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Article(id=1153986653568885445, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986642063905290, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20240925003, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1727193600000, receivedDateStr=2024-09-25, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1753061458245, onlineDateStr=2025-07-21, pubDate=1739548800000, pubDateStr=2025-02-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1753061458245, onlineIssueDateStr=2025-07-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1753061458245, creator=13701087609, updateTime=1753061458245, updator=13701087609, issue=Issue{id=1153986642063905290, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='3', pageStart='1', pageEnd='316', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1753061455502, creator=13701087609, updateTime=1760070725729, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1183385652272968023, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986642063905290, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1183385652272968024, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986642063905290, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=25, endPage=34, ext={EN=ArticleExt(id=1153986654630044377, articleId=1153986653568885445, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Application of zearalenone structural analogues in real-time monitoring of column capacity in immunoaffinity columns, columnId=1151895321526759957, journalTitle=Journal of Food Safety & Quality, columnName=Special Topic: Detection and Prevention and Control of Biotoxins in Food, runingTitle=null, highlight=null, articleAbstract=

Objective To explore a method that can monitor the immunoaffinity column capacity of zearalenone (ZEN) in real time during detection. Methods The 2 kinds of artificial structural analogues of ZEN (α-ZEL-G and β-ZEL-G) were synthesized by esterification of α-zearalanol (α-ZEL) and β-zearalanol (β-ZEL) with glutaric anhydride in this study. The products were purified and identified by fluorescence, ultraviolet and high-resolution mass spectrometry. A competitive enzyme-linked immunosorbent assay (ELISA) curve was established using the same antibody with immunoaffinity columns, and their affinity with the antibody was compared by half maximal inhibitory concentration (IC50). α-ZEL-G and β-ZEL-G were used as column capacity tracers and added to the immunoaffinity column together with different concentrations of ZEN to explore a method for real-time monitoring of immunoaffinity column capacity. Results The IC50 of the competitive ELISA curves of ZEN, α-ZEL-G and β-ZEL-G were 2.0, 1.3 and 10.0 ng/mL, respectively, indicating that the affinity of α-ZEL-G with antibody was slightly higher than that of ZEN, while the affinity of β-ZEL-G with antibody was significantly lower than that of ZEN with antibody. The experimental results showed that: α-ZEL-G in immunoaffinity columns with different column sizes would affect the recovery rates of ZEN. Whereas the recovery rates of ZEN with different concentration were more than 80% while the adding concentrations of β-ZEL-G was 50% of the column capacity. Conclusion β-ZEL-G with lower antibody affinity is more suitable for real-time monitoring of column volume. It shows good application results in the test of actual samples.

, correspAuthors=Ren-Rong LIU, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Zi-Yao WU, Hai-Ling MA, Guang-Jie WU, Jia-Ying ZHAO, Li-Ping WANG, Ren-Rong LIU), CN=ArticleExt(id=1153986684556403464, articleId=1153986653568885445, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=玉米赤霉烯酮结构类似物在免疫亲和柱柱容量实时监测中的应用, columnId=1151895321669366295, journalTitle=食品安全质量检测学报, columnName=本期专题：食品中生物毒素检测与防控, runingTitle=null, highlight=null, articleAbstract=

目的 探索一种可以在检测过程中实时监测玉米赤霉烯酮(zearalenone, ZEN)免疫亲和柱柱容量的方法。方法 将两种ZEN的天然结构类似物: α-玉米赤霉烯醇(α-zearalanol, α-ZEL)、β-玉米赤霉烯醇(β-zearalanol, β-ZEL)与戊二酸酐酯化反应合成了ZEN的两种人工结构类似物(α-ZEL-G、β-ZEL-G), 样品纯化后, 通过荧光、紫外、高分辨率质谱法对其进行了鉴定; 利用免疫亲和柱的抗体建立了竞争酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)曲线, 通过半抑制浓度(half maximal inhibitory concentration, IC50)比较它们与抗体之间的亲和力。分别将α-ZEL-G和β-ZEL-G作为柱容量示踪物, 与不同浓度的ZEN一起加入免疫亲和柱结合, 探索建立一种免疫亲和柱柱容量实时监测的方法。结果 ZEN、α-ZEL-G和β-ZEL-G的竞争ELISA曲线的IC50分别为: 2.0、1.3和10.0 ng/mL, 表明α-ZEL-G与抗体的亲和力略高于ZEN, 而β-ZEL-G与抗体的亲和力则明显低于ZEN与抗体的亲和力。实验结果表明: α-ZEL-G在不同柱容量的免疫亲和柱中会影响ZEN的回收率, 而β-ZEL-G的加入量在柱容量50%时, 不同浓度的ZEN的回收率大于80%。结论 与抗体亲和力更低的β-ZEL-G更适合用于柱容量实时监测, 在实际样品的检测中显示出良好的应用效果。

, correspAuthors=刘仁荣, authorNote=null, correspAuthorsNote=
* 刘仁荣(1969—), 男, 博士, 教授, 主要研究方向为微生物学、食品分析与安全检测。E-mail:
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#吴子尧和马海灵为共同第一作者

吴子尧(1999—), 女, 硕士研究生, 主要研究方向为食品安全检测。E-mail:

马海灵(1998—), 女, 硕士研究生, 主要研究方向为食品安全检测。E-mail:

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吴子尧(1999—), 女, 硕士研究生, 主要研究方向为食品安全检测。E-mail:

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吴子尧(1999—), 女, 硕士研究生, 主要研究方向为食品安全检测。E-mail:

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马海灵(1998—), 女, 硕士研究生, 主要研究方向为食品安全检测。E-mail:

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马海灵(1998—), 女, 硕士研究生, 主要研究方向为食品安全检测。E-mail:

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Development and preparation of immunoaffinity column for aflatoxin B1 based on chitosan microspheres[J]. Journal of Food Safety & Quality, 2024, 15(7): 217-224., articleTitle=Development and preparation of immunoaffinity column for aflatoxin B1 based on chitosan microspheres, refAbstract=null), Reference(id=1183428233434382501, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986653568885445, doi=null, pmid=null, pmcid=null, year=2024, volume=18, issue=null, pageStart=7224, pageEnd=7233, url=null, language=null, rfNumber=[31], rfOrder=36, authorNames=TIAN Y, LIU Z, SUN M, journalName=Journal of Food Measurement and Characterization, refType=null, unstructuredReference=TIAN Y, LIU Z, SUN M, et al. Establishment, application and comparison of three immunoaffinity pretreatment techniques for mycotoxins systematically[J]. 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Effects of α-ZEL-G on the recovery of ZEN (n=3)

, figureFileSmall=null, figureFileBig=null, tableContent=
IAC柱容量/ng 添加量/ng 平均回收率/% RSDs/% 实时柱容量/ng
α-ZEL-G ZEN α-ZEL-G ZEN α-ZEL-G ZEN
4000 6000 4000 48.57 46.66 4.20 6.95 >4000
2000 56.18 67.36 3.12 2.00 >4000
500 71.09 84.98 2.43 5.99 >4000
100 77.54 93.25 2.23 1.30 >4000
20 79.23 99.34 2.94 11.93 >4000
4000 4000 76.98 67.81 3.35 4.50 >4000
2000 92.49 86.29 2.24 3.23 >4000
500 90.11 91.20 5.62 4.76 >4000
100 93.28 94.38 1.75 2.10 >4000
20 90.48 102.40 3.72 14.82 >4000
2000 4000 83.37 74.34 1.93 8.23 >4000
2000 86.37 87.38 3.86 4.26 >3000
500 90.37 94.56 2.00 2.43 >2000
100 87.43 98.30 3.34 2.67 >2000
20 94.24 103.40 2.31 8.93 >2000
1000 1500 1000 50.34 47.46 6.23 3.03 >1000
500 57.17 63.50 4.14 7.08 >1000
100 74.14 86.53 5.63 1.54 >1000
20 84.23 98.23 2.30 9.12 >1000
1000 1000 61.70 62.47 4.81 4.35 >1000
500 86.23 79.40 7.93 4.75 >1000
100 91.09 92.88 2.34 3.30 >1000
20 93.12 97.25 5.91 9.43 >1000
500 1000 73.40 82.30 5.65 4.65 >1000
500 90.10 93.30 2.62 3.24 >800
100 92.10 100.23 1.77 2.75 >500
20 94.12 104.20 2.34 6.13 >400
), ArticleFig(id=1183428229038751758, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986653568885445, language=CN, label=表1, caption=

α-ZEL-G对ZEN回收率的影响(n=3)

, figureFileSmall=null, figureFileBig=null, tableContent=
IAC柱容量/ng 添加量/ng 平均回收率/% RSDs/% 实时柱容量/ng
α-ZEL-G ZEN α-ZEL-G ZEN α-ZEL-G ZEN
4000 6000 4000 48.57 46.66 4.20 6.95 >4000
2000 56.18 67.36 3.12 2.00 >4000
500 71.09 84.98 2.43 5.99 >4000
100 77.54 93.25 2.23 1.30 >4000
20 79.23 99.34 2.94 11.93 >4000
4000 4000 76.98 67.81 3.35 4.50 >4000
2000 92.49 86.29 2.24 3.23 >4000
500 90.11 91.20 5.62 4.76 >4000
100 93.28 94.38 1.75 2.10 >4000
20 90.48 102.40 3.72 14.82 >4000
2000 4000 83.37 74.34 1.93 8.23 >4000
2000 86.37 87.38 3.86 4.26 >3000
500 90.37 94.56 2.00 2.43 >2000
100 87.43 98.30 3.34 2.67 >2000
20 94.24 103.40 2.31 8.93 >2000
1000 1500 1000 50.34 47.46 6.23 3.03 >1000
500 57.17 63.50 4.14 7.08 >1000
100 74.14 86.53 5.63 1.54 >1000
20 84.23 98.23 2.30 9.12 >1000
1000 1000 61.70 62.47 4.81 4.35 >1000
500 86.23 79.40 7.93 4.75 >1000
100 91.09 92.88 2.34 3.30 >1000
20 93.12 97.25 5.91 9.43 >1000
500 1000 73.40 82.30 5.65 4.65 >1000
500 90.10 93.30 2.62 3.24 >800
100 92.10 100.23 1.77 2.75 >500
20 94.12 104.20 2.34 6.13 >400
), ArticleFig(id=1183428229147803664, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986653568885445, language=EN, label=Table 2, caption=

Effects of β-ZEL-G on the recovery of ZEN (n=3)

, figureFileSmall=null, figureFileBig=null, tableContent=
IAC柱容量/ng 添加量/ng 平均回收率/% RSDs/% 实时柱容量/ng
β-ZEL-G ZEN β-ZEL-G ZEN β-ZEL-G ZEN
4000 6000 4000 58.04 62.57 3.23 2.84 >4000
2000 55.67 88.77 6.65 2.45 >4000
500 73.73 90.53 2.93 4.54 >4000
100 82.18 92.59 1.86 4.70 >4000
20 69.19 97.88 10.56 8.76 >4000
4000 4000 65.08 76.74 5.75 7.56 >4000
2000 61.01 95.53 2.13 2.54 >4000
500 81.97 91.08 2.54 5.94 >4000
100 96.64 114.71 6.56 3.23 >4000
20 79.26 102.92 3.43 6.60 >4000
2000 4000 59.39 85.34 6.56 6.80 >4000
2000 83.13 90.63 5.43 2.92 >3000
500 89.41 104.37 3.91 2.77 >2000
100 94.64 101.37 5.53 6.45 >1000
20 93.64 110.72 9.91 4.56 >1000
1000 1500 1000 59.83 61.66 2.65 3.65 >1000
500 58.22 74.65 5.24 6.75 >1000
100 65.68 98.94 10.65 2.35 >1000
20 81.29 102.65 3.02 8.14 >1000
1000 1000 65.64 72.88 6.54 5.45 >1000
500 65.44 91.67 1.65 3.65 >1000
100 86.36 105.62 3.46 5.60 >1000
20 91.23 99.12 5.75 7.58 >1000
500 1000 73.36 83.77 3.54 3.95 >1000
500 90.49 93.04 2.75 1.94 >900
100 88.25 103.01 6.46 3.45 >500
20 93.68 104.01 5.45 12.18 >500
), ArticleFig(id=1183428229294604307, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986653568885445, language=CN, label=表2, caption=

β-ZEL-G对ZEN回收率的影响(n=3)

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IAC柱容量/ng 添加量/ng 平均回收率/% RSDs/% 实时柱容量/ng
β-ZEL-G ZEN β-ZEL-G ZEN β-ZEL-G ZEN
4000 6000 4000 58.04 62.57 3.23 2.84 >4000
2000 55.67 88.77 6.65 2.45 >4000
500 73.73 90.53 2.93 4.54 >4000
100 82.18 92.59 1.86 4.70 >4000
20 69.19 97.88 10.56 8.76 >4000
4000 4000 65.08 76.74 5.75 7.56 >4000
2000 61.01 95.53 2.13 2.54 >4000
500 81.97 91.08 2.54 5.94 >4000
100 96.64 114.71 6.56 3.23 >4000
20 79.26 102.92 3.43 6.60 >4000
2000 4000 59.39 85.34 6.56 6.80 >4000
2000 83.13 90.63 5.43 2.92 >3000
500 89.41 104.37 3.91 2.77 >2000
100 94.64 101.37 5.53 6.45 >1000
20 93.64 110.72 9.91 4.56 >1000
1000 1500 1000 59.83 61.66 2.65 3.65 >1000
500 58.22 74.65 5.24 6.75 >1000
100 65.68 98.94 10.65 2.35 >1000
20 81.29 102.65 3.02 8.14 >1000
1000 1000 65.64 72.88 6.54 5.45 >1000
500 65.44 91.67 1.65 3.65 >1000
100 86.36 105.62 3.46 5.60 >1000
20 91.23 99.12 5.75 7.58 >1000
500 1000 73.36 83.77 3.54 3.95 >1000
500 90.49 93.04 2.75 1.94 >900
100 88.25 103.01 6.46 3.45 >500
20 93.68 104.01 5.45 12.18 >500
), ArticleFig(id=1183428229466570774, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986653568885445, language=EN, label=Table 3, caption=

Spiked recovery experiments of β-ZEL-G column capacity real-time monitoring sample (n=5)

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样品 ZEN加标量/ng 平均回收率/% RSDs/% 实时柱容量/ng
β-ZEL-G ZEN β-ZEL-G ZEN
小麦 4000 61.57 86.34 8.75 5.10 >4000
2000 84.56 94.97 4.35 7.41 >3000
500 91.28 92.84 7.88 0.92 >1500
100 88.82 93.36 2.16 2.31 >1000
20 79.28 139.12 5.65 13.74 >1000
玉米 4000 56.32 88.59 2.39 2.53 >4000
2000 69.17 99.22 3.84 2.30 >3000
500 65.97 93.82 11.37 2.72 >1000
100 88.02 98.13 1.37 6.07 >1000
20 71.54 115.64 4.56 1.81 >1000
大米 4000 63.83 84.23 5.57 3.24 >4000
2000 80.09 93.15 2.55 9.32 >3000
500 86.52 92.51 8.37 7.41 >1500
100 88.38 97.87 6.43 3.12 >1000
20 83.65 108.56 10.66 7.41 >1000
), ArticleFig(id=1183428229575622680, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986653568885445, language=CN, label=表3, caption=

β-ZEL-G柱容量实时监测样品加标回收实验(n=5)

, figureFileSmall=null, figureFileBig=null, tableContent=
样品 ZEN加标量/ng 平均回收率/% RSDs/% 实时柱容量/ng
β-ZEL-G ZEN β-ZEL-G ZEN
小麦 4000 61.57 86.34 8.75 5.10 >4000
2000 84.56 94.97 4.35 7.41 >3000
500 91.28 92.84 7.88 0.92 >1500
100 88.82 93.36 2.16 2.31 >1000
20 79.28 139.12 5.65 13.74 >1000
玉米 4000 56.32 88.59 2.39 2.53 >4000
2000 69.17 99.22 3.84 2.30 >3000
500 65.97 93.82 11.37 2.72 >1000
100 88.02 98.13 1.37 6.07 >1000
20 71.54 115.64 4.56 1.81 >1000
大米 4000 63.83 84.23 5.57 3.24 >4000
2000 80.09 93.15 2.55 9.32 >3000
500 86.52 92.51 8.37 7.41 >1500
100 88.38 97.87 6.43 3.12 >1000
20 83.65 108.56 10.66 7.41 >1000
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玉米赤霉烯酮结构类似物在免疫亲和柱柱容量实时监测中的应用
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吴子尧 # , 马海灵 # , 吴光杰 , 赵佳莹 , 王李平 , 刘仁荣 *
食品安全质量检测学报 | 本期专题：食品中生物毒素检测与防控 2025,16(3): 25-34
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食品安全质量检测学报 | 本期专题：食品中生物毒素检测与防控 2025, 16(3): 25-34
玉米赤霉烯酮结构类似物在免疫亲和柱柱容量实时监测中的应用
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吴子尧# , 马海灵# , 吴光杰, 赵佳莹, 王李平, 刘仁荣*
作者信息
  • 江西科技师范大学生命科学学院, 南昌 330000

    • 吴子尧(1999—), 女, 硕士研究生, 主要研究方向为食品安全检测。E-mail:

    马海灵(1998—), 女, 硕士研究生, 主要研究方向为食品安全检测。E-mail:

通讯作者:

* 刘仁荣(1969—), 男, 博士, 教授, 主要研究方向为微生物学、食品分析与安全检测。E-mail:
Application of zearalenone structural analogues in real-time monitoring of column capacity in immunoaffinity columns
Zi-Yao WU , Hai-Ling MA , Guang-Jie WU, Jia-Ying ZHAO, Li-Ping WANG, Ren-Rong LIU*
Affiliations
  • College of Life Sciences, Jiangxi Science & Technology Normal University, Nanchang 330000, China
出版时间: 2025-02-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20240925003
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摘要
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目的 探索一种可以在检测过程中实时监测玉米赤霉烯酮(zearalenone, ZEN)免疫亲和柱柱容量的方法。方法 将两种ZEN的天然结构类似物: α-玉米赤霉烯醇(α-zearalanol, α-ZEL)、β-玉米赤霉烯醇(β-zearalanol, β-ZEL)与戊二酸酐酯化反应合成了ZEN的两种人工结构类似物(α-ZEL-G、β-ZEL-G), 样品纯化后, 通过荧光、紫外、高分辨率质谱法对其进行了鉴定; 利用免疫亲和柱的抗体建立了竞争酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)曲线, 通过半抑制浓度(half maximal inhibitory concentration, IC50)比较它们与抗体之间的亲和力。分别将α-ZEL-G和β-ZEL-G作为柱容量示踪物, 与不同浓度的ZEN一起加入免疫亲和柱结合, 探索建立一种免疫亲和柱柱容量实时监测的方法。结果 ZEN、α-ZEL-G和β-ZEL-G的竞争ELISA曲线的IC50分别为: 2.0、1.3和10.0 ng/mL, 表明α-ZEL-G与抗体的亲和力略高于ZEN, 而β-ZEL-G与抗体的亲和力则明显低于ZEN与抗体的亲和力。实验结果表明: α-ZEL-G在不同柱容量的免疫亲和柱中会影响ZEN的回收率, 而β-ZEL-G的加入量在柱容量50%时, 不同浓度的ZEN的回收率大于80%。结论 与抗体亲和力更低的β-ZEL-G更适合用于柱容量实时监测, 在实际样品的检测中显示出良好的应用效果。

玉米赤霉烯酮  /  免疫亲和柱  /  高效液相色谱法  /  柱容量  /  实时监测

Objective To explore a method that can monitor the immunoaffinity column capacity of zearalenone (ZEN) in real time during detection. Methods The 2 kinds of artificial structural analogues of ZEN (α-ZEL-G and β-ZEL-G) were synthesized by esterification of α-zearalanol (α-ZEL) and β-zearalanol (β-ZEL) with glutaric anhydride in this study. The products were purified and identified by fluorescence, ultraviolet and high-resolution mass spectrometry. A competitive enzyme-linked immunosorbent assay (ELISA) curve was established using the same antibody with immunoaffinity columns, and their affinity with the antibody was compared by half maximal inhibitory concentration (IC50). α-ZEL-G and β-ZEL-G were used as column capacity tracers and added to the immunoaffinity column together with different concentrations of ZEN to explore a method for real-time monitoring of immunoaffinity column capacity. Results The IC50 of the competitive ELISA curves of ZEN, α-ZEL-G and β-ZEL-G were 2.0, 1.3 and 10.0 ng/mL, respectively, indicating that the affinity of α-ZEL-G with antibody was slightly higher than that of ZEN, while the affinity of β-ZEL-G with antibody was significantly lower than that of ZEN with antibody. The experimental results showed that: α-ZEL-G in immunoaffinity columns with different column sizes would affect the recovery rates of ZEN. Whereas the recovery rates of ZEN with different concentration were more than 80% while the adding concentrations of β-ZEL-G was 50% of the column capacity. Conclusion β-ZEL-G with lower antibody affinity is more suitable for real-time monitoring of column volume. It shows good application results in the test of actual samples.

zearalenone  /  immunoaffinity column  /  high performance liquid chromatography  /  column capacity  /  real-time monitoring
吴子尧, 马海灵, 吴光杰, 赵佳莹, 王李平, 刘仁荣. 玉米赤霉烯酮结构类似物在免疫亲和柱柱容量实时监测中的应用. 食品安全质量检测学报, 2025 , 16 (3) : 25 -34 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20240925003
Zi-Yao WU, Hai-Ling MA, Guang-Jie WU, Jia-Ying ZHAO, Li-Ping WANG, Ren-Rong LIU. Application of zearalenone structural analogues in real-time monitoring of column capacity in immunoaffinity columns[J]. Journal of Food Safety & Quality, 2025 , 16 (3) : 25 -34 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20240925003
正文
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0 引言
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玉米赤霉烯酮(zearalenone, ZEN)是一种大环β-再环酸内酯, 由镰刀菌属真菌产生, 主要是禾谷镰刀菌(Fusarium graminearum)和Fusarium semiectum[1]。由于其结构与天然存在的雌激素相似, 在人类和动物中可引起明显的雌激素效应[2], 被国际癌症研究机构(International Agency for Research on Cancer, IARC)列为第三类致癌物。ZEN分子式是C18H22O5, 在一些动物体内, ZEN可在肝脏和肠黏膜中被3-OH-类固醇脱氢酶还原为玉米赤霉烯醇(zearalanol, ZEL), ZEL的两种非对应异构体为α-ZEL和β-ZEL。其化学结构式如图1所示。
ZEN的毒性主要表现在免疫毒性、生殖毒性、遗传毒性和细胞毒性4个方面[3]。ZEN主要存在于玉米、大米、小麦和豆类等农作物中, 对人类和动物的健康造成严重的危害和巨大的经济损失, 世界卫生组织、欧盟和世界各国对各粮食农油产品中ZEN及其代谢物的含量制定了严格的标准[4]。GB 2761—2017《食品安全国家标准 食品中真菌毒素限量》规定小麦、玉米及其粉质品中ZEN含量应≤60 µg/kg, 相比欧盟分类较少[5]。高效准确检测粮食中ZEN的方法是近年来研究的重点, 目前国内外对于ZEN有各种检测方法, 这些检测方法各有利弊, 其中免疫学检测方法能大量快速检测霉菌毒素, 但其精密度和准确性不高, 可用作大量样品霉菌毒素初筛, 免疫学检测方法因其灵敏度高、简单、耗时少等优点而受到更多关注, 例如酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)和其他一些新兴的光学、比色或电化学免疫传感器方法[6-8]。2023年HUANG等[9]研究了一种基于双功能金纳米颗粒(gold nanoparticles, AuNPs)探针的酶联免疫吸附测定(nanoparticles-enzyme-linked immunosorbent assay, nano- ELISA), 用于检测流行的食用和药用食品薏苡仁种子中的ZEN, 利用了AuNPs方便的标记特性, 允许过氧化物酶和抗体通过静电吸附相继固定在AuNPs上, 构建用于鉴定和信号放大的双功能探针以达到更高的灵敏度, 所提出的方法的检出限和定量限比传统ELISA提高了约7倍和5倍。对ZEN进行精确的定量需要用仪器检测法, 如高效液相色谱法(high performance liquid chromatography, HPLC)[10]、气相色谱-质谱法(gas chromatography-mass spectrometry, GC-MS)[11]和高效液相色谱-串联质谱法(high performance liquid chromatography-tandem mass spectrometry, HPLC-MS/MS)[12]。2023年FATEMEH等[13]采用免疫亲和柱和高效液相色谱法(immune affinity column high performance liquid chromatography, IAC-HPLC)同时测定和分析了150份水稻、小麦和玉米样品中的ZEN。在最佳条件下, ZEN的线性范围0.2~350 ng/g, 检出限0.015~0.061 ng/g, 定性限为0.052~0.203 ng/g。2020年XU等[14]通过一种具有高适配体负载和快速制备亲和整体色谱柱, 与HPLC偶联对ZEN的进行高效特异性识别和灵敏检测。在使用现代设备技术进行分析之前, 需要对样品进行前处理, 分离方法和样品制备过程对定量分析结果影响很大。ZEN的样品前处理有免疫亲和柱净化(immune affinity column, IAC)、固相萃取(solid phase extraction, SPE)、液液萃取(liquid-liquid extraction, LLE)、QuEChERS法等[15-16]。近年来有许多新兴的前处理技术, 如基于纳米吸附剂的分散固/微固相萃取(dispersed solid phase extraction, D-SPE/ dispersive micro-solid phase extraction, D-μ-SPE)[17-22], 基于介孔结构泡沫的D-SPE[7]和中空纤维基液相微萃取[8]。免疫亲和柱作为ZEN检测的GB 2761—2017第一法(IAC-HPLC)前处理方法, 能够快速、高效、专一地从样本中分离出ZEN[23-25], 这种方法是利用抗原抗体的特异性结合, 降低基质干扰效应, 从样本中分离出待测物[26]。胡云等[27]对免疫亲和柱提取液进行了优化, 准确分析食品中ZEN的含量, 回收率较优化前有了提高, 方法灵敏度高, 定量结果准确。LUO等[28]建立了一种GC-MS测定饲料中ZEN及其5种衍生物, 前处理选择制备有效的免疫亲和柱用于样品纯化, 然后在通过GC-MS对目标化合物进行免疫亲和色谱分析, 该方法经证明可靠, 适用于测定饲料中的ZEN及其衍生物。BRENN-STRUCKHOFOVA等[29]研究了一种分离ZEN的样品净化方法, 通过将抗ZEN抗体固定在多孔溶胶-凝胶玻璃中制备的免疫亲和柱。该方法证明可靠, 适用于小麦和小麦制品加标实验。顾一丹等[30]通过优化免疫亲和柱的亲和基质, 制备出粒径大小合适且均一的壳聚糖微球并以此为载体偶联毒素的单克隆抗体制备免疫亲和柱。经验证, 用于实际样品检测回收率为87.90%~96.37%。该方法证明可靠, 适用于多种毒素实际样品加标实验。
IAC的柱容量是保证检测准确性的重要指标, 若柱容量不足, 将会影响检测结果的准确性。因此, 所有标准方法都对IAC的柱容量有严格的规定。但目前IAC柱容量的测定通常是通过抽检的方式确定, 由于IAC是一次性耗材, 抽检并不能确保每一支IAC都是合格的, 对检测结果造成一定的隐患。如果可以在检测过程中实时监测免疫亲和柱柱容量, 将有效克服这一弊端[31], 但目前相关研究鲜见报道。若要实现这一目标, 需要研究一种柱容量示踪物, 该示踪物的理化性质应与ZEN接近且能在同一色谱条件下有效分离和检测, 同时不影响ZEN的回收率和检测结果。鉴于此, 本研究拟通过化学方法对ZEN的2种天然结构类似物α-ZEL、β-ZEL进行修饰, 获得两种ZEN人工结构类似物, 并探索其作为柱容量实时监测示踪物的可行性和规律, 探索建立ZEN免疫亲和柱柱容量的实时监测方法, 对于基于免疫亲和柱前处理技术的食品安全检测方法具有较高的科学价值和较好的应用前景。
1 材料与方法
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1.1 材料与试剂
ZEN (100 µg/mL)、α-ZEL (5 mg)、β-ZEL (5 mg)标准品(纯度98%, 青岛普瑞邦生物工程有限公司); ZEN免疫亲和柱、5G5ZEN抗体(深圳易瑞生物技术股份有限公司); CW01025羊抗鼠酶标二抗(100 µL, 1 mg/mL, 江苏康为世纪生物科技股份有限公司); 戊二酸酐(纯度98%)、4-二甲氨基吡啶(dimethylaminopyridine, DMAP)(纯度98%)(上海阿拉丁生化科技股份有限公司); 四甲基联苯胺(tetramethyl benzidine, TMB)(分析纯, 湖州英创生物科技有限公司); 硫酸(H2SO4)(分析纯)、氯化钙(CaCl2)(纯度98%)、氯化钠(NaCl)(纯度99.5%)、磷酸二氢钾(KH2PO4)(纯度99.5%)、十二水合磷酸氢二钠(Na2HPO4·12H2O)(纯度99%)、氯化钾(KCl)(纯度99.5%)、四氢呋喃(tetrahydrofuran, THF)(分析纯)(西陇科学股份有限公司); 吐温-20 (tween-20)(分析纯, 天津大茂化学试剂合伙企业); 甲醇、乙腈(色谱纯, 西班牙ScharLab S.L公司); 高效液相色谱级水用MicroPure UV/UF超纯水仪(德国Thermo Fisher Scientific公司)制备, 符合国家标准方法GB/T 6682—2008《实验室用水规格和试验方法》标准规定的I类水规格。
1.2 仪器与设备
Agilent 1260 infiniteⅡ高效液相色谱仪、Pribolab C18柱(4.6 mm×250 mm, 5 μm)(美国Agilent公司); BSP-400半制备液相色谱(美国Water公司); BCD-216YH旋转蒸发仪(青岛海尔有限公司); JHD-006氮吹仪(上海极恒实业有限公司); Centrifuge5804R高速冷冻离心机(美国Eppendorf公司); Vortex-5-Kylin-Bell漩涡仪(青岛启麟电子有限公司); MicroPure UV/UF超纯水仪(德国Thermo Fisher Scientific公司); FW100高速粉碎机(天津市泰斯特仪器有限公司)。
1.3 溶液配制
无水THF: 用CaCl2在隔绝潮气下回流, 除去其中的水和过氧化物, 然后蒸馏, 收集66 ℃的馏分蒸馏时不要蒸干, 将剩余少量残液即倒出。
20 mg/mL DMAP溶液: 称取20 mg DMAP粉末, 加入1 mL无水THF, 摇晃均匀, 静置。
20 mg/mL戊二酸酐溶液: 称取20 mg戊二酸酐粉末, 加入1 mL无水THF, 摇晃均匀, 静置。
10 nmol/L磷酸盐缓冲液(phosphate buffer solution, PBS): 称取8 g NaCl、0.2 g KH2PO4、2.9 g Na2HPO4·12H2O、0.2 g KCl、溶于约0.8 L超纯水中, 将pH调到7.4, 后用超纯水定容至1 L。室温放置。
0.05%磷酸盐缓冲液-吐温20洗液(phosphate buffer solution-tween-20, PBST): 在1 L PBS溶液中加入500 µL吐温-20, 摇晃均匀, 静置。
1.4 实验方法
配制20 mg/mL溶于无水THF的DMAP溶液和20 mg/mL溶于无水THF的戊二酸酐溶液。在5 mg α-ZEN/β-ZEN粉末样品中加入500 µL上述DMAP溶液和500 µL戊二酸酐溶液, 25 ℃、180 r/min振荡反应约72 h, 制备原理如图2所示。待反应完成后, 反应液真空抽干, 用500 µL乙腈复溶, 半制备型液相色谱仪纯化。
1.5 色谱条件
ZEN的GB 5009.209—2016《食品安全国家标准 食品中玉米赤霉烯酮的测定》中第一法IAC-HPLC的液相色谱条件为参考, 通过α-ZEL-G、β-ZEL-G峰形改善对HPLC条件进行优化, 探索不同流动相对峰形, 出峰时间的影响, 优化流动相。
1.6 α-ZEL-G、β-ZEL-G紫外光谱、荧光光谱和质谱鉴定
将α-ZEL-G、β-ZEL-G纯品分别用乙腈溶解至10 µg/mL, 在紫外光度计中扫描200~400 nm的紫外吸光光谱。在荧光分光光度仪中扫描检测α-ZEL-G、β-ZEL-G纯品荧光性质, 确定其最大激发波长和最大发射波长, 通过改变激发波长: 260、270、280 nm获得其最大发射波长在440 nm, 通过最大发射波长确定最大激发波长为274 nm, 确定了荧光峰的波长范围和峰值。将α-ZEL-G、β-ZEL-G样品送科学指南针高分辨质谱仪检测, 检测条件如下: 以电喷雾离子源(electrospray ionization, ESI)检测, 毛细管电压, +2.5 kV; 源温度, 150 °C; 去溶剂温度, 500 ℃; 锥形气体流量, 150 L/h; 去溶剂气体流量, 1000 L/h。检测在多反应监测(multiple reaction monitoring, MRM)模式下进行。Mass LynxTM 4.1软件(Waters)用于数据采集和处理。
1.7 α-ZEL-G、β-ZEL-G竞争ELISA方法的建立
用PBS稀释ZEN人工抗原质量浓度为1 µg/mL, 并在96孔聚苯乙烯微孔板(120 µL/孔)上于4 ℃包被过夜。用0.05% PBST洗液在洗板机洗板3次并拍干板上的残留液体, 后采用5%脱脂奶粉37 ℃保湿封闭2 h (350 µL/孔)。0.05% PBST洗板3次拍干, 每孔加入倍比稀释的50 µL ZEN标准品、α-ZEL-G纯品、β-ZEL-G纯品和50 µL 1:8000体积比稀释的ZEN抗体(与免疫亲和柱的抗体相同), 37 ℃保湿恒温结合40 min。0.05% PBST洗涤6次并拍干。ZEN/α-ZEL-G/β-ZEL-G稀释浓度从2000 ng/mL开始2.5倍倍比稀释。每孔加入工作浓度的酶标二抗100 µL, 37 ℃保湿恒温结合40 min, 0.05% PBST洗涤6次并拍干水分。每孔加入100 µL TMB显色液, 37 ℃静置显色8 min, 取出后每孔迅速加入50 µL 2 mol/L H2SO4终止反应, 用酶标仪测定各孔的450 nm吸光值。
以ZEN/α-ZEL-G/β-ZEL-G浓度对数为横坐标, 结合率(B/B0×100%, B0为不加ZEN标准品的OD值, B为加ZEN标准品的OD值)为纵坐标, 建立竞争ELISA标准曲线。
1.8 α-ZEL-G、β-ZEL-G IAC-HPLC方法建立及加标回收率测定
考虑到α-ZEL-G、β-ZEL-G和ZEN分子量不同, 为了方便比较, 以下α-ZEL-G、β-ZEL-G的添加量通过摩尔数折算为ZEN的质量, 将α-ZEL-G、β-ZEL-G用流动相稀释为6个不同质量浓度: 10、50、100、200、500、1000 ng/mL进行HPLC检测, 计算其3次的峰面积平均值为纵坐标, 横坐标为浓度, 建立其线性回归方程, 即建立α-ZEL-G、β-ZEL-G的HPLC标准曲线。
从4 ℃冰箱取10支柱容量为4000 ng ZEN免疫亲和柱放置在不低于20 ℃的室温复温30 min, 取下塞头, 待柱内保护液自然流干后, 用10 mL 10 nmol/L PBS淋洗免疫亲和柱。用10%的乙腈水配制100 ng/mL β-ZEL-G与α-ZEL-G的上样液20 mL各5只分别过柱, 控制液体以每秒有1~2滴的速度流出, 然后用10 mL 10 nmol/L PBS淋洗免疫亲和柱, 再用10 mL超纯水淋洗。立刻用吹干免疫亲和柱内液体, 取下上样管, 向免疫亲和柱中投入2 mL甲醇洗脱, 若在1~2 min内没有液体流出, 通过对亲和柱施加压力直至有液体流出, 随后不再施加压力, 让液体在重力作用下自然流出。收集洗脱液并混匀, 55 ℃以下氮气吹干后, 用1 mL HPLC流动相溶解样品, 液相色谱定量分析测定α-ZEL-G、β-ZEL-的回收率。
1.9 ZEN IAC饱和柱容量检测
根据GB 5009.209—2016中第一法, 建立其线性回归方程, 即建立ZEN的HPLC标准曲线。
从4 ℃冰箱取5支标称柱容量为4000 ng和5支标称柱容量为1000 ng的ZEN免疫亲和柱, 放置在室温复温30 min, 取下塞头, 待柱内保护液自然流干后, 用10 mL 10 nmol/L PBS淋洗免疫亲和柱。用10%的乙腈水配制含400 ng/mL与200 ng/mL的ZEN标准品上样液各20 mL分别过4000 ng和1000 ng规格的亲和柱, 控制液体以每秒有1~2滴的速度流出, 然后用10 mL 10 nmol/L PBS淋洗免疫亲和柱, 再用10 mL超纯水淋洗。吹干免疫亲和柱内液体, 向免疫亲和柱中加入2 mL甲醇洗脱。收集洗脱液并混匀, 55 ℃以下氮气吹干后, 用1 mL HPLC流动相溶解样品, 液相色谱仪定量分析测定ZEN的回收率。
1.10 α-ZEL-G、β-ZEL-G建立IAC-HPLC柱容量实时监测方法
从4 ℃冰箱取一批标称柱容量为4000 ng和标称柱容量为1000 ng的ZEN免疫亲和柱, 放置在室温复温30 min备用。用10%乙腈水配制含量分别为300、200、100、75、50和25 ng/mL质量浓度的β-ZEL-G或α-ZEL-G上样液20 mL; 同时用10%甲醇-水配制含量分别为200、100、50、25、5、1 ng/mL的ZEN上样液20 mL。取下塞头, 让免疫亲和柱内保护液自然流干, 用10 mL 10 nmol/L PBS淋洗免疫亲和柱。按照具体浓度将配制好的β-ZEL-G或α-ZEL-G上样液20 mL过柱, 之后ZEN上样液继续过柱, 控制液体以每秒有1~2滴的速度流出, 然后用10 mL 10 nmol/L PBS淋洗IAC, 再用10 mL超纯水淋洗。吹干免疫亲和柱内液体, 向IAC中加入2 mL甲醇洗脱。收集洗脱液, 55 ℃以下氮气吹干后, 用1 mL HPLC流动相溶解样品, 液相色谱定量分析测定ZEN的回收率。
1.11 β-ZEL-G IAC柱容量实时检测方法样品加标回收验证
将建立的柱容量实时监测IAC-HPLC方法应用于ZEN最常污染的3种类粮食(玉米、大米和小麦)的样品提取液的加标回收实验中, 验证方法的准确性, 可重复性。
购买市售小麦、大米和玉米3种样品, 在用GB 5009.209—2016方法确认不含ZEN后, 各组(每组5份)分别称取20.0 g粉碎小麦、大米和玉米试样(精确到0.1 g)于均质杯中, 以均质器高速搅拌粉碎, 过筛(粒径小于2 mm), 混合均匀后加入100 mL 20%乙腈水提取液, 涡旋振荡提取30 min, 静置离心后定量滤纸过滤。每份样品移取20 mL滤液加入80 mL超纯水水稀释混匀, 经玻璃纤维滤纸过滤至滤液澄清, 获得小麦、大米和玉米3种样品的待测样品提取液, 将3种待测样品提取液分别添加200、100、25、5和1 ng/ mL的ZEN制备加标液, 取一批柱容量为4000 ng的ZEN IAC, 每支加入2000 ng的β-ZEL-G作为柱容量实时监测的示踪物, 再加入不同ZEN加标量的加标液验证柱容量实时检测方法在实际样品中的准确度和精确性。
1.12 数据处理
数据表达为平均值±标准偏差。采用Excel 2016软件处理数据, Graphpad pism9.5作图。
2 结果与分析
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2.1 α-ZEL-G、β-ZEL-G色谱条件的优化
对α-ZEL-G、β-ZEL-G HPLC条件优化方法如下: 色谱分离采用Pribolab C18柱(4.6 mm×250 mm, 5 μm)反相分析柱, 并保持在30 ℃。HPLC分析的流动相由两种溶液组成: 50% A相(乙腈)和50% B相(90%超纯水+10%甲醇)。所有分析的流速为1 mL/min, 进样体积为20 μL。分析前, 柱子先用0.5 mL/min的流动相冲洗30 min, 然后用1.0 mL/min的流速冲洗30 min。β-ZEL-G使用二极管阵列检测器(diode array detector, DAD)检测, 检测波长为238 nm, α-ZEL-G使用荧光检测器(fluorescence detector, FLD)检测器, 激发波长: 274 nm, 发射波长440 nm。用优化过后的色谱条件对α-ZEL-G、β-ZEL-G进行测定, 得到了峰形良好的色谱峰, 优化后的色谱法如图3所示。
2.2 α-ZEL-G、β-ZEL-G的纯化和定量
α-ZEL-G、β-ZEL-G使用半制备型液相分离纯化样品后, 在对应保留时间的峰接样获得约20 mL样品溶液, 经60 ℃旋转蒸发浓缩后, 得到纯化的α-ZEL-G、β-ZEL-G纯品称重得到其质量, 其中α-ZEL-G在投入5 mg后反应得到2.36 mg纯品, 得率为47.2%。β-ZEL-G在投入5 mg后反应得到2.18 mg纯品, 得率为43.6%。
2.3 α-ZEL-G、β-ZEL-G紫外光谱、荧光光谱和质谱鉴定
ZEN标准品及α-ZEL-G、β-ZEL-G的紫外扫描图谱如图4所示。ZEN与α-ZEL-G、β-ZEL-G也有基本相同的紫外吸收光谱, 都在238、274、316 nm有紫外吸收峰。
ZEN和α-ZEL-G、β-ZEL-G有相似的荧光特性, 但荧光强度不同, 用激发波长扫描274 nm扫描得到发射光谱结果如图4所示, ZEN标准品和α-ZEL-G、β-ZEL-G都在同一波长有荧光峰, α-ZEL-G荧光强度高略高于ZEN, 而β-ZEL-G荧光吸收强度为ZEN与α-ZEL-G的十分之一。
α-ZEL-G、β-ZEL-G的质谱检测结果如图5所示, 高分辨质谱 (ESI) m/z [M+H]+ C23H30O8理论值434.19; 实测值434.19证明成功获得目标反应物。
2.4 α-ZEL-G、β-ZEL-G ELISA竞争抑制曲线及交叉反应率
通过竞争ELISA探究α-ZEL-G, β-ZEL-G及ZEN与抗体亲和力。得到ZEN、α-ZEL-G、β-ZEL-G的ELISA竞争抑制曲线如图6所示, 在线性范围内, ZEN的线性方程为Y=-0.427X+0.6141, 相关系数r2=0.9923, 半抑制浓度(half maximal inhibitory concentration, IC50)为2.0 ng/mL; α-ZEL-G的线性方程为Y=-0.467X+0.5521, 相关系数r2=0.9928, IC50为1.3 ng/mL; β-ZEL-G的线性方程为Y=-0.3958X+0.8936, 相关系数r2=0.9912, IC50为10.0 ng/mL。α-ZEL-G与抗体的亲和力略高于ZEN, 而β-ZEL-G与抗体的亲和力则明显低于ZEN与抗体的亲和力。
2.5 α-ZEL-G、β-ZEL-G的IAC-HPLC方法建立及加标回收率测定
通过优化的HPLC色谱条件建立α-ZEL-G、β-ZEL-G线性回归方程, α-ZEL-G的线性方程为Y=0.019X-0.1231, 相关系数r2=0.9995, β-ZEL-G的线性方程为Y=0.2107X, 相关系数r2=0.9997, 用于过柱后的α-ZEL-G、β-ZEL-G定量, 测定其加标回收率。
在标称柱容量为4000 ng的ZEN IAC中分别加入2000 ng α-ZEL-G、β-ZEL-G, HPLC测定过柱后的α-ZEL-G、β-ZEL-G回收率。经过比较α-ZEL-G、β-ZEL-G过柱前后的HPLC色谱图, 过柱后分别得到1845.80 ng α-ZEL-G、1828.21 ng β-ZEL-G, 均值回收率分别为93.29%、91.41%, 相对标准偏差(relative standard deviation, RSD, n=5)分别为1.76%、0.97%, 结果良好(回收率>85%, RSD<5%), 证明α-ZEL-G、β-ZEL-G、都可以被免疫亲和柱截留, 与亲和柱内抗体有较好的亲和力。
2.6 ZEN IAC饱和柱容量检测
在4000 ng规格的ZEN IAC中投入8000 ng ZEN, 通过液相色谱仪检测得到ZEN平均回收率为52.41%, RSD为2.06% (n=5), 结果表明4000 ng规格的ZEN IAC饱和柱容量为4193 ng, 即每支4000 ng规格ZEN IAC实际可以结合4193 ng ZEN。在1000 ng规格的ZEN IAC中投入4000 ng ZEN, 通过液相检测得到ZEN平均回收率为26.9%, RSD为1.23% (n=5), 结果表明1000 ng规格的ZEN IAC饱和柱容量为1076 ng, 即每支1000 ng规格ZEN IAC实际可以结合1076 ng ZEN。
2.7 α-ZEL-G、β-ZEL-G建立IAC-HPLC柱容量实时监测方法
以4000 ng和1000 ng柱容量的ZEN IAC为实验材料, 研究不同浓度的α-ZEL-G、β-ZEL-G作为IAC-HPLC的柱容量实时监测方法中示踪物与不同浓度的ZEN上柱, HPLC可同时测定得到α-ZEL-G、β-ZEL-G和ZEN在不同添加量条件下的回收率。
如表1、2所示, 结果表明在α-ZEL-G的投入量为50%、100%和150% ZEN IAC标称柱容量时, 会影响ZEN与IAC内的抗体结合, 降低ZEN的回收率, 无法作为柱容量监测物; β-ZEL-G投入上样液中, 若超过柱容量, 也会对ZEN的回收率有影响, 在ZEN标称柱容量范围内, β-ZEL-G对ZEN回收率的影响明显小于α-ZEL-G, 在β-ZEL-G添加量为标称柱容量的50%时, 所有浓度的ZEN回收率大于80%, 高低两种柱容量规格的亲和柱回收率情况有同样的趋势, ZEN与α-ZEL-G(或β-ZEL-G)二者之和(实时柱容量=α-ZEL-G/β-ZEL-G添加量×α-ZEL-G/β-ZEL-G回收率+ZEN添加量×ZEN回收率)为实际测得的柱容量, 从而在测定ZEN的同时得到IAC柱容量。
2.8 β-ZEL-G IAC柱容量实时检测方法样品加标回收验证
选用柱容量为4000 ng的ZEN IAC, 以2000 ng的β-ZEL-G为柱容量实时监测示踪物, 建立柱容量实时监测IAC-HPLC方法。将建立的方法应用于ZEN最常污染的3种类粮食(玉米、大米和小麦)的样品提取液的加标回收实验中, 验证方法的准确性和可重复性。结果如表3所示, 小麦样品柱容量实时检测方法的平均ZEN加标回收率为92.84%~139.12%, RSDs为0.92%~13.74%, 玉米为88.59%~115.64%, RSDs为1.81%~6.07%, 大米为84.23%~108.56%, RSDs为3.12%~9.32%。说明在实际样品的加标回收实验验证中, ZEN的回收率和RSD基本在有效范围之内, 检测结果正常。在小麦样本中的回收率和RSD略偏高, 可能是由于基质干扰, 提取方法需要进一步优化。
3 结论
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为了提高ZEN的检测的准确度, 简化柱容量监测程序, 本研究旨在研发一种便捷、高效应用于IAC柱容量检测的示踪物。本研究通过添加β-ZEL-G到样本中, 作为每次检测的内标物, 可保证在未知样本中对ZEN含量检测结果的准确性和IAC柱容量质量检测的有效性, 若未知样本中没有检测出ZEN, 但β-ZEL-G添加量的回收率大于80%, 表明IAC柱容量满足质量要求, 检测结果准确有效; 当未知样本中测出ZEN, 且ZEN检出量加上β-ZEL-G检出量的总量大于标称柱容量80%, 表明IAC柱容量满足质量要求, 检测结果准确有效。这种方法初步实现了免疫亲和柱柱容量的实时检测, 对食品安全检测方法具有较高的科学价值和应用前景。通过人工的方法合成的结构类似物不会与天然存在的结构类似物混淆而影响检测结果, 具有作为柱容量示踪物的潜力。由于柱容量一般影响高浓度样品的回收率, 所以高浓度样品更需要监测柱容量, 2000 ng β-ZEL-G在加标样品中不会对ZEN的回收率造成影响, 不仅可以监测高浓度样品检测时的柱容量, 也可以作为所有浓度样品的阳性对照, 保障柱容量和检测过程的有效性。通过研究发现: 结构类似物与IAC抗体的亲和力是选择合适的柱容量实时监测示踪物的关键, 后续会继续研究ZEN衍生物的改造, 进一步降低其与IAC抗体的亲和力, 研究更适用于免疫亲和柱柱容量示踪物的结构类似物。
基金
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  • 国家自然科学基金项目(31960501)
  • 江西省教育厅科技落地计划项目(KJLD14067)
文献
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2025年第16卷第3期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20240925003
  • 接收时间：2024-09-25
  • 首发时间：2025-07-21
  • 出版时间：2025-02-15
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  • 收稿日期：2024-09-25
基金
国家自然科学基金项目(31960501)
江西省教育厅科技落地计划项目(KJLD14067)
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    江西科技师范大学生命科学学院, 南昌 330000

通讯作者:

* 刘仁荣(1969—), 男, 博士, 教授, 主要研究方向为微生物学、食品分析与安全检测。E-mail:
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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