用于检测生物胺组胺的高亲和力适体。,Analytical Chemistry

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用于检测生物胺组胺的高亲和力适体。
Analytical Chemistry ( IF 6.7 ) Pub Date : 2019-05-01 00:00:00 , DOI: 10.1021/acs.analchem.9b00075
Teresa Mairal Lerga ₁ , Miriam Jauset-Rubio ₁ , Vasso Skouridou ₁ , Abdulaziz S Bashammakh ₂ , Mohammad S El-Shahawi ₂ , Abdulrahman O Alyoubi ₂ , Ciara K O'Sullivan _{1,

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Affiliation

组胺在各种生理功能中的重要性及其在变应原反应中的作用使这种小分子成为研究最多的生物胺之一。尽管已描述了各种基于色谱的方法进行分析测定，但它们在成本，分析时间和低便携性方面的缺点限制了它们在原位常规测试中的适用性。在这项工作中，我们试图确定组胺结合适体，然后将其用于开发快速，简便，灵敏的组胺检测分析方法，以适合需要的分析。设计经典的SELEX工艺，使用磁珠固定靶标，十轮后选择完成。从阳性和反向选择磁珠进行最后一轮的下一代测序后，鉴定了几个序列，并使用apta-PCR亲和力测定（APAA）进行了初步筛选。候选人的结构和功能表征导致H2适体的鉴定。H4适体对组胺的高结合亲和力已通过四种独立的测定方法进行了验证（K _D为3–34 nM）。最后，H2适体用于基于磁珠的竞争性测定方法的开发，用于检测缓冲液和合成尿液中的组胺，分别达到非常低的检测限18 pM和76 pM，而没有基质效应观测到的。这些结果强调了用于鉴定小分子结合适体的策略的适用性，以及所选择的H2适体与生物样品分析的相容性，从而促进了常规测试的现场护理设备的开发。正在进行的工作集中在将H2适体的应用扩展到肉，鱼和饮料中腐败变质的检测上，以及评估截短形式的适体的亲和力。

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High Affinity Aptamer for the Detection of the Biogenic Amine Histamine.

The importance of histamine in various physiological functions and its involvement in allergenic responses make this small molecule one of the most studied biogenic amines. Even though a variety of chromatography-based methods have been described for its analytical determination, the disadvantages they present in terms of cost, analysis time, and low portability limit their suitability for in situ routine testing. In this work, we sought to identify histamine-binding aptamers that could then be exploited for the development of rapid, facile, and sensitive assays for histamine detection suitable for point-of-need analysis. A classic SELEX process was designed employing magnetic beads for target immobilization and the selection was completed after ten rounds. Following Next Generation Sequencing of the last selection rounds from both positive and counter selection magnetic beads, several sequences were identified and initially screened using an apta-PCR affinity assay (APAA). Structural and functional characterization of the candidates resulted in the identification of the H2 aptamer. The high binding affinity of the H2 aptamer to histamine was validated using four independent assays (K_D of 3–34 nM). Finally, the H2 aptamer was used for the development of a magnetic beads-based competitive assay for the detection of histamine in both buffer and synthetic urine, achieving very low limits of detection of 18 pM and 76 pM, respectively, while no matrix effects were observed. These results highlight the suitability of the strategy followed for identifying small molecule-binding aptamers and the compatibility of the selected H2 aptamer with the analysis of biological samples, thus facilitating the development of point-of-care devices for routine testing. Ongoing work is focused on extending the application of the H2 aptamer to the detection of spoilage in meat, fish, and beverages, as well as evaluating the affinity of truncated forms of the aptamer.

更新日期：2019-05-01

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