Detection of aflatoxin B1 by immunochromatographic test strip based on CuS nanoflowers and nanobody
-
摘要: 黄曲霉毒素B1(AFB1)是有毒代谢产物,不仅污染农作物,还容易通过食物链进入人体,影响人类健康。为了及时、快速、方便地检测出农作物中的AFB1,需要一种低成本、用户友好、高效的检测方法。构建以硫化铜纳米花(CuS Nanoflowers,CuS NFs)为信号标签,纳米抗体G8-DIG为识别探针的免疫层析试纸条法用于检测黄曲霉毒素B1。对CuS NFs的质量浓度、G8-DIG的添加量、T线质量浓度、探针量进行优化。结果表明:该方法的检出限(LOD)为2.4 ng/mL,检测范围为6.87~96.21 ng/mL,加标回收率在86%~110 %之间,变异系数(CV)≤10.9 %;对玉米质控样进行检测,回收率为111.8 %。选用的CuS NFs和G8-DIG作为信号探针,能够在检测AFB1的同时降低试纸条的成本。Abstract: Aflatoxin B1 (AFB1) is a toxic metabolite typically produced by Aspergillus flavus and parasitic fungi, not only contaminates crops but also readily enters the human body through the food chain, affecting human health. Given the widespread distribution, severe contamination, significant hazards to humans and animals, as well as the difficulty in removal of AFB1, there is an urgent need for a convenient and sensitive detection method. In contrast to instrumental analysis methods, immunochromatographic test strip (ICTS) offers advantages such as short detection times, high efficiency, and no requirement for specialized personnel. In view of the wide applications of ICTS and the excessive variety and quantity of crops tested, it is necessary to reduce the cost of ICTS. Nanobody G8 was fused with an anti-digoxin antibody to construct the expression vector pET25b-G8-DIG, which was then transformed into E. coli BL21 for prokaryotic expression. Following nickel column purification, the synthesized G8-DIG fusion protein was confirmed to have a molecular weight of approximately 30 kDa through SDS-PAGE gel electrophoresis. The multifunctional fusion protein G8-DIG was able to bind to both AFB1 antigen and digoxin (DIG) antigen. Copper sulfide nanoflowers (CuS Nanoflowers, CuS NFs) were synthesized by the reaction of copper nitrate trihydrate and thiourea, and the nanomaterial was confirmed to possess a floral structure with dimensions around 300 nm through TEM and SEM analysis. Optimization of the ICTS including the optimal mass concentration of CuS NFs solution, the amount of G8-DIG added, the mass concentration of the T-line, and the probe quantity revealed that the detection limit (LOD) of this method was 2.4 ng/mL, with a detection range of 6.87-96.21 ng/mL. The spiked recovery rate ranged from 86% to 110%, and the coefficient of variation (CV) was less than 10.9%. When tested on corn quality control samples, the recovery rate was 111.8%. Furthermore, the specificity of the method was assessed by detecting OTA, T-2, DON, and ZEN as interfering samples on the test strip, demonstrating excellent specificity of the developed method. Utilizing DIG-BSA in place of the secondary antibody traditionally used for the control line on test strip improved the stability and cost-effectiveness of the test strip. The selected CuS NFs and G8-DIG, serving as signal probes, not only enable the detection of AFB1 but also reduce the cost of the test strip.
-
Keywords:
- CuS nanoflowers /
- nanobody /
- immunochromatographic test strip /
- aflatoxin B1
-
-
[1] AHMADI M, SHAHBAHRAMI R, KHAJEH F, et al. Aflatoxin B1 and viruses’ combined pathogenesis: a mini systematics review of invitro and invivo studies [J]. Acta histochemica, 2024, 126(1): 152116.
[2] 闫兆凤. 中国主粮中真菌毒素污染现状 [J]. 卫生研究, 2022, 51(4): 685-690. [3] 韩宇. 高效液相色谱法检测农产品中黄曲霉毒素B1的含量 [J]. 食品安全导刊, 2023(14): 67-69. [4] WANG Y Z, HOU C, DAI Y Q, et al. Determination of aflatoxin B1 by novel nanofiber-packed solid-phase extraction coupled with a high performance liquid chromatography-fluorescence detector [J]. Analytical methods, 2023, 15(4): 472-481.
[5] 王鑫, 张凤枰, 杨娟. 高效液相色谱-串联质谱法测定玉米蛋白粉中黄曲霉毒素B1测量不确定度评定 [J]. 中国粮油学报, 2024, 39(05): 181-188. [6] 徐加兵, 叶慧, 陈骁鹏, 等. UPLC-MS/MS法与ELISA法测定胖大海中黄曲霉毒素的比较研究 [J]. 中国药师, 2023, 26(10): 168-175. [7] 黄周梅, 武旭悦, 黄金发, 等. 基于金属有机骨架(ZIF-8)的酶联免疫法灵敏检测米粉中的黄曲霉毒素B1 [J]. 现代食品科技, 2022, 38(03): 299-306, 327. [8] 张光胤, 蔡爱丽, 邓放明, 等. 双模态改良型酶联免疫吸附测定法用于黄曲霉毒素B1的检测研究 [J]. 食品与发酵工业, 2021, 47(12): 224-230. [9] 郝小童, 朱正伟, 曲文莉, 等. 基于金标抗体的电化学免疫传感器检测米糠油中黄曲霉毒素B1 [J]. 食品安全质量检测学报, 2023, 14(20): 252-259. [10] 李宗达, 陆康, 裴强, 等. 基于金掺杂的锆基金属有机框架电化学适配体传感器检测玉米中黄曲霉毒素B1 [J]. 食品安全质量检测学报, 2023, 14(21): 132-140. [11] 温慧娇, 廖芷晴, 靳贵英. 真菌毒素检测方法进展 [J]. 中国药品标准, 2023, 24(5): 465-475. [12] WANG C Y, FANG Z, FAN F, et al. Facile synthesis of size-tunable Cu39S28 micro/nano-crystals and small-sized configuration enhanced visible-light photocatalytic activity [J]. CrystEngComm, 2013, 15(29): 5792-5798.
[13] SHU R, LIU S J, XU J K, et al. Galvanic replacement inspired signal amplification: background-free and antibody-thrift in situ growth immunochromatography [J]. Chemical engineering journal, 2022, 437: 135362.
[14] SHU R, LIU S J, WANG M L, et al. Dual-plasmonic CuS@Au heterojunctions synergistic enhanced photothermal and colorimetric dual signal for sensitive multiplexed LFIA [J]. Biosensors and bioelectronics, 2024, 255: 116235.
[15] ZHANG M, BU T, TIAN Y M, et al. Fe3O4@CuS-based immunochromatographic test strips and their application to label-free and dual-readout detection of Escherichia coli O157: H7 in food [J]. Food chemistry, 2020, 332: 127398.
[16] JI Y W, REN M L, LI Y P, et al. Detection of aflatoxin B1 with immunochromatographic test strips: enhanced signal sensitivity using gold nanoflowers [J]. Talanta, 2015, 142: 206-212.
[17] HAMERS-CASTERMAN C, ATARHOUCH T, MUYLDERMANS S, et al. Naturally occurring antibodies devoid of light chains [J]. Nature, 1993, 363(6428): 446-448.
[18] LIU X, TANG Z W, DUAN Z H, et al. Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference [J]. Talanta, 2017, 164: 154-158.
[19] PAN D, LI G H, HU H Z, et al. Direct immunoassay for facile and sensitive detection of small molecule aflatoxin B1 based on nanobody [J]. Chemistry, 2018, 24(39): 9869-9876.
[20] HE T, WANG Y R, LI P W, et al. Nanobody-based enzyme immunoassay for aflatoxin in agro-products with high tolerance to cosolvent methanol [J]. Analytical chemistry, 2014, 86(17): 8873-8880.
[21] 蔡冲, 闫洪林, 唐晓倩, 等. 纳米抗体特性及其在农产品真菌毒素检测中的应用 [J]. 中国油料作物学报, 2022, 44(2): 451-459. [22] SALVADOR J P, VASYLIEVA N, GONZALEZ-GARCIA I, et al. Nanobody-based lateral flow immunoassay for the rapid detection of aflatoxin B1 in almond milk [J]. ACS food science & technology, 2022, 2(8): 1276-1282.
[23] 孔静, 郑楠, 王加启, 等. 纳米抗体结构特征与制备研究进展 [J]. 农业生物技术学报, 2024, 32(1): 209-219. -
期刊类型引用(3)
1. 朱紫琪，赵仁勇，田双起，李月，田昊，姬豫. 冻结方式对冷冻生坯包子品质的影响. 河南工业大学学报(自然科学版). 2025(01): 1-10 . 
百度学术
2. 王立峰，厉珺，徐斐然，闫宇轩，肖香，王世杰，艾连中，王海鸥. 副干酪乳杆菌对小麦发酵面团流变特性与风味的影响. 中国食品学报. 2021(07): 225-233 . 百度学术
3. 程强，陈迪，王金水. Lactobacillus plantarum M616生长及代谢特性研究. 河南工业大学学报(自然科学版). 2020(06): 20-25+34 . 百度学术
其他类型引用(1)
计量
- 文章访问数: 322
- HTML全文浏览量: 5
- PDF下载量: 14
- 被引次数: 4
下载:
