*2.5. Microelectrode Array-Based Electrochemical Sensors*

Microelectrode arrays refer to micromaterials in which multiple micro-scale electrodes are arranged on a single substrate [116]. Microelectrode arrays are based on a 3D structure that improves and increases the electrochemically active area in which analytes participate in the redox reaction, providing a high sensing performance [117]. In addition, considering that a plurality of electrodes is spatially arranged in a microelectrode array, it is possible to analyse signals of cells by position on a single substrate and to analyse single cells and spheroids electrochemically [118].

The literature reports that microelectrode array-based electrochemical sensors that can non-invasively monitor stem cell differentiation have been developed. For example, a microelectrode array that electrochemically monitors cardiomyocyte differentiation has been described [119]. This platform was designed to culture iPSCs for an extended period and to perform qualitative and quantitative analyses of the differentiated cells' maturity for

efficient cardiomyocyte differentiation. The platform was used to monitor the cardiomyocyte differentiation of iPSCs for 119 days using electrochemical impedance spectroscopy. In addition, the effect of 2D and 3D culture environments on cardiomyocyte differentiation was evaluated non-invasively by analysing the differentiated cells' impedance. The results confirmed that more mature cardiomyocytes were produced in a 3D environment.

Gao et al. [120] decribed a microelectrode array-based sensing platform that monitors neuronal differentiation by electrochemically detecting neurotransmitters released from olfactory bulb neurons. The olfactory bulb neurons were cultured in a microelectrode array located on the corresponding platform; cells were stimulated using glutamate and gammaaminobutyric acid (GABA) to detect electrochemical signals from the cells. As a result of detecting cells at various concentrations of glutamate and GABA, it was confirmed that cell signals depended on the concentration of the stimulant and that neurotoxicity occurred at high concentrations. The sensing ability of the platform to detect neurotransmitters through GABA stimulation can be used to detect neural differentiation of stem cells non-invasively and label-free.

Similarly, a sensing platform capable of electrochemically analysing DA exocytosis in dopaminergic neurons differentiated from human ESCs was developed in 2020 [121]. The microelectrode array in the platform was composed of reduced GO and poly(3,4 ethylene dioxythiophene):polystyrene sulfonate (PEDOT:PSS) nanocomposites. Due to the nanocomposites' excellent electrochemical properties, the device's LOD for DA was calculated to be 2 nM. Moreover, it could detect DA released from neurons in a locationby-amperometric manner thanks to the intrinsic advantage of the microelectrode array (Figure 6). The platform's superior dopaminergic sensing ability allowed dopaminergic differentiation to be detected in real-time.

**Figure 6.** A microelectrode array-based electrochemical sensor. (**a**) Analysis of dopaminergic differentiation of hESCs cultured on the platform. (**b**) Detection of DA exocytosis on the platform. Reprinted with permission from [121]. Copyright 2022, Elsevier. DA, dopamine; DAPI, 4 ,6-diamidino-2 phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hESCs, human embryonic stem cells; mRNA, messenger ribonucleic acid; TH, tyrosine hydroxylase. "n.d.", indicates non-detected; "n.s.", indicates non-significant; \*\* *p* < 0.01, \*\*\* *p* < 0.001.

A review of the current literature supports that microelectrode arrays have several advantages as electrochemical sensors. First, the 3D regularly arranged electrodes could considerably improve existing electrodes' electrochemical sensing ability by increasing the electrochemically active area. Second, the microelectrode array can be used as a stem cell cultivation platform to analyse cells on a single substrate by location. In addition, single cells and spheroids can be effectively detected.
