*2.4. Microfluidic System-Based Electrochemical Sensors*

Microfluidic systems refer to systems in which fluid can flow through micro-scale channels fabricated on a substrate [107]. The microfluidic system has the advantage that it can be performed on a single chip under various conditions in a short time using only a small volume of reagents. Due to these structural features, microfluidic systems have been actively applied as biosensors [108–110]. Furthermore, given that the microchannel in microfluidic systems enables electrochemical species in the analytes to be confined near the electrochemical electrodes, they are advantageous to electrochemical sensing with high performance [111]. Above all, microfluidic systems can mimic the cellular microenvironment, which can be applied as a stem cell cultivation platform [112].

An example of an electrochemical sensor based on a droplet microfluidic system capable of sensing osteogenic differentiation was described by Fan et al. in 2019 [113]. This sensor could detect the impedance of a single cell; therefore, it was possible to analyse the differentiation of stem cells non-invasively without a label. The sensor detected a difference between the impedance of undifferentiated and differentiated cells. As osteogenic differentiation progressed, the variation in cell impedance decreased. In addition, the average impedance decreased as the differentiation progressed. Conversely, the capacity of the cells analysed on the sensor gradually increased with differentiation. These results were consistent with the fact that calcium ion channels were gradually formed on the cell membrane following osteogenic differentiation.

In 2020, a brain-on-a-chip device based on a microfluidic system capable of analysing neural differentiation was developed [114]. This platform's design structure included three isolated compartments, suggesting that this structure was suitable for pharmacological manipulations, and a plastic lid and a specific gas supply chamber to build a gas supply system. It was validated that neurons could be cultured and maintained for up to 98 days on the platform. In addition, it was confirmed that neurons were differentiated normally on the device with positive expression of axonal and dendritic markers and that their neuronal networks were formed normally. Furthermore, the spike train tiling coefficient was successfully measured from the neuronal network of neurons differentiated on the platform.

Another study by Lee et al. described a microfluidic system-based sensing and cultivation platform capable of electrochemically analysing the cellular function of cardiomyocytes differentiated from iPSCs [115] (Figure 5a). Interestingly, the platform developed contained an aptamer and a gold-based microfluidic system. The functionality of cardiomyocytes could be electrochemically monitored by selectively sensing markers, such as troponin T, creatine kinase, and human epidermal growth factor receptor 2; these markers are related to the functionality of cardiomyocytes (Figure 5b). In addition, to investigate the interaction between cardiac and heart cancer tissues cultured on the platform, troponin secreted from each cell was detected after single or dual interaction with the platform on which each

tissue was cultured (Figure 5c). Results showed that troponin released from healthy cardiac tissues increased in the single and dual platforms. In contrast, troponin released from healthy tissues on the dual platform was lower than from cells that did not interact with heart cancer tissues. In addition, cell functionality was evaluated electrochemically by measuring biomarkers released from healthy cardiomyocytes and heart cancer cells on the aptamer and microfluidic system-based platforms. The results obtained on the platform were consistent with ELISA results.

**Figure 5.** A microfluidic system-based electrochemical sensor for monitoring cardiomyocyte differentiation. (**a**) A schematic illustration showing an aptamer and microfluidic system-based electrochemical sensor. (**b**) Analysis of the important role of interaction between cardiac and heart cancer tissues through biomarkers sensing. (**c**) Monitoring of cardiotoxicity-associated biomarkers using the aptamer and microfluidic system-based electrochemical sensor. Reprinted with permission from [115]. Copyright 2020, Wiley Online Library. DOX, doxorubicin; HER2, human epidermal growth factor receptor 2; TGFβ1, transforming growth factor beta 1.\* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001.

Therefore, it can be concluded that microfluidic systems are effective micromaterials for constructing sensing platforms for the non-invasive and label-free monitoring of stem cell differentiation. In particular, microfluidic systems are suitable for application as a stem cell culture platform and can considerably improve the performance of existing electrochemical sensors.
