**1. Introduction**

MicroRNAs (miRNAs) are important regulators of cell proliferation, division, differentiation, and apoptosis. They can regulate the expression levels of various genes in DNA post-transcriptionally. The abnormal expression of miRNAs has been associated with many diseases (cancer, tumors, and diabetes) [1], and miRNAs can be obtained through blood, urine, etc. [2], which has the advantage of being non-invasive. Therefore, miRNAs are considered important biomarkers for tumors [3]. Patryk Krzeminski [4] et al. demonstrated that DNA methylation contributes to miRNA-155 expression, and the survival data of myeloma cells show a correlation between miR-155 expression and multiple myeloma outcomes. A number of studies have shown that miRNA-155 is closely related to MM, and the overexpression of miRNA 155 in blood is an important signal for the diagnosis of MM [5–7]. Therefore, the detection of miRNA-155 to diagnose early MM is an important and efficient method.

The concept of upconversion luminescence was first proposed by Auzel and Ovsyankin et al. [8]. The radiation process is a nonlinear anti-Stokes emission, excited by the effective absorption of two or more low-energy photons. Then, it transitions from the ground state to the excited state through a multi-step process, and finally, it returns

**Citation:** Ding, Z.; He, Y.; Rao, H.; Zhang, L.; Nguyen, W.; Wang, J.; Wu, Y.; Han, C.; Xing, C.; Yan, C.; et al. Novel Fluorescent Probe Based on Rare-Earth Doped Upconversion Nanomaterials and Its Applications in Early Cancer Detection. *Nanomaterials* **2022**, *12*, 1787. https:// doi.org/10.3390/nano12111787

Academic Editor: Antonios Kelarakis

Received: 1 April 2022 Accepted: 20 May 2022 Published: 24 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to the ground state energy level in radiative transition, realizing the conversion of longwavelength excitation light into short-wavelength emission light. The excitation wavelength (980 nm) of upconverted nanomaterials is in the "optical transmission window" of biological tissues [9–12] due to the least light absorbed by biological tissues [13–16]. This special luminescence process makes upconversion nanomaterials have incomparable advantages over traditional organic dyes, quantum dots, fluorescent proteins, and other biomolecular markers [17–19], such as near-infrared light as excitation light, resulting in less photo-damage, lower auto-fluorescence background of biological tissue, and a deep penetration depth [9–19]. In addition, rare-earth-doped upconversion nanoparticles have multi-wavelength emissions, high-fluorescence intensity and high stability, good water solubility, as well as good biocompatibility [20–31]. Moreover, rare-earth-doped nanoparticles also have strong scintillation luminescence that can be used for X-ray-induced photodynamic therapy, which is a well-captivated area, as this new therapy can be used for deep as well as skin cancer treatment [20–22], radiation dosimetry [23,24] and temperature sensing [25]. These irreplaceable advantages establish upconversion luminescent nanomaterials as a potential fluorescent marker in biological detection [28–31] and have developed rapidly. For example, Mao et al. [32] prepared NaYF4:Yb,Er upconversion nanoparticles using the hydrothermal method, and discovered their specific detection of miRNA by using the principle of base stacking on the surface. Kowalik et al. [33] linked IgG antibodies to the PEG-NHS-modified NaYF4:Yb,Tm@SiO2 surface to achieve specific labeling to demonstrate the grea<sup>t</sup> potential of photodynamic targeted therapy.

The main body of rare-earth-doped upconversion nanoparticle materials is composed of host materials, sensitizers, and activators [34]. The commonly used host materials themselves have no fluorescence but can provide a suitable crystal field for the activated ions, so that the luminescent centers produce specific emissions. Y3+, Gd3+, Lu3+, La3+ are usually selected as host elements [35]. The high-quality host material plays a decisive role in the entire upconversion luminescence efficiency. The spacing between rare-earth ions, the symmetry distribution, and the number of coordination ions would affect the crystal structure of the host material. The most common NaYF4 is currently recognized as the most effective blue-green light upconversion luminescent host material [36,37]. Because the hexagonal NaYF4 has a higher symmetry than the cubic NaYF4, the luminous efficiency of the upconversion in hexagonal NaYF4 is 10-times higher than in the cubic NaYF4 [38]. Nd3+ and Yb3+ ions are the most common sensitizers in the upconversion systems [39,40]. Yb3+ has a large absorption cross-section at 950–1000 nm, which is related to the only excited state and can absorb the excited infrared photons as well as effectively obtain the energy. The energy is then transferred to the activators. Yb3+ is more often chosen as the sensitizer of nanomaterials because its upconversion luminescence efficiency is higher than that of Nd3+. The activators are rare-earth ions, such as Er3+, Tm3+, Ho3+, Nd3+, Pr3+, etc. [41–44], among which the Yb3+/Er3+ (Tm3+, Ho3+) pairs are recognized as the most efficient [45].

The standard preparation methods of rare-earth-doped upconversion nanoparticles include the co-precipitation method, high-temperature thermal decomposition method, solvothermal method, microemulsion method, sol–gel method, etc. Nanoparticles with tunable morphology, high-fluorescence intensity, and good dispersion have advantages and disadvantages. For example, Gao et al. [46] compared the effects of factors, such as the concentration of reactants, the ratio of reactants and ligand solvents, and the types of ligand solvents on the synthesis of nanoparticles, respectively. Hydrothermal synthesis has been widely used for NaYF4:Yb,Er nanoparticle synthesis. This method has a fast reaction and precipitation rate and crystallinity; however, the fluorescence efficiency of the nanoparticles synthesized is relatively low. Amphiphilic ligands can help the particles to be well dispersed in various polar solvents. However, the surface of the obtained upconversion nanoparticles is usually coated with hydrophobic organic ligands, such as oleic acid, oleylamine, and octadecene, which make them less water soluble and difficult for biological applications. To improve the water solubility and biocompatibility, the surface of upconvertion nanoparticles must be modified with bioactive ligands or biomolecules.

Surface ligand exchange, surface ligand oxidation, and surface ligand assembly are common surface modification methods [47–49]. Jiang et al. [48] used polymeric anhydride to interact with octadecene and oleic acid ligands on the surface of nanoparticles by coordination adsorption, and then cross linked with dicyclohexanetriamine to obtain stable water-soluble upconverted nanoparticles. The effective modification of upconverted materials has become a key factor in their applications in biological detections.

In this paper, high-temperature thermal decomposition was used to prepare NaYF4:Yb3+/Tm3+ upconverted nanoparticle materials and it was found that the optimal Yb3+/Tm3+ ratio is 40:1, in terms of the luminescence efficiency. These new types of upconverted nanoparticles were modified by surface coating and tested for protein detection for the purpose of early cancer diagnosis.

#### **2. Experimental Materials and Methods**

## *2.1. Reagents and Instruments*

Chemicals and Reagents: These chemicals were purchased from Shanghai Aladdin Biochemical Technology Co. Ltd. (Shanghai, China): yttrium chloride hexahydrate (YCl3·6H2O), ytterbium chloride hexahydrate (YbCl3·6H2O), thulium chloride hexahydrate (TmCl3·6H2O) gadolinium chloride hexahydrate (GdCl3·6H2O), oleic acid (OA), 1-octadecene (ODE), ammonium fluoride (NH4F), cyclohexane (C6H12), methanol (MeOH). These Chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China): hydrochloric acid (HCL), succinic acid (C6H10O4), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), acetonitrile (C2H3N), and 1-ethyl-(3-Dimethylaminopropyl) carbonate diimide hydrochloride (EDC).

Anhydrous ethanol (C2H6O) and sodium hydroxide (NaOH) were purchased from Xilong Chemical Co., Ltd. (Guangzhou, Guangdong, China). Bovine serum albumin (BSA), N,N-dimethylformamide (DMF), and N-hydroxysuccinimide (NHS) were purchased from Shanghai Macklin Biochemical Co. Ltd. (Shanghai, China). Nitronium tetrafluoroborate (NOBF4) was purchased from Hebei Bailingwei Superfine Materials Co., Ltd. (Langfang, Hebei, China), PBS buffer and DNA with amino and FAM were purchased from Sangon Biotech Co. Ltd. (Shanghai, China), corresponding base sequence is 5-NH2- CCCCCCCCCCCCACCCCTATCACGATTAGCATTAA-6-FAM-3. The ultrapure water was produced by a water purification system (H20BASIC-B, Sartorius, Germany).

Instrumentation: the intelligent digital magnetic stirring electric heating mantle (ZNCL-TS-250 mL, Shanghai Anchun Instrument Co., Ltd., Shanghai, China) was used to prepare upconverted nanomaterials and carboxylated proteins. Fluorescence was measured using a fluorescence spectrometer (F-4600, Hitachi, Hitachi, Ltd., Tokyo, Japan). Phase analysis of upconverted nanomaterials was carried out by X-ray powder diffractometer (X-ray Diffraction, XRD, D8 ADVANCE, Bruker, Germany). The morphology of the upconverted nanomaterials was characterized by field emission scanning electron microscope (Scanning Electron Microscope, SEM, SU8010, Hitachi Co., Ltd., Tokyo, Japan) and scanning transmission electron microscope (Transmission Electron Microscopy, TEM, FEI TECNAI G2 F20, Hitachi Co., Ltd., Tokyo, Japan). A Fourier transform infrared spectrometer measured infrared absorption (Tensor 27, Bruker, Germany). The luminescence was measured with a 980 nm fiber laser (BOT980-5W, Xi'an Leize Electronic Technology Co., Ltd., Xi'an, China).

#### *2.2. Preparation of Upconverted Nanomaterials*

Rare-earth-doped upconverted luminescent nanomaterials NaYF4:Yb,Tm were synthesized by high-temperature thermal decomposition. The preparation of 2 mmol NaYF4:20% Yb3+, 0.5% Tm3+ nanomaterials was performed by first charging 1.39 mmol YCl3·6H2O, 0.6 mmol YbCl3·6H2O, and 0.01 mmol TmCl3·6H2O into a three-neck round-bottom flask. Then, oleic acid (12 mL) and octadecene (30 mL) were added, and the flask flowed with nitrogen gas for 10 min to ensure no oxygen in the flask. Next, under nitrogen protection with magnetic stirring, the mixture was heated to 160 ◦C and reacted for 1 h to obtain a pale-yellow solution. The mixture was cooled to 50 ◦C and then 10 mL of a methanol solu-

tion containing 8.0 mmol of ammonium fluoride and 5.0 mmol of sodium hydroxide was added dropwise to the mixture. The reaction was continuously stirred at 50 ◦C for 30 min to ensure a complete integration. Then, the temperature was raised to 80 ◦C to evaporate the methanol. During the evaporation of methanol, the solution continued to bubble, and the mixture was continuously heated to 120 ◦C for 30 min until no more bubbles were generated in the solution. Finally, the sample was heated to 300 ◦C for 90 min. After the reaction was over, the solution was naturally cooled to room temperature and cyclohexane was added to disperse the mixture. The mixture was centrifuged at 8000 rpm/min for 5 min to obtain a precipitate, which was then washed with cyclohexane. The above steps were centrifuged and washed three times, and the NaYF4:Yb,Tm upconverted nanomaterial finally obtained was dispersed in cyclohexane and stored. In addition, the experimental steps for the preparation of NaGdF4:Yb,Tm upconverted nanomaterials are the same as above. The YCl3·6H2O in the experimental material was replaced by GdCl3·6H2O, and the experimental steps were repeated to obtain NaGdF4:Yb,Tm upconverted nanomaterials.
