以亚甲基蓝为杂交指示剂的电化学适配体鼠伤寒沙门氏菌传感技术

doi:10.3864/j.issn.0578-1752.2018.06.017

摘要/Abstract

摘要： 【目的】构建一种更具实用性的可定量检测鼠伤寒沙门氏菌的新型电化学适配体传感器，克服传统沙门氏菌检测方法在时效性、灵敏度和操作简便性等方面的不足。【方法】将反应制得的氧化石墨烯（GO）滴涂在玻碳电极（GCE）表面，于PBS缓冲液中采用电化学还原法将其还原为还原氧化石墨烯（rGO），随后放置电极于氯金酸溶液中进行电沉积，于表面修饰一层纳米金（AuNPs）。依靠金硫键的作用将鼠伤寒沙门氏菌适配体互补链（S）固定在电极上，滴加封闭剂巯基己醇（MCH）占据电极表面空余位点，保证电极无非特异性吸附后，再将鼠伤寒沙门氏菌适配体（Apt）滴涂于电极表面，使S与Apt杂交形成DNA双链结构。将电极于37℃下同鼠伤寒沙门氏菌与核酸外切酶I（Exo I）的混合液孵育，依靠鼠伤寒沙门氏菌与Apt高度特异性结合，将Apt从电极表面带离，再基于Exo I对单链DNA的剪切作用，使鼠伤寒沙门氏菌得以循环重复作用于适配体，再将电极浸入亚甲基蓝（MB）溶液一段时间后，通过监测电极表面的电信号，并对其在菌液中的孵育时间及Exo I的浓度进行优化，构建检测鼠伤寒沙门氏菌的新型电化学适配体传感器。使用构建的传感器对大肠杆菌、金黄色葡萄球菌、志贺氏菌、单增李斯特菌以及副溶血弧菌进行检测，以确定该传感器的特异性；对2×10²—2×10⁷ cfu/mL的鼠伤寒沙门氏菌进行检测，以确定该传感器的敏感性；对猪肉样品进行鼠伤寒沙门氏菌的定量检测，以确定该传感器的实用性。【结果】所构建的鼠伤寒沙门氏菌电化学适配体传感器在菌液中的最适孵育时间为40 min，Exo I的最适浓度为0.8 U·µL^-1。特异性试验结果表明，该传感器仅在鼠伤寒沙门氏菌存在时有电信号响应，而对非目标菌无响应。敏感性试验结果表明，该传感器具有很高的敏感性，对鼠伤寒沙门氏菌的敏感性可达67 cfu/mL。使用构建的鼠伤寒沙门氏菌电化学适配体传感器对猪肉中的鼠伤寒沙门氏菌进行定量检测，加标回收率在97.3%—106.7%。【结论】所构建的新型鼠伤寒沙门氏菌电化学适配体传感器具备灵敏度高、特异性强、易于操作、检测迅速及成本低廉等优点，有望应用于食品工业中鼠伤寒沙门氏菌的现场快速定量检测。

关键词: 鼠伤寒沙门氏菌, 电化学适配体传感器, 还原氧化石墨烯, 纳米金, 亚甲基蓝, 核酸外切酶I

Abstract: 【Objective】A novel assay of electrochemical aptasensor for quantitative detection of Salmonella typhimurium with better practicability was constructed and investigated in order to overcome the shortcomings of traditional Salmonella detection methods, such as time-saving, sensitivity, simplicity, and etc.【Method】The prepared graphene oxide (GO) solution was dropped onto the glassy carbon electrode (GCE) surface and was reduced in PBS buffer by electrochemical reduction method to obtain reduced graphene oxide (rGO), and then Au nanoparticles (AuNPs) were electrochemically deposited onto the electrode by submersion in HAuCl_4.The complementary strands of the aptamers of Salmonella typhimurium (S) were attached to the surface of rGO/AuNPs GCE by Au-S bond, and then the electrode surface was blocked with MCH. Subsequently, the aptamers of Salmonella typhimurium (Apt) were dripped onto the modified electrode to make Apt bind with S. The modified electrode was immersed into the mixture containing Salmonella typhimurium and exonuclease I (Exo I) at 37 °C. In terms of the characteristics of Exo I that could amplify electrical signals and the aptamers that could exclusively bind with Salmonella typhimurium, the aptamers were taken away from S circularly. Then, the modified electrode was immersed in methylene blue (MB) solution for a while. Finally, the conditions of the incubation time in bacteria liquid, the Exo I concentration were optimized and the electrical signals of the electrode surface was monitored to construct the aptasensor. This electrochemical aptasensor was used to test Escherichia coli, Staphylococcus aureus, Shigella, Listeria monocytogenes and Vibrio parahaemolyticus to ensure the electrochemical aptasensor’s specificity. The electrochemical aptasensor was used to detect 2×10²-2×10⁷ cfu/mL Salmonella typhimurium to ensure the electrochemical aptasensor’s sensitivity. Then this electrochemical aptasensor was used to detect the pork to evaluate the practical use of electrochemical aptasensor.【Result】The optimization of the electrochemical aptasensor incubation time in bacterial liquid and the Exo I concentration were studied in detail, and the optimal conditions were 40 min and 0.8 U·μL^-1. The developed aptasensor was specific to Salmonella typhimurium and doesn’t react with non-target bacteria. The electrochemical aptasensor can successfully detect the Salmonella typhimurium target down to 67 cfu/mL. A good recovery of Salmonella typhimurium in the range of 97.3%-106.7% was obtained in pork by electrochemical aptasensor assays developed.【Conclusion】This electrochemical aptasensor can detect Salmonella typhimurium with a high sensitivity, a high specificity, an easy operation, a rapid detection and a low cost, which provide a good application prospect in the field of rapid quantitative detection of Salmonella typhimurium.

Key words: Salmonella typhimurium, electrochemical aptasensor, reduced graphene oxide, Au nanoparticles, methylene blue, exonuclease I

徐连应，彭海霞，邵玉宇，王毕妮，张富新. 以亚甲基蓝为杂交指示剂的电化学适配体鼠伤寒沙门氏菌传感技术[J]. 中国农业科学, 2018, 51(6): 1192-1201.

XU LianYing, PENG HaiXia, SHAO YuYu, WANG BiNi, ZHANG FuXin. An Electrochemical Aptasensor for Detection of Samonella typhimurium with Methylene Blue as Hybridization Indicator[J]. Scientia Agricultura Sinica, 2018, 51(6): 1192-1201.

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参考文献

[1] SHIM W B, SONG J E, MUN H, CHUNG D H, KIM M G. Rapid colorimetric detection of Salmonella typhimurium using a selective

filtration technique combined with antibody-magnetic nanoparticle nanocomposites. Analytical and Bioanalytical Chemistry, 2014, 406(3): 859-866.

[2] 李庆德, 原志伟, 沈巍. 沙门氏菌的危害及其快速检测方法的研究进展. 湖北畜牧兽医, 2010(1): 10-12.

LI D Q, YUAN Z W, SHEN W. Progress in the hazards of Salmonella and its rapid detection methods. Hubei Journal of Animal and Veterinary Science, 2010(1): 10-12. (in Chinese)

[3] WANG Z P, XU H, WU J, YE J, YANG Z. Sensitive detection of Salmonella with fluorescent bioconjugated nanoparticles probe. Food Chemistry, 2011, 125(2): 779-784.

[4] NIU K L, ZHENG X P, HUANG C S, XU K, ZHI Y, SHEN H B, JIA N Q. A colloidal gold nanoparticle-based immunochromatographic test strip for rapid and convenient detection of Staphylococcus aureus. Journal of Nanoscience and Nanotechnology, 2014, 14(7): 5151-5156.

[5] 牛凯莉. 基于胶体金免疫层析法的食源性致病菌检测技术的研究[D]. 上海: 上海师范大学, 2013.

NIU K L. The research of detecting methods of for the food-borne pathogenic bacteria based on the Colloidal gold immunochromatography assay [D]. Shanghai: Shanghai Normal University, 2013. (in Chinese)

[6] 户桂涛, 范云场, 董兴, 李云, 缪娟. 电化学传感器在食品分析中的应用进展. 材料导报, 2015, 19: 40-45.

HU G T, FAN Y C, DONG X, LI Y, MIAO J. Progress in application of electrochemical sensors in food analysis. Materials Review, 2015, 19: 40-45. (in Chinese)

[7] 张义红, 许文静, 杨坤. 电化学生物传感器的研究进展. 集成技术, 2014, 3(5): 19-27.

ZHANG Y H, XU W J, YANG K. Progress in electrochemical biosensors. Journal of Integration Technology, 2014, 3(5): 19-27. (in Chinese)

[8] XU Y, CHENG G F, HE P G, FANG Y Z. A review: Electrochemical aptasensors with various detection strategies. Electroanalysis, 2009, 21(11): 1251-1259.

[9] GILBERT S D, STODDARD C D, WISE S J, BATEY R T. Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain. Journal of Molecular Biology, 2006, 359(3): 754-768.

[10] PADMANABHAN K, PADMANABHAN K P, FERRARA J D, SADLER J E, TULINSKY A. The structure of alpha-thrombin inhibited by a 15-mer single-stranded DNA aptamer. The Journal of Biological Chemistry, 1993, 268(24): 17651-17654.

[11] FANG Z Y, WU W, LU X W, ZENG L W. Lateral flow biosensor for DNA extraction-free detection of Salmonella based on aptamer mediated strand displacement amplification. Biosensors and Bioelectronics, 2014, 56: 192-197.

[12] FLOREA A, TALEAT Z, CRISTEA C, MAZLOUM-ARDAKANI M, S?NDULESCU R. Label free MUC1 aptasensors based on electrodeposition of gold nanoparticles on screen printed electrodes. Electrochemistry Communications, 2013, 33: 127-130.

[13] WANG J L, WANG F A, DONG S J. Methylene blue as an indicator for sensitive electrochemical detection of adenosine based on aptamer switch. Journal of Electroanalytical Chemistry, 2009, 626(1/2): 1-5.

[14] DU Y, LI B L, WANG F, DONG S J. Au nanoparticles grafted sandwich platform used amplified small molecule electrochemical aptasensor. Biosensors and Bioelectronics, 2009, 24(7): 1979-1983.

[15] GEIM A K, NOVOSELOV K S. The rise of grapheme. Nature Materials, 2007, 6(3): 183-191.

[16] 徐连应, 王毕妮, 张富新. 基于复合纳米材料和酶切信号放大电化学适体传感器检测沙门氏菌. 中国农业科学, 2017, 50(21): 4208-4217.

XU L Y, WANG B N, ZHANG F X. An electrochemical aptasensor for detection of Salmonella based on composite nanomaterial and enzymatic recycling for amplification. Scientia Agricultura Sinica, 2017, 50(21): 4208-4217. (in Chinese)

[17] FRENS G. Controlled nucleation for regulation of particle-size in monodisperse gold suspensions. Nature Physical Science, 1973, 241(105): 20-22.

[18] TURKEVICH J, STEVENSON P C, HILLIER J. A study of the nucleation and growth processes in the synthesis of colloidal gold. Discussion of the Faraday Society, 1951, 11: 55-75.

[19] HUMMERS W S, OFFEMAN R E. Preparation of graphitic oxide. Journal of the American Chemical Society, 1958, 80(6): 1339-1340.

[20] AIIEN M J, TUNG V C, KANER R B. Honeycomb carbon: a review of graphene. Chemical Reviews, 2010, 110(1): 132-145.

[21] CHEN J R, JIAO X X, LUO H Q, LI N B. Probe-label-free electrochemical aptasensor based on methylene blue-znchored grapheme oxide amplification. Journal of Materials Chemistry B, 2013, 1: 861-864.

[22] CHEN D, FENG H B, LI J H. Graphene oxide: Preparation, functionalization, and electrochemical applications. Chemical Reviews, 2012, 112(11): 6027-6053.

[23] LABIB M, ZAMAY A S, KOLOVSKAYA O S, RESHETNEVA I T, ZAMAY G S, KIBBEE R J, SATTAR S A, ZAMAY T N, BEREZOVSKI M V. Aptamer-based viability impedimetric sensor for bacteria. Analytical Chemistry, 2012, 84(21): 8966-8969.

[24] MA X, JIANG Y, JIA F, YU Y, CHEN J, WANG Z. An aptamer- based electrochemical biosensor for the detection of Salmonella. Journal of Microbiological Methods, 2014, 98: 94-98.

[25] KARA P, KERMAN K, OZKAN D, MERIC B, ERDEM A, OZKAN Z, OZSOZ M. Electrochemical genosensor for the detection of interaction between methylene blue and DNA. Electrochemistry Communications, 2002, 4(9): 705-709.

[26] ERDEM A, KERMAN K, MERIC B, OZSOZ M. Methylene blue as a novel electrochemical hybridization indicator. Electroanalysis, 2001, 13(3): 219-223.

[27] ROHS R, SKLENAR H, LAVERY R, RODER B. Methylene blue binding to DNA with alternating GC base sequence: a modeling study. Journal of American Chemical Society, 2000, 122(12): 2860-2866.

[28] ZHAO G C, ZHU J J, CHEN H Y. Spectroscopic studies of the interactive model of methylene blue with DNA by means of β-cyclodextrin. Spectrochimca Acta. Part A: Molecular and Biomolecular Spectroscopy, 1999, 55(5): 1109-1117.

[29] OZKAN D, KARA P, KERMAN K, MERIC B, ERDEM A, JELEN F, NIELSEN P E, OZSOZ M. DNA and PNA sensing on mercury and carbon electrode by using methylene blue as an electrochemical label. Bioelectrochemistry, 2002, 58(1): 119-126.

[30] JIANG B Y, WANG M, LI C, XIE J Q. Label-free and amplified aptasensor for thrombin detection based on background reduction and direct electron transfer of hemin. Biosensors and Bioelectronics, 2013, 43: 289-292.

[31] ZHENG D M, ZOU R X, LOU X H. Label-free fluorescent detection of ions, proteins, and small molecules using structure-switching aptamers, SYBR Gold, and exonuclease I. Analytical chemistry, 2012, 84(8): 3554-3560.