Biosensors and Bioelectronics 54 (2014) 207–210
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
FRET-based dimeric aptamer probe for selective and sensitive Lup an 1 allergen detection T. Mairal a, P. Nadal a, M. Svobodova a,n, C.K. O'Sullivan a,b,nn a b
Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira i Virgili, 43007, Tarragona, Spain Institució Catalana de Recerca I Estudis Avancats, Passeig Lluís Companys 23, 08010 Barcelona, Spain
art ic l e i nf o
a b s t r a c t
Article history: Received 13 August 2013 Received in revised form 14 October 2013 Accepted 22 October 2013 Available online 7 November 2013
A sensitive method for the rapid and sensitive detection of the anaphylactic food allergen Lup an 1 (β-conglutin) exploiting fluorescence resonance energy transfer (FRET) has been developed. A high affinity dimeric form of a truncated 11-mer aptamer against β-conglutin was used, with each monomeric aptamer being flanked by donor/acceptor moieties. The dimeric form in the absence of target yields fluorescence emission due to the FRET from the excited fluorophore to the proximal second fluorophore. However, upon addition of β-conglutin, the specific interaction induces a change in the bi-aptameric structure resulting in an increase in fluorescence emission. The method is highly specific and sensitive, with a detection limit of 150 pM, providing an effective tool for the direct detection of the toxic β-conglutin subunit in foodstuffs in just 1 min at room temperature. & 2013 Elsevier B.V. All rights reserved.
Keywords: FRET 11 mer β-conglutin aptamer Lup an 1 allergen
1. Introduction Lupin is an herbaceous plant of the leguminous family. This plant belongs to the genus Lupinus, which includes more than 450 species. Lupin seeds have been used as human food and animal feed since ancient times (Ballester et al., 1984) and lupin products have been demonstrated to have an added value over numerous bakery products and have advantages in comparison to other legumes like soy (Erbas et al., 2005). Biological activities have been attributed to lupin proteins, including plasma cholesterol and triglyceride lowering effects (Sirtori et al., 2004), and anti-hypertensive properties (Pilvi et al., 2006). However, a number of severe food allergy reactions to lupin have been reported (Parisot et al., 2001, Jappe and Vieth, 2010). Lupin allergy apparently arises by either primary sensization (Novembre et al., 1999; Smith et al., 2004; Holden et al., 2005) or clinical cross-reactivity in individuals who are allergic to peanut (Parisot et al., 2001; Foss et al., 2006; Faeste and Namork, 2010). These cross-allergic clinical reactions of peanut to other members of the leguminous family, such as soy, peas, beans, and lentils, occur in about 5% of peanut-allergic individuals but were found in 68% of those with lupin allergy (Faeste et al., 2004). Lupin flour and lupin products thus require mandatory advisory labeling
n
Corresponding author. Corresponding author at: Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira i Virgili, 43007, Tarragona, Spain. Fax: þ34 977 559621. E-mail addresses: [email protected] (M. Svobodova), [email protected], [email protected] (C.K. O'Sullivan). nn
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.070
on foodstuff sold in the European Union (2006/142/EC, 2006), and recently the International Union of Immunological Societies (IUIS) allergen nomenclature subcommittee designated β-conglutin as the Lup an 1 allergen (Goggin et al., 2008). There are several techniques used to detect lupin allergens, and the majority is based on antibodies used in Enzyme Linked Immunosorbent Assay (ELISA) kits. The currently available commercial ELISAs exploit polyclonal antibodies that are not specific to each of the conglutin subunits and reports in the literature detail only monoclonal IgG antibodies against α-conglutin and IgM antibodies against β-conglutin (Dooper et al., 2007; Holden et al., 2007), and there are no reports or commercial ELISAs for the specific detection of β-conglutin. Aptamers are DNA or RNA oligonucleotides that bind to their cognate target with high affinity and specificity and are selected through a SELEX (Systematic Evolution of Ligands by Exponential Enrichment; Ellington and Szostak, 1990; Robertson and Joyce, 1990; Tuerk and Gold, 1990) from a randomly generated population of sequences for their ability to bind a desired molecular target. The specific ability of aptamers, which undergo significant conformational changes upon target binding, offers enhanced flexibility in the design of novel biosensors, where aptamer–target recognition is transduced into a detectable optical/electrochemical signal (Nutiu and Li, 2005; Yang et al., 2005; He et al., 2005; Nutiu and Li, 2004; Rupcich et al., 2006a, 2006b; Katilius et al., 2006; White and Holcombe, 2007; Li et al., 2008). Taking advantage of the ligand induced conformational changes of aptamers, dualfluorophore-labeled aptamers have been developed to give targetdependent fluorescence changes through Fluorescence Resonance Energy Transfer (FRET; Ueyama et al., 2002; Wang et al., 2005),
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T. Mairal et al. / Biosensors and Bioelectronics 54 (2014) 207–210
the radiation-free transfer of energy from an excited donor dye to a suitable acceptor dye, a physical process that depends on spectral overlap and correct dipole alignment of the two dyes. We have recently reported on the selection of an aptamer against β-conglutin, the lupin subunit that has been identified by the IUIS as the Lup an 1 allergen (Nadal et al., 2012). This selected aptamer was subsequently truncated from a original 93 mer with KD 3.6 10 7 to a 11 mer with KD of 1.7 10 9 (Nadal et al., 2013). This truncated 11 mer is guanine rich and predicted to fold into G-quadruplex structures, composed of stacked guanine tetrads, which are stabilized by Hoogsteen-type hydrogen bonds between the guanines and by interactions with cations located between the tetrads. Indeed NMR confirmed the presence of a quadruplex structure within the truncated 11 mer and further studies with RP/HPLC showed this 11-mer quadruplex to have a retention time notably longer than expected, which was attributed to the presence of a dimeric structure (Nadal, 2012). The majority of dimeric based aptamer assays previously reported are based on dimeric structures constructed with different linkers with the aim of increasing affinity in comparison to the use of a simple monomer (Davis et al., 1996; Ringquist and Parma, 1998; Hasegawa et al., 2008; Boyacioglu et al., 2013). The truncated 11-mer β-conglutin aptamer reported here forms a dimeric structure naturally without the use of any linker similar to previous reports of cyclic oligonucleotides that form dimeric quadruplex structures, having a profound influence on the global topology and stability of the structure (Casals et al., 2012). The aim of this work was the use of a truncated 11-mer aptamer, which exists naturally in a dimeric form, to construct dimeric aptamer FRET probe for the detection of β-conglutin. In the work reported here, a sensitive method for rapid Lup an 1 allergen detection using FRET detection based on dimeric aptamer probe has been developed. A truncated 11-mer aptamer was flanked on either the 5′ or 3′ with a donor dye, as well as being flanked on either the 5′ or 3′ end with an acceptor dye to construct dimeric FRET-based probes. Following addition of β-conglutin (the Lup an 1 anaphylatic allergen) the change in fluorescence signal was monitored and specificity of the induced change in FRET demonstrated using control proteins. This is the first report on the use of the naturally occurring dimeric form of the 11-mer β-conglutin aptamer for the detection of β-conglutin. 2. Material and methods 2.1. Material Lyophilized fluorophore labeled oligonucleotides (donor 1 (D1)) 5′-ggt ggg ggt gg AF488-3′; donor 2 (D2) 5′-AF488 ggt ggg ggt gg-3′; acceptor 1 (A1) 5′-ggt ggg ggt gg AF555-3′; and acceptor 2 (A2) 5′-AF555 ggt ggg ggt gg-3′ were synthesized and purified by reverse phase high-performance liquid chromatography by Ella Biotech GmbH (Martinsried, Germany). Proteins from Lupinus albus seeds were extracted, purified and characterized as previously described (Nadal et al., 2011), obtaining a pure isolate of α, β, γ and δ conglutins. The binding buffer was phosphate buffered saline (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.4) containing 1.5 mM of MgCl2. All solutions were prepared in high purity water obtained from a Milli-Q RG system (Spain) and passed through a filter (0.45 nm). 2.2. Formation of FRET based dimeric aptamer probe The truncated 11-mer aptamer was flanked on either the 5′ or the 3′ end with Alexa Fluor 488 (λEX ¼492 nm, λEM ¼ 519 nm), as well as on either the 5′ or 3′-end with the Alexa Fluor 555 (λEX ¼533 nm, λEM ¼568 nm). The Alexa Fluor 488 labeled oligonucleotide (100 nM) was mixed with the Alexa Fluor 555 labeled
oligonucleotide (100 nM) in order to form dimeric aptamer FRET probe. FRET takes place as the donor Alexa Fluor 488 is excited at 492 nm and emits at 519 nm, exciting the Alexa Fluor 555, with subsequent emission at 568 nm. All fluorescent measurements were performed in a Cary Fluorimeter at 22 1C constant temperature. 2.3. FRET based aptamer detection of
β-conglutin
β-conglutin protein (0–200 nM) was mixed with the previously prepared FRET based dimeric aptamer probe (100 nM) and incubated together at 4 1C, 22 1C and 37 1C for 1 and 15 min in binding buffer of total volume 120 ml. Changes in FRET decay, following the addition of β-conglutin, were measured through a variable emission scan (500–700) at a fixed excitation wavelength (492 nm). 2.4. Detection of
β-conglutin from lupin protein extract
For preparation of lupin protein extract, 1 g of lupine flour was defatted with n-hexane, then centrifuged at 6000 g for 10 min at room temperature, and the pellet was dried under vacuum for 48 h. Proteins were extracted from the dried pellet using 100 mM Tris-HCl (pH ¼7.5) containing 10% NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM of ethylene glycol tetraacetic acid (EGTA) and centrifuged at 30,000 g for 10 min at room temperature. The pellet containing the prolamine fraction was removed and the supernatant containing the globulin fractions was stored at 20 1C. The protein concentrations of the lupin extract was determined with a BCA assay using BSA as a standard protein. The protein extracts were diluted in B&W buffer to obtain different dilutions and used in detection studies as described above.
3. Results and discussion 3.1. FRET based dimeric aptamer probe A series of experiments combining different FRET pairs (D1A1, D1A2, D2A1, D2A2) were carried out in order to confirm a dimeric structure of 11-aptamer and to elucidate the favoured FRET couple. The highest FRET was observed for couples D1A2 (5′–3′:3′–5′) and D2A2 (5′–3′:5′–3′), obtaining very similar results (Fig. 1A). Ongoing work to elucidate the structure of the dimeric aptamer using circular dichroism suggests that the 11-mer forms different dimeric structures of both anti-parallel and parallel binding, which is clearly observed in the results reported here. 3.2. FRET detection of
β-conglutin
For the detection of β-conglutin using labeled dimeric oligonucleotide probe, different concentrations β-conglutin was mixed with the oligonucleotide probe solution. It was found that FRET occurs (with corresponding emission at 568 nm) on increasing the concentration of β-conglutin (Fig. 1B), which indicates that the designed dimeric aptamer probes can be used for β-conglutin sensing. The fluorescence intensity of the labeled oligonucleotide probes induced by the presence of β-conglutin was monitored. In the absence of β-conglutin there is a fluorescence emission at 568 nm, but with increasing concentration of β-conglutin, the fluorescence intensity gradually increases. In Fig. 2(A) the relationship of the fluorescence change of the aptamer probe and β-conglutin concentration for both the D1A2 and D2A2 dimer couples can be observed and for both couples the fluorescence intensity increased linearly over the β-conglutin concentration range from 5 10 9 to 20 10 9 M, (r2 ¼ 0.99). The detection limit (L.O.D.) was estimated to be 150 pM and 158 pM for the D1A2 and
T. Mairal et al. / Biosensors and Bioelectronics 54 (2014) 207–210
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Fig. 1. (A) Truncated 11-mer aptamer flanked on either the 5′ or 3′ end with the Alexa Fluor 488 or Alexa Fluor 555 and increments in intensity at 568 nm for the different FRET couples (Δ intensity ¼λEX of FRET couples λEX of corresponding acceptor). (B) FRET effect produced by the combination of donor 1 and acceptor 2 before and after the addition of β-conglutin.
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Fig. 2. (A) The standard curves of the fluorescence change of the D1A2 (i) and D2A2 (ii) aptamer probe and β-conglutin concentration ranging from 0 to 200 nM. Data obtained using an excitation 492 nm and emission at 568 nm. (B) The specificity of FRET detection for β-conglutin allergen using D1A2 (i) and D2A2 (ii) aptamer probe. Serial dilutions (0–200 nM) of each of the lupin conglutin subunits were used for cross-reactivity studies. The error bars represent the standard deviation of 3 repetitions.
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4. Conclusions Dimeric aptamer FRET probes for the detection of β-conglutin were developed. These probes offered an effective signal transduction method for the rapid and quantitative recognition of β-conglutin, requiring less than a minute incubation at room temperature. The FRET probes were demonstrated to be highly specific to the β-conglutin target as well as being very sensitive, achieving a detection limit of E150 pM, which is markedly lower than the LOD of E3 nM currently achievable with antibody based ELISAs. Acknowledgments This work was supported by funding from the National Project RecerCaixa (CO074670 APTALUP). References Fig. 3. The effect of temperature and incubation time on the developed FRET based dimeric aptamer assay.
D2A2 dimers, respectively, based on 5 repetitions, 3 orders of magnitude better than in case of S 40 β-conglutin aptamer before its truncation, where the L.O.D. obtained by competitive ELONA was 153 nM (Nadal, 2012).
3.3. Cross-reactivity studies with other lupin conglutins The selectivity of the dimeric probes to detect β-conglutin was evaluated by observing whether there was any interaction with other lupin conglutins, namely the α, γ, and δ conglutin subunits (Fig. 2B). In the presence of β-conglutin, fluorescence emission increased, but for both dimeric probes (D1A2, D2A2) no fluorescence changes were observed when other lupin conglutins were mixed with the aptamer probe, demonstrating the excellent selectivity of the labeled dimeric oligonucleotide probe. The parameters that can affect the response, such as aptamer concentration, incubation time, temperature and pH, were optimized. Room temperature (22 1C) and neutral pH of commonly used buffers (7.4) were optimum, which can be attributed to the fact that these were the conditions used in the SELEX process for selection of the aptamer. The effect of aptamer concentration on the response was studied from 0 to 300 nM (data not shown) and an increasing response with increasing aptamer concentrations was observed; 100 nM was the lowest concentration that could be used for the detection of a change in signal upon addition of the β-conglutin and as the objective of the work was to achieve as low a detection limit as possible 100 nM of aptamer was used. The effect of incubation time was also studied and the maximum response was observed after just 1 min incubation and no increment in response was observed with increasing incubation times (Fig. 3), highlighting markedly improved assay performance as compared with previous reports of aptamer based assays that typically require 10–30 min to achieve maximum binding (Zhu et al., 2012; Yildirim et al., 2012; Bai et al., 2012). To test if the developed method is workable in a realistic sample, the detection of β-conglutin allergen directly from lupin protein extract was studied. The amount of β-conglutin in lupin protein extract was determined by analyzing 3 different protein lupin extracts and interpolated to the calibration curve of β-conglutin. The concentration of β-conglutin in 100 μg/ml of lupin protein extract was observed to be 2279 μg/ml. These results demonstrate the viability of this method for the direct detection of β-conglutin in real samples.
Ballester, D., Zacarías, I., García, E., Yánez, E., 1984. J. Food Sci. 49 (1), 14–16. Bai, H., Wang, R., Hargis, B., Lu, H., Li, Y., 2012. Sensors (Basel) 12 (9), 12506–12518. Boyacioglu, O., Stuart, C.H., et al., 2013. Mol. Ther. Nucleic Acids 2. (e107). Casals, J., Viladoms, J., Pedroso, E., Gonzáles, C., 2012. J. Nucleic Acids , http://dx.doi. org/10.4061/2010/468017. Davis, K.A., Abrams, B., et al., 1996. Nucleic Acid Res. 23 (4), 702–706. Dooper, M.M., Holden, L., Faeste, C.K., Thompson, K.M., Egaas, E., 2007. Int. Arch. Allergy Immunol. 143 (1), 49–58. Ellington, A.D., Szostak, J.W., 1990. Nature 346 (6287), 818–822. Erbas, M., Certel, M., Uslu, M.K., 2005. Food Chem. 89 (3). (5-5). Faeste, C., Namork, E., 2010. Food Anal. Methods 3 (4), 357–362. Faeste, C.K., Lovik, M., Wiker, H.G., Egaas, E., 2004. Int. Arch. Allergy Immunol. 135 (1), 36–39. Foss, N., Duranti, M., Magni, C., Frokiaer, H., 2006. Int. Arch. Allergy Immunol. 141 (2), 141–150. Goggin, D.E., Mir, G., Smith, W.B., Stuckey, M., Smith, P.M., 2008. J. Agric. Food Chem. 56 (15), 6370–6377. Hasegawa, H., Taira, K.-i., et al., 2008. Sensors 8 (2), 1090–1098. He, F., Tang, Y., Wang, S., Li, Y., Zhu, D., 2005. J. Am. Chem. Soc. 127 (35), 12343–12346. Holden, L., Faeste, C.K., Egaas, E., 2005. J. Agric. Food Chem. 53 (15), 5866–5871. Holden, L., Moen, L.H., Sletten, G.B., Dooper, M.M., 2007. J. Agric. Food Chem. 55 (7), 2536–2542. Jappe, U., Vieths, S., 2010. Mol. Nutr. Food Res. 54 (1), 113–126. Katilius, E., Katiliene, Z., Woodbury, N.W., 2006. Anal. Chem. 78 (18), 6484–6489. Li, W., Yang, X., Wang, K., Tan, W., Li, H., Ma, C., 2008. Talanta 75 (3), 770–774. Nadal, P., Canela, N., Katakis, I., O'Sullivan, C.K., 2011. J. Agric. Food Chem. 59 (6), 2752–2758. Nadal, P., Pinto, A., Svobodova, M., Canela, N., O'Sullivan, C.K., 2012. PLoS One 7 (4), e35253. Nadal, P., 2012. Selection, Characterization and Analytical Application of DNA Aptamer Against the Anaphylactic Toxic Alergen, β -conglutin, Lup an 1 (Ph.D. thesis). Nadal, P., Svobodova, M., Mairal, T., O'Sullivan, C.K., 2013. Anal. Bioanal. Chem. , http://dx.doi.org/10.1007/s00216-013-7385-0. Novembre, E., Moriondo, M., Bernardini, R., Azzari, C., Rossi, M.E., Vierucci, A., 1999. J. Allergy Clin. Immunol. 103 (6), 1214–1216. Nutiu, R., Li, Y., 2004. Chemistry 10 (8), 1868–1876. Nutiu, R., Li, Y., 2005. Angew. Chem. Int. Ed. Engl. 44 (34), 5464–5467. Parisot, L., Aparicio, C., Moneret-Vautrin, D.A., Guerin, L., 2001. Allergy 56 (9), 918–919. Pilvi, T.K., Jauhiainen, T., Cheng, Z.J., Mervaala, E.M., Vapaatalo, H., Korpela, R., 2006. J. Physiol. Pharmacol. 57 (2), 167–176. Ringquist, S., Parma, D., 1998. Cytometry 33 (4), 394–405. Robertson, D.L., Joyce, G.F., 1990. Nature 344 (6265), 467–468. Rupcich, N., Chiuman, W., Nutiu, R., Mei, S., Flora, K.K., Li, Y., Brennan, J.D., 2006a. J. Am. Chem. Soc. 128 (3), 780–790. Rupcich, N., Nutiu, R., Li, Y., Brennan, J.D., 2006b. Angew. Chem. Int. Ed. Engl. 45 (20), 3295–3299. Sirtori, C.R., Lovati, M.R., Manzoni, C., Castiglioni, S., Duranti, M., Magni, C., Morandi, S., D'Agostina, A., Arnoldi, A., 2004. J. Nutr. 134 (1), 18–23. Smith, W.B., Gillis, D., Kette, F.E., 2004. Med. J. Aust. 181 (4), 219–220. Tuerk, C., Gold, L., 1990. Science 249 (4968), 505–510. Ueyama, H., Takagi, M., Takenaka, S., 2002. J. Am. Chem. Soc. 124 (48), 14286–14287. Wang, J., Li, T., Guo, X., Lu, Z., 2005. Nucleic Acids Res. 33 (2), e23. White, B.R., Holcombe, J.A., 2007. Talanta 71 (5), 2015–2020. Yang, C.J., Jockusch, S., Vicens, M., Turro, N.J., Tan, W., 2005. Proc. Natl .Acad. Sci. USA 102 (48), 17278–17283. Yildirim, N., Long, F., Gao, C., He, M., Shi, H.C., Gu, A.Z., 2012. Environ. Sci. Technol. 46 (6), 3288–3294. Zhu, Y., Chandra, P., Song, K.M., Ban, C., Shim, Y.B., 2012. Biosens. Bioelectron. 36 (1), 29–34.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
FRET-based dimeric aptamer probe for selective and sensitive Lup an 1 allergen detection T. Mairal a, P. Nadal a, M. Svobodova a,n, C.K. O'Sullivan a,b,nn a b
Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira i Virgili, 43007, Tarragona, Spain Institució Catalana de Recerca I Estudis Avancats, Passeig Lluís Companys 23, 08010 Barcelona, Spain
art ic l e i nf o
a b s t r a c t
Article history: Received 13 August 2013 Received in revised form 14 October 2013 Accepted 22 October 2013 Available online 7 November 2013
A sensitive method for the rapid and sensitive detection of the anaphylactic food allergen Lup an 1 (β-conglutin) exploiting fluorescence resonance energy transfer (FRET) has been developed. A high affinity dimeric form of a truncated 11-mer aptamer against β-conglutin was used, with each monomeric aptamer being flanked by donor/acceptor moieties. The dimeric form in the absence of target yields fluorescence emission due to the FRET from the excited fluorophore to the proximal second fluorophore. However, upon addition of β-conglutin, the specific interaction induces a change in the bi-aptameric structure resulting in an increase in fluorescence emission. The method is highly specific and sensitive, with a detection limit of 150 pM, providing an effective tool for the direct detection of the toxic β-conglutin subunit in foodstuffs in just 1 min at room temperature. & 2013 Elsevier B.V. All rights reserved.
Keywords: FRET 11 mer β-conglutin aptamer Lup an 1 allergen
1. Introduction Lupin is an herbaceous plant of the leguminous family. This plant belongs to the genus Lupinus, which includes more than 450 species. Lupin seeds have been used as human food and animal feed since ancient times (Ballester et al., 1984) and lupin products have been demonstrated to have an added value over numerous bakery products and have advantages in comparison to other legumes like soy (Erbas et al., 2005). Biological activities have been attributed to lupin proteins, including plasma cholesterol and triglyceride lowering effects (Sirtori et al., 2004), and anti-hypertensive properties (Pilvi et al., 2006). However, a number of severe food allergy reactions to lupin have been reported (Parisot et al., 2001, Jappe and Vieth, 2010). Lupin allergy apparently arises by either primary sensization (Novembre et al., 1999; Smith et al., 2004; Holden et al., 2005) or clinical cross-reactivity in individuals who are allergic to peanut (Parisot et al., 2001; Foss et al., 2006; Faeste and Namork, 2010). These cross-allergic clinical reactions of peanut to other members of the leguminous family, such as soy, peas, beans, and lentils, occur in about 5% of peanut-allergic individuals but were found in 68% of those with lupin allergy (Faeste et al., 2004). Lupin flour and lupin products thus require mandatory advisory labeling
n
Corresponding author. Corresponding author at: Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira i Virgili, 43007, Tarragona, Spain. Fax: þ34 977 559621. E-mail addresses: [email protected] (M. Svobodova), [email protected], [email protected] (C.K. O'Sullivan). nn
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.070
on foodstuff sold in the European Union (2006/142/EC, 2006), and recently the International Union of Immunological Societies (IUIS) allergen nomenclature subcommittee designated β-conglutin as the Lup an 1 allergen (Goggin et al., 2008). There are several techniques used to detect lupin allergens, and the majority is based on antibodies used in Enzyme Linked Immunosorbent Assay (ELISA) kits. The currently available commercial ELISAs exploit polyclonal antibodies that are not specific to each of the conglutin subunits and reports in the literature detail only monoclonal IgG antibodies against α-conglutin and IgM antibodies against β-conglutin (Dooper et al., 2007; Holden et al., 2007), and there are no reports or commercial ELISAs for the specific detection of β-conglutin. Aptamers are DNA or RNA oligonucleotides that bind to their cognate target with high affinity and specificity and are selected through a SELEX (Systematic Evolution of Ligands by Exponential Enrichment; Ellington and Szostak, 1990; Robertson and Joyce, 1990; Tuerk and Gold, 1990) from a randomly generated population of sequences for their ability to bind a desired molecular target. The specific ability of aptamers, which undergo significant conformational changes upon target binding, offers enhanced flexibility in the design of novel biosensors, where aptamer–target recognition is transduced into a detectable optical/electrochemical signal (Nutiu and Li, 2005; Yang et al., 2005; He et al., 2005; Nutiu and Li, 2004; Rupcich et al., 2006a, 2006b; Katilius et al., 2006; White and Holcombe, 2007; Li et al., 2008). Taking advantage of the ligand induced conformational changes of aptamers, dualfluorophore-labeled aptamers have been developed to give targetdependent fluorescence changes through Fluorescence Resonance Energy Transfer (FRET; Ueyama et al., 2002; Wang et al., 2005),
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the radiation-free transfer of energy from an excited donor dye to a suitable acceptor dye, a physical process that depends on spectral overlap and correct dipole alignment of the two dyes. We have recently reported on the selection of an aptamer against β-conglutin, the lupin subunit that has been identified by the IUIS as the Lup an 1 allergen (Nadal et al., 2012). This selected aptamer was subsequently truncated from a original 93 mer with KD 3.6 10 7 to a 11 mer with KD of 1.7 10 9 (Nadal et al., 2013). This truncated 11 mer is guanine rich and predicted to fold into G-quadruplex structures, composed of stacked guanine tetrads, which are stabilized by Hoogsteen-type hydrogen bonds between the guanines and by interactions with cations located between the tetrads. Indeed NMR confirmed the presence of a quadruplex structure within the truncated 11 mer and further studies with RP/HPLC showed this 11-mer quadruplex to have a retention time notably longer than expected, which was attributed to the presence of a dimeric structure (Nadal, 2012). The majority of dimeric based aptamer assays previously reported are based on dimeric structures constructed with different linkers with the aim of increasing affinity in comparison to the use of a simple monomer (Davis et al., 1996; Ringquist and Parma, 1998; Hasegawa et al., 2008; Boyacioglu et al., 2013). The truncated 11-mer β-conglutin aptamer reported here forms a dimeric structure naturally without the use of any linker similar to previous reports of cyclic oligonucleotides that form dimeric quadruplex structures, having a profound influence on the global topology and stability of the structure (Casals et al., 2012). The aim of this work was the use of a truncated 11-mer aptamer, which exists naturally in a dimeric form, to construct dimeric aptamer FRET probe for the detection of β-conglutin. In the work reported here, a sensitive method for rapid Lup an 1 allergen detection using FRET detection based on dimeric aptamer probe has been developed. A truncated 11-mer aptamer was flanked on either the 5′ or 3′ with a donor dye, as well as being flanked on either the 5′ or 3′ end with an acceptor dye to construct dimeric FRET-based probes. Following addition of β-conglutin (the Lup an 1 anaphylatic allergen) the change in fluorescence signal was monitored and specificity of the induced change in FRET demonstrated using control proteins. This is the first report on the use of the naturally occurring dimeric form of the 11-mer β-conglutin aptamer for the detection of β-conglutin. 2. Material and methods 2.1. Material Lyophilized fluorophore labeled oligonucleotides (donor 1 (D1)) 5′-ggt ggg ggt gg AF488-3′; donor 2 (D2) 5′-AF488 ggt ggg ggt gg-3′; acceptor 1 (A1) 5′-ggt ggg ggt gg AF555-3′; and acceptor 2 (A2) 5′-AF555 ggt ggg ggt gg-3′ were synthesized and purified by reverse phase high-performance liquid chromatography by Ella Biotech GmbH (Martinsried, Germany). Proteins from Lupinus albus seeds were extracted, purified and characterized as previously described (Nadal et al., 2011), obtaining a pure isolate of α, β, γ and δ conglutins. The binding buffer was phosphate buffered saline (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.4) containing 1.5 mM of MgCl2. All solutions were prepared in high purity water obtained from a Milli-Q RG system (Spain) and passed through a filter (0.45 nm). 2.2. Formation of FRET based dimeric aptamer probe The truncated 11-mer aptamer was flanked on either the 5′ or the 3′ end with Alexa Fluor 488 (λEX ¼492 nm, λEM ¼ 519 nm), as well as on either the 5′ or 3′-end with the Alexa Fluor 555 (λEX ¼533 nm, λEM ¼568 nm). The Alexa Fluor 488 labeled oligonucleotide (100 nM) was mixed with the Alexa Fluor 555 labeled
oligonucleotide (100 nM) in order to form dimeric aptamer FRET probe. FRET takes place as the donor Alexa Fluor 488 is excited at 492 nm and emits at 519 nm, exciting the Alexa Fluor 555, with subsequent emission at 568 nm. All fluorescent measurements were performed in a Cary Fluorimeter at 22 1C constant temperature. 2.3. FRET based aptamer detection of
β-conglutin
β-conglutin protein (0–200 nM) was mixed with the previously prepared FRET based dimeric aptamer probe (100 nM) and incubated together at 4 1C, 22 1C and 37 1C for 1 and 15 min in binding buffer of total volume 120 ml. Changes in FRET decay, following the addition of β-conglutin, were measured through a variable emission scan (500–700) at a fixed excitation wavelength (492 nm). 2.4. Detection of
β-conglutin from lupin protein extract
For preparation of lupin protein extract, 1 g of lupine flour was defatted with n-hexane, then centrifuged at 6000 g for 10 min at room temperature, and the pellet was dried under vacuum for 48 h. Proteins were extracted from the dried pellet using 100 mM Tris-HCl (pH ¼7.5) containing 10% NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM of ethylene glycol tetraacetic acid (EGTA) and centrifuged at 30,000 g for 10 min at room temperature. The pellet containing the prolamine fraction was removed and the supernatant containing the globulin fractions was stored at 20 1C. The protein concentrations of the lupin extract was determined with a BCA assay using BSA as a standard protein. The protein extracts were diluted in B&W buffer to obtain different dilutions and used in detection studies as described above.
3. Results and discussion 3.1. FRET based dimeric aptamer probe A series of experiments combining different FRET pairs (D1A1, D1A2, D2A1, D2A2) were carried out in order to confirm a dimeric structure of 11-aptamer and to elucidate the favoured FRET couple. The highest FRET was observed for couples D1A2 (5′–3′:3′–5′) and D2A2 (5′–3′:5′–3′), obtaining very similar results (Fig. 1A). Ongoing work to elucidate the structure of the dimeric aptamer using circular dichroism suggests that the 11-mer forms different dimeric structures of both anti-parallel and parallel binding, which is clearly observed in the results reported here. 3.2. FRET detection of
β-conglutin
For the detection of β-conglutin using labeled dimeric oligonucleotide probe, different concentrations β-conglutin was mixed with the oligonucleotide probe solution. It was found that FRET occurs (with corresponding emission at 568 nm) on increasing the concentration of β-conglutin (Fig. 1B), which indicates that the designed dimeric aptamer probes can be used for β-conglutin sensing. The fluorescence intensity of the labeled oligonucleotide probes induced by the presence of β-conglutin was monitored. In the absence of β-conglutin there is a fluorescence emission at 568 nm, but with increasing concentration of β-conglutin, the fluorescence intensity gradually increases. In Fig. 2(A) the relationship of the fluorescence change of the aptamer probe and β-conglutin concentration for both the D1A2 and D2A2 dimer couples can be observed and for both couples the fluorescence intensity increased linearly over the β-conglutin concentration range from 5 10 9 to 20 10 9 M, (r2 ¼ 0.99). The detection limit (L.O.D.) was estimated to be 150 pM and 158 pM for the D1A2 and
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Fig. 1. (A) Truncated 11-mer aptamer flanked on either the 5′ or 3′ end with the Alexa Fluor 488 or Alexa Fluor 555 and increments in intensity at 568 nm for the different FRET couples (Δ intensity ¼λEX of FRET couples λEX of corresponding acceptor). (B) FRET effect produced by the combination of donor 1 and acceptor 2 before and after the addition of β-conglutin.
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Fig. 2. (A) The standard curves of the fluorescence change of the D1A2 (i) and D2A2 (ii) aptamer probe and β-conglutin concentration ranging from 0 to 200 nM. Data obtained using an excitation 492 nm and emission at 568 nm. (B) The specificity of FRET detection for β-conglutin allergen using D1A2 (i) and D2A2 (ii) aptamer probe. Serial dilutions (0–200 nM) of each of the lupin conglutin subunits were used for cross-reactivity studies. The error bars represent the standard deviation of 3 repetitions.
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4. Conclusions Dimeric aptamer FRET probes for the detection of β-conglutin were developed. These probes offered an effective signal transduction method for the rapid and quantitative recognition of β-conglutin, requiring less than a minute incubation at room temperature. The FRET probes were demonstrated to be highly specific to the β-conglutin target as well as being very sensitive, achieving a detection limit of E150 pM, which is markedly lower than the LOD of E3 nM currently achievable with antibody based ELISAs. Acknowledgments This work was supported by funding from the National Project RecerCaixa (CO074670 APTALUP). References Fig. 3. The effect of temperature and incubation time on the developed FRET based dimeric aptamer assay.
D2A2 dimers, respectively, based on 5 repetitions, 3 orders of magnitude better than in case of S 40 β-conglutin aptamer before its truncation, where the L.O.D. obtained by competitive ELONA was 153 nM (Nadal, 2012).
3.3. Cross-reactivity studies with other lupin conglutins The selectivity of the dimeric probes to detect β-conglutin was evaluated by observing whether there was any interaction with other lupin conglutins, namely the α, γ, and δ conglutin subunits (Fig. 2B). In the presence of β-conglutin, fluorescence emission increased, but for both dimeric probes (D1A2, D2A2) no fluorescence changes were observed when other lupin conglutins were mixed with the aptamer probe, demonstrating the excellent selectivity of the labeled dimeric oligonucleotide probe. The parameters that can affect the response, such as aptamer concentration, incubation time, temperature and pH, were optimized. Room temperature (22 1C) and neutral pH of commonly used buffers (7.4) were optimum, which can be attributed to the fact that these were the conditions used in the SELEX process for selection of the aptamer. The effect of aptamer concentration on the response was studied from 0 to 300 nM (data not shown) and an increasing response with increasing aptamer concentrations was observed; 100 nM was the lowest concentration that could be used for the detection of a change in signal upon addition of the β-conglutin and as the objective of the work was to achieve as low a detection limit as possible 100 nM of aptamer was used. The effect of incubation time was also studied and the maximum response was observed after just 1 min incubation and no increment in response was observed with increasing incubation times (Fig. 3), highlighting markedly improved assay performance as compared with previous reports of aptamer based assays that typically require 10–30 min to achieve maximum binding (Zhu et al., 2012; Yildirim et al., 2012; Bai et al., 2012). To test if the developed method is workable in a realistic sample, the detection of β-conglutin allergen directly from lupin protein extract was studied. The amount of β-conglutin in lupin protein extract was determined by analyzing 3 different protein lupin extracts and interpolated to the calibration curve of β-conglutin. The concentration of β-conglutin in 100 μg/ml of lupin protein extract was observed to be 2279 μg/ml. These results demonstrate the viability of this method for the direct detection of β-conglutin in real samples.
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