基于石墨烯材料的黄曲霉毒素液相色谱检测方法研究
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摘要
黄曲霉毒素是一类由真菌（主要为黄曲霉菌和寄生曲霉菌）产生的高毒次生代谢产物，以高毒性和强致癌性著称，早在1993年黄曲霉毒素B1就被世界卫生组织（World Health Organization,WHO）的癌症研究机构认定为1类致癌物。自然界中，黄曲霉毒素的产毒菌株分布广泛，因此，黄曲霉毒素广泛存在于玉米、花生、大米、植物油脂等农产品中，严重威胁农产品消费安全。近年来，世界各国设置了严格的限量标准来保障农产品质量安全，对黄曲霉毒素检测方法的要求也越来越高。因此，新型快速的黄曲霉毒素检测方法研究就变得尤为重要。
在众多黄曲霉毒素的分析方法中，样品前处理仍然被认为是整个分析过程里，一个非常重要且需要重点研究的环节，尤其是对于复杂样品，它甚至成为制约整个分析过程的决定性因素。农产品中黄曲霉毒素的含量极低，加之很多农产品含油量高，基质复杂，这些都进一步增加了前处理的难度。
本研究针对以上迫切需要解决的样品前处理难题，将新型纳米材料——石墨烯及其衍生物应用于黄曲霉毒素的检测研究中，并建立了基于新材料萃取的黄曲霉毒素液相色谱检测方法，为农产品中黄曲霉毒素污染监测提供了技术支撑。本论文的主要研究内容和创新点如下：
1.成功研制出三种石墨烯纳米材料，并对其形貌和结构进行分析。以石墨粉为原料，经化学氧化制备出氧化石墨烯，合成的氧化石墨烯呈绸缎状，厚度在0.8~1.1nm之间，片层大小在0.1~2μm之间。进一步通过两种合成方式，制备出氧化石墨烯-二氧化硅复合材料，SEM分析表明，氧化石墨烯被成功包覆在二氧化硅纳米颗粒上，并将小颗粒的二氧化硅颗粒聚集，一定程度上起到粘合剂作用。通过在氧化石墨烯的分散溶液中引入铁基前驱物，采用溶剂热法一步还原，制备出有磁性的石墨烯-四氧化三铁复合材料。XRD和SEM的结果表明，直径在100nm的四氧化三铁磁珠被均匀附着在了石墨烯的片层上。
2.研究探明了氧化石墨烯对黄曲霉毒素B1的吸附性能，并建立了利用氧化石墨烯作为吸附剂的食用植物油中黄曲霉毒素B1脱除方法。研究发现，黄曲霉毒素B1在氧化石墨烯上的吸附平衡时间为4h，最大吸附容量为62.5ng/mg。氧化石墨烯不仅可以吸附黄曲霉毒素B1，还可以吸附黄曲霉毒素B2。氧化石墨烯对植物油脂样品中黄曲霉毒素的实际吸附率在97.8~98.8%之间，说明其可以用于实际植物油样品中黄曲霉毒素的脱除。
3.通过优选氧化石墨烯，建立了基于氧化石墨烯萃取花生中黄曲霉毒素HPLC检测方法。新方法评价结果表明，四种黄曲霉毒素的检出限在0.08~0.65μg/kg之间，定量限在0.25~2.0μg/kg之间,在检测范围内表现出良好的线性相关性，相关系数在0.99217~0.99995之间，花生样品中四种黄曲霉毒素的加标回收率在85.1~100.8%之间，相对标准偏差在2.1~7.9%之间，证明本方法适用花生实际样品检测。
4.创建了氧化石墨烯-二氧化硅复合材料的黄曲霉毒素SPE前处理技术，研究建立了与之配套的黄曲霉毒素HPLC检测方法，并成功用于大米和玉米粮油产品黄曲霉毒素检测中。新方法检测的四种黄曲霉毒素在0.5~50μg/kg的范围内均具有良好的线性，其相关系数在0.99956~0.99994之间，检出限在0.05~0.5μg/kg之间，定量限在0.2~2.0μg/kg之间。
5.针对植物油基质粘性大，萃取材料固液相难分离的缺陷，成功建立了以石墨烯-四氧化三铁复合材料作为萃取材料的植物油中黄曲霉毒素MSPE的前处理方法与配套HPLC检测方法。该方法黄曲霉毒素B1的检测范围在0.85~51μg/kg之间，检出限为0.85μg/kg；黄曲霉毒素B2的检测范围相对较大，在0.22~66.99μg/kg之间，检出限为0.22μg/kg。方法中黄曲霉毒素B1和B2的定量限分别为2.64μg/kg和0.66μg/kg。
Aflatoxins are a group of mycotoxins produced as highly toxic secondary metabolites by manyspecies of Aspergillus, a fungus, the most notable ones being Aspergillus flavus and Aspergillusparasiticus. Aflatoxins are highly toxic and carcinogenic. Especially, aflatoxin B1is classified as aGroup1carcinogen by the World Health Organisation (WHO) in1993. The aflatoxin-producing fungiare widespread in nature, which leads to contamination of aflatoxins in a variety of agro products, suchas corn, peanuts, rice, vegetable oils and so on. Thus, it is a huge threat to comsuption safety of agroproducts. In recent years, strict standards and control measurements have been established to ensureagro-products quality and safety in different countries successively, which means that the requirementfor the methods for the detection of aflatoxins is getting higher the higher. Therefore, the researches onnovel methods for rapid detection of aflatoxins become more and more important.
In many methods for detection and quantification of aflatoxins, sample pretreatment is stillconsidered as an important part in whole analytical procedure, which needs to pay much attention.Especially for complex samples, it may even become a bottleneck point in the whole analysis process.Many agro products with low content of aflatoxins have a high oil content and complex matrix, whichfurther increases the difficulty of aflatoxins analysis.
For solving such problems, in this study, new nanomaterials--graphene and its derivatives areused as adsorbents for aflatoxins coupled with HPLC to establish new methods for detecting aflatoxins.The new methods can be the technical support for monitoring studies of aflatoxins in agro products. Themain research contents and innovative points are as follows:
1. Three kinds of nanomaterials were successfully synthesized. And their morphology and structurewere also studied. Graphene oxide (GO) was synthesized by chemical oxidation of graphite powder.Silk-like GO sheets showed a thickness of0.8~1.1nm, with a layer size of0.1~2μm. A series ofGO-SiO2nanocomposites were prepared by two ways. The SEM results proved that GO wassuccessfully grafted on the silica nanoparticles. The results also indicated that GO acted as inorganicglue to bind the small silica nanoparticles together. A magnetic graphene material with a one-stepsolvothermal method was synthesized by introducing a ferrous precursor in GO solution. The XRD andSEM results showed that the Fe3O4magnetic beads with a diameter of100nm are uniformly attachedon the graphene sheets.
2. The tests were performed to evaluate the ability of graphene oxide (GO) as the adsorbent toadsorb aflatoxins B1. And the GO was successfully applied in detoxifying of aflatoxins in ediblevegetable oils. The research also found that it took4hours for GO to adsorb aflatoxin B1to achieveadsorption equilibrium. The maximum adsorption capacity for GO is62.5ng/mg. The GO can notonly adsorb aflatoxin B1but also a certain content of aflatoxin B2. The recoveries of the GO adsorptionof aflatoxin B1in actual oil samples ranged from97.8to98.8%, indicating that GO was suitable for removing aflatoxins in edible vegetable oils.
3. Graphene oxide was synthesized and selected to be applied as an effective adsorbent fordetermination and quantification of aflatoxins in peanuts by HPLC. The limit of detection of thismethod ranges from0.08to0.65μg/kg. The limit of quantification of this method ranges from0.25to2.0μg/kg. Good linear relationships have been obtained with the correlation coefficient (r) ranging from0.99217to0.99995. The recoveries of the four aflatoxins in peanut samples are in the range from85.1%to100.8%with the relative standard deviations between2.1%and7.9%, indicating that this method wassuitable for real sample analysis.
4. A method for detection aflatoxins in rice and corn has been developed by using GO-SiO2nanocomposites as the extraction material in SPE. In this method, four aflatoxins can be detected with agood linear in the range of0.5~50μg/kg. The values of the correlation coefficient between0.99956~0.99994have been achieved. The limits of detection of this method for four aflatoxins are inthe range of0.05~0.5μg/kg with the limits of quantitation in the range of0.2~2.0μg/kg.
5. The vegetable oils were extremely viscous. Therefore, the solid materials for extraction aredifficult to separate from the liquid sample solution. A MSPE method was optimized to successfullyextract two aflatoxins in vegetable oils. In this method, for aflatoxin B1, a linear detection range of0.85~51μg/kg was found with a detection limit of0.85μg/kg. For aflatoxin B2, the new detectionmethod has a larger linear detection ranging from0.22~66.99μg/kg with a detection limit of0.22μg/kg.The limits of quantitation for aflatoxin B1and B2were2.64and0.66μg/kg respectively.
在众多黄曲霉毒素的分析方法中，样品前处理仍然被认为是整个分析过程里，一个非常重要且需要重点研究的环节，尤其是对于复杂样品，它甚至成为制约整个分析过程的决定性因素。农产品中黄曲霉毒素的含量极低，加之很多农产品含油量高，基质复杂，这些都进一步增加了前处理的难度。
本研究针对以上迫切需要解决的样品前处理难题，将新型纳米材料——石墨烯及其衍生物应用于黄曲霉毒素的检测研究中，并建立了基于新材料萃取的黄曲霉毒素液相色谱检测方法，为农产品中黄曲霉毒素污染监测提供了技术支撑。本论文的主要研究内容和创新点如下：
1.成功研制出三种石墨烯纳米材料，并对其形貌和结构进行分析。以石墨粉为原料，经化学氧化制备出氧化石墨烯，合成的氧化石墨烯呈绸缎状，厚度在0.8~1.1nm之间，片层大小在0.1~2μm之间。进一步通过两种合成方式，制备出氧化石墨烯-二氧化硅复合材料，SEM分析表明，氧化石墨烯被成功包覆在二氧化硅纳米颗粒上，并将小颗粒的二氧化硅颗粒聚集，一定程度上起到粘合剂作用。通过在氧化石墨烯的分散溶液中引入铁基前驱物，采用溶剂热法一步还原，制备出有磁性的石墨烯-四氧化三铁复合材料。XRD和SEM的结果表明，直径在100nm的四氧化三铁磁珠被均匀附着在了石墨烯的片层上。
2.研究探明了氧化石墨烯对黄曲霉毒素B1的吸附性能，并建立了利用氧化石墨烯作为吸附剂的食用植物油中黄曲霉毒素B1脱除方法。研究发现，黄曲霉毒素B1在氧化石墨烯上的吸附平衡时间为4h，最大吸附容量为62.5ng/mg。氧化石墨烯不仅可以吸附黄曲霉毒素B1，还可以吸附黄曲霉毒素B2。氧化石墨烯对植物油脂样品中黄曲霉毒素的实际吸附率在97.8~98.8%之间，说明其可以用于实际植物油样品中黄曲霉毒素的脱除。
3.通过优选氧化石墨烯，建立了基于氧化石墨烯萃取花生中黄曲霉毒素HPLC检测方法。新方法评价结果表明，四种黄曲霉毒素的检出限在0.08~0.65μg/kg之间，定量限在0.25~2.0μg/kg之间,在检测范围内表现出良好的线性相关性，相关系数在0.99217~0.99995之间，花生样品中四种黄曲霉毒素的加标回收率在85.1~100.8%之间，相对标准偏差在2.1~7.9%之间，证明本方法适用花生实际样品检测。
4.创建了氧化石墨烯-二氧化硅复合材料的黄曲霉毒素SPE前处理技术，研究建立了与之配套的黄曲霉毒素HPLC检测方法，并成功用于大米和玉米粮油产品黄曲霉毒素检测中。新方法检测的四种黄曲霉毒素在0.5~50μg/kg的范围内均具有良好的线性，其相关系数在0.99956~0.99994之间，检出限在0.05~0.5μg/kg之间，定量限在0.2~2.0μg/kg之间。
5.针对植物油基质粘性大，萃取材料固液相难分离的缺陷，成功建立了以石墨烯-四氧化三铁复合材料作为萃取材料的植物油中黄曲霉毒素MSPE的前处理方法与配套HPLC检测方法。该方法黄曲霉毒素B1的检测范围在0.85~51μg/kg之间，检出限为0.85μg/kg；黄曲霉毒素B2的检测范围相对较大，在0.22~66.99μg/kg之间，检出限为0.22μg/kg。方法中黄曲霉毒素B1和B2的定量限分别为2.64μg/kg和0.66μg/kg。
Aflatoxins are a group of mycotoxins produced as highly toxic secondary metabolites by manyspecies of Aspergillus, a fungus, the most notable ones being Aspergillus flavus and Aspergillusparasiticus. Aflatoxins are highly toxic and carcinogenic. Especially, aflatoxin B1is classified as aGroup1carcinogen by the World Health Organisation (WHO) in1993. The aflatoxin-producing fungiare widespread in nature, which leads to contamination of aflatoxins in a variety of agro products, suchas corn, peanuts, rice, vegetable oils and so on. Thus, it is a huge threat to comsuption safety of agroproducts. In recent years, strict standards and control measurements have been established to ensureagro-products quality and safety in different countries successively, which means that the requirementfor the methods for the detection of aflatoxins is getting higher the higher. Therefore, the researches onnovel methods for rapid detection of aflatoxins become more and more important.
In many methods for detection and quantification of aflatoxins, sample pretreatment is stillconsidered as an important part in whole analytical procedure, which needs to pay much attention.Especially for complex samples, it may even become a bottleneck point in the whole analysis process.Many agro products with low content of aflatoxins have a high oil content and complex matrix, whichfurther increases the difficulty of aflatoxins analysis.
For solving such problems, in this study, new nanomaterials--graphene and its derivatives areused as adsorbents for aflatoxins coupled with HPLC to establish new methods for detecting aflatoxins.The new methods can be the technical support for monitoring studies of aflatoxins in agro products. Themain research contents and innovative points are as follows:
1. Three kinds of nanomaterials were successfully synthesized. And their morphology and structurewere also studied. Graphene oxide (GO) was synthesized by chemical oxidation of graphite powder.Silk-like GO sheets showed a thickness of0.8~1.1nm, with a layer size of0.1~2μm. A series ofGO-SiO2nanocomposites were prepared by two ways. The SEM results proved that GO wassuccessfully grafted on the silica nanoparticles. The results also indicated that GO acted as inorganicglue to bind the small silica nanoparticles together. A magnetic graphene material with a one-stepsolvothermal method was synthesized by introducing a ferrous precursor in GO solution. The XRD andSEM results showed that the Fe3O4magnetic beads with a diameter of100nm are uniformly attachedon the graphene sheets.
2. The tests were performed to evaluate the ability of graphene oxide (GO) as the adsorbent toadsorb aflatoxins B1. And the GO was successfully applied in detoxifying of aflatoxins in ediblevegetable oils. The research also found that it took4hours for GO to adsorb aflatoxin B1to achieveadsorption equilibrium. The maximum adsorption capacity for GO is62.5ng/mg. The GO can notonly adsorb aflatoxin B1but also a certain content of aflatoxin B2. The recoveries of the GO adsorptionof aflatoxin B1in actual oil samples ranged from97.8to98.8%, indicating that GO was suitable for removing aflatoxins in edible vegetable oils.
3. Graphene oxide was synthesized and selected to be applied as an effective adsorbent fordetermination and quantification of aflatoxins in peanuts by HPLC. The limit of detection of thismethod ranges from0.08to0.65μg/kg. The limit of quantification of this method ranges from0.25to2.0μg/kg. Good linear relationships have been obtained with the correlation coefficient (r) ranging from0.99217to0.99995. The recoveries of the four aflatoxins in peanut samples are in the range from85.1%to100.8%with the relative standard deviations between2.1%and7.9%, indicating that this method wassuitable for real sample analysis.
4. A method for detection aflatoxins in rice and corn has been developed by using GO-SiO2nanocomposites as the extraction material in SPE. In this method, four aflatoxins can be detected with agood linear in the range of0.5~50μg/kg. The values of the correlation coefficient between0.99956~0.99994have been achieved. The limits of detection of this method for four aflatoxins are inthe range of0.05~0.5μg/kg with the limits of quantitation in the range of0.2~2.0μg/kg.
5. The vegetable oils were extremely viscous. Therefore, the solid materials for extraction aredifficult to separate from the liquid sample solution. A MSPE method was optimized to successfullyextract two aflatoxins in vegetable oils. In this method, for aflatoxin B1, a linear detection range of0.85~51μg/kg was found with a detection limit of0.85μg/kg. For aflatoxin B2, the new detectionmethod has a larger linear detection ranging from0.22~66.99μg/kg with a detection limit of0.22μg/kg.The limits of quantitation for aflatoxin B1and B2were2.64and0.66μg/kg respectively.
引文
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