Generation of Aptamers Against Natural Toxins and Their Application as Biosensors

  • Yuji Morita
  • Daisuke Fujiwara


RNA or ssDNA aptamers, which are capable of binding to target molecules with high affinity and specificity, are selected in vitro from large combinatorial nucleic acid libraries by a process known as systematic evolution of ligands by exponential enrichment (SELEX). Using this SELEX technology, many aptamers have been generated against a wide range of target molecules, including proteins, nucleic acids, small molecules, and whole cells. In this chapter, we described various methods for generating aptamers, including methods that do not require target immobilization. Among these aptamers, ones that are specific for natural toxins, such as mycotoxins, are of great interest to the food industry, as they can used in developing tools (biosensors) for ensuring food safety. We also summarized several aptamer-based detection strategies. Lastly, we described biosensor applications of aptamers for natural toxins.


RNA Aptamer SELEX Aptasensor Natural toxin 



We thank Dr. Yasuyuki Tomita for helpful discussions.


  1. Andrade MA, Lancas FM (2017) Determination of Ochratoxin A in wine by packed in-tube solid phase microextraction followed by high performance liquid chromatography coupled to tandem mass spectrometry. J Chromatogr A 1493:41–48CrossRefGoogle Scholar
  2. Aquino-Jarquin G, Toscano-Garibay JD (2011) RNA aptamer evolution: two decades of SELEction. Int J Mol Sci 12:9155–9171CrossRefGoogle Scholar
  3. Breidbach A (2017) A greener, quick and comprehensive extraction approach for LC-MS of multiple mycotoxins. Toxins 9:E91CrossRefGoogle Scholar
  4. Bunka DH, Stockley PG (2006) Aptamers come of age – at last. Nat Rev Microbiol 4:588–596CrossRefGoogle Scholar
  5. Cerchia L, Duconge F, Pestourie C, Boulay J, Aissouni Y, Gombert K, Tavitian B, de Franciscis V, Libri D (2005) Neutralizing aptamers from whole-cell SELEX inhibit the RET receptor tyrosine kinase. PLoS Biol 3:e123CrossRefGoogle Scholar
  6. Chang TW, Janardhanan P, Mello CM, Singh BR, Cai S (2016) Selection of RNA aptamers against botulinum neurotoxin type A light chain through a non-radioactive approach. Appl Biochem Biotechnol 180:10–25CrossRefGoogle Scholar
  7. Chen X, Huang Y, Duan N, Wu S, Xia Y, Ma X, Zhu C, Jiang Y, Wang Z (2014) Screening and identification of DNA aptamers against T-2 toxin assisted by graphene oxide. J Agric Food Chem 62:10368–10374CrossRefGoogle Scholar
  8. Chiuman W, Li Y (2007) Simple fluorescent sensors engineered with catalytic DNA ‘MgZ’ based on a non-classic allosteric design. PLoS One 2:e1224CrossRefGoogle Scholar
  9. Cho M, Xiao Y, Nie J, Stewart R, Csordas AT, Oh SS, Thomson JA, Soh HT (2010) Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing. Proc Natl Acad Sci USA 107:15373–15378CrossRefGoogle Scholar
  10. Crosby NT (1984) Review of current and future analytical methods for the determination of mycotoxins. Food Addit Contam 1:39–44CrossRefGoogle Scholar
  11. Cruz-Aguado JA, Penner G (2008) Determination of ochratoxin a with a DNA aptamer. J Agric Food Chem 56:10456–10461CrossRefGoogle Scholar
  12. Daniels DA, Chen H, Hicke BJ, Swiderek KM, Gold L (2003) A tenascin-C aptamer identified by tumor cell SELEX: systematic evolution of ligands by exponential enrichment. Proc Natl Acad Sci USA 100:15416–15421CrossRefGoogle Scholar
  13. de Silva C, Walter NG (2009) Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme. RNA 15:76–84CrossRefGoogle Scholar
  14. Dobbelstein M, Shenk T (1995) In vitro selection of RNA ligands for the ribosomal L22 protein associated with Epstein-Barr virus-expressed RNA by using randomized and cDNA-derived RNA libraries. J Virol 69:8027–8034PubMedPubMedCentralGoogle Scholar
  15. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822CrossRefGoogle Scholar
  16. El-Moghazy AY, Soliman EA, Ibrahim HZ, Noguer T, Marty JL, Istamboulie G (2016) Ultra-sensitive biosensor based on genetically engineered acetylcholinesterase immobilized in poly (vinyl alcohol)/Fe-Ni alloy nanocomposite for phosmet detection in olive oil. Food Chem 203:73–78CrossRefGoogle Scholar
  17. Fang X, Tan W (2010) Aptamers generated from cell-SELEX for molecular medicine: a chemical biology approach. Acc Chem Res 43:48–57CrossRefGoogle Scholar
  18. Fedor MJ, Uhlenbeck OC (1992) Kinetics of intermolecular cleavage by hammerhead ribozymes. Biochemistry 31:12042–12054CrossRefGoogle Scholar
  19. Feng C, Dai S, Wang L (2014) Optical aptasensors for quantitative detection of small biomolecules: a review. Biosens Bioelectron 59:64–74CrossRefGoogle Scholar
  20. Forster AC, Symons RH (1987) Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site. Cell 50:9–16CrossRefGoogle Scholar
  21. Frauendorf C, Jaschke A (2001) Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem 9:2521–2524CrossRefGoogle Scholar
  22. Furukawa K, Gu H, Breaker RR (2014) In vitro selection of allosteric ribozymes that sense the bacterial second messenger c-di-GMP. Methods Mol Biol 1111:209–220CrossRefGoogle Scholar
  23. Gu H, Furukawa K, Breaker RR (2012) Engineered allosteric ribozymes that sense the bacterial second messenger cyclic diguanosyl 5′-monophosphate. Anal Chem 84:4935–4941CrossRefGoogle Scholar
  24. Gu H, Duan N, Wu S, Hao L, Xia Y, Ma X, Wang Z (2016) Graphene oxide-assisted non-immobilized SELEX of okdaic acid aptamer and the analytical application of aptasensor. Sci Rep 6:21665CrossRefGoogle Scholar
  25. Guo X, Wen F, Zheng N, Luo Q, Wang H, Wang H, Li S, Wang J (2014) Development of an ultrasensitive aptasensor for the detection of aflatoxin B1. Biosens Bioelectron 56:340–344CrossRefGoogle Scholar
  26. Guo X, Wen F, Zheng N, Li S, Fauconnier ML, Wang J (2016) A qPCR aptasensor for sensitive detection of aflatoxin M1. Anal Bioanal Chem 408:5577–5584CrossRefGoogle Scholar
  27. Ha TH (2015) Recent advances for the detection of Ochratoxin A. Toxins 7:5276–5300CrossRefGoogle Scholar
  28. He J, Liu Y, Fan M, Liu X (2011) Isolation and identification of the DNA aptamer target to acetamiprid. J Agric Food Chem 59:1582–1586CrossRefGoogle Scholar
  29. Hu W, Li X, He G, Zhang Z, Zheng X, Li P, Li CM (2013) Sensitive competitive immunoassay of multiple mycotoxins with non-fouling antigen microarray. Biosens Bioelectron 50:338–344CrossRefGoogle Scholar
  30. Huang CJ, Lin HI, Shiesh SC, Lee GB (2010) Integrated microfluidic system for rapid screening of CRP aptamers utilizing systematic evolution of ligands by exponential enrichment (SELEX). Biosens Bioelectron 25:1761–1766CrossRefGoogle Scholar
  31. Janardhanan P, Mello CM, Singh BR, Lou J, Marks JD, Cai S (2013) RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin. Talanta 117:273–280CrossRefGoogle Scholar
  32. Jenison RD, Gill SC, Pardi A, Polisky B (1994) High-resolution molecular discrimination by RNA. Science 263:1425–1429CrossRefGoogle Scholar
  33. Jenne A, Hartig JS, Piganeau N, Tauer A, Samarsky DA, Green MR, Davies J, Famulok M (2001) Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology. Nat Biotechnol 19:56–61CrossRefGoogle Scholar
  34. Jensen KB, Atkinson BL, Willis MC, Koch TH, Gold L (1995) Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands. Proc Natl Acad Sci USA 92:12220–12224CrossRefGoogle Scholar
  35. Kim HK, Liu J, Li J, Nagraj N, Li M, Pavot CM, Lu Y (2007) Metal-dependent global folding and activity of the 8-17 DNAzyme studied by fluorescence resonance energy transfer. J Am Chem Soc 129:6896–6902CrossRefGoogle Scholar
  36. Klug SJ, Famulok M (1994) All you wanted to know about SELEX. Mol Biol Rep 20:97–107CrossRefGoogle Scholar
  37. Klussmann S, Nolte A, Bald R, Erdmann VA, Furste JP (1996) Mirror-image RNA that binds D-adenosine. Nat Biotechnol 14:1112–1115CrossRefGoogle Scholar
  38. Koizumi M, Soukup GA, Kerr JN, Breaker RR (1999) Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP. Nat Struct Biol 6:1062–1071CrossRefGoogle Scholar
  39. Kulbachinskiy AV (2007) Methods for selection of aptamers to protein targets. Biochem Biokhim 72:1505–1518CrossRefGoogle Scholar
  40. Lai HC, Wang CH, Liou TM, Lee GB (2014) Influenza A virus-specific aptamers screened by using an integrated microfluidic system. Lab Chip 14:2002–2013CrossRefGoogle Scholar
  41. Lipi F, Chen S, Chakravarthy M, Rakesh S, Veedu RN (2016) In vitro evolution of chemically-modified nucleic acid aptamers: pros and cons, and comprehensive selection strategies. RNA Biol 13:1232–1245CrossRefGoogle Scholar
  42. Liu J, Lu Y (2006) Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes. Nat Protoc 1:246–252CrossRefGoogle Scholar
  43. Lou X, Qian J, Xiao Y, Viel L, Gerdon AE, Lagally ET, Atzberger P, Tarasow TM, Heeger AJ, Soh HT (2009) Micromagnetic selection of aptamers in microfluidic channels. Proc Natl Acad Sci USA 106:2989–2994CrossRefGoogle Scholar
  44. Luan Y, Chen J, Li C, Xie G, Fu H, Ma Z, Lu A (2015a) Highly sensitive colorimetric detection of Ochratoxin A by a label-free aptamer and gold nanoparticles. Toxins 7:5377–5385CrossRefGoogle Scholar
  45. Luan Y, Chen Z, Xie G, Chen J, Lu A, Li C, Fu H, Ma Z, Wang J (2015b) Rapid visual detection of Aflatoxin B1 by label-free aptasensor using unmodified gold nanoparticles. J Nanosci Nanotechnol 15:1357–1361CrossRefGoogle Scholar
  46. Malhotra S, Pandey AK, Rajput YS, Sharma R (2014) Selection of aptamers for aflatoxin M1 and their characterization. J Mol Recognit 27:493–500CrossRefGoogle Scholar
  47. Marton S, Cleto F, Krieger MA, Cardoso J (2016) Isolation of an aptamer that binds specifically to E. coli. PLoS One 11:e0153637CrossRefGoogle Scholar
  48. Mayer G, Ahmed MS, Dolf A, Endl E, Knolle PA, Famulok M (2010) Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures. Nat Protoc 5:1993–2004CrossRefGoogle Scholar
  49. McKeague M, Bradley CR, De Girolamo A, Visconti A, Miller JD, Derosa MC (2010) Screening and initial binding assessment of fumonisin b(1) aptamers. Int J Mol Sci 11:4864–4881CrossRefGoogle Scholar
  50. Mendonsa SD, Bowser MT (2004) In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis. Anal Chem 76:5387–5392CrossRefGoogle Scholar
  51. Morris KN, Jensen KB, Julin CM, Weil M, Gold L (1998) High affinity ligands from in vitro selection: complex targets. Proc Natl Acad Sci USA 95:2902–2907CrossRefGoogle Scholar
  52. Nguyen VT, Kwon YS, Kim JH, Gu MB (2014) Multiple GO-SELEX for efficient screening of flexible aptamers. Chem Commun 50:10513–10516CrossRefGoogle Scholar
  53. Nutiu R, Li Y (2003) Structure-switching signaling aptamers. J Am Chem Soc 125:4771–4778CrossRefGoogle Scholar
  54. Nutiu R, Li Y (2005a) Aptamers with fluorescence-signaling properties. Methods 37:16–25CrossRefGoogle Scholar
  55. Nutiu R, Li Y (2005b) In vitro selection of structure-switching signaling aptamers. Angew Chem Int Ed 44:1061–1065CrossRefGoogle Scholar
  56. Ogihara K, Savory N, Abe K, Yoshida W, Asahi M, Kamohara S, Ikebukuro K (2015) DNA aptamers against the Cry j 2 allergen of Japanese cedar pollen for biosensing applications. Biosens Bioelectron 63:159–165CrossRefGoogle Scholar
  57. Oh SS, Qian J, Lou X, Zhang Y, Xiao Y, Soh HT (2009) Generation of highly specific aptamers via micromagnetic selection. Anal Chem 81:5490–5495CrossRefGoogle Scholar
  58. Park JW, Tatavarty R, Kim DW, Jung HT, Gu MB (2012) Immobilization-free screening of aptamers assisted by graphene oxide. Chem Commun 48:2071–2073CrossRefGoogle Scholar
  59. Park JW, Jin Lee S, Choi EJ, Kim J, Song JY, Bock Gu M (2014) An ultra-sensitive detection of a whole virus using dual aptamers developed by immobilization-free screening. Biosens Bioelectron 51:324–329CrossRefGoogle Scholar
  60. Pelossof G, Tel-Vered R, Elbaz J, Willner I (2010) Amplified biosensing using the horseradish peroxidase-mimicking DNAzyme as an electrocatalyst. Anal Chem 82:4396–4402CrossRefGoogle Scholar
  61. Raddatz MS, Dolf A, Endl E, Knolle P, Famulok M, Mayer G (2008) Enrichment of cell-targeting and population-specific aptamers by fluorescence-activated cell sorting. Angew Chem Int Ed 47:5190–5193CrossRefGoogle Scholar
  62. Rhouati A, Yang C, Hayat A, Marty JL (2013) Aptamers: a promising tool for ochratoxin A detection in food analysis. Toxins 5:1988–2008CrossRefGoogle Scholar
  63. Richard JL (2007) Some major mycotoxins and their mycotoxicoses – an overview. Int J Food Microbiol 119:3–10CrossRefGoogle Scholar
  64. Robertson DL, Joyce GF (1990) Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 344:467–468CrossRefGoogle Scholar
  65. Roll R, Matthiaschk G, Korte A (1990) Embryotoxicity and mutagenicity of mycotoxins. J Environ Pathol Toxicol Oncol 10:1–7PubMedGoogle Scholar
  66. Sassanfar M, Szostak JW (1993) An RNA motif that binds ATP. Nature 364:550–553CrossRefGoogle Scholar
  67. Seok Y, Byun JY, Shim WB, Kim MG (2015) A structure-switchable aptasensor for aflatoxin B1 detection based on assembly of an aptamer/split DNAzyme. Anal Chim Acta 886:182–187CrossRefGoogle Scholar
  68. Shangguan D, Li Y, Tang Z, Cao ZC, Chen HW, Mallikaratchy P, Sefah K, Yang CJ, Tan W (2006) Aptamers evolved from live cells as effective molecular probes for cancer study. Proc Natl Acad Sci USA 103:11838–11843CrossRefGoogle Scholar
  69. Sharma A, Catanante G, Hayat A, Istamboulie G, Ben Rejeb I, Bhand S, Marty JL (2016) Development of structure switching aptamer assay for detection of aflatoxin M1 in milk sample. Talanta 158:35–41CrossRefGoogle Scholar
  70. Singh KK, Parwaresch R, Krupp G (1999) Rapid kinetic characterization of hammerhead ribozymes by real-time monitoring of fluorescence resonance energy transfer (FRET). RNA 5:1348–1356CrossRefGoogle Scholar
  71. Soh JH, Lin Y, Rana S, Ying JY, Stevens MM (2015) Colorimetric detection of small molecules in complex matrixes via target-mediated growth of aptamer-functionalized gold nanoparticles. Anal Chem 87:7644–7652CrossRefGoogle Scholar
  72. Soukup GA, Breaker RR (1999) Engineering precision RNA molecular switches. Proc Natl Acad Sci USA 96:3584–3589CrossRefGoogle Scholar
  73. Soukup GA, Emilsson GA, Breaker RR (2000) Altering molecular recognition of RNA aptamers by allosteric selection. J Mol Biol 298:623–632CrossRefGoogle Scholar
  74. Stoltenburg R, Reinemann C, Strehlitz B (2005) FluMag-SELEX as an advantageous method for DNA aptamer selection. Anal Bioanal Chem 383:83–91CrossRefGoogle Scholar
  75. Stoltenburg R, Reinemann C, Strehlitz B (2007) SELEX – a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng 24:381–403CrossRefGoogle Scholar
  76. Tang Z, Shangguan D, Wang K, Shi H, Sefah K, Mallikratchy P, Chen HW, Li Y, Tan W (2007) Selection of aptamers for molecular recognition and characterization of cancer cells. Anal Chem 79:4900–4907CrossRefGoogle Scholar
  77. Tomita Y, Morita Y, Suga H, Fujiwara D (2016) DNA module platform for developing colorimetric aptamer sensors. BioTechniques 60:285–292CrossRefGoogle Scholar
  78. Travascio P, Li Y, Sen D (1998) DNA-enhanced peroxidase activity of a DNA-aptamer-hemin complex. Chem Biol 5:505–517CrossRefGoogle Scholar
  79. Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510CrossRefGoogle Scholar
  80. Turner NW, Subrahmanyam S, Piletsky SA (2009) Analytical methods for determination of mycotoxins: a review. Anal Chim Acta 632:168–180CrossRefGoogle Scholar
  81. Vater A, Klussmann S (2003) Toward third-generation aptamers: Spiegelmers and their therapeutic prospects. Curr Opin Drug Discov Dev 6:253–261Google Scholar
  82. Wang L, Liu X, Zhang Q, Zhang C, Liu Y, Tu K, Tu J (2012) Selection of DNA aptamers that bind to four organophosphorus pesticides. Biotechnol Lett 34:869–874CrossRefGoogle Scholar
  83. Wang B, Chen Y, Wu Y, Weng B, Liu Y, Lu Z, Li CM, Yu C (2016) Aptamer induced assembly of fluorescent nitrogen-doped carbon dots on gold nanoparticles for sensitive detection of AFB1. Biosens Bioelectron 78:23–30CrossRefGoogle Scholar
  84. Watanabe M, Shimizu H (2005) Detection of patulin in apple juices marketed in the Tohoku district, Japan. J Food Protect 68:610–612CrossRefGoogle Scholar
  85. Wu ZS, Lu H, Liu X, Hu R, Zhou H, Shen G, Yu RQ (2010) Inhibitory effect of target binding on hairpin aptamer sticky-end pairing-induced gold nanoparticle assembly for light-up colorimetric protein assay. Anal Chem 82:3890–3898CrossRefGoogle Scholar
  86. Wu S, Duan N, Li X, Tan G, Ma X, Xia Y, Wang Z, Wang H (2013) Homogenous detection of fumonisin B(1) with a molecular beacon based on fluorescence resonance energy transfer between NaYF4: Yb, Ho upconversion nanoparticles and gold nanoparticles. Talanta 116:611–618CrossRefGoogle Scholar
  87. Xiang Y, Wang Z, Xing H, Wong NY, Lu Y (2010) Label-free fluorescent functional DNA sensors using unmodified DNA: a vacant site approach. Anal Chem 82:4122–4129CrossRefGoogle Scholar
  88. Xu Y, Yang X, Wang E (2010) Review: aptamers in microfluidic chips. Anal Chim Acta 683:12–20CrossRefGoogle Scholar
  89. Xu L, Zhang Z, Zhang Q, Li P (2016) Mycotoxin determination in foods using advanced sensors based on antibodies or aptamers. Toxins 8:E239CrossRefGoogle Scholar
  90. Yang J, Bowser MT (2013) Capillary electrophoresis-SELEX selection of catalytic DNA aptamers for a small-molecule porphyrin target. Anal Chem 85:1525–1530CrossRefGoogle Scholar
  91. Yang Y, Yang D, Schluesener HJ, Zhang Z (2007) Advances in SELEX and application of aptamers in the central nervous system. Biomol Eng 24:583–592CrossRefGoogle Scholar
  92. Yang C, Wang Y, Marty JL, Yang X (2011) Aptamer-based colorimetric biosensing of Ochratoxin A using unmodified gold nanoparticles indicator. Biosens Bioelectron 26:2724–2727CrossRefGoogle Scholar
  93. Zhao W, Chiuman W, Brook MA, Li Y (2007) Simple and rapid colorimetric biosensors based on DNA aptamer and noncrosslinking gold nanoparticle aggregation. Chembiochem 8:727–731CrossRefGoogle Scholar
  94. Zhao W, Brook MA, Li Y (2008) Design of gold nanoparticle-based colorimetric biosensing assays. Chembiochem 9:2363–2371CrossRefGoogle Scholar
  95. Zivarts M, Liu Y, Breaker RR (2005) Engineered allosteric ribozymes that respond to specific divalent metal ions. Nucleic Acids Res 33:622–631CrossRefGoogle Scholar
  96. Zollner P, Mayer-Helm B (2006) Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography-atmospheric pressure ionisation mass spectrometry. J Chromatogr A 1136:123–169CrossRefGoogle Scholar

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© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Research Laboratories for Health Science & Food Technologies, Research and Development DivisionKirin Company LimitedYokohamaJapan
  2. 2.Central Laboratories for Key Technologies, Research and Development DivisionKirin Company LimitedYokohamaJapan

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