Label‐Free Impedance Aptasensor for Major Peanut Allergen Ara h 1

Abstract

[a] Aptamers (synthetic oligonucleotides with an affinity to a given target) hold great potential as bio-recognition elements in biosensors [1]. Selected through the systematic evolution of ligands by exponential enrichment (SELEX) process [2] from a random DNA library, aptamers have shown good affinity to all kinds of analytes: drugs, toxins, proteins, low-molecular metabolites, and so forth [3].Although aptamers and their targets are generally redox inactive, label-free electrochemical detection is possible when a soluble redox indicator is present in the measuring solution.[4] Particularly, this strategy has been successfully applied for the detection of thrombin using the 15-base thrombin-binding aptamer (TBA) [5] and to detect immunoglobulin E (IgE) using a 37-base aptamer [6]. The detection principle states that bulky proteins insulate the electrode surface, owing to capturing by the immobilized aptamer. This results in a decreased electrontransfer rate between a soluble redox indicator and an electrode. It is clear that the insulating effect will be noticeable when the target is relatively large compared to the aptamer. For instance, thrombin (MW 37 000) has a molecular weight about eight times greater than TBA (MW 4700). But such a small aptamer is rather unique. Binding domains of recently developed aptamers are unknown, that forces to use a full SELEX sequence, which normally includes two primer regions of 20 bases and a variable sequence of about 40 bases. In the present work, we challenge electrochemical detection of a major peanut allergen Ara h 1 that appears to be an appropriate bulky target for a recently developed 80-base aptamer [7].Ara h 1 is a stable trimeric protein (about eight times larger than its aptamer) which belongs to vicilin family of seed storage globulins found in nuts, seeds, and soy. These foods are listed as major food allergens and must be indicated on packages, according to European Direc- Simplicity of electrochemical sensing, along with ease of aptamer production and immobilization, allow feature elaboration of simple, cheap, and miniaturized sensing devices, which offer great possibilities in food quality control.To prepare electrodes sensitive to Ara h 1, the gold electrodes were modified by the aptamer through the thiol functional group and treated with 2-mercaptoethanol (ME) as a blocking agent. It is well known that the presence of negatively charged oligonucleotides on an electrode causes a decrease in the rate of interfacial electron transfer for the [Fe(CN) 6 ] 4À/3À redox couple [8]. The effect can be quantitatively determined using EIS when spectra are fitted by a simple equivalent circuit (insert in Figure 1), which consists of electron-transfer resistance (R ET ), a constant phase element (CPE), a Warburg element (Z W ) and the resistance of the solution (R S ). The parameter R ET provides necessary information about electron-transfer kinetics and can be visually estimated from EIS in a complex plane diagram (Nyquist plot) as the diameter of the distorted s...

Electrochemical Affinity Biosensors in Food Safety

“…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].…”

“…The method allows the determination of as low as 0.5 ppb gliadin standard (0.5 ppm of gluten). Moreover, a direct approach for the impedimetric determination of Ara h 1 was developed by self-assembling a thiolated-80-base DNA aptamer on a gold-disk electrode [111]. This approach provided a relatively narrow linear range (0.1-1.8 µM) and a C50 value of 1.8 µM.…”

“…MBs-based biosensors have also been described for food allergens determination [94,97,99,103,104] making use of the advantages of these magnetic carriers in the final performance of electrochemical biosensors in terms of simplicity, sensitivity, reduced assay time and minimization of matrix effects, essential for the analysis of complex food extracts. Regarding the assay formats, competitive formats [94,102,109] have been scarcely employed and direct [93,95,96,105,106,111,112] and sandwich [97][98][99][100][101]103,104,107,108,112] configurations have been the most commonly used. Key: AuNPs: gold nanoparticles; ASV: anodic stripping voltammetry; CHOM: chicken ovomucoid; Con A: Concanavalin A; CV: cyclic voltammetry; GCE: glassy carbon electrode; DPV: differential pulse voltammetry; EIS: electrochemical impedance spectroscopy; LSV: linear sweep voltammetry; MBs: magnetic beads; m-GEC: magnetic graphite-epoxy composite; OTA: ochratoxin; OVA: ovalbumin; A P-L-Arg: poly(l-Arginine); MWCNTs: multi-walled carbon nanotubes; QDs: quantum dots; SPCE: screen-printed carbon electrode; SPdCE: screen-printed dual carbon electrodes.…”

Electrochemical Affinity Biosensors Based on Disposable Screen-Printed Electrodes for Detection of Food Allergens

“…The aptamer was soon used as biorecognition element in biosensors, including electrochemical ones. Electrochemical screen-printed biosensors for peanut allergens have been developed so far for Ara h 1 [12], Ara h 2 and Ara h 6 (Table 1). A magnetoimmunosensor for the dual detection of Ara h 1 and Ara h 2 was reported by Ruiz-Montiel et al [90].…”

“…The reader is referred to excellent recent reviews [7,10] for a detailed illustration of the many challenges associated with allergen detection. Electrochemical biosensors have been shown to provide simple, cost-effective analysis with significant reduction in the time per analysis compared to classic ELISA [10,11,12,13]. Among various types of electrodes in electrochemical biosensors, the flexibility in design allowed by the screen-printing process enables different sensor configurations [14,15], various possibilities for attaching the biorecognition element, to change electrode material by simply changing the ink to be printed, thus allowing to include mediators, nanoparticles, graphene etc.…”