*3.2.1.1 Electrochemical biosensors*

The first proposed and commercialized biosensors were electrochemical biosensors, which is why they are most commonly reported. The basic principle of this class of biosensors is that the interaction between the biomolecule (bioreceptor) and the target analyte results in a chemical reaction that produces or consumes ions or electrons and in turn changes the electrical properties of the analyte solution, such as electrical current or potential. Transducer detects these changes by producing an electrochemical signal which is correlated with the amount of analyte present in the sample solution.

Advantages of electrochemical biosensors include minimal requirements for sample preparation and sensitivity at small sample volumes. It is also possible to perform sample analysis directly, which enables automation. Drawbacks of detections are poor reproducibility and stability [27].

**Figure 2.** *Schematic illustration of the common classification of biosensors.*

Electrochemical biosensors are classified according to the type of measured signal into subclasses: potentiometric, amperometric, conductometric, and biosensors based on ion-selective field-effect transistors (ISFETs). Different measurement principles always require a specific design of an electrochemical cell [21].

*Potentiometric biosensors* are based on the use of ion-selective electrode (ISEs) at the top of which an ion-selective membrane is placed which is responsible for selectivity to target ions in the presence of interfering ions in the sample. These devices measure the difference between the potential of the working and reference electrodes at essentially zero current, and this difference corresponds to the concentration of the analyte.

*Amperometric biosensors* are the most widespread class of electrochemical biosensors. Amperometric biosensors are more sensitive and faster than potentiometric but have poor selectivity and are susceptible to the interference of electroactive species that are not of interest [22, 28].

*Conductometric biosensors* are based on measurement of electrical conductivity in sample solution between two electrodes, as a consequence of the biochemical reaction. Conductometric biosensors operate at sufficiently low driving voltage, are not sensitive to light, do not require the use of a reference electrode, and can be produced using inexpensive technology [23, 24].

*Biosensors based on the ion-selective field-effect transistors (ISFET)* are the fourth class of electrochemical biosensors, suitable for the direct detection of ions. Change of activity of ions of a sample causes a change in the potential of the gate electrode that is brought into contact with the analyte solution. The change of the electric potential is then measured.

#### *3.2.1.2 Optical biosensors*

Optical biosensors are a biosensor class in which the transducer detects optical changes in the input light resulting from the interaction of the bioreceptor and the target analyte, and the amplitude of these changes is in correlation with the concentration of the present analyte in the analyzed sample. Among the significant advantages of these optical devices are insensitivity to electromagnetic interference, small instrumentation, simplicity, and noninvasiveness of measurement, as well as the possibility of application in vivo, since they are non-electrical biosensors. According to the optical configuration, biosensors can be intrinsic or extrinsic. In intrinsic biosensors, the incident light wave is closed in a wave guide or an optical fiber, along which it propagates, but the design of the structure in which the wave is closed is such that it allows the interaction of the wave with the analyte. In extrinsic biosensors, the light wave passes directly through the sample phase and reacts with it, and the optical fiber serves as a means of transmitting the signal.

*Absorption-based biosensors* are simple and inexpensive devices that allow the determination of concentrations of different analytes, based on the fact that each type of analyte absorbs a certain wavelength of light emitted into the sample. Guiding the light from the light source to the sample and from the sample to the detector can be performed using the same optical fiber or different fibers [29].

*Surface plasmon resonance (SPR) biosensors* use an optical detection technique where on the interface of metal and dielectrics, the amplified incident light hits the metal surface and excites the electrons, thereby generating electromagnetic waves (plasmons). Plasmon propagation is very sensitive to the changes in the refractive index of the material near the metal surface, which are caused by biomolecular interaction, such as, for example, specific binding of the analyte [30, 31].

*Fluorescence-based optical biosensors* can directly detect target atoms or molecules by measuring the change in the frequency of electromagnetic radiation emitted by

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converter.

*Biosensors for Determination of Heavy Metals in Waters DOI: http://dx.doi.org/10.5772/intechopen.84139*

(FRET).

*3.2.1.3 Piezoelectric biosensors*

*3.2.1.4 Thermal biosensors*

nature of the reactions catalyzed by enzymes.

*3.2.2 Classification by type of biocomponent/bioreceptor*

interaction between biological material and analyte.

them. The frequency change is stimulated by the absorption of radiation and the consequent appearance of the excited state of the target species. Detection can also be carried out indirectly, using fluorescence labels or fluorescence energy transfer

*Luminescence-based biosensors* can be classified into chemiluminescent and bioluminescent optical biosensors. Unlike biosensors based on fluorescence, in these sensor devices, the triggered state of the target atoms or molecules is obtained as a result of their exothermic chemical reaction, and while returning to the ground state, the excited species emit light without or with minimal heat. When such a chemical reaction occurs within a biological organism, then it is a bioluminescence.

Piezoelectric biosensors are devices in which the biorecognition element is integrated with a piezoelectric material used as a transducer. Among many types of natural and synthetic materials that exhibit a piezoelectric effect, quartz crystals are most commonly used [28, 32] because of their availability, as well as high temperature resistance and chemical stability in aqueous solution. The basic principle of measurement for this type of biosensor is based on the ability of a piezoelectric material to generate electrical potential when deformed under the applied mechanical stress, and vice versa, to elastically deform when exposed to an electric field.

Thermal biosensors, also called calorimetric or thermometric, are a biosensor class in which the transducer detects interactions between bioreceptors and analyte resulting in a change of temperature, which is in correlation with the concentration of the analyte. As thermal transducers in these devices, thermistors or thermopiles are used [21, 33]. Some of the advantages of thermal biosensors are detection without the need for labeling of reactants, not requiring frequent recalibration, and no disturbances by electrochemical and optical properties of the sample [21, 34]. In most research papers published about this type of sensor, described experiments were carried out using enzyme-based thermal biosensors, due to the exothermic

Biocomponent/bioreceptor is responsible for the detection and interaction with

Biocatalytic biosensors, also known as metabolism sensors, comprise a biological component that catalyzes the chemical conversion of the analyte with which it interacts and detect the magnitude of the resulting changes such as product formation, reactant disappearance, or inhibition of the reaction, which are correlated with the concentration of the analyte [35]. Affinity biosensors are based on selective interaction between the analyte and the biological component through their irreversible binding, resulting in a physicochemical change detected by the

the analyte and therefore is a very important part of any type of biosensor. The receptor is responsible for the selective and sensitive recognitions of the analyte, and the energy liberated during the interaction of the analyte and the receptor is converted into an electrical signal that is suitable for measurement. The most commonly used biological elements are enzymes and antibodies. Biosensors can be divided into two main categories: biocatalytic and affinity sensors based on the *Biosensors for Determination of Heavy Metals in Waters DOI: http://dx.doi.org/10.5772/intechopen.84139*

them. The frequency change is stimulated by the absorption of radiation and the consequent appearance of the excited state of the target species. Detection can also be carried out indirectly, using fluorescence labels or fluorescence energy transfer (FRET).

*Luminescence-based biosensors* can be classified into chemiluminescent and bioluminescent optical biosensors. Unlike biosensors based on fluorescence, in these sensor devices, the triggered state of the target atoms or molecules is obtained as a result of their exothermic chemical reaction, and while returning to the ground state, the excited species emit light without or with minimal heat. When such a chemical reaction occurs within a biological organism, then it is a bioluminescence.

### *3.2.1.3 Piezoelectric biosensors*

*Biosensors for Environmental Monitoring*

tration of the analyte.

potential is then measured.

*3.2.1.2 Optical biosensors*

species that are not of interest [22, 28].

produced using inexpensive technology [23, 24].

Electrochemical biosensors are classified according to the type of measured signal into subclasses: potentiometric, amperometric, conductometric, and biosensors based on ion-selective field-effect transistors (ISFETs). Different measurement

*Potentiometric biosensors* are based on the use of ion-selective electrode (ISEs) at the top of which an ion-selective membrane is placed which is responsible for selectivity to target ions in the presence of interfering ions in the sample. These devices measure the difference between the potential of the working and reference electrodes at essentially zero current, and this difference corresponds to the concen-

*Amperometric biosensors* are the most widespread class of electrochemical biosensors. Amperometric biosensors are more sensitive and faster than potentiometric but have poor selectivity and are susceptible to the interference of electroactive

*Conductometric biosensors* are based on measurement of electrical conductivity in sample solution between two electrodes, as a consequence of the biochemical reaction. Conductometric biosensors operate at sufficiently low driving voltage, are not sensitive to light, do not require the use of a reference electrode, and can be

*Biosensors based on the ion-selective field-effect transistors (ISFET)* are the fourth class of electrochemical biosensors, suitable for the direct detection of ions. Change of activity of ions of a sample causes a change in the potential of the gate electrode that is brought into contact with the analyte solution. The change of the electric

Optical biosensors are a biosensor class in which the transducer detects optical changes in the input light resulting from the interaction of the bioreceptor and the target analyte, and the amplitude of these changes is in correlation with the concentration of the present analyte in the analyzed sample. Among the significant advantages of these optical devices are insensitivity to electromagnetic interference, small instrumentation, simplicity, and noninvasiveness of measurement, as well as the possibility of application in vivo, since they are non-electrical biosensors. According to the optical configuration, biosensors can be intrinsic or extrinsic. In intrinsic biosensors, the incident light wave is closed in a wave guide or an optical fiber, along which it propagates, but the design of the structure in which the wave is closed is such that it allows the interaction of the wave with the analyte. In extrinsic biosensors, the light wave passes directly through the sample phase and reacts with

*Absorption-based biosensors* are simple and inexpensive devices that allow the determination of concentrations of different analytes, based on the fact that each type of analyte absorbs a certain wavelength of light emitted into the sample. Guiding the light from the light source to the sample and from the sample to the detector can be performed using the same optical fiber or different fibers [29]. *Surface plasmon resonance (SPR) biosensors* use an optical detection technique where on the interface of metal and dielectrics, the amplified incident light hits the metal surface and excites the electrons, thereby generating electromagnetic waves (plasmons). Plasmon propagation is very sensitive to the changes in the refractive index of the material near the metal surface, which are caused by biomolecular interaction, such as, for example, specific binding of the analyte [30, 31].

*Fluorescence-based optical biosensors* can directly detect target atoms or molecules by measuring the change in the frequency of electromagnetic radiation emitted by

it, and the optical fiber serves as a means of transmitting the signal.

principles always require a specific design of an electrochemical cell [21].

**146**

Piezoelectric biosensors are devices in which the biorecognition element is integrated with a piezoelectric material used as a transducer. Among many types of natural and synthetic materials that exhibit a piezoelectric effect, quartz crystals are most commonly used [28, 32] because of their availability, as well as high temperature resistance and chemical stability in aqueous solution. The basic principle of measurement for this type of biosensor is based on the ability of a piezoelectric material to generate electrical potential when deformed under the applied mechanical stress, and vice versa, to elastically deform when exposed to an electric field.

#### *3.2.1.4 Thermal biosensors*

Thermal biosensors, also called calorimetric or thermometric, are a biosensor class in which the transducer detects interactions between bioreceptors and analyte resulting in a change of temperature, which is in correlation with the concentration of the analyte. As thermal transducers in these devices, thermistors or thermopiles are used [21, 33]. Some of the advantages of thermal biosensors are detection without the need for labeling of reactants, not requiring frequent recalibration, and no disturbances by electrochemical and optical properties of the sample [21, 34]. In most research papers published about this type of sensor, described experiments were carried out using enzyme-based thermal biosensors, due to the exothermic nature of the reactions catalyzed by enzymes.

#### *3.2.2 Classification by type of biocomponent/bioreceptor*

Biocomponent/bioreceptor is responsible for the detection and interaction with the analyte and therefore is a very important part of any type of biosensor. The receptor is responsible for the selective and sensitive recognitions of the analyte, and the energy liberated during the interaction of the analyte and the receptor is converted into an electrical signal that is suitable for measurement. The most commonly used biological elements are enzymes and antibodies. Biosensors can be divided into two main categories: biocatalytic and affinity sensors based on the interaction between biological material and analyte.

Biocatalytic biosensors, also known as metabolism sensors, comprise a biological component that catalyzes the chemical conversion of the analyte with which it interacts and detect the magnitude of the resulting changes such as product formation, reactant disappearance, or inhibition of the reaction, which are correlated with the concentration of the analyte [35]. Affinity biosensors are based on selective interaction between the analyte and the biological component through their irreversible binding, resulting in a physicochemical change detected by the converter.

### *3.2.2.1 Antibody-based biosensors*

Antibodies are proteins produced by the immune system in response to a foreign substance in the body. Also known as immunoglobulins (Ig), they are Y-shaped proteins generated by a type of white blood cells called B lymphocytes (B cells). Their ability to recognize specific molecules makes them suitable for use as biorecognition component in biosensors. During the process of biological recognition, the antibodies bind tightly to antigens forming complexes. There are five classes of antibodies, based on their structure and function: IgA, IgD, IgE, IgG, and IgM. Among them, IgG is the class most frequently used for heavy metal detection, because of their higher affinity and specificity compared to other classes. Antibodies such as monoclonal, polyclonal, or recombinant can be utilized in biosensors. Monoclonal antibodies are homogeneous antibodies, derived from single B cell; thus they all have the same specificity, i.e., to bind to one unique epitope (binding site) on a specific antigen. Unlike monoclonal antibodies, polyclonal antibodies are produced from different B cells against the same antigen and therefore have affinity for various binding sites of that antigen. This feature of polyclonal antibodies results in their stronger binding to the target species, but due to the recognition of multiple epitopes, they have higher potential for cross-reactivity, i.e., specificity for nontargeted antigens with similar structural regions as the targeted one. The production of recombinant antibodies is enabled by genetic engineering. Important properties of antibodies for providing accurate results for detection and measurement using biosensors are high sensitivity and specificity, with minimal cross-reactivity [36].

Different types of approaches have been developed and used for immobilization of Abs onto a sensor surface, such as covalent binding, non-covalent immobilization, and coupling by affinity interactions, because the immobilization is the crucial step which can affect the optimal performance of an antibody-based biosensor [37]. Reaction conditions, such as temperature, pH, and ionic strength, can also affect the activity of the antibodies [38].

#### *3.2.2.2 Enzyme-based biosensors*

Enzymes are biocatalysts that catalyze chemical reactions. Their task is to translate the characteristic substance (substrate) into a product. Enzymes are highly selective for the particular substrate which makes them suitable sensor material. Detection mechanism of enzyme-based biosensors is based on activation or inhibition of their activities as a response caused by heavy metals. Usually the metal ion reacts with the thiol groups present in enzymatic structures that result in conformational changes and thus affect the catalytic activity. Different enzymes have been used for the structure of biosensors based on inhibition. Enzymes such as glucose oxidase, urease, glutathione S-transferase, alkaline phosphatase, lactate dehydrogenase, acid phosphatase, and invertase have been utilized to detect metals such as cadmium, lead, copper, mercury, zinc, etc. However, inhibition-based biosensors have an important disadvantage, which is insufficient selectivity because some of the enzymes simultaneously inhibit several metals.

Biosensors based on immobilized enzymes are also used, and they show several advantages compared to free enzymes:

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biosensors.

of heavy metals.

*Biosensors for Determination of Heavy Metals in Waters DOI: http://dx.doi.org/10.5772/intechopen.84139*

• In the case of reversible inhibition, sometimes reactivation of the enzyme

The problem with biosensors based on enzymatic inhibition is that only a few

Proteins, such as phytochelatins or metallothioneins, can be used as biological components in biosensors when immobilized on the surface of the transducer [39]. The interaction of proteins and metals in the biosensor is realized through the formation of complexes, and the detection technique does not require labeling. The resulting changes in the protein layer are detected by measuring the electrical capacity or impedance by the relevant transducer. Using the protein biosensor enabled the assessment of bioavailable concentrations of heavy metals. In addition, using capacitive sensors, which belong to the class of electrochemical biosensors, it is possible to achieve much higher sensitivity to low concentrations of heavy metals,

Whole cell-based biosensors are based on using biosensing cells, such as microorganisms, plant cells, algae, fungi, protozoa, etc., which can be natural or recombinant [40]. The use of whole cells as biological elements of recognition has many advantages. Whole cell-based biosensors are usually cheaper than biosensors based on enzymes, because the whole cells can be easily cultivated and are easier to isolate and purify compared with enzymes. Whole cells are more tolerant to a significant change in pH, temperature, or ionic strength. A multistep reaction is possible because one cell can contain all the enzymes and cofactors needed to detect the analyte. Biosensors of this type can easily be regenerated or maintained by allowing cells to regrow while working in situ. Preparation of samples is usually not necessary. Compared to enzyme-based biosensors, the disadvantages of these devices are that they are susceptible to interference of contaminants that are not targeted analytes. They also have a relatively slow response, compared to other types of

**3.3 Application of biosensors in detection and monitoring of heavy metals**

The unique biosensor features make them widely applicable in the field of water quality control, from the point of view of detecting and determining the concentration of heavy metals. The use of biosensors for individual or continuous measurements is dependent on the type of biologically active element. Since biological compounds such as cholesterol, glucose, urea, etc. are generally not electroactive, the combination of reactions is needed for obtaining an electroactive element, which leads to a change of current intensity [41]. **Table 2** shows the classification of biosensors based on the recognition component that was utilized for the detection

A proper immobilization of the biosensing element onto the transducer surface maintains biomaterial functionality while ensuring accessibility of the receptor cells toward analytes and proximity of the bioreceptor and transducer. The factors which determine the choice of a suitable physical or chemical immobilization method

• Faster analysis, less than 5 min.

enzymes are sensitive to heavy metals.

activity is not necessary.

*3.2.2.3 Protein-based biosensors*

compared to cell-based devices.

*3.2.2.4 Whole cell-based biosensors*


• Faster analysis, less than 5 min.

*Biosensors for Environmental Monitoring*

*3.2.2.1 Antibody-based biosensors*

the activity of the antibodies [38].

the enzymes simultaneously inhibit several metals.

• Reduced interferences in differential mode.

advantages compared to free enzymes:

• No preincubation is required.

*3.2.2.2 Enzyme-based biosensors*

Antibodies are proteins produced by the immune system in response to a foreign substance in the body. Also known as immunoglobulins (Ig), they are Y-shaped proteins generated by a type of white blood cells called B lymphocytes (B cells). Their ability to recognize specific molecules makes them suitable for use as biorecognition component in biosensors. During the process of biological recognition, the antibodies bind tightly to antigens forming complexes. There are five classes of antibodies, based on their structure and function: IgA, IgD, IgE, IgG, and IgM. Among them, IgG is the class most frequently used for heavy metal detection, because of their higher affinity and specificity compared to other classes. Antibodies such as monoclonal, polyclonal, or recombinant can be utilized in biosensors. Monoclonal antibodies are homogeneous antibodies, derived from single B cell; thus they all have the same specificity, i.e., to bind to one unique epitope (binding site) on a specific antigen. Unlike monoclonal antibodies, polyclonal antibodies are produced from different B cells against the same antigen and therefore have affinity for various binding sites of that antigen. This feature of polyclonal antibodies results in their stronger binding to the target species, but due to the recognition of multiple epitopes, they have higher potential for cross-reactivity, i.e., specificity for nontargeted antigens with similar structural regions as the targeted one. The production of recombinant antibodies is enabled by genetic engineering. Important properties of antibodies for providing accurate results for detection and measurement using biosensors are high sensitivity and specificity, with minimal cross-reactivity [36]. Different types of approaches have been developed and used for immobilization of Abs onto a sensor surface, such as covalent binding, non-covalent immobilization, and coupling by affinity interactions, because the immobilization is the crucial step which can affect the optimal performance of an antibody-based biosensor [37]. Reaction conditions, such as temperature, pH, and ionic strength, can also affect

Enzymes are biocatalysts that catalyze chemical reactions. Their task is to translate the characteristic substance (substrate) into a product. Enzymes are highly selective for the particular substrate which makes them suitable sensor material. Detection mechanism of enzyme-based biosensors is based on activation or inhibition of their activities as a response caused by heavy metals. Usually the metal ion reacts with the thiol groups present in enzymatic structures that result in conformational changes and thus affect the catalytic activity. Different enzymes have been used for the structure of biosensors based on inhibition. Enzymes such as glucose oxidase, urease, glutathione S-transferase, alkaline phosphatase, lactate dehydrogenase, acid phosphatase, and invertase have been utilized to detect metals such as cadmium, lead, copper, mercury, zinc, etc. However, inhibition-based biosensors have an important disadvantage, which is insufficient selectivity because some of

Biosensors based on immobilized enzymes are also used, and they show several

• A thousand times lower consumption of immobilized enzymes.

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• In the case of reversible inhibition, sometimes reactivation of the enzyme activity is not necessary.

The problem with biosensors based on enzymatic inhibition is that only a few enzymes are sensitive to heavy metals.
