**2.1 DNA and RNA usage in biosensor technology**

In studies where DNA is used as a biorecognition agent, biosensor systems can be designed by measuring molecules that interact directly or indirectly with DNA. Especially in genetic analysis, DNA determinations have been performed based on complementary base pairings (**Figure 2**).

Chen et al. [13] developed a DNA biosensor whose signal was increased in triplicate for the determination of transgenic soybeans. Signal enhancement was performed as rolling circle amplification (RCA). In the biosensor system Chronocoulometry was used to detect DNAs via electrode surface charges. Firstly, Fe3O4@Au magnetic nanoparticles were produced and the electrode was modified with SH modified DNAs to capture the target DNA sequence. After this immobilization on carbon electrode and incubated by dropping target DNA, complementary DNA forms a double structure with the target DNA. Afterwards, the double helix was cut and removed with ExoIII and 4 single-stranded ssDNA with a gold nanoparticle cube in the center was added to the free cutted ends, and binding was achieved with the help of Phi29 DNA polymerase and T4. [Ru(NH3)6] 3+, which forms a complex with anionic phosphates, is used as a complex mediator specifically bound on this quaternary structure. The amount of DNA was determined chronocoulometrically over the electrochemical signals generated by the ruthenium complex. A linear calibration curve was obtained between 10−16 and 10−7 M, and LOD was calculated as 4.5 × 10−17 M. Target DNA analysis took 2 hours. By the help of this complex DNA analysis, the authors managed to develop an ultra-sensitive system. Sensitivity is increased with the DNA immobilization steps used here. However, since the study requires a repetitive enzyme treatment, it seems costly and time consuming in developmental process.

Nucleic acid biosensors can also be developed by using enzymes used in DNA analysis. In recent years, studies using the CRISPR-Cas9 system are used in biotechnological studies as a genetic editing molecule. Uygun et al. [14] developed an impedimetric graphene oxide electrode modified with CRISPR-dCas9 for the determination of the target circulating tumor DNA molecules containing the PIK3CA exon 9 mutation, which is used as a biomarker in breast cancer and is referred to as a liquid biopsy biomarker. In this study, deactivated Cas9 (dCas9) proteins without exonuclease activity, were used as biorecognition receptor by modifying them with a sgRNA that would recognize the target ctDNA sequence. Graphene oxide electrodes were modified with dCas9 and sgRNA, consequently. In this way, the modification for ctDNA analysis is completed. The short analysis time (40 seconds) needed was a great advantage. The impedimetric measurement method with a 1.92 nM LOD in the range of 2–20 nM ctDNA concentration has been developed.

**Figure 2.** *Schematic representation of the DNA as biorecognition receptor.*

Because of the charges on the DNA, analysis of cations, where the bases show selectivity, can be detected. In another biosensor study, due to its negatively charged structure, the ability of chelating properties of DNAs for metal ions

#### *Nucleic Acids for Electrochemical Biosensor Technology DOI: http://dx.doi.org/10.5772/intechopen.93968*

is used. Zhang et al. [15] used DNA molecules for the determination of mercury ions. DNA probes modified with the DNA sulfur group formed a self-assembly monolayer on the gold electrode. Afterwards, the electrode modified with the reporter DNA, which has a gold nanoparticle in the center, was measured by binding mercury ions. Both impedance and CV were used in the study. The linear measuring range was set as 1–200 nM and the LOD was calculated as 0.05 nM. By this study, thanks to the use of DNA molecules, the mercury molecule was determined with a relative standard deviation of 3% within real samples.

To observe the complementary base pairing and post translational modifications, secondary conjugates such as antibody or enzyme can be used in the DNA based biosensor studies. Huang et al. [16] developed a biosensor to determine DNA methylation levels. In this study, the methylation level was determined with the help of a bioconjugate modified by a secondary antibody. This conjugate is formed on graphene oxide layers that target CpG and have horseradish peroxidase (HRP) labeled anti-5-methylcytosine antibodies. The signal generated by HRP is directly proportional to the presence of methyl groups, and DPV was used in this enzymatic reaction-based study. Hydroquinone as a mediator, i.e. electron transmitter, also reduced the reaction voltage and made it more selective. Then, the measurement was carried out on the complex of the bioconjugate with cytosine containing methyl groups. While the biosensor reaches 1 fM LOD value, it can measure in the linear range between 1 fM and 10 nM. The preparation process of the biosensor took approximately 5 hours. In this study, since DNA methylation is performed by measuring the enzyme activity on the bioconjugate, the measurement efficiency is based on the performance of the antibody, enzyme and the mediator.

Jahandari et al. [17] developed a biosensor modified with gold nanoparticles for DNA-based Temodal (anticancer drug) determination. They also modified dsDNA molecules electrochemically on the gold nanoparticles deposited on the pencil graphite electrode. The binding of the Temodal to the DNA on the biosensor was measured by DPV. Here, the main measurement is not the Temodal measurement directly. As the intercalator agent binds to the DNA, the reduction potential of the guanine base on the DNA decreases due to the amount of Temodal that is bound to DNA. Temodal performed intercalation by the interaction of the minor groove on the DNA. In this system with a maximum measurement time of 8 minutes, a maximum recovery deviation of 5% was observed in real samples. Linearly, it showed 1 nM LOD with measurement performance between 5 nM and 45 μM. The performance and sensitivity of this study are directly proportional to the presence of guanine bases. The base ratio on DNA is important in the design of such studies.

Ebrahimi et al. [18] determined cadmium ions using ethyl green (EG) on a simple DNA-based biosensor system. EG can be used as a hybridization indicator in DNA studies. Cadmium is known as a toxic heavy metal and this heavy metal measurement is especially important for biological samples. Cadmium destabilizes the double helix by forming a bond through the N7 atom of the guanine base on DNA. Similarly, in this study, measurement was performed using differential pulse voltammetry signals of EG. The signal generated by DPV is the oxidation signal that occurs as the result of the release of EG by destabilizing double helix. In other words, EG among the DNA helix is released as the result of the binding of cadmium to DNA, and it forms a destabilized DNA signal with cadmium. Measurement of cadmium ions showed 0.3 pM LOD with a linear measurement between 1 pM and 1 nM and between 10 nM and 1 μM. The limitation of the study is the accumulation performance of EG on DNA. The degree of this accumulation determines the sensitivity. It is difficult to determine the low amounts of cadmium in drinking water with a relative standard deviation of 8%.

In biosensors developed with DNA, the properties of DNA hybrids can be used. Yang et al. [19] have developed an electrochemical biosensor for the determination of MCF-7 cells, which are breast cancer cells. In this biosensor, DNA molecules were used for modification. The system provided linear measurement between 100 and 1 million cells in 1 mL and the LOD was found to be 80 cells/mL. The biosensor system was developed as a sandwich-type and the signal was made more sensitive with nanomaterials on antibodies labeled with DNA. With the 3D nanomaterials used in the design of the system, the modification steps are quite highly complex and difficult in terms of workload. In the study, the electrode was first modified with 3D-graphenes and then was modified with gold nanocages with antibody on the carbon nanotubes and immobilized on the electrode. DNA fragments labeled with a secondary antibody bonded on MCF-7 bound on these antibodies, and the measurement was performed with DPV. Here, MCF-7 measurement was performed by DNA hybridization, that is, by measuring the degree of binding of the complement bound to the ssDNA labeled with the last bound antibody.

Saeedfar et al. [20] developed a biosensor using multi-walled carbon nanotubes modified with gold nanoparticles to determine the sex of Arowana fish. The process of determining the sex of the Arowana fish before maturation is quite difficult. Arowana is an ancient and very expensive fish. This biosensor was developed as it is a very advantageous approach for the fish farming industry to distinguish the gender of the fish in time. For this, a hybridization-based approach has been adopted. Carbon nanotubes were used as a hybridization agent by complexing with ruthenium (III) chloride hex ammoniate in this study. DNA determination was performed between 10−21 M and 10−9 M and 1.55 × 10−21 M was the lowest detection limit.

Apart from DNA and proteins used in DNA determination, uncharged DNA variant known as Peptide Nucleic Acid (PNA) is also used. Unlike DNA, instead of a phosphate backbone, this molecule contains a backbone made of neutral amino acids. PNA of this nature can be used as potential probes for both DNA and RNA determination. The advantage of PNA is that it provides a great advantage in reducing the determination time. Tian et al. [21] used graphene field-effected transistors in their PNA-modified electrochemical biosensor study. In the DNA analysis study by measuring the gate voltage, it reached the level of 0.1 aM LOD and performed between 0.1 aM and 1 pM.

Due to their structural features, RNA biosensors can also be used in biosensor development, just like DNA biosensors. The structural differences of RNA with DNA were mentioned above. Apart from their metabolic functions, RNA and DNA are similar except for the difference in oxidation in ribose sugar. Studies have also been carried out which DNA and RNA can be used together in biosensor studies, especially using complementary DNAs for RNA analysis. In this section, RNA-based biosensors and types of studies are summarized.

Luo et al. [22] have developed an electrochemical DNA biosensor for exosomal miRNA analysis. The study was generally modified by adding a DNA probe modified with methylene blue (MB) over amino groups after the lysine amino acid was electrochemically coated on the glassy carbon electrode. Complementary to this probe, a ferrocene-modified secondary probe is attached. The biosensor was prepared for sample measurement by exosome extraction that releases miRNA to hybridize them to complementary chain and detected by labeled with Ferrocene (Fc). The variation of the Fc signal with the MB folded on itself indicates the miRNA and probe measurement, consequently. It shows performance at 2.3 fM level LOD and linear measurement range between 10 and 70 fM.

As a result, DNA and RNA have been used both as a recognition agent and as a target molecule with successful results. In some of these studies, the determination was carried out by measuring a secondary molecule on the basis of DNA hybridization. Besides, DNA analysis with the use of Cas proteins and DNA analysis with PNA were performed by using different methods and extremely low detection limits were reached.
