Analytical and Bioanalytical Chemistry

, Volume 408, Issue 3, pp 875–884 | Cite as

β-Conglutin dual aptamers binding distinct aptatopes

  • Miriam Jauset Rubio
  • Markéta Svobodová
  • Teresa Mairal
  • Thomas Schubert
  • Stefan Künne
  • Günter Mayer
  • Ciara K. O’SullivanEmail author
Research Paper


An aptamer was previously selected against the anaphylactic allergen β-conglutin (β-CBA I), which was subsequently truncated to an 11-mer and the affinity improved by two orders of magnitude. The work reported here details the selection and characterisation of a second aptamer (β-CBA II) selected against a second aptatope on the β-conglutin target. The affinity of this second aptamer was similar to that of the 11-mer, and its affinity was confirmed by three different techniques at three independent laboratories. This β-CBA II aptamer in combination with the previously selected β-CBA I was then exploited to a dual-aptamer approach. The specific and simultaneous binding of the dual aptamer (β-CBA I and β-CBA II) to different sites of β-conglutin was confirmed using both microscale thermophoresis and surface plasmon resonance where β-CBA II serves as the primary capturing aptamer and β-CBA I or the truncated β-CBA I (11-mer) as the secondary signalling aptamer, which can be further exploited in enzyme-linked aptamer assays and aptasensors.


β-Conglutin Dual aptamer SELEX Sandwich assay Truncation studies 



This work was supported by funding from the national project RecerCaixa (CO074670 APTALUP).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2015_9179_MOESM1_ESM.pdf (1.9 mb)
ESM 1 (PDF 1971 kb)


  1. 1.
    Adams GP, Schier R (1999) Generating improved single-chain Fv molecules for tumor targeting. J Immunol Methods 231(1–2):249–260CrossRefGoogle Scholar
  2. 2.
    Neri D, Momo M, Prospero T, Winter G (1995) High-affinity antigen binding by chelating recombinant antibodies (CRAbs). J Mol Biol 246(3):367–373CrossRefGoogle Scholar
  3. 3.
    Viti F, Tarli L, Giovannoni L, Zardi L, Neri D (1999) Increased binding affinity and valence of recombinant antibody fragments lead to improved targeting of tumoral angiogenesis. Cancer Res 59(2):347–352Google Scholar
  4. 4.
    Han K, Liang Z, Zhou N (2010) Design strategies for aptamer-based biosensors. Sens (Basel, Switzerland) 10(5):4541–4557CrossRefGoogle Scholar
  5. 5.
    Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346(6287):818–822CrossRefGoogle Scholar
  6. 6.
    Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Sci (New York, NY) 249(4968):505–510CrossRefGoogle Scholar
  7. 7.
    Tasset DM, Kubik MF, Steiner W (1997) Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. J Mol Biol 272(5):688–698CrossRefGoogle Scholar
  8. 8.
    Bai Y, Feng F, Zhao L, Wang C, Wang H, Tian M, Qin J, Duan Y, He X (2013) Aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance sensor for the detection of subnanomolar thrombin. Biosens Bioelectron 47:265–270CrossRefGoogle Scholar
  9. 9.
    Daniel C, Melaine F, Roupioz Y, Livache T, Buhot A (2013) Real time monitoring of thrombin interactions with its aptamers: insights into the sandwich complex formation. Biosens Bioelectron 40(1):186–192CrossRefGoogle Scholar
  10. 10.
    Park JH, Cho YS, Kang S, Lee EJ, Lee GH, Hah SS (2014) A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay. Anal Biochem 462:10–12CrossRefGoogle Scholar
  11. 11.
    Pinto A, Bermudo Redondo MC, Ozalp VC, O'Sullivan CK (2009) Real-time apta-PCR for 20 000-fold improvement in detection limit. Mol BioSyst 5(5):548–553CrossRefGoogle Scholar
  12. 12.
    Romhildt L, Pahlke C, Zorgiebel F, Braun HG, Opitz J, Baraban L, Cuniberti G (2013) Patterned biochemical functionalization improves aptamer-based detection of unlabeled thrombin in a sandwich assay. ACS Appl Mater Interfaces 5(22):12029–12035CrossRefGoogle Scholar
  13. 13.
    Sosic A, Meneghello A, Antognoli A, Cretaio E, Gatto B (2013) Development of a multiplex sandwich aptamer microarray for the detection of VEGF165 and thrombin. Sens (Basel, Switzerland) 13(10):13425–13438CrossRefGoogle Scholar
  14. 14.
    Sosic A, Meneghello A, Cretaio E, Gatto B (2011) Human thrombin detection through a sandwich aptamer microarray: interaction analysis in solution and in solid phase. Sens (Basel, Switzerland) 11(10):9426–9441CrossRefGoogle Scholar
  15. 15.
    Lee J-e, Kim J, Lee S, Kim J, Mah S, Gu M (2013) In-situ on-fabric one-touch colorimetric detection using aptamer-conjugated gold nanoparticles. BioChip J 7(2):180–187CrossRefGoogle Scholar
  16. 16.
    Liu J, Yang X, Wang K, Wang Q, Liu W, Wang D (2013) Solid-phase single molecule biosensing using dual-color colocalization of fluorescent quantum dot nanoprobes. Nanoscale 5(22):11257–11264CrossRefGoogle Scholar
  17. 17.
    Vinkenborg JL, Karnowski N, Famulok M (2011) Aptamers for allosteric regulation. Nat Chem Biol 7(8):519–527CrossRefGoogle Scholar
  18. 18.
    Wang Q, Zhou C, Yang X, Liu L, Wang K (2014) Probing interactions between human lung adenocarcinoma A549 cell and its aptamers at single-molecule resolution. J Mol Recognit JMR 27(11):676–682CrossRefGoogle Scholar
  19. 19.
    Min K, Jo H, Song K, Cho M, Chun Y-S, Jon S, Kim WJ, Ban C (2011) Dual-aptamer-based delivery vehicle of doxorubicin to both PSMA (+) and PSMA (−) prostate cancers. Biomaterials 32(8):2124–2132CrossRefGoogle Scholar
  20. 20.
    Min K, Song KM, Cho M, Chun YS, Shim YB, Ku JK, Ban C (2010) Simultaneous electrochemical detection of both PSMA (+) and PSMA (−) prostate cancer cells using an RNA/peptide dual-aptamer probe. Chem Commun (Camb) 46(30):5566–5568CrossRefGoogle Scholar
  21. 21.
    Jo H, Youn H, Lee S, Ban C (2014) Ultra-effective photothermal therapy for prostate cancer cells using dual aptamer-modified gold nanostars. J Mater Chem B 2(30):4862–4867CrossRefGoogle Scholar
  22. 22.
    Jo H, Her J, Ban C (2015) Dual aptamer-functionalized silica nanoparticles for the highly sensitive detection of breast cancer. Biosens Bioelectron 71:129–136CrossRefGoogle Scholar
  23. 23.
    Fang LX, Huang KJ, Liu Y (2015) Novel electrochemical dual-aptamer-based sandwich biosensor using molybdenum disulfide/carbon aerogel composites and Au nanoparticles for signal amplification. Biosens Bioelectron 71:171–178CrossRefGoogle Scholar
  24. 24.
    Ruslinda AR, Penmatsa V, Ishii Y, Tajima S, Kawarada H (2012) Highly sensitive detection of platelet-derived growth factor on a functionalized diamond surface using aptamer sandwich design. Analyst 137(7):1692–1697CrossRefGoogle Scholar
  25. 25.
    Abbaspour A, Norouz-Sarvestani F, Noori A, Soltani N (2015) Aptamer-conjugated silver nanoparticles for electrochemical dual-aptamer-based sandwich detection of Staphylococcus aureus. Biosens Bioelectron 68:149–155CrossRefGoogle Scholar
  26. 26.
    Hu PP, Liu H, Zhan L, Zheng LL, Huang CZ (2015) Coomassie brilliant blue R-250 as a new surface-enhanced Raman scattering probe for prion protein through a dual-aptamer mechanism. Talanta 139:35–39CrossRefGoogle Scholar
  27. 27.
    Ahmad Raston NH, Gu MB (2015) Highly amplified detection of visceral adipose tissue-derived serpin (vaspin) using a cognate aptamer duo. Biosens Bioelectron 70:261–267CrossRefGoogle Scholar
  28. 28.
    Rinker S, Ke Y, Liu Y, Chhabra R, Yan H (2008) Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding. Nat Nano 3(7):418–422CrossRefGoogle Scholar
  29. 29.
    Zhao J, Zhang Y, Li H, Wen Y, Fan X, Lin F, Tan L, Yao S (2011) Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer–AuNPs–HRP conjugates. Biosens Bioelectron 26(5):2297–2303CrossRefGoogle Scholar
  30. 30.
    Ogawa A, Samoto M, Takahashi K (2000) Soybean allergens and hypoallergenic soybean products. J Nutr Sci Vitaminol 46(6):271–279CrossRefGoogle Scholar
  31. 31.
    Sanchez-Monge R, Lopez-Torrejon G, Pascual CY, Varela J, Martin-Esteban M, Salcedo G (2004) Vicilin and convicilin are potential major allergens from pea. Clin Exp Allergy J Br Soc Allergy Clin Immunol 34(11):1747–1753CrossRefGoogle Scholar
  32. 32.
    Scurlock AM, Burks AW (2004) Peanut allergenicity. Ann Allergy Asthma Immunol Off Publ Am Coll Allergy Asthma Immunol 93(5 Suppl 3):S12–S18CrossRefGoogle Scholar
  33. 33.
    Teuber SS, Sathe SK, Peterson WR, Roux KH (2002) Characterization of the soluble allergenic proteins of cashew nut (Anacardium occidentale L.). J Agric Food Chem 50(22):6543–6549CrossRefGoogle Scholar
  34. 34.
    Shewry PR, Napier JA, Tatham AS (1995) Seed storage proteins: structures and biosynthesis. Plant Cell 7(7):945–956CrossRefGoogle Scholar
  35. 35.
    Holden L, Sletten GB, Lindvik H, Faeste CK, Dooper MM (2008) Characterization of IgE binding to lupin, peanut and almond with sera from lupin-allergic patients. Int Arch Allergy Immunol 146(4):267–276CrossRefGoogle Scholar
  36. 36.
    Magni C, Herndl A, Sironi E, Scarafoni A, Ballabio C, Restani P, Bernardini R, Novembre E, Vierucci A, Duranti M (2005) One- and two-dimensional electrophoretic identification of IgE-binding polypeptides of Lupinus albus and other legume seeds. J Agric Food Chem 53(11):4567–4571CrossRefGoogle Scholar
  37. 37.
    Campbell CP, Yates DH (2010) Lupin allergy: a hidden killer at home, a menace at work; occupational disease due to lupin allergy. Clin Exp Allergy J Br Soc Allergy Clin Immunol 40(10):1467–1472CrossRefGoogle Scholar
  38. 38.
    Koplin JJ, Martin PE, Allen KJ (2011) An update on epidemiology of anaphylaxis in children and adults. Curr Opin Allergy Clin Immunol 11(5):492–496CrossRefGoogle Scholar
  39. 39.
    Sanz ML, de Las Marinas MD, Fernandez J, Gamboa PM (2010) Lupin allergy: a hidden killer in the home. Clin Exp Allergy J Br Soc Allergy Clin Immunol 40(10):1461–1466CrossRefGoogle Scholar
  40. 40.
    Goggin DE, Mir G, Smith WB, Stuckey M, Smith PM (2008) Proteomic analysis of lupin seed proteins to identify conglutin Beta as an allergen, Lup an 1. J Agric Food Chem 56(15):6370–6377CrossRefGoogle Scholar
  41. 41.
    Nadal P, Pinto A, Svobodova M, Canela N, O'Sullivan CK (2012) DNA aptamers against the Lup an 1 food allergen. PLoS One 7(4):e35253CrossRefGoogle Scholar
  42. 42.
    Svobodova M, Mairal T, Nadal P, Bermudo MC, O'Sullivan CK (2014) Ultrasensitive aptamer based detection of beta-conglutin food allergen. Food Chem 165:419–423CrossRefGoogle Scholar
  43. 43.
    Nadal P, Svobodova M, Mairal T, O'Sullivan CK (2013) Probing high-affinity 11-mer DNA aptamer against Lup an 1 (beta-conglutin). Anal Bioanal Chem 405(29):9343–9349CrossRefGoogle Scholar
  44. 44.
    Mairal T, Nadal P, Svobodova M, O'Sullivan CK (2014) FRET-based dimeric aptamer probe for selective and sensitive Lup an 1 allergen detection. Biosens Bioelectron 54:207–210CrossRefGoogle Scholar
  45. 45.
    Svobodova M, Pinto A, Nadal P, OS CK (2012) Comparison of different methods for generation of single-stranded DNA for SELEX processes. Anal Bioanal Chem 404(3):835–842CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Miriam Jauset Rubio
    • 1
  • Markéta Svobodová
    • 1
  • Teresa Mairal
    • 1
  • Thomas Schubert
    • 2
  • Stefan Künne
    • 3
  • Günter Mayer
    • 3
  • Ciara K. O’Sullivan
    • 1
    • 4
    Email author
  1. 1.Nanobiotechnology and Bioanalysis Group, Department of Chemical EngineeringUniversitat Rovira I VirgiliTarragonaSpain
  2. 2.2bind GmbHRegensburgGermany
  3. 3.Life and Medical Sciences InstituteUniversity of BonnBonnGermany
  4. 4.Institució Catalana de Recerca I Estudis AvancatsBarcelonaSpain

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