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Campylobacter, 3rd edn ASM Press, Washington, DC, pp 263–276 857 Zhang W et al 2008 A new immuno-PCR assay for the detection of low concentration of shiga toxin and its variants J Clin Microbiol 46: 1292–1297 Zhang Y et al 2007 Genome evolution in major Escherichia coli O157:H7 lineages BMC Genomics 16: 121 P1: SFK/UKS BLBS102-c45 P2: SFK BLBS102-Simpson March 21, 2012 14:38 Trim: 276mm X 219mm Printer Name: Yet to Come 45 Biosensors for Sensitive Detection of Agricultural Contaminants, Pathogens and Food-Borne Toxins Barry Byrne, Edwina Stack, and Richard O’Kennedy Introduction Contaminant Monitoring Traditional Means of Assessment Instrumentation-Based Analysis Biosensors Biacore Sensor Surfaces Assay Configuration Antibodies Antibody Production Strategies Optical Immunosensors for Quality Determination Electrochemical Sensors Alternative Biosensor Formats Pathogens Bacterial Pathogens Fungal Pathogens Toxins Mycotoxins Water and Marine Toxins Legislation Conclusion Acknowledgements Websites of Interest References Abstract: Immunosensors permit the rapid and sensitive analysis of a range of analytes Here, we provide a critical assessment of how such formats can be implemented, with emphasis on the detection of bacterial and fungal pathogens, agricultural contaminants (e.g pesticides and herbicides) and toxins INTRODUCTION The monitoring of quality of food destined for human consumption is a key consideration for farmers, the food industry, legislators and, most importantly, for consumers (Karlsson 2004) Hence, it is an absolute necessity to ensure that any contaminants that may have a deleterious effect on human health are monitored qualitatively and quantitatively in a sensitive and reliable manner For example, herbicides and pesticides are extremely effective at suppressing the growth of plant and insect populations on agricultural produce, such as tomatoes and strawberries However, the extensive use of such potentially toxic compounds may compromise the quality of the product, and prolonged exposure may manifest itself as chronic toxicity in human hosts (Keay and McNeil 1998) Furthermore, bacterial strains such as Salmonella typhimurium and Listeria monocytogenes, which are causative agents of salmonellosis and listeriosis, respectively, can act as opportunistic pathogens and cause death through the ingestion of contaminated produce Consequently, the development of suitable methods for their rapid detection is an absolute necessity Finally, there is also an urgent need to accurately monitor the distribution of toxins, including fungal (e.g mycotoxins) and water-borne toxins (e.g phycotoxins), which cause severe illness through the consumption of contaminated food (e.g nuts, shellfish meat) In summary, rapid, sensitive and accurate methodologies are essential for the evaluation of product quality and for satisfying legislative requirements CONTAMINANT MONITORING Traditional Means of Assessment There are several standard methods that are currently used to monitor the quality of food As an example, fruit and vegetable produce may be inspected by monitoring the colour, gloss, firmness, shape and size of the product, as well as noting the presence or absence of visible defects This visual inspection may be performed alongside more invasive methods, including the analysis of the soluble solid content of the product and determining the acidity, and is particularly useful for produce such as apples, pears and berries The main advantage of such tests relates to the fact that they may be carried out immediately post-harvest Food Biochemistry and Food Processing, Second Edition Edited by Benjamin K Simpson, Leo M.L Nollet, Fidel Toldr´a, Soottawat Benjakul, Gopinadhan Paliyath and Y.H Hui C 2012 John Wiley & Sons, Inc Published 2012 by John Wiley & Sons, Inc 858 P1: SFK/UKS BLBS102-c45 P2: SFK BLBS102-Simpson March 21, 2012 14:38 Trim: 276mm X 219mm Printer Name: Yet to Come 45 Biosensors for Sensitive Detection of Agricultural Contaminants, Pathogens and Food-Borne Toxins by the farmer, and at a minimal cost (Mitchum et al 1996) As further examples, grain and nuts may be inspected for the presence of fungal contamination, while bacterial spoilage may be indicated by the presence of an uncharacteristically strong odour, such as in coleslaw and milk However, these tests are not sufficient for providing confirmation to the consumer that the product satisfies regulations with respect to acceptable maximum residue limits (MRLs) Furthermore, it is not possible to provide accurate quantitative or qualitative analysis of individual contaminants through these methodologies, including those that cannot be seen by visual inspection (e.g mycotoxins) Hence, it is common practice for food samples to be removed and sent to an external laboratory where comprehensive in situ analysis may be performed (Giraudi and Baggiani 1994) Instrumentation-Based Analysis There is a selection of different methodologies available for quality determination, and some of the more relevant examples are discussed in this section Product firmness can be determined through the implementation of the Magness-Taylor test, namely a destructive method that assesses the maximum force required to perforate the product in a specific way (Abbott 2004) This has been applied for the analysis of fruit, including pears (G´omez et al 2005) The non-destructive determination of elasticity may also be permitted through the measurement of acoustic responses, with the signal being interrogated using fast Fourier transform-based analysis Shmulevich et al (2003) demonstrated the efficacy of this approach for evaluating the firmness of apples, and monitored product softening over time in a controlled atmosphere environment While these methods are suitable for monitoring the structural properties of the product in question, they not permit rigorous quality evaluation More suitable analytical methods include near-infrared (IR) spectroscopy, implemented by Berardo et al (2005) for the detection of mycotoxigenic fungi and associated toxic metabolites, and scanning electron microscopy, applied for the inspection of ultrastructural changes of the epicuticular layer of oranges treated with fludioxonil, a pesticide used to control the growth of two Penicillium species (Penicillium digitatum and Penicillium italicum) (Schirra et al 2005) Furthermore, gas chromatography (GS) or mass spectrometry (MS) and high-performance liquid chromatography (HPLC) are accurate and highly sensitive methods for the detection of an array of contaminants, including pesticide, herbicide and toxins residues The latter method may also be used to detect the presence of indicator molecules that are representative of product freshness, including flavonoids (MacLean et al 2006), while liquid chromatography coupled with mass spectrometry (LC-MS) can accurately monitor product bitterness This was demonstrated by Dourtoglou et al (2006) for the analysis of olives (Olea europaea) In spite of the efficacy of these analytical platforms, the instrumentation needed to perform this analysis is expensive and bulky and may require extensive operator training In addition, analysis times may also be extensive, as many contaminants require lengthy sample pre-treatment prior to assessment Here, we focus on the application of biosensor-based platforms that are rapid, sensitive and reliable and are frequently used for the detection of herbicide and pesticide residues, bacterial and fungal pathogens and toxins (fungal and water-borne) BIOSENSORS A biosensor can be defined as an analytical device that incorporates a biological element for promoting biorecognition of an analyte of interest (e.g herbicide, bacterial cell or toxin) A schematic representation of a biosensor, illustrating the three main components of the system, namely a bioligand, a transducer and a readout device, is shown in Figure 45.1 Biosensor-based platforms can use a wide selection of different recognition elements, including nucleic acid probes, lectins and antibodies (Table 45.1) The focus in this chapter will be placed on the use of antibodies for detection of contaminants that are of interest to the food industry with examples of enzyme- and nucleic acid-based detection also provided Analytical matrix Antigen Antibody Sensor surface Transducer 859 Computational data analysis Figure 45.1 General format of a biosensor The biorecognition element is in contact with the transducer, which converts the signal to an output shown on the computer For illustrative purposes, an antibody-based platform is shown, with non-specific antigens represented by squares and triangles, respectively P1: SFK/UKS BLBS102-c45 P2: SFK BLBS102-Simpson March 21, 2012 14:38 Trim: 276mm X 219mm 860 Printer Name: Yet to Come Part 8: Food Safety and Food Allergens Table 45.1 The Recognition Elements Commonly Used in Sensor Systems Antibodies and antibody fragments: Derived by enzyme digestion or genetic engineering (Fab fragment, scFv and diabody) Lectins, namely carbohydrate-binding proteins Only suitable for the detection of glycosylated entities, such as glycoproteins Enzymes: e.g those specific for one particular substrate (i.e horseradish peroxidase for hydrogen peroxide) Cell membrane receptors A living cell: eukaryotic or prokaryotic Nucleic acid-based probes: DNA and RNA or peptide nucleic acid Aptamers Chemically generated recognition surfaces: Including plastibodies (artificial antibodies or molecularly imprinted polymers) Fab, fragment antigen binding; scFv, single-chain variable fragment A transducer is a device, such as a piezoelectric crystal or photoelectric cell that converts input energy of one form into output energy of another, with the output signal generated proportional to the concentration of the target analyte (Luong et al 1995) There are many different types of transducers that can be used in biosensor-based platforms, and a selection of these is shown in Table 45.2 Finally, a readout device typically consists of a computer-linked monitor that presents the data in a form that can easily be interpreted by the end-user Many different biosensor platforms have dedicated software packages that can facilitate in the interpretation of biomolecular interactions For example, Biacore, a frequently used com- mercial optical biosensor based on surface plasmon resonance (SPR), which is discussed in more detail later, uses Biaevaluation software that presents data in the form of a sensorgram (Fig 45.2) Here, interactions between immobilised and free entities can be easily visualised by monitoring changes in refractive index (RI) This correlates to a change in mass imparted by the interaction between the two binding elements (e.g antibody and cognate antigen), with units of measurement referred to as response units (RU) In Biacore analysis, a response of 1000 RU is representative of a change in resonance angle of 0.1◦ , which corresponds to an alteration in the surface coverage of the sensor surface of approximately ng/mm2 While Biacore is an excellent example of a biosensor that is commonly used for the evaluation of quality of food (which is discussed in more detail later) other biosensor platforms are also applicable that are based on electrochemical, piezoelectric, magnetic and thermal detection (Byrne et al 2009) Many of these platforms are developed ‘in-house’ and have their own dedicated software packages for the interpretation of data, and key examples of these novel sensors are also described later Finally, for a biosensor to be applicable in any field, including in the monitoring of quality of food produce, it must have certain characteristics These are listed in Table 45.3 Biacore Biacore (GE Healthcare) uses an optical-based transducer system for the measurement of analytes based on the principle of SPR SPR works on the principle of total internal reflection (TIR), a phenomenon that occurs at the interface between two non-absorbing materials, such as water and a solid When a source of light is directed at such an interface from a medium with a higher RI to a medium of lower RI (such as light travelling through glass and water), the light is refracted to the interface Table 45.2 Examples of Transducers Commonly Used in Biosensor Systems Type Electrochemical Example Conductimetric Potentiometric Voltammetric Field effect transistor-based Field effect transistor Optical Surface plasmon resonance Thermal Calorimetry Surface acoustic wave Piezoelectric Rayleigh surface wave Electrochemical quartz crystal microbalance Principle of Use Solutions containing ions conduct electricity Depending on the reaction, the change in conductance is measured Measurement of the potential of a cell when there is no current flowing to determine the concentration of an analyte A changing potential is applied to a system and the resulting change in current is measured A current flows along a semi-conductor from a source gate to a drain A small change in gate voltage can cause a large variation in the current from the source to the drain Surface plasmon resonance (a detailed explanation is given in this chapter) Heat exchange is detected by thermistors and related to the rate of a reaction An immobilised sample on the surface of a crystal affects the transmission of a wave to a detector A vibrating crystal generates current that is affected by a material adsorbed onto its surface ... by squares and triangles, respectively P1: SFK/UKS BLBS102-c45 P2: SFK BLBS102-Simpson March 21, 2012 14: 38 Trim: 276mm X 219mm 86 0 Printer Name: Yet to Come Part 8: Food Safety and Food Allergens... that are rapid, sensitive and reliable and are frequently used for the detection of herbicide and pesticide residues, bacterial and fungal pathogens and toxins (fungal and water-borne) BIOSENSORS... immediately post-harvest Food Biochemistry and Food Processing, Second Edition Edited by Benjamin K Simpson, Leo M.L Nollet, Fidel Toldr´a, Soottawat Benjakul, Gopinadhan Paliyath and Y.H Hui C 2012