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玉米赤霉烯酮(Zearalenone,ZEN),又称F2毒素,是由镰刀菌属产生的一种非甾体霉菌毒素。小麦、玉米、大麦等谷物在生产、加工、储存过程中均可能被镰刀菌属污染[1],同时也有研究表明部分粮食副产品如饲料、啤酒[2]、牛奶[3]中也被发现存在玉米赤霉烯酮污染。ZEN是一种内分泌干扰物,对动物和人类有外源性雌激素作用,具有生殖毒性[4],此外,ZEN还具有细胞毒性[5]、免疫毒性[6]以及致癌性[7],目前已经被国际癌症研究机构列为Ⅲ类致癌物。目前,多个国家和地区都对ZEN进行了含量限制,欧盟对谷物中玉米赤霉烯酮的最高限量为100 μg·kg−1,我国对谷物及其制品中玉米赤霉烯酮的最高限量为60 μg·kg−1[8],对饲料中玉米赤霉烯酮的最高限量为500 μg·kg−1[9]。
高效液相色谱法、液相色谱-串联质谱法及免疫亲和柱-荧光光度法等技术由于具有高灵敏度和高准确性的优点,是目前ZEN检测最常用的方法,但是存在预处理复杂、设备昂贵,对操作人员要求高等缺点,不适用于大量样品的快速筛选。新的生物传感分析技术,如电化学生物传感器法[10-11],模拟肽表位ELISA传感器法[12-13],分子印迹传感器法[14],蛋白质芯片光学检测技术[15]等,由于高灵敏度和高特异性已被用于食品中ZEN检测。但是,这些传感技术也存在一些问题,如电化学方法的重复性差,电极容易被污染,ELISA方法检测周期长、试剂消耗量大,分子印迹法易出现包埋现象,不易洗脱等。因此,亟需开发用于食品中ZEN快速、灵敏、准确的新型分析技术,对确保食品安全具有重要意义。
倏逝波光纤免疫传感器是基于倏逝波原理和荧光免疫分析原理,激发光在光纤探头以全内反射方式传播时,光纤探头表面形成的倏逝波激发结合在光纤探头表面荧光标记的生物识别分子(抗体或核酸适体)发光,利用检测到的荧光信号与样品中待测物浓度的线性关系实现定量检测。由于倏逝波距光纤探头界面呈指数式衰减,其有效渗透深度有限(<100 nm),不需要清洗步骤即可鉴别出结合在生物传感表面的荧光标记抗体和溶液中游离的荧光标记抗体,可以有效降低检测时间。本课题组研发的倏逝波光纤免疫传感器结合了倏逝波生物传感技术和微流控技术的优点,具有试剂消耗低、检测快速、重复性好、携带方便等特点,在检测各种污染物方面具有很大的潜力,本研究首次将其应用于食品中ZEN的快速检测,通过优化检测条件,实现了实际样品中ZEN的快速、高灵敏检测。
用于食品中玉米赤霉烯酮高灵敏、快速检测的倏逝波免疫传感技术
Rapid and sensitive detection of ZEN in cereal using an evanescent wave optofluidic immunosensor
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摘要: 玉米赤霉烯酮(zearalenone,ZEN)是一种由镰刀菌属产生的次级代谢产物,利用课题组自主研发的倏逝波光纤免疫传感器,以玉米赤霉烯酮包被抗原修饰的光纤探头作为生物识别元件,基于间接竞争免疫分析原理,建立了ZEN的高灵敏、快速定量检测方法。在最佳检测条件下,ZEN的检测限为0.35 μg·L−1,线性区间为0.75—7.48 μg·L−1,单个检测周期只需8 min。包被抗原修饰的光纤探头可重复使用100次以上而没有明显的性能损失,可以保证检测结果的准确性并降低成本。对小麦、玉米、猪饲料进行了不同浓度的加标回收测试,结果显示加标回收率在80.86%—113.92%范围内,相对标准偏差小于7.90%,表明该传感器具有很好的稳定性和精确度,可以用于食品中ZEN的快速检测,为食品安全评价提供有力分析工具。Abstract: Zearalenone (ZEN) is a secondary metabolite produced by Fusarium. Based on the indirect competitive immunoassay principle, an portable and reusable evanescent wave optofluidic immunosensor is at the first time reported for rapid and sensitive detection of ZEN using zearalenone antigen modified fiber optic bio-probe as a biological recognition element. Under optimal conditions, the detection limit of ZEN is 0.35 μg·L−1, and the linear range is 0.75—7.48 μg·L−1, one detection cycle is within 8 min. The antigen modified fiber optic bio-probe can be reused more than 100 times without obvious performance loss, which ensures the accuracy of the detection results and reduce cost. The recovery of wheat, corn and pig feed with different concentrations was tested. The results showed that the recovery rate was in the range of 80.86%—113.92%, the relative standard deviation is less than 7.90%. These results indicated that this immunosensor has good stability and accuracy and can be employed for the rapid detection of ZEN in food and provides a powerful analysis tool for food safety evaluation.
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Key words:
- zearalenone /
- evanescent wave /
- immunosensor /
- detection
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图 6 抗体选择性和光纤重复性(a)抗体选择性。AT、DON、OTA和AFB1浓度为1000 μg·L−1,ZEN浓度为10 μg·L−1;(b)光纤重复性。空白样品多次循环检测的荧光信号值
Figure 6. Selectivity of antibody and reusability of the optic fiber probe:(a)Selectivity of the antibody for detection of ZEN. The concentrations of AT, DON, OTA, and AFB1 were 1000 μg·L−1, respectively, and the concentration of ZEN was 10 μg·L−1;(b)Reusability of the fiber optic probe. Maximum signal for immunoreaction of the multiple tests in the absence of ZEN
表 1 ZEN不同检测方法比较
Table 1. Comparison of different detection methods for ZEN
方法
Methods线性范围/(μg·L−1)
Analysis range检测限/(μg·L−1)
LOD检测时间/min
Detection time参考文献
ReferenceHPLC 5—1000 1.0 29.6 [18] 磁性固相萃取联合HPLC 1—100 0.24 40 [19] 荧光偏振免疫传感器 3—1052 0.54 15 [20] 分子印迹法 50—1000 18 60 [14] 基于磁珠的酶联免疫 — 0.13 65 [21] 酶联免疫法 0.28—6.84 0.26 140 [12] 表面等离子体共振 2.8—75 — 20 [22] 量子点标记层析试纸条 0.78—25 0.58 8 [23] 胶体金试纸条 — 1.25 5 [24] 倏逝波光纤免疫传感器 0.75 — 7.48 0.35 8 本研究 表 2 倏逝波光纤免疫传感器检测实际样品的加标回收率及变异系数
Table 2. The recovery rate and coefficient of variation of the real samples detected by the evanescent wave optofluidic immunosensor
实际样品
Real samples加标浓度/(μg·kg−1)
Spiked concentration检测浓度/(μg·kg−1)
Detected concentration加标回收率/%
Recovery变异系数/%
RSD小麦 Wheat 50 56.96 113.92 1.25 100 80.86 80.86 1.43 150 165.27 110.18 0.19 玉米 Corn 50 55.65 111.29 1.12 100 93.62 93.62 6.88 150 132.07 88.05 0.29 猪饲料 Pig feed 50 50.78 101.56 5.21 100 81.55 81.55 2.94 150 143.18 95.45 7.90 -
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