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Abstract
Introduction
Protocol
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Chemistry

邻苯二甲酸酯结合 DNA 核酸适体的选择、表征及其在电化学 Aptasensor 中的应用

Published: March 21, 2018
doi:
10.3791/56814
, , , , ,
1Department of Chemistry,Capital Normal University, 2College of Life Sciences,Capital Normal University

Summary

本文提出了一种体外的协议, 用于对组特定邻苯二甲酸酯结合 DNA 适配子的筛选和表征。在电化学 aptasensor 中也包括了所选核酸适体的应用。

Abstract

邻苯二甲酸酯 (PAEs) areone 主要群体的持久性有机污染物。由于同源性的快速增长, PAEs 的群特异检测非常理想。DNA 适配子已越来越多地应用于生物传感器平台上的识别元素, 但很少报道适配子对高度疏水性的小分子靶点 (如 PAEs) 的选择。这项工作描述了一种基于珠子的方法, 用于选择组特定的 DNA 适配子 PAEs。将氨基甲酸二丁酯 (DBP-NH2) 合成并固定在环氧活化的琼脂糖珠上, 使其在固定基质表面显示邻苯乙酯基团, 并因此选择组特定的粘合剂。通过定量聚合链反应和磁分离, 确定了核酸适体候选者的离解常数。适配子对其他 PAEs 的相对亲和性和选择性由竞争性化验确定, 核酸适体候选者预先限定在 DBP– NH 2 附着的磁性珠子上, 并在孵化时释放到上清被测试的 PAEs 或其他潜在的干扰物质。竞争试验的应用, 因为它提供了一个简便的亲和力比较 PAEs 之间没有功能组的表面固定化。最后, 我们演示了电化学 aptasensor 的制备方法, 并将其应用于邻苯二甲酸二 (2-己酯) 的超灵敏和选择性检测。该协议为核酸适体发现其他疏水小分子提供了见解。

Introduction

随着经济的快速发展, 工业化的加速, 城市建设的不断加快, 环境污染问题也日益严重。典型的环境污染物包括重金属离子、毒素、抗生素、杀虫剂、内分泌干扰物和持久性有机污染物 (pop)。除了金属离子和毒素, 其他污染物是小分子, 通常由各种同源物组成。例如, 最有毒的持久性有机污染物包括多氯联苯、多环芳烃 (多环芳烃)、多溴联联苯醚 (二苯醚)、聚氯氯苯并 (PCDDs)、多氯氧芴 (呋喃) 和苯二甲酸酸酯 (PAEs)1,2, 它们都由许多同源项组成。小分子检测主要是以色谱/质谱技术为基础进行的, 因为其应用的多样性3,4,5,6。对于现场检测, 最近开发了基于抗体的方法7,8,9。但是, 由于这些方法对某些同类具有高度的特异特性, 因此必须执行多个测试。更严重的是, 新的同源生长得如此之快, 以至于他们的抗体不能及时生成。因此, 在一项试验中, 开发组特定的生物传感器来监测所有同源物的总水平, 可为评估环境污染状况提供一个宝贵的指标。

近年来, 核酸适配子在各种生物传感平台中被广泛应用为识别元素, 因为它们能够识别各种各样的目标, 从离子和小分子到蛋白质和细胞10, 11 ,12。适配子是通过一个体外方法来识别的, 所谓的配位系统进化的指数富集 (SELEX) 13, 14.SELEX 从随机合成单链寡核苷酸库开始, 其中包含大约 1014-1015序列。随机库的大小确保了 RNA 或 DNA 候选结构的多样性。典型的 SELEX 过程包括多轮的浓缩, 直到图书馆丰富的序列, 高度亲和性和特异性的目标。最后的浓缩池然后排序, 离解常数 (Kd) 和选择性反对潜在的干扰物质是由不同的技术, 如过滤器结合, 亲和层析, 表面等离子共振 (SPR),等。15

由于水的溶解度极差, 且缺乏用于表面固定的功能基团, 因此在理论上很难核酸适体选择持久性有机污染物。SELEX 的重大进展加速了适配子的发现。但是, 尚未报告持久性有机污染物的组特定适配子的选择。到目前为止, 只有对某些同类的高特异性的 PCB 结合 DNA 适配子被识别为 16.PAEs 主要应用于聚氯乙烯材料中, 将聚氯乙烯从硬塑料改成弹性塑料, 从而充当增塑剂。一些 PAEs 已被认定为内分泌干扰物质, 可对肝肾功能造成严重损害, 减少男性精子的运动, 并可能导致畸形精子形态和睾丸癌的发生17。未报告复合类或组特定的 PAE 绑定适配子。

这项工作的目标是提供一个代表性的协议, 以选择组特定的 DNA 适配子高疏水性小分子, 如 PAEs, 一个代表性的持久性有机污染物组。我们还演示了选定的核酸适体在环境污染检测中的应用。该协议为核酸适体发现其他疏水小分子提供了指导和见解。

Protocol

1. 图书馆及底漆设计与合成 设计初始库和引物。库 (池0): 5 ‘-TCCCACGCATTCTCCACATC-N40-CCTTTCTGTCCTTCCGTCAC-3 ‘正向底漆 (FP): 5 ‘-TCCCACGCATTCTCCACATC-3 ‘磷酸化反向底漆 (PO4-RP): 5 ‘-PO4-GTGACGGAAGGACAGAAAGG-3 ‘ 综合池0, FP 和 PO4-RP 使用标准的 phosphoramidite 化学18,19,20, 并通过标…

Representative Results

我们设计并合成了氨基甲酸二丁酯 (DBP-NH2) 作为定位靶 (图 1F)。然后, 我们进行 DNA 核酸适体选择 PAEs 使用 DBP-NH 2 作为锚定目标和遵循经典的目标固定化的方法(图 2)。在每一个回合中, 使用变性页来优化 pcr 的周期数 (图 3) 进行了先导 pcr。强烈建议变性页面, 而不是本机页, 因为我们和其他?…

Discussion

适配子的一个显著好处是, 它们通过体外方法 SELEX 识别, 而抗体是通过体内免疫生成的. 因此, 在设计良好的实验条件下, 适配子可以选择理想的靶向特异性, 而抗体仅限于生理条件。

为了便于从自由序列分离绑定序列, 最近报告了几种修改后的 SELEX, 其中毛细管电泳30, 微流体 31, 磁性/丙烯酸珠/琼脂糖珠 14, …

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢国家自然科学基金 (21675112)、北京教育委员会科技计划重点项目 (KZ201710028027) 和首都师范大学燕京青年学者项目的资助。

Materials

UV-2550 Shimadzu,Japan protocol, section 3.8.2
DNA Engnine Thermal cycler,PTC0200 BIO-RAD section 3.5.1.2 and 3.5.2
C1000 Touch BIO-RAD section 5.3.6 and 6.3
VMP3 multichannel potentiostat Bio-Logic Science, Claix, France section 7.4,7.8 and 7.11
Epoxy-activated Sepharose 6B GE Healthcare (Piscataway, NJ, USA) 10220020 argarose beads, section 2.3 and 3.3
Dynabeads M-270 carboxylic acid magnetic beads Invitrogen, USA 420420 magnetic beads,section 5.2. and 5.3
Premix Taq Hot Start Version Takara,Dalian,China R028A polymerase, section 3.5.1.1
PARAFILM Sealing Membrane Bemis, USA PM-996 section 3.6.5
Lambda Exonuclease Invitrogen, USA EN0561 section3.7.1.2.The 10 × reaction buffer is provided along with λ exonuclease by the provider.
Dr. GenTLE
Precipitation Carrier		 Takara,Dalian,China 9094 section 3.6.2 and 3.8.1
UNIQ-10 PAGE DNA recovery kit Sangon Biotech (Shanghai) B511135 section 4.2
SYBR Gold nucleic acid gel stain Invitrogen, USA 1811838 nucelic acid stain dye, section 3.5.1.5
SYBR Premix Ex Taq II Takara,Dalian,China RR820A polymerase mix contaning polymerase and dNTPs, section 5.3.5
2-(N-Morpholino)ethanesulfonic acid (MES) Sigma-Aldrich CAS: 1132-61-2 section 5.2.1
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Invitrogen, USA CAS: 25952-53-8 section 5.2.2
N-hydroxysuccinimide (NHS) Sigma-Aldrich 6066-82-6 section 5.2.3
mercaptohexanol (MCH) Sigma-Aldrich CAS: 1633-78-9 section 7.7
Gold electrode Shanghai Chenhua CHI101 section 7.4. – 7.11
tris(2-carboxyethyl) phosphine hydrochloride (TCEP) Sigma-Aldrich CAS: 51805-45-9 section 7.5
O-(2-Mercaptoethyl)-O'-methyl-hexa-(ethylene glycol) Sigma-Aldrich CAS: 651042-82-9 section 7.7
diethylhexyl phthalate (DEHP) National Institute of Metrology, China CAS: 117-81-7 section 7.11
Tween 20 Sigma-Aldrich CAS: 9005-64-5 polyoxyethy-lene(20) sorbaitan monolaurate
Triton X-100 Sigma-Aldrich CAS: 9002-93-1 non-ionic surface active agent
PBS Sigma-Aldrich P5368 10 mM phosphate buffer containing 1 M NaCl, pH 7.4
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References

  1. Vorkamp, K., Riget, F. F. A review of new and current-use contaminants in the Arctic environment: Evidence of long-range transport and indications of bioaccumulation. Chemosphere. 111, 379-395 (2014).
  2. Net, S., Sempere, R., Delmont, A., Paluselli, A., Ouddane, B. Occurrence, fate, behavior and ecotoxicological state of phthalates in different environmental matrices. Environ Sci Technol. 49 (7), 4019-4035 (2015).
  3. Xie, Q. L., Liu, S. H., Fan, Y. Y., Sun, J. Z., Zhang, X. K. Determination of phthalate esters in edible oils by use of QuEChERS coupled with ionic-liquid-based dispersive liquid-liquid microextraction before high-performance liquid chromatography. Anal BioanalChem. 406 (18), 4563-4569 (2014).
  4. Ierapetritis, I., Lioupis, A., Lampi, E. Determination of phthalates into vegetable oils by isotopic dilution gas chromatography mass spectrometry. Food Anal Methods. 7 (7), 1451-1457 (2014).
  5. Sun, J. Z., He, H., Liu, S. H. Determination of phthalic acid esters in Chinese white spirit using dispersive liquid-liquid microextraction coupled with sweeping beta-cyclodextrin-modified micellar electrokinetic chromatography. J Sep Sci. 37 (13), 1679-1686 (2014).
  6. Yilmaz, P. K., Ertas, A., Kolak, U. Simultaneous determination of seven phthalic acid esters in beverages using ultrasound and vortex-assisted dispersive liquid-liquid microextraction followed by high-performance liquid chromatography. J Sep Sci. 37 (16), 2111-2117 (2014).
  7. Sun, R., Zhuang, H. An ultrasensitive gold nanoparticles improved real-time immuno-PCR assay for detecting diethyl phthalate in foodstuff samples. Anal Biochem. 480, 49-57 (2015).
  8. Sun, R. Y., Zhuang, H. S. A sensitive heterogeneous biotin-streptavidin enzyme-linked immunosorbent assay for the determination of di-(2-ethylhexyl)phthalate (DEHP) in beverages using a specific polyclonal antibody. Anal Methods. 6 (24), 9807-9815 (2014).
  9. Zhou, L., Lei, Y., Zhang, D., Ahmed, S., Chen, S. An ultra-sensitive monoclonal antibody-based enzyme-linked immunosobent assay for dibutyl phthalate in human urinary. Sci Total Environ. 541, 570-578 (2016).
  10. Shen, J., Li, Y., Gu, H., Xia, F., Zuo, X. Recent development of sandwich assay based on the nanobiotechnologies for proteins, nucleic Acids, small Molecules, and ions. Chem Rev. 114 (15), 7631-7677 (2014).
  11. Yin, X. -. B. Functional nucleic acids for electrochemical and electrochemiluminescent sensing applications. TrAC, Trends Anal Chem. 33, 81-94 (2012).
  12. Nguyen, V. -. T., Kwon, Y. S., Gu, M. B. Aptamer-based environmental biosensors for small molecule contaminants. Curr Opin Biotechnol. 45, 15-23 (2017).
  13. Groher, F., Suess, B. In vitro selection of antibiotic-binding aptamers. Methods. 106, 42-50 (2016).
  14. Yang, K. -. A., Pei, R., Stojanovic, M. N. In vitro selection and amplification protocols for isolation of aptameric sensors for small molecules. Methods. 106, 58-65 (2016).
  15. Jing, M., Bowser, M. T. Methods for measuring aptamer-protein equilibria: a review. Anal. Chim. Acta. 686 (1-2), 9-18 (2011).
  16. Mehta, J., et al. Selection and characterization of PCB-binding DNA aptamers. Anal Chem. 84 (3), 1669-1676 (2012).
  17. Matsumoto, M., Hirata-Koizumi, M., Ema, M. Potential adverse effects of phthalic acid esters on human health: A review of recent studies on reproduction. Regul Toxicol Pharm. 50 (1), 37-49 (2008).
  18. Goodchild, J. Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconjug Chem. 1 (3), 165-187 (1990).
  19. Brown, D. M. A brief history of oligonucleotide synthesis. Protocols for Oligonucleotides and Analogs: Synthesis and Properties. , 1-17 (1993).
  20. Reese, C. B. Oligo-and poly-nucleotides: 50 years of chemical synthesis. Org Biomol Chem. 3 (21), 3851-3868 (2005).
  21. Sproat, B., Colonna, F., Mullah, B., et al. An efficient method for the isolation and purification of oligoribonucleotides. Nucleos Nucleot Nucl. 14 (1-2), 255-273 (1995).
  22. Han, Y., et al. Selection of group-specific phthalic acid esters binding DNA aptamers via rationally designed target immobilization and applications for ultrasensitive and highly selective detection of phthalic acid esters. Anal Chem. 89 (10), 5270-5277 (2017).
  23. Bartlett, J. M. S., Stirling, D. A short history of the polymerase chain reaction. PCR protocols. , 3-6 (2003).
  24. Saiki, R. K., Scharf, S., Faloona, F., et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 230, 1350-1354 (1985).
  25. Albright, L. M., Slatko, B. E. Denaturing polyacrylamide gel electrophoresis. Current Protocols in Nucleic Acid Chemistry. , A. 3B 1-A. 3B 5 (2001).
  26. Summer, H., Grämer, R., Dröge, P. Denaturing urea polyacrylamide gel electrophoresis (Urea PAGE). JoVE. (32), e1485 (2009).
  27. Jing, M., Bowser, M. T. Methods for measuring aptamer-protein equilibria: a review. Anal Chim Acta. 686 (1), 9-18 (2011).
  28. Sharma, T. K., Bruno, J. G., Dhiman, A. ABCs of DNA aptamer and related assay development. Biotechnol Adv. 35 (2), 275-301 (2017).
  29. Liu, R., et al. Signaling-probe displacement electrochemical aptamer-based sensor (SD-EAB) for detection of nanomolar kanamycin A. Electrochim Acta. 182, 516-523 (2015).
  30. Mendonsa, S. D., Bowser, M. T. In vitro evolution of functional DNA using capillary electrophoresis. J Am Chem Soc. 126, 20-21 (2004).
  31. Lou, X. H., et al. Micromagnetic selection of aptamers in microfluidic channels. Proc Natl Acad Sci USA. 106 (9), 2989-2994 (2009).
  32. Cho, M., et al. Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing. Proc. Natl. Acad. Sci. U.S.A. 107, 15373-15378 (2010).
  33. Cho, M., et al. Quantitative selection and parallel characterization of aptamers. Proc Nat Acad Sci USA. 110 (46), 18460-18465 (2013).
  34. Song, K. -. M., et al. Gold nanoparticle-based colorimetric detection of kanamycin using a DNA aptamer. Anal Biochem. 415 (2), 175-181 (2011).
  35. Yang, Z., Ding, X., Guo, Q., et al. Second generation of signaling-probe displacement electrochemical aptasensor for detection of picomolar ampicillin and sulfadimethoxine. Sens Actuators B. 253 (2017), 1129-1136 (2017).
  36. Lou, X., Zhao, T., Liu, R., Ma, J., Xiao, Y. Self-assembled DNA monolayer buffered dynamic ranges of mercuric electrochemical sensor. Anal Chem. 85 (15), 7574-7580 (2013).
  37. Zhao, T., et al. Nanoprobe-enhanced, split aptamer-based electrochemical sandwich assay for ultrasensitive detection of small molecules. Anal Chem. 87 (15), 7712-7719 (2015).
  38. Lou, X., He, L. A. Surface passivation using oligo(ethylene glycol) in ATRP-assisted DNA detection. Sens Actuators,B. 129 (1), 225-230 (2008).

Tags

Keywords: Phthalic Acid EsterDNA AptamerElectrochemical AptasensorHydrophobic MoleculesGroup-specific AptamersLambda ExonucleaseNative PAGE GelMagnetic BeadsPAE Binding Buffer
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Wu, X., Diao, D., Lu, Z., Han, Y., Xu, S., Lou, X. Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor. J. Vis. Exp. (133), e56814, doi:10.3791/56814 (2018).
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