*3.2.2.3 Protein-based biosensors*

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, compared to cell-based devices.

## *3.2.2.4 Whole cell-based biosensors*

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 biosensors.

#### **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 of heavy metals.

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


**Table 2.**

*Classification of biosensors based on the recognition component that was utilized for the detection of heavy metals*

are physicochemical properties of the analyte, nature of the chosen biosensing element, the type of used transducer, and the operating conditions of biosensor. Antibody-based biosensors can be used as an alternative approach for the detection of metal ions, due to antibody features such as high specificity and binding affinity for antigens harmful for the organism. Detection mechanism of these devices is based on antibody-metal ion complex formation. The resulted response of their immunochemical interaction is converted by a transducer to measurable values and processed to readable values. Antibodies are capable for antigen detection in very low concentrations [38], but if their cross-reactivity is high, they can yield falsepositive results of an assay of heavy metals in water [55].

A monoclonal antibody that recognizes 16 different metal-EDTA complexes has been produced and evaluated in terms of its binding affinity. The obtained results showed that the antibody has a maximum binding affinity for cadmium and mercury-EDTA complexes. [56]. In the inhibition immunoassay where the measurement of Cd2+ in water samples was carried out using monoclonal antibodies firmly bound to the cadmium-EDTA complex, but not to EDTA without metal [42], the biosensor showed satisfactory insensitivity to cations Ca2+, Na2+, and K1+ it encountered and achieved a reliable measurement in the presence of 1 mM of excess Fe3+, Mg2+, and Pb2+.

Monoclonal antibodies were used to detect Pb2+ without labeling, in a localized surface plasmon resonance-based optical biosensor [57]. The results of the experiment showed that at optimal monoclonal antibody immobilizing conditions, absorbability increased to 12.2% for detecting 10–100 ppb Pb(II)-EDTA complex with a limit of detection of 0.27 ppb.

Kulkarni et al. were the first to develop acid phosphatase-based fluorescence biosensor for the analysis of heavy metal ions Hg2+, Cr2+, and Cu2+. Increased concentration of metal ions resulted in increased enzyme inhibition and therefore decreased fluorescence. The enzyme was stable for more than 2 months at 4°C [58]. They also observed that mixture of heavy metal ions exhibit positive effect on the performance of biosensor.

The urease enzyme has been widely investigated as a possible biocomponent in heavy metal detection biosensors. Urease has been tested single and in combination with other enzymes. Electrochemical biosensor based on urease and glutamic dehydrogenase (GLDH) was developed for detecting heavy metals in water samples [59]. Also, a disposable potentiometric biosensor based on pure urease was developed, with the ability to detect copper and silver at sub-ppm level. For the detection of Pb and

**151**

**4. Conclusions**

such as pacemakers to the detection of drugs.

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

femtomolar concentration.

conformation [51].

Cd in liquid samples, biosensors based on the combination of urease and acetylcholinesterase (Ache) were developed as a biocomponent with a detection limit of 1 ppb in water samples. It is known that ions of heavy metals inhibit alkaline phosphatase which was used for forming the biosensor with alkaline phosphatase as a biocomponent. It was found that the sensitivity of the developed biosensor to Cd2+ and Zn2+ was

Two protein-based biosensors were developed on the basis of GST-SmtA and MerR [60] proteins, and their sensitivity and selectivity for heavy metal ions (Cd2+, Cu2+, Hg2+, and Zn2+) were measured using a capacitance transducer. Both types of biosensors have shown high sensitivity, enabling detection of metal ions up to

Capacitance protein-based biosensor using synthetic phytochelatins (ECs) was developed for the detection of heavy metal ions (Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+), and the results of the experiments showed a lower sensitivity for all metal ions except for Zn2+ compared to systems based on SmtA and MerR, which can be explained by conformational changes in the protein, taking into account that the change in capacitance is function of the resulting change in protein

In cell-based biosensors, bioelement is fused with reporter gene. The detection mechanism is based on the activation of the reporter gene upon the contact between bioreceptor and target analyte, yielding an output measurable signal that is a cor-

Various cell-based biosensors have been used for the detection of heavy metals in water due to their ease of production and field testing, the ability to perform fast single measurement, as well as continuous measurements, and the ease of identifying bioavailable concentrations of toxicants that allows estimation of effects that

The advantage of bacterial cells is resistance to environmental conditions that could destroy the sensory element if exposed to them, supplying it with a relatively stable environment. Due to specific metabolic pathways used in microorganisms, compared to isolated enzymes, microbial sensors have the potential for more selective analysis of heavy metals which cannot be measured by simple enzyme reactions [61]. In order to be available for any sensing mechanism that is based inside the cell, there is a need for analytes to be able to enter the cell via diffusion, nonspecific uptake, or active transport. Alternative approaches are implemented in the cases when membrane permeability for an analyte is not sufficient. These approaches include allocation of the recognition element to the outside of the cell or the introduction of an appropriate transport mechanism for importing the analyte [61]. A large number of studies in which performances of whole cell-based biosensors were tested have utilized electrochemical and optical transducers. For detection of heavy metal ions (Cd2+, Cu2+, Fe3+, Hg2+, Pb2+,and Zn2+) at concentrations of 10μM, a mammalian heart cells-based biosensor was developed [54], with excellent performance in terms of frequency selection, amplitude and duration of detection within 15 min. Biosensor, based on immobilized engineered bacteria *Alcaligenes eutrophus* (AE1239) and optical transducer, was utilized for monitoring the bioavailable copper ions in synthetic water samples, wherein the lowest limit of detection was 1 μM [62].

Biosensors have a very wide range of applications, from environmental monitoring, food safety, detection of various diseases, use in artificial implantable devices

relation with bioavailable concentration of heavy metal.

heavy metals have on living organisms.

10 ppb, whereas, with regard to ion Pb2+, there was no significant inhibition.

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

*Biosensors for Environmental Monitoring*

2A81G5 Antibody ISB4 12F6

Alkaline Phosphatase Pyruvate enzymes Oxidase Urease

**Table 2.**

*of heavy metals*

Glutathione S-transferase Mer R proteins Metallothionein

are physicochemical properties of the analyte, nature of the chosen biosensing element, the type of used transducer, and the operating conditions of biosensor. Antibody-based biosensors can be used as an alternative approach for the detection of metal ions, due to antibody features such as high specificity and binding affinity for antigens harmful for the organism. Detection mechanism of these devices is based on antibody-metal ion complex formation. The resulted response of their immunochemical interaction is converted by a transducer to measurable values and processed to readable values. Antibodies are capable for antigen detection in very low concentrations [38], but if their cross-reactivity is high, they can yield false-

*Classification of biosensors based on the recognition component that was utilized for the detection* 

Whole cells and cardiac cells Hg, Pb, Cd, Fe, Cu, Zn [54]

**Type of bioreceptor Analyzed heavy metal Reference**

Cd Cd U

Zn Hg Cd Hg Hg, Ag

Cd, Zn

Hg, Cu, Cd, Zn, Pb Cd, Zn, Ni

[42] [43] [44]

[45] [46] [47] [48]

[49, 50] [51, 52] [53]

A monoclonal antibody that recognizes 16 different metal-EDTA complexes has been produced and evaluated in terms of its binding affinity. The obtained results showed that the antibody has a maximum binding affinity for cadmium and mercury-EDTA complexes. [56]. In the inhibition immunoassay where the measurement of Cd2+ in water samples was carried out using monoclonal antibodies firmly bound to the cadmium-EDTA complex, but not to EDTA without metal [42], the biosensor showed satisfactory insensitivity to cations Ca2+, Na2+, and K1+ it encountered and achieved a

reliable measurement in the presence of 1 mM of excess Fe3+, Mg2+, and Pb2+.

Monoclonal antibodies were used to detect Pb2+ without labeling, in a localized surface plasmon resonance-based optical biosensor [57]. The results of the experiment showed that at optimal monoclonal antibody immobilizing conditions, absorbability increased to 12.2% for detecting 10–100 ppb Pb(II)-EDTA complex

Kulkarni et al. were the first to develop acid phosphatase-based fluorescence biosensor for the analysis of heavy metal ions Hg2+, Cr2+, and Cu2+. Increased concentration of metal ions resulted in increased enzyme inhibition and therefore decreased fluorescence. The enzyme was stable for more than 2 months at 4°C [58]. They also observed that mixture of heavy metal ions exhibit positive effect on the

The urease enzyme has been widely investigated as a possible biocomponent in heavy metal detection biosensors. Urease has been tested single and in combination with other enzymes. Electrochemical biosensor based on urease and glutamic dehydrogenase (GLDH) was developed for detecting heavy metals in water samples [59]. Also, a disposable potentiometric biosensor based on pure urease was developed, with the ability to detect copper and silver at sub-ppm level. For the detection of Pb and

positive results of an assay of heavy metals in water [55].

with a limit of detection of 0.27 ppb.

performance of biosensor.

**150**

Cd in liquid samples, biosensors based on the combination of urease and acetylcholinesterase (Ache) were developed as a biocomponent with a detection limit of 1 ppb in water samples. It is known that ions of heavy metals inhibit alkaline phosphatase which was used for forming the biosensor with alkaline phosphatase as a biocomponent. It was found that the sensitivity of the developed biosensor to Cd2+ and Zn2+ was 10 ppb, whereas, with regard to ion Pb2+, there was no significant inhibition.

Two protein-based biosensors were developed on the basis of GST-SmtA and MerR [60] proteins, and their sensitivity and selectivity for heavy metal ions (Cd2+, Cu2+, Hg2+, and Zn2+) were measured using a capacitance transducer. Both types of biosensors have shown high sensitivity, enabling detection of metal ions up to femtomolar concentration.

Capacitance protein-based biosensor using synthetic phytochelatins (ECs) was developed for the detection of heavy metal ions (Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+), and the results of the experiments showed a lower sensitivity for all metal ions except for Zn2+ compared to systems based on SmtA and MerR, which can be explained by conformational changes in the protein, taking into account that the change in capacitance is function of the resulting change in protein conformation [51].

In cell-based biosensors, bioelement is fused with reporter gene. The detection mechanism is based on the activation of the reporter gene upon the contact between bioreceptor and target analyte, yielding an output measurable signal that is a correlation with bioavailable concentration of heavy metal.

Various cell-based biosensors have been used for the detection of heavy metals in water due to their ease of production and field testing, the ability to perform fast single measurement, as well as continuous measurements, and the ease of identifying bioavailable concentrations of toxicants that allows estimation of effects that heavy metals have on living organisms.

The advantage of bacterial cells is resistance to environmental conditions that could destroy the sensory element if exposed to them, supplying it with a relatively stable environment. Due to specific metabolic pathways used in microorganisms, compared to isolated enzymes, microbial sensors have the potential for more selective analysis of heavy metals which cannot be measured by simple enzyme reactions [61].

In order to be available for any sensing mechanism that is based inside the cell, there is a need for analytes to be able to enter the cell via diffusion, nonspecific uptake, or active transport. Alternative approaches are implemented in the cases when membrane permeability for an analyte is not sufficient. These approaches include allocation of the recognition element to the outside of the cell or the introduction of an appropriate transport mechanism for importing the analyte [61].

A large number of studies in which performances of whole cell-based biosensors were tested have utilized electrochemical and optical transducers. For detection of heavy metal ions (Cd2+, Cu2+, Fe3+, Hg2+, Pb2+,and Zn2+) at concentrations of 10μM, a mammalian heart cells-based biosensor was developed [54], with excellent performance in terms of frequency selection, amplitude and duration of detection within 15 min.

Biosensor, based on immobilized engineered bacteria *Alcaligenes eutrophus* (AE1239) and optical transducer, was utilized for monitoring the bioavailable copper ions in synthetic water samples, wherein the lowest limit of detection was 1 μM [62].

## **4. Conclusions**

Biosensors have a very wide range of applications, from environmental monitoring, food safety, detection of various diseases, use in artificial implantable devices such as pacemakers to the detection of drugs.

Application for pollution monitoring requires the biosensor to work from several hours to several days. Such biosensors are a tool for "long-term monitoring." Whether it is a long-term follow-up or analysis of individual shots, biosensors are used as technologically advanced devices both in settings with limited resources and in sophisticated medical settings.

Considering the complex and critical situation in the field of environmental protection, and the state of natural waters from the aspect of pollution with heavy metals, and taking into account the toxicity of heavy metal ions, it is necessary to continuously work on finding new efficient techniques for their detection. Conventional analytical techniques can no longer satisfy the needs of constant monitoring and frequent field analysis of water because they are expensive, often with bulky equipment and a long analysis time, and require well-trained analysts. Biosensors can be used to overcome the limitations of conventional methods. In the future, designing a biosensor with the appropriate material will surely help in the selective identification of metal ions not only from water but also from any other matrix.
