*3.2. Upconversion Nanoparticle-Based Optical Sensors*

Upconversion refers to a phenomenon in which external energy is changed into higher energy through a phosphor [135]. Considering that UCNPs show unique optical properties different from conventional phosphors, they have been applied to bioimaging and the optical applications of conventional phosphors. Specifically, UCNPs do not quench and are chemically stable. Furthermore, unlike conventional quantum dots, the maximum emission wavelength does not depend on particle size. In addition, UCNPs can easily emit multi-colour emissions by changing doping materials. In particular, UCNPs doped with lanthanum elements are excited by long wavelengths and have very low cytotoxicity; therefore, they are very useful for use in cell-based sensors [136]. To date, various UCNP-based sensors capable of monitoring stem cell differentiation have been reported in the literature.

For example, Wang et al. [137] developed a multifunctional UCNP capable of real-time detection and control of osteogenic differentiation in MSCs using NIR. The researchers synthesised thulium/erbium-doped core-shell UCNPs coated with mesoporous silica for drug loading and to install photomechanical azobenzene that acted as an agitator. Then, the RGD peptide and matrix metalloproteinase 13 (MMP13) sensitive peptide-black hole quencher-3 group were conjugated to the UCNP surface responsible for cell targeting and detecting differentiation. Finally, icariin, a drug that can induce MSCs' osteogenic differentiation, was loaded onto the UCNPs to form a nanocomplex. The drug was released from the fabricated UCNP nanocomplexes using NIR light in a controlled way, which was based on trans-azobenzene being converted to a cis isomer under UV and visible light. According to the results of reverse transcription (RT)-PCR, WB, and autonomously replicating sequence (ARS), the UCNP nanocomplex efficiently induced osteogenic differentiation of MSCs under NIR light at a wavelength of 980 nm and successfully detected MMP13 produced by osteogenesis. Therefore, this developed multifunctional UCNP could control the osteogenesis of MSCs and detect cell differentiation in real time, making it a potential tool for progressing regenerative medicine.

Similarly, Yan et al. [138] reported on the controlled osteogenic differentiation of MSCs with a light-responsive nanoplatform to treat osteoporosis (OP). The nanoplatform was a modification of that of Wang et al. described previously. Like Wang et al.'s method, the UCNPs were first doped with thulium/erbium and coated with mesoporous silica. Then photocaged linker 4-(hydroxymethyl)-3-nitrobenzoic acid and polyethylene glycol linker were linked to the surface to conjugate to the cap β-cyclodextrin and the RGD-targeted peptide/MMP13-sensitive peptide-black hole quencher, creating a drug loading nanoplatform. According to the RT-PCR, WB, ALP/ARS/immunofluorescence staining, and ALP activity results, the release of icariin by NIR light at 980 nm induced controlled osteogenic differentiation of MSCs for OP treatment. In addition, MMP13 produced by the MSCs' osteogenic differentiation cleaved the MMP13-sensitive peptide, removing the peptideblack hole quencher and allowing the UCNPs to fluoresce; this allowed real-time detection of osteogenic differentiation. The results of haematoxylin and eosin, Masson's trichrome, immunohistochemical, tartrate-resistant acid phosphatase, and toluidine blue staining of a femoral terminal section showed that significant bone remodelling had occurred in the OP rat model. This study's results suggested that the synthesised UCNP nanoplatform enabled remote control and real-time detection of osteogenic differentiation for OP treatment by NIR and could be a potential alternative to current OP treatment.

Non-destructive stem cell differentiation control and monitoring using UCNPs has also been studied for neural differentiation from MSCs. However, conventional UCNPs have shortcomings, such as low emission intensity due to undesirable energy transfer paths. Low power density excitations can minimise detrimental energy reverse transitions and produce bright visible emissions. Therefore, Rabie et al. [139] developed a core–shell–shell sandwich-structured UCNP with enhanced luminescent output relative to conventional UCNPs. This core–shell–shell UCNP was then used to construct a biosensor to detect DA released from stell cell-derived dopaminergic neurons (Figure 8). This UCNP detected DA released in vivo during the differentiation of stem cells into specific neurons at the single cell level in a highly selective, real-time, and non-invasive manner, with a sensitivity of at least three times higher than similarly designed systems. This sensor was demonstrated to detect DA at low concentrations. The developed NIR-based neurotransmitter detection method has significant potential for the diagnosis of diseases related to neurodegenerative diseases and stem cell treatment strategies.

**Figure 8.** Upconversion nanoparticle-based optical sensor capable of monitoring neurogenesis. (**a**) Upconversion luminescence profiles and analysis of the operating mechanism and characteristics of UCNPs for each condition. (**b**) Monitoring of neuronal differentiation of hNSCs using the UCNPs-based optical sensor. Reprinted with permission from [139]. Copyright 2019, Wiley Online Library. cAMP, cyclic adenosine monophosphate; DA, dopamine; GDNF, neurotrophic factor; GO, graphene oxide; hNSCs, human neural stem cells; NIR, near-infrared; UCNPs, upconversion nanoparticles. \*\* *p* < 0.01.

The literature identified several UCNPs with high optical properties and low cytotoxicity. Moreover, UCNP-based sensors can monitor stem cell differentiation in real-time, non-invasively, without a label. In addition to cell imaging, UCNPs are potential drug delivery systems to control stem cell differentiation.
