Biosensors in food processing are a major research thrust area in the present scenario of accurate and fast detection of pathogens. The inherent specificity, selectivity, adaptability of biosensors and advantages such as simple and low-cost instrumentation, fast response times, minimum sample pre-treatment and high sample throughput make them ideal for use throughout food processing, analysis, quality and safety. Potential applications within the supply chain range from testing of foodstuffs for maximum pesticide residue verification through to the routine analysis of analytes concentrations, such as, glucose, sucrose, alcohol, etc., which may be indicators of food quality/ acceptability. There is a growing need for biosensors in the food industry. Potential markets include microbial food safety, food defense, pesticide residue screening, testing for genetically modified organisms, government inspection agencies, and anyone else seeking a diagnostic tool for detecting pathogens quickly and accurately. These markets are expected to grow annually as legislation creates new standards for microbial monitoring. Products with quicker detection times and reusable features will be much coveted by those interested in real-time diagnostics of disease-causing pathogens. As the world becomes more concerned with safe and secure food, the demand for rapid biosensors will only increase and thus for new materials to be used in making biosensors for food processing.
Keywords: Biosensors, Microbial Food Safety, Pathogen Detection, Food Processing
TOC of Figures
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Introduction
Food safety and quality control is the prime concern both for consumer and food industry. The global efforts to improve consumer protection and public health have lead to an increasing number of analytical approaches applicable to food analysis and process control. Biosensors are applicable to clinical diagnostics, food analysis, cell culture monitoring, environmental control, and various military situations. The inherent specificity, selectivity, and adaptability of biosensors make them ideal candidates for use throughout the food industry. Biosensor systems are practical, efficient and convenient tools in the area of routine analysis, monitor production processes or storage of nutrition and to control contamination outbreaks with minimal time and effort on sample preparation. Adapting the biosensor technology for food industry could lead to immense improvements in quality control, food safety, and traceability.
Biosensor
A biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component.
It consists of 3 parts:
Sensitive biological element (biological material (e.g. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.), a biologically derived material or biomimic). The sensitive elements can be created by biological engineering.
Transducer or the detector element (works in a physicochemical way, optical, piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte with the biological element into another signal (i.e., transducers) that can be more easily measured and quantified. Some common transducers used for fabrication of biosensor are listed in .
Associated electronics or signal processors that are primarily responsible for the display of the results in a user-friendly way. This sometimes accounts for the most expensive part of the sensor device, however it is possible to generate a user friendly display that includes transducer and sensitive element.
Figure Basic Principle of Biosensors
A common example of a commercial biosensor is the blood glucose biosensor, which uses the enzyme glucose oxidase to break blood glucose down. In doing so it first oxidizes glucose and uses two electrons to reduce the FAD (a component of the enzyme) to FADH2. This in turn is oxidized by the electrode (accepting two electrons from the electrode) in a number of steps. The resulting current is a measure of the concentration of glucose. In this case, the electrode is the transducer and the enzyme is the biologically active component.
Recently, arrays of many different detector molecules have been applied in so called electronic nose devices, where the pattern of response from the detectors is used to fingerprint a substance. Current commercial electronic noses, however, do not use biological elements. A canary in a cage, as used by miners to warn of gas, could be considered a biosensor. Many of today's biosensor applications are similar, in that they use organisms which respond to toxic substances at much lower concentrations than humans can detect to warn of the presence of the toxin. Such devices can be used in environmental monitoring, trace gas detection and in water treatment facilities.
Table Main Transduction Systems Used for Biosensor Fabrication
Transducer type
Examples
Electrochemical
Clark electrode, Mediated electrodes, Ion selective electrodes (ISES), Field effect transistor (FET) based devices, Light addressable potentiometric sensors (LAPS)
Optical
Absorbance, Luminescence, Fluorescence, Photodiodes, Waveguide systems, Integrated optical devices
Piezoelectric
Quartz crystals, Surface acoustic wave (saw) devices
Calorimetric
Thermometric
Types of Biosensor
Electrochemical Biosensor
Electrochemical biosensors are normally based on enzymatic catalysis of a reaction that produces or consumes electrons (such enzymes are rightly called redox enzymes). The sensor substrate usually contains three electrodes, a reference electrode, a working electrode and a sink electrode. An auxiliary electrode (also known as a counter electrode) may also be present as an ion source. The target analyte is involved in the reaction that takes place on the active electrode surface, and the ions produced create a potential which is subtracted from that of the reference electrode to give a signal. We can either measure the current (rate of flow of electrons is now proportional to the analyte concentration) at a fixed potential or the potential can be measured at zero current (this gives a logarithmic response). Note that potential of the working or active electrode is space charge sensitive and this is often used. Further, the label-free and direct electrical detection of small peptides and proteins is possible by their intrinsic charges using biofunctionalized ion-sensitive field-effect transistors.
Figure Basic Principle of Electrochemical Biosensors
Basically electrochemical biosensors can be based on amperometric, potentiometric, conductimetric, or impedimetric transducers . Typically in (bio-)electrochemistry, the reaction under investigation would either generate a measurable current (amperometric), a measurable potential or charge accumulation (potentiometric) or measurably alter the conductive properties of a medium (conductometric) between electrodes. References are also made to other types of electrochemical detection techniques, such as impedimetric, which measures impedance (both resistance and reactance), and field-effect, which uses transistor technology to measure current as a result of a potentiometric effect at a gate electrode .
Ion Channel Switch Biosensor
The use of ion channels has been shown to offer highly sensitive detection of target biological molecules. By imbedding the ion channels in supported or tethered bilayer membranes (t-BLM) attached to a gold electrode, an electrical circuit is created. Capture molecules such as antibodies can be bound to the ion channel so that the binding of the target molecule controls the ion flow through the channel. This results in a measurable change in the electrical conduction which is proportional to the concentration of the target.
ICS - channel open ICS - channel closed
Figure Basic Principle of Ion Channel Switch
The magnitude of the change in electrical signal is greatly increased by separating the membrane from the metal surface using a hydrophilic spacer. Quantitative detection of an extensive class of target species, including proteins, bacteria, drug and toxins has been demonstrated using different membrane and capture configurations.
Piezoelectric Biosensor
The resonant frequency of an oscillating piezoelectric crystal can be affected by a change in mass at the crystal surface. Piezoelectric immunosensors are able to measure a small change in mass. Piezoelectric sensors utilise crystals which undergo an elastic deformation when an electrical potential is applied to them. An alternating potential produces a standing wave in the crystal at a characteristic frequency. This frequency is highly dependent on the elastic properties of the crystal, such that if a crystal is coated with a biological recognition element the binding of a (large) target analyte to a receptor will produce a change in the resonance frequency, which gives a binding signal.
Figure Basic Principle of Piezoelectric Sensor
Potentiometric Biosensors
Potentiometric biosensors are based on monitoring the potential of a system at a working electrode, with respect to an accurate reference electrode, under conditions of essentially zero current flow. In operation, potentiometric measurements are related to the analyte activity (of a target species). The use of ion selective and gas sensitive membranes coupled to enzyme systems, linked to the potentiometric sensor, allows the fabrication of a biosensor device specific to the enzyme substrate or product. By measuring either the ions or the gases that are generated or consumed as a result of the enzyme activity, an effective method for measuring the concentration of the target analyte can be realized. Potentiometric biosensors can operate over a wide range (usually several orders of magnitude) of concentrations. The use of potentiometric biosensors for food analysis has not been as widely reported as for amperometric sensors. However, examples of where this approach has been used, for food analysis, include estimating monophenolase activity in apple juice , determining the concentration of sucrose in soft drinks , measuring isocitrate concentrations in fruit juices , and determining urea levels in milk .
Amperometric Bisensors
The use of amperometry as the method of transduction has proved to be the most widely reported using an electrochemical approach. Amperometric or voltammetric biosensors typically rely on an enzyme system that catalytically converts electrochemically nonactive analytes into products that can be oxidized or reduced at a working electrode. In contrast to potentiometric devices, the principle operation of amperometric biosensors is defined by a constant potential applied between a working and a reference electrode. The imposed potential encourages redox reactions to take place, causing a net current to flow. The magnitude of this current is proportional to the concentration of electroactive species present in solution. Both cathodic (reducing) and anodic (oxidizing) reactions can be monitored amperometrically. Many amperometric biosensors described to date have been based on the use of enzymes. Typically, oxidase enzymes have been the most frequently exploited catalysts used for these biosensor formats. In operation, amperometric biosensors tend to monitor either the oxygen consumed or the hydrogen peroxide generated. Both are electrochemically active, oxygen can be electrochemically reduced, and hydrogen peroxide can be oxidized. The current generated is proportional to the concentration of the enzyme substrate (i.e., the target analyte) present. Commercially available instruments, based on amperometric enzyme biosensors, are available. An example of this includes the range of analyzers manufactured and sold by YSI Inc. (Yellow Springs, OH). These instruments are designed for use in clinical diagnostics, environmental monitoring, and the food processing industries. The YSI 2700 SELECT Biochemistry Analyzer is designed to measure common food components such as glucose (dextrose), sucrose, lactose, lactate, galactose, glutamate, choline, glutamine ethanol, hydrogen peroxide, and starch. This is a fully automated instrument, at the heart of which is an electrochemical amperometric biosensor.
Calorimetric Biosensors
Sensors based on calorimetric transduction are designed to detect heat generated or consumed during a biological reaction. Many biochemical reactions are accompanied by either heat absorption or production, by using sensitive heat detection devices, biosensors for specific target analytes have been constructed. In the field of food analysis, several reports have described the use of such biosensors to detect metabolites. described a thermometric biosensor system to determine sucrose in sugarcane. To measure sucrose, the enzyme invertase was immobilized on a thermistor system and the heat generated during the enzyme reaction was used to calculate the sucrose content of cane sugar.
Optical Biosensors
Optical-based biosensor systems have proved to be the most widely reported. These sensors are based on measuring responses to illumination or to light emission. Optical biosensors can employ a number of techniques to detect the presence of a target analyte and are based on well-founded methods including chemiluminescence, light absorbance, fluorescence, phosphoresence, photothermal techniques, light polarization and rotation, surface plasmon resonance (SPR), and total internal reflectance. Optical-based biosensors offer a number of advantages including speed and reproducibility of the measurement. Commercially, one of the most successful optical-based biosensor systems introduced has been the range of instruments supplied by BIAcore (Uppsala, Sweden). This instrument can be employed to study a wide range of biological interactions, both automatically and in real-time. The instrument is based on SPR, whereby biomolecular binding events cause changes at a metal/ liquid interface, usually involving a complex that includes a specific antibody against a target analyte. On binding, these changes (in the refractive index) are recognized by a shift in the SPR signal, indicating a presence of the target analyte in a sample solution. SPR sensor systems have been used extensively to investigate the presence of harmful contaminating microorganisms in food and to determine food quality. For example, an optically based biosensor was recently used to screen poultry liver and eggs for the presence of the drug nicarbazin, a feed additive used to prevent outbreaks of coccidiosis in boiler chickens . The limits of detection for the sensor system were 17 and 19 ng g-1 for liver and eggs, respectively.
Acoustic Biosensors
Piezoelectric quartz crystals can be affected by a change of mass at the crystal surface, this phenomenon has been successfully exploited and used to develop biosensors. For practical applications, the surface of the crystal can be modified with recognition elements (e.g., antibodies) that can bind specifically to a target analyte. If the crystal is placed in an alternating electric field, the crystals are subjected to mechanical deformations. At a particular frequency, a mechanical or acoustic resonance is induced. The frequency of this response will be dependent on the size and mass of the crystal. Hence, any change in mass (e.g., binding of the target analyte to the recognition element) is detected by the change in oscillation frequency of the crystal. Biosensors based on acoustic transduction have tended to be used mainly for the detection of contaminating microorganisms. Nonetheless, such sensor systems have been used to monitor other aspects of food production. described the use of an acoustic sensor to detect genetically modified organisms. Such devices could pave the way to providing efficient screening tools in food analysis.
Immunosensors
Immunosensors are based on exploiting the specific interaction of antibodies with antigens. Typically, immunoassays (such as the widely used enzyme-linked immunosorbent assay technique) employ a label (e.g., enzyme, fluorescent marker) to detect the immunological reaction. The use of biosensor platforms, linked to an immunoassay format, offers a route to rapid quantitative measurements of target analytes. Both electrochemical and optical transduction systems have been exploited. For example, immunochromatographic methods can be coupled with electrochemical or optical detectors to yield simple dipstick style devices, combining the speed and convenience of sensors with the specificity and sensitivity of immunoassays. To use labelled antibodies, a number of detection strategies are available including competitive competition and displacement assays . An example of where this approach has been exploited is illustrated by the detection of bovine progesterone during milking . With reproductive management a major financial concern of the dairy industry, these biosensor systems were designed to provide a rapid means for determining the onset of oestrus in dairy cattle. The sensor systems were designed to be operated in the dairy parlor during milking. Both approaches adopted a liquid handling system linked to a suitable immunoassay sensor. Relevant targets of immunosensors implemented to food safety are prevalent bacterial toxins (staphylococcal enterotoxins and clostridial toxins), plant toxins (Ricin), mycotoxins (aflatoxins and ochratoxin A), marine toxins, and other pathogenic bacterial contaminations (Listeria, Salmonella, Staphylococcus aureus, or Escherichia coli). These cause acute intoxication and also chronic diseases in humans consuming contaminated food .
Sensor Arrays
The ability to measure several analytes at the same time, using a single biosensor element, is becoming a reality. A one-shot disposable biosensor, comprised of individual sensor elements printed on one support matrix, has been described . The sensor array simultaneously measured glucose, sucrose, and ascorbic acid concentrations in tropical fruits such as pineapples. Such measurements can be used to determine the status of fruit maturation and ripening, hence, allowing growers, food transport operatives and retailers the opportunity to rapidly determine the quality of the produce. The electronic nose is a specific kind of sensor array that can discriminate several volatile compounds according to the electronic response (e.g., voltage, resistance, conductivity) arising from the different gas sensors, usually metaloxide chemosensors. After exposure of the volatile compounds to the sensor array, a signal pattern is collected, and results are evaluated with multivariate analysis or processed by an artificial neural network.
Whole Cell Biosensors
The use of immobilized whole cells (usually bacteria) as the recognition element for biosensor applications has been widely described in . Typically, electrochemical transduction methods have been used, particularly, the Clark oxygen electrode. These sensor systems rely on the interaction of a particular microorganism in the presence of a target analyte. By monitoring the respiratory activity of the microorganism, it has proved possible to detect and quantify the target analyte in a range of food matrices. However, traditional whole cell biosensors are inherently nonspecific in their action and, thus, may not be appropriate for some analytes that are associated with fresh produce flavor or taste. Improved specificity of whole cell biosensors has been achieved using recombinant microorganisms , but this has mainly been for detection of pollutants and/or toxins.
Table Whole cell Biosensors to Detect some Analytes in Food
Analyte
Microorganism
Reference
Short chain fatty acids
Arthrobacter nicotianae
Vitamin B-6
Saccharomyces uvarum
Phenylalanine
Proteus vulgarius
Sulfite
Thibacillus thiooxidans
Glucose
Aspergillus niger
Amino Acid Bioavailability
Escherichia coli
Methionine
Escherichia coli
Considerations in Biosensor Development
Once a target analyte has been identified, the major tasks in developing a biosensor involve:
Selection of a suitable bioreceptor molecule
Selection of a suitable immobilization method
Selection of a suitable transducer
Designing of biosensor considering measurement range, linearity, and minimization of interference
Packaging of biosensor
The item 1 requires knowledge in biochemistry and biology, the item 2 requires knowledge in chemistry, the item 3 requires knowledge in electrochemistry and physics, and the item 4 requires knowledge in kinetics and mass transfer. Once a biosensor has been designed, it has to be put into a package for convenience manufacturing and use. The current trend is miniaturization and mass production. Modern IC (integrated circuit) fabrication technology and micromachining technology are used increasingly in fabricating biosensors. Therefore, interdisciplinary cooperation is essential for a successful development of a biosensor.
Requirements for Sensors
To be commercially successful, a biosensor has to meet the general requirements of commercial sensors. These are:
Table General requirements for commercial biosensors
Relevance of output signal to measurement environment
Accuracy and repeatability
Sensitivity and resolution
Dynamic range
Speed of response
Insensitivity to temperature (or temperature compensation)
Insensitive to electrical and other environmental interference
Amenable to testing and calibration
Reliability and Self-Checking Capability
Physical robustness
Service requirements
Capital cost
Running costs and life
Acceptability by user
Product safety-sample host system must not be contaminated by sensor
Applications
There are many potential applications of biosensors of various types. The analytical technology based on sensors is an extremely wide field, which impacts on all the major industrial sectors, such as pharmaceutical, healthcare, food, agriculture, environment and water. The main requirements for a biosensor approach to be valuable in terms of research and commercial applications are the identification of a target molecule, availability of a suitable biological recognition element, and the potential for disposable portable detection systems to be preferred to sensitive laboratory-based techniques in some situations. Some examples are given below:
Glucose monitoring in diabetes patients.
Other medical health related targets
Environmental applications e.g. the detection of pesticides and river water contaminants
Remote sensing of airborne bacteria e.g. in counter-bioterrorist activities
Detection of pathogens
Determining levels of toxic substances before and after bioremediation
Detection and determining of organophosphate
Routine analytical measurement of folic acid, biotin, vitamin B12 and pantothenic acid as an alternative to microbiological assay
Determination of drug residues in food, such as antibiotics and growth promoters, particularly meat and honey.
Drug discovery and evaluation of biological activity of new compounds.
Protein engineering in biosensors.
Detection of toxic metabolites such as mycotoxins
Food and beverage industry applications of biosensors
Food Safety- Rapid tests for diseases, bacteria, BSE
Electronic nose and taste
Food decay detection
Process quality
Future Prospects
Data Processing and Pattern Recognition. If we compare the present biosensors with the natural ones (of, for example, the nose or the eye), they are very crude and simplistic. The recognition molecules in the 'natural sensors' are not necessarily highly specific but the signal transduction via the biomolecules is sophisticated. The specificity often comes from processing of the data collected and recognizing the pattern via a continuous learning process. This mode of operation using the data collected from multiple biosensors is expected to be exploited in the future because the ever increasing capability of microprocessors will provide fast computation.
Micro Instrument. The third generation biosensors have built-in signal processing circuitry. When such sensors are combined with the micro scale valves and actuators currently under development (utilizing micromachining technology), a whole analytical instrument can be built on a silicon wafer. Such an instrument can be mass produced and used in a variety of applications including homes, hospitals, automobiles, toxic dump sites, etc.
Molecular Electronics. The effort to continuously increase the density of electronic components to obtain ever smaller 'packages' will be limited eventually, not by the microlithographic technique employed but by the minimum size allowable for a transistor (note that 'transistor' is the building block of microprocessors and memory chips). Many biological molecules are able to synthesize complex self-organizing molecules with apparently just the required electronic properties. This suggests that the solution to this problem may be found in replacing silicon with biomolecular components. This idea has led to the proposition of many molecular electronic systems. In the past, materials and processing methods developed for microelectronic applications have been exploited in sensor developments. Therefore, any future developments in molecular electronics are expected to be imported into biosensor technology.
Biosensors in food analysis
Application of the biosensor technique in the field of food processing and quality control is promising. Biosensors offer advantages as alternatives to conventional methods due to their inherent specificity, simplicity and quick response. There are several applications of biosensors in food analysis. The use of biosensors in foods is prevalent for analysis of metabolites, pathogenic bacteria, toxins, antibiotics and pesticides. Food microbiologists are constantly seeking rapid and reliable automated systems for the detection of biological activity. Immunodiagnostics and enzyme biosensors are two of the leading technologies that have had the greatest impact on the food industry. The use of these two systems has reduced the time for detection of pathogens such as Salmonella to 24 h and has provided detection of biological compounds such as cholesterol or chymotrypsin. Biosensor technology has the potential to speed up the detection, increase specificity and sensitivity, enable high-throughput analysis, and to be used for monitoring of critical control points in food production and online quality assessment and control like cooking process in food industry . There is an excellent scope for the application of biosensors in the seafood industry for fast assessment of quality . reported the applications of Carbon Nano Tubes -based biosensors in the field of food quality and control, waste water treatment and bioprocessing Biosensor systems can be designed such that they can be operated at-site on a real-time basis, removing the reliance on expensive centralized laboratory-based testing. Moreover, the process of miniaturization can be adapted to biosensors. Hence, an array of sensors can be integrated into a small portable device for multiple parameter determination for use by non-specialized persons with a minimum of manual manipulation. This is one of the major advantages of using biosensors, as measurements can be made either during raw material preparation, food processing (e.g., as QC devices), or for checking the reliability of storage conditions. Hence, these devices can act as cost effective tools for QC, for process control, and for the determination of food safety.
Analysis of food components or other metabolites
New advances in the biosensors are related to the development of smaller devices (pocketsize) capable of on-the-spot measurements of a wide range of analytes. These apparatus find use in the food industry for evaluating the freshness of fish or meat via the biogenic amines generated during food decomposition . Since biosensors can provide continuous data on a specific analyte, they are also well suited for optimization of food processing . Another type of biosensor is the cell sensor that uses microorganisms, cells and animals, or plant tissues in intimate contact with the transducer device, converting the biochemical reaction into an electric response signal. These are very useful for determination of complex variables, such as the determination of freshness in meat and fish, and for the detection of mutagenic substances. The development of inexpensive, reliable, and more robust biosensors capable of working under harsh conditions is nowadays one of the main fields of research in this area . Some amperometric biosensors have been developed for the determination of concentration of metabolites such as glucose, sucrose, lactate, alcohol, glutamate, and ascorbic acid, typically found in many food items as described in . Tyrosinase-based biosensors show promise as a rapid and portable tool for monitoring total phenol levels in the field and during wine processing and aging . Some recently reported applications of amperometric biosensors include determination of glucose in some food samples of dairy industry (Grassino, Milardovi, Grabari, HYPERLINK "#_ENREF_12"&HYPERLINK "#_ENREF_12" Grabari, 2011), lactose in milk and dairy products , total polyphenolic content in wine and tea samples (Goriushkina, Soldatkin, HYPERLINK "#_ENREF_10"&HYPERLINK "#_ENREF_10" Dzyadevych, 2009) and wine and must analysis (Granero, Fernández, Agostini, HYPERLINK "#_ENREF_11"&HYPERLINK "#_ENREF_11" Zón, 2010).
Electronic nose and tongue
The electronic nose, a specific kind of sensor array has been applied to classify cereal grains, to discriminate moldy, weakly musty, and strongly musty oat samples to predict ergosterol levels and fungal CFU in wheat , to predict deoxynivalenol and ochratoxin A levels in barley grains (the latter as below or above 5 lg/kg), to indicate ochratoxin A, citrinin, and ergosterol production in wheat, and to indicate mycotoxin formation by Fusarium strains . An electronic tongue comprising 30 potentiometric chemical sensors and pattern recognition tools for data processing was used for the analysis of mineral waters, coffee, soft drinks, and flesh food, namely fish. For detection of odorant compounds, an impedimetric sensor based on fullerene-modified supported lipid membrane and thin layer potentiometry were successfully applied. The electronic tongue is capable of distinguishing among different sorts of beverages: natural and artificial mineral waters, individual and commercial brands of coffee, and commercial and experimental samples of soft drinks containing different sweeteners .
Genetically modified organisms in food
The term genetically modified (GM) foods or genetically modified organisms (GMO) is most commonly used to refer to crop plants created for human or animal consumption using the latest molecular biology techniques. GMO technology is deemed to be an excellent tool to overcome the food shortage for booming population in many countries. There are widespread suspicions regarding health implications of GMO-based foods. Therefore, a device is necessary to detect GMO in food, and electrochemical sensors have emerged as viable candidates. Surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors are utilized in GMO screening . An electrochemical method for measurement of recombinant protein levels using transgenic avidin maize as a model GMO was reported by . A disposable genosensor for GMO has been reported by through detecting the nearly ubiquitous genetic element of transgenic organisms- Nopalin synthethase (NOS) terminator. The piezoelectric sensor suitable for determination of genetically modified soybean roundup ready in the genomic DNA without PCR amplification was developed by .
Drug, Pesticides residues and other toxins
The widespread administration of antibiotics raises significant food safety issues since antibiotic resistance can be transferred to humans on ingestion of affected meat and milk products. Therefore, most of the biosensors developed are aimed at determining antibiotics in biological or food samples, although their application for environmental monitoring can be considered. . Biosensors and arrays for mycotoxins are promising biotechnological tools for mycotoxin detection in food Several different sensors like enzyme sensors, optical immunosensors, electrochemical sensors, quartz crystal sensors, and surface plasmon resonance biosensors for the determination of mycotoxins and other small molecule neurotoxins have been reported. Acetyicholinesterase-biosensors have been applied for the detection of Organophosphorus pesticides like chloropyrifos, carbamate insecticides in water and food (Hildebrandt, Bragós, Lacorte, HYPERLINK "#_ENREF_14"&HYPERLINK "#_ENREF_14" Marty, 2008) and neurotoxic insecticides in food.
Veterinary drugs are generally used in farm animals for therapeutic and prophylactic purposes, and they include a large number of different types of compounds that can be administered in the feed or in the drinking water. In some cases, the residues may proceed from contaminated animal feedstuffs. In order to protect consumer health, maximum residue limits of veterinary medical products in foodstuffs of animal origin (liver, milk, egg, kidney, muscle, fat, etc.) have been established according to European Union regulation (2377/90/CEE). Rigorous screening procedures have been implemented in EU member states to detect the illegal administration of steroid hormones as growth promoters. A sensitive biosensor for chloropyrifos (CPF), an organophosphorus pesticide, was developed by immobilizing acetylcholinesterase (AChE) through covalent bonding to an oxidized exfoliated graphite nanoplatelet (xGnPs) chitosan cross-linked composite . Surface plasmon resonance biosensor are also applied for detection of ochratoxin A in cereals and beverages , Tetrodotoxin in Food Matrices
Microbial contaminants in food
The detection and identification of pathogenic microorganisms based on conventional culturing techniques is very elaborate, time-consuming, and have to be completed in a microbiology laboratory and are therefore not suitable for on-site monitoring. Food contamination by pathogenic bacteria, such as E. coli, Salmonella typhimurium, Campylobacter jejuni, Legionella pneumophila, Staphylococcus aureus, Bacillus cereus, Streptococci, etc., causes numerous food-borne diseases The demand for a fast, reliable, and sensitive method for food analysis has paved the path for electrochemical biosensors for this field . In monitoring food-borne bacteria, the PCR-gene probe-based sensor has great potential , and there are reports of detection of fewer than 40 cells per gram of food sample by this method without an enrichment step . Examples of the use of biosensors for detection of contaminating microorganisms in food are given in .
Biosensors merit special mention due to their sensitivity, accuracy, cost-effectiveness and simplicity, not only of the construction, but also of the sample pre-treatment, if necessary, and the measurement step. Furthermore, Biosensor arrays offer additional advantages, such as the possibility to measure multiple samples and provide multi-mycotoxin profiles in one assay. In this case, apart from shortening the analysis time, accuracy is improved by the assessment of matrix interferences and synergistic effects among mycotoxins. Biosensors and arrays for mycotoxins are thus promising biotechnological tools for mycotoxin detection in food .
Table Use of Biosensors for detection of contaminating microorganisms in food
Target Organism
Food Matrix
Detection method
Detection Limit
Reference
Escherichia coli
Dairy products
Acoustic
3 Ã- 105 to 6.2 Ã- 107/mL
Staphylococcal enterotoxin A
Hot dogs, potato salad, milk, and mushrooms
Optical
10-100 ng/g
Salmonella typhimurium
Chicken carcass wash fluid
Optical
1 Ã- 105 to 1 Ã- 107/mL
Staphylococcal enterotoxin B
Milk
Optical
0.5 ng/mL
Salmonella groups B, D, and E
Range of foods
Optical
1 Ã- 107CFU/mL
E. coli
Range of foods
QCM (acoustic)
1.7 Ã- 105 to 8.7 Ã- 107CFU/mL
E. coli 0157:H7
Range of foods
Optical
1 Ã- 103 CFU/mL
Bacillus cereus
Food matrices
Conductometric
35-88CFU/mL
Table Various Analytes Monitored in Food by Biosensors
Analyte
Food matrix
Enzyme
Detection limit
Reference
Fructose
Citrus fruits
Fructose dehydrogenase
10 μM
Amines
Apricots and cherries
Diamine oxidase and polyamine oxidase
2 Ã- 10-6mol/L
L-ascorbic acid
Fruit juices
Ascorbate oxidase
5.0 Ã- 10-5M
Sucrose
Fruit juices
Sucrose phosphorylase, phosphoglutaminase, Glucose-6-phosphate 1-dehydrogenase
9.25 g/L in
pineapple juice
Malic acid
Apples, potatoes, and tomatoes
Malate dehydrogenase
0.028 mM
Polyphenols
Vegetables
Horseradish peroxidase
1 μmol/L
β-D-glucose, total D-glucose, sucrose, L-ascorbic acid
Tropical fruits (mango, Pineapple, and papaw)
Glucose oxidase, mutarotase, Invertase, mutarotase, and Ascorbate oxidase
7 mM
Cysteine sulfoxides
Alliums (e.g., onion and garlic)
Allinase
5.9 Ã- 10-6M
Pyruvic acid
Onion
Pyruvate oxidase
2 μmol/g
Essential fatty acids
Fats and oils
Lipoxygenase, lipase, and esterase
0.04 mM in an FIA system
Lysine
Range of foods
Lysine oxidase
1 Ã- 10-5mol/L
Glucose and maltose
Beer
Glucose oxidase and
Amyloglucosidase
40 mM to glucose
Rancification indicators
Olive oils
Tyrosinase
0.2-2.0 μM in different oils
Lactate
Wine and yoghurt
Lactate oxidase
1.4Ã- 10-6mol/L
D- and L-amino acids
Foods
Amino acid oxidase
0.1 or 0.2 mM for L- and D-amino acids
Choline
Dairy produce
Choline oxidase
5 μmol/L
Organophosphorus pesticide
Range of foods
Acetyl cholinesterase
0.2-1.8 ppm
Insecticide residues
Infant food
Acetyl cholinesterase
5 μg/kg
Laminarin
Seaweed
1,3-glucanase and glucose oxidase
50 μg/mL
Alcohol
Beer and wine
Alcohol oxidase and horseradish peroxidase
5.3Ã- 10-6mol/L
L-lactate
Alcoholic beverages and dairy products
Lactate oxidase
3.8 Ã- 106M
D,L-lactic acid and L-malic acid
Wines
L(+) lactate oxidase (LOD), D( ) lactate dehydrogenase (D-LDH) and horseradish peroxidase (HRP),
Phytic acid and phytase
Food
phytase and pyruvate oxidase
Utilization of Biosensors in Industrial Process Control
The development of cost-effective on-site analytical methods is an urgent need to monitor food safety. The continued development of biosensor technology will soon make available "on-line quality control" of food production, which will not only reduce cost of food production but will also provide greater safety and increased food quality. Partially disposable biosensors for the quick assessment of damage in foodstuff after thermal treatment have been reported .
Process Control
Real-time monitoring of carbon sources, dissolved gases, in fermentation processes could lead to optimization of the procedure giving increased yields at decreased materials cost. While real-time monitoring with feedback control involving automated systems does exist, currently only a few common variables are measured on-line (e.g. pH, temperature, CO2, O2)) which are often only indirectly related with the process under control.
Three different methods of controlling a process are:
1. Off-line distant: central laboratory coarse control with significant time lapse
2. Off-line local: fine control with short time lapse
3. On-line: real-time monitoring and control
On-Line Control
On-Line control is most desirable as it allows the process to follow an ideal pre-programmed fermentation profile to give maximum output. Some factors like in situ sterilization, sensor life-time, sensor fouling, etc. may affect the On-line control system. However, Some of the problems can be overcome if the sensor is situated so that the sample is run to waste, but this causes a volume loss, which can be particularly critical with small volume fermentations.
Off-Line Control
On-Line control may be the ultimate aim, considerable advantage can be gained in moving from Off-line distant to Off-line local method for process control as it ensure rapid analysis and enables finer control of the fermentation. The demands of the sensor are also not as stringent.
Benefits of Process Control
The benefits with process-control technology include:
Improved product quality, reduction in rejection rate following manufacture
Increased product yield, process tuned in real time to maintain optimum conditions throughout and not just for limited periods
Increased tolerance in quality variation of some raw materials. These variations can be compensated in the process-control management
Reduced reliance on human 'seventh sense' to control process
Improved plant performance-processing rate and line speed automated, so no unnecessary dead-time allocated to plant
Optimized energy efficiency
The use of biosensors in industrial process in general could facilitate plant automation, cut analysis costs and improve quality control of the product.
Conclusion:
The future of detection methods for microorganisms shall be guided by biosensor, which has already contributed enormously in sensing and detection technology. Sensors specificity, sensitivity, reproducibility and analysis stability should all be improved in future work.
