High affinity aptamer for the detection of biogenic amine histamine

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High affinity aptamer for the detection of biogenic amine histamine Teresa Mairal Lerga, Miriam Jauset-Rubio, Vasso Skouridou, Abdulaziz Saleh Omar Bashammakh, Mohammad Soror El-Shahawi, Abdulrahman Obaid Al-Youbi, and Ciara K. O' Sullivan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00075 • Publication Date (Web): 01 May 2019 Downloaded from http://pubs.acs.org on May 1, 2019

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Analytical Chemistry

High Affinity Aptamer for the Detection of the Biogenic Amine Histamine Teresa Mairal Lergaa,*, Miriam Jauset-Rubioa, Vasso Skouridoua, Abdulaziz S. Bashammakhb, Mohammad S. El-Shahawib, Abdulrahman O. Alyoubib, Ciara K. O’Sullivana,c,* a Nanobiotechnology&

Bioanalysis Group, INTERFIBIO Consolidated Research Group, Departament d’EnginyeriaQuimica, Universitat Rovira I Virgili, Avinguda Paı̈sos Catalans 26, 43007 Tarragona, Spain b Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, 21589 Jeddah, Saudi Arabia c Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, 08010 Barcelona, Spain

ABSTRACT:The importance of histamine in various physiological functions and its involvement in allergenic responses, make this small molecule one of the most studied biogenic amines. Even though a variety of chromatography-based methods have been described for its analytical determination, the disadvantages they present in terms of cost, analysis time and low portability limit their suitability for in situ routine testing. In this work, we sought to identify histamine-binding aptamers that could then be exploited for the development of rapid, facile and sensitive assays for histamine detection suitable for point-of-need analysis. A classic SELEX process was designed employing magnetic beads for target immobilization and the selection was completed after ten rounds. Following Next Generation Sequencing of the last selection rounds from both positive and counter selection magnetic beads, several sequences were identified and initially screened using an apta-PCR affinity assay (APAA). Structural and functional characterization of the candidates resulted in the identification of the H2 aptamer. The high binding affinity of the H2 aptamer to histamine was validated using four independent assays (KD of 3 –34 nM). Finally, the H2 aptamer was used for the development of a magnetic beads-based competitive assay for the detection of histamine in both buffer and synthetic urine, achieving very low limits of detection of 18 pM and 76pM, respectively, whilst no matrix effects were observed. These results highlight the suitability of the strategy followed for identifying small molecule-binding aptamers and the compatibility of the selected H2 aptamer with the analysis of biological samples, thus facilitating the development of point-of-care devices for routine testing. Ongoing work is focused on extending the application of the H2 aptamer to the detection of spoilage in meat, fish and beverages, as well as evaluating the affinity of truncated forms of the aptamer.

INTRODUCTION Biogenic amines (BAs) are low-molecular organic compounds formed by the enzymatic or thermal decarboxylation of amino acids and they are present in numerous types of food including fish, meat, cheese, fermented products and beverages1as well as in biological fluids such as blood, urine and saliva2. The presence of BAs in the human body is essential for the biological function of the active cells3 and their unbalanced secretion can be used as a biomarker for some types of cancer (e.g. neuroblastoma and pheochromocytoma4). Furthermore, since some BAs act as neurotransmitters, they can potentially affect neurotransmitter levels causing a disruption in neurological signal transmission and producing psychiatric5 and neurological disorders such as Alzheimer’s disease6. Of all the biogenic amines acting as neurotransmitters, histamine plays an important role in gastric secretion, related with the narcolepsy disorder, cellular differentiation and cell growth, neurotransmission and neuromodulation7. Histamine has also been extensively studied because of its involvement in allergenic response8. When the concentration of BAs in the

body is excessive, either by intake or due to histamine intolerance, adverse reactions including headaches, migraines, nausea, rashes, hypertension and blood pressure changes can be provoked9. Histamine is also increased in salivary samples in periodontal disease, and salivary histamine levels can thus be used as a marker of periodontitis10. The importance of the concentration of BAs in tissues and biological fluids and their use as diseases markers is thus well known but the instability of BAs and their low concentration in biological fluids hinder their accurate detection. Histamine is considered to be the most relevant BA due to its biological toxicity, which is enhanced in the presence of putrescine, cadaverine, tyramine and phenylethylamine due to the inhibition of histamine-detoxifying enzymes11,12. The analytical determination of histamine is a complicated task due to the complexity of the matrices13,14, the structural similarities between the different BAs and the low concentration in biological fluids, with the normal concentration of histamine in blood, being 4.01 ± 3.4 nM and in urine 213.5 ± 178.9 nM15. Current methods of detection are based on chromatography,

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including High Performance Liquid Chromatography coupled with mass spectrometry16 and fluorescence detection17, as well as gas chromatography and thin layer chromatography18. Less frequently, BAs are detected using ELISA, radioimmunoassays (RIA)19and capillary electrophoresis coupled with mass spectrometry or spectrofluorimetry. These techniques are inherently laboratory based, requiring expensive instrumentation as well as skilled personnel20, and can thus not be deployed to the point-of-need for in situ analysis. Commercial ELISA detection kits are based on the use of antibodies, which are not only expensive but also frequently exhibit cross-reactivity with other BAs. There are also a few studies reporting the use of (bio)sensors for the detection of biogenic amines with the majority of those reported using enzymes as biorecognition elements13. Degefu et al.,21developed an enzyme-free sensor, using ligninmodified glassy carbon electrode for the detection of histamine in spiked human urine obtaining recoveries in the range of 93 100%, thus demonstrating the possibility of using these sensors for real sample analysis but the interference from other compounds such as uric acid and histidine considerably affected the sensor performance. The majority of the reports focus on the detection of an individual biogenic amine, but there are a few that describe the detection of multiple BAs, including that reported by Justino et al.,22for the simultaneous detection of histamine and putrescine. These BAs were also detected by Escobar and coworkers23using anamperometric dual enzymatic biosensor, whilst Leonardo et al.,24 developed an enzymatic electrochemical biosensor for the simultaneous detection of histamine, putrescine and cadaverine. In recent years the use of aptamers for the bio-recognition of small molecules for diverse applications has garnered increasing interest25,26. Aptamers are synthetic oligonucleotides that fold into three-dimensional structures and can bind a huge range of targets from proteins to whole cells and small molecules with very high affinity and specificity. Aptamers are synthesized in vitro using the SELEX (systematic evolution of ligands by exponential enrichment) process27 and provide many advantages compared to antibodies, mainly in terms stability, lower cost and specificity. To date, only one aptamer has been reported against a biogenic amine, namely tyramine, and the aptamer was demonstrated to have a KD of 0.2 µM. This aptamer also exhibited some crossreactivity with histamine (5%) and tryptamine (15%)28. The use of magnetic beads (MBs) for the development of aptasensors has also gained considerable interest with applications in the detection of proteins29,30, small molecules31 and drugs32. The use of MBs or the immobilization of aptamers for target capture facilitates a greater efficiency in target capture, leading to signal amplification effects33. The overall goal of this work was the selection of aptamers against histamine, which could then be exploited in a battery of assays for use with biological samples (blood, urine, saliva) as well as with food extracts. To achieve this objective, the

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selection of histamine-binding aptamers was performed using SELEX in combination with Next Generation Sequencing for the identification of aptamer candidates. The selected sequences were initially screened by an apta-PCR affinity assay (APAA) and the binding affinity of the most promising candidate was confirmed by three additional methods, demonstrating the high affinity of the selected aptamer for histamine. This aptamer was then exploited in a magnetic bead-based competitive assay for the ultrasensitive detection of histamine in both buffer and synthetic urine, achieving detection limits in the low picomolar range. EXPERIMENTAL SECTION SELEX strategy The DNA library used for aptamer selection consisted of a 94mer DNA sequence containing a random region of 50 nucleotides flanked by primer binding regions: 5′agctccagaagataaattacagg-N(50)-caactaggatactatgacccc-3′. In the initial round of positive selection, the DNA library pool (300 pmol) in Tris-binding buffer was denatured at 95 °C for 5 minutes and immediately cooled to 4 °C, after which 5 μl of histamine-magnetic beads, prepared as detailed in the Supporting Information (S2), were added and the volume was adjusted to 100 μl with Tris-binding buffer. The mixture was left to incubate for 30 minutes at 22°C using tilt rotation and the beads were then washed several times with 200 μl of Trisbinding buffer, re-suspended in 20 μl of water and the bound ssDNA library pool was amplified by PCR. Single-stranded DNA (ssDNA) was generated by a combination of asymmetric PCR and exonuclease digestion using phosphorylated reverse primer and lambda exonuclease, as previously described34. Highly purified ssDNA was obtained using the Oligo Clean & Concentrator kit and was used as the initial ssDNA pool for the following round of SELEX. After the first round, a negative selection step was introduced prior to the positive selection step, in order to remove non-specific binders. Each ssDNA pool was incubated with 5 μl of control beads (non-functionalized carboxylic acid magnetic beads blocked with Tris-HCl buffer), and as before, the volume was brought to 100μl using Trisbinding buffer, then incubated at 22°C for 30 minutes, and finally the unbound ssDNA was subsequently used for the positive selection step. In the 5th round, a counter selection step was implemented following the negative selection and prior to the positive selection step, in order to eliminate any sequences with cross-reactivity to other related biogenic amines. Tyramine and tryptamine were individually immobilized on carboxylic acid magnetic beads (see SI for details) and used for counter selection together with amine-magnetic beads containing an aliphatic carbon spacer on their surface in an effort to mimic the structures of the aliphatic biogenic amines (i.e. cadaverine, spermine, spermidine and putrescine). The evolution of the selection process was monitored by a bead-PCR based assay. Specifically, 2 µl of negative beads (naked beads without immobilized histamine), positive beads (histamine-magnetic beads) and counter beads (related molecules-coated magnetic beads) were incubated with 50 µl of 100 nM of ssDNA pool from different selection rounds in binding buffer for 30 minutes at 22°C under tilt rotation. After a washing step, the bound ssDNA was amplified using PCR and the products were analyzed using agarose gel electrophoresis and ImageJ


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software. The SELEX pool from the tenth round was finally sequenced using Next Generation Ion Torrent sequencing (Centre for OmicSciences, Eurecat, Reus, Spain) and the raw data was imported into the Galaxy server (http:// usegalaxy.org/) for analysis.

Structural studies by Circular Dichroism spectroscopy Circular dichroism (CD) studies were performed on a Chirascan CD spectrometer (Applied Photophysics, UK) using a 10 mm path length quartz microcell in the range 220 – 340 nm with a 1 nm step and adaptive sampling. Three scans were acquired per sample after auto-subtraction of the background (binding buffer), which were averaged and smoothed using the Pro-Data Chirascan software. Each aptamer candidate was prepared at 1 μM and histamine at 100 μM in PBS-binding buffer (10 mM PBS, 2mM KCl, 1.5 mM MgCl2, pH 7.4). Binding mixtures of each aptamer with histamine using the same final concentrations were also prepared and incubated for 30 minutes at 25°C before spectra acquisition. Binding affinity studies Apta-PCR affinity assay (APAA) The binding properties and the specificity of the histamine aptamer candidates were evaluated with the APAA. Specifically, 2 µl of positive, negative and counter magnetic beads were individually incubated with 50 µl of 1nM of each aptamer candidate in Tris-binding buffer for 30 minutes at 22 °C under tilt rotation, as described before25,35. The bound aptamer was amplified using PCR, the products were visualized using agarose gel electrophoresis and the intensity of the bands was estimated using the ImageJ software. To determine the affinity dissociation constants (KD), serial dilutions of each aptamer in the range of 39 pM to 10 nM were used to perform the APAA. Following PCR amplification and gel electrophoresis, the intensity of the bands was plotted and fitted to the one site-specific binding model using with the GraphPad Prism software for the calculation of the KD values. Direct assay using histamine-magnetic beads The 5’-biotin-modified H2 histamine aptamer (50 µl of serial dilutions in the range of 1.56 – 200 nM in PBS binding buffer) was incubated with 2µl of histamine- magnetic beads for 30 minutes at room temperature, followed by blocking with 5% skimmed milk in PBS-Tween. After washing the beads with PBS-Tween, 50 µl of streptavidin poly-HRP conjugate (1/20,000 dilution in PBS-Tween) were added to the beads and incubated for a further 30 minutes. Finally, following a final washing step, 50 µl of TMB substrate were added to the beads, and color development was stopped after 5 minutes by the addition of 50 µl of 1 M H2SO4. The absorbance was read at 450 nm and the data was plotted using the GraphPad Prism software for the calculation of the KD as explained above. Direct assay using histamine-sepharose resin Histamine was immobilized on sepharose resin via epoxy chemistry (see S3 for details). For the determination of the KD,

50 µl of different concentrations of biotinylated H2 aptamer (1.5-100 nM) in PBS-binding buffer were mixed with 10 µl of histamine-sepharose resin in individual tubes and incubated for 30 minutes at room temperature. The resin was then blocked with 5% w/v skimmed milk in PBS-Tween and following thorough washing, 50 µl of streptavidin poly-HRP (1/20,000 in PBS-Tween) were added and incubated during 30 minutes at room temperature. Finally, the resin was washed with PBSTween and 50 µl of TMB substrate were added followed by the addition of 50 µl of 1 M H2SO4 10 minutes later to stop color development. The absorbance was read at 450 nm and the KD was calculated. Unmodified epoxy-activated sepharose 6B resin was used as negative control. Direct assay using histamine immobilized on microtiter plate Histamine was immobilized on maleimide-activated microplate wells using 11-mercaptoundecanoic acid (MUA) as a crosslinker (see S4 for details). Different concentrations of the biotinylated H2 aptamer in PBS-binding buffer were prepared in the range of 1.5 –100 nM and 50 µl of each solution were directly added to the wells of the histamine-modified maleimide plate. The plate was incubated for 30 minutes followed by washing with PBS-Tween. Streptavidin poly-HRP (50 µl of 1/20,000 dilution in PBS-Tween) was then added and following a 30-minute incubation and a subsequent washing step, 50 µl of TMB substrate were added. The absorbance was read at 450 nm after 5 minutes color development and the addition of 50 µl of 1 M H2SO4. All incubation steps were performed at room temperature unless otherwise specified. Unmodified maleimide activated wells were used as negative controls. Histamine detection with a bead-based competition assay A competitive assay for the ultrasensitive detection of histamine was developed. A range of different concentrations of histamine (50 µl of 0-10 mM in PBS-binding buffer) were pre-incubated in individual tubes with 50 µl of 100 nM biotinylated H2 aptamer (also prepared in PBS-binding buffer) for 30 minutes at 22 °C. Subsequently, 2 µl of histamine-magnetic beads were added to each tube, followed by 20 minutes incubation at 22 °C. The tubes were then placed in a magnet holder to capture the magnetic beads, which were then rigorously washed with PBSbinding buffer, followed by blocking with 5% w/v skimmed milk in PBS-Tween. The beads were then re-suspended in 50 µl of streptavidin poly-HRP (1/20,000 dilution in PBS-Tween) and incubated for 30 minutes at 22 °C. Following a final washing step with PBS-Tween, the beads were re-suspended and 50 µl of TMB substrate added, and the color development was then stopped 5 minutes later via addition of 50 µl of 1 M H2SO4. The absorbance was read at 450 nm and the raw data was plotted using the GraphPad Prism software for the calculation of the limit of detection (LOD) using the sigmoidal dose response (variable slope) model. All incubation steps were performed under tilt rotation. Various parameters affecting the performance of the assay were previously optimized as detailed in the supplementary material (Figure S6).The feasibility of the assay in biological fluids was demonstrated by using synthetic urine to prepare the histamine solutions (0 – 10 mM). The assay was performed as described above and the LOD calculated.



RESULTS AND DISCUSSION Histamine aptamer selection strategy Selection of histamine-binding aptamers was performed using magnetic partitioning25,36–38. Prior to the selection, two types of carboxyl-modified magnetic beads were compared and evaluated for their immobilization efficiencies. Dynabeads M270 have a diameter of 2.8 μm, whilst Chemicell SiMAG beads are almost three times smaller with a diameter of 1 μm. Immobilization of histamine was successfully achieved using both types of beads, as detected using a polyclonal antihistamine antibody. Whilst similar levels of antibody binding were achieved for both of the functionalized beads, the polyclonal antibody was observed to non-specifically react with the Chemicell beads, and from that it could be assumed that a higher level of histamine immobilization was achieved on the Dynabeads M-270 (Figure S1a), and thus these beads were chosen for implementation in the SELEX process. Tyramine and tryptamine were immobilized in a similar manner on the same type of beads for use in the counter selection steps. The designed strategy was based on a positive selection performed during the 1st round, followed by the incorporation of negative selection in the 2nd round and counter selection in the 5th round. The negative selection was performed using naked-carboxyl magnetic beads to eliminate any sequences with affinity to the bead matrix alone, and tyramine and tryptamine immobilized on carboxyl magnetic beads, were used for the counter selection to remove sequences with crossreactivity to other biologically relevant BAs. To represent the group of aliphatic BAs that share a high structural similarity (cadaverine, spermine, spermidine and putrescine), aminemodified magnetic beads with an aliphatic carbon linker on their surface were used. The selection process performed for the isolation of histamine-binding aptamers was completed after ten rounds. This is reflected by the evolution studies performed for the entire selection by using the same volume of each bead type used in all selection rounds to amplify bound DNA (Figure S2). When the negative selection was introduced during the 2nd round, the intensity of the PCR product after the amplification of the negative beads using library-specific primers was higher than the one observed from the positive beads (histaminemagnetic beads) since the highly diverse library used for the selection (with approximately 1015 different variants) contains many sequences that bind non-specifically to the beads matrix. As the selection progressed, the non-specific and cross-reactive sequences with other BAs were eliminated and towards the end of the selection process, only specific sequences appeared to be present in the enriched DNA pool (R9-R10 in Figure S2). The evolution of the selection process was also monitored using APAA, a bead-PCR based assay. Histamine-functionalized magnetic beads were incubated with equal concentrations of ssDNA prepared from individual selection rounds. Bound DNA was amplified using PCR and resolved using gel electrophoresis. The intensity of the bands from each round was measured using the ImageJ program. The histamine-binding sequences in the ssDNA pools were enriched round by round, as an increasing amount of ssDNA was amplified from the histamine-magnetic beads, particularly during the last rounds of the SELEX process. After the 8th round of selection, a band

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corresponding to DNA sequences longer than the original library (94nt) appeared, which can be attributed to non-specific DNA amplification, a common problem during the amplification of random ssDNA pools. To eliminate any selection bias and ensure that only the correct size sequences were used, the band corresponding to the library length (approximately 100 bp of dsDNA after PCR amplification) was extracted from the agarose gel, purified and used for the preparation of ssDNA, thus resulting in a lower amount of input ssDNA used for the following rounds 9 and 10. The positive evolution of the selection process was also reflected by the decreasing number of PCR cycles required for the amplification of bound ssDNA using the histamine-magnetic beads (Figure S3a). Concomitantly, the number of PCR cycles required for the amplification of bound DNA using the negative and counter selection beads increased, suggesting that by the end of the selection (round 10), the majority of the sequences present in the ssDNA pool was specifically binding to the target molecule. The specificity of the final ssDNA pool from the 10th round was confirmed. DNA from the 10th round of selection was prepared and separately incubated with the histamine-magnetic beads and the beads used for the counter selection (i.e. with immobilized tryptamine and tyramine and the amine-modified beads used for the removal of sequences binding to aliphatic amines as a general model for the small aliphatic BAs). The ssDNA from the last selection round bound specifically only to the histamine-immobilized beads, indicating that the pool selected displayed very low to no cross-reactivity with the other BAs tested (Figure S3b). Identification of aptamer candidates by Next Generation Sequencing (NGS) The DNA pools from the 10th round of selection from both positive histamine and counter tryptamine beads were used for Ion Torrent Next-Generation Sequencing. The tryptamine beads were also analyzed in order to eliminate any histaminebinding sequences with cross-reactivity with tryptamine if present in both pools. DNA was prepared using different modified forward primers containing adaptor and barcode sequences and a universal reverse primer. The raw data was imported into the Galaxy server (http:// usegalaxy.org/) and analyzed (Table S1). The raw data was filtered in order to obtain the sequences of library length and remove shorter or longer sequences resulting from non-specific amplification. These filtered sequences were finally collapsed in order to identify unique sequences. The 8 most over-represented sequences from the top 100 unique sequences found in the histamine pool were aligned by multiple sequence alignment using ClustalWin an effort to reveal any common sequence motif. Six aptamer candidates from the histamine-binding pool were chosen for further analysis (Table S2). The criteria for this choice were based on their high representation and their absence from the tryptamine-binding pool. Evaluation of the aptamer candidates by apta-PCR affinity assay (APAA)


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There are several techniques and methodologies used for the evaluation of the affinity of post-SELEX aptamers to their cognate targets, including surface plasmon resonance (SPR)39, surface plasmon resonance imaging (SPRi)40, biolayer interferometry (BLI), isothermal titration calorimetry (ITC), electrophoretic mobility shift assay (EMSA), microscale thermophoresis (MST) and fluorescence anisotropy, among others. When the targets are small molecules, this analysis is more complicated as it is difficult to the reach the required levels of sensitivity41. We have previously reported the use of APAA for the evaluation of 17β-estradiol35 and testosteronebinding aptamers25 and demonstrated the suitability of this approach to study the binding of aptamers to small molecules. Using large molecules, the results from APAA show a good correlation with results obtained using standard techniques like SPR and MST, and therefore the APAA methodology was also used in this work to evaluate the binding properties of the selected aptamer candidates. Initially, a screening of all the histamine aptamer candidates was performed by individually incubating the histamine, counter (mixture of tryptamine, tyramine and amine beads) and naked magnetic beads with a constant concentration of each aptamer. Bound aptamers were detected following PCR amplification and gel electrophoresis analysis (Figure 1a). The intensity of the bands was estimated with the ImageJ software (Figure 1b). All candidates exhibited higher apparent affinity for the histamine-magnetic beads compared to the other bead types, with the exception of H3, which was excluded from further analysis due to its high cross-reactivity with the counter beads. The affinity dissociation constants (KD) of the remainder of the candidates were determined with the same assay using a range of aptamer concentrations from 39 pM to 10 nM and a constant level of histamine-magnetic beads. KD values were determined by plotting the intensity of the agarose gel bands against aptamer concentration using the GraphPad Prism software and the one site specific binding model. Aptamer candidates H1, H2 and H9 exhibited very high affinity for their target histamine with KD values in the low nanomolar range (Figure S4).The KD values of the histamine aptamers obtained are in the same range as the affinity constants of aptamers calculated for other small molecules using the same methodology25 or other assays42, thus demonstrating the suitability and efficiency of the APAA approach for the study of aptamer-small molecule interactions.

Figure 1: APAA assay: (a) Schematic representation (b) Evaluation of the histamine aptamer candidates.

Structural evaluation of the aptamer candidates by CD The aptamer candidates H1, H2, H4, H5 and H9 were also evaluated using CD spectroscopy (the H3 candidate was omitted because of its high cross-reactivity with the counter selection beads indicated with the APAA). Initially, the aptamers were analyzed alone to study their folding state. As shown in Figure 2 (panels H1, H2, H4, H9), a CD maximum at 273 - 278 nm and a CD minimum at 244 - 248 nm were recorded for the H1, H2, H4 and H9 aptamers, which are characteristic of B-type double-stranded DNA43, indicating hairpin formation, also in accordance with the predicted structures of the aptamers (Figure S5) obtained with the m-fold web server44. On the other hand, a hypsochromic shift of the CD maximum in the spectrum of the H5 aptamer to 270 nm was observed (Figure 2, H5), suggesting a parallel G-quadruplex sub-structure (CD maximum at 260 nm) within the entire aptamer folding, also suggested by the G-score of 20 calculated for this sequence using the QGRS Mapper online tool45. Subsequently, the CD spectra of the aptamers were recorded in binding mixtures with histamine to identify any possible changes indicative of conformational changes upon histamine binding. No (significant) changes were observed in the spectra of the aptamer candidates H1, H4, H5 and H9 in the presence of histamine, suggesting that no structural switch occurs when these aptamers bind their target. In contrast, changes in both the CD maximum and CD minimum peaks of the H2 aptamer were observed, suggesting that this aptamer undergoes some conformational change to facilitate histamine binding.



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to validate the binding of the H2 aptamer to histamine. This aptamer was chosen as the ideal candidate for further characterization due to its high specificity and affinity for histamine and, combined with the target-induced conformational change as suggested by CD, which could be exploited in subsequent assay development.

Figure 2: Structural evaluation of histamine aptamer candidates H1, H2, H4, H5 and H9with CD. Validation of the binding properties of the H2 aptamer As mentioned already, the small size of histamine limits the methods which can be reliably used to monitor its interactions with aptamers. Also as with all aptamers, it is desirable to use different methodologies to confirm the specificity and the affinity of the aptamer candidates, as well as validating the performance of the selected aptamer taking its final application into consideration37. Four different approaches were thus used

As discussed above, with the APAA using unmodified aptamer and histamine immobilized on magnetic beads the KD of H2 was calculated at 3.08 ± 1.13nM (Figure 3a). A direct binding assay was also developed exploiting the same histamine-magnetic beads but with a modified version of the aptamer containing a 5’ biotin. Streptavidin poly-HRP was used for final detection. Very similar results to the APAA were obtained, with the KD calculated to be 5.61 ± 0.27nM (Figure 3b). Using the same principle but a different matrix to eliminate the possibility of matrix participation in the binding event, sepharose resin was used for histamine immobilization, and again using biotinylated H2 aptamer. In this format a KD of 34.21 ± 8.59nM was obtained (Figure 3c), and the small difference in KD observed can be attributed to different immobilization levels of histamine on the resin as well as the spacing between histamine and the matrix. The approaches for determination of KD based on the sepharose resin and magnetic beads are both based on the same principle,

Figure 3: Binding affinity assays: (a) APAA methodology, representing the intensity of the band as visualized using gel electrophoresis using Image J software vs. the concentration of the aptamer; (b)Different concentrations of the biotinylated aptamer vs. the absorbance signal at 450 nm using magnetic beads, or (c) sepharose resin, or (d) maleimide coated microtiter plates, to immobilized histamine.


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simply using difference matrices. To demonstrate the interaction of the aptamer with histamine in an alternative format, the use of histamine immobilized on maleimidemodified microtiter plates was explored. Different thiocarboxylic acids were evaluated as spacers for the immobilization of histamine, including thioctic acid, 6mercaptohexanoic acid, 3-mercaptopropionic acid, 4mercaptophenylpropionic acid, 11-mercapto-1-undecanoic acid (MUA). MUA resulted in lower levels of non-specific binding as compared to the other ones tested (data not shown). The histamine-coated microplate was exposed to different concentrations of the biotinylated H2 aptamer, followed by addition of streptavidin-polyHRP, and the KD was calculated to be 10.48 ± 0.93nM (Figure 3d), in good correlation with the other techniques studied. Overall, the results from the four different methods used to study the binding of the H2 aptamer were in agreement, verifying its high affinity for histamine with KD in the range of 3 – 34 nM.

Competitive assay for the analytical determination of histamine Following characterization and confirmation of the high affinity of the H2 aptamer, a competitive assay as illustrated in Figure 4, was developed for the detection of histamine in urine. The assay was based on the pre-incubation of the biotinylated H2 aptamer with free histamine representing the unknown sample to be quantified. After a defined incubation time, histaminemagnetic beads were added to capture any available unbound aptamer. Magnetic separation was used to remove the aptamerfree histamine (i.e. any histamine present in sample under interrogation). Following removal of the aptamer-free histamine complexes in the supernatant, the captured magnetic beads were rigorously washed, and finally the bound biotinylated aptamer was detected using streptavidin poly-HRP and TMB substrate. The absorbance measured from the beadbound aptamer is thus inversely proportional to the concentration of the free histamine present in the sample.

Figure 4: Schematic representation of the detection of histamine using a competitive assay on beads.

Several parameters were initially optimized to improve the assay performance, including assay temperature, length of preincubation and incubation steps, as well as length of incubation of the reporter streptavidin poly-HRP conjugate with the aptamer-histamine magnetic bead complex29,32. Initially, the effect of temperature on the assay performance was evaluated, comparing room temperature (22 – 25°C) to physiological temperature of 37°C, where all assay steps were carried out at the same temperature. The LOD obtained at room temperature was 54 nM as compared to 535 nM obtained using 37°C, and room temperature was thus chosen for further experiments (Figure S6a). The length of pre-incubation of the H2 aptamer with the sample being interrogated was then evaluated, and an increase in preincubation time resulted in increased sensitivity, reaching a plateau at 30 minutes, and this was used in further experiments (Figure S6b). The incubation of this solution with histamine-

modified magnetic beads was then optimized, and again increasing the time of incubation led to an improvement of the LOD, reaching a plateau are 20 minutes, which was thus chosen as optimal (Figure S6c). Finally, the last step of the assay involving the incubation of the aptamer-histamine magnetic beads complex with the reporter streptavidin poly-HRP was optimized by evaluating the assay performance at 15 minutes and 30 minutes, with higher signals observed for the longer incubation (Figure S6d). With the optimized parameters of the competition assay, the specificity of the H2 aptamer was evaluated using potentially interfering molecules. Tyramine was selected as a representative aromatic biogenic amine, whilst spermidine was selected as a model for the aliphatic BAs. Histidine on the other hand was chosen as precursor of histamine and its presence in biological fluids could seriously interfere with the detection of histamine46,47. As shown in Figure 5a, the signals obtained from the binding of the H2 aptamer on the histamine-magnetic beads during the incubation step was not affected by the concentration



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of the potential interferents. The results highlights the high specificity of the aptamer and the developed assay for histamine detection as well as its compatibility with biological fluids containing structurally related molecules. A calibration curve using a range of histamine concentrations (10 fM to 10mM) was then constructed, achieving an LOD of 18pMthus compatible with the detection of histamine in biological fluids. This was demonstrated with the detection of histamine in synthetic urine and a LOD of 76 pM was achieved, suggesting low matrix effect (Figure 5b).There are very few reports detailing the detection of histamine in urine, or other biological fluids16, and the majority of them describe the use of chromatography-based methods, with a wide range of detection limits reported, for example a LOD of 225 nM reported by Comas-Bastéet al.,48 and 40 pM by Hogan et al.,16 using the same technique.

Electrochemical competitive assays exploiting antibodies and histamine conjugates have been reported for detection in biological fluids. An LOD of 4.5 pM was reported by Yang et al.,47 in serum whilst Lim and co-workers,48 demonstrated the detection of histamine in whole blood in the range of 1.8 – 18 µM. There is, thus, a mature need for a cost-effective, easy-touse, reliable and accurate method that can be deployed to use at the point-of-need. CONCLUSIONS In this work, we describe the selection of an aptamer against histamine and its application in an analytical method for histamine detection in biological samples. This task is very challenging due to the small size of the target and the high structural similarity with other biogenic amines. A classic SELEX strategy was designed, based on the use of magnetic beads for the immobilization of histamine while negative and counter selections were implemented in the process in order to increase aptamer specificity. Next Generation Sequencing of the last selection rounds from both histamine and counter selection beads enabled the elimination of sequences present in both pools indicating cross-reactivity with the other biogenic amines comprising the mixture of the counter selection beads. Among the six aptamer candidates selected for further characterization, aptamer H2 was selected for assay development because of its high affinity and specificity as well as its structure-switching property after binding suggested by circular dichroism studies. The dissociation constant of this aptamer was calculated using four independent approaches confirming its high affinity with KD in the range of 3 – 34 nM, depending on the method used. Strikingly, the aptamer did not

Figure 5: Competitive assay (a) Cross-reactivity with potential interferents, tyramine (representing aromatic biogenic amines); spermidine (representing aliphatic biogenic amines) and histidine (the amino acid precursor of histamine); (b) Calibration curves performed in buffer and synthetic urine. exhibit any cross-reactivity with the other biogenic amines studied including tyramine, tryptamine and aliphatic amines or with its amino acid precursor, histidine. Finally, an ultrasensitive and rapid methodology was developed for the detection of histamine in urine based on a competition assay exploiting the H2 aptamer and magnetic beads, which can be completed in under two hours. Low limits of detection were achieved, 18 pM in buffer and 76pM in synthetic urine, thus demonstrating the applicability of the assay to biological samples exhibiting the required sensitivity for the analytical determination of histamine in biological fluids. To date, the histamine aptamer we report here, along with a previously reported tyramine aptamer28 are the only ones developed against biogenic amines. Further work is in progress to simplify the assay and render it compatible with lower cost material such as nitrocellulose membranes for the paper-based lateral flow detection of histamine in saliva, blood and urine, as well as in food extracts. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Details about the materials, methodology and evaluation of histamine immobilization on different matrixes; evolution of SELEX process; predicted structures of the aptamer candidates; KD determination of aptamer candidates by APAA assay; optimization of the competitive assay, summary of the results from NGS and histamine aptamers sequences (PDF).

AUTHOR INFORMATION


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Corresponding Author

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* Dr. Ciara K. O’Sullivan “[email protected]” * Dr. Teresa Mairal Lerga “[email protected]”

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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT The authors are grateful to King Abdulaziz University, under the financing of the collaborative project “Ultrasensitive, extremely rapid lateral flow assays exploiting nanoparticles for the detection of biogenic amines, detection of adulteration of food with meat products and identification of contaminating meat and viruses” for funding.

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High affinity aptamer for the detection of biogenic amine histamine

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