Department of Chemical Engineering and Analytical Chemistry, Institute for Research on Nutrition and Food Safety (INSA•UB), University of Barcelona, C/Martí i Franquès 1-11, 08028, Barcelona, Spain.
Department of Analytical Chemistry, University of Valencia, C/Doctor Moliner 50E, 46100, Burjassot, Valencia, Spain.
Mikrochim Acta. 2023 May 13;190(6):219. doi: 10.1007/s00604-023-05795-y.
An aptamer-functionalized stir bar sorptive extraction (SBSE) coating is described for the first time devoted to selective isolation and preconcentration of an allergenic food protein, concavanalin A (Con A), followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) determination. For this purpose, the polytetrafluoroethylene surface of commercial magnetic stir bars was properly modified and vinylized to immobilize a thiol-modified aptamer against Con A via straightforward "thiol-ene" click chemistry. The aptamer-functionalized stir bar was employed as SBSE sorbent to isolate Con A, and several parameters that can affect the extraction efficiency were investigated. Under the optimized conditions, Con A was extracted and desorbed during 30 and 45 min, respectively, at 25 °C and 600 rpm. The SBSE MALDI-TOF-MS method provided limits of detection of 0.5 μg mL for Con A. Furthermore, the SBSE coating was highly selective to Con A compared to other lectins. The developed method was successfully applied to the determination of low levels of Con A in several food matrices (i.e., white beans as well as chickpea, lentils, and wheat flours). Recoveries ranged from 81 to 97% with relative standard deviations below 7%. The aptamer-based stir bars presented suitable physical and chemical long-term stability (1 month) and a reusability of 10 and 5 extraction cycles with standards and food extracts, respectively. The developed aptamer-affinity extraction devices open up the possibility of developing novel highly selective SBSE coatings for the extraction of proteins and peptides from complex samples.
一种适体功能化搅拌棒吸附萃取 (SBSE) 涂层首次被描述，用于选择性分离和浓缩过敏原蛋白，即伴大豆球蛋白 A (Con A)，随后进行基质辅助激光解吸/电离飞行时间质谱 (MALDI-TOF-MS) 测定。为此，商用磁性搅拌棒的聚四氟乙烯表面经过适当修饰和乙烯化，通过简单的“硫醇-烯”点击化学将硫醇修饰的适体固定在表面上。将适体功能化的搅拌棒用作 SBSE 吸附剂来分离 Con A，并研究了影响萃取效率的几个参数。在优化条件下，Con A 在 25°C 和 600 rpm 下分别萃取和洗脱 30 和 45 min。SBSE-MALDI-TOF-MS 方法对 Con A 的检测限为 0.5 μg·mL。此外，与其他凝集素相比，SBSE 涂层对 Con A 具有更高的选择性。该方法成功应用于几种食品基质（即白豆以及鹰嘴豆、小扁豆和小麦粉）中低水平 Con A 的测定。回收率在 81%至 97%之间，相对标准偏差低于 7%。基于适体的搅拌棒具有合适的物理和化学长期稳定性（1 个月），以及分别用于标准品和食品提取物的 10 次和 5 次萃取循环的可重复使用性。开发的适体亲和萃取装置为从复杂样品中提取蛋白质和肽开发新型高度选择性的 SBSE 涂层开辟了可能性。
