*3.1. Gold and Silver Nanoparticle-Based Optical Sensors*

Nanomaterials, such as AuNPs and AgNPs, have excellent optical properties [122], which are unique depending on their particle size [123,124]. In addition, AuNPs can be modified through chemical conjugation with various other nanomaterials and probes to form new types of hybrid nanomaterials; this allows sensors with higher selectivity and sensitivity to target analytes to be developed [125,126]. In particular, AuNPs can greatly enhance the surface plasmon effect because they are mainly applied to optical sensing Raman spectroscopy methods [127].

Various AuNP-based optical sensors capable of monitoring stem cell differentiation non-invasively and in real-time have been reported. For example, Cao et al. report a gold-based surface-enhanced Raman spectroscopy (SERS) sensor for monitoring osteogenic differentiation [128]. This sensor had a hybrid structure based on AuNPs and nucleic acids and sensitively detectable micro-RNAs (miRs), such as miR-144-3p, associated with osteogenesis. The DNA nucleic acid binds site-specific targets via its complementary interaction. Therefore, hybrid nanostructures in which a specific DNA strand is conjugated on the surface of AuNPs are highly selective. Furthermore, the gold-based nanostructures selectively detected the Raman signals from the target miR. In addition, the probe showed high optical properties and enhanced Raman signals. These advantageous features allowed the sensor to be used in stem cell cultivation and long-term monitoring of their osteogenic differentiation.

In another study, Sun et al. developed a smart gold nanoprobe for detecting alkaline phosphatase (ALP) activity during bone marrow MSCs' osteogenic differentiation [129]. The smart nanoprobe was designed by decorating the surface of AuNPs with 5-bromo-4-chloro-3-indolyl phosphate (Au@BCIP), which is suitable for use as a SERS nanoprobe. This probe allowed non-invasive and living-cell permeable monitoring of ALP activity with high sensitivity and selectivity. Moreover, this probe could detect a single cell without cell deformation, and the preparation process was simple, so time and effort could be saved compared to conventional methods. Therefore, the non-invasive detection of ALP activity associated with bone disease in vivo models and osteogenic differentiation of bone marrow MSCs can be more fully understood from the perspective of ALP activity.

A 2021 article by Hua et al. describes the development of an imaging probe consisting of gold nanostars (AuStar) and silver sulphide quantum dots for labelling and accurately tracking MSCs in a hypodermic and myocardial infarction model with deep tissue penetration [130] (Figure 7). The probe's AuStar-disseminated tumour cell cluster section enabled high-resolution Raman imaging, effectively delineating stem cells in surrounding normal tissues at a single-cell resolution scale. In addition, the labelling agents were biocompatible and did not alter the MSCs' biological properties, compensating for existing invasive monitoring methods used for tracking stem cells' shortcomings.

Lee et al. [131] fabricated magneto-plasmonic nanorods that detected the expression level of miRNA-124 and characterised the neurogenesis of human-iPSC-derived hNSCs in a non-destructive and efficient way. The plasmonic (gold) parts of the nanorod selectively and sensitively recognised target exosomal miRNAs using a molecular beacon (MB), which was on the gold component. The MB and miRNA hybridised to form an MB–miRNA complex with an increased fluorescence signal, increasing the signal-to-noise ratio through the metal-enhanced fluorescence effect. This system was non-destructive and could possibly advance the transplantation of differentiated stem cells.

**Figure 7.** A gold-based NIR sensor for monitoring of chondrogenic differentiation. (**a**) Characterisation of the AuStar-DTTC-Ag2S (GDS) nanoparticles. (**b**) Ex vivo Raman imaging of GDS-MSCs in a myocardial infarction model. Reprinted with permission from [130]. Copyright 2020, Wiley Online Library. Ag, silver; Ag2S, silver sulphide; Au, gold; AuStar, gold nanostar; BSA, bovine serum albumen; DMEM, Dulbecco's modified eagle medium; DTTC, 3.3 -diethylthiatricarbocyanine iodide; GFP, green fluorescent protein; MSCs, mesenchymal stem cells; NIR, near-infrared; PBS, phosphate-buffered saline; S, sulphur; SERS, surface-enhanced Raman spectroscopy.

An ultrasensitive nanosensor consisting of a Au-coated nanopore thin film was reported by Yang et al. in 2021 [132]. This nanosensor was developed for the detection of N27 cells' Glial cell-derived neurotrophic factor (GDNF) secretion [132]. The GDNF is a small protein that strongly promotes the survival of dopaminergic and motor neurons, and the evaluation of its magnetic stimulation is valuable. Due to the characteristics of gold, the nanosensor's conversion signal appeared as an optical signal (optical interference fringes) reflected from the nanopore thin film. The optical signal shift occurred because of changes in the effective optical thickness when the GDNF bound to its antibody. The nanostructure helped coat more gold, greatly increasing the sensor's sensitivity; this was demonstrated by a significantly improved GDNF LOD (2 pg/mL) compared with the rat ELISA kit assay (32 pg/mL). In addition, it was inexpensive, easy to use, and suitable for measuring GDNF secretion with ultra-high sensitivity. Furthermore, it can potentially be used for other highly secreted substances.

In a study on AgNP-based optical sensors, Koh et al. [133] developed a 3D cell culture scaffold and SERS-based biosensor to detect multiple differentiated markers from adiposederived MSCs. Scaffolds were composed of electrospun nanofibres with hydrogel patterns. Moreover, the sensing scaffolds were coated with AgNPs, which can be conjugated with specific antibodies for SERS analysis. This type of scaffold culture platform successfully supported adipose-derived MSCs' proliferation and differentiation in osteogenic differentiation media. In addition, the SERS-capture substrate detected various differentiation markers with SERS tags made of Au-Ag alloy nanoboxes. The time-dependent release of three different osteogenic differentiation markers (ALP, osteocalcin, and fibronectin) were detected up to the pg/mL levels without interference or crosstalk for three weeks. Therefore, the platform was sufficiently sensitive to monitor markers during osteogenic differentiation. This platform was suggested to overcome the limitations of existing stem cell differentiation monitoring methods, as it did not require cell pre-processing, enabled continuous analysis with a single platform, and the multi-sensing scaffold could detect various biomarkers.

Similarly, Li et al. [134] developed a SERS sensor for accurate and quantitative detection of DA in blood. The sensor consisted of zipper-like ortho-nanodimers and AgNPs with a uniform 1 nm gap. The AgNPs were electrostatically self-assembled onto a glass slide; then, the complementary DNA of the DA aptamer was bound to the surface of the AgNPs. The SERS probe was synthesised by decorating AgNPs with DA aptamers and the Raman reporter 5,5'-dithiobis-(2-nitrobenzoic acid). When these SERS probes were added to a substrate, they combined with the complementary DNA forming zipper-like ortho nanodimers with a 1 nm gap between the probe and AgNPs on the substrate in a state of equilibrium between electrostatic repulsive force and hybridisation contractility. The uniform gap allowed the SERS sensors to detect DA with ultra-high sensitivity (LOD = 10 aM) while maintaining signal uniformity (relative standard deviation < 5%). Even in a complex serum environment, the sensor maintained excellent validity and stability from 1 pM to 10 nM, which was about two times lower than conventional methods. In addition, monitoring the DA of cells released from living neurons was performed for the first time. This was achieved by introducing a single microfluidic chip containing a 3D cell culture device. The DA quantification in human blood samples showed recoveries ranging from 87.5% to 123.7%. Given the difficulty of DA quantification in complex physiological samples, this SERS sensor may provide a powerful tool for the in vitro investigation of neurological processes and clinical examination of dopaminergic disorders.

Based on the literature reviewed, gold- and silver nanomaterials have excellent optical properties and the advantage of being easily transformable into various 3D nanostructures. In addition, AuNPs can be widely applied to optical sensing methods because of their easy surface modification with other nanomaterials.
