Enzymeâ based Sensors

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9 Enzyme-based Sensors Anastasios Economou1, Stephanos K. Karapetis2, Georgia-Paraskevi Nikoleli2, Dimitrios P. Nikolelis3, Spyridoula Bratakou2 and Theodoros H. Varzakas4 1

Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, Greece Laboratory of Inorganic and Analytical Chemistry, School of Chemical Engineering, National Technical University of Athens, Athens, Greece 3 Laboratory of Environmental Chemistry, Department of Chemistry, University of Athens, Athens, Greece 4 Higher Technological Educational Institute of Peloponnese, Department of Food Technology, School of Agricultural Technology, Food Technology and Nutrition, Kalamata, Greece 2

9.1 ­Introduction to enzymatic biosensors The term ‘biosensor’ was introduced by Cammann (1977). A stricter definition of the term was set later by the International Union of Pure and Applied Chemistry (IUPAC) (Thevenot et al., 2001). A biosensor is defined as ‘a self-contained integrated device that is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is in direct spatial contact with a transduction element.’ Therefore, a biosensor consists of two main components: a bioreceptor and a transducer. The bioreceptor is composed of a biomolecule recognition element (an enzyme, an antibody, a protein receptor, DNA, or whole cells) that recognizes the target analyte, whereas the transducer converts the recognition event into a measurable signal (electrical, optical, thermal, and so on). A typical biosensor construct also normally incorporates signal-processing elements (amplification, filtering, data processing, and storage) and a display of the final result. A schematic diagram of a typical biosensor is illustrated in Figure 9.1. The specificity of every biosensor is ultimately dictated to the specificity of the bioreceptor molecule used. Enzymes have been the most widely used bioreceptor molecules in biosensor applications. Enzymes are proteins that act as catalysts for biochemical reactions. Their activity is determined by their chemical composition and 3D structure. An enzyme is capable of recognizing only a specific substrate (or a class of substrates) and, therefore, is able to catalyze only a specific chemical reaction. In terms of the mechanism used in catalysis, enzymes can be categorized in six classes (Vargas-Bernal et al., 2012). Among these, oxidoreductases (dehydrogenases, oxidases, peroxidases and oxygenases), which catalyze oxidation/reduction reactions of the substrate via transfer Advances in Food Diagnostics, Second Edition. Edited by Fidel Toldrá and Leo M.L. Nollet. © 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.

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–

+

–

–

+

–

Biocompatible layer

Transducer (electrical, optical, physical)

Amplifier Signal

Microelectronics data Processing Analytes

Immobilized biomolecules

Urea, HMs, etc. Triglycerides Glucose

Antibody Enzymes

Cholesterol Antigens Target DNA

Probe DNA Organisms, bacteria

Figure 9.1  Schematic diagram describing the parts and operation of a typical biosensing device (reprinted with permission from Turdean, 2011).

of hydrogen or electrons, are the most analytically useful enzymes in biosensor applications (Vargas-Bernal et al., 2012). The popularity of enzymes as bioreceptor molecules in biosensors is due to (Borgmann et al., 2011; Marazuela and Moreno-Bondi, 2002): a) the large number of enzymatic reactions that can be exploited for analytical purposes; b) the wide array of detectable species – in addition to direct detection of the substrate and product of the enzymatic reaction, inhibitors (compounds that inhibit the

9  Enzyme-based Sensors

enzymatic reaction) and mediators (compounds that enhance the catalytic activity) can be monitored indirectly; c) the flexibility in detection – different types of transduction (electrochemical, optical, thermal) can be used to detect the analyte of interest; d) the low consumption of enzymes, since they are not consumed during the analysis; e) the high selectivity of enzymatic reactions; f ) the commercial availability of enzymes at high purity. The disadvantages of using enzymes in biosensors devices are the following: a) Enzymes are bulky proteins and, often, the active site of the enzyme is not readily accessible to the substrate; therefore, the activity of the enzyme is reduced. b) Enzymes have an inherently limited lifetime, and can be deactivated by components in the sample, or by extreme chemical and physical conditions prevailing in the sample. Therefore, biosensors exhibit only limited long-term stability. c) The enzymatic activity is dependent on pH, ionic strength, chemical inhibitors and temperature. d) The cost of some commercial enzymes is often high. Common enzymatic reactions forming the basis of biosensors in food diagnostics are listed in Table 9.1. Historically, the first biosensor was described by Clark and Lyon in 1962, when the term ‘enzyme electrode’ was actually adopted (Clark and Lyon, 1962). The development of an enzymatic biosensor involves: a) selection of a suitable enzyme; b) selection of a suitable immobilization method; Table 9.1  Common enzymes used in food diagnostics and their reactions. Enzyme

Enzymatic reaction

alcohol oxidase

ethanol + O2 → acetaldehyde + H2O2

glucose oxidase

glucose + O2 → gluconic acid + H2O2

lactose oxidase

lactose+ O2 → pyruvate + H2O2

alcohol dehydrogenase

ethanol + NAD+ → acetaldehyde + NADH + H+ acetaldehyde + NAD+ → acetic acid + NADH + H+

lactate dehydrogenase

lactate + NAD+ → pyruvate + NADH

Acetylcholinesterase cholineoxidase

acetylcholine + H2O → choline + acetic acid choline + O2 → betaine aldehyde + H2O2

horseradish peroxidase

H2O2+ electron donor → 2H2O + oxidized donor (electron donor = phenols, aromatic amines, luminol etc)

tyrosinase

phenol + 1/2O2 → quinone + H2O

lipase

triglycerides + 3H2O → glycerol + 3 fatty acids

nitrate reductase

NO3– + NADH + H+→ NO2– + NAD+ + H2O

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c) selection of a suitable transducer; d) optimization of the biosensor in terms of dynamic range, linearity, and minimization of interferences; e) packaging of the biosensor; f ) commercialization. The features of an ideal biosensor are (Vaddiraju et al., 2010; Preda et al., 2011): a) wide applicability to many sample matrices; b) high accuracy and precision; c) excellent sensitivity and specificity; d) wide dynamic range; e) rapid response time for real-time monitoring; f ) high operational and physical robustness (i.e. insensitivity to variations of pH, ionic strength, temperature, pressure etc); g) long-term stability, lifetime and reliability; h) amenability to testing and calibration; i) low service requirements, running and capital costs; j) product safety (biocompatibility if the biosensor is to be used for invasive monitoring in clinical situations, and in environmental applications the host system must not be contaminated by the sensor); k) small size, portability and low power requirements. As noted earlier, a main requirement for a biosensor is that the bioreceptor must be placed in close proximity to the transducer. In enzymatic biosensors, this is normally achieved by immobilization of the enzymes on the surface of the transducer. Immobilized enzymes offer some unique advantages in comparison with soluble enzymes (Ansari and Husain, 2012; Twyman, 2005): a) Reproducibility and low consumption of the enzyme; the same amount of immobilized enzyme can be used repeatedly for several measurements, ensuring high shortterm reproducibility and low consumption. b) Stability: in many cases, enzymes are stabilized by the immobilization process, being active at a wider range of conditions. c) Simplicity: the analysis procedure is simplified and the analysis time shortened, because the use of additional solutions is not required. d) Sensitivity: since the transduction step is performed in situ, the detected species is not able to diffuse away from the transducer enhancing the sensitivity. An ideal immobilization method should ensure: a) Chemical stability: the activity of the enzyme over time should remain constant. b) Protection: the catalytic activity of the enzyme should not be affected by matrix components. c) Mechanical stability: the immobilized enzyme should remain tightly bound to the surface of the transducer.

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E

E

E

E

E E

E

E E

E

E Entrapment

E

Adsorption

E

E P

E

E

E

E

E

E

E E

Covalence

E

E

P

E Cross-linking

Affinity

Figure 9.2  Schematic representation of the main immobilization methods. E – enzyme; P – inert protein (reprinted with permission from Sassolas et al., 2012).

Since the type of immobilization method dramatically affects the analytical characteristics of enzymatic biosensors (sensitivity, accuracy, precision, lifetime, etc.), the development of suitable immobilization strategies is of critical importance. There are five main immobilization methods: adsorption, entrapment, cross-linking, covalent bonding and affinity attachment. The advantages and drawbacks of these methods have been extensively reviewed (Sassolas et al., 2012; Choi, 2004; Balasubramanian and Burghard, 2006). Figure 9.2 schematically illustrates the main enzyme immobilization methods.

9.2 ­Types of transducers The majority of existing enzymatic biosensors are based on either electrochemical (amperometric and potentiometric) or optical (absorbance, fluorescence, chemiluminescence) transduction (D’Orazio, 2003), while other types of transducers (thermal, piezoelectric etc) are less frequently used. The principle of these techniques are listed in Table 9.2, whereas typical examples of the electrochemical and optical enzymatic biosensors are listed in Table 9.3. Electrochemical biosensors can operate in turbid environments, exhibit satisfactory sensitivity, have low power requirements and are suitable for miniaturization. The two most important classes of electrochemical transducers for biosensing include the amperometric and potentiometric/ISFET devices (Zhang et al., 2008; Grieshaber et al., 2008).

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Table 9.2  The operational principles of the main transducers used in biosensors. Transduction system

Technique/parameter

Electrochemical

Amperometric/current at a constant voltage Potentiometric/voltage

Electrical

Conductometric/conductivity

Optical

Photometry/absorption Fluorescence/fluorescence intensity Chemiluminescence/chemiluminescence intensity

Thermal

Calorimetry/heat

Piezoelectric

Surface acoustic waves/mass absorption at the surface of a crystal Bulk acoustic waves/mass absorption at the total mass of a crystal

Table 9.3  Examples of electrochemical and optical detection in food diagnostics. Analyte

Enzyme

Detection

aspartame

carboxylesterase, alcoholoxidase, carboxypeptidase, L-aspartase, peptidase, aspartateaminotransferase, glutamateoxidase and α-chymotrypsin

amperometric

sorbitol

sorbitol dehydrogenase, NAD+

amperometric

benzoic acid

Tyrosinase

amperometric

sulphites

sulphite oxidase

amperometric

parathion

parathionhydrolase

amperometric

propoxur and carbaryl

acetylocholinesterase

optical

diazinon and dichlorvos

tyrosinase

amperometric

paraoxon

alkaline phosphatase

optical

nitrate

nitratereductase

amperometric

nitritereductase

optical

phosphate

polyphenol oxidase and alkaline phosphatase, phosphorylase, phosphoglucomutase and glucose-6-phosphate dehydrogenase

amperometric

arsenic, cadmium, bismuth

cholinesterase

electrochemical

cadmium, copper, chrome, nickel, zinc

urease

optical

copper and mercury

glucoseoxidase

amperometric

fructose

fructosedehydrogenase

amperometric

Lactose

β-Galactosidase

amperometric

l-aminoacids

D-aminoacidoxidase

amperometric

l-glutamate

L-glutamateoxidase

amperometric

ethanol

alcohol oxidase, alcohol dehydrogenase

amperometric

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Table 9.3  (Continued) Analyte

Enzyme

Detection

cholesterol

cholesteroloxidase and peroxidase

amperometric

a-solanine

boutyrylcholinesterase

potentiometric

oxalate

oxalate oxidase, catalase

optical

polyphenols

tyrosinase, laccase

amperometric

fatty acids

lipase

amperometric

biogenic amines

amine oxidase and peroxidase

amperometric

hypoxanthine

xanthineoxidase

amperometric

inosine

xanthineoxidase

amperometric

lactic acid

xanthine oxidase, diamine oxidase, polymide oxidase

amperometric

Amperometric transduction is based on the application of a suitable potential to a working electrode, which initiates oxidation or reduction of an electroactive species. The resulting current is proportional to concentration of the target species. Enzymatic reactions often generate chemical species (e.g. H2O2, NADH, quinone) that can be measured by amperometry. Depending on the electrical communication between the enzyme and the transducer, amperometric enzymatic biosensors can be classified into three generations (Borgmann et al., 2011; Wang, 2008). In biosensors of the first generation, the reactant or product of the enzymatic reaction diffuses to the transducer and causes the response. In the second generation of biosensors, intermediate compounds, called mediators, are utilized to transfer electrons between the enzyme and the transducer. Mediators can be immobilized on the surface of the transducer, together with the enzyme (Sarma et al., 2008; Grieshaber et al., 2008; Malhotra and Chaubey, 2003). In the case of the third generation of biosensors, direct electron transfer is possible between the active side of the enzyme and the transducer without the need of a mediator (Borgmann et al., 2011; Preda et al., 2011; Sarma et al., 2008; Wang, 2008). Electrodes are usually made of noble metals and different forms of carbon (carbon paste, glassy carbon, carbon fibres, nanotubes or screen-printed graphite (Sarma et al., 2008; Albareda-Sirvent et al., 2000). In potentiometry, the potential developed across an ion-selective membrane, forming part of an indicator electrode (hence the term ‘ion-selective electrode’) is measured against a reference electrode. The relationship between membrane potential and the concentration of the target species is governed by the Nernst equation (Grieshaber, et  al., 2008). Enzymatic potentiometric biosensors are conveniently fabricated by immobilising the enzyme on the surface of the ion-selective membrane. Field-effect transistors (FETs) are a type of transistor that uses an electric field to control the conductivity of a channel (i.e. a region depleted of charge carriers) between two electrodes (i.e. the source and drain) in a semiconducting material. Control of the conductivity is achieved by varying the electric field potential, relative to the source and drain electrode, at a third electrode, known as the gate. Depending on the configuration and doping of the semiconducting material, the presence of a sufficient positive or

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negative potential at the gate electrode would either attract charge carriers (e.g. electrons) or repel charge carriers in the conduction channel. The current focus in biosensing applications is on ISFET (ion-selective field-effect transistor) and EnFET (enzyme field-effect transistor) devices. One of the most popular methods for the construction of FET-based biosensing devices is the immobilization of  enzymes at the gate surface of ion-selective ISFET devices, creating an EnFET (Grieshaber et al., 2008; Pijanowska and Torbicz, 2005; Pohanka, 2009). The majority of potentiometric and EnFET enzymatic transducers rely on the measurement of H+ or NH4+ ions produced in various enzymatic reactions, by making use of pH or NH4+ sensitive membranes, respectively. Optical enzymatic biosensors can be used in combination with different types of spectroscopic technique, including absorption, fluorescence and chemiluminescence (Marazuela and Moreno-Bondi, 2002; Choi, 2004; Monk and Walt, 2004). The enzymes commonly applied in optical biosensors are oxidases and oxidoreductases (which catalyze the oxidation of compounds using oxygen or NAD+), esterases (which produce acids), decarboxylases (which produce CO2) and deaminases (which produce NH3). The biological receptor is immobilized directly on the surface of an optical fibre. NADH has a strong absorbance at 340–360 nm, which can be used to monitor the concentration of substrates of dehydrogenase enzymes, such as pyruvate or lactate. Many enzymatic sensors for the determination of organophoshorous pesticides are based on the use of acetylcholinesterase, an enzyme involved in neurochemical reactions, which is inhibited by pesticides. Acetylcholinesterase can be immobilized in optical fibre biosensors, and detection is based on measuring the pH changes caused by release of acetic acid, using pH-sensitive dyes (Marazuela and Moreno-Bondi, 2002; Balasubramanian and Burghard, 2006). Fluorescence is often applied in combination with oxidase enzymes, and the decrease in the oxygen concentration is measured by the luminescence quenching of different transition metal complexes. In addition, several fibre-optic sensors have been based on detection of the fluorophore NADH at 455 nm, produced as a result of the action on different substrates of dehydrogenases enzymes (e.g. glutamate or alcohol dehydrogenases) immobilised on optical fibres (Borgmann et al., 2011). Chemiluminescence sensors, in which the analyte induces emission of light on interaction with a bioreceptor, can also be used. The popular chemiluminescence reaction between luminol and hydrogen peroxide, catalyzed by horseradish peroxidase, has served as the basis for different biosensors (Borgmann et al., 2011). Electrochemi­ luminescence (electrogenerated chemiluminescence) biosensor systems mainly include detection of NADH using dehydrogenases and H2O2 using oxidases. Since many enzymes can produce H2O2 during their substrate-specific enzymatic reaction, electrochemiluminescence enzyme biosensors are made possible by coupling the luminol light-emitting reaction with enzyme-catalyzed reactions generating H2O2. Ru(bpy)23+based enzyme electrochemiluminescence biosensors operate on the Ru(bpy)23+/NADH co-reactant systems, in conjunction with deoxygenases.

9.3 ­Enzymatic biosensors and the food industry Current analytical methods in the food industry are laborious, expensive, usually slow, and require bulky instrumentation. However, the food industry requires pocket-sized

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devices capable of fast, on-site measurements of undiluted samples during the production or processing of foods, or for quality control purposes. Most of these drawbacks can be conveniently addressed by enzymatic biosensors. Enzymatic biosensors are used to measure food ingredients (sugars, acids, amino acids, inorganic ions, alcohols and carbohydrates), contaminants (residues of pesticides and heavy metals), food additives (sorbitol, benzoic acid, sulfites) and indicators of food ‘freshness’ (such as biogenic amines). The utility of enzymatic biosensors in food analysis has been reviewed in detail (Barthemlebs et al., 2010; Prodromidis and Karayannis, 2002; Cock et al., 2009); VargasBernal et al., 2012; Jaffrezic-Renault, 2001; Upadhyaya and Verma, 2013; Sharma et al., 2003).

9.4 ­Biosensors for the analysis of main food components 9.4.1 Sugars

Electrochemical biosensors are often used for sugar detection. In these devices, either single or multiple enzymes are immobilized directly on different types of electrochemical transducers (carbon, screen-printed, etc.) using a variety of methods (Monosik et al., 2012). Glucose biosensors are important in monitoring fermentation processes, and in the dairy, wine, beer, and sugar industry (Mao et al., 2008). The detection of glucose is based on biosensors that utilize glucose oxidase, which catalyzes the oxidation of glucose into gluconic acid. Usually, the hydrogen peroxide produced is monitored:

oxidase D - Glucose + O2 Glucose  → D - gluconic acid + H 2O2

Hydrogen peroxide can then be measured by means of electrochemiluminescence reaction with luminol, or via amperometry with a suitable mediator such as Prussian blue:

H 2O2 + [ Med ]red Peroxidase → [ Med ]ox + H 2O



A flow injection with optical fibre biosensors for glucose, based on luminol electrochemiluminescence, has been described. The sol-gel method is introduced to immobilize glucose oxidase (GOx) on the surface of a glassy carbon electrode. Glucose could be quantified in the concentration range between 50 μM and 10 mM, with a detection limit around 26 μM. The proposed method can be applied for the determination of glucose in soft drink samples. Zhu et al. (2002) used an amperometric probe-type glucose sensor with Pt working electrode, and an Ag/AgCl reference one, polarized at +650 mV. The results showed that cellulose acetate membranes treated with amylamine were the most convenient structures to establish single-membrane recognition layers. The linearity and response time of this electrode were found to be up to 320 mM of glucose and 500 sec, respectively, at pH 4. An electrode was used to determine the glucose content in real samples. Results were in a good agreement with the conventional measurement method (Alp et al., 2000). D-Fructose is a monosaccharide found in three main forms in the food: as free fructose (present in fruits and honey), in sucrose (a disaccharide with sucrose), or as

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fructans  –  oligosaccharides (present in some vegetables and wheat). Because of its higher sweetening ability compared to sucrose and glucose, fructose is used as a diet sweetener in diabetic foods. Enzyme electrodes for fructose determination are often based on D-fructose-5-dehydrogenase, using an electron acceptor serving as an electrochemical mediator, such as ferrocene or ferricyanide:

-Fructose dehydrogenase D - Fructose + [ Med ]ox D → 5 - keto - D - Fructose + [ Med ]red



An improved amperometric biosensor, based on a SBM composite transducer, has been used for the determination of D-fructose in some food samples. The enzyme, D-fructose dehydrogenase, was incorporated directly into a solid composite transducer containing both 2-hexadecanone as a SBM and chemically modified graphite. Ferri­ cyanide was used as a redox mediator, and the amperometric signals were linearly proportional to D-fructose concentrations in the range 5 × 10−5 to 1 × 10−2 M. The use of chemically modified graphite by a mild oxidation step was shown to improve the biosensor selectivity against anionic interferents, such as L-ascorbate. The assay of D-fructose by this electrode was not influenced by the presence of sugars or other interferents. The results agreed well with those obtained with the commercial kit (Stredansky et al., 1999). Sucrose determination requires a multienzyme system, in which sucrose is first hydrolyzed to fructose and glucose by the enzyme invertase:

D - Sucrose + H 2O Invertase  → D - fructose + a - D - glucose

α-D-glucose is converted to its β-isomer by the enzyme mutarotase, and glucose oxidase can then be used for the determination of glucose, as explained above. It must be noted that the possibility to immobilize multiple enzymes on electrodes enables multi-enzyme determinations. One example of this is the development of a multi-enzyme electrode obtained by a two-step immobilization of the enzymes GOx, mutarotase and invertase. GOx was entrapped in a poly-1,3-diaminobenzene film on a platinum electrode by electrochemical polymerization, and a combination of mutarotase and invertase was cross-linked over the electrode via bovine serum albumin and glutaraldehyde. The sucrose con­ centration was determined from hydrogen peroxide oxidation. This immobilization method minimized interference from ascorbic acid. A second electrode, for glucose determination only, was constructed using an inactive invertase. The biosensor showed a good agreement with the standard LC method for sucrose and glucose analysis in soft drinks (Surareungchai et al., 1999). Lactose is the main disaccharide present in milk and its content is closely related to the quality of milk and dairy products. It is hydrolyzed by the enzyme β-galactosidase to galactose and glucose. Galactose can be further oxidized to galacto-hexodialdose:

-galactosidase D - Lactose + H 2O b → D - galactose + D - glucose



-galactose oxidase D - galactose + O2 D → D - galacto - hexoldialdose + H 2O2

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For estimation of lactose in milk and its products, an amperometric lactose biosensor was developed by immobilizing galactosidase and galactose oxidase in LangmuirBlodgett films of poly(3-hexyl thiophene)/stearic acid. The working electrode may be used for the estimation of lactose/galactose in food and biological fluids (Sharma et al., 2004). Another research team immobilized β-galactosidase into polyelectrolyte membranes, using organic solvents and perfluorosulfonated polymer. The results of the analysis of milk whey, in a flow-injection system that included lactose biosensor based on Berlin blue (as a signal transducer) and polyelectrolyte membranes, correlated well with measurement data obtained by a standard chromatographic technique (Lukacheva et al., 2007). 9.4.2 Acids

Organic acids constitute another important class of main food components. Optical, electrochemical and calorimetric biosensors can be used for their determination (Monosik et al., 2012). Lactic acid is an indicator of the fermentative processes and is related to the freshness, stability, and storage quality of several products such as tomato sauces, fruits, juices, wine, and milk. Typically lactate biosensors are based on the following reactions:

-lactate oxidase L - lactate + O2 L → pyruvate + H 2O2



-lactate dehydrogenase L - lactate + NAD + L → pyruvate + NADH + H +



NADH + H + + [ Med ]ox Diaphorase → [ Med ]red + NAD +



The NAD+ can be monitored amperometrically or photometrically. A biosensor for the selective determination of lactate in wine, based on robust solid composite transducers, was developed by Katrlik et al. Transducers were comprised of a solid binding matrix having a hydrophobic skeleton, with the enzymes L-lactate dehydrogenase (LDH) and diaphorase (DP) placed onto the transducer surface and covered by a dialysis membrane, which substantially reduced interferences derived from easily oxidisable compounds of wine, such as polyphenols. As a mediator, ferricyanide was used. The results obtained by the biosensors were in good agreement with those obtained by liquid chromatography (Katrlik et al., 1999). Another paper described an amperometric lactate biosensor based on a conducting polymer, poly-5,20-50,200-terthiophene-30-carboxylic acid (pTTCA), and multi-walled carbon nanotube (MWNT) composite present on a gold electrode. LDH and the oxidized form of nicotinamide adenine dinucleotide (NAD+) were subsequently immobilized onto the pTTCA/MWNT composite film. The detection signal was amplified by the pTTCA/MWNT assembly, with immobilized enzyme. The applicability of the biosensor in commercial milk and human serum samples was demonstrated successfully (Rahman et al., 2009).

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Malic acid is present mainly in fruit and vegetables, juices and other commodities. This acid is a very important parameter characterizing quality of wine, and it is often used as an acidulant. Its determination using biosensors is based on the following reactions:

dehydrogenase L - Malate + NAD + Malate  → oxalacetate + NADH + H + Malic enzyme ( pyruvic-malic carboxylase )

L - Malate + NADP +   → pyruvate + CO2 + NADPH + H + L-Ascorbic acid occurs naturally in some foods, but is also frequently added to processed foods as an antioxidant. The ascorbic acid content in food materials is an indicator of its freshness. L-Ascorbate is oxidized to dehydroascorbate, with production of hydrogen peroxide, which is ultimately detected:

- Ascorbate oxidase 2L - ascorbate + O2 L → 2L - dehydroascorbate + 2H 2O2



Acetic acid is produced during the fermentation of alcoholic drinks, and found in soy sauce and vinegar. Among others, a widely used tri-enzymes system can be used for developing acetate biosensors (e.g. measuring the consumption of oxygen in the last reaction):

kinase Acetate + ATP Acetic  → acetyl - P + ADP



kinase Phosphoenolpyruvate + ADP Pyruvate   → Pyruvate + ATP



oxidase Pyruvate + phosphate + O2 Pyruvate  → acetylphosphate

Citric acid exists in several fresh fruits such as lemons and limes and is also used as an additive in the food industry, mainly as a preservative and an acidulant. Enzymatic biosensors for citrate are based on the following scheme:

lyase Citrate Citrate  → oxaloacetate + acetic acid



decarboxylase Oxaloacetate Oxaloacetate  → Pyruvate + CO2



oxidase Pyruvate + phosphate + O2 Pyruvate  → acetylphosphate + CO2 + H 2O2

The hydrogen peroxide generated can be determined electrochemically, but the enzyme used, citrate lyase, is unstable, so this methodology is rarely used. 9.4.3  Amino acids

The presence of D-amino acids in foods is associated with a decrease in protein digestibility, thus affecting the bio-availability of essential amino acids and seriously impairing the nutritional value of the food. D-amino acids are also generally considered to be important markers of bacterial contamination of the food products. D-amino acid

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oxidase (DAAO) is a peroxisomal enzyme, containing FAD as a cofactor, that is expressed in a wide range of species, from yeasts to human, but not in bacteria nor in plants. Its function is to catalyze the oxidative deamination of D-isomer of amino acids to the corresponding 2-oxoacid and ammonia. During this step, the FAD is reduced, and the  catalytic cycle is completed through its reoxidation by O2 to yield hydrogen peroxide (Monosik et al., 2012).

-amino acid oxidase D - amino acid + O2 + H 2O2 D → 2 - oxoacid + NH 4 + +H 2O2

L-amino acids ca be determined using L-amino acid oxidase (LAAO) ,while enzymatic biosensors for specific amino acids (e.g. L-glutamate) can be developed using by immobilization of L-glutamate oxidase and L-glutamate dehydrogenase on amperometric transducers. 9.4.4 Alcohols

The determination and control of ethanol is important in the brewing, wine making and distilling industries. Tax regulations also require exact determination of the ethanol content, especially in spirits. Ethanol biosensors are based mainly on immobilized alcohol oxidase or dehydrogenase, catalyzing the following reactions (Prodromidis and Karayannis, 2002; Monosik et al., 2012):

dehydrogenase Ethanol + NAD + Alcohol  → acetaldehyde + NADH + H +



oxidase Ethanol + O2 + Alcohol  → acetaldehyde + H 2O2

In foods and beverages, glycerol serves as a humectant, solvent and sweetener, and may help to preserve foods. It is also used as a filler in commercially prepared low-fat foods (e.g. cookies), and as a thickening agent in liqueurs. Glycerol is a secondary fermentation product of alcoholic secondary fermentation, contributing to the viscosity and smoothness of a wine, with a favourable effect on the taste. Glycerol determination by an amperometric system can be based on the enzymatic reactions (Prodromidis and Karayannis, 2002; Monosik et al., 2012), as follows:

dehydrogenase Glycerol + NAD + Glycerol  → dihydroxyacetone - glycerol dehydrogenase Glycerol + [ Med ]ox Poo  → dihydroxyacetone + [ Med ]red

kinase Glycerol + ATP + Glycerol  → L - glycerolphosphate + ADP





-Glycerolphosphate oxidase L - Glycerolphosphate + O2 + L  → dihydroxyacetonephosphate + H 2 O2

The same reactions could be used for triglyceride detection, after hydrolysis and detection of the glycerol released.

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9.5 ­Biosensors for contaminants 9.5.1 Pesticides

Pesticides are chemicals used in agricultural production to destroy or control weeds, insects, fungi and other pests. Pesticides are considered to be some of the most dangerous contaminants, because of their ability to accumulate, and their long-term effects on living organisms. Currently, over 800 active ingredients, which belong to more than 100 substance classes, can be classified as pesticides. Organochlorine, organophosphorus and organonitrogen pesticides (carbamates, triazines and their derivatives) are the most important of these. There are two approaches to detecting pesticides using biosensors: an indirect method based, on inhibition of enzyme activity; and a direct approach, based on measurement of the enzymatic reactants or products (Sassolas et al., 2012). The general principle of enzymatic inhibition biosensors is based on the correlation between the toxicity of pesticides and the decrease of enzyme activity. Therefore, the development of these biosensing systems relies on a quantitative measurement of the enzyme activity, before and after exposure to a target analyte. Typically, the enzyme inhibition-based biosensor includes the following steps: determination of initial enzymatic activity; incubation of a biosensor in the sample (potentially containing the target pesticides); and, finally, measurement of the residual activity. This activity can be measured using different transduction techniques, including amperometry, potentiometry, spectrometry, fluorimetry and calorimetry, depending on the enzymatic reaction used (Sassolas et al., 2012; Carlo and Compagnone, 2010; Verma and Bhardwaj, 2015; McGrath et al., 2012). In enzyme inhibition-based pesticide biosensors, the biological receptors are usually cholinestereases, such as acetylcholinesterase (AChE), butyrylcholinesterase (BChE) or urease. For instance, AChE catalyzes the following reaction (Liu et al., 2013): AChE



Acetylcholine + H 2O → Choline + Acetic acid



Organophosphate and carbamate pesticides selectively inhibit cholinesterases by blocking the serine in the active site, through nucleophilic attack, to produce a serine phosphoester (via phosphorylation). The pH variation produced by the acid formation can be measured using electrochemical methods, such as potentiometry. This pH change can also be measured using pH-sensitive spectrophotometric indicators, or pHsensitive fluorescence indicators. In many cases, a two-enzyme system can be used in which acetylcholine is coupled to choline oxidase (Sassolas et al., 2012). AChE



Acetylcholine + H 2O → Choline + Acetic acid



ChoD



Choline + O2 → Betaine + H 2O2 Tyrosinase oxidizes monophenol in 2-consecutive steps:



activity Monophenol + O2 Cresolate  → Catechol activity Catechol + O2 Catecholase  → O - quinone



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Tyrosinase is inhibited by different compounds, such as carbamate pesticides and atrazine. Numerous electro-chemical biosensors based on the inhibition of tyrosinase activity have been reported (Sassolas et al., 2012). Alkaline phosphatase (ALP) catalyzes the following reaction:

Phosphate monoester + H 2O → alcohol + phosphate

ALP is inhibited by different compounds. The substrate (phosphate monoester) can be selected so that the product (alcohol) is optically or electrochemical active. For instance, phenyl phosphate forms electrochemically active phenol The second strategy for pesticide detection is based on organophosphorus hydrolase (OPH), which is able to hydrolyze a number of organophosphate pesticides with the formation of organophosphorus acid and alcohol (Liu et al., 2013; Serna-Cock and Perenguez-Verdugo, 2011). OPH



OP + H 2O → OP - ACID + R - OH

The alcohol formed can be electroactive or chromophoric, and can be measured by electrochemical or optical techniques, respectively. The main weakness of the enzymatic approach for pesticide detection is the limited qualitative information acquired, since the response is related to the total amount of pesticides in the sample. Also, the interpretation of results is further complicated by the fact that each pesticide exhibits a different inhibitory effect of the enzymatic activity. Finally, the specificity of these biosensors is rather limited, as they are prone to interference by other compounds in the sample (e.g. heavy metals). 9.5.2  Heavy metals

Heavy metals and their ions are ubiquitous and, by definition, are metals having atomic weights between 63.5 and 200.6 g mol−1 and a specific gravity greater than 5 g cm−3. Heavy metals constitute one of the most serious group of pollutants. Even in small concentrations, they are a threat to the environment and human health, because they are non-biodegradable. Metals leaching from eating utensils and cookware lead to metallic contamination of food and water. Additional sources of hazardous exposure are metallic constituents of pesticides and therapeutic agents, fossil fuels, industrial activity (metal-plating factories, mining industries, tanning, dye and chemical manufacturing industries) (Verma and Singh, 2005). The commonest operational strategy in heavy metal biosensors is inhibition of the enzymatic activity, based on the interaction of metal ions with exposed thiol or methylthiol-­ groups of the enzyme’s amino acids. Different enzymes, such as acetylcholine­sterase, alkaline phosphatase, urease, invertase, peroxidase, L-lactate dehydrogenase, tyrosinase, and nitrate reductase, have been used. The inhibition of the immobilized enzyme can be detected via electrochemical (amperometric, potentiometric, and conductometric) or optical measurements (Upadhyaya and Vermaa, 2013; Verma and Singh, 2005; Turdean, 2011). Heavy metal enzymatic biosensors exhibit the same weaknesses as pesticide biosensors (i.e. limited selectivity), but are useful for fast screening purposes.

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9.6 ­Food freshness indicators, antinutrients and additives Biogenic amines (BA) are basic nitrogenous compounds, formed mainly by decarboxylation of amino acids, or by amination and transamination of aldehydes and ketones. By chemical structure, biogenic amines can either be aliphatic (putrescine, cadaverine, spermine, spermidine), aromatic (tyramine, phenylethylamine) or heterocyclic (histamine, tryptamine) compounds. The detection of biogenic amines is a valuable tool for assessing the freshness and quality of a wide variety of protein-containing products, such as fish, meat, wine, beer, vegetables, fruits, nuts, and chocolate, where BAs accumulate in the process of food spoilage. The safety of fish and fish products has been mostly assessed by determining the histamine content, although cadaverine and putrescine have been found to potentiate histamine toxicity. The degree of histamine toxicity depends on the efficiency of the detoxification system of a body. If histamine alone is present in food intake, it is eliminated with the help of a metabolizing enzyme, diamine oxidase (DAO). However, in the presence of cadaverine and putrescine, this enzyme is inhibited. The allowed maximum residue level of histamine in food is 50 ppm (450 mM) in US and 100 ppm (900 mM) in Europe. Fish freshness has also been estimated by the level of trimethylamine (TMA). Biosensors for biogenic amines analysis are most commonly based on diamine oxidases, although other enzymes, such as putrescine oxidase, monoamine oxidase, methylamine dehydrogenase and flavin-containing mono-oxygenase type-3 (FMO3), have been also employed (Kivirand and Rinken, 2011). DAO



RCH 2 NH 2 + O2 + H 2O → RCHO + NH 3 + H 2O2 HRP



H 2O2 + Fe ( II ) → H 2O + Fe ( III )



Ethanol accumulation in vegetables, and during storage of fruits, can be detected using immobilized alcohol oxidase and alcohol peroxidase, together with a chromogene. Alcohol oxidase catalyzes the oxidation of ethanol in acetaldehyde and H2O2 in the presence of O2, and the peroxidase catalyzes the oxidation of the chromogene, causing a change in colour. The maturity of fruits can also be evaluated using biosensors, by measuring the levels of glucose or sucrose. There are some compounds that give rise to disagreeable smell in food commodities that can be detected with biosensors (such as 2,4,6-tricloroanisole in wine (due to corks). Polyphenols, which are strong antioxidants, can be measured in olive oil using immobilized lacase or tyrosinase enzymes and amperometric detection. Antinutrients are compounds that can generate health disorders to the consumer, by slowing down absorption of certain nutrients and causing deficiencies. For instance, oxalate, amygdalin and glucoalcaloids can be measured using biosensors incorporating oxalate oxidase, β-glucosidase and cholinesterase, respectively (Cock et al., 2009). The use of additives and preservatives has become an increasingly common practice in a food industry seeking increased lifetime, longer shelf-life, and better organoleptic properties. Many food additives can be detected using biosensors. Typical examples are aspartame, with catalysis by carboxyl esterase, alcohol oxidase, caboxypeptidase, L-aspartase,

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peptidase, aspartate aminotransferase, glutamate oxidase and α-chymotrypsin, sorbitol with sorbitol dehydrogenase and nicotinamide adenine dinucleotide (NAD+), and benzoic acid with tyrosine of sulphites with sulphite oxidase, all relying on amperometric transduction (Cock et al., 2009).

9.7 ­Future perspectives This work has demonstrated the wide applicability of enzymatic biosensors in food diagnostics. Multiplexed multidetection of several analytes (by incorporation of different enzymes on the same transducer or an array of transducers) is a promising field of research (Barthemlebs et al., 2010). Although there is a vast number of research articles on biosensors, only a few commercial devices are available (Table 9.4; Barthemlebs et  al., 2010). These devices are mainly based on the use of oxidases, and oxygen or hydrogen peroxide-mediated detection. The development of commercial enzymatic biosensors at a large scale must tackle some important challenges, such as the limited lifetime of enzymes, standardization in calibration, long-term stability, portability, elimination of interferences by using advanced immobilization protocols, and so on. Table 9.4  Commercial biosensors for food monitoring. Biosensor name

Analyte

Biocomponent

Company and reference

AM2 & AM3

Ethanol

AOX

Analox instruments

AM5

Methanol

AOX

(UK & USA)

GL6

Glucose

GOX,AOX,LOX

www.analox.com

Ethanol

GK + GPOX

Lactate Methanol Glycerol LM5

Lactate

LOX

Answer 8000

Glucose

GOX + HRP

Gwent sensors(UK) www.g-s-l.co.uk

Microzyme

Lactate

n.r

Biosentec (France) www.biosentec.fr

OLGA

Glucose

n.r

Sensolytics (Germany)

Lactate

www.sensolytics.com

Sucrose Ethanol Glutamate Per Bacco 2000

Glucose

GOX

Biofutura s.r.l (Italy)

Per Bacco 2002

Lactate

LDH

www.biofutura.it

Malate

MDH

Glucose

n.r

Senzytech one

Lactate Malate

Tectronik (Italy) www.tectronik.it

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