An Electrochemical Aptasensor for Detection of Aflatoxin M1 Based on Reduced Graphene Oxide
-
摘要: 本实验基于还原氧化石墨烯（RGO）构建了一种用于黄曲霉毒素M1（AFM1）检测的电化学适配体传感器。采用红枣汁还原氧化石墨烯（GO）制备RGO，RGO通过滴涂法修饰在玻碳电极（GCE）表面，利用电沉积法将纳米金修饰在RGO/GCE上，AFM1的适配体（Apt）通过Au-S键固定在AuNPs/RGO/GCE电极表面用于靶标AFM1的捕获。当AFM1存在时，AFM1与适配体特异性结合形成AFM1-Apt复合物，该复合物阻碍了电子的传递，导致电化学信号减弱。对RGO的制备条件进行优化，利用差示脉冲伏安法（DPV）监测电极表面的电化学信号，并对不同类型的毒素（黄曲霉毒素B1、黄曲霉毒素B2、赭曲霉毒素A和伏马毒素B1）、不同浓度的AFM1（1×10−7~5×10−4 ng/mL）以及羊乳样品进行检测以确定电化学适配体传感器的特异性、灵敏性和实用性。结果表明，GO:红枣汁=2:1（V:V），pH=11时所制备的RGO的导电能力最强。传感器的电信号与AFM1浓度的对数呈线性关系，检测范围为1×10−7~5×10−4 ng/mL，检测限为3.3×10−5 pg/mL，同时所建立的方法仅对AFM1的检测有响应，而对干扰毒素无响应，说明电化学适配体传感器的特异性良好。使用建立的AFM1电化学适配体传感器对羊奶中的AFM1含量进行测定，发现所构建的传感器具有很高的灵敏性和良好的选择性，有望应用于食品工业中真菌毒素的快速、准确检测当中。Abstract: In this study, a fast and sensitive electrochemical aptasensor for sensitive determination of AFM1 was successfully established based on reduced graphene oxide (RGO). RGO was prepared by reducing graphene oxide with jujube juice. The synthesized RGO was dropped onto the surface of GCE. AuNPs was modified on the surface of the RGO/GCE via electrodepositio. The thiolated aptamer (SH-Apt) of the AFM1 was immobilized on the surface of the AuNPs/RGO/GCE through strong Au-S bond. When AFM1 was present, AFM1 bound specifically to the aptamer forming Apt-AFM1 conjugates. The conjugates hindered electron transfer, causing a decrease of current signal. Differential pulse voltammetry (DPV) was used to monitor electrochemical signal. This electrochemical aptasensor was used to test aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), ochratoxin A (OTA) and fumonisin B1 (FB1) to ensure the electrochemical aptasensor’s specificity. This electrochemical aptasensor was used to detect 1×10−7~5×10−4 ng/mL AFM1 to ensure the electrochemical aptasensor’s sensitivity. And this electrochemical aptasensor was used to detect goat milk to evaluate the practical use of electrochemical aptasensor. The results showed that RGO had the strongest conductivity when GO to jujube juice was 2:1 (V:V) and pH value was about 11. There was a good linear relationship between electrochemical signal and logarithm of AFM1 concentration in the range of 1×10−7~5×10−4 ng/mL with a low detection limit of 3.3×10−5 pg/mL. What’s more, the developed aptasensor was specific to AFM1 and did not respond to interfering mycotoxins, which suggested that the electrochemical aptasensor possessed an excellent selectivity for AFM1 detection. AFM1 electrochemical aptamer sensor was used to determine the content of AFM1 in goat milk. It was found that the sensor had high sensitivity and good selectivity, and it was expected to be applied to the rapid and accurate detection of mycotoxins in food industry.
-
Key words:
- aflatoxin M1 /
- electrochemical aptasensor /
- reduced graphene oxide
-
图 1 基于还原氧化石墨烯的电化学适配体传感器检测AFM1原理图
Figure 1. Schematic of the electrochemical aptasensor for the detection of AFM1 based on RGO
图 2 不同材料还原对RGO导电性的影响
Figure 2. Effects of different materials reduction on RGO conductivity
注：（a，a'）发酵枣汁中的多酚；（b，b'）原枣汁中的多酚；（c，c'）发酵枣汁；（d，d'）原枣汁；A图表示使用CV法对不同材料还原的RGO导电性的测定；B图表示使用SWV法对不同材料还原的RGO导电性的测定。
图 4 电化学适配体传感器在5 mmoL/L [Fe（CN）6]3−/4−溶液中的CV和SWV表征图
Figure 4. CV and SWV characterization of electrochemical aptasensor in 5 mmoL/L [Fe(CN)6]3-/4- solution
注：（a，a'）：裸GCE；（b，b'）：还原氧化石墨烯修饰后的电极；（c，c'）：纳米金修饰后电极；d，d'：适配体修饰后的电极；e：AFM1孵育后的电极；A表示CV表征图谱；B表示SWV表征图谱。
图 5 不同条件下制备的RGO的导电能力
Figure 5. The conductivity of RGO prepared under different conditions
注：（A）GO和红枣汁的比例；（B）不同的pH（比例是2:1）。
图 6 不同浓度的AFM1所对应的DPV响应曲线图
Figure 6. Differential pulse voltammograms corresponding to different concentrations of AFM1
注：a~g分别代表0，1×10−7，1×10−6，1×10−5，1×10−4， 5×10−4，1×10−3 ng/mL的AFM1。
图 7 DPV峰电流值与不同浓度的AFM1的校准曲线
Figure 7. Calibration curves of DPV peak current and AFM1 with different concentrations
图 8 电化学适配体传感器对不同干扰物的特异性评估
Figure 8. Evaluation of the specificity of the aptasensor toward different interferences
注：所有毒素的浓度均为1×10−5 ng/mL。
表 1 本研究与报道的检测AFB1的方法的比较
Table 1. Comparison of reported methods for the detection of AFB1
黄曲霉毒素 检测方法 检测范围（ng/mL） 检测限（ng/mL） 参考文献 AFB1 高效液相色谱法 0.1~20 0.08 [32] 电化学适配体传感器 1×10−8~1×10−6 2×10−9 [33] 电化学适配体传感器 5~200 0.035 [34] 电化学适配体传感器 0.05~20 0.016 [35] 表面增强拉曼适配体传感器 0.0001~100 4×10-4 [36] AFM1 高效液相色谱法 0.05~5 0.02 [37] 电化学适配体传感器 0.002~0.6 9×10-4 [22] 电化学适配体传感器 0.002~0.15 1.15×10−3 [38] 电化学适配体传感器 0.006~0.06 1.98×10−3 [5] 荧光适配体传感器 0.0001~0.5 0.0194×10−3 [39] 电化学适配体传感器 1×10−7~5×10−4 3.3×10−8 本工作 
下载: 导出CSV
表 2 羊乳样品中AFM1的检测回收率（n=3）
Table 2. Recovery of AFM1 detection in goat milk (n=3)
样品 添加量AFM1
（ng/mL）测得量
（ng/mL）回收率
（%）相对标准偏差
（%）1 1×10−4 1.03×10−4 103.0 6.82 2 1×10−5 8.58×10−6 85.8 7.87 3 1×10−6 9.36×10−7 93.6 5.05
下载: 导出CSV
-
[1] Yugender G K, Catanante G L, Hayat A, et al. Disposable and portable electrochemical aptasensor for label free detection of aflatoxin B1 in alcoholic beverages[J]. Sensors and Actuators B Chemical,2015,235:466−473. [2] Inoue T, Nagatomi Y, Uyama A, et al. Degradation of aflatoxin B1 during the fermentation of alcoholic beverages[J]. Toxins,2013,5(7):1219−1229. doi: 10.3390/toxins5071219 [3] Sugiyama K, Hiraoka H, Sugitakonishi Y. Aflatoxin M1 contamination in raw bulk milk and the presence of aflatoxin B1 in corn supplied to dairy cattle in Japan[J]. Shokuhinseigaku Zasshi Journal of the Food Hygienic Society of Japan,2008,49(5):352−359. doi: 10.3358/shokueishi.49.352 [4] Rodríguez-Blanco M, Ramos A J, Prim M, et al. Usefulness of the analytical control of aflatoxins in feedstuffs for dairy cows for the prevention of aflatoxin M1 in milk[J]. Mycotoxin Research,2019,36:11−22. [5] Nguyen B H, Tran L D, Do Q P, et al. Label-free detection of aflatoxin M1 with electrochemical Fe3O4/polyaniline-based aptasensor[J]. Materials Science and Engineering: C,2013,33(4):2229−2234. doi: 10.1016/j.msec.2013.01.044 [6] Mollarasouli F, Asadpour-Zeynali K, Campuzano S, et al. Non-enzymatic hydrogen peroxide sensor based on graphene quantum dots-chitosan/methylene blue hybrid nanostructures[J]. Electrochimica Acta,2017,246:303−314. doi: 10.1016/j.electacta.2017.06.003 [7] Linting Z, Ruiyi L, Zaijun L, et al. An immunosensor for ultrasensitive detection of aflatoxin B1 with an enhanced electrochemical performance based on graphene/conducting polymer/gold nanoparticles/the ionic liquid composite film on modified gold electrode with electrodeposition[J]. Sensors and Actuators B: Chemical,2012,174:359−365. doi: 10.1016/j.snb.2012.06.051 [8] 凌强. 石墨烯负载金属氧化物复合材料的制备及应用研究[D]. 南京: 南京大学, 2014. [9] Agharkar M, Kochrekar S, Hidouri S, et al. Trends in green reduction of graphene oxides, issues and challenges: A review[J]. Materials Research Bulletin,2014,59:323−328. doi: 10.1016/j.materresbull.2014.07.051 [10] Iravani S. Green synthesis of metal nanoparticles using plants[J]. Green Chemistry,2011,13(10):2638−2642. doi: 10.1039/c1gc15386b [11] Weng X, Wu J, Ma L, et al. Impact of synthesis conditions on Pb(II) removal efficiency from aqueous solution by green tea extract reduced graphene oxide[J]. Chemical Engineering Journal,2019,359(2):976−981. [12] Kuila T, Bose S, Khanra P, et al. A green approach for the reduction of graphene oxide by wild carrot root[J]. Carbon,2012,50(3):914−921. doi: 10.1016/j.carbon.2011.09.053 [13] Kartick B, Srivastava S K, Srivastava. Green synthesis of graphene[J]. Journal of Nanoence & Nanotechnology,2013,13(6):4320. [14] Mahmoud A T, Behzad H. Green-synthesis of reduced graphene oxide nanosheets using rose water and a survey on their characteristics and applications[J]. Rsc Advances,2013,3(32):13365−13370. doi: 10.1039/c3ra40856f [15] Song J, Bi J, Chen Q, et al. Assessment of sugar content, fatty acids, free amino acids, and volatile profiles in jujube fruits at different ripening stages[J]. Food Chemistry,2019,270(11):344−352. [16] 王丽玲, 蒲云峰, 李雁琴, 等. 红枣中γ-氨基丁酸的研究进展[J]. 食品科学技术学报,2019,37(6):23−28. doi: 10.3969/j.issn.2095-6002.2019.06.004 [17] Wojdy O A, Figiel A, Legua P, et al. Chemical composition, antioxidant capacity, and sensory quality of dried jujube fruits as affected by cultivar and drying method[J]. Food Chemistry,2016,207:170−179. doi: 10.1016/j.foodchem.2016.03.099 [18] Wang B N, Cao W, Gao H, et al. Simultaneous determination of six phenolic compounds in jujube by lc-ecd[J]. Chromatographia,2010,71(7-8):703−707. doi: 10.1365/s10337-010-1485-1 [19] Pawlowska A M, Camangi F, Bader A, et al. Flavonoids of Zizyphus jujuba L. and Zizyphus spina-christi (L.) willd (rhamnaceae) fruits[J]. Food Chemistry,2009,112(4):858−862. doi: 10.1016/j.foodchem.2008.06.053 [20] 黄婉玉. 超滤对红枣汁理化性质和抗氧化活性的影响[D]. 西安: 西北大学, 2010. [21] 王毕妮. 红枣多酚的种类及抗氧化活性研究[D]. 杨凌: 西北农林科技大学, 2011. [22] Hamid J S, Mohammad R, Mohammad D N, et al. A novel electrochemical aptasensor for detection of aflatoxin M1 based on target-induced immobilization of gold nanoparticles on the surface of electrode[J]. Biosensors and Bioelectronics,2018,117:487−492. doi: 10.1016/j.bios.2018.06.055 [23] Hummers W S, Offeman R E. Preparation of Graphitic Oxide[J]. Journal of the American Chemical Society,1958,208:1334−1339. [24] Allen M J, Tung V C, Kaner R B. Honeycomb carbon: A review of graphene[J]. Chemical Reviews,2009,110(1):132−145. [25] Sharma A, Catanante G, Hayat A, et al. Development of structure switching aptamer assay for detection of aflatoxin M1 in milk sample[J]. Talanta,2016,158:35−41. doi: 10.1016/j.talanta.2016.05.043 [26] Dinçkaya E, Kınık Ö, Sezgintürk M K, et al. Development of an impedimetric aflatoxin m1 biosensor based on a DNA probe and gold nanoparticles[J]. Biosensors and Bioelectronics,2011,26(9):3806−3811. doi: 10.1016/j.bios.2011.02.038 [27] 陈超, 段芳莉. 氧化石墨烯褶皱行为与结构的分子模拟研究[J]. 物理学报,2020,69(19):147−154. [28] Goud K Y, Hayat A, Catanante G, et al. An electrochemical aptasensor based on functionalized graphene oxide assisted electrocatalytic signal amplification of methylene blue for aflatoxin B1 detection[J]. Electrochimica Acta,2017,244:96−103. doi: 10.1016/j.electacta.2017.05.089 [29] Park Y, Hong Y N, Weyers A, et al. Polysaccharides and phytochemicals: A natural reservoir for the green synthesis of gold and silver nanoparticles[J]. Nanobiotechnology Iet,2012,43(6):69−78. [30] Iravani S. Green synthesis of metal nanoparticles using plants[J]. Green Chemistry,2014(13):2638−2650. [31] Zeng Y Y, Zheng A X, Wu J, et al. Horseradish peroxidase and aptamer dual-functionalized nanoprobe for the amplification detection of alpha-methylacyl-CoA racemase[J] Analytica Chimica Acta, 2015, 899: 100−105. [32] Alguel I, Kara D. Determination and chemometric evaluation of total aflatoxin, aflatoxin B1, ochratoxin A and heavy metals content in corn flours from Turkey[J]. Food Chemistry,2014,157(15):70−76. [33] Geleta G S, Zhao Z, Wang Z. A novel reduced graphene oxide/molybdenum disulfide/polyaniline nanocomposite-based electrochemical aptasensor for detection of aflatoxin B1[J]. Analyst,2018,143(7):1644−1649. doi: 10.1039/C7AN02050C [34] Qian J, Ren C C, Wang C Q, et al. Gold nanoparticles mediated designing of versatile aptasensor for colorimetric/electrochemical dual-channel detection of aflatoxin B1[J]. Biosensors and Bioelectronics,2020,166:112443. doi: 10.1016/j.bios.2020.112443 [35] Li Y Y, Liu D, Zhu C X, et al. Sensitivity programmable ratiometric electrochemical aptasensor based on signal engineering for the detection of aflatoxin b1 in peanut[J]. Journal of Hazardous Materials,2020,387:122001−122008. doi: 10.1016/j.jhazmat.2019.122001 [36] He H R, Sun D W, Pu H B, et al. Bridging Fe3O4@Au nanoflowers and Au@Ag nanospheres with aptamer for ultrasensitive sers detection of aflatoxin B1[J]. Food Chemistry,2020,324:126832. doi: 10.1016/j.foodchem.2020.126832 [37] 华宇, 高和杨, 聂兴娜, 周旌, 张大伟. 同位素内标-高效液相色谱-串联质谱法检测牛奶及奶粉中黄曲霉毒素M1[J]. 食品安全质量检测学报,2020,11(6):1978−1984. [38] Istamboulié G, Paniel N, Zara L, et al. Development of an impedimetric aptasensor for the determination of aflatoxin M1 in milk[J]. Talanta,2016,146:464−469. doi: 10.1016/j.talanta.2015.09.012 [39] Niazi S, Khan I M, Yu Y. et al. et al. A novel fluorescent aptasensor for aflatoxin m1 detection using rolling circle amplification and g-C3N4 as fluorescence quencher[J]. Sensors and Actuators B-Chemical,2020,315:128049. doi: 10.1016/j.snb.2020.128049 -
点击查看大图
图(8) / 表(2)
计量
- 文章访问数: 137
- HTML全文浏览量: 65
- PDF下载量: 23
- 被引次数: 0
