Disposable electrochemical aptasensor for gluten determination in food

Abstract

Reliable detection of decreasing amounts of gluten in food is the only way of ensuring the safety of all coeliac patients. Results obtained with the method of choice, immunochemical assays, are not entirely comparable and many of them are sandwich assays that cannot recognize hydrolyzed proteins. In this work, we propose a competitive electrochemical sensor based on a recently described aptamer targeting the gliadin immunodominant peptide 33-mer that triggers the coeliac disease. The sensing layer is built on the surface of a screen-printed carbon electrode (SPCE) by adsorption of streptavidin and subsequent peptide immobilization. A competition between the peptide and gluten proteins from samples for a defined concentration of biotinylated Gli 4 aptamer is established. The aptamer bound to the peptide on the surface is finally measured after enzyme labelling and chronoamperometric detection of an enzymatically obtained electrochemically active product. This method is able to detect as low as 0.113 µg L-1 of gliadin, which corresponds to 380 µg kg-1 of gluten in food, taking all dilutions and conversion factors into consideration, with a reproducibility lower than 11%. The aptasensor was applied to food samples with gluten contents above and below the legislated threshold for gluten-free labelling in the EU, obtaining good agreement with the official R5 immunochemical method. mg kg -1Gluten Threshold gluten-free S P eaptasensor Graphical Abstract (for review) Highlights An electrochemical aptamer-based sensor on peptide-modified carbon screen-printed electrodes for gluten determination  Sensitivity is improved compared with reference enzyme-linked immunosorbent assays  The sensor was successfully applied to detect gluten in various food samples AbstractReliable detection of decreasing amounts of gluten in food is the only way of ensuring the safety of all coeliac patients. Results obtained with the method of choice, immunochemical assays, are not entirely comparable and many of them are sandwich assays that cannot recognize hydrolyzed proteins. In this work, we propose a competitive electrochemical sensor based on a recently described aptamer targeting the gliadin immunodominant peptide 33-mer that triggers the coeliac disease. The sensing layer is built on the surface of a screen-printed carbon electrode (SPCE) by adsorption of streptavidin and subsequent peptide immobilization. A competition between the peptide and gluten proteins from samples for a defined concentration of biotinylated Gli 4 aptamer is established. The aptamer bound to the peptide on the surface is finally measured after enzyme labelling and chronoamperometric detection of an enzymatically obtained electrochemically active product. This method is able to detect as low as 0.113 µg L -1 of gliadin, which corresponds to 380 µg kg -1 of gluten in food, taking all dilutions and conversion factors into consideration, with a reproducibility lower than 11%. The aptasensor was applied to food samples with gluten contents above and below the legislat...

Cutting-Edge Advances in Electrochemical Affinity Biosensing at Different Molecular Level of Emerging Food Allergens and Adulterants

“…A large number of commercial ELISAs kits are currently available and for a wide variety of food allergens [13], involving mainly sandwich but also competitive type formats. As disadvantages, these tests can be slow (up to 3.5 h), are prone to the appearance of cross-reactions, they do not provide fully comparable results due to the use of different immunoreagents [16] and require relatively expensive instrumentation (ELISA plate readers), hardly portable and miniaturizable. It should also be noted that only a limited number of kits have been validated by the international Association of Analytical Communities (AOAC).…”

“…Table 2 shows as a label-free aptasensor has been developed for the detection of lysozyme using a direct format implemented on electrodes nanostructured with AuNPs [33]. The methods developed for the detection of gluten, requiring a high sensitivity, involved competitive formats between gluten proteins (gliadin) and a synthetic biotinylated peptide immobilized on the surface of a SPCE (Figure 6a,b) [16] or Strep-MBs [34]. The biotinylated aptamers attached to the immobilized peptide was labeled enzymatically with Strep-HRP to perform chronoamperometric transduction with the H 2 O 2 /TMB system (Figure 6c).…”

Gluten Detection Methods and Their Critical Role in Assuring Safe Diets for Celiac Patients

“…However, to further improve the detection efficiency, selection of a suitable labeling system, such as 6-FAM labeling of biotinylated aptamer was tested [140]. The development of electrochemical competitive apta-sensors for gluten detection relied on the immobilization of biotinylated 33-mer peptide onto a streptavidin carbon surface, with LOD as low as 380 µg/kg [141]. When gluten content was measured in food samples, such as rolled oats or fit-snacks extracted with ethanol, with the apta-sensor, it tolerated up to 1.2% of ethanol without compromising reproducibility and performance of the assay.…”

Structure-informed detection and quantification of peptides in food and biological fluids

“…Current techniques utilized in detecting this biomarker in food samples is typically incapacitated in detecting the maximum acceptable detection limit of 20 ppm. Aptamers have been developed in recent years with superior sensitivity compared to antibody‐based sensors for the detection of 33‐mer peptide, the most immunodominant fragment of α2‐gliadin, the allergenic protein found in some cereals which trigger celiac diseases (López‐López, Miranda‐Castro, de‐los‐Santos‐Alvarez, Miranda‐Ordieres, & Lobo‐Castañón, ; Malvano, Albanese, Pilloton, & Di Matteo, ). Further, Wang, Wang, Wang, and Chen () recently developed an aptamer‐based resonance light scattering approach for the detection of kanamycin in milk samples.…”