Biosensor Group, Korea Institute of Science and Technology Europe Forschungsgesellschaft mbH, Saarbrücken, Germany.
Biotechnol J. 2024 Jan;19(1):e2300482. doi: 10.1002/biot.202300482. Epub 2023 Dec 4.
Phage display technology employs a library of engineered filamentous M13 viruses infecting only bacteria. In this study, the genuine phage display selection technique was applied to identify a Pb(II) specific peptide. After three rounds of positive selection against Pb(II) coated agarose-based beads and the consecutive negative screenings against interfering metal ions (Al(III), Co(II), Fe(III), Ni(II), and Zn(II)), a final phage library with enhanced Pb(II) binding was obtained. Enzyme Linked Immunosorbent Assay (ELISA) analyses confirmed the selective Pb(II) binding of the enriched viruses. Twenty monoclonal phage plaques were randomly selected, single stranded DNAs (ssDNAs) were isolated and sequenced. Sequencing results revealed four different peptide sequences. Pb9 peptide (KASPYIT) showing the most specific Pb(II) binding was selected for detection studies. Pb9 was synthetically synthesized with additional three cysteine (3xCys) units at C-terminal. Twenty nanometers AuNPs were functionalized with Pb9-3xCys peptides through Au-thiol (Au-S) interaction. A colorimetric Pb(II) detection system was validated using the engineered peptide-AuNP complex at a calculated LOD of around 11 nM (3σ/k, n = 6) for the case study. The detection system was Pb(II) selective over various metal ions (Ag(II), Al(III), Au(III), Cd(II), Co(II), Cr (III), Cu(II), Fe(III), Hg(II), Mg(II), Mn(II), Ni(II), and Zn(II)). Such metal ion specific peptides can be further studied to develop simple, user friendly and cost-effective tools to design alternative detection and bioremediation systems for a circular economy.
噬菌体展示技术采用了一种仅感染细菌的工程丝状 M13 噬菌体文库。在这项研究中，真正的噬菌体展示选择技术被应用于鉴定一种 Pb(II) 特异性肽。经过三轮针对 Pb(II)涂覆琼脂糖珠的阳性选择和对干扰金属离子（Al(III)、Co(II)、Fe(III)、Ni(II)和 Zn(II)）的连续阴性筛选，获得了具有增强 Pb(II)结合能力的最终噬菌体文库。酶联免疫吸附分析（ELISA）分析证实了富集病毒对 Pb(II)的选择性结合。随机选择了 20 个单克隆噬菌体斑，分离并测序单链 DNA（ssDNA）。测序结果揭示了四个不同的肽序列。Pb9 肽（KASPYIT）显示出最特异的 Pb(II)结合被选为检测研究。Pb9 肽在 C 端添加了三个半胱氨酸（3xCys）单元被合成。二十纳米的 AuNPs 通过 Au-硫醇（Au-S）相互作用被 Pb9-3xCys 肽功能化。使用工程肽-AuNP 复合物验证了比色法 Pb(II)检测系统，对于案例研究，计算出的检测限约为 11 nM（3σ/k，n=6）。该检测系统对各种金属离子（Ag(II)、Al(III)、Au(III)、Cd(II)、Co(II)、Cr(III)、Cu(II)、Fe(III)、Hg(II)、Mg(II)、Mn(II)、Ni(II)和 Zn(II)）具有选择性。可以进一步研究这些金属离子特异性肽，以开发简单、用户友好且具有成本效益的工具，用于设计替代的检测和生物修复系统，以实现循环经济。
