*2.2. Nucleic Acid-Based Electrochemical Sensors*

Nucleic acids, including DNA and RNA, are biopolymers composed of nucleotide units [79]. Nucleic acid molecules have specific nucleotide sequences, which comprise of the bases of adenine, thymine, cytosine, and guanine, that can bind strongly with complementary base-pair sequences. The intrinsic properties of nucleic acids have the potential as nanomaterials for biosensors [74,80–83], as they impart high selectivity in terms of the capability to detect target molecules selectively. In addition, nucleic acidbased aptamers can be developed as ligands for target materials through the systematic evolution of ligands by exponential enrichment technology [84,85]. These aptamers can be developed faster and cheaper than antibodies. Moreover, aptamers have unique 3D structures (e.g., loop, stem, quadruplex, bulge, hairpin, and pseudoknot) through the nucleotide sequence alignment of the nucleic acids; this allows the aptamers to bind more strongly and selectively to the target [86–88]. In addition, nucleic acid materials can be

chemically conjugated to fluorescent or electrochemical probes, giving a stronger signal and higher affinity toward analytes.

Park et al. [89] described a microelectrode based on carboxylated polypyrrole nanotubes conjugated with aptamers capable of evaluating the neuronal maturation of neurons. These authors showed that the aptamer-based sensor could sensitively and selectively measure dopamine (DA) exocytosis as a neuronal function. To improve the sensor's performance, the DA sensitivity according to the carboxylated polypyrrole nanotube diameter was first analysed. Then, the optimised aptamer-based sensor was evaluated for DAsensing performance using amperometry, which showed an excellent limit of detection (LOD) of 100 pM. Furthermore, the sensor could electrochemically distinguish DA in the presence of other neurotransmitters, such as norepinephrine, serotonin, and phenethylamine. This study demonstrated that the developed sensor could electrochemically detect exocytotic DA released from neuronal cells due to DA's high sensitivity and selectivity. The study's results suggested the possibility of aptamer-based electrochemical sensors to monitor the neural differentiation process of stem cells.

A nucleic acid-based electrochemical sensor capable of monitoring cardiomyocyte differentiation was reported in 2021 [90]. This nucleic acid-based sensor contained hybrid materials, including short DNA domains and peptide motifs that bind complementarily to cardiomyocyte-specific regulatory proteins. Notably, this sensor showed a low level of LOD of 0.42 pg/mL. In addition, the sensor selectively detected electrochemical signals from cardiac troponin (cTnl) as a target molecule in the presence of other proteins, including human serum albumin and human brain natriuretic peptide. Due to the sensor's high affinity and sensitivity to cTnl, it was possible to determine the cTnl expression level through electrochemical signals measured from cardiomyocytes differentiated from MSCs. In addition, the electrochemical signal for cTnl obtained while monitoring the cardiomyocyte differentiation process was consistent with the result of flow cytometry, validating the high reliability of this sensor.

In another study, an aptamer-based electrochemical sensor was developed to evaluate the neuronal function at the single cell level [91]. This sensor comprised micro-wells, DA aptamers, and co-reactant-embedded polymer dots (Pdots). The sensor's embedded Pdots provided electrochemical luminescence signals, which served to visualise the electrochemical DA signal (Figure 3a,b). The hybrid structure of this sensor allowed it to capture a single or a small number of differentiated cells inside a micro-well, which then selectively detected the DA released from the captured neurons using the DA aptamer. This sensor exhibited stable cell viability as a cell cultivation and differentiation platform with low cell toxicity. In addition, this sensor demonstrated a low LOD of 53 pM DA (Figure 3c,d) and was capable of evaluating the amount of DA exocytosis.

Nakatsuka et al. reported a DNA aptamer-based nanopipette capable of monitoring the differentiation process of iPSCs into serotonin neurons [92]. This sensor was used to electrochemically detect 5-hydroxytryptamine (5-HT) release from serotonin neurons as differentiated cells. In addition, the nanopipette form of the sensor allowed size exclusion of non-specific proteins in complex culture medium environments, further enhancing the sensor's 5-HT selectivity. Specifically, this sensor was able to detect 5-HT of less than 3 nM using fast scan CV, which is an excellent sensing capability for DA, similar to the sensing capability of enzyme-linked immunosorbent assay (ELISA). Above all, this sensor was able to detect 5-HT released at the cellular level through the introduction of an aptamer capable of binding specifically to 5-HT.

These studies support that nucleic acid materials can greatly improve electrochemical sensing ability, especially target selectivity, through their specific binding sites. In addition, nucleic acid materials can be combined with other types of biomolecules or sensing probes through chemical conjugation to provide various 3D ligands, which can improve electrochemical sensors' capabilities.

**Figure 3.** Aptamer-based electrochemical sensors. (**a**,**b**) ECL images of neurons injected in the micro-well and DA aptamer-based electrochemical sensor. (**c**,**d**) Analysis of DA sensing capability of micro-well and DA aptamer-based electrochemical sensor. Reprinted with permission from [91]. 5-HT, 5-hydroxytryptamine; Ag, silver; AgCl silver chloride; Au, gold; DA, dopamine; ECL, electrochemical luminescence; RSD, relative standard deviation.

#### *2.3. Carbon Nanomaterial-Based Electrochemical Sensors*

Carbon nanomaterials (CNPs), such as graphene and its derivatives, fullerene and carbon nanotubes (CNTs), and nanofibres are composed of chemical structures in which carbon atoms are combined through sp<sup>2</sup> hybridisation, resulting in the delocalisation of electrons [93]. The intrinsic properties of CNPs depend on their configuration and include good thermal stability, mechanical properties and chemical resistance. Furthermore, due to their structural characteristics, they have good electrical properties, including electron mobility and electrical conductivity. Moreover, CNPs can be synthesised into specific 3D structures, such as CNTs, and sheet forms, such as graphene. In addition, CNPs are biocompatible and can improve cellular functions, for example, cell adhesion and proliferation, making them suitable for stem cell cultivation platforms [94–103].

In a 2021 article, Castagnola et al. describe a graphene-based electrochemical nanosensor to evaluate neuronal function [104]. This sensor comprised graphene flakes with a 3D arrangement via photolithographic processes. This sensor showed high electrochemically active area enhancement. Specifically, the electrochemically active area of the sensor was about 88 times higher than that of conventional carbon fibre electrodes. The sensor sensitively and selectively detected DA as a target analyte using fast scan CV to evaluate neuronal function; the LOD for DA was calculated to be approximately 364.44 nM. Furthermore, the sensor was demonstrated to discriminate between serotonin and DA. Overall, this sensor's enhanced electrochemical properties for sensitive and selective sensing of DA

suggested that neurogenesis could be monitored in real-time by sensing DA released from neurons in vitro.

Vasudevan et al. [105] developed a nanosensor based on CNPs (Figure 4a). Specifically, this sensor was composed of carbon fibre, which detected DA released from neural stem cell-derived dopaminergic neurons and promoted neurogenesis in vivo based on optogenetics as an optical fibre. To improve the electrochemically active surface area of the sensor, a 15 μm thick polyimide buffer layer was coated onto the surface of a silica-based optical fibre; then, this layer was processed to form of 8 μm thick pyrolytic carbon fibre surrounded by cladding. Subsequently, human neural stem cells (hNSCs) were cultured on the sensor surface and differentiated into dopaminergic neurons to detect DA exocytosis electrochemically using an amperometric method. According to the amperometric results, the electrochemical signal towards DA was not confirmed from undifferentiated cells on the sensor surface. However, a clear electrochemical current peak towards DA was confirmed from the differentiated cells (Figure 4b,c). Moreover, as a result of monitoring the DA signals on the sensor surface during the 10-day differentiation period, it was observed that the DA signal gradually improved according to dopaminergic differentiation. These results suggested that the sensor could monitor dopaminergic differentiation non-invasively.

**Figure 4.** A carbon fibre-based electrochemical sensor for monitoring in vitro neurogenesis. (**a**) Immunocytochemistry images of hNSCs-derived dopaminergic neurons cultured on the sensor. (**b**) Amperometry graph of non-differentiated and differentiated cells after stimulating DA exocytosis. (**c**) Electrochemical current peaks toward DA for time-dependent monitoring of the dopaminergic differentiation on the sensor. Reprinted with permission from [105]. Copyright 2019, Wiley Online Library. DA, dopamine; DNA, deoxyribonucleic acid; hNSCs, human neural stem cells; TH, tyrosine hydroxylase.

Similarly, Pham Ba et al. constructed a CNT-based nanosensor to monitor neuronal differentiation [106]. This sensor's Nafion®-radical layer was composed of CNT transistors and was demonstrated to selectively detect DA in the presence of the interfering molecules acetylcholine and glutamine. In addition, it was confirmed that neuronal cells could be normally attached and cultured on the sensor surface. Moreover, an amperometric response to DA was observed immediately after potassium chloride (KCl) stimulation from neurons cultured on the sensor surface. Furthermore, the sensor obtained different amperometric responses to DA by adding different concentrations of KCl, suggesting that the sensor could discriminate between different degrees of DA exocytosis.

The previously mentioned studies support that carbon nanomaterials, including graphene, carbon fibre, and CNT, can be actively utilised to construct excellent sensing platforms with high electrical properties. In particular, nanosensors based on the graphene family have been demonstrated to monitor neuronal differentiation by sensitively and selectively sensing DA through π-π stacking. Moreover, CNPs' biocompatibility and high sensing capability make them suitable for stem cell cultivation platforms and non-invasive monitoring of various types of stem cells, such as ESCs and MSCs.
