**1. Introduction**

Luminescent materials are functional materials that absorb incident energy and subsequently emit incident energy in photons. Most luminescent materials, including organic dyes and quantum dots (QDs) [1], follow Stokes Law. They absorb high-energy photons and emit low-energy photons (energy reduction), known as downconversion materials [2–7]. On the contrary, UCNPs relate to the conversion of long-wavelength NIR light with lower energy into ultraviolet or visible light with higher energy. This process is also known as the anti-stokes process, which is so-called the UCL phenomenon.

When almost all the trivalent rare earth ions (RE3+) are excited externally, the rare earth elements of 15 lanthanides, yttrium, and scandium can move between different energy levels, because they have a unique 4f electron configuration energy level, such as shown in Figure 1. In addition, due to the shielding of the secondary 5s25p6 shell, the external environment has little effect on rare earth ions [8–11]. Under near infrared (NIR) laser irradiation, typical lanthanide-doped upconversion nanoparticles exhibit anti-Stokes shifted visible light and ultraviolet light emission, while the autofluorescence background is minimal, and the light scattering of biological tissues is greatly reduced. The reduction in light scattering caused by this near-infrared excitation will make the penetration depth of biological tissues far greater than the penetration depth under ultraviolet or visible

**Citation:** Gao, C.; Zheng, P.; Liu, Q.; Han, S.; Li, D.; Luo, S.; Temple, H.; Xing, C.; Wang, J.; Wei, Y.; et al. Recent Advances of Upconversion Nanomaterials in the Biological Field. *Nanomaterials* **2021**, *11*, 2474. https:// doi.org/10.3390/nano11102474

Academic Editor: Julia Pérez-Prieto

Received: 4 September 2021 Accepted: 15 September 2021 Published: 22 September 2021

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light excitation, which makes UCNP have grea<sup>t</sup> potential in biological applications [12–21], including biological tissues imaging [22–24], biological detection [25–30], photodynamic therapy, and other biomedical fields [31–36].

In this review, we briefly describe the luminescence mechanism and synthetic methods of UCNPs. However, the surface of UCNPs obtained through conventional surface modification methods lacks the active groups that are binding to biomolecules, which limits their application in the biological field to a certain extent. Here, we introduce several surface modification methods and systematically discuss the latest progress of UCNPS in biological imaging and photodynamic therapy.

**Figure 1.** Energy-level diagrams of rare-earth ions. Typical upconversion emissive excited states are highlighted by a red bold line. Reprinted with permission from ref. [11]. Copyright 2015 Annual Reviews.

## **2. Mechanism of Upconversion**

In general, UCNPs are composed of inorganic crystal matrix and rare-earth ions, which do not constitute the light-emitting energy level. The main function is to provide the lattice structure of rare-earth ions to ensure they have appropriate luminescent conditions. According to current research, there are five upconversion mechanisms: excited-state absorption (ESA), energy transfer upconversion (ETU), cooperative sensitization upconversion (CSU), cross-relaxation (CR), and photon avalanche (PA) [37,38].

#### *2.1. Excited State Absorption (ESA)*

Excited state absorption (ESA) occurs in the form of continuous absorption of pump photons by a single ion through the trapezoidal structure of a simple multistage system. As shown in Figure 2a, it is realized by a three-level system that continuously absorbs two photons. This mechanism is due to the equal separation degree of E1 and E2, E2 and E3, and the storage capacity of intermediate E2. When an ion is excited from the ground state to the E2 level, because the lifetime of the E2 level is very long, the other pump photon is likely to absorb another photon before it decays to the ground state, so as to promote the ion from the E2 level to the higher E3 level, resulting in the upconversion emission of the E3 level. In order to meet the above process and achieve efficient ESA, the energy states of lanthanide elements need to be arranged in a trapezoid, and only a few lanthanide ions, such as Er3+, Ho3+, Tm3+ and Nd3+ have such energy level structure [39]. In addition, it is found that the output wavelength of commercial diode lasers (about 975 nm and/or 808 nm) can well match the excitation wavelength of these energy level structures.

**Figure 2.** Upconversion mechanisms of RE-doped UCNPs: (**a**) ESA, (**b**) ETU, (**c**) CSU, (**d**) CR and (**e**) PA.

## *2.2. Energy Transfer Upconversion (ETU)*

Unlike ESA, Energy transfer upconversion (ETU) involves two identical or different ions. In this photophysical process, ion 1 transitions from the ground state to E2 by absorbing excitation light; then the absorbed photon energy is transferred to the ground state E1 and excited state E2 of ion 2, so that they are excited again, while ion 1 relaxes back to ground state E1. In this process, the quantum yield of upconversion largely depends on the average distance between ion 1 and ion 2, and is mainly determined by the concentration of doped rare earth ions. The ETU process is the most important photophysical process in upconversion luminescence, because so far, the use of upconversion nanoparticles for therapeutic diagnostics and other applications is through the sensitizer Yb3+, which has a strong absorption of the excitation light at 975 nm, thus making activators (Er3+, etc.) produce more efficient fluorescence emission [40–45].

Moreover, the scattering and absorption of biological tissues at 975 nm are relatively small, and no optical interference occurs. Here, Yb3+ has a very good effect as a sensitizer because it has a sufficiently large absorption cross-section in the near-infrared region of about 975 nm. In addition, since Yb3+ has only two energy levels, its optimal concentration can be maintained at a high level (20–100% for fluorinated nanoparticles) without causing harmful cross-relaxation. To date, most research has focused on the development of Yb3+ sensitized upconversion nanoparticles pumped at about 975 nm. Using lanthanide ions themselves as sensitizers, high-efficiency ETU can also be observed in single lanthanide doping systems, for example, long-wavelength 1490 nm excitation of Er3+-doped LiYF4 [46]; or doping under 1200 nm excitation NaGdF4 nanoparticles doped with Ho3+ [47]. The use of other sensitizers can be used to quench and enhance the luminous intensity of certain emission bands. For example, Nd3+, Ce3+, and Ho3+ are used as sensitizers to enhance the blue emission band of Tm3+, the red emission band of Ho3+, and the near-infrared emission band of Tm3+, respectively [48–51].

#### *2.3. Cooperative Sensitization Upconversion (CSU)*

Cooperative sensitization upconversion (CSU) in Figure 2c is a photophysical process of the interaction of three rare earth ions (two types). Ion 1 and ion 3 usually belong to the same sensitizer, such as Yb3+. After being excited by light, ion 1 and ion 3 transition to an excited state. However, ion 1 or ion 3 alone cannot excite ion 2, because their excited state energy levels are quite different, so ion 1 and ion 3 need to be co-excited to produce a virtual excited state energy level that can be compared with the excited state energy level of ion 2. The ion 2 absorbs the energy of cooperative sensitization and emits a higher energy photon. The photophysical process of CSU is often uncommon, because the energy transfer efficiency is low, and the para-virtual pair energy levels in the transfer process are involved. These energy levels must be described by quantum mechanics in higher disturbances. Nevertheless, limiting the excitation to compensate for the low efficiency provides a possibility to achieve high-resolution imaging, which is not possible with other upconversion mechanisms. At present, the CSU mechanism of Yb3+/Tb3+ [52], Yb3+/Eu3+ [53], Yb3+/Pr3+ [54] ion pairs has been reported.
