Heavy metal contamination is an urgent environmental issue that poses a significant threat to human health and the ecosystem. To mitigate the adverse impacts of heavy metal pollution, the aim of this research was to develop genetically engineered zebrafish as biosensors, which offer a promising alternative for detecting heavy metal exposure, specifically Cd²⁺ and Zn²⁺. A novel heavy metal-sensitive gene construct metallothionine 2 promoter with DsRed reporter gene (mt2-DsRed2) was synthesized and integrated into zebrafish embryos using a Tol2 transposon transposase system with the transgenic zebrafish line subjected to biosensing applications for Cd²⁺ and Zn²⁺. The biosensor showed specific responses with linear correlation heavy metal concentration and DsRed fluorescence signal for Cd²⁺ and Zn²⁺ with (p < 0.01) a minimum detection limit of 4 ppb for each metal ion but not for the non-specific metal ion Ni²⁺, which makes it suitable for laboratory-based heavy metal assessment assays in the low heavy metal concentration ranges of 0–10 ppb. Additionally, the study investigated the toxicity of heavy metals on zebrafish early developmental stages applying a modified version of the OECD Fish Embryo Toxicity (FET-236) test. Accordingly, Cd²⁺, Zn²⁺, and Ni²⁺ exhibited no significant toxicity effects on zebrafish embryos within the low dose range of 2–10 ppb confirming the biocompatibility of the transgenic zebrafish biosensor for heavy metal sensing applications. Thus, the developed transgenic zebrafish line can accurately sense heavy metals Cd²⁺ and Zn²⁺ within the low dose range, making it a promising alternative laboratory assay for environmental monitoring and risk assessment.

中文翻译：

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用于重金属检测的创新型转基因斑马鱼生物传感器


重金属污染是一个紧迫的环境问题，对人类健康和生态系统构成重大威胁。为了减轻重金属污染的不利影响，本研究的目的是开发基因工程斑马鱼作为生物传感器，这为检测重金属暴露提供了一种有前途的替代方案，特别是 Cd ² ⁺ 和 Zn ² ⁺。合成了一种具有 DsRed 报告基因 （mt2-DsRed2） 的新型重金属敏感基因构建体金属硫氨酸 2 启动子，并使用 Tol2 转座子转座子转座酶系统整合到斑马鱼胚胎中，转基因斑马鱼系经受 Cd ²⁺ 和 Zn ²⁺ 的生物传感应用。生物传感器显示出线性相关重金属浓度和 Cd ²⁺ 和 Zn ²⁺ 的 DsRed 荧光信号的特异性响应，（p < 0.01） 每种金属离子的最小检测限为 4 ppb ²⁺ ，但非特异性金属离子 Ni 则不然，这使其适用于 0-10 ppb 的低重金属浓度范围内的基于实验室的重金属评估测定。此外，该研究还应用了经合组织鱼胚胎毒性 （FET-236） 测试的修改版本，调查了重金属对斑马鱼早期发育阶段的毒性。因此，在 2-10 ppb 的低剂量范围内，Cd ²⁺ 、 Zn ²⁺ 和 Ni 对斑马鱼胚胎没有 ²⁺ 表现出显着的毒性影响，证实了转基因斑马鱼生物传感器在重金属传感应用中的生物相容性。 因此，开发的转基因斑马鱼系可以准确感应低剂量范围内的重金属 Cd ²⁺ 和 Zn ²⁺ ，使其成为环境监测和风险评估的有前途的替代实验室检测方法。