High Affinity Aptamer for the Detection of the Biogenic Amine Histamine

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 (K D 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 76 pM, respectively, while no matrix effects were observed. These results highlight the suitability of the strategy followed for identifying small moleculebinding 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.

Development of a histamine aptasensor for food safety monitoring

“…Selection of DNA histamine aptamers have been recently reported by Mairal Lerga et al . 26 . A notable difference in their selection method was the use of histamine immobilized via the amino group whereas we immobilized L-histidine leaving both the imidazole and the amine available for the aptamers to interact with.…”

Aptamers Against the β-Conglutin Allergen: Insights into the Behavior of the Shortest Multimeric(Intra)Molecular DNA G-Quadruplex

“…In recent years, aptamers have been increasingly employed for food safety monitoring [22,23], and an emerging area of application is the detection of food allergens. In 2012, we reported the selection of a high affinity aptamer against β-conglutin [24], a lupine protein that has been identified by the International Union of Immunological Societies as the Lup an 1 allergen.…”

A Bottom-Up Approach for Developing Aptasensors for Abused Drugs: Biosensors in Forensics

“…Finally, the resulting pool is sequenced, and the selected aptamers are characterized for their binding affinity. Since the first introduction of the SELEX method in 1990 by two independent groups [24,25], a large number of aptamers have been developed for various targets ranging from small molecules [26,27] to proteins [28,29], whole cells [30], and tissues [31,32].…”