 与传统分析方法相比,灵敏度有2-3级的改善.
结论:
- 开发的WCMS方法提供了一种新,高度敏感和高效的方法,用于评估在分子层面上具有多种DNA损伤机制的GTIs.
- 这些工程 WCMS 系统有助于高通量选和敏感的风险评估各种矩阵中的基因毒污染物.
- 这项研究通过提供比传统的GTI评估技术更准确,更安全和更具成本效益的替代方案,推动了毒理学测试领域的发展.
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