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 861 Table 45.3 The Ideal Characteristics of a Biosensor Resonance signal (RU) P1: SFK/UKS BLBS102-c45 10 11 The biorecognition element must be highly specific for the substrate or antigen Re-usable, with multiple readings permitted on a single device High sensitivity Cost-effective Permit rapid or ‘real-time’ analysis of biomolecular interactions Use of ‘on-line’ or ‘in-situ’ measurement Good signal to noise ratio The device should be robust The ability to measure samples in a high throughput fashion, if required Fast turnaround time on analysis Ease of use Time (s) Immobilised surface Association Dissociation Regeneration Figure 45.2 Schematic representation of a Biacore sensorgram, which provides information relating to the biomolecular interaction between the immobilised antibody and its cognate antigen (relevant analyte) (Markey 2000) However, when the light is above a particular angle of incidence, no light is refracted across the interface and TIR occurs Even though the incident light is reflected back from the interface, an electromagnetic field (called an evanescent wave) penetrates a distance of the order of one wavelength travelling into the less optically dense medium A key advantage of SPR over other optical methods of detection is that SPR measures the interaction between immobilised molecules on a surface (e.g an antigen, or an antibody immobilised onto a sensor chip) and the corresponding ligand in solution passing over this matrix This means that the reaction can be measured in a coloured solution or in a turbid complex matrix, such as in fruit juice or vegetable preparations Hence, there is no requirement for food samples to be pre-treated to remove contaminating coloured species prior to analysis The Biacore system has a lower limit of detection (LOD) of approximately 10 RU (which is approximately 10 pg/mm2 ), which is suitable for the detection of trace amounts of analytes of interest, such as pesticide or herbicide residues There are several platforms available for use The Biacore Q instrument has major potential for quality control analysis, whereas the Biacore 3000 is particularly useful for high sensitivity antibodybased analyte detection and kinetic analysis The T100 and the more recently developed A100, permit high-throughput analysis of protein–protein and protein–ligand interactions that may be useful in the judicious selection of antibodies for specific diagnostic applications Sensor Surfaces When selecting a biosensor-based platform, such as Biacore, for the evaluation of quality of food produce, several key considerations should be made The first of these relates to the nature of the analytical surface that will be used in the assay One of the primary reasons why Biacore is commonly selected for food analysis (aside from the excellent sensitivity and flexibility for designing a suitable assay) relates to the fact that many different surface chemistries are available for conjugating molecules onto the sensor chip surface to perform the assay Biacore uses sensor chips, such as those mentioned in Table 45.4, and the choice of chip chemistry is dependent on a number of different factors, including the desired application of the assay, the physical and chemical properties of the binding partners and the nature of the biomolecular interaction The CM5 chip is the most versatile Biacore chip currently available and is the most frequently used for food analysis Its matrix consists of a completely modified carboxy-methylated dextran covalently attached to a gold surface, and is ideally suited for the analysis of a variety of different binding events, ranging from those involving small organic molecules to multidomain proteins Analytes of interest can readily be coupled to the sensor chip surface through the application of a number of different cross-linking chemistries (e.g amine, thiol or aldehyde coupling), and a stable surface can be produced that permits accurate and repeated analysis on a single surface (Dillon et al 2005) as non-covalently bound entities can readily be removed from the surface by a process termed regeneration Typically, this is achieved with low concentrations of acid (HCl) or base (NaOH) As an example, for the detection of a bacterial strain in a complex sample matrix (e.g coleslaw), a pathogen-specific antibody may be immobilised on a CM5 surface by amine P1: SFK/UKS BLBS102-c45 P2: SFK BLBS102-Simpson March 21, 2012 14:38 Trim: 276mm X 219mm 862 Printer Name: Yet to Come Part 8: Food Safety and Food Allergens Table 45.4 The Surface Chemistries of Available Biacore Chips Chip Type Modification Type Applications CM5 100% carboxylation of dextran surface CM4 CM3 C1 30% carboxylation of dextran surface 100% carboxylation of dextran surface 100% carboxylation of dextran surface L1 SA NTA HPA Au and SIA Lipophilic Streptavidin surface Nickel – nitrilotriacetic acid Flat hydrophobic surface None General use Routinely selected for antibody-antigen and protein-protein interaction analysis Serum, cell extracts Serum, cell extracts Permits binding events to occur closer to the sensor surface, which is advantageous in situations where multivalent interactions occur, or where analytes are large Lipid capturing Detection of biotin-containing molecules Useful for immobilisation Detection of histidine-tagged molecules Used for membrane-associated interactions Useful for self-assembled monolayer-based interactions analysis coupling This covalently captured immunoglobulin may then be used as a bioligand to capture the pathogen from the food sample, which is injected over the sensor surface and, therefore, is in solution The change in mass resulting from the biomolecular interaction introduces a change in RI, as seen by an increase in RU, and regeneration can subsequently be used to liberate the bacterial cell(s), so that the immobilised antibody is available for subsequent analysis This method of analysis can be used for detection purposes and can be used to establish LODs Furthermore, where required, kinetic analysis can be used to determine the affinity of the antibody for its cognate antigen Assay Configuration For food-based biosensor analysis, analytes of interest range in size from large (intact bacterial and fungal cells) to small (pesticide and toxin residues), requiring that the assay format selected must be capable of providing accurate and quantitative detection Biacore-based assays that monitor interactions between large biomolecules, such as antibodies and proteinaceous antigens, can be developed by immobilising either the antibody or antigen on the surface, as the mass change introduced by the binding of the other entity is sufficient to cause a recordable change in RU that can be seen on a sensorgram However, as contaminants such as herbicides, pesticides and toxins have low molecular weights, it is often necessary to employ an indirect measurement method where the analyte is immobilised directly on the sensor surface, and the larger of the binding elements is subsequently introduced The resultant change in mass introduced by the binding of the larger element (in solution) to the smaller, immobilised ligand can therefore be easily seen as a change in RU If the assay was to be performed in reverse (e.g the larger entity is immobilised and the small hapten or toxin is free in solution), a small change in mass would be introduced during an interaction event, which may be difficult to detect and quantify reliably Many of the examples that are discussed in this chapter employ antibody-based competition or inhibition assay formats (Fig 45.3) An inhibition assay involves the combination of the sam- ple of interest with the specific antibody before injection onto a biosensor chip containing an immobilised target molecule There is competition between the immobilised and free antigen (from the sample to be analysed) for antibody binding A change in signal (e.g RU) is recorded, and this is inversely proportional to the amount of target analyte that remains free in solution An alternative method to detect analytes of interest requires a competitive assay format In this format, the antibody, specific to the analyte of interest, is immobilised on the surface The sample, containing the analyte to be determined, is mixed with a known concentration of standard consisting of the target analyte that has been conjugated to a large carrier protein This results in the analyte and the conjugated standard competing for the immobilised antibody on the surface of the biochip An increase in signal is caused by the binding of the large analyte-carrier conjugate and the data generated are similar to the inhibition assay since the signal recorded is inversely proportional to the amount of target analyte present in the sample However, to perform these assays, it is necessary to have a suitable antibody, and methods for antibody production are discussed in Section ‘Antibody Production Strategies’ ANTIBODIES Antibodies are the key biorecognition elements of the immune system A large variety of antibodies and antibody-derived fragments have been produced with the capability of detecting an array of structurally diverse analytes, ranging from proteins to haptens, and these have been implemented in a number of different biosensor formats Antibodies are globular glycoproteins (sugar-containing proteins), and five main classes (or serotypes) exist in nature, namely IgA, IgM, IgE, IgD and IgG Single antibody molecules typically have molecular weights of approximately 150–200 kilodaltons (kDa) IgG antibodies (Fig 45.4) have a Y-shaped backbone with four polypeptide chains located in two identical chains that are covalently attached through disulphide bonds The innermost chains are referred to as the heavy chains because they are approximately double the molecular weight of the outer arms (termed the light chains) The 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 863 Dextran matrix Linker layer Gold layer Glass layer Inhibition assay Antigen Competition assay Antibody Conjugated antigen Figure 45.3 Formats for inhibition and competition assays For illustration, a Biacore surface is represented Inhibition: Free analyte inhibits binding of the antibody to the immobilised analyte on the chip The signal generated when the antibody binds to the immobilised analyte is inversely proportional to the concentration of free analyte in the sample Competition: Free ( ) and conjugated ( ) analyte compete for binding to the immobilised antibody The signal generated is inversely proportional to the amount of free analyte in the sample Heavyy chain Complementarity determining regions Light chain VH VH VL Intrachain disulphide bond CH1 CL Hinge region Carbohydrate CH2 Interchain disulphide bond CH3 Figure 45.4 Structure of an IgG antibody CH1–3 refers to constant heavy regions to 3, respectively VH , variable heavy; VL , variable light P1: SFK/UKS BLBS102-c45 P2: SFK BLBS102-Simpson March 21, 2012 14:38 Trim: 276mm X 219mm 864 Printer Name: Yet to Come Part 8: Food Safety and Food Allergens recognition sites of the antibody, which interact with an epitope on an antigen of interest, are located at the ends of the variable heavy (VH ) and variable light (VL ) regions of the heavy and light chains, respectively They are commonly referred to as the complementarity determining regions, or CDRs Each arm of an antibody can bind to one antigen, so one IgG molecule can theoretically bind to two antigens VVH H VH CH1 Antibody Production Strategies There are three main methods for generating antibodies that may be used in biosensor-based platforms for quality evaluation, and these are discussed in this section Polyclonal antibodies are produced through the immunisation of animal hosts with a particular antigen The immunogen is typically administered in the presence of a suitable adjuvant, which elicits an immune response in the host Small molecules, such as toxins or haptens, may have to be conjugated to larger carrier molecules, such as bovine serum albumin (BSA), to enhance immunorecognition Serum titres are typically analysed by enzyme-linked immunosorbent assay (ELISA) to quantify the host-based response If this is deemed to be suitable, blood samples are subsequently collected and the antibodies generated are purified from the serum Polyclonal antibodies typically consist of a variety of different serotypes with varying affinities/specificities towards the analyte in question Animals often used for polyclonal antibody generation include guinea pigs, rabbits, goats, sheep and donkeys (Leenaars and Hendriksen 2005) The second method involves the use of hybridoma technology to produce monoclonal antibodies (Kăohler and Milstein 1975, Nelson et al 2000, Hudson and Souriau 2003) These are generated by immunising an animal (typically, a mouse) with the antigen of interest in the presence of a suitable adjuvant Once a sufficient immune response is detected by ELISA, the spleen, bone marrow from long bones (femur and humerus) or primary lymphoid organs (such as lymph nodes) are removed from the sacrificed animal and the antibody-producing B-cells are harvested These cells can then be fused to immortal myeloma cells by using an electrical current or polyethylene glycol The resulting hybrid cells (hybridomas), which secrete antibodies that are directed towards the desired antigen, are then selected and cloned out to ensure monoclonality The advantage of this approach is that there is a constant supply of the antibody that is required for analysis However, there is a significant cost involved in the production and the screening of these antibodies Recombinant antibodies are the third form of antibodies that are increasingly being used They are often produced in bacterial strains such as Escherichia coli and are expressed in a phage display format Libraries, with the capacity to express a large number of antibodies, are generated, and they are referred to as naive, synthetic or immune depending on their mode of production (Bradbury and Marks 2004) Immune libraries are constructed though the administration of the immunogen of interest to a suitable host (e.g mouse, rabbit, chicken), which is monitored for antibody production Antibody-encoding nucleic acid is then purified from lymphoid organs, such as the spleen, and cloned into a phage or phagemid vector that, in turn, is VL VL CL scFv Fab Figure 45.5 Different antibody fragments available for biosensorbased analysis CH1–3 refers to constant heavy region 1; VH , variable heavy; VL , variable light; scFv, single-chain variable fragment; Fab, fragment antigen binding propagated in E coli Phage display libraries can subsequently be screened against targets of interest by biopanning This is feasible since the phage express the active recombinant antibodies on their surface and those that bind to the specific antigen immobilised on a capture surface can be differentiated from nonbinding antibodies, and characterised further to determine their affinity for the target antigen A key advantage with recombinant antibodies is the ability to increase their affinity for an antigen of interest through site-directed mutagenesis or other approaches such as chain shuffling or error-prone PCR, which is not possible for monoclonal or polyclonal antibodies (Conroy et al 2009, O’Kennedy et al 2010) The main types of antibody fragments produced by phage display are the fragment antigen binding (Fab) and single-chain variable fragment (scFv), whose structures are shown in Figure 45.5 The presence of the constant regions in a Fab is thought to aid in the stabilisation of the antibody variable regions, which might not function efficiently when expressed in the monomeric scFv format (Răothlisberger et al 2005) It was previously shown in our laboratory that the Fab antibody format is the most reliable and sensitive for use in small molecule competition biosensor assays involving haptens The strict monovalency of this format can lead to a significant enhancement in assay sensitivity in both ELISA and competition SPR assays (Townsend et al 2006) The scFv is the most widely used antibody fragment The variable regions (VH and VL ) of the antibody are linked by a flexible peptide linker The most frequently used are based on glycine-serine repeat structures, with the length of the linker related to the intended valency of the molecule When a short linker is used, the stability and folding of the scFv does not occur properly This is caused by the insufficient juxtaposing of the VH and VL regions in the single chain for the monomer to function ScFvs selected in this format invariably form bivalent dimers, or diabodies, which often have increased avidity for an antigen over the monomeric forms typically observed when long-linker systems are used (Holliger et al 1993, Kortt et al 1997, Atwell 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 et al 1999) Long linkers (ranging from 18–21 amino acids) favour the production of scFv formats, which are predominantly monomeric (Holliger et al 1993, Perisic et al 1994, McGuinness et al 1996) In summary, recombinant antibodies are an excellent alternative to polyclonal and monoclonal antibodies for the detection of analytes of interest, and have great potential for application in the monitoring of quality in the food industry (Hudson and Souriau 2003, O’Kennedy et al 2010) Optical Immunosensors for Quality Determination There have been several excellent examples of biosensor-based analysis for the detection of pesticides, herbicides, toxins and bacterial cells, and some of the more pertinent observations that employ antibody-based recognition (immunosensors) are discussed in this section One of the earliest examples demonstrating the use of antibody-based biosensing for detecting herbicide residues was described by Minunni and Mascini (1993) Here, Biacore was selected as a platform to facilitate the detection of traces (50 pg/mL) of the herbicide atrazine in water samples Another optical biosensor was developed to detect and quantify carbamate residues in vegetables It was observed that changes in the concentration of carbamate could be monitored using chlorophenol red (Xavier et al 2000) Moran et al (2002) used SPR to characterise antibodies that were subsequently used to detect the presence of 2-(4-thiazolyl)benzimidazole, a molecule that is used as a food preservative and an agricultural fungicide Schlecht et al (2002) implemented a C1 four-channel sensor chip in a study to quantifiably detect the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), an organochlorine herbicide, and monitor cross-reactivity of polyclonal antibodies with a structurally related analogue, namely 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) This assay was permitted by immobilising 2,4-D analogues onto the surface of a C1 sensor chip surface through a thiol-carboxyl group reaction, and had a sensitivity of 0.1µg/mL Finally, Caldow et al (2005) used a Biacore Q instrument to detect the bacteriostatic antibiotic, tylosin, in bees’ honey This polyketide is active against most gram-positive bacteria, mycoplasma, and certain gram-negative bacteria They were able to detect tylosin at the level of 2.5µg/kg in honey, demonstrating the ability of biosensor platforms, such as Biacore, to detect analytes in complex sample matrices These five key examples demonstrate early applications of using immunosensors for the detection of low-molecular weight analytes that have a deleterious effect on the quality of agricultural produce More recently, miniaturised biosensor platforms have been developed for in situ analysis of pesticide and herbicide residues A portable immunosensor for the detection of 2,4-D was described by Kim et al (2007) Here, murine hosts were immunised with a 2,4-D-BSA conjugate, and the resultant monoclonal antibodies were implemented for detection purposes When tested on spiked river water samples, this assay format had excellent sensitivity (0.1 ppb of 2,4-D) and permitted the parallel analysis of multiple samples This example also demonstrates how modification of the assay format can greatly improve sensitivity, which is a key 865 consideration for the detection of analytes, such as 2,4-D, which may reside in food or water samples in trace amounts A sandwich assay format, incorporating a second biotinylated antibody, was subsequently developed whose sensitivity was significantly enhanced (0.1 ppt of 2,4-D) Competitive and inhibition assay formats can be utilised effectively in a number of different biosensor formats for the detection of pesticide and herbicide residues In some cases, this can be applied where the direct monitoring of the interaction between an antigen and its cognate antibody is not sufficiently sensitive To illustrate this, Gouzy et al (2009) developed a Biacore-based SPR competition assay for the detection of the herbicide isoproturon, as employing a direct detection assay was deemed to be inadequate Here, a rat-derived anti-isoproturon monoclonal antibody was selected for biorecognition, and the competition assay had a good LOD (0.1µg/L) Salmain et al (2008) developed an indirect competition immunoassay format for the detection of atrazine The biosensor format implemented was an IR optical platform that had nanomolar sensitivity Salmain et al (2008) subsequently performed comparative sensitivity analysis in an ELISA-based assay format, and similar observations were made ELISA assays are routinely selected for immunodetection purposes and, depending on the quality of the antibody, have excellent sensitivity However, a major drawback relates to the fact that analysis times are often lengthy, with multiple incubation and washing stages required for assay completion This is in contrast to biosensor-based assays that permit rapid analysis and facilitate the detection of multiple analytes on a single sensor surface, as opposed to using multiple wells Therefore, ELISA formats may be initially used to validate an assay format before transferring this to a biosensor platform This has been demonstrated by Herranz et al (2008) for the evaluation of leporine polyclonal antibodies specific for simazine derivatives prior to their implementation in an immunosensor platform The resultant assay had a LOD of 1.3 ng/L in contaminated water samples, and results were obtained in 30 minutes In the biosensor assay described by Herranz et al (2008), it was also possible to monitor for cross-reactivity with other molecules, including structurally-related triazines (propazine and atrazine), and demonstrate the absence of non-specific binding to unrelated entities, such as 2,4-D Biosensors permit rapid cross-reactivity analysis to be performed, where multiple analytes may be tested on a single antibody-immobilised surface Alternatively, where small analytes are to be detected, numerous structural analogues may be immobilised on different surfaces (e.g in a CM5 sensor chip, four individual flow cells are available) and tested with a panel of antibodies This parallel immunosensing approach was recently demonstrated by Gao et al (2009) for the detection of atrazine and four additional chemicals, namely paraverine, 17-β-estradiol, chloramphenicol and nonylphenol The biorecognition elements implemented during this analysis were either monoclonal (anti-atrazine, anti17-β-estradiol and anti-chloramphenicol) or polyclonal (antinonylphenol and anti-paraverine) antibodies generated in murine and leporine hosts, respectively Optical biosensor-based platforms that can sensitively detect multiple contaminants, such as herbicide or pesticide residues, suggest the way forward for ... SFK/UKS BLBS102-c45 P2: SFK BLBS102-Simpson March 21, 2012 14: 38 Trim: 276mm X 219mm 86 2 Printer Name: Yet to Come Part 8: Food Safety and Food Allergens Table 45.4 The Surface Chemistries of Available... SFK/UKS BLBS102-c45 P2: SFK BLBS102-Simpson March 21, 2012 14: 38 Trim: 276mm X 219mm 86 4 Printer Name: Yet to Come Part 8: Food Safety and Food Allergens recognition sites of the antibody, which interact... 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 86 3 Dextran matrix Linker