Kuo Fang-Yin, Chang Bo-Yao, Wu Ching-Yi, Mong Kwok-Kong Tony, Chen Yu-Chie
Department of Applied Chemistry, National Chiao Tung University , Hsinchu 300, Taiwan.
Anal Chem. 2015 Oct 20;87(20):10513-20. doi: 10.1021/acs.analchem.5b02712. Epub 2015 Oct 8.
Foodborne illness outbreaks resulting from contamination of Escherichia coli O157:H7 remain a serious concern in food safety. E. coli O157:H7 can cause bloody diarrhea, hemolytic uremic syndrome, or even death. The pathogenicity of E. coli O157:H7 is mainly caused by the expression of Shiga-like toxins (SLTs), i.e., SLT-1 and SLT-2. SLTs are pentamers composed of a single A and five B subunits. In this study, we propose a magnetic nanoparticle (MNP)-based platform to rapidly identify SLT-1 from the complex cell lysate of E. coli O157:H7. The core of the MNPs is made of iron oxide, whereas the surface of the core is coated with a thin layer of alumina (Fe3O4@Al2O3 MNPs). The Fe3O4@Al2O3 MNPs are functionalized with pigeon ovalbumin (POA), which contains Gal-α(1→4)-Gal-β(1→4)-GlcNAc termini that can bind SLT-1B selectively. Furthermore, POA is a phosphate protein. Thus, it can be easily immobilized on the surface of the Fe3O4@Al2O3 MNPs through aluminum phosphate chelation under microwave heating within 1.5 min. The generated POA-Fe3O4@Al2O3 MNPs are capable of effectively enriching SLT-1B from complex cell lysates simply by pipetting 20 μL of the sample in and out of the tip in a vial for ∼1 min. To release SLT-1 from the MNPs, Gal-α(1→4)-Gal disaccharides were used for displacement. The released target species are sufficient to be identified by matrix-assisted laser desorption/ionization mass spectrometry. Although the sample volume used in this approach is small (20 μL) and the enrichment time is short (1 min), the selectivity of this approach toward SLT-1B is quite good. We have demonstrated the effectiveness of this approach for rapid determination of the presence of SLT-1 from complex cell lysates and ham/juice samples based on the detection of SLT-1B.
由大肠杆菌O157:H7污染导致的食源性疾病暴发仍是食品安全领域的一个严重问题。大肠杆菌O157:H7可引起血性腹泻、溶血尿毒综合征，甚至死亡。大肠杆菌O157:H7的致病性主要由志贺样毒素（SLTs）即SLT-1和SLT-2的表达引起。SLTs是由一个A亚基和五个B亚基组成的五聚体。在本研究中，我们提出了一种基于磁性纳米颗粒（MNP）的平台，用于从大肠杆菌O157:H7的复杂细胞裂解物中快速鉴定SLT-1。MNPs的核心由氧化铁制成，而核心表面涂有一层薄薄的氧化铝（Fe3O4@Al2O3 MNPs）。Fe3O4@Al2O3 MNPs用鸽卵清蛋白（POA）进行功能化，POA含有可选择性结合SLT-1B的Gal-α(1→4)-Gal-β(1→4)-GlcNAc末端。此外，POA是一种磷酸化蛋白。因此，在微波加热1.5分钟内，通过磷酸铝螯合作用，它可以很容易地固定在Fe3O4@Al2O3 MNPs的表面。所生成的POA-Fe3O4@Al2O3 MNPs能够通过在小瓶的吸头中简单地吸入和排出20 μL样品约1分钟，有效地从复杂细胞裂解物中富集SLT-1B。为了从MNPs中释放SLT-1，使用Gal-α(1→4)-Gal二糖进行置换。释放的目标物质足以通过基质辅助激光解吸/电离质谱法进行鉴定。尽管该方法使用的样品体积小（20 μL）且富集时间短（1分钟），但该方法对SLT-1B的选择性相当好。我们已经证明了基于对SLT-1B的检测，该方法对于从复杂细胞裂解物和火腿/果汁样品中快速测定SLT-1的存在是有效的。
