*2.1. Gold Nanoparticle-Based Electrochemical Sensors*

Gold's conductivity is 4.11 × 107 S/m, which is highly favourable for electrochemical sensors [54]. Moreover, gold is colloidal in AuNPs, which has various advantageous features for electrochemical sensors [55–63]. For example, AuNPs are easy to synthesise and can be conjugated with multiple biomolecules, such as protein ligands, nucleic acids, and antibodies, which can enforce the intrinsic properties of the AuNPs [64–66]. Moreover, it has been reported that AuNPs are non-cytotoxic, cell-friendly materials with great potential for sensing biomolecules in a cell-based environment and for cell cultivation platforms [67–72]. Therefore, AuNPs have been widely used to develop electrochemical sensors with new nanostructures or modify existing electrochemical electrode surfaces at the nanoscale.

Many studies have described AuNP-based electrochemical sensors capable of sensing stem cell differentiation. For example, Suhito et al. developed an electrochemical AuNP-based electrochemical nano-biosensor to identify the differentiation of embryonic stem cells (ESCs) [59]. To fabricate this sensor, AuNPs were densely deposited on a transparent indium–tin oxide-coated glass electrode through electrochemical deposition. The study's cell detection results using differential pulse voltammetry (DPV) showed that the undifferentiated ESCs generated relatively strong electrochemical signals compared with differentiated ESCs-derived endothelial cells. Interestingly, this sensor could sensitively measure ESCs based on the high electrical conductivity of gold, detecting at least 12,500 cells on one platform. Moreover, it was shown that this sensor could ensure ESCs' adhesion and long-term cell growth, suggesting its application as an ESCs' cultivation platform and a platform for electrochemically monitoring various stem cell differentiation.

In another study, Lee et al. described a AuNP-based nano-biosensor for non-invasive, real-time monitoring of the osteogenesis of mesenchymal stem cells (MSCs) [73]. Specifically, this sensor comprised a 3D AuNP-based nanoarray; the surface of the gold nanoarray was modified with GO. This nanostructure efficiently increased gold's electrical conductivity and electron transfer rate through GO modification, allowing the detection of paminophenol (PAP) produced by the enzymatic reaction occurring in MSCs' osteogenesis. Furthermore, this study observed that this nanoarray provided physicochemical cues beneficial to cellular adhesion and osteogenic differentiation. As a result, this gold-based electrochemical sensing platform detected the anodic signals of PAP using cyclic voltammetry (CV), quantitatively monitoring differentiation during 3 weeks of osteogenesis.

In 2021, a AuNP-based sensing platform was developed that monitored the differentiation of stem cells and controlled their cell differentiation by regulating cellular adhesion (Figure 2a,b) [74]. This sensor was based on a nanoassembly in which AuNPs and Arg-Gly-Asp peptide (RGD) ligands were conjugated on the surface of magnetic iron (II, III) oxide (Fe3O4) nanoparticles. Specifically, the magnetite mediated the control of falling and rising ligand movements via linker compression and stretching, thereby regulating MSCs' integrin expression pattern, cellular adhesion, and consequent osteogenic differentiation. Additionally, due to the high electrical conductivity of the AuNP nanoassembly-based sensor, osteogenic differentiation could be monitored by sensitively measuring PAP's redox using CV.

In 2022, a AuNP-based electrochemical nano-biosensor capable of sensing the generation process and maturity of kidney organoids produced through the differentiation of induced pluripotent stem cells (iPSCs) was developed (Figure 2c,d) [75]. Moreover, the variations of organoids led to the need for a non-destructive evaluation of their maturity [76–78]. Therefore, this sensor was developed to assess the iPSCs differentiation into kidney organoid by sensitively detecting the electrochemical signals originating from the organoids through a gold film structure on which there was electrochemically deposited AuNPs. Interestingly, while monitoring the kidney organoid generation on this sensor, two peaks were detected in the DPV results. Specifically, it was confirmed that the first peak corresponded to cell outgrowth, while the second peak differed depending on the maturity of kidney organoids. A strong second peak was observed for organoids with distinct tubular structures. These results demonstrate that this electrochemical sensor could detect the successful production of kidney organoids in a label-free, non-destructive manner.

In general, studies have shown that AuNPs can be applied to develop electrochemical sensors that sensitively detect the target analytes involved in stem cell differentiation because of AuNPs' excellent electrical conductivity, versatility, and ease of synthetic manipulation with various biomolecules and nanomaterials.

**Figure 2.** Gold nanoparticle-based electrochemical sensors. (**a**) Characterisation of gold nanoassembly and magnetic nanoparticles. (**b**) Time-dependent monitoring of osteogenesis using the gold nanoassembly-based electrochemical sensors. (**c**,**d**) DPV results of iPSCs differentiation and organoid generation on gold-based electrochemical electrode. Reprinted with permission from [74]. Copyright 2021, Wiley Online Library; Reprinted with permission from [75]. Copyright 2022, Wiley Online Library. AuNPs, gold nanoparticles; DPV, differential pulse voltammetry; Fe3O4, iron (II, III) oxide; iPSCs, induced pluripotent stem cells. N.S. indicates "not significant." \*\* *p* < 0.01, and \*\*\* *p* < 0.001.
