Abstract：
In this study, a magnetic nanomaterial antibody (Ab)-SiO2@Fe3O4 was synthesized, which was employed to absorb aflatoxin B1 (AFB1) in complicated grain matrices. The Ab-SiO2@Fe3O4 material was then paired with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for subsequent accurate detection. The Ab-SiO2@Fe3O4 material has a specific adsorption capacity for AFB1 because of the stable and specific biological binding between antigen and antibody. This process can achieve the identification between the material and food matrix quickly, thereby completing the separation and enrichment process. Then, high sensitivity and high accuracy HPLC-MS/MS were employed for signal readout and actual quantification, which can significantly increase the detection efficiency and enable high-throughput detection of numerous samples. In the pretreatment process, Fe3O4 was first synthesized by microwave-assisted hydrothermal synthesis within 1 h, and Ab-SiO2@Fe3O4 was then produced using the enhanced Stober\'s approach. This material with high adsorption performance was synthesized under relatively mild conditions and short time. To obtain Ab-SiO2@Fe3O4 materials with uniform particle size, magnetic properties, and dispersibility that met the requirements, synthesis conditions of Ab-SiO2@Fe3O4 and conditions for capturing the AFB1 target were analyzed. The findings demonstrated that the best effect was obtained when the dosage of FeCl3·6H2O was 10.0 mmol, the heating time was 40 min, and 100 μL tetraethoxysilane was employed for SiO2 coating. The AFB1 antibody was then combined with the surface of SiO2@Fe3O4 under several conditions. The findings revealed that the best coupling efficiency of Ab could be obtained when the concentration of 2-morpholinoethanesulfonic acid monohydrate (MES) was 10 mmol/L, pH was 6.5, and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)∶N-hydroxysuccinimide substances (NHS) was 2∶1. The coupling buffer was then selected as phosphate buffer (PBS) with pH=7.4, and 8 mg Ab-SiO2@Fe3O4 was employed to separate and enrich AFB1 at 37 ℃ for 10 min. In the actual detection, acetonitrile-water-formic acid (85∶10∶5, v/v/v) was employed as the extraction solution. After ultrasonic extraction for 10 min, Ab-SiO2@Fe3O4 was employed to separate and enrich AFB1 in the extract. The supernatant was dried with nitrogen and reconstituted with 1-mL acetonitrile. The solution was then filtered through a 0.22 μm filter and detected using HPLC-MS/MS, thereby realizing the quick and quantitative detection of AFB1. AFB1 had an excellent linear relationship in the range of 2-50 μg/L under the optimal analytical conditions, and the correlation coefficient was less than 0.99. The LOD was 0.04 μg/kg, and the LOQ was 0.13 μg/kg. The spiked recoveries of AFB1 in three grain matrices ranged from 76.21% to 92.85% with RSD≤5.29% at four different spiked levels. The approach was applied to the determination and analysis of AFB1 in 30 real grain samples of rice, corn, and wheat. The findings demonstrated that AFB1 was detected in one wheat sample and two corn samples, and its content was 0.38, 0.13, and 0.47 μg/kg, respectively, and no toxins were found in other samples. The approach combined Ab-SiO2@Fe3O4 magnetic nanomaterials with HPLC-MS/MS, which could obtain high-efficiency separation and enrichment of AFB1. Furthermore, the low-cost Ab-SiO2@Fe3O4 could be stored for more than a week and complete the pretreatment process within 30 min. This effective pretreatment process combined with HPLC-MS/MS could realize the analysis of several samples within a short time, and had a promising application prospect in the detection of AFB1 in grains.
摘要：
在这项研究中,合成了一种磁性纳米材料抗体(Ab)-SiO2@Fe3O4,用于在复杂的谷物基质中吸收黄曲霉毒素B1（AFB1）。然后将Ab-SiO2@Fe3O4材料与高效液相色谱-串联质谱(HPLC-MS/MS)配对,用于随后的准确检测。Ab-SiO2@Fe3O4材料由于抗原和抗体之间的稳定和特异性生物结合而对AFB1具有特异性吸附能力。此过程可以快速实现物料与食品基质之间的识别，从而完成分离和富集过程。然后，高灵敏度和高精度HPLC-MS/MS用于信号读出和实际定量，可以显著提高检测效率,实现大量样品的高通量检测。在预处理过程中,首先在1h内通过微波辅助水热合成合成Fe3O4，然后使用增强的Stober方法生产Ab-SiO2@Fe3O4。在相对温和的条件和较短的时间下合成了具有高吸附性能的材料。获得粒径均匀的Ab-SiO2@Fe3O4材料,磁性，和满足要求的分散性，分析了Ab-SiO2@Fe3O4的合成条件和捕获AFB1靶标的条件。结果表明，当FeCl3·6H2O的用量为10.0mmol时，效果最好。加热时间为40分钟，和100μL四乙氧基硅烷用于SiO2涂层。然后在几种条件下将AFB1抗体与SiO2@Fe3O4的表面结合。结果表明，当2-吗啉代乙磺酸一水合物（MES）的浓度为10mmol/L时，Ab的偶联效率最好。pH为6.5,1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)∶N-羟基琥珀酰亚胺物质(NHS)的摩尔比为2∶1。然后选择偶联缓冲液为pH=7.4的磷酸盐缓冲液（PBS），并使用8mgAb-SiO2@Fe3O4在37℃下分离和富集AFB110分钟。在实际检测中,采用乙腈-水-甲酸(85∶10∶5,v/v/v)作为提取液。超声提取10分钟后，Ab-SiO2@Fe3O4用于分离和富集提取物中的AFB1。用氮气干燥上清液并用1-mL乙腈重构。然后将溶液通过0.22μm过滤器过滤并使用HPLC-MS/MS检测。从而实现AFB1的快速定量检测。在最佳分析条件下,AFB1在2-50μg/L范围内具有良好的线性关系,相关系数小于0.99。LOD为0.04μg/kg,LOQ为0.13μg/kg。在四个不同的加标水平下，三种谷物基质中AFB1的加标回收率为76.21％至92.85％，RSD≤5.29％。将该方法应用于30个水稻实粒样品中AFB1的测定和分析,玉米,和小麦。结果表明，在一个小麦样品和两个玉米样品中检测到AFB1，其含量为0.38、0.13和0.47μg/kg，分别,在其他样本中没有发现毒素。该方法将Ab-SiO2@Fe3O4磁性纳米材料与HPLC-MS/MS联用,获得高效分离富集的AFB1。此外，低成本的Ab-SiO2@Fe3O4可以储存一周以上，并在30分钟内完成预处理过程。这种有效的预处理工艺与HPLC-MS/MS相结合,可以在短时间内实现对多个样品的分析,在谷物中AFB1的检测中具有广阔的应用前景。
