Siderophores are metal chelators produced by microorganisms to facilitate binding and uptake of iron. The isolation and characterization of siderophores are impeded by typically low siderophore yields and the complexity of siderophore-containing extracts generated with traditional purification methods. We investigated titanium dioxide nanoparticle solid-phase extraction (TiO₂ NP SPE) as a technique to selectively concentrate and purify siderophores from complex matrices for subsequent LC-MS detection and identification. TiO₂ NP SPE showed a high binding capacity (15.7 ± 0.2 μmol mg⁻¹ TiO₂) for the model siderophore desferrioxamine B (DFOB) and proved robust to pH changes and the presence of EDTA. These are significant advances in comparison to immobilized metal affinity chromatography (IMAC). The TiO₂ NP SPE was highly selective and recovered 77.6 ± 6.2% of DFOB spiked to a compositionally complex bacterial culture supernatant. The simple clean-up procedure removed the majority of contaminants and allowed direct detection of siderophores from the LC-MS base peak chromatogram. The ‘untargeted’ purification and analysis of an untreated supernatant of iron-deprived bacterial culture allowed for the direct identification of two known and three novel ferrioxamines. Thus, TiO₂ NP SPE in combination with LC-MS offers great potential as a discovery platform for the purification and subsequent quantification or identification of novel siderophores of microbial origin.

中文翻译：

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用二氧化钛纳米粒子固相萃取纯化铁载体。

铁载体是微生物产生的金属螯合剂，以促进铁的结合和吸收。典型的低铁载体产量和传统纯化方法产生的含铁载体提取物的复杂性阻碍了铁载体的分离和表征。我们研究了二氧化钛纳米颗粒固相萃取（TiO ₂ NP SPE）作为从复杂基质中选择性富集和纯化铁载体以用于后续LC-MS检测和鉴定的技术。TiO ₂ NP SPE的结合能力高（15.7±0.2μmolmg ^-1 TiO ₂），用于模型铁载体去铁胺B（DFOB），并被证明对pH值变化和EDTA的存在很强。与固定金属亲和色谱法（IMAC）相比，这些都是重要的进步。TiO ₂ NP SPE具有很高的选择性，回收了掺入组成复杂的细菌培养上清液的DFOB的77.6±6.2％。简单的清理程序去除了大多数污染物，并允许从LC-MS基础峰色谱图中直接检测铁载体。脱铁细菌培养物未经处理的上清液的“非目标”纯化和分析可直接鉴定出两种已知和三种新型亚铁胺。因此，TiO ₂ NP SPE与LC-MS组合具有广阔的潜力，可作为发现平台，用于纯化，后续定量或鉴定微生物来源的新型铁载体。