Huang Shengfeng, Gan Ning, Liu Haibo, Zhou You, Chen Yinji, Cao Yuting
Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo, 315211, China.
Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo, 315211, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Aug 15;1060:247-254. doi: 10.1016/j.jchromb.2017.05.035. Epub 2017 Jun 12.
In this work, a novel aptamer functionalized magnetic adsorbent was developed and combined with magnetic dispersive solid phase extraction (MDSPE) for selective enrichment of several amphenicol antibiotics residues (chloramphenicol(CAP), thiamphenicol(TAP) and florphenicol(FF)) in foodstuff then determined by High Performance Liquid Chromatography (HPLC)-Diode array detector(DAD). Firstly, a magnetic silica-coated FeO microsphere(FeO@SiO) was synthetized by sol-gel method, then it was functionalized by amino groups through 3-Aminopropyltriethoxysilane (APTES) reagent to form FeO@SiO-NH; Thirdly, the amino group on FeO@SiO-NH was transferred to carboxylic group via the succinic anhydride to form FeO@SiO-COOH. Finally a kind of DNA aptamer with amino group which can simultaneously recognize CAP, TAP, FF, was functionalized on FeO@SiO-COOH through adding the EDC/sulfo-NHS coupling reagent (FeO@SiO@Apt). Therefore, a very stable and useful adsorbent was synthesized for the detection of chloramphenicol series antibiotics. This adsorbent can specifically and simultaneously recognize and enrich CAP, TAP, and FF with high adsorption amount from some complicated food matrix, e.g. milk based on the high affinity of aptamer towards the analytes. The saturated extraction capacities for CAP, TAP and FF by the adsorbent were 2.82, 2.56, 2.72μg/g (mass of target/adsorbent)respectively and the enrichment folds were more than 100 times. Afterwards, the target analytes were washed away by pH 8.5 0.1M Tris-HCl buffer solution and detected by HPLC-DAD. The parameters including extraction temperature, extraction capacity, extraction & desorption pH, extraction & desorption time were investigated. With the optimized conditions, the limits of detection (LOD) and limits of quantitation (LOQ) were 0.12-0.17ng/ml and 0.40-0.55ng/ml for the amphenicols in milk. The adsorbent also has good reproducibility for extraction which can be reused at least for 60 cycles with the recovery over 80% (Fig. S2). The MDSPE combined with HPLC-DAD detection possessed the advantages of high selectivity, extraction capacity and very convenient for magnetic separation. In addition, this method is environment friendly and employed no organic solution in the period of pretreatment and extracting. It is a universal platform which can be extended to selective enrichment other organic pollutants residues if changing the modified aptamers.
在本研究中，开发了一种新型适配体功能化磁性吸附剂，并将其与磁性分散固相萃取（MDSPE）相结合，用于选择性富集食品中的几种氯霉素类抗生素残留（氯霉素（CAP）、甲砜霉素（TAP）和氟苯尼考（FF）），然后通过高效液相色谱（HPLC）-二极管阵列检测器（DAD）进行测定。首先，采用溶胶-凝胶法合成了磁性二氧化硅包覆的Fe3O4微球（Fe3O4@SiO2），然后通过3-氨丙基三乙氧基硅烷（APTES）试剂对其进行氨基功能化，形成Fe3O4@SiO2-NH2；第三步，通过丁二酸酐将Fe3O4@SiO2-NH2上的氨基转化为羧基，形成Fe3O4@SiO2-COOH。最后，通过加入EDC/sulfo-NHS偶联试剂，将一种带有氨基且能同时识别CAP、TAP、FF的DNA适配体功能化到Fe3O4@SiO2-COOH上（Fe3O4@SiO2@Apt）。因此，合成了一种非常稳定且有用的吸附剂用于氯霉素类抗生素的检测。基于适配体对分析物的高亲和力，该吸附剂能够特异性地同时识别和富集CAP、TAP和FF，且从一些复杂的食品基质（如牛奶）中具有高吸附量。该吸附剂对CAP、TAP和FF的饱和萃取容量分别为2.82、2.56、2.72μg/g（目标物质量/吸附剂质量），富集倍数超过100倍。之后，用pH 8.5的0.1M Tris-HCl缓冲溶液洗脱出目标分析物，并用HPLC-DAD进行检测。考察了萃取温度、萃取容量、萃取和解吸pH值、萃取和解吸时间等参数。在优化条件下，牛奶中氯霉素类抗生素的检测限（LOD）和定量限（LOQ）分别为0.12 - 0.17ng/ml和0.40 - 0.55ng/ml。该吸附剂的萃取重现性良好，至少可重复使用60次，回收率超过80%（图S2）。MDSPE与HPLC-DAD检测相结合具有选择性高、萃取容量大、磁分离非常方便等优点。此外，该方法环境友好，在预处理和萃取过程中不使用有机溶剂。这是一个通用平台，如果更换修饰的适配体，可扩展到选择性富集其他有机污染物残留。
