Reduced Graphene Oxide Modified Electrodes for Sensitive Sensing

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Reduced Graphene Oxide Modified Electrodes For Sensitive Sensing of Gliadin in Food Samples Fereshteh Chekin, Santosh K. Singh, Alina Mihaela Vasilescu, Vishal M. Dhavale, Sreekumar Kurungot, Rabah Boukherroub, and Sabine Szunerits ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00608 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 1, 2016

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Reduced Graphene Oxide Modified Electrodes For Sensitive Sensing of Gliadin in Food Samples Fereshteh Chekin,a,b* Santosh K. Singh,c,d Alina Vasilescu,e Vishal M. Dhavale,f Sreekumar Kurungot,c,d Rabah Boukherrouba and Sabine Szuneritsa* a

Institute of Electronics, Microelectronics and Nanotechnology (IEMN), UMR CNRS 8520, Lille1 University, Avenue Poincaré-CS60069, 59652 Villeneuve d’Ascq, France b

c

Department of Chemistry, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran

Physical and Materials Chemistry Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India d

Academy of Scientific and Innovative Research, Anusandhan Bhawan, 2 RafiMarg, New Delhi 110 001, India e International Center of Biodynamics, 1B Intrarea Portocalelor, Sector 6, Bucharest 060101, Romania f

Chemical Resources Laboratory, Tokyo Institute of Technology, R1-17, 4259, Nagatsuta, Midori-ku, Japan

ABSTRACT Incidences of food allergies are on the rise, which can greatly affect the well-being of children as well as adults. Intolerance to gluten, a protein composite of gliadin and glutenin, present in wheat, barley and rye and several cereals, can be the causative agent of coeliac disease (CD) and other allergic reactions. Gluten-free diet became essential for people affected by CD, and consequently, the amount of gluten in food products needs to be strictly controlled. In this paper, we report an electrochemical label-free immunosensor for ultrasensitive and speciﬁc detection of gliadin. The sensor takes benefit of the specific properties of porous reduced graphene oxide (prGO) covalently functionalized with anti-gliadin antibodies using 1pyrenecarboxylic acid as linker molecule. Using differential pulse voltammetry (DPV) and [Fe(CN)6]3-/4- as redox probe, a decrease of current is linked to the presence of gliadin. The sensor achieved a detection limit of 1.2 ng mL-1 over 1.2-34 ng mL-1 linear range with high selectivity. The advantages offered by this sensor are the possibility to regenerate the surface of the immunosensor, its rapid and ease of production, as well as it applicability for the screening of gliadin concentrations in real food samples, as shown here. KEWORDS: gliadin; anti-gliadin antibody, immunosensor; porous reduced graphene oxide; food samples

*

Corresponding authors: [email protected] and [email protected]

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Allergic reactions to food, affecting skin, the respiratory and/or gastrointestinal tract, and in some cases the cardiovascular systems, are on the steady increase. Most food allergens, defined as the substrates that cause an adverse immune response,1 are proteins and in the most majority of cases are due to food from nuts, fish, wheat, egg or milk.2 While the EU has strict labeling rules for 14 allergens, there are currently no threshold levels for food allergens.3 A common allergen found to be responsible for allergic reaction is gluten. The two main components of the gluten protein are gliadin, an alcohol-soluble 36 kDa protein, and glutenin protein aggregates of high molecular mass and low molecular mass subunits (200.000-few millions kDa).4-5 People with coeliac disease (CD) display a severe immune response upon the ingestion of gluten containing food (e. g. wheat, oats, barley, etc), which ultimately results in chronic inflammation and damage of the upper part of the small intestine.6 As the only treatment of CD is a gluten-free diet, the reliable labeling of gluten-free products became essential. Currently, there is no general agreement yet on the term of “gluten-free”.7 Based on the study by Catasse et al.,8 a maximum content of 20 ppm (20 mg gluten/1 kg food) is allowed since January 2013 in “gluten-free” products and considered safe in Europe, as per Codex Alimentarius9. However, the Spanish Federation of Celiac Associations (FACE) recommends a 10 ppm maximum in order to deem a food product as gluten-free. In Japan, 10 ppm gluten is the current maximum limit,10 while in Australia, the gluten limit currently allowed in glutenfree food is only 3 ppm and is determined by the current detection limit of analytical methods there. One limitation linked to the establishment of the maximum gluten level allowed in “allergen–free” products is linked to the sensitivity of the analytical methods in place. As a minimal amount of allergens in food can induce severe immunological reactions in some people, successful allergy management requires the development of highly sensitive detection schemes for allergens in food products. Consequently, the quantification of gliadin for proper food labeling and consumer protection became of high importance. The current trend in allergen detection is to lower the detection limit of the analytical methods. Enzyme based immunoassays are currently the CODEX standard method for gluten analysis in foods labeled as “gluten free”. Different antibodies are used in such assays and their degree of specificity has prevented the establishment of a single standardized method.7 One of the commercial available gliadin kids such as the one from R-Biopharam (Table 1) uses monoclonal R5 antibodies, which react with the gliadin fractions from wheat and corresponding prolamines form rye and barley has a detection limits for gliadin of 0.5 ppm and a limit of quantification of 2.5 ppm. Independent on the assay used, enzyme amplification is costly and the reaction is time consuming, making the search for alternative analytical platforms timely. Over the years, electrochemical biosensors have become accepted platforms for fast and efficient sensing of proteins,11-12 and have emerged as potential alternatives, either for the diagnosis of CD or for the detection of gliadin in food.7, 13-25 In most cases the final goal was the detection of antibodies considered as biomarkers of CD; however similar sensor architectures can be used to detect gliadin in food (Table 1). Most electrochemical gliadin sensors use amplification strategies to obtain reasonable detection limits. The only two direct assays are those proposed by Nassef et al.21 and Eksin et al.23 The label-free electrochemical gliadin immunosensor proposed by Nassef et al.21 is based on the self-assembly of anti-gliadin Fab fragments onto gold electrodes with a detection limit of 420 ng mL-1 (11 nM). Eksin et al proposed a disposable pencil graphite electrode with a detection limit of 7.11 µg mL-1 using the oxidation signal of gluten. 23 Porous carbon materials have been proposed in the literature as excellent electrode materials for the development of sensitive immunosensors.26 Porous electrode materials display unique 2 ACS Paragon Plus Environment

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advantages for electrochemical sensors as the resident porosity increases the specific surface area of the electrode allowing the immobilization of a high amount of ligands, which will increase the sensitivity of the sensor. In addition, it promotes diffusion of the analyte through interconnected pores, allowing fast sensing. Porous materials from graphene have several advantages over other porous carbon structures such as high mechanical strength which can help to prevent the collapse of the porous structures, excellent chemical stability and excellent electrochemical properties.27-28 Porous reduced graphene oxide (prGO) possesses thus all the advantages of rGO nanosheets for electrochemical sensing29-34 such as increased active area and facilitated mass transport, both being advantageous for sensitive sensing. While prGO architectures have been widely used for the fabrication of high-performance energy storage devices,27, 35-36 the use of prGO for sensing is limited to some reports. Han et al. demonstrated the benefits of prGO for gas sensing.37 The interest for non-enzymatic glucose sensing using metallic nanoparticle decorated prGO has been reported by Li et al.38 We demonstrate here, that rGO modified glassy carbon electrodes (GCE), chemically modified with 1pyrenecarboxylic acid39 onto which anti-gliadin antibodies were covalently linked show high sensitive and selectivity to gliadin and can be used for the detection of gliadin in food samples. In this work, we demonstrate the interest of a porous reduced graphene oxide (prGO) modified electrode, formed by using hydrogen peroxide as etching agent, for the direct electrochemical detection of gliadin. To exemplify the potential of a prGO based gliadin sensor, commercial available polyclonal anti-gliadin antibodies were immobilized onto the prGO modified electrodes (Figure 1A). In this configuration, the sensor is particularly useful for the verification of gluten contamination of unprocessed foods such as different flours.

Experimental Materials Gliadin, polyclonal rabbit anti-gliadin antibody, 1-pyrenecarboxylic acid (PCOOH), Nhydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and potassium hexacyanoferrate(II) ([K4Fe(CN)6]), gliadin, hydrogen peroxide (H2O2), hydrazine monohydrate, phosphate buffer tablets (PBS, 0.1 M) and bovine serum albumin were purchased from Aldrich and used as received. Graphene oxide (GO) powder was purchased from Graphenia, Spain. Seven different food samples (wheat flour, pasta, quaker oats, cereal, rice flour, corn flour, gluten-labeled food) were purchased from local supermarket (Lille, France). Preparation of Porous Reduced Graphene Oxide (prGO) Reduced graphene oxide (rGO) was prepared from GO precursor using hydrazine reduction. Briefly, to 5 mL GO aqueous suspension (0.5 mg/mL) was added hydrazine hydrate (0.50 mL, 32.1 mM) and was heated in an oil bath at 100 °C for 24 h over which the reduced GO gradually precipitated out the solution. The product was isolated by filtration over a PVDF membrane with a 0.45 µm pore size, washed copiously with water (5×20 mL) and methanol (5×20 mL) and dried in an oven at 100°C over night. The synthesis of prGO is based on a previous work by some of us.40 rGO power (100 mg) was dispersed in H2O2 (100 ml ; 30 %), ultrasonicated for 30 min and the mixture was refluxed for 12 h at 60 0C. The obtained solution was filtered and the recovered prGO powder was dialysed to remove H2O2 and to separate from small sized graphene quantum dots. Fabrication of Gliadin Sensitive Electrochemical Immunosensor 3 ACS Paragon Plus Environment

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Prior to surface functionalization, glassy carbon (GC) electrodes were polished (aqueous slurries of α-alumina (0.05 µm) on a polishing cloth) and sonicated in deionized water for 10 min, before being dried under nitrogen. An aqueous suspension of prGO (1 mg mL-1; 5 µL) was drop-casted onto the GCE and allowed to dry. The GC-prGO electrode was further immersed into an aqueous solution of 1-pyrenecarboxylic acid (1 mM) for 1 h at room temperature. Anti-gliadin antibodies were immobilized on GCprGO by first activating the carboxyl groups via immersion into a solution of EDC (15 mM)/NHS (15 mM) in PBS (0.1 M pH 7.4) for 30 min, followed by covalent coupling of the anti-gliadin antibodies (6 µL, 7.5 mg/mL concentration in PBS) by incubation for 40 min and washing (3 times) with PBS. The GC-prGO-antibody interface was then immersed into a solution of BSA (2 w/v %) in PBS (0.1 M, pH 7.0) for 1 h and washed (3 times) with PBS (0.1 M; pH 7.0). Electrochemical Measurements Electrochemical measurements were performed with a potentiostat/galvanostat (Autolab, The Netherlands). A conventional three-electrode configuration was employed using a silver wire, and a platinum mesh as reference and auxiliary electrodes, respectively. Differential pulse voltammograms of 5 mM Fe(CN)6]3-/4- solution in 0.1 M PBS were recorded within the potential range from -0.1 V to 0.5 V under modulation amplitude of 25 mV with a step potential of 5 mV. Characterization Transmission electron microscopy (TEM) analysis of the samples was performed by dispersing 1 mg of the material in 5 mL of isopropyl alcohol (IPA) and drop-casting 5 µL of the resulting solution on a 200 mesh Cu grid. The material coated Cu grid was dried under ambient conditions and imaged using a FEI-TECNAI G2 F20 TEM instrument operated at an accelerated voltage of 200 kV (Cs = 0.6 mm, resolution 1.7 Å). Raman analysis was performed on a HR 800 Raman spectrometer (Jobin Yvon, Horiba, France) using a 632 nm red laser (NRS 1500 W) as excitation source. X-ray photoelectron spectroscopy (XPS) was performed in a PHl 5000 VersaProbe-Scanning ESCA Microprobe (ULVAC-PHI, Japan/ USA) instrument at a base pressure below 5×10-9 mbar. Core level spectra were acquired at pass energy of 23.5 eV with a 0.1 eV energy step. All spectra were acquired with 90° between X-ray source and analyzer and with the use of low energy electrons and low energy argon ions for charge neutralization. After subtraction of the linear background, the core-level spectra were decomposed into their components with mixed Gaussian–Lorentzian (30:70) shape lines using the CasaXPS software. Quantification calculations were conducted using sensitivity factors supplied by PHI. Scanning electron microscopic images of the films were obtained using an electron microscope ULTRA 55 (Zeiss). Extraction procedure Two different extraction procedures were used. For the extraction of flours and no heatprocessed food, ethanolic extraction can be used.21Food samples to be analyzed were first crushed using a mortar and a pestle, and the wheat proteins extracted twice with 60% ethanol solution. Flour samples (1g) were mixed with water (10 mL) for 1 h under stirring. After centrifugation at 5000 rpm for 10 min a pellet was formed. Gliadin was extracted from this pellet by immersion into ethanol (60%, 10 mL) for 1 h, centrifuged (30 min) and the supernatant used for analysis. The ethanolic solutions were further diluted with PBS buffer before analysis with the immunosensor.


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The other approach consisted in using the Mendez extraction cocktail to extract gliadin from different food samples. This approach can be used for the extraction of flours as well as for heat-processed food. The cocktail consist of 60% ethanol (v/v) , 5 mM dithiothreitol and 6 % SDS in PBS pH 7. 1g of the grounded sample was mixed with 10 mL of this cocktail and incubated in an oven at 50°C for 40 min. The samples were centrifuged for 30 min and the supernatant used for analysis. The ethanolic solutions were further diluted with PBS buffer before analysis with the immunosensor. The sample solution was incubated with the immunosensor for 30 min. After incubation the immunosensor was washed with 0.1 M PBS buffer and immersed in a solution of 5 mM [Fe(CN)6]3-/4- in 0.1 M PBS for the electrochemical measurement. A calibration curve using gliadin (Sigma-Aldrich) standards was established to determine the concentration. The results were compared to a commercial ELSIA kit for gluten (RBiopharm, Germany).

RESULTS AND DISCUSSION Porous Reduced Graphene Oxide Modified Glassy Carbon Electrodes (GC-prGO) Chemical etching using KOH, HNO3 and other bases are widely used for the preparation of porous carbon materials.28 As illustrated in Figure 1B, we opted for the treatment of reduced graphene oxide (rGO) with hydrogen peroxide (H2O2) at elevated temperature, generating oxidizing free radicals such as OḢ•, O• and HO2• which result in the formation of an increasing amount of epoxy groups on rGO. Subsequent hydrolysis of the epoxy groups to hydroxyl groups with subsequent breaking of C-C bonds results in porous structures as evidenced in the TEM images in Figure 1C. While rGO displays transparent continuous nanosheets, uniformly distributed nanopores (4-6 nm in diameter) are observed after H2O2 treatment. Raman analysis of rGO and prGO was in addition performed (Figure 1D) and revealed the introduction of defects in the graphene framework after H2O2 treatment. While for rGO a ID/IG ≈ 0.90 was determined, the alue increased to ≈ 0.98 in the case of prGO due to the creation of pores. XPS was further employed to identify the change in chemical composition of rGO upon H2O2 treatment. Figure 1E shows the C1s high-resolution image of rGO and prGO. The C1s core level XPS spectrum of rGO displays the characteristic bands at 284.6 eV (sp2-hybridized carbon), 286.2 eV (C-O) and 288.5 eV (C=O) with a C/O ratio of 3.3. In the case of prGO, the C1s can be deconvoluted into bands at 284.6 eV (sp2-hybridized carbon), 285.9 eV (C-O), 288.3 eV (C=O) and an additional small component at 291.5 eV (OC=O). Figure 2A exhibits the cyclic voltammetric responses of GC electrode before and after coating with rGO and prGO using [Fe(CN)6]3-/4- as the redox couple. The redox current increased considerably in the presence of one layer of drop-casted prGO, being higher than that recorded for rGO formed under the same conditions. The increased current correlates with an increased surface area and good electronic properties of prGO. Interestingly, deposition of several layers of prGO by drop-casting did not show a positive effect on the electrochemical behavior (Figure S1). This suggests that for thicker films, the advantage of the porous material is masked due to an overlap of prGO nanosheets. The SEM images in Figure 2B indicate indeed that the morphology changes considerably depending on the amount of prGO layers deposited. In the following, one layer of prGO drop-casted onto GCE was selected for the sensor development. Gliadin Immunosensor Design Aromatic hydrocarbons such as pyrene derivatives tend to strongly adsorb on graphene based structures through π-π interactions.39, 41 We used 1-pyrenecarboxylic acid (PCOOH) for the 5 ACS Paragon Plus Environment

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integration of carboxylic functions onto GC-prGO modified electrodes (Figure 1A) as previously reported by An et al.41 While the lypophilic pyrene part of PCOOH adheres strongly to prGO without disruption the C-C bonds of prGO, keeping thus the electronic conductivity unchanged, the presence of hydrophilic COOH functions increase the wettability of the interface in aqueous media and allows further surface modification to take place The successful functionalization of prGO with PCOOH is confirmed by cyclic voltammetry in acidic medium (Figure 2C).42-43 From integration of the cathodic band, a surface coverage of (1.3±0.5) nmol cm-2 could be determined. The presence of larger amount of COOH is also in agreement with a decreased current response by cyclic voltammetry using the negatively charged [Fe(CN)6]3-/4- redox couple, due to electrostatic repulsion (Figure 2D). In addition, comparing the high-resolution C1s spectrum of prGO and when modified with PCOOH shows a significant increase of the band at 290.2 due to the incorporation of the COOH groups onto prGO (Figure 2E). The other bans at 284.1 (Csp2), 285.0 (C-C/C-H), 286.0 (C-O) and 287.9 (C=O) are comparable, with an increase in the band at 285.0 due to the C-C/C-H bands of pyrene. To make the sensor interface specific for gliadin, anti-gliadin antibodies were covalently linked to the carboxylic acid functions using EDC/NHS chemistry (Figure 1A). A major concern inherent to any detection biosensors is the possibility of background interferences due to non-specific adsorption of other analytes. This is even more pronounced on graphene-based materials because of their large surface area and rich π-conjugation structure. Before using the sensor for gliadin sensing, the interface was further immersed into a bovine serum albumin (BSA) solution (2 wt. %).30 The cyclic voltammogram of the GC-prGO-antibody electrode after BSA blocking is depicted in Figure 2D. Covalent integration of anti-gliadin antibodies results in a decrease in charge transfer due to the formation of an insulating barrier by the antibodies. Electrochemical Sensing of Gliadin To determine whether the GC-prGO-antibody electrode can be used for analysis of gliadin, the change in peak current upon gliadin addition was measured using differential pulse voltammetry (DPV) (Figure 3A). DPV offers the advantage that the measurement of the faradic current is performed after the double layer charging process has subsided, resulting in a favorable signal-to-noise rate for low analyte concentrations. When gliadin binds to the surface functionalized with anti-gliadin antibodies, a significant reduction in redox signal is observed (Figure 3A). This decrease in current is stronger on GC modified with prGO than with rGO (Figure 3B), indicating the advantage of the prGO material. In the case or GC-prGO-antibody electrodes under optimized conditions, a linear relationship in the range of 1.2-34 ng mL-1 gliadin was recorded with a correlation coefficient of 0.998 according to i(µA) = 501 – 11.5 [gliadin] (ng mL-1) (Figure 3C). The limit of detection (LOD) for gliadin was ≈1.2±0.5 ng mL-1 from five blank noise signals. For a GC electrode modified with rGO the LOD was 2.1±0.6 ng mL-1. Moreover, this electrode shows a decreased linear range (12.1-25 ng mL-1) with correlation coefficient of 0.998 according to i(µA) = 504 – 5.4 [gliadin] (ng mL-1) (Figure 3C). The performance of the sensor is comparable to other electrochemical sensors using different signal amplification strategies (Table 1). Compared to the label-free impedimetric approach proposed by Nassef et al. using anti-gliadin Fab fragments20 attached to gold electrodes (LOD of 0.42 µg mL-1), the GCprGO-antibody electrode exhibits improved sensitivity (Table 1). The gliadin sensor is also competitive compared to another gliadin immunosensor proposed by the same group,21 where depending on the thiol linker used to attach the capture antibody 6 ACS Paragon Plus Environment

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gliadin, detection limits of 5.5 and 11.6 ng mL-1 could be obtained. However, such a detection limit is only achieved using signal amplification. Compared to this and other sandwich assays, our approach presented here relies on the direct detection of gliadin, eliminating the necessity of enzymatic amplification, which brings improvement in analysis time and costs. The sensing interface can be regenerated for multiple testing, decreasing furthermore the costs of the analysis. Regeneration of the electrode is achieved upon immersion of the immunosensor in NaOH-H3PO4 (pH 12.0) solution for 10 min. The gliadin interaction with the anti-gliadin antibody is disrupted under these conditions, without damaging the performance of the sensor (Figure 4A). After 5 regeneration/sensing cycles, the DPV response to a 20 ng mL-1 gliadin solution decreased by only 7 %. The good sensitivity of the biosensors proposed allows a sample dilution factor of 1:500 to 1:1000, which is reasonable with regards to introduction of errors and which allows avoiding both electrochemical interferences and problems due to reagents in the extraction cocktail potentially affecting the gliadin-antibody affinity recognition. The long-term stability test of the sensor when stored at 4 °C for 2 months showed a loss of 5 % when tested upon the addition of 20 ng mL-1 gliadin. The reproducibility of the electrode fabrication for gliadin sensing, expressed in terms of relative standard deviation, was determined to be 5.2 % at a gliadin concentration of 20 ng mL-1. To evaluate the effect of potential interfering compounds, we compared the sensor’s response towards gliadin with the signal obtained with lysozyme, casein, as well as other allergen proteins. As seen from Figure 4B, the biosensor displayed excellent selectivity. Gliadin Sensing in Real Samples To test the feasibility of the developed gliadin sensor for the analysis of non-fermented food, rice flour and gluten-free labeled wheat flour were spiked with different gliadin concentrations and analyzed using the GC-prGO-antibody electrode (Table 2). The results were compared to those recorded using a commercial ELISA kit. The recovery values presented in Table 2 show excellent results using Mendez extraction followed by both detection approaches. Comparable results were obtained using ethanolic extraction (Table S1) The applicability of the electrochemical assay was tested on other commercial samples (Table 3). Significant gliadin levels were detected in wheat flour, pasta, quaker oats and cereal as expected. In particular, the sensor could distinguish between gluten free wheat flour and gluten containing wheat flour making the sensor well adapted for gliadin sensing. CONCLUSIONS In conclusion, the interest of porous reduced graphene oxide (prGO) modified electrodes for gliadin sensing was demonstrated. Covalent integration of anti-gliadin antibodies using carboxylated pyrene as a linker molecule resulted in a selective gliadin interface with a ≈1.2±0.5 ng mL-1 detection limit for gliadin, appropriate for sensing in real food samples labeled as gluten-free. The possibility of directly measuring gliadin concentrations without enzymatic or other amplifications makes the proposed sensors of interest and brings improvement in analysis time and costs related to amplification-base assays. Manual steps such as drop-casting the prGO are amendable to mass-production using techniques such as ink-jet printing, which will furthermore reduce the time and costs of the sensor fabrication. An additional advantage of this sensor is its reusability, making the immunosensor fabrication and the use or prGO justifiable.


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While the aim of this work was to show the interest of a prGO modified electrode for gliadin sensing, in the future monoclonal rather than the commercially available polyclonal gliadin antibodies, as used here, can be covalently integrated onto the sensing electrode using the same chemistry developed here. Furthermore, aptamers for intact gliadin and an immunotoxic gliadin peptide, have recently been described,44,45 and might be considered as alternative selectivity elements. Undoubtedly, combining stable and specific biorecognition elements such as aptamers with the sensitive detection offered by GC-prGO electrodes could lead to extending the applications of gliadin detection from simple matrices to heated and hydrolyzed foods and beverages. Acknowledgements R.B. and S.S. gratefully acknowledge financial support from the Centre National de la Recherche Scientifique (CNRS), the Lille1 University and the Hauts-de-France Region. S.S thanks the Institut Universitaire de France (IUF) for financial support. Supporting Information Supporting information available: The following files are available free of charge: Figure S1 showing the influence on the number of drop-casted prGO layers on the cyclic voltammograms. Table S1 reporting the recovery values of gliadin from spiked rice and gluten-free flour using ethanolic extraction.

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12. Leca-Bouvier, B.; Blum, L. J., Biosensors for Protein Detection. Anal. Lett. 2005, 38, 1491-1517. 13. Dulay, S.; Lozano-Sanchez, P.; Iwuoha, E.; Katakis, I.; O’Sullivan, C. K., Electrochemical detection of celiac disease related antitissue transglutaminase antibodies using thiol based surface chemistry. . Biosens. Bioelectron. 2011, 26, 3852-3856. 14. Martin-Fernandez, B.; Miranda-Ordiers, A.; Lobo-Castanon, M. J.; Frutos-Cabanillas, G.; Santos-Alvarez., d.-l.; Lopez-Ruis, B., Colorimetric sensor strips for lead (II) assay utilizing nanogold probes immobilized polyamide-6/nitrocellulose nano-fibers/nets. . Biosens. Bioelectron. 2014, 60, 244-251. 15. Neves, M. M. P. S.; Gonzalez-Garcia, M. B.; Nouws, H. P. A.; Costa-Garcia, A., Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen printed carbon electrodes. Biosens. Bioelectron. 2012, 31, 95-100. 16. Neves, M. P. S.; Gonzalez-Garcia, M. B.; Nouws, H. P. A.; Costa-Garcia, A., An electrochemical deamidated gliadin antibody immunosensor for celiac disease clinical diagnosis. . Analyst 2013, 138, 1956–1958. 17. Pividori, M. I.; Lermo, A.; Bonanni, A.; Algegret, S.; delValle, M., Electrochemical immunosensor for the diagnosis of celiac disease. . Anal. Biochem. 2009, 388, 229-234. 18. Puiu, M.; Moscone, D.; Ricci, F.; Bala, C., A modular electrochemical peptide-based sensor for antibody detection. Chem. Commun. 2014, 50, 8962. 19. Rosales-Rivera, L. C.; Acero-Sanchez, J. L.; Lozano-Sanchez, P.; Katakis, I.; O'Sullivan, C. K., Electrochemical immunosensor detection of antigliadin antibodies from real human serum. Biosens. Bioelectron. 2011, 26, 4471-4476. 20. Nassef, H. M.; Civit, L.; Fragoso, A.; OSullivan, C. K., Amperometric Immunosensor for Detection of Celiac Disease Toxic Gliadin Based on Fab Fragments. . Anal. Chem. 2009, 81, 5299–5307. 21. Nassef, H. M.; Bermudo Redondo, M. C.; Ciclitira, P. J.; Ellis, H. J.; Fragoso, A.; Osullivan, C. K., Electrochemical immunosensor for detection of Celiac disease toxic gliadin in foodstuff. Anal. Chem. 2008, 80, 9265–9271. 22. Amaya-Gonzalez, S.; de-los-Santos-Alvarez, N.; Lobo-Castanon, M. J.; MirandaOrdieres, A. J.; Tunon-Blanco, P., Amperometric Quantification of Gluten in Food Samples Using an ELISA Competitive Assay and Flow Injection Analysis. Electroanalysis 2011, 23, 108 – 114. 23. Eksin, E.; Congur, G.; Erdem, A., Electrochemical assay for determination of gluten in flour samples. Food Chem. 2015, 184, 183-187. 24. Laube, T.; Kergaravat, S. V.; Fabiano, S. N.; Hernandez, S. R.; Alegret, S.; Pividori, M. I., Magneto immunosensor for gliadin detection in gluten-free foodstuff: Towards food safety for celiac patients. Biosens. Bioelectron. 2011, 27, 46-52. 25. Peres, A. M.; Dias, L. G.; Veloso, A. C.; Meirinho, S. G.; Morais, J. S.; Machado, A. A., An electronic tongue for gliadins semi-quantitative detection in foodstuffs. . Talanta 2011, 83, 857–864. 26. Walcarius, A., Electrocatalysis, sensors and biosensors in analytical chemistry based on ordered mesoporous and macroporous carbon-modified electrodes. Trends Anal. Chem. 2012, 38, 79-97. 27. Han, S.; Wu, D.; Li, S.; Zhang, F., Porous graphene materials for advanced electrochemical energy storage and conversion devices. Adv. Mater. 2014, 26, 849-864. 28. Jiang, L.; Fan, Z., Design of advanced porous graphene materials: from graphene nanomesh to 3D architectures. . Nanoscale 2014, 6, 1922. 29. Chen, D.; Feng, H.; Li, J., Graphene oxide: preparation, functionalization, and electrochemical applications. Chem. Rev. 2012, 112 6027–6053.


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30. He, L.; Wang, Q.; Mandler, D.; Li, M.; Boukherroub, R.; Szunerits, S., Detecition of folic acid protein in human serum using reduced graphene oxide electrodes modiifed by folicacid. Biosens. Bioelectron. 2016, 75, 389-395. 31. Wu, S.; He, Q.; Tan, C.; Wang, Y.; Zhang, H., Graphene-Based Electrochemical Sensors. Small 2013, 9, 1160-1172. 32. Ambrosi, A.; Chua, C. K.; Latiff, N. M.; Loo, A. H.; Wong, C. H. A.; Eng, A. Y. S.; Bonannia, A.; Pumera, M., Graphene and its electrochemistry – an update. Chem. Soc. Rev. 2016, 45, 2458-2493 33. Joshi, R. K.; Alwarappan, S.; Yoshimura, M.; Sahajwalla, V.; Nishina, Y., Graphene oxide: the new membrane material, App. Mater. Today 2015, 1, 1-12. 34. Seah, T. H.; Poh, H. L.; Chua, C. K.; Sofer, Z.; Pumera, M., Towards Graphene Applications in Security: The Electrochemical Detection of Trinitrotoluene in Seawater on Hydrogenated Graphene. Electroanal. 2014, 26, 62. 35. Ning, G.; Fan, Z.; Wang, G.; Gao, J.; Qian, W.; Wei, F., Gram-scale synthesis of nanomesh graphene with high surface area and its application in supercapacitor electrodes. . Chem. Commun. 2011, 47, 5976. 36. Ren, L.; Hui, K. N.; Huio, K. S.; Liu, Y.; Qi, X.; Zhong, J.; Du, Y.; Yang, J., D hierarchical porous graphene aerogel with tunable meso-pores on graphene nanosheets for high-performance energy storage. Sci. Rep. 2015, 5, 14229. 37. Han, T. H.; Huang, Y.-K.; Tan, A. T. L.; Dravid, V. P.; Huand, J., Steam etched porous graphene oxide network for chemical sensing. J. Am. Chem. Soc. 2011, 133, 15264– 15267. 38. Li, G.; Huo, H.; Xu, C., Ni0.31Co0.69S2 nanoparticles uniformly anchored on a porous reduced graphene oxide framework for a high-performance non-enzymatic glucose sensor. . J. Mater. Chem. A 2015, 3, 4922. 39. Liu, J.; Tang, J.; Gooding, J. J., Strategies for chemical modification of graphene and applications of chemically modified graphene. J. Mater. Chem. 2012, 22, 12435-12452. 40. Palaniselvam, T.; Kashyap, V.; Bhange, S. N.; Baek, J.-B.; Kurungot, S., Nanoporous Graphene Enriched with Fe/Co‐N Active Sites as a Promising Oxygen Reduction Electrocatalyst for Anion Exchange Membrane Fuel Cells. Adv. Funct. Mater. 2016, 23, 2150-2162 41. An, X.; Butler, T. W.; Washington, M.; Nayak, S. K.; Swastik, K., Optical and sensing properties of 1-Pyrenecarboxylic acid-functionalized graphene films laminated on polydimethylsiloxane membranes. . ACS Nano, 2011, 5, 1003–1011. 42. Bachman, J., C.; Kavian, R.; Graham, D. J.; Kim, D. Y.; Noda, S.; Nocera, D. G.; Shao-Horn, Y.; Lee, S. W., Electrochemical polymerization of pyrene derivatives on functionalized carbon nanotubes for pseudocapacitive electr. Nat. Commun. 2015, 6, 7040. 43. Reuillard, B.; Le Goff, A.; Cosnier, S., Non-covalent double functionalization of carbon nanotubes with a NADH oxidation Ru(II)-based molecular catalyst and a NADdependent glucose dehydrogenase. . Chem. Commun. 2014, 50, 11731. 44. Weng, X. Gaur, G.; Neethirajan, S., Rapid Detection of Food Allergens by Microfluidics ELISA-Based Optical Sensor. Biosensors 2016, 6, 24-34. 45. Chu, P. T.; Wen, H. W., Sensitive detection and quantification of gliadin contamination in gluten-free food with immunomagnetic beads based liposomal fluorescence immunoassay. . Anal. Chim. Acta 2013, 787, 246–253 46. Chu, P. T.; Lin, C. S.; Chen, W. J.; Chen, C. F.; Wen, H. W., 2012. 60, , Detection of gliadin in foods using a quartz crystal microbalance biosensor that incorporates gold nanoparticles. . J. Agric. Food. Chem. 2012, 60, 6483–6492.


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47. Pinto, A.; Nadal-Polo, P.; Henry, O.; Bermudo-Redondo, M. C.; Svobodova, M.; O′Sullivan, C. K., Label-free detection of gliadin food allergen mediated by real-time aptaPCR. Anal. Bioanal. Chem. 2014, 406, 515−524.


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Table 1. Analytical performance of various (bio)analytical assays and sensors for gliadin. Method Type of assay Linear range LOD Ref. a Colorimetric Sandwich R5 ELISA, 0.5 ppm antibody 20 EIS Direct detection, 1-20 µg mL-1 420 ng mL-1 antigliadin Fab fragments 21 DPV Sandwich, antibody 10-60 ng mL-1 5.5/11.6 ng mL-1 -1 23 DPV Direct detection 7.11 µg mL 22 CA Competitive ELISA in a 1.8–27.4 ppb 1.0 ppb FIA setup, antibody 7 CA Competitive aptamer 4.9 ng mL-1 (Gli 1) -1 based assay 0.5 ng mL (Gli 4) 24 CA Competitive magneto 12.5–329.3 ng 5.1 ng mL-1 immunoassay mL-1 20 CA Direct detection, 5-30 ng mL-1 3.29 ng mL-1 antigliadin Fab fragments 25 Potentiometry E-tongue (set of 36 1-2 ppm polymeric membranes) 44 Colorimetric Sandwich; microfluidic 6.25-50 ng mL-1 4.77 ng mL-1 ELISA; antibody 45 Fluorescence Magnetic beads, antibody 10-500 µg mL-1 600 ng mL-1 46 5 QCM Direct detection, 8 ppb 10-2×10 ppb antibody 47 Quantitative Competitive aptamer100 ng mL-1 PCR based assay DPV Direct detection 1.2-34 ng mL-1 1.2±0.5 ng mL-1 This (0.0012 ppm) work a

Product specifications available at http://www.r-biopharm.com/products/food-feed-analysis/allergens/gliadingluten/item/ridascreen-gliadin CA: chronoamperometry; DPV: differential pulse voltammetry; EIS: Electrochemical Impedance Spectroscopy; QCM: quartz microbalance


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Table 2: Recovery values of spiked rice and gluten free flour extracted using Mendez extraction, analysed with the CG-prGO-antibody electrode and using the commercial sandwich ELISA kit (R-Biopharm). In all cases, n=3 replicate measurements.

Spiking con. Final con. ng mL-1 ng mL-1 Electrochemical sensor 0 9.8 10 19.8 20 29.8 30 39.8 Sandwich ELISA (R-Biopharm) 0 10.4 10 20.4 20 30.4 30 40.4 Spiking con. Final con. ng mL-1 ng mL-1 Electrochemical sensor 0 1.5 10 11.5 20 21.5 30 31.5 Sandwich ELISA (R-Biopharm) 0 2.0 10 12.0 20 22.0 30 32.0

Rice flour sample found concentration / ng mL-1

Recovery / %

9.8±2.1 19.5±2.2 29.9±1.9 39.4±2.4

N/A 98 100 98

10.4±4.1 20.7±3.2 30.2±3.9 40.5±4.1

N/A 1014 99 100

Gluten-free flour found concentration / ng mL-1

Recovery / %

1.5± 0.5 11.9± 0.9 22.0± 0.5 31.2± 0.5

N/A 103 102 99

2.4±1.1 12.8±1.5 22.8±1.1 32.1±1.8

N/A 106 104 100

Table 3. Concentration of gliadin in food samples determined using the developed GC-prGOantibody gliadin sensor or a commercial ELISA kit. Gliadin concentration Food sample CG-prG-antibody ELISA Wheat flour 52±3 µg mL-1 56±2 µg mL-1 Pasta 40±1 µg mL-1 42±4 µg mL-1 -1 Quaker oats 18±3 µg mL 17±1 µg mL-1 Cereal 39±3 µg mL-1 42±3 µg mL-1 -1 Gluten-free wheat flour 1.5± 0.5 ng mL 2.2±1.1 ng mL-1


ACS Sensors

(A)

prGO

GC

PCOOH

GC-prGO

GC-prGO-COOH 1. EDC/NHS 2. anti-gliadin 3. BSA

initial

Gliadin

+ gliadin

GC-prGO-antibody

(B)

(C) rGO rGO

(D) prGO

prGO

intensity / a. u.

1000

2000

3000

4000

100 nm

Raman shift / cm

100 nm

-1

Figure 1: (A) Concept of the label-free electrochemical gliadin immunosensor; (B) Schematic illustration of the synthesis of porous reduced graphene oxide (prGO); (C) Transmission electron microscopy (TEM) images of (a) reduced graphene oxide (rGO), (b) porous reduced graphene oxide (prGO); (D) Raman spectra of rGO (black), prGO (blue) (A) 100 50 i / µA

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GC G C -rG O G C -prG O

0 -50

-100

-0,2

0

0,2

0,4

0,6

E /V

(B)

(C)

(D)

(E)


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100

1 00

G C -prG O G C -prG O -PC O O H 50 G C -prG O -antib ody

i / µA

50 i / µA

0

prGO-PCOOH

0 -50

-50

prGO -1 00

-100 0

0,1 0,2

0,3 0,4 0,5 0,6 E/V

-0 ,2

0,7

0

0 ,2

0,4

0,6

292 290 288 286 284 282 280 binding energy / eV

E /V

Figure 2: (A) Cyclic voltammograms recorded on GC (black), GC-rGO (grey) and GC-prGO (green) using (A) [Fe(CN)6]3-/4- (5mM)/PBS (0.1 M), scan rate = 50 mV s-1; (B) SEM images of prGO modified GC electrodes; (C) Cyclic voltammogram of GC-prGO-PCOOH in HClO4 (0.1M) scan rate = 50 mV s-1; (D) Influence of the surface chemical steps on the electrochemical behaviour of the interface: cyclic voltammograms recorded on GC-prGO (green), GC-prGO-PCOOH (grey) and GC-prGO-antibody (black) using [Fe(CN)6]3-/4(5mM)/PBS (0.1 M); scan rate = 50 mV s-1; (E) C1s core level spectra for GC-prGO and GCprGO-PCOOH (A)

(B)

G C-prG O -antibody

500

G C-rG O -antibod y 500

-1

400

+ 10 ng m L

300

+ 20 ng m L

-1

-1

+ 1 ng m L -1 + 10 n g m L

400

-1

-1

i / µA

i / µA

+ 1 ng m L

200

+ 20 ng m L -1 + 40 ng m L

300 200

+ 40 ng m L

100 0 -0,1

0

0,1

0,2

0,3

0,4

-1

100 0 -0,1

0,5

0

0,1

0,2

E /V

0,3

0,4

0 ,5

E /V

(C)

(D)

600 GC-prGO-antib ody GC-rG O-antibo dy

480

400 i / µA

i / µA

500

300 200

440 400 G C-prGO -antibody G C-rG O -antibody

100 0

360

0

10

20

30

40

50

60

[gliadin] / ng mL

70

0

80

2

-1

4

6

8

[gliadin] / ng mL

10

-1

Figure 3: (A) Differential pulse voltammograms of GC-prGO-antibody electrodes in [Fe(CN)6]3-/4- (5 mM)/PBS (0.1 M) upon the addition of gliadin; (B) Differential pulse voltammograms of GC-rGO-antibody electrodes in [Fe(CN)6]3-/4- (5 mM)/PBS (0.1 M) upon the addition of gliadin; (C) Change of current as a function of gliadin concentration for GCprGO-antibody (•) and GC-prGO-antibody (•), (D) calibration curve zoomed to low concentrations

(B) 300

250

250

200

200

50

0

rice flour

100

50

casein

150

100

lysozyme

150

gliadin

i / µA

300

corn flour

(A) i / µA

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0

0 1 2 3 4 5 regeneration/sensor cycles

0

1 2 3 different sam ples

4

Figure 4. (A) Influence of regeneration step on the oxidation current detected on GC-prGOantibody electrodes using [Fe(CN)6]3-/4- (15mM)/PBS (0.1 M) upon addition of 20 ng mL-1 gliadin; (B) Detected current by DPV on GC-prGO-antibody electrodes in [Fe(CN)6]3-/4- (5 mM)/PBS (0.1 M) upon the addition of gliadin (20 ng mL-1), lysozyme (20 ng mL-1); casein 15 ACS Paragon Plus Environment

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(20 ng mL-1); rice flour (20 ng mL-1); corn flour (20 ng mL-1) extracted in Mendez extraction cocktail.


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TOC graphic

no gluten

+ gluten


Reduced Graphene Oxide Modified Electrodes for Sensitive Sensing

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