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
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
References (47)
- et al.
Mammalian cell-based biosensors for pathogens and toxins
Trends in Biotechnology
(2009) - et al.
Characterisation of the cellular reduction of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT): Subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction
Archives of Biochemistry and Biophysics
(1993) - et al.
Human skin penetration of selected model mycotoxins
Toxicology
(2012) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Analytical Biochemistry
(1976)Update of survey, regulation and toxic effects of mycotoxins in Europe
Toxicology Letters
(2002)- et al.
Application of the assay of aflatoxins by liquid chromatography with fluorescence detection in food analysis
Journal of Chromatography A
(2000) - et al.
High throughput cellular biosensor for the ultra-sensitive, ultra-rapid detection of aflatoxin M1
Food Control
(2013) - et al.
Phenylpyrrole-resistance and aflatoxin production in Aspergillus parasiticus Speare
International Journal of Food Microbiology
(2008) - et al.
A non-enzymatic nanomagnetic electro-immunosensor for determination of Aflatoxin B1 as a model antigen
Sensors and Actuators B: Chemical
(2013) - et al.
Application of biomarkers to assessment of risk to human health from exposure to mycotoxins
The Microchemical Journal
(1996)
Application of “membrane-engineering” to bioelectric recognition cell sensors for the ultra-sensitive detection of superoxide radical: A novel biosensor principle
Analytica Chimica Acta
Superoxide determination using membrane-engineered cells: An example of a novel concept for the construction of cell sensors with customized target recognition properties
Sensors and Actuators B: Chemical
Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays
The Journal of Immunological Methods
Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles
Cell
Effect of cell electroporation on the conductivity of a cell suspension
Biophysical Journal
Enzyme-Linked Immunosorbent Assay (ELISA) based on superparamagnetic nanoparticles for aflatoxin M1 detection
Talanta
Immunoassay based on monoclonal antibody for aflatoxin detection in poultry feed
Food Chemistry
Aflatoxin B1 and total aflatoxins in peanut butter, pistachio paste, fig paste and paprika powder – Immunoaffinity column liquid chromatography with post-column derivatization
Aflatoxins in corn, raw peanuts and peanut butter, liquid chromatography with post-column photochemical derivatization
Electrochemical Methods: Fundamentals and Applications
Analysis of simulated reversible cyclic voltammetric responses for a charged redox species in the absence of added electrolyte
The Journal of Physical Chemistry
Handbook of toxic fungal metabolites
Aflatoxins in food: Occurrence, biosynthesis, effects on organisms, detection and methods of control
Food Science & Nutrition
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