Elsevier

Food Control

Volume 73, Part A, March 2017, Pages 64-70
Food Control

Development of a cellular biosensor for the detection of aflatoxin B1, based on the interaction of membrane engineered Vero cells with anti-AFB1 antibodies on the surface of gold nanoparticle screen printed electrodes

https://doi.org/10.1016/j.foodcont.2016.06.002Get rights and content

Highlights

  • Aflatoxin B1 cytotoxicity on Vero cells was assessed.

  • Cell modification with the anti-Aflatoxin B1 antibody through membrane engineering.

  • Cell culture on Screen Printed Electrodes surface.

  • Development of a mammalian cell biosensor for aflatoxin B1 detection.

Abstract

The development of methods for the detection of aflatoxin B1 (AFB1) in foods is a very important practice for ensuring food quality and safety. Most tests of AFB1 are still conducted with conventional methods (i.e. antibody-based ELISA tests, high performance liquid chromatography – HPLC); however biosensor methods are being developed to date as screening tools for field analysis. Compared to immunology/ELISA-like tests or chromatography methods, biosensors are able to provide rapid, sensitive, robust and cost effective quantitative methods for on-site testing. In this work we propose a cellular biosensor based on Vero cells, membrane engineered with anti-AFB1 antibody as the biological recognition element reacting with AFB1 molecules on gold nanoparticle/screen printed electrodes (SPEs) (three electrode system). In order to culture the cells on the SPEs surfaces the working electrodes were coated with poly-l-lysine to facilitate cell adhesion. The SPEs were connected to a potentiostat device through a transducer and chronoamperometric (CA) and cyclic voltammetric (CV) measurements were performed. Quantitative results obtained using the cellular biosensor method for AFB1 were compared to those obtained using the HPLC method in pistachio samples spiked with AFB1. The method displayed good sensitivity (r2 = 0.87) and detection limit (0.5 ng/mL).

Introduction

Mycotoxins are toxic secondary metabolites that can occur in food and feed in conditions that promote mould growth (Boonen et al., 2012). For the purpose of the present study aflatoxin B1 (AFB1) was chosen as it belongs to a group of highly toxic difuranocoumarin derivatives that are produced by many strains of Aspergillus flavus and Aspergillus parasiticus and can contaminate a wide range of foods and animal feedstuffs stored under temperature and humidity conditions favourable to mould growth (Cole and Cox, 1981, Creppy, 2002;; Waliyas & Reddy, 2009). Specifically peanuts, corn and cereal crops, before or after harvest are susceptible to AFB1 contamination (Ellis et al., 1991, Radoi et al., 2008). AFB1 is particularly interesting for analysis because of the current regulatory decisions (European Commission regulation (EC) No 1881/2006, 2006, FAO, 2001, FAO, 2004 and FAO/WHO, 2007) according to which the allowable level for aflatoxins in Europe is just 4 mg/kg (ppm) of total aflatoxin or 2 ppm of aflatoxin B1. The International Agency for Research on Cancer (IARC) has classified aflatoxin B1 as a group 1 human carcinogen (IARC, 1993). This toxin exhibits carcinogenic, teratogenic and mutagenic properties and has been isolated from a wide variety of agricultural products (Moss, 2002). AFB1 can enter the food chain mainly by ingestion via the dietary route in humans and animals; it has been shown that the intake of AFB1 over a long period of time, even at very low concentrations may be highly dangerous (Miraglia, Brera, & Colatosti, 1996).

Many analytical methods have been developed for the determination of aflatoxins including thin-layer chromatography (TLC) (Fernandez, Belio, Ramos, Sanz, & Saez, 1997;; Ramesh, Sarathchandra, & Sureshkumar, 2013), antibody-based, indirect Enzyme Linked Immunosorbent Assays (ELISA) (Rossi et al., 2012;; Sun, Gu, Li, & Dong, 2015), often coupled with lateral flow devices (Maragos & Busman, 2010) and high-performance liquid chromatography (HPLC) (Jaimez et al., 2000;; Yazdanpanah et al., 2013). According to Kovacs, cell-based sensors utilize the ability of cells to selectively respond to complex mixtures of signals in a way that makes them highly attractive for detection of chemical and biological analytes, for detection of environmental toxins and for drug screening (Kovacs, 2003). A previous study from Rasooly et al. has shown that low levels of AFB1 stimulate monkey Vero kidney cells, whereas high levels kill the cells (Rasooly, Hernlem, & Friedman, 2013).

The purpose of the study was the development of a cell based biosensor with Vero cells (plain or membrane engineered with anti-AFB1 antibodies) as the biosensor’s biological part for the detection of AFB1 in pistachio matrices. In the present work, anti-AFB1 antibodies were inserted to Vero cell membranes through electroinsertion and osmotic insertion, in order to be used as the biosensor’s biological part.

The results were compared afterwards with the cellular biosensors’ results. The biosensor was based on a potentiometric method (cyclic voltammetry, chronoamperometry) employing gold nanoparticle-modified carbon screen printed electrodes for the determination of AFB1 with plain Vero and Vero-anti-AFB1 based cellular biorecognition elements. In the near future they will play a key role in the field of cellular biosensors of high selectivity and specificity. In addition, Vero cell viability and total protein concentration have been assessed. In the second part, an analytical method was developed for the determination of AFB1 in pistachio and nut matrices after extraction by the immunoaffinity column (IAC) method.

Section snippets

Reagents

Vero cell culture was originally purchased from LGC Promochem (Teddington, UK). Dulbecco’s Modified Eagle’s Medium, fetal bovine serum, l-glutamine, penicillin/streptomycin, and trypsin/EDTA were purchased from Biochrom AG (Berlin, Germany). Dimethylthiazol-2-2,5 diphenyl tetrazolium bromide (MTT) was provided from MP Biomedicals (Solon, OH) and anti-AFB1 mouse monoclonal IgG (200 μg/mL) from Santa Cruz Biotechnology Inc (Dallas, TX). The Immunoaffinity columns (IAC) AflaClean™ (3 mL widebore)

Cell viability after anti-AFB1 antibodies insertion

The viability of Vero cells after modification with anti-AFB1 antibodies with the methods of electroporation (A) and osmotic insertion (C) are shown in Fig. 2. The cell viability was measured with the MTT assay (Mosmann, 1983) after the modification with six different anti-AFB1 antibody concentrations (0, 1, 1.4, 2, 6 and 10 μg/mL). Cells that did not undergo any modification were used for comparison (-elec/ion, -osm.ins.) with the electroporated and the osmotically modified cells. The results

Conclusions

Cell based detection systems nowadays are promising approaches for sensing the functionality or biological activity of an analyte (e.g. toxin) by monitoring perturbations in physiological activities of mammalian cells after exposure (Banerjee & Bhunia, 2009). This study shows that the prototype cell-based biosensor is a potentially very useful device that offers the unique possibility to detect and semi-quantify one of the most potent environmental toxins AFB1. The LOQ and LOD of the cell

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This research project is funded under the Action “Research & Technology Development Innovation projects (AgroETAK)”, MIS 453350, in the framework of the Operational Program “Human Resources Development”. It is co-funded by the European Social Fund and by National Resources through the National Strategic Reference Framework 2007–2013 (NSRF 2007–2013) coordinated by the Hellenic Agricultural Organisation “DEMETER”.

Glossary

AFB1
Aflatoxin B1
AuNPs
Gold Nanoparticles
BERA
Bioelectric Recognition Assay, Bioelectric Recognition Assay
CA
Chronoamperometry
CV
Cyclic Voltammetry
DMEM
Dulbecco’s Modified Eagle’s Medium
DMSO
Dimethyl Sulfoxide
ELISA
Enzyme Linked Immunosorbent Assay
FBS
Foetal Bovine Serum
GNP-Carbon electrode
Gold Nanoparticle- Carbon electrode
HPLC
High Performance Liquid Chromatography
IAC
Immunoaffinity Column Method
IARC
International Agency for Research on Cancer
LOD
Limit of Detection
LOQ
Limit of Quantification
MTT

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