一种简单灵敏的基于适配体的黄曲霉毒素B1电化学传感器

doi:10.13208/j.electrochem.190219

摘要/Abstract

摘要：

黄曲霉毒素B1（AFB1）以其高毒性和致癌性成为食品安全隐患而备受关注. 本文拟构建一种新颖、简单、快速、灵敏的传感器用于谷物食品中AFB1的痕量检测. 将介孔碳（CMK）修饰在工作电极表面来增大电极的表面积,再将工作电极恒电位沉积金纳米粒子（AuNPs）,提高电信号的同时,为下一步巯基化适配体的连接提供位点. 检测过程中,AFB1可以竞争性地去除吸附在适配体链上的亚甲基蓝（MB）引起电信号的变化,对AFB1进行定量检测. 修饰的工作电极导电性能得到改善,灵敏度大大提高,对AFB1的线性响应范围为0.1 ~ 75 μg·L^-1,检出限低至36 ng·L^-1. 在对不同谷物食品（大米、玉米、糯米）进行加标回收实验中,回收率在92.3% ~ 103.6%范围之间,实现对目标物的定量检测. 本文为食品中AFB1快速检测方法提供了一种新思路和新方法.

关键词: 黄曲霉毒素B1, 适配体, 传感器, 检测方法

Abstract:

Aflatoxin B1 has attracted much attention because of its high toxicity and carcinogenicity, which has become a great concern in food safety. Based on the principle of specific binding between Aflatoxin B1 and its aptamer, an aptamer-based electrochemical sensor had been designed and developed for the determination of minor Aflatoxin B1 contained in grain. The mesoporous carbons were first modified on the surface of the working electrode, and then the gold nanoparticles were on-site electrodeposited at a constant potential. Each modified electrode was characteritised by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). As a result, the surface area and the electrochemical signal of the modified electrode were all greatly increased, providing more attachment sites for the following conjugation of the aptamer. During the detecting process, Aflatoxin B1 could compete with methylene blue on the aptamer chain to cause methylene blue shedding and the electrochemical signals were changed which could be used to quantify the concentration of Aflatoxin B1. The surface modifications could evidently improve the conductivity and sensitivity of the sensor. A linear response in current to Aflatoxin B1 was found ranging from 0.1 to 75 μg·L^-1 with the detection limit as low as 36 ng·L^-1 (S/N = 3). The spiked recovery tests of different grains (rice, corn, glutinous rice) revealed that the recovery rates were between 92.3 and 103.6%, showing excellent accuracy, sensitive quantitative detection of the target substance and good reproducibility. This work has demonstrated a new method to develop a novel, simple, fast and sensitive sensor for the detection of trace amount of Aflatoxin B1 in grains.

Key words: Aflatoxin B1, aptamer, sensor, detection method

中图分类号:

O646

刘冰, 赵耀帅, 秦月, 王怡, 朱艳杰, 王硕. 一种简单灵敏的基于适配体的黄曲霉毒素B1电化学传感器[J]. 电化学（中英文）, 2020, 26(3): 422-430.

LIU Bing, ZHAO Yao-shuai, QIN Yue, WANG Yi, ZHU Yan-jie, WANG Shuo. A Simple and Sensitive Aptamer-Based Electrochmical Sensor for Determination of Aflatoxin B1[J]. Journal of Electrochemistry, 2020, 26(3): 422-430.

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参考文献 32

[1]	Zhang Z, Nie D, Fan K, et al. A systematic review of plant-conjugated masked mycotoxins: Occurrence, toxicology, and metabolism[J]. Critical Reviews in Food Science and Nutrition, 2019,60(9):1523-1537. URL pmid: 30806521
[2]	Li P W( 李培武), Ding X X( 丁小霞), Bai Y Z( 白艺珍), et al. Advance in research on risk assessment of aflatoxin in agricultural products[J]. Scientia Agricultura Sinical( 中国农业科学) , 2013,46(12):2534-2542. doi: 10.3864/j.issn.0578-1752.2013.12.014 URL
[3]	Salati S, D Imporzano G, Panseri S, et al. Degradation of Aflatoxin B1 during anaerobic digestion and its effect on process stability[J]. International Biodeterioration & Biodegradation, 2014,94:19-23.
[4]	Ma Y L, Kong Q B, Hua H, et al. Aflatoxin B1 up-regulates insulin receptor substrate 2 and stimulates hepatoma cell migration[J]. PLOS ONE, 2012,7(10):e47961. doi: 10.1371/journal.pone.0047961 URL pmid: 23112878
[5]	Moon J, Kim G, Lee S. A Gold Nanoparticle and Aflatoxin B1-BSA conjugates based lateral flow assay method for the analysis of Aflatoxin B1[J]. Materials, 2012,5(12):634-643. doi: 10.3390/ma5040634 URL
[6]	Kotinagu K, Mohanamba T, Kumari L R. Assessment of Aflatoxin B1 in livestock feed and feed ingredients by high-performance thin layer chromatography[J]. Veterinary World, 2015,8(12):1396-1399. doi: 10.14202/vetworld.2015.1396-1399 URL pmid: 27047050
[7]	Amirkhizi B, Arefhosseini S R, Ansarin M, et al. Aflatoxin B1 in eggs and chicken livers by dispersive liquid-liquid microextraction and HPLC[J]. Food Additives & Contaminants: Part B, 2015,8(4):245-249.
[8]	Zhou G H, Chen Y J, Kong Q, et al. Detoxification of Aflatoxin B1 by zygosaccharomyces rouxii with solid state fermentation in peanut meal[J]. Toxins, 2017,9(1):42. doi: 10.3390/toxins9010042 URL
[9]	Luo D Q( 罗定强), Huang Y( 黄艳), Fan B J( 樊宝娟), et al. Determination of aflatoxin B1, B2, G1, G2 in nelumbinissemen by LC-MS/MSLC-MS/MS[J]. Anhui Medical and Pharmaceutical Journall( 安徽医药), 2014,18(1):41-44.
[10]	Zhang M H( 张铭函), Jiang J Q( 姜俊巧), Ge J J( 葛君杰), et al. Acetylcholinesterase biosensor platform based on BP2000 for the detection of carbaryl[J]. Journal of Ele-ctrochemistryl( 电化学), 2018,24(4):4-9.
[11]	Li W J( 李文进), Liu X( 刘霞), Li R Z( 李蓉卓), et al. Progress on pesticide residues detection by electrochemical sensor[J]. Food & Machineryl( 食品与机械), 2013,29(4):241-245.
[12]	Rawal R, Chawla S, Devender, et al. An amperometric biosensor based on laccase immobilized onto Fe₃O₄NPs/cMWCNT/PANI/Au electrode for determination of phenolic content in tea leaves extract[J]. Enzyme and Microbial Technology, 2012,51(4):179-185. doi: 10.1016/j.enzmictec.2012.06.001 URL pmid: 22883551
[13]	Talemi R P, Mousavi S M, Afruzi H. Using gold nanostars modified pencil graphite electrode as a novel substrate for design a sensitive and selective dopamine aptasensor[J]. Materials Science & Engineering C - Materials for Biological Applications, 2017,73:700-708. doi: 10.1016/j.msec.2016.12.119 URL pmid: 28183663
[14]	Xue X Y, Cheng R, Shi L, et al. Nanomaterials for water pollution monitoring and remediation[J]. Environmental Chemistry Letters, 2017,15(1):23-27. doi: 10.1007/s10311-016-0595-x URL
[15]	Liu W, Luo J, Zhao M, et al. Effect of amino compounds on luminol-H₂O₂-gold nanoparticle chemiluminescence system[J]. Analytical and Bioanalytical Chemistry, 2016,408(30):8821-8830. URL pmid: 27473431
[16]	Cheng C E, Lin C Y, Chang H Y, et al. Surface-enhanced Raman scattering of graphene with photo-assisted-synthesized gold nanoparticles[J]. Optics Express, 2013,21(5):6547-6554.
[17]	Lang J W, Yan X B, Yuan X Y, et al. Study on the electrochemical properties of cubic ordered mesoporous carbon for supercapacitors[J]. Journal of Power Sources, 2011,196(23):10472-10478. doi: 10.1016/j.jpowsour.2011.08.017 URL
[18]	Liu F L, Guo Z B, Ling H G, et al. Effect of pore structure on the adsorption of aqueous dyes to ordered mesoporous carbons[J]. Microporous and Mesoporous Materials, 2016,227:104-111. doi: 10.1016/j.micromeso.2016.02.051 URL
[19]	Cho M S, Kim Y W, Han S Y, et al. Detection for folding of the thrombin binding aptamer using label-free electrochemical methods[J]. BMB Reports, 2008,41(2):126-131. URL pmid: 18315948
[20]	Nimjee S M, Rusconi C P, Sullenger B A. APTAMERS: an emerging class of therapeutics[J]. Annual Review of Medicine, 2005,56(1):555-583. doi: 10.1146/annurev.med.56.062904.144915 URL
[21]	Wang J, Wang F, Dong S. Methylene blue as an indicator for sensitive electrochemical detection of adenosine based on aptamer switch[J]. Journal of Electroanalytical Chemistry, 2009,626(1/2):1-5. doi: 10.1016/j.jelechem.2008.08.008 URL
[22]	Bang G S, Cho S, Kim B G. A novel electrochemical detection method for aptamer biosensors[J]. Biosensors & Bioelectronics, 2005,21(6):863-870. doi: 10.1016/j.bios.2005.02.002 URL pmid: 16257654
[23]	Wang J, Wang F, Dong S. Methylene blue as an indicator for sensitive electrochemical detection of adenosine based on aptamer switch[J]. Journal of Electroanalytical Chemistry, 2009,626(1/2):1-5. doi: 10.1016/j.jelechem.2008.08.008 URL
[24]	Erdem A, Kerman K, Meric B, et al. Novel hybridization indicator methylene blue for the electrochemical detection of short DNA sequences related to the hepatitis B virus[J]. Analytica Chimica Acta, 2000,422(2):139-149. doi: 10.1016/S0003-2670(00)01058-8 URL
[25]	Bagheri H, Talemi R P, Afkhami A. Gold nanoparticles deposited on fluorine-doped tin oxide surface as an effective platform for fabricating a highly sensitive and specific digoxin aptasensor[J]. RSC Advances, 2015,5(72):58491-58498. doi: 10.1039/C5RA09402J URL
[26]	Mashhadizadeh M H, Talemi R P. Application of diazo-thiourea and gold nano-particles in the design of a highly sensitive and selective DNA biosensor[J]. Chinese Chemical Letters, 2015,26(1):160-166. doi: 10.1016/j.cclet.2014.09.004 URL
[27]	Yu X Y( 于雪云). The application of cyclic voltammetry experimental technique is briefly described[J]. Journal of Dezhou Universityl( 德州学院学报), 2012,28(S1):204-205.
[28]	Qi Q D( 乔庆东), Li Q( 李琪). Electrochemical behaviors of ferrocene using cyclic voltammetry[J]. Journal of Liaoning Shihua Universityl( 辽宁石油化工大学学报), 2013,34(3):5-7.
[29]	Park J S, Kim H J, Lee J H, et al. Amyloid beta detection by faradaic electrochemical impedance spectroscopy using interdigitated microelectrodes[J]. Sensors, 2018,18(2):426. doi: 10.3390/s18020426 URL
[30]	Sabet F S, Khabbaz H, Hosseini M, et al. FRET-based aptamer biosensor for selective and sensitive detection of Aflatoxin B1 in peanut and rice[J]. Food Chemistry, 2016,220:527-532. doi: 10.1016/j.foodchem.2016.10.004 URL pmid: 27855935
[31]	Seok Y, Byun J Y, Shim W B, et al. A structure-switchable aptasensor for aflatoxin B1 detection based on assembly of an aptamer/split DNAzyme[J]. Analytica Chimica Acta, 2015,886:182-187. URL pmid: 26320651
[32]	Sergeyeva T, Yarynka D, Piletska E, et al. Fluorescent sensor systems based on nanostructuerd polymeric membranes for selective recognition of Aflatoxin B1[J]. Talanta, 2017,175:101-107. doi: 10.1016/j.talanta.2017.07.030 URL pmid: 28841965

Sensor	Linear range	Detection limit	Reference
Aptamer/QDs conjugates sensor	10 ~ 400 nmol·L^-1	3.4 nmol·L^-1	[30]
Aptamer/split DNAzyme sensor	0.1 ~ 1×10⁴ ng·mL^-1	0.1 ng·mL^-1	[31]
Molecularly imprinted polymer sensor	14 ~ 500 ng·mL^-1	14 ng·mL^-1	[32]
Aptamer-based electrochmical sensor	75 μg·L^-1 ~ 100 ng·L^-1	36 ng·L^-1	This work

Sample	Initial concentration/(μg·kg^-1)	Proposed sensor		HPLC
Sample	Initial concentration/(μg·kg^-1)	Amount/ (μg·kg^-1)	Recovery/% (means±SD, n=3)	Amount/ (μg·kg^-1)	Recovery/% (means±SD, n=3)
Rice	10.0	10.36	103.6±2.4	10.15	101.7±0.8
	25.0	23.76	95.1±3.0	23.38	93.3±1.6
	50.0	46.31	92.7±5.6	48.91	97.8±0.2
Glutinous rice	10.0	9.75	97.9±1.3	9.42	93.6±0.8
	25.0	24.23	97.2±3.8	23.64	94.3±7.8
	50.0	48.04	96.2±4.3	45.08	90.0±0.6
Corn	10.0	9.52	95.2±2.8	10.56	106.3±1.3
	25.0	23.73	95.0±3.8	21.35	84.8±0.4
	50.0	46.49	92.3±1.8	44.95	89.9±3.3