*4.3. Nucleic Acid-Based Bio-Recognition Materials*

Compared with enzyme and antibody, nucleic acid (DNA or RNA)-based electrochemical biosensor was reported later, but their various applications are increased exponentially due to their multiple advantages. Their conformation is more robust than antibodies or enzymes. They can be entrapped in the biosensor assembly and bind with a wide range of specific targets with elevated affinity and sensitivity [65]. The interaction between immobilized nucleic acid and the analyte can change structures and electrochemical properties. One of the analytes are aptamers, which have artificial functional single-stranded DNA or RNA structures that can bind various target molecules, such as amino acids, small molecules, proteins, and cells, with high specificity affinity [66]. Aptamers can be obtained through an in the vitro selection procedure, followed by the classical methodology of

systematic evolution of ligands by exponential enrichment (SELEX). Tuerk and Gold first proposed it in 1990 [67]. Figure 1f showed a highly sensitive impedimetric aptasensor for the selective detection of acetamiprid and atrazine [57].

Many nucleic acid-based electrochemical biosensor configurations have been extensively studied in gene analysis, clinical diagnostics, and environmental monitoring due to their fast, low-cost, sensitive, and selective responses to numerous analytes [68]. The most crucial step in preparing the nucleic acid-based electrochemical biosensor is the surface immobilization of the oligonucleotide strands. A terminal modification (sulfhydryl and amino groups) is the most common method to immobilise nucleic acid. Its most significant advantage lies in efficiently achieving directional and stable fixation. It is easy to prepare DNA arrays and realize high-throughput determination combined with lab-on-a-chip technology. The electrochemical detection of nucleic acid can be divided into direct and indirect methods. The electroactivity of oligonucleotide strands can be changed in the direct methods, which also changes the interfacial properties of the oligonucleotide strands-modified electrode in terms of conductivity, capacitance, or impedance. The indirect methods depend on the usage of electrochemical active nucleic acid labels [69] or intercalators [70] (e.g., methylene blue) [71].

Nucleic acids have been most widely used in metal ion detection, mainly consisting of the following four types: metal ion-specific DNAs, aptamers, DNAzymes, and guanine (G)-rich oligonucleotides, which can be related to G-quadruplexes [72]. Heavy metal ions can generate the partial disordering of oligonucleotide strands and reduce base stacking and base pairing after forming a metal–base complex. DPV studied the evaluation of the interaction of Pb2+, Cd2+, Ni2+, and Pd2+ with dsDNA, including hydrogen bonding cleavage, double helix conformation, and oxidative damage to DNA bases at GCE. [73]. Hg2+ can combine with two thymine bases (T) and mediate T–T mismatch to form a stable T–Hg2+–T structure which is more durable than the natural adenine–thymine (A– T) base pair with a binding constant close to 10<sup>6</sup> M−<sup>1</sup> [74]. Ag<sup>+</sup> can selectively interact with cytosine (C)-rich oligonucleotide strands to form C–Ag+–C mismatch [75]. These impressive mismatches belong to coordination bonds, and on these principles, significant efforts have been made for high selectivity and sensitivity to determine Hg2+ [40,76] and Ag<sup>+</sup> [77,78]. As for the detection of Pb2+ ion, the G-rich DNA sequences are widely used due to their ability to fold to form a most compact G-quadruplex structure, especially in the presence of Pb2+ ion [79]. For example, the simultaneous detection and determination of mercury (II) and lead (II) ions were implemented by Wang et al. [80]. The biosensor functionality was improved by placing the amino-modified reduced graphene oxide (NH2 rGO) nanofilm on a gold electrode as an excellent anchorage for the DNAzyme and the DNA strands. The presence of target ions could be recognized through the difference in charge-transfer resistance values before and after DNA interactions with Hg2+ and Pb2+ ions.

#### *4.4. Whole Cell-Based Bio-Recognition Materials*

Whole cells or microorganisms used for environmental biosensing can be classified as bacteria, yeasts, and fewer algae. The whole cell-based biosensor combines cells and transducers, generating a measurable electrical signal against the specific or target analytes [81]. In recent years, the whole cell has become an excellent alternative to the traditional bio-recognition elements due to their easy cultivation and manipulation, hosting many enzymes to catalyze reactions and good compatibility with various types of transducers. Substantial efforts have been made, from commercial to well-characterized cells with robust and specific enzymatic properties [82]. Moreover, they can give information on the pollutants' bioavailability and toxicity toward eukaryotic or prokaryotic cells [83].

Whole cells played an essential role in detecting heavy metal ions as the carrier to adsorb, precipitate or metabolize heavy metal ions. The whole cell was integrated into biosensors for low cost, low toxicity, high adsorption, and feasible fabrication based on the complexation, ion exchange, and physical adsorption between the whole cell and metal ions. Alpat et al., used green microalgae (Tetraselmis chuii) for the biosorption, preconcentration, and determination of Cu2+ in an easy, inexpensive, sensitive, and effective way [84]. The working electrode was fabricated by mixing green microalgae and carbon paste. Different pulse cathode differential voltammetry showed good linearity in the range of 5.0 × <sup>10</sup><sup>−</sup>8–1.0 × <sup>10</sup>−<sup>6</sup> M with the L.O.D. of 4.6 × <sup>10</sup>−<sup>10</sup> mol L−1. A Phormidium sp. modified voltammetric sensor for Pb2+ detection from aqueous solutions was also developed. Possible functional groups involved in Pb2+ accumulation were carboxyl, sulphoxide, and alcoholic groups. The developed microbial biosensor's analytical properties and selectivity were investigated comprehensively, with a detection limit of 2.5 × <sup>10</sup>−<sup>8</sup> M [85].

The oxygen consumption estimates the biological oxygen demand (BOD) during the biodegradation with the aerobic whole cells as the catalysts. It is known that the biosensors need biorecognition elements with minimal selectivity and high activity of bio-oxidation for a wide range of organics, which ensures their application in the practical water samples containing water nutrients and complex organics. Xia's group developed the fast detection method of BOD by selecting the Bacillus subtilis as a biorecognition element for its resistance in extreme conditions. They created a single-microbial-layered structure on the gold surface where the Bacillus subtilis bonded covalently. However, the conductivity was low due to the microbial electrode, and the biocompatibility was also poor [86]. In a second study, they improved the performance by creating the rough electrode surface with the microbial layer, and the carboxyl graphene and Au nanoparticles' electrodeposition was used for creating this roughness [87]. In a third study, they used magnetite-functionalized Bacillus subtilis as the element of this BOD microsensor that can be regenerated and immobilized on an ultramicroelectrode array (UMEA). Modification and regeneration of the electrodes array are controlled magnetically. The assay can be performed in a short time (5 min) with vastly improved sensitivity. The calibration plot is linear in 2–15 mg·L−1. The developed biosensor was also applied successfully to determine BOD in spiked water samples [88]. Khor et al., constructed a two-electrode sensor system using calcium alginate to immobilize microorganisms for BO detection [89]. Ferrocyanide can dissolve in the carrier solution and be fixed into the membrane to participate in a biochemical reaction. Ultramicroelectrode has the advantages of small size, fast diffusion and mass transfer, and a fast, stable state. In the combination of optimized microbial-sensitive film thickness, the rapid detection of BOD is realized.

