enzyme-based biosensors for the detection of metal contaminants in the environment

CHAPTER 12

Advances in protein/enzyme-based biosensors for the detection of metal contaminants in the environment Carlos S. Osorio-González*, Krishnamoorthy Hegde*, Satinder Kaur Brar*, Beatriz Delgado-Cano†, Natali Gómez-Falcón‡, Antonio Avalos-Ramírez† * INRS-ETE, University of Quebec, Quebec, QC, Canada National Center in Environmental Technology and Electrochemistry, Shawinigan, QC, Canada ‡ Higher Technological Institute of Tierra Blanca (ITSTB), Tierra Blanca, Veracruz, Mexico †

Contents 1. Introduction 2. Metallo-polypeptides 3. Effect of metal-peptide binding on polypeptide structure 4. Enzymes and proteins with potential as metal biosensors 5. Inhibitors and interferences on metal-polypeptide binding 6. Recent progress in polypeptide-based biosensors 7. Advantages of polypeptide-based biosensors 8. Trends in the development and use of polypeptide-based metallic sensors 9. Conclusions Acknowledgments References

245 246 249 250 252 254 255 256 257 257 257

1. Introduction Since the beginning of industrial revolution, the usage of heavy metals has been increased accordingly to the population growth and socioeconomic improvement of several countries [1, 2]. These metal ions come from natural (stones and soil mainly) and anthropogenic sources (electronic components, batteries, mining, glassware, preservatives, paints, etc.). The main reason for monitoring the presence of heavy metals in the environment is their effects on human health, crops, and wildlife. Generally, the toxic effect of these compounds is due to their bioaccumulation in the tissues, affecting plants, animals, and humans [3]. The metals causing main concern and as consequence the most studied are lead (Pb), cadmium (Cd), zinc (Zn), mercury (Hg), silver (Ag), chromium (Cr), copper (Cu), iron (Fe), platinum (Pt), and Arsenic’s (As) group elements, due to their impact on human health and environment [4].

Tools, Techniques and Protocols for Monitoring Environmental Contaminants https://doi.org/10.1016/B978-0-12-814679-8.00012-1

Copyright © 2019 Elsevier Inc. All rights reserved.

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A wide variety of techniques to monitor and quantify heavy metals has been developed. Among the most used are mass spectrometry, ion chromatography, and optical mass spectrometry coupled to inductive plasma [5]. However, these types of techniques present a series of disadvantages to carrying out the monitoring or detection of heavy metals. One of the most important is the cost of analysis equipment and need of specialized personnel to perform samplings and analysis, the possibility of damage or lost samples during their transfer to laboratory, the complete solubilization of metal ions for analysis, and the time necessary to condition samples and analyze them [6]. As an alternative, to effectively monitor and quantify heavy metals in the environment, the use of biosensors is an interesting option. These biosensors are simpler devices than sophisticated equipment and it can be used in situ (directly in a contaminated area) to determine presence and concentration of these metals [7]. A biosensor is a device in which biological elements such as proteins, enzymes, DNA, antibodies, and microorganisms are integrated into a transducer (electrical, electrochemical, optical, mass sensitive, or thermometrics), with the purpose of transmitting a digital signal which corresponds to metal ion concentration [8]. An enzymatic biosensor is mainly based on the catalytic or inhibition response of enzymes to specific metal ions. This kind of biosensor using an enzyme or an enzymatic complex is based on the quantification of a substrate consumed or a product formed over time when the inhibitory compound is present [9]. The bioreceptors used in the preparation of enzymatic/protein biosensors to monitor heavy metals in the environment are enzymes such as alkaline phosphatase, L-glycerol phosphate oxidase, pyruvate oxidase, L-lactate dehydrogenase, peroxidase, and urease or some proteins as glutathione-S-transferase-SmtA, synthetic phytochelatin, metallothioneins (MTs), and human carbonic anhydrase II variant [10]. This chapter provides an overview of the various enzymes/proteins used to construct biosensors for heavy metals monitoring in the environment.

2. Metallo-polypeptides Peptides are short chains of amino acid monomers which are linked by peptide (amide) bonds. They are covalent chemical bonds between the amine group of one amino acid and the carboxyl group of another. A polypeptide corresponds with a peptide chain constituted by 10 or more amino acids. In order to conform the polypeptides with three-dimensional and stable structure (secondary and ternary structure), peptides can be connected by covalent and no covalent bonds: disulfide bridges (between two thiol groups), intra and intermolecular hydrogen bonds, electrostatic interactions, and metal coordination interactions [11, 12]. A metallo-polypeptide is a polypeptide that contains a metal ion cofactor which defines the reactivity and structure of the molecule, acting as catalytic center (in proteins and enzymes) or forming coordination complex. Metallo-polypeptides play a key role in

Detection of metal contaminants in the environment

different biological processes. The binding of metal ions with peptide structure depends specifically on the location of their stereoscopic site, which is composed of different adjacent peptide chains. In general, this kind of polypeptide has a symbiotic effect with different metal ions, allowing them to function as a type of storage or transport for ions. In turn, the ions confer special functionalities to polypeptides [13]. The coordination complex between atoms of amino acid residues of peptide chain (nitrogen, oxygen, or sulfur) and metal ion stabilizes the peptide structure, for example, metal-peptide interactions naturally occur with amino acids (histidine, cysteine, glutaric acid, lysine, serine, and tyrosine) and some metal ions such as silver, cadmium, cooper, and zinc. Polypeptides chains have also been chemically synthesized in order to create specific interactions between metals and peptides in order to facilitate the metal-peptide interactions, control metallo-polypeptide interactions, structure, functions, and reactivity. The most used coordination ligands which favored metal-polypeptide bindings are presented in Table 12.1. There are peptides of low molecular weight such as MTs and phytochelatins (PCs), which have a wide potential to bind with heavy metals, through the thiol group. In a natural way, these peptides are present in cellular organisms, where their presence is a response to intoxication with heavy metals [22]. These characteristics led to use them as bioreceptors in biosensors for monitoring and quantifying heavy metals in the environment by the metal binding on specific sites [23]. In environments with neutral pHs, MTs bind strongly to metal ions such as zinc and copper, whereas at low pH (<3.5) they can also bind to arsenic ions (As3+) [10]. Fig. 12.1 shows the most common binding of this kind of peptide with heavy metal ions through the metal-thiolate group [5]. Nevertheless, unlike MTs, PCs have a higher affinity for heavy metal ions. The main reason for this affinity is the presence of Glu-Cys units with the ability to bind a different metal ion [24, 25].

Table 12.1 Coordination ligands and binding metals to form metal-polypeptides Ligands (naturals/artificial)

Cysteine Histidine Tryptophan Glutamic acid Catecholate Pyridine Bipyridine Terpyridine

Reactive group

Sulfhydryl group Nitrogen of the imidazole Indole ring

Binding metals 2+

2+

References 2+

Cd , Pb , and Zn Ag+, Cd2+, Cu2+, Ni2+, Zn2+ Pb2+, Cu2+, Ni2+, Hg2+, and Zn2+ Carboxylate group Zn2+, Cd2+, Ni2+, and Co2+ Hydroxyl group Mo6+, Ti4+, and Fe3+ Nitrogen from pyridine ring Cu2+, Cd2+, and Cr2+ Nitrogen from pyridine ring Pd2+, Cu2+, Hg2+, and Pb2+ Nitrogen from pyridine ring Cu2+, and Zn2+

[14] [15] [16] [17] [18] [19] [20] [21]

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248

O

O

O C

H

NH

H2N

OH

N H

CH2 C

NH2

C CH2

OH COOH

H

C NH

Glycine

CH2

O

Phytochelatin

Inhibition site

Heavy metal ions

Zn2+ Cd2+ O

CH2SH

H CH2

NH C COOH

C CH2

H

Ni2+

C NH

COOH

NH C

CH2

O

n

Phytochelain

Fig. 12.1 Phytochelatin binding with different heavy metal ions through the metal-thiolate group.

COOH

NH C

n

Glutamylcysteinyl

H

CH2SH

H

O

Tools, techniques and protocols for monitoring environmental contaminants

O

HS

Detection of metal contaminants in the environment

3. Effect of metal-peptide binding on polypeptide structure The peptides are ligands for a wide range of metal ions, due to their high potential to donate electrons through their chains of amino acids; this makes them effective and specific at the time of forming the so-called metal-peptide complexes. For example in a single chain of these ligands, two atoms that can donate electrons are oxygen from the carbonyl group and nitrogen from the amide group or the amine termination [23]. To form these bonds, the peptides depend to a large extent on the metal ion and the amino acid sequence (presence of residues) present in the protein. These interactions are critical in the self-assembly, structure, folding, stability, and activity of the polypeptides [12]. In this sense, the metal-peptide interactions are given by specific ligands present in the peptide chain. These interactions are generally linked by noncovalent bonds and not by the bond between the nitrogen, oxygen, or sulfur ions present in the amino acids of peptides. There are many factors that influence the affinity and formation of bonds between metallic ion and peptide. Some of these factors are the geometry of the complex, the level of hardness or softness of the ion, the sequence of amino acids, the pH, the type of chain or residue present in the amino acid at the time of forming the bond, and the potential of the metal ion to deprotonate nitrogens from amide group to form the peptide-metal complex [23]. A strategy to improve the efficiency of the interaction between the peptide and metal ions is the use of different matrix (glutaraldehyde, chitosan, polymers, carbon paste, and polysaccharides) to immobilize the peptides and increase the reaction levels. Fig. 12.2 shows five different methods of immobilization used in biosensors for the detection of heavy metals in the environment. As example of these strategies is the research done by Ghica and Brett [26], where on a carbon film immobilized through of cross-linking method, two different enzymes [glucose oxidase (GOD) and bovine serum albumin

Fig. 12.2 Different methods to immobilize enzymes/proteins in biosensors.

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(BSA)] mixed with glutaraldehyde. After that, the film was used in an electrode for the determination of metal cations such as cadmium, copper, lead, and zinc. The response of the biosensor to the inhibition of glucose increased linearly up to 1.8 mM and reached a limit of detection of metal ions of 20 μM. On the other hand, with this study, they reaffirmed the assumption that the influence of oxygen mediator on the inhibition factor of the enzyme does not show changes when it was immobilized by the cross-linking method. The immobilization of enzymes or proteins on the surface of the transducers is of high importance to design the biosensor. Some of the advantages of biosensors using immobilized enzymes in comparison with other methods are less time to do the analysis, less amount of sample is needed, and the interferences decrease [27]. Several works using immobilized enzymes for heavy metals detection in the environment have been carried out. Guo et al. [28] developed a magnetoelastic biosensor (ME) functionalized with BSA for the detection of Pb2+, Cd2+, and Cu2+. The main feature of this biosensor was the immobilization of enzyme in a gold magnetoelastic band that allows to activate, control, and measure the oscillation, resonance, and frequency of the metal ions through magnetic fields. In this sense, the combination of the metal ions and enzyme cause their precipitation, followed by their binding to the surface of the biosensor. The above generates an increase in the mass load, which in turn leads to the reduction of the frequency of its resonance, which belongs to the concentration of each metal ion. Finally, the best results were founded to highest sensitivity of this biosensor to metals with high molecular weight and low concentrations thereof. On the another hand, Elsebai et al. [29] investigated different factors (enzyme and substrate concentration, pH, and incubation time) in two immobilized enzymes by the method of cross-linking with glutaraldehyde in a conventional system of three electrodes (glassy carbon, platinum wire, and Ag/AgCl) for Hg2+ detection. The highest percentage of inhibition was obtained with a concentration of 5 mg mL
1 of enzyme and 0.1 mM of H2O2; these correspond to the lowest substrate value. Thereby, the degree of inhibition is greatly influenced by the concentration of the substrate since if the inhibitor competes with the enzyme, the increase in substrate concentration decreases the inhibition activity of the metal ion. On the other hand, the highest percentage of inhibition (35.7%) and sensitivity (1.23 μAcm
2 mM
1) with a pH value of 7 was obtained. This result agrees with the pH value where catalase shows the best enzymatic activity. Finally, the highest percentage of inhibition was with the higher incubation time.

4. Enzymes and proteins with potential as metal biosensors Enzymes are proteins used to catalyze specific reactions. This specificity is given by the active site and the chemical structure of the molecule. Nevertheless, some enzymes do not depend on the presence of an active site to be able to carry out the union with some metallic ion [27].

Detection of metal contaminants in the environment

Among the uses that can be given to enzymes, bioreceptors in biosensors for the monitoring of heavy metals in the environment can be mentioned, thanks to its high selectivity and specificity with metal ions [30]. These molecules are highly selective with fast reaction times and compatible with other enzymes. They are more efficient to bind metal than other biological receptors, such as microorganisms, or chemical reactions [31]. The biosensors based on enzymes or proteins can be classified into direct and indirect bioreceptors. The main difference is the way in which metal ions are detected during the enzymatic reaction [32]. The main mechanism of action of these molecules is the conversion of the metal ion into a product that can be quantifiable and they can detect an ion by different mechanisms (inhibition or activation of enzymes) after reacting or interacting with the metal ion [33]. In this sense, there are several groups of enzymes (phosphatases, oxidases, dehydrogenases, peroxidases) that have been used as bioreceptors in biosensors for the monitoring of heavy metals in the environment. Ogo nczyk et al. [33] evaluated the use of potentiometric electrodes with immobilized urease for the detection of heavy metals through the development of screen-printing technology. Por otro lado Berezhetskyy et al. [34] using alkaline phosphatase immobilized on membranes, and a conductometric biosensor with interdigitated gold electrodes quantified Cd2+, Co2+, Zn2+, Ni2+, Pb2+ in water. In this work, the quantifying was through to inhibition of enzymatic activity caused by metal ions. Similarly (enzymatic inhibition) but through the use of GOD immobilized in carbon films and electrosynthesized poly-o-phenylenediamine, Ghica and Brett [26] and Guascito et al. [35] evaluated the enzymatic inhibition caused by heavy metals such as Hg2+, Ag+, Cu2+, Cd2+, Pb2+, Cr3+, Fe3+, Co2+, Ni2+, Zn2+, Mn2+, and CrO4 2
through of electrochemical and amperometric biosensors, respectively. Fig. 12.3 shows the general components of a biosensor, where the basic principle is the molecular recognition of a compound or element through an inhibition or activation reaction with the bioreceptor to later transform it into a signal through the transducer [36]. The final product of the combination of these three components is the biosensor with a high level of specificity for the metal ion of interest.

Fig. 12.3 General scheme for heavy metal detection using biosensors based on enzyme/proteins.

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Basically, the biosensor binding a metal emits a signal captured by the transducer which converts it into quantifiable information [37]. An important part to consider in the construction of biosensors based on enzymes/proteins is the control of the enzymatic activity generated in the matrix where the bioreceptors are immersed. For example, the enzyme interacts directly with the detection layer of the metal ion which is given as a function of its concentration (zero-order kinetic regime) [2]. An example of this enzyme-based biosensors is the work done by Chey et al. [38] through the development of a potentiometric biosensor based on the physical immobilization (adsorption) of GOD on the surface of ZnO nanorods (ZnO-NRs) for mercury ions determination. The results showed linear ranges of up to 0.5  10
4 at 20 mM mercury concentrations at fixed glucose concentrations of 1–10 mM. Additionally, the enzyme retained its original activity by 90% after three weeks, showing a high sensitivity, selective and stable response to mercury ions. Likewise, Attar et al. [39] evaluated horseradish peroxidase (HRP) immobilized on the surface of carbon films for the detection of Cr3+ and Cr4+ ions. They reported that when H2O2 was used as a substrate, mixed binding with Cr3+ had an inhibition value of 11.4 μM and the non-competitive binding with Cr4+ ions had an inhibition value of 0.78 μM. Additionally, no interference was found with other ions, therefore, the selectivity was high for the two chromium ions.

5. Inhibitors and interferences on metal-polypeptide binding The medium containing metal ions can present compounds which could decrease or inhibit the activity of enzymes, or activate their activity and performance. In the first case, the inhibition effect of the metal ion allows to quantify the concentration of the inhibitor ion and in the second case it works in an inverse manner, activating the reaction according to the amount of the ion bound with the active site of the enzyme [40]. In this sense, the mechanisms of inhibition can be classified into two groups, namely, those of reversible inhibition and those of irreversible inhibition. In the reversible inhibition, the metal ions are bound to the enzymes by the interaction of noncovalent bonds at fast association rates, where the chemical equilibrium of the substrate-enzyme complex allows the enzyme to recover [27]. This reversible effect can occur when the enzyme is in a free state or as a substrate-enzyme complex and the inhibitors can form two inactive complexes (binary or ternary), which allow quantifying the presence of the metal ion [42]. On the other hand, the operation of the reversible inhibition is limited by the effectiveness of the metal ion that is inhibiting the enzyme. Nomngongo et al. [42] evaluated through reversible and noncompetitive HRP inhibition activity for Cd2+, Cu2+, and Pb2+. As support for the enzyme was used polyaniline (PANI) film, and was immobilized through electrostatically method in a platinum electrode. The inhibition kinetics showed a slow answer in the presence of Cd2+; these are due to the slow formation of links with the enzyme and the changes that this

Detection of metal contaminants in the environment

metal causes in the structure of the enzyme. In general, the results obtained with this biosensor through reversible and noncompetitive HRP inhibition were highest selectivity for the detection of Cu2+ and Pb2+ with 1.33 and 2.28 Ki,j amp. Irreversible inhibition is primarily due to the break of the three-dimensional structure of the enzyme. The foregoing is caused by strong binding of the inhibitor with the enzyme. The advantage of this bond (enzyme-substrate) is providing an indirect quantification of the metal ion, because of the loss of enzymatic activity, which is directly proportional to the concentration of metal ions [43–45]. Inhibitors can be classified into three main groups: competitive, noncompetitive, and mixed. Do and Lin [46] evaluated a biosensor with urease immobilized in a polyaniline nanostructure (PANi) in order to detect lead (Pb2+) and mercury (Hg2+) by irreversible inhibition of enzymatic activity. The dissociation values of the absorbed enzyme as well as of each of the metal ions evaluated (Kd) were 0.45 and 8.67 ppm, respectively. The values obtained for the constant of the inhibition reaction rate of the enzyme (Kirr) were 1.66  10
3 s
1 for Pb2+ and 3.16  10
3 s
1 for Hg2+. Finally, the highest detection sensitivity by the biosensor was for the mercury ion. Competitive inhibitors are those having chemical structure very similar to that of the substrate, so that both inhibitor and substrate compete for the active site of the enzyme in a chemical equilibrium process (enzymatic activity can be restored), without an effect on vmax; however, KM is replaced by qKM, which is the constant for substrate competition. A reversible inhibition can be carried out by increasing the substrate/inhibitor ratio [47, 48]. The impact of these inhibitors is the decrease of the catalytic activity of the enzyme, reducing the catalyst concentration available for the substrate. Varjovi et al. [49] developed an electrochemical biosensor in combination with GOD immobilized on a carbon nanotube film and a Nile-blue nanocomposite (Nb-SWCNTs) with glutaraldehyde on a graphite electrode. The study was conducted for the detection of Hg2+ and Pb2+. Kinetics of inhibition for these heavy metals were competitive. Where GOD catalyzes the oxidation of β-D-glucose producing gluconic acid in the presence of O2, allowing greater amperometric signals due to the competitive reaction of enzymes with the substrate. The percentages of inhibition reached with the biosensor for each metal were 44% for Hg2+ and 37.2% for Pb2+ with a concentration of 100 μM for both metals. Noncompetitive inhibitors not only bind to the enzyme, but also to the enzymesubstrate complex. Unlike competitive inhibitors, they do not bind to the active site of the enzyme. This causes a deformation of the chemical structure of the enzyme, which leads to the inhibitor binding only to the enzyme-substrate complex and not to the free enzyme. This promotes the formation of a ternary complex, which prevents the formation of a final product. The kinetic effect of this type of inhibitors occurs in the reduction of vmax and KM, due to the effect of competitive inhibition [26, 50]. The inhibition by the presence of several competitive inhibitors cannot be reversed by increasing the concentration of the substrate. On the contrary, the degree of inhibition increases and consequently the formation of the enzyme-substrate-inhibitor complex [51]. Finally, mixed

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inhibitors can take the role of a competitive or noncompetitive inhibitor generating both inhibition effect, and this inhibition cannot be overcome with the increase in the ratio of the substrate to the enzyme as in the case of competitive inhibitors [52].

6. Recent progress in polypeptide-based biosensors Nowadays, the development of enzyme/protein-based biosensors for monitoring and detecting heavy metals in the environment has been increasing. The main feature of this development is based on some factors that are fundamental to the efficiency of these devices. Two of these characteristics could be the use of nanomaterials as catalytic supports and the production and availability of high-quality enzymes (tolerant to high temperatures and rapid changes in pH). Additionally, the development of economic and reusable biosensors is sought. Other factors have also been considered for the development of these devices, for example, the protein-metal interactions are still difficult to understand because of multiple factors involved in this process, such as the affinity of a ligand with a metal ion, the polarity, the matrix protein, the pH, and the temperature [53]. Table 12.2 presents some of the work that has been performed during the last decade regarding the use of enzymes/proteins for the development of heavy metal biosensors. Table 12.2 Recent works in biosensor based on enzyme/protein for heavy metal detection Enzyme

Heavy metal

Limited detection

Support used

References

Horseradish peroxidase (HRP)

Pb2+ and Cu2+

Pb2+ ¼ 2.5 μg L
1 Cu2+ ¼ 4.2 μg L
1

[54]

Hg2+, Pb2+ and Cd2+

Maize tasselmultiwalled carbon nanotube (MT-MWCNT) Pt/PANI-coPDTDA/HRP

Cd2+ ¼ 8  10
4 μg L
1 Pb2+ ¼9.38  10
4 μg L
1 Hg2+ ¼7.89  10
4 μg L
1 Interdigitated gold Cd2+ ¼ 0.5 ppm electrodes and Pb2+ ¼ 2.0 ppm enzyme Zn2+ ¼ 5.0 ppm membranes Ni2+ ¼ 40.0 ppm Cd2+¼10
19 M Ceramic part of gold Hg2+ ¼ 10
17 M interdigitated transducers Gold nanorods Cd2+ ¼ 2 mM Hg2+ ¼ 1 mM Cu2+ ¼ 2 mM Graphene-oxide and Hg2+ ¼ 0.0075 ppb poly-vinyl alcohol Pb2+ ¼ 0.015 ppb Cd2+ ¼ 0.0312 ppb

Alkaline phosphatase

Cd2+, Co2+, Zn2+, Ni2+ and Pb2+ Cd2+ and Hg2+ Hg2+, Pb2+ and Cu2+ Hg2+, Pb2+ and Cd2+

[55]

[56]

[57]

[58]

[59]

Detection of metal contaminants in the environment

Table 12.2 Recent works in biosensor based on enzyme/protein for heavy metal detection—cont’d Enzyme

Heavy metal

Glucose oxidase Cd2+, Co2+, Cu2+ and Ni2+ Hg2+ Cu2+, Hg2+, Pb2+ and Cd2+ Ag+, Cu2+ and Co2+ Urease

Hg2+ and Pb2+

Limited detection

Support used

References

Cd2+ ¼ 1.5–6.0 Co2+ ¼ 5.0–35 Cu2+ ¼ 0.2–3.0 Ni2+ ¼ 20–120 0.076 μM L
1

Cobalt or copper hexacyanoferrate modified carbon film Liposome and chitosan Ultrathin polypyrrole

[50]

Cu2+ ¼ 0.079 μM Hg2+ ¼ 0.025 μM Pb2+ ¼ 0.024 μM Cd2+ ¼ 0.044 μM Cu2+ ¼ 0.19 ppb Cd2+ ¼ ND Ag+ ¼ ND Hg2+ ¼ 0.45 ppm Pb2+ ¼ 8.67 ppm

Nitrogen-doped carbon nanotubes (N-CNTs) Nano-structured polyaniline (PANi)-Nafion (NSPN)/ Au/Al2O Glassy carbon Agarose-guar gum

Hg2+ 1.8  10
11 M 2+ + 2+ Hg , Ag , Pb Hg2+ ¼ 1  10
8 M 2+ Pb2+ ¼ 11.2  10
8 M and Cd Cd2+ ¼ 1  10
7 M Ag+ ¼ 1  10
7 M 2+ 6+ β-Galactosidase Cd and Cr Cr6+ ¼ 91.7 ng L
1 Immobilization by cross-linking with Cd2+ ¼ 6.95 ng L
1 glutaraldehyde Recombinant As3+ and Hg2+ As3+ ¼ 13 ppb Screen-printed human MT Hg2+ ¼ 45 ppb carbon electrodes isoform 1a (SPCEs) Phytochelatins/ As3+, Cd2+, Pb2+ ¼ 0.18 ppb nm
1 Porous silicon lysine and Pb2+ multilayers and flat As3+ ¼ 0.25 ppb nm
1 gold surfaces Cd2+ ¼ 0.22 ppb nm
1

Catalase Invertase and glucose oxidase

[60] [61]

[62]

[48]

[29] [45]

[63]

[10]

[25]

Currently, enzymes such as GOD, alkaline phosphatase, MTs, and PCs are the most commonly used bioreceptors in the development of biosensors for the detection of heavy metals in the environment. Technologies advances to produce enzyme supports are based mainly in screen-printed and nanomaterials [59, 64, 65].

7. Advantages of polypeptide-based biosensors Among the main advantages offered by biosensors based on enzymes or proteins for the detection of heavy metals is the wide variety of enzymatic reactions that can be exploited,

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detecting directly the substrate and the product of the reaction that takes place. Likewise, the inhibitors and mediators of the reaction can be controlled; there is a great variety of detection systems for this purpose, a low consumption of peptides during the reaction is needed, peptides present high selectivity and specificity, and they are available in the market [66]. On the other hand, the advantages that these devices offer in comparison with the usual techniques for the detection of heavy metals are their size and their portability, which makes them efficient in the sense that they can be carried without any problem to the sampling in situ [7]. Another advantage that this type of device offers in relation to techniques that are commonly used for the detection and quantification of metal ions in the absence of a pretreatment such as filtration or precipitation to analyze the sample [9]. In summary, the main advantages in the use of proteins/enzymes in the development of biosensors for heavy metals in the environment are as follows: (i) fast screening and identification of metals, incorporating a protein/enzyme to detector support with high affinity to the metal ion of interest; (ii) easy detection and analysis; (iii) specific interaction of the protein/ enzyme with the metal; (iv) specific affinity of protein/enzyme for the metal, can be used to improve the processes of immobilization; and (v) affinity of the proteins/enzymes can be combined to obtain a double affinity, to improve its stability and selectivity toward metals.

8. Trends in the development and use of polypeptide-based metallic sensors The development of biosensors for the monitoring and detection of heavy metals in the environment is a great challenge since continuous monitoring is needed in situ and in real time. Intensive research into the development of new biotechnological, nanotechnological, and nanoengineering tools, implemented in biosensors, has improved the efficiency of these devices by detecting relatively low concentrations of metal ions in the environment. On the other hand, the implementation of these technologies in biosensors makes them more competitive and above all friendly to the environment. Additionally, this type of methodologies must be competitive (resilient, low cost, portable, and easy to use) with those currently used in the market. In general, the ability to develop an efficient enzyme/ protein-based biosensor depends to a large extent on the availability of these bioreceptors and their quality. The use of genetic tools is of great importance, since currently specific enzymes/proteins from microorganisms or genetically improved plants can be obtained. Protein engineering is working in the production and the structural chemical design of enzymes/proteins such as site-specific placement of reactive groups, fluorescent labels, receptive biomolecules, and improved stability of long-term biomolecules. For the development of biosensors based on enzymes/proteins, it is necessary to demonstrate their viability regarding their efficiency, construction, and operation. This is necessary to determine if it is feasible to commercialize them on a large scale. In this sense, the development of this type of technology and devices has a wide commercial potential.

Detection of metal contaminants in the environment

According to Suryan [2] the market for these devices for 2020 is estimated at 21,000 million dollars with around 500 companies worldwide developing this type of devices.

9. Conclusions The specificity of enzymes to perform chemical reactions under suitable conditions is widely used in the development of biosensors for the detection and quantification of metal ions in the environment. The integration of enzymes on the surface of supports is achieved through their immobilization in different materials. The operating principle of heavy metal biosensors is a dynamic process, which is characterized mainly by its rapid reaction speed, its easy portability and handling, and the low amount of sample to carry out the determination. The presence of inhibitors and their mechanisms to affect biosensors must be considered to avoid wrong determinations. The market for these devices increases, justifying the continuous research and development to improve their quality and costs. The advances of some scientific sectors, such as material sciences and protein engineering contribute to produce more specific and economic heavy metal biosensors. With this type of characteristics, the biosensors based on enzyme/protein are very useful for monitoring different heavy metals. However, greater technological development is needed to improve the efficiency and stability of these devices to the changing environmental systems in which heavy metals are present.

Acknowledgments Financial support from NSERC (No. 284111, Discovery; No. 476649-14, Strategic Research Grant), from FRQNT (No. 2018-CO-204655, College researcher) and INRS-ETE has been acknowledged.

References [1] M.B. Gumpu, S. Sethuraman, U.M. Krishnan, J.B.B. Rayappan, A review on detection of heavy metal ions in water—an electrochemical approach, Sensors Actuators B Chem. 213 (2015) 515–533, https:// doi.org/10.1016/j.snb.2015.02.122. [2] S.K. Suryan, Biosensors: a tool for environmental monitoring and analysis, in: R. Kumar, A.K. Sharma, S.S. Ahluwalia (Eds.), Advances in Environmental Biotechnology, Springer Singapore, Singapore, 2017, pp. 265–288, https://doi.org/10.1007/978-981-10-4041-2_16. [3] S. Dzyadevych, N. Jaffrezic-Renault, Conductometric biosensors, in: Biological Identification, Elsevier, Cambridge, UK, 2014, pp. 153–193, https://doi.org/10.1533/9780857099167.2.153. [4] S. Zhan, Y. Wu, L. Wang, X. Zhan, P. Zhou, A mini-review on functional nucleic acids-based heavy metal ion detection, Biosens. Bioelectron. 86 (2016) 353–368, https://doi.org/10.1016/j.bios. 2016.06.075. [5] J.C. Gutierrez, F. Amaro, A. Martı´n-Gonza´lez, Microbial biosensors for metal(loid)s, in: C. CravoLaureau, C. Cagnon, B. Lauga, R. Duran (Eds.), Microbial Ecotoxicology, Springer International Publishing, Cham, 2017, pp. 313–336, https://doi.org/10.1007/978-3-319-61795-4_13. [6] Fosso-Kankeu, Microbial metalloproteins-based responses in the development of biosensors for the monitoring of metal pollutants in the environment, in: Heavy Metals in the Environment Microorganisms and Bioremediation, CRC Press, Boca Raton, FL, USA, 2018, pp. 1–30.

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