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Enzyme immunoassays are currently the methods of choice for gluten control in foods labelled as gluten free, providing a mechanism for assessing food safety for consumption by coeliac and other allergic patients. However, their limitations, many of them associated to the reactivity of the different antibodies used and their degree of specificity, have prevented the establishment of a standardised method of analysis. We explore new methods for quantitatively determining gluten content in foods based on the use of two recently described aptamers, raised against a 33-mer peptide recognised as the immunodominant fragment from α2-gliadin. The assays use the target peptide immobilised onto streptavidin-coated magnetic beads in combination with a limited amount of biotin-aptamer in a competitive format, followed by streptavidin-peroxidase labelling of the aptamer that remains bound to the magnetic beads. The enzyme activity onto the beads, measured by chronoamperometry in disposable screen-printed electrodes, is inversely related to the target concentration in the test solution. We find that while the assay using the aptamer with the highest affinity towards the target (Gli 4) achieves low detection limits (~0.5 ppm) and excellent analytical performance, when challenged in samples containing the intact protein, gliadin, it fails in detecting the peptide in solution. This problem is circumvented by employing another aptamer (Gli 1), the most abundant one in the SELEX pool, as a receptor. The proposed assays allow the convenient detection of the allergen in different kinds of food samples, including heat-treated and hydrolysed ones. The obtained results correlate with those of commercially available antibody-based assays, providing an alternative for ensuring the safety and quality of nominally gluten-free foods. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: The design and performance of an inhibition assay for the electrochemical detection of tobramycin is reported. This platform uses a monovalent system for introducing the enzyme conjugate on a tagged-aptamer that specifically recognizes the aminoglycoside antibiotic tobramycin. Compared with multivalent systems such as biotin–streptavidin, the sensitivity is greatly improved reducing the limit of detection from 5μM to 0.1μM in aqueous solution. This labeling strategy along with the use of a nuclease-resistant RNA aptamer allowed the determination of tobramycin in pre-treated human serum samples within the therapeutic range, using magnetic microparticles as a solid support for the aptassay, and differential pulse voltammetry as detection technique. [Copyright &y& Elsevier]

Affinity characterization is essential to develop reliable aptamers for tumor biomarker detection. For alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma (HCC), two DNA aptamers were described with very different affinity. In this work, we estimate the dissociation constant of both of them by means of a direct assay on magnetic beads modified with AFP and electrochemical detection on carbon screen-printed electrodes (SPCE). Unlike previous works, both aptamers showed similar dissociation constant (Kd) values, in the subµM range. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers by both terminal deoxynucleotidyl transferase (TdT) and rolling circle amplification (RCA). Both DNA amplifications improved the sensitivity and also the apparent binding constants from 713 nM to 189 nM for the short aptamer and from 526 nM to 32 nM for the long aptamer. This improvement depends on the true affinity of the binding pair, which ultimately limits the analytical usefulness. [ABSTRACT FROM AUTHOR]

Abstract The development of chemical sensors capable of detecting the specific glycosylation patterns of proteins offers a powerful mean for the early detection of cancer. Unfortunately, this strategy is scarcely explored because receptors recognizing the glycans linked to proteins are challenging to discover. In this work, we describe a simple method for directing the selection of aptamers toward the glycan structure of the glycoproteins, with prostate-specific antigen (PSA) as a model target. Using this strategy, we identified one aptamer (PSA-1) that binds the glycan moiety of PSA with reasonable affinity (a dissociation constant of 177 ± 65 nM). Interestingly, an electrochemical sensor with a sandwich format employing the identified aptamer as a signaling receptor, provides a tool of discriminating human PSA from the unglycosylated protein, with a limit of detection of 0.66 ng/mL. The sensor responds to different levels of PSA in serum, correlating well with chemiluminescence ELISA used in hospitals even with higher potential to discriminate clinically meaningful prostate cancer. Although validation on a larger cohort is needed, this is the first demonstration of an aptamer-based sensor to detect PSA by focusing in its glycan moiety Highlights • A SELEX approach to direct aptamers toward de glycan structure of glycoproteins. • Aptamers recognizing the glycan moiety of prostate-specific antigen. • An aptamer-based electrochemical sandwich biosensor to detect PSA in serum. • A promising approach to aid prostate cancer diagnosis, distinguishing it from benign prostate hyperplasia. [ABSTRACT FROM AUTHOR]

The tunability of SELEX procedure is an essential feature to supply bioaffinity receptors (aptamers) almost on demand for analytical and therapeutic purposes. This longstanding ambition is, however, not straightforward. Non-invasive cancer diagnosis, so called liquid biopsy, requires collection of body fluids with minimal or no sample pretreatment. In those raw matrices, aptamers must recognize minute amounts of biomarkers that are not unique entities but large sets of variants evolving with the disease stage. The susceptibility of aptasensors to assay conditions has driven the selection of aptamers to natural environments to ensure their optimum performance in clinical samples. We present herein a compilation of the SELEX procedures in natural milieus. By revising the electrochemical aptasensors applied to clinical samples for cancer diagnosis and tracing back to the original SELEX we analyze whether aptamers raised using these SELEX strategies are being incorporated to the diagnostic devices and how aptasensors are finding their way to a market dominated by antibody-based assays. Image 1 • Electrochemical aptasensors tested in clinical samples for cancer biomarkers. • Critical revision of aptasensors to detect CTCs, exosomes and proteins. • Relevance of natural environment during SELEX on aptamer performance. • Prospects of in-vitro diagnostic devices based on electrochemical aptasensors. [ABSTRACT FROM AUTHOR]

Aptamers are starting to increase the reagents tool box to develop more sensitive and reliable methods for food allergens. In most of these assays, aptamers have to be modified for detection and/or immobilization purposes. To take full advantage of their affinity, which decisively influence the detectability, these modifications must be faced rationally. In this work, a recently developed aptamer for an immunotoxic peptide of gliadin associated to celiac disease is used in different configurations and modified with various markers and anchored groups to evaluate the influence of such modifications on the real affinity. The interaction in solution with the peptide is strong for a relatively small molecule ( K d = 45 ± 10 nM, 17 °C) and slightly stronger than that for the immobilized intact protein due to a cooperative binding effect. Comparatively, while only minor differences were found when the peptide or the aptamer were immobilized, labeling with a biotin resulted preferable over fluorescein ( K d = 102 ± 11 vs 208 ± 54 nM, 25 °C). These findings are of prime importance for the design of an aptamer-based analytical method for gluten quantification. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) plays an essential role in tumor progression and invasion through its continuous remodeling. The growth of most carcinomas is associated with an excessive collagen deposition that provides the proper environment for tumor development and chemoresistance. The α1 chain of a minor human collagen, type XI, is overexpressed in some tumor stroma, but not found in normal stroma. To test the clinical utility of this collagen as a cancer biomarker, specific receptors are needed. Available antibodies do not show enough selectivity or are directed toward the propeptide region that is cleaved when the protein is released to the ECM. Here we show the selection of an aptamer for the specific C-telopeptide region using a 16-mer peptide as the target for the SELEX. The aptamer selected with a K d of ∼25 nM was able to capture the collagen XI from cell lysates. It was also used for target detection in a mixed antibody-aptamer sandwich assay showing it can be useful for diagnostic purposes in biological fluids. [Display omitted] • Extracellular matrix components are an understudied source of cancer biomarkers. • Detection of collagen XI, overexpressed in some cancers, needs suitable probes. • A 16mer peptide was the target for the first aptamer for α chain of human collagen XI. • Mass spectrometry verifies collagen XI as the target of D1 aptamer from cell lysates. • Antibody-aptamer sandwich assay detects colXIα1 in cell lysates. [ABSTRACT FROM AUTHOR]

Enzyme-linked immunosorbent assays are currently the most popular methods to quantify gluten in foods. Unfortunately, the antibodies used as specific receptors in such methods are not compatible with the usual solvents for the extraction of gluten proteins. In consequence, commercial tests require a high dilution of the sample after the extraction, increasing the limit of quantification and decreasing convenience. In this work, we have rationally truncated an aptamer capable of recognizing gliadin in a deep eutectic solvent (DES). The truncated aptamer is a 19-nucleotides-long DNA that minimizes self-hybridization, allowing the development of an electrochemical sandwich-based sensor for the quantification of gluten in the DES ethaline. The sensor incorporates two identical biotin-labeled truncated aptamers, one of which is immobilized on a carbon screen-printed electrode and the other reports the binding of gliadin after incubation in streptavidin-peroxidase. This sensor can detect gliadin in DES, with a dynamic range between 1 and 100 μg/L and an intra-assay coefficient of variation of 11%. This analytical performance allows the quantification of 20 μg of gluten/kg of food when 1 g of food is extracted with 10 mL of ethaline. We demonstrate the ability of this method to achieve the measurement of gluten in food samples, after the extraction with pure ethaline. The assay is useful for the analysis of residual gluten levels in foods, thus facilitating the evaluation of any potential health risk associated with the consumption of such food by people with celiac disease or other gluten-related disorders. Image 1 • First aptamer-based sensor directly deployed in a deep-eutectic solvent. • A truncated aptamer is designed to be applied in a sandwich format. • Electrochemical sensor exhibits a limit of quantification of 20 μg gluten/kg sample. • Detection of residual gluten levels in food for highly-sensitive celiac patients. [ABSTRACT FROM AUTHOR]