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.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Adams GP, Schier R (1999) Generating improved single-chain Fv molecules for tumor targeting. J Immunol Methods 231(1–2):249–260
Neri D, Momo M, Prospero T, Winter G (1995) High-affinity antigen binding by chelating recombinant antibodies (CRAbs). J Mol Biol 246(3):367–373
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):347–352
Han K, Liang Z, Zhou N (2010) Design strategies for aptamer-based biosensors. Sens (Basel, Switzerland) 10(5):4541–4557
Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346(6287):818–822
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):505–510
Tasset DM, Kubik MF, Steiner W (1997) Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. J Mol Biol 272(5):688–698
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 47:265–270
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):186–192
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 462:10–12
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):548–553
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):12029–12035
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):13425–13438
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):9426–9441
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):180–187
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):11257–11264
Vinkenborg JL, Karnowski N, Famulok M (2011) Aptamers for allosteric regulation. Nat Chem Biol 7(8):519–527
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):676–682
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):2124–2132
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):5566–5568
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):4862–4867
Jo H, Her J, Ban C (2015) Dual aptamer-functionalized silica nanoparticles for the highly sensitive detection of breast cancer. Biosens Bioelectron 71:129–136
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 71:171–178
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):1692–1697
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 68:149–155
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 139:35–39
Ahmad Raston NH, Gu MB (2015) Highly amplified detection of visceral adipose tissue-derived serpin (vaspin) using a cognate aptamer duo. Biosens Bioelectron 70:261–267
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):418–422
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):2297–2303
Ogawa A, Samoto M, Takahashi K (2000) Soybean allergens and hypoallergenic soybean products. J Nutr Sci Vitaminol 46(6):271–279
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):1747–1753
Scurlock AM, Burks AW (2004) Peanut allergenicity. Ann Allergy Asthma Immunol Off Publ Am Coll Allergy Asthma Immunol 93(5 Suppl 3):S12–S18
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):6543–6549
Shewry PR, Napier JA, Tatham AS (1995) Seed storage proteins: structures and biosynthesis. Plant Cell 7(7):945–956
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):267–276
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):4567–4571
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):1467–1472
Koplin JJ, Martin PE, Allen KJ (2011) An update on epidemiology of anaphylaxis in children and adults. Curr Opin Allergy Clin Immunol 11(5):492–496
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):1461–1466
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):6370–6377
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):e35253
Svobodova M, Mairal T, Nadal P, Bermudo MC, O'Sullivan CK (2014) Ultrasensitive aptamer based detection of beta-conglutin food allergen. Food Chem 165:419–423
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):9343–9349
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 54:207–210
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):835–842
This work was supported by funding from the national project RecerCaixa (CO074670 APTALUP).
Conflict of interest
The authors declare that they have no competing interests.
Electronic supplementary material
Below is the link to the electronic supplementary material.
(PDF 1971 kb)
About this article
Cite this article
Jauset Rubio, M., Svobodová, M., Mairal, T. et al. β-Conglutin dual aptamers binding distinct aptatopes. Anal Bioanal Chem 408, 875–884 (2016). https://doi.org/10.1007/s00216-015-9179-z
- Dual aptamer
- Sandwich assay
- Truncation studies