Introduction to Food Biosensors

SYAZANA ABDULLAH LIM AND MINHAZ UDDIN AHMED

1.1 Overview

Food quality and safety surveillance is an important global issue. Monitoring the safety and quality level of foods is of critical importance to ensure that food reaching consumers is safe to eat. Food is often preserved at a desired level of characteristics and qualities to ensure its beneficial properties are maintained. Since overall food quality and safety are also determined by proper handling, preparation and storage it is therefore imperative to fully understand the effect of food preservation methods and each step involved in food processing. Information about food contents can also be found on food packaging to provide assurance to consumers that food has been tested and is free of harmful and undesirable substances. If they fail to follow international food guidelines, manufacturers can face serious legal actions with economic consequences. Currently, traditional analytical techniques such as high-performance liquid chromatography and gas chromatography are well accepted and taken as gold standards for food quality and safety monitoring, but these conventional procedures are cumbersome and time consuming, requiring expensive instrumentation and skilled operators. Alternatively, biosensors can provide an invaluable method for agro-food diagnostics since they are convenient, portable and do not need particular skills to operate. Biosensors in the food industry may be used to analyze nutrients, to detect natural toxins and antinutrients, for monitoring of food processing, and for detection of genetically modified organisms. Through enzymatic and immunogenic reactions, biosensors can be used to determine the level of pesticides, antibiotics, proteins, vitamins B complex and fatty acids found in foods. Figure 1.1 depicts the different applications of biosensors used in food industries. As shown in Figures 1.2 and 1.3, research and development in food biosensors have been remarkably rapid in the past decade and are set to expand further with the advances in material science and biotechnology. The various types of instruments required for the agro-food diagnostics market can be mainly categorized as large multi-analyzers, bench-top portable instruments and single-use disposable sensors. Table 1.1 lists the desirable characteristics for biosensor commercialization.

A biosensor is a measurement device constituting of a biological element that functions as a target recognition entity, in conjunction with a transducer that converts a biological recognition episode to a measurable signal (Figure 1.4). A biosensor setup is basically comprised of three parts:

• a biological receptor for biomolecular recognition of sample analyte

• a transducer to translate recognition event into an appropriate signal

• a detection technique for signal analysis and processes.

Typical biological recognition elements may be tissue, living cells, enzymes, antibody or antigen. Signal transduction can be in the format of electric current, electric potential, intensity and phase of electromagnetic radiation, mass, conductance, impedance, magnetic, temperature and viscosity, or a combination of these techniques. A biosensor's experimental performance is based on its sensitivity, limit of detection, linear range, reproducibility, selectivity, interference response, response time, easy operation, portability, storage, and operational stability. This introductory chapter aims to provide readers with an overview of different types of biosensor with sections dedicated to discussions of the important components of biosensors.

Biosensors can be categorized either on the basis of their biological receptors (e.g., aptasensor or immunosensor), their signal transduction mechanism (e.g., amperometric or optical sensors), or their final application (e.g., clinical sensors).

1.2 Receptors for Biosensing

Biomolecular recognition is essential in biosensor application. In early biosensor development, recognition receptors were primarily obtained from living organisms. Now, with the emergence of modern recombinant techniques, the engineering and manipulation of synthetic receptors in the laboratory have opened up endless possibilities for biosensing design beyond what is possible in nature.

Receptor-based biosensors are categorized into two types: biocatalytic and bioaffinity-based biosensors. A biocatalytic biosensor predominantly uses enzymes as the biological mediator that catalyzes a signalling biochemical reaction, whereas a bioaffinity-based biosensor monitors the binding episode itself. In the latter type, biomolecular recognition uses specific binding proteins, lectins, receptors, nucleic acids, membranes, whole cells, antibodies, or antibody-related substances.

1.2.1 Natural Receptors

1.2.1.1 Enzyme-Based Bioreceptors

The unique attributes of enzymes, with their ability to specifically recognize their substrates and catalyze their transformation, makes enzymes a powerful tool for use in analytical devices. This distinctive characteristic is due to the complementary structures of the substrate and its binding site on the enzyme, which is the particular region of the enzyme where enzyme-substrate interactions occur. The concentration of target analyte can then be determined by measuring the catalytic transformation of the analyte by the enzyme. Conversely, an enzyme can also be inhibited by its target analyte and this can be used to determine the concentration of target analyte in correlation with a decrease in enzymatic product formed. Not only are enzymatic reactions substrate-specific but they are also product-specific, in contrast to uncatalyzed reactions or reactions catalyzed by chemical catalysts which often generate by-products. Experimental variables such as substrate concentration, temperature, pH, ionic strength, and the availability of a competitive or non-competitive inhibitor can influence the catalytic performance of an enzyme and consequently its stability. Although some enzymes are stable in their native state when implemented in different environments, most enzymes are less stable and this hampers their effective application as biomolecular recognition units in biosensors. This instability is caused by changes in an enzyme's three-dimensional configuration, which in effect disfigures the enzyme's active site due to the disruption of non-covalent bonds holding the native protein in place. The main contributing factors determining the conformation stability of the enzyme are hydrophobic interactions, intrapeptide hydrogen bonds, and the ability to recover the original conformation during dehydration-hydration activities. These three interactions need to be preserved for an enzyme to play a roles as biological recognition tool under harsh operating conditions.

Another important issue related to enzyme-based immunosensors is immobilization of the enzyme on the transducer surface. The selection of immobilization strategy must be based on a number of factors:

• The enzyme has to be stable during the reaction.

• The reagents forming crosslinking bonds should not reach the enzyme's active center, which must be protected.

• The washing of unbound enzyme must not be detrimental to the immobilized enzyme.

• The mechanical properties of the carrier should be considered.

However, enzymes are now often used for labeling purposes than as actual bioreceptors, especially with the massive improvement in enzyme-labeling techniques during the past decade.

1.2.1.2 Antibodies as Bioreceptors

Antibodies, which can be polyclonal or monoclonal, are regarded as the prime choice for use in the biomolecular recognition component of biosensors, due to their target specificity and affinity. Polyclonal antibodies derived from the serum of an immunized animal form an array of molecular populations (each arising from a separate cell line) that recognize various regions (haptens) on the immunogen. Monoclonal antibodies of predetermined specificity, derived from cell hybrids made by fusing normal spleen cells with malignantly transformed antibody-secreting cells, typically recognize a more specific region of the immunogen than their polyclonal counterparts. The use of monoclonal antibodies is more common in immunosensor studies because they are quite homogeneous in their molecular structure, have similar binding characteristics, and can be produced in large quantities. Monoclonal antibodies also eliminate the problem associated with the density of binding sites that can be immobilized on the surface of the signal transducer due to the absence of serum proteins and other non-analyte-specific antibodies.

Using antibodies as bioreceptors has its own limitations including high cost, limited lifespan, and susceptibility to high temperature. To improve the robustness of antibodies for biosensor applications, antibodies composed of only heavy chains with very small antigen binding sites, obtained from sharks and camelids, have been explored. These single domains are stable at high temperature (up to 90 °C), stable to detergents, and very soluble. Another advantage of using antibody fragments is that they can be tailored and engineered to further improve their affinity and stability to environment stress.

1.2.1.3 Nucleic Acids

Nucleic acids are employed as bioreceptors through the immobilization of a single-stranded oligonucleotide onto a transducer surface to detect its complementary target sequence. The DNA hybridization event is then translated into a signal. The overall performance of a DNA-based biosensor predominantly depends on the immobilization of nucleic acid with the receptors oriented so that they can be readily accessed by the target. A DNA probe can be attachment to a transducer surface using various strategies to ensure optimal probe orientation for the target recognition event. These schemes include straightforward adsorption on carbon surfaces, thiolated DNA to form a self-assembly monolayer on a gold surface, the use of functional alkanethiol-based monolayers for covalent attachment to a gold surface, biotylated DNA coupling with avidin or strepvadin, and carbodiimide attachment to functional groups on carbon electrodes.

The introduction of engineered peptide nucleic acid has provided a new direction in nucleic acid recognition, contributing to impressive sequence specificity for DNA biosensors. Tree-like DNA dendrimers consist of many single-stranded branches that can hybridize to their complementary DNA sequence and this can also be immobilized on a transducer to achieve higher sensitivity. A greatly increased hybridization capacity and hence a substantially amplified response is achieved by immobilizing these dendritic nucleic acids onto the physical transducer.

1.2.1.4 Whole Cells as Receptors

Whole cells, developed from bacterial strains, can be used as the molecular biorecognition constituent in a biosensor due to the ability of microorganisms to identify and respond to a number of stimuli. Microorganisms recognize and absorb the analyte of interest and this either increases or inhibits their respiratory activities. Products such as protons and ammonia liberated during metabolic processes can then be detected and converted to an electronic signal by a transducer. Whole-cell based biosensors can also be used to observe changes in the vicinity of the cells by examining their electrical properties, thus making this type of biosensor a reliable tool for the detection of pathogens in food samples.

Immobilization is a crucial in whole-cell biosensor fabrication. Different methods of immobilization protocols such as adsorption, encapsulation, entrapment, covalent interaction, and crosslinking can be employed. There are, however, a number of problems associated with the current immobilization techniques, for example:

• Cell viability and function are affected significantly by covalent binding and crosslinking.

• Physical adsorption can cause desorption of microbial cells.

• Immobilization via entrapment may suffer from poor sensitivity as a result of the extra diffusion resistance caused by the entrapment material.

Microbes have the advantages of long lifetime, cost effectiveness, and being able to work in a wide range of pH and temperature. Nevertheless whole cell-based sensors are not a popular choice in this market since microbial cells are complex, have long response times due to diffusional problems, are less sensitive, show poor detection limits and poor selectivity in multiplex detection, non-homogenous intrinsic cellular genotype and phenotype, and stochastic protein expression. This situation has slowly started to change due to the rapid development of recombinant DNA technology, to improve the activity of whole cells, allowing customization and tailoring of microorganisms for detection of particular targets. This makes microbes an excellent source to consume or degrade new substrates under certain cultivation conditions.

1.2.2 Engineered Receptors

Despite the excellent affinity and selectivity displayed by natural bioreceptors, e.g., enzymes and antibodies, their use in biosensor applications remains a challenge. For instance, in antibody production variations can occur in the quality and concentration of the antibodies in each batch. Other obstacles to the use of antibodies include high cost, stability, renewability, and immunity against low molecular weight chemicals and toxins. A study has shown that only 49% (2726 out of 5436) of commercial antibodies obtained from animals could be validated to recognize only their targets. Additionally, a considerable amount of money has been spent on protein-binding reagents for use on antibodies, where these antibodies themselves may be the laboratory tool most typically causing irreproducible research. Thus, new types of replacement for antibody-antigen binding molecules that mimic natural bioreceptors have been developed in recent years to overcome the drawback of these bioreceptors in biosensor applications. The most common synthetic ligands are aptamers, i.e. single-stranded DNA or RNA oligonucleotides that fold into a three-dimensional secondary structure with binding affinity for a target molecule. Other synthetic recognition ligands include molecularly imprinted polymers (MIPs), scaffolded peptides, combined binding agents derived from low-affinity ligands, and combinatorial chemistry ligands. With the exception of aptamers, many of these synthetic ligands have a major drawback, which is their low KD (~10-6 to 10-7 mol L-1), 2-3 orders of magnitude lower than that of antibodies.

1.2.2.1 Aptamers

Aptamers, also known as "chemical antibodies," are acquired from screening large combinatorial libraries through an in vitro selection and amplification process known as systematic evolution of ligands through exponential enrichment (SELEX), which has allowed the isolation of oligonucleotide sequences capable of recognizing targeted antigens with high affinity and specificity. The basic principle of SELEX is to simulate (and stimulate) evolution (systematic evolution of ligands). Evolution in nature is a slow and complex process, but it can be artificially speeded up by the incorporation of a number of selection rounds (usually 10-15 rounds) and exponential amplification of ligands by polymerase chain reaction (PCR) at each round of selection (exponential enrichment). Unlike antibodies, nucleic acid-based aptamers are able to withstand harsh operational conditions due to the comparatively rigid backbones and limited flexibility of nucleic acids in comparison to proteins, which have more torsional freedom and multiple conformational states with respect to their backbones and side chains.

The outstanding molecular recognition of aptamers is due to their ability to adopt specific and complex three-dimensional shapes characterized as stems, loops, bulges, hairpins, pseudoknots, triplexes, or quadruplexes. These three-dimensional configurations allow the binding of targets ranging from small molecules to large ones such as peptides. Another advantage of nucleic acid aptamers is their excellent affinities for their targets, typically with dissociation constants ranging from picomolar to millimolar. Figure 1.5 shows the different recognition schemes for aptasensing according to their level of integration.