**2.2 Aptamers as synthetic biorecognition receptors**

Aptamers have caught a very rapid trend after their discovery. These artificial nucleic acids are produced by the systematic evolution of ligands by exponential enrichment (SELEX) method [5]. By being artificially produced, they show affinity for a wide range of target molecules. They achieve this affinity with easy physical changes. Aptamer sequences designed in the correct sequence for the target molecule work by binding to the target site by undergoing conformational change when the target molecule approaches. The conformational change can be measured by using a label or surface resistance, shown in **Figures 3** and **4**, respectively.

Ohno et al. [23] developed a label-free immunosensor-based aptamer-modified graphene FET. The researchers, who modified the G-FET with IgE aptamers, determined IgG by measuring the gate voltage on the FET. With a protein concentration measurement between 0.29 and 340 nM, a LOD value of 0.29 nM was reached. 47 nM isotherm value was observed as the Kd value. Since nonspecific binding of different proteins except this molecule was not observed.

In another study, Chen et al., [24] developed an aptamer-based biosensor for the electrochemical determination of Human epidermal growth factor (HER2). Differential pulse voltammetry was chosen as a measurement technique. In the study, after modifying the gold nanorod nanomaterials with palladium, they formed a bioconjugate for analysis by modifying it with an aptamer containing SH group and horse radish peroxidase (HRP). Later, they modified the gold electrode with DNA tetrahedrons. DNA tetrahedron is a nanoscale structure designed by using the complementary base pairing reactions of nucleic acids. They modified the single-stranded DNA aptamer from one corner of the DNA tetrahedron structure.

**Figure 3.** *Labeled-aptamer based electrochemical biosensor technology.*

**Figure 4.** *Schematic representation of the impedimetric aptamer biosensor.*

DNA tetrahedron is preferred because of its mechanical structural rigidity and high affinity (5000 times higher for ssDNA aptamer). The DNA was dropped onto the tetrahedron-aptamer modified gold electrode, aptamer's unbound ends were modified with BSA. After HER2 molecules were captured by the aptamer on the electrode, the bioconjugate was added on this modification and the biosensor was constructed???. HER2 measurement was performed indirectly through the conversion of HRP to hydrogen peroxide and the measurement of hydroquinone (mediator) added to the environment. In this study, researchers used the DNA tetrahedron structure and aptamer composite, thus HER2 recognition capacity was increased with this modification. The biosensor, with a working range of 10–200 ng/mL, provided the opportunity to make the analysis in 60 minutes. LOD was found to be 0.15 ng/mL.

Apart from large proteins, aptamer molecules are also used to identify small molecules. Aptamer molecules are particularly useful for the determination of molecules which are extremely difficult and demanding to identify easily. Swensen et al. [25] conducted real-time cocaine measurement in their biosensor study. This determination was carried out in approximately one minute. The microfluidic chip has been modified with cocaine-specific aptamers for cocaine binding. One end of the aptamer is modified with methylene blue. The sample, injected into the microfluidic chamber, was added to the fetal bovine serum with a flow, and the measurement was performed by binding to the aptamer. Rapid measurement is important for the rapid determination of this drug, which has a half-life of 60–90 minutes in serum. Besides the speed advantage, this system has a 20 pM LOD with a linear detection range of 10–2000 μM.

Apart from organic molecules, aptamer-based biosensors have also been developed for the determination of ions. Radi et al. [26] developed a biosensor system for the aptamer-based potassium determination. The gold electrode was made ready

for potassium ion measurement after being modified with aptamers with SH at one end and Fc at the other. The biosensor had a measurement limit between 0.1 and 1 mM and reached a value of 0.015 LOD by using electrochemical impedance spectroscopy.

Besides the principle of binding aptamer molecules to a molecule, a bifunctional aptamer-based biosensor for determination of adenosine and lysozyme was developed by Deng et al. [27]. In their study, they performed the determination of adenosine and lysozyme on DNA/DNA duplex. The gold electrode was first modified by a probe with SH group (adenosine selective) and then a secondary probe (lysozyme selective) attached to this probe, and the modification was completed with a DNA loaded nanomaterial with a central gold nanoparticle. Finally, ruthenium complexes were added as electroactive species on this structure. After the primary probe captures adenosine with adenosine binding, the gold nanoparticle structure together with the secondary probe is separated from the biosensor. With lysozyme binding, the secondary structure is separated from the main structure by binding lysozyme. All these bonding and separation reactions were followed by the electrochemical activity of ruthenium complexes. Electrochemical measurement


*DPV: differential pulse voltammetry, EIS: electrochemical impedance spectroscopy, CV: cyclic voltammetry, Coul: coulometry, fc: ferrocene, PIK3CA: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, AuNPs: gold nanoparticles, G-FET: graphene field effect transistor, MB: methylene blue, HER2: human epidermal growth factor, AC: alternative current.*

#### **Table 1.**

*Comparison of the nucleic acid based biosensors.*

was carried out by CV. Lysozyme and adenosine bifunctional biosensor reached the LOD limits of 0.01 μg/mL and 0.02 nM, respectively. Measurements were made between 0.02 and 40 nM for adenosine and 10–60 μg/mL for lysozyme.

With aptamer nucleic acids, target biomolecules can be easily identified. Although aptamer molecules work similarly to antibodies, they can easily be modified with a secondary biomolecule. Molecules that do not have denaturation problems unlike proteins can be very useful in the development of biosensors, especially in the development of electrochemical biosensors. Electrochemical activation of electroactive species or direct aptamer binding to the target molecule can be measured impedimetrically. As a result, aptamer systems are suitable for affinity-based sensor development in biosensors (**Table 1**).
