Development of a genosensor for peanut allergen ARA h 2 detection and its optimization by surface response methodology

Abstract

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. bealopru@ucm.es AbstractA new selective electrochemical genosensor has been developed for the detection of an 86-mer DNA peanut sequence encoding part of the allergen Ara h 2 (conglutinhomologue protein). The method is based on a sandwich format, which presents two advantages: it permits shortening the capture probe and avoids labeling of the target.Screen-printed gold electrodes have been used as platform for the immobilization of oligonucleotides by the well-known S-Au bond. Mixed self-assembled monolayers (SAM), including thiol-modified capture probe and mercaptohexanol, were prepared to achieve an organized, homogeneous and not too compact SAM in which unspecific adsorption of the capture probe would be prevented. The optimization of the sensing phase was carried out using the Design of Experiments (DoE) approach.Traditionally, response optimization is achieved by changing the value of one factor at a time until there is no further improvement. However, DoE involves regulating the important factors so that the result becomes optimal. Optimized conditions were found to be 1.34 µM for capture probe concentration and 3.15 mM for 2 mercaptohexanol (spacer) concentration. When the optimal conditions were employed the analytical performance of the proposed genosensor improved significantly, showing a sensitivity as high as 3 µA/nM, with a linear range from 5 10 -11 to 5 10 -8 M and a detection limit of 10 pM.

Electrochemical Affinity Biosensors in Food Safety

“…Worth to mention also the increasing tendency to fabricate these electrochemical biosensors onto screen-printed electrodes (SPEs) [85,[95][96][97][98][99][100][101][102][103][104]107,109,112,113] since they are suitable to be fabricated with different materials, to be drawn in diverse geometries and multiplexed formats and Recently, Sugawara et al [116] developed an electrochemical biosensor to detect ovalbumin (OVA) by adsorbing a specific peptide probe (peptide-1) conjugated with the electroactive daunomycin on a GCE. In the presence of OVA, the peptide probe was released from the electrode surface and the DPV peak current of the daunomycin moiety decreased.…”

“…Regarding DNA sensors, all the reported works use specific thiolated probes self-assembled on gold electrodes [105] or AuNPs-modified electrodes [106][107][108]. While linear DNA probes were used for the implementation of sandwich hybridization assays for the determination of Ara h 2 [107] and α2-gliadin [108], direct assays were implemented for Ara h 1 determination using dual-labeled stem-loop probes [105,106].…”

“…While linear DNA probes were used for the implementation of sandwich hybridization assays for the determination of Ara h 2 [107] and α2-gliadin [108], direct assays were implemented for Ara h 1 determination using dual-labeled stem-loop probes [105,106]. The strategy developed by Martin-Fernández et al [108] is based on sandwich hybridization of target DNA with a thiolated capture probe immobilized together with MCH onto Au-SPEs and a fluorescein isothiocyanate (FITC)-modified detector probe.…”

“…In addition, given the sensitivity required for some allergens detection, many biosensor devices with improved sensitivity based on the use of nanomaterials, such as QDs [115], AuNPs [93,100,101,[106][107][108], MWCNTs [93] and graphene [95,96,106] have been developed. Worth to mention also the increasing tendency to fabricate these electrochemical biosensors onto screen-printed electrodes (SPEs) [85,[95][96][97][98][99][100][101][102][103][104]107,109,112,113] since they are suitable to be fabricated with different materials, to be drawn in diverse geometries and multiplexed formats and Recently, Sugawara et al [116] developed an electrochemical biosensor to detect ovalbumin (OVA) by adsorbing a specific peptide probe (peptide-1) conjugated with the electroactive daunomycin on a GCE.…”

“…They are classified according to the type of used bioreceptor. The bioreceptors most commonly used are specific antibodies [93][94][95][96][97][98][99][100][101][102][103][104], single-stranded DNA molecules [105][106][107][108], aptamers [109][110][111][112], and other affinity receptors like lectins [113] and peptides [114]. The affinity reaction between these bioreceptors, which are immobilized onto electrodes or solid supports like magnetic beads (MBs), and the target allergen has been monitored using (chrono)amperometry [68,94,97,99,[101][102][103][104]109], voltammetry [93,95,96,98,102,103,106,107,[112][113][114] and electrochemical impedance spectroscopy [105,110,111].…”