2.1.

Preparation of ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ Nanoparticles

${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanoparticles were prepared following a precipitation process previously reported with some modifications.⁵⁶ ${\mathrm{ZrOCl}}_2·8{\mathrm{H}}_2\mathrm{O}$ and ${\mathrm{YbCl}}_3·6{\mathrm{H}}_2\mathrm{O}$ (99.9%) were purchased from Aldrich, and ${\mathrm{ErCl}}_3·6{\mathrm{H}}_2\mathrm{O}$ (99.99%) was acquired from RE Acton. Ammonium hydroxide (${\mathrm{NH}}_4\mathrm{OH}$) at 30 vol% was supplied by Karal. In a typical experiment, ${\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ co-doped ${\mathrm{ZrO}}_2$ with a molar ratio of $2:1$ for ${\mathrm{Yb}}^{3+}:{\mathrm{Er}}^{3+}$ was prepared by dissolving 2.6333 g of ${\mathrm{ZrOCl}}_2$, 0.2362 g of ${\mathrm{YbCl}}_3·6{\mathrm{H}}_2\mathrm{O}$ (2 mol % of ${\mathrm{Yb}}_2{\mathrm{O}}_3$), and 0.1351 g of ${\mathrm{ErCl}}_3·6{\mathrm{H}}_2\mathrm{O}$ (1 mol % of ${\mathrm{Er}}_2{\mathrm{O}}_3$) in 50 ml of a mixture of $H_{2}O/EtOH$ ($1:1$ wt%). After 15 min under stirring, the nonionic surfactant Pluronic F127 was introduced in the mixture at a molar ratio of $\mathrm{F}127/{\mathrm{ZrO}}_2=0.0082$. Afterward, 30 ml of ${\mathrm{NH}}_4\mathrm{OH}$ was added to precipitate the salts. The resulted suspensions were transferred into a sealed autoclave and a hydrothermal treatment was carried out at 80°C for 12 h. After this, the autoclave was allowed to cool down for 30 min and the solutions were washed twice with absolute ethanol and water in a centrifuge at 4000 rpm for 10 min. Subsequently, the powders were put in a ceramic crucible and dried at 80°C for 12 h. Finally, all samples were annealed at 1000°C using a heating rate of $5°\mathrm{C}/\min$.

2.2.

Conjugation and Functionalization of ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ Nanoparticles

The conjugation of ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanoparticles with the Ki-67 protein was carried out by following a previously reported method with some modifications.¹⁵ This process was performed as follows: 0.1 g of ${\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ doped ${\mathrm{ZrO}}_2$ nanoparticles were stirred with $490\mu \mathrm{l}$ of APTES for 24 hours. This bifunctional compound has amine- and alkoxysilane groups. The alkoxysilane reacts with the OH moieties on the ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanoparticles surface, leaving the amino groups exposed for further functionalization. The samples were washed once with ethanol and water to eliminate the excess of residues and centrifuged at 6000 rpm for 10 min. The samples were dried at 40°C for 12 h. The ${\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ co-doped ${\mathrm{ZrO}}_2$ nanoparticles coated with APTES were dispersed in $670\mu \mathrm{l}$ of phosphate buffered saline (PBS) $1\times$ ($\mathrm{pH}=7.4$) and then $200\mu \mathrm{l}$ of $1:500$ Biotin-Anti-rabbit (mouse IgG) from BIOCARE was added to the suspension to bind the carboxylic acid groups of the IgG with the amino groups exposed in the nanoparticles; this suspension was kept at 4°C for 12 h. After that, the nanoparticles were washed with distilled water and centrifuged at 6000 rpm for 10 min to remove the supernatant. The conjugated material was kept at 37°C for 12 h. Subsequently, $300\mu \mathrm{l}$ of PBS $1\times$ and $10\mu \mathrm{l}$ of antigen Ki-67-rabbit antibody from BIOCARE were added to the nanoparticles and stored for another 12 h at 4°C. Finally, the conjugated ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanoparticles were washed with distilled water and centrifuged at 6000 rpm for 10 min. The final material was dispersed and stored in distilled water.

2.3.

Structural Characterization (X-Ray Diffraction, Raman, HRTEM, SEM, Fourier Transform Infrared)

X-ray diffraction (XRD) patterns were obtained using a SIEMENS D-5005 equipment using a Cu tube with $\mathrm{K}\alpha$ radiation at 1.5405 Å, scanning in the 20 to 80 deg $2\theta$ range with increments of 0.02 deg and a sweep time of 2 s. Raman patterns were obtained using a Renishaw Raman System (inVia Raman Microscope), which uses a 785-nm laser and a $50\times$ objective. The nanoparticles were suspended in isopropyl alcohol at room temperature and dispersed with ultrasonication. Afterward, the solution of nanoparticles was dropped on 3-mm-diameter lacey carbon copper grids to obtain the HRTEM images in an FEI Titan 80-300 with an accelerating voltage set to 300 kV. In addition, the nanoparticle micrographs were obtained by an SEM Hitachi SU8010 at 30.0 kV. The Fourier transform infrared (FTIR) spectra were obtained using a Perkin-Elmer spectrophotometer with a deuterated triglycine sulfate detector and a spectral resolution of $4{\mathrm{cm}}^{-1}$. The samples were prepared using the KBr pellet method and the spectra were obtained in the range of 1000 to $4000{\mathrm{cm}}^{-1}$.

2.4.

Photoluminescence Characterization

Photoluminescence characterization was performed using a continuous wave semiconductor laser diode with an excitation power of 350 mW and centered at 970 nm. The luminescence emission was analyzed with a Spectrograph Spectra Pro 2300i and a R955 photomultiplier tube from Hamamatsu. The system was PC controlled with Spectra Sense software. The samples were supported in 1 mm capillary tubes in order to guarantee the same quantity of excited material. Special care was taken to maintain the alignment of the setup in order to compare the intensities between different characterized samples. All measurements were performed at room temperature.

2.5.

Incubation and Confocal Microscopy

HeLa cells were grown at a density of $5\times {10}^4\text{cells}/\mathrm{mL}$ in six-well culture plates with coverslips at the bottom and incubated in 3 mL of RPMI-1640 cell media for 24 h at 37°C under 5% ${\mathrm{CO}}_2$. After this, the cell media was replaced by 3 ml of ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanoparticles, ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}\text{-}\mathrm{APTES}$, and ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}\text{-}\mathrm{APTES}$-Biotin-Anti-rabbit/rabbit antibody-AntiKi-67 with a concentration of $100\mu \mathrm{g}/\mathrm{mL}$ and was incubated for 6 h. Finally, the cell-plated coverslips corresponding to each sample were washed twice with PBS buffer (1 mM, pH 7.4) and stained with nuclei-staining NucBlue® Live solution for 15 min. All the cell-plated coverslips were fixed with a solution of 4% formaldehyde. The fixed and stained coverslips were placed in microscope slides and analyzed under a two-photon Olympus FV1000 MPE SIM laser scanning confocal microscope.

2.6.

Zeta Potential and Dynamic Light Scattering Measurements

Dynamic light scattering (DLS) and zeta potential measurements were carried out using a Malvern Instrument Zetasizer Nano (red laser 633 nm). The samples were dispersed in PBS (1 mM, $\mathrm{pH}=7.4$) with a concentration of $1\mathrm{mg}/\mathrm{mL}$. The DLS and zeta potential were analyzed at 25°C.

3.1.

Crystalline Structure and Morphology

The XRD pattern of the ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanopowder is shown in Fig. 1(a). This plot shows peaks corresponding to (1,0,1), (0,1,1), (2,1,1), and (1,1,2) planes, respectively. All the peaks are associated with the tetragonal phase of zirconia, according to the JCPDS 37-1413 card.⁵⁷ The ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanopowder obtained by the precipitation method was analyzed by Raman spectroscopy, see Fig. 1(b). The peaks at 626, 552, 525, 445, 336, 260, 238, and $185{\mathrm{cm}}^{-1}$ represent the spectrum. The peaks located at 445 and $626{\mathrm{cm}}^{-1}$ as well as the shoulders located at 185 and $260{\mathrm{cm}}^{-1}$ are in agreement with the tetragonal phase of zirconia.⁵⁸ The nanocrystal sizes were determined by TEM and a representative micrograph is presented in Fig. 2(a). The nanocrystals have an average size of 20 nm and spherical shape. Besides, Fig. 2(b) is an SEM image, which shows well-dispersed nanocrystals, and this was caused by the introduction of PF127 during the synthesis process.⁵⁹ The size and dispersion of the co-doped ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanocrystals was controlled from the nucleation process due to the presence of ammonia, water/ethanol, and surfactant Pluronic PF127.⁵⁶ To promote the efficient internalization in HeLa cells, it is important to have particles in the nanoscale size regime. In addition, the colloidal stability of the nanoparticles is also significant to avoid the formation of aggregates, which may prevent the effective interaction between the nanoparticles and the cell surface.

Fig. 1

Structural characterization of ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanocrystals: (a) x-ray diffraction and (b) Raman spectroscopy, using a laser of 785 nm.

Fig. 2

(a) HRTEM image and (b) SEM image of ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanoparticles.

3.2.

FTIR, Zeta-Potential, and DLS

Figure 3 shows the FTIR spectra of ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanocrystals and ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ prepared with APTES, Biotin-Anti-rabbit (mouse IgG), and rabbit antibody-AntiKi-67, respectively. These spectra provide information regarding functional groups and impurities on the surface of nanoparticles. They also corroborated that the process of functionalization and conjugation was successfully achieved. Figure 3(a) shows the FTIR spectra of nonfunctionalized nanoparticles ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$. It depicts small peaks associated with OH groups in the range of 3200 to $3600{\mathrm{cm}}^{-1}$. Moreover, a peak is also observed at $450{\mathrm{cm}}^{-1}$, which is related to the $\mathrm{Zr}─\mathrm{O}$ stretching vibrations.⁶⁰ The spectrum in Fig. 3(b) shows a broadening of the bands centered at 3600 and $564{\mathrm{cm}}^{-1}$ due to the presence of $\mathrm{Si}─\mathrm{OH}$ and $\mathrm{Si}─\mathrm{O}─\mathrm{Si}$ bonds, respectively.^61,62 Other peaks at $\sim 2923$ and $2351{\mathrm{cm}}^{-1}$ are related to $\mathrm{C}─\mathrm{H}$ bonds and ${\mathrm{CO}}_2$ impurities, respectively. The band located in the range of 3000 to $3400{\mathrm{cm}}^{-1}$ is associated with amine groups.^63,64 The ${\mathrm{CO}}_2$ impurities adsorbed in the surface of the nanoparticles can come from the synthesis and/or the environment during the measurement process, which was probably caused by the granular characteristic of the nanopowder. The OH groups were introduced during the hydrolysis and condensation process where the M-OH ($\mathrm{M}=\mathrm{Zr}$, Er, and Yb) bond was formed due to the excess of hydroxyls in solution. According to the FTIR spectra in Figs. 3(a) and 3(b), the contamination produced by those hydroxyl groups is very low. Biotin-Anti-rabbit (mouse IgG) protein is conjugated to the nanoparticles containing APTES by forming an amide bond between the free amino groups located at the surface of ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}$ and the carboxylic acid groups exposed in the IgG protein. The ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}/\mathrm{Biotin}$ FTIR spectrum is shown in Fig. 3(c); the spectrum illustrates a new band associated with the amide bond at $1770{\mathrm{cm}}^{-1}$. Moreover, a peak centered at $658{\mathrm{cm}}^{-1}$ is also associated with Biotin according to literature.^65,66 These data further confirm the functionalization of nanoparticles. The next step is to analyze the process of conjugation with the antigen Ki-67-rabbit antibody (${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}/\mathrm{Biotin}/\mathrm{AntiKi}\text{-}67$), see Fig. 3(d). The bands related to Biotin are still observed, and there is a general decrease of the peaks related to impurities such as ${\mathrm{CO}}_2$ and OH radicals. However, it is observed that there is a widening of the $658{\mathrm{cm}}^{-1}$ band when AntiKi-67 is added. Based on this information, it is expected that AntiKi-67 is readily available to interact with HeLa cells.

Fig. 3

Fourier transform infrared spectra of (a) ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$, (b) ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$/(3-aminopropyl)triethoxysilane (APTES), (c) ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}/\mathrm{Biotin}$, and (d) ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}/\mathrm{Biotin}/\mathrm{AntiKi}67$.

The conjugation of the nanoparticles was also analyzed by zeta potential and DLS measurements, see Table 1. The zeta potential changed from negative to positive when the ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanoparticles surface is modified with APTES, which is an indication that the amino groups are covering the nanoparticles surface. Moreover, the zeta potential was shifted from positive to negative after Biotin and antigen Ki-67 proteins were chemically attached to the surface of the material, suggesting the presence of carboxylate groups.^67,68 A value of $-36\mathrm{mV}$ obtained in ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+/}\mathrm{APTES}/\mathrm{Biotin}/\mathrm{AntiKi}\text{-}67$ also indicates that nanoparticles can be stable in PBS due to their high electrostatic repulsion, which is suitable for bioapplications. A high negative value also suggests a high adsorption of nanoparticles on the nucleus of HeLa cells.⁶⁹ Moreover, DLS measurements showed that the hydrodynamic diameter of the nanoparticles increased when the different molecules were added; the average sizes for ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$, ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}$, and ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}/\mathrm{Biotin}/\mathrm{AntiKi}\text{-}67$ were 748, 1232, and 4694 nm, see Table 1. The size of the nanoparticles in ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ does not coincide with the one measured with TEM, probably due to the agglomeration of nanoparticles that were dispersed in PBS.

Table 1

Nanoparticle characterization using dynamic light scattering (DLS) and zeta potential for ZrO2:Yb3+-Er3+, ZrO2:Yb3+-Er3+-(3-aminopropyl)triethoxysilane (APTES), and ZrO2:Yb3+-Er3+-APTES-Biotin-AntiKi67 nanocrystals.

Figure 4 shows a schematic representation for the functionalization and conjugation of the nanoparticles. The OH moieties produced after ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ synthesis react with the alkoxysilane groups of APTES to afford a silica shell on the nanoparticles leaving the amine groups exposed on the surface of the material. In the next step of the reaction, the Biotin-Anti-rabbit (mouse IgG) molecule is conjugated to the amino groups by using the COOH moieties of the IgG. At this point, the Anti-rabbit can interact with the biomolecule AntiKi-67-rabbit antibody.

Fig. 4

Schematic representation of the functionalization and conjugation of ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanoparticles.

3.3.

Luminescent Properties

The mechanism of UPC emission in ${\mathrm{Er}}^{3+}\text{-}{\mathrm{Yb}}^{3+}$ co-doped ${\mathrm{ZrO}}_2$ is well established in the literature.⁷⁰ Figure 5 shows a strong red emission band with peaks at 653 and 657 nm as well as a weak green band after excitation at 970 nm. Green and red emission bands are assigned to ${{}^2\mathrm{H}}_{11/2}+{{}^4\mathrm{S}}_{3/2}\to {{}^4\mathrm{I}}_{15/2}$ and ${}^4{\mathrm{F}}_{9/2}\to {{}^4\mathrm{I}}_{15/2}$ transitions of the ${\mathrm{Er}}^{3+}$ ion, and they are caused by the successive absorption of two photons after energy transfer from ${\mathrm{Yb}}^{3+}$ ions.⁵⁹ According to previous works, the emission is predominantly red because OH groups have a vibrational energy (3000 to $4000{\mathrm{cm}}^{-1}$) which produces nonradiative relaxations from the mixed level ${{}^2\mathrm{H}}_{11/2}+{{}^4\mathrm{S}}_{3/2}$ toward the ${}^4{\mathrm{F}}_{9/2}$ level.⁷¹ In our case, the presence of OH moieties in all samples is corroborated by the FTIR spectra in Fig. 3. The inset in Fig. 5 shows that the integrated emission corresponds to the red band. Moreover, it is observed that the red emission of the samples ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}$ (Z-A) and ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}/\mathrm{Biotin}$ (Z-A-B) decreases progressively with respect to the sample of reference without conjugated (${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$) (Z). Nevertheless, the emission was improved when the nanoparticles were conjugated with AntiKi-67 (APTES-Biotin-AntiKi-67) (Z-A-B-K). The integrated red emission diminished with the addition of APTES and Biotin molecules because other contaminants, such as ${\mathrm{CO}}_2$, $\mathrm{C}─\mathrm{H}$, and amine groups, appeared and the presence of hydroxyls increased. These elements may act as quenching centers of luminescence and also create defects, which behave as traps for luminescence.^71,72 It is important to point out that the sample with APTES-Biotin had the highest levels of impurities [see Fig. 3(c)]; therefore, it showed the lowest luminescence. In contrast, the sample with APTES-Biotin-AntiKi-67 had the lowest amount of contaminants (OH, ${\mathrm{CO}}_2$, and $\mathrm{C}─\mathrm{H}$) [see Fig. 3(d)]; therefore, it presented the highest red emission, see Fig. 5.

Fig. 5

Photoluminescence spectra of ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$, ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}$, ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}/\mathrm{Biotin}$, and ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}/\mathrm{Biotin}/\mathrm{AntiKi}67$. Inset shows the integrated emission of the samples.

3.4.

Imaging of ${\mathrm{ZrO}}_2:\mathrm{Yb}\text{-}\mathrm{Er}$ Nanocrystals Incubated in HeLa Cells

Figure 6 shows the images obtained by the two-photon confocal microscope after HeLa cells were incubated with the different materials synthesized in this work. Figure 6(a) shows that the ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$ nanoparticles are situated out of the cell, probably due to the negative charge on the surface of the nanoparticles, which limits the internalization in HeLa cells. Figure 6(b) depicts ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}$ nanoparticles located on the cytoplasm of HeLa cells; these nanoparticles have no AntiKi-67, but they have APTES on their surface; this indicates that the positive charge on the surface of the nanoparticles enhances the internalization in HeLa cells. Figure 6(c) is an image of HeLa cells with nanoparticles conjugated with AntiKi-67; it is observed that 6 h of incubation is sufficient to reach the cytoplasm of HeLa cells. It is observed that there are a greater number of particles within the cell and near the nucleus. The most accepted theory is that nanoparticles are internalized via endosome-mediated transport or through ribosome exchanges.³³ In general, the red emission from nanoparticles is strong in all images, which demonstrates the efficient luminescence generated by the nanoparticles synthesized in this work. It is worth noting that there was no autofluorescence from the cells after exciting the UPC nanoparticles with 970 nm. Furthermore, these images denote different sizes of emission points; this is probably induced by the nanoparticle agglomeration.

Fig. 6

Confocal microscopy images of (a) ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}$, (b) ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}$, (c) ${\mathrm{ZrO}}_2:{\mathrm{Yb}}^{3+}\text{-}{\mathrm{Er}}^{3+}/\mathrm{APTES}/\mathrm{Biotin}/\mathrm{AntiKi}67$ nanoparticles after 6 h of incubation in HeLa cells.

Compared to other methods for the conjugation of nanoparticles, our technique avoids the use of other elements, such as carbon and citrate, which are relatively toxic.^32,34 Moreover, it uses biomolecules (antigen and antibody) to lead our nanoparticle toward a targeted organelle; to the best of our knowledge, this kind of molecule has not been used on luminescent nanoparticles. Finally, further research is needed not only to improve the distribution and internalization of nanoparticles, but also to label specific organelles inside the HeLa cells. Those studies are in progress, and they will be presented in a subsequent work.