Synthesis and In Vitro Testing of YVO4:Eu3+@silica-NH-GDA-IgG Bio-Nano Complexes for Labelling MCF-7 Breast Cancer Cells

We present a visual tool and facile method to detect MCF-7 breast cancer cells by using YVO4:Eu3+@silica-NH-GDA-IgG bio-nanocomplexes. To obtain these complexes, YVO4:Eu3+ nanoparticles with a uniform size of 10–25 nm have been prepared firstly by the hydrothermal process, followed by surface functionalization to be bio-compatible and conjugated with cancer cells. The YVO4:Eu3+@silica-NH-GDA-IgG nanoparticles exhibited an enhanced red emission at 618 nm under an excitation wavelength of 355 nm and were strongly coupled with MCF-7 breast cancer cells via biological conjugation. These bio-nanocomplexes showed a superior sensitiveness for MCF-7 cancer cell labelling with a detection percentage as high as 82%, while no HEK-293A healthy cells were probed under the same conditions of in vitro experiments. In addition, the detection percentage of MCF-7 breast cancer cells increased significantly via the functionalization and conjugation of YVO4:Eu3+ nanoparticles. The experimental results demonstrated that the YVO4:Eu3+@silica-NH-GDA-IgG bio-nanocomplexes can be used as a promising labelling agent for biomedical imaging and diagnostics.


Introduction
Nanostructured materials containing rare earth elements with numerous advantages such as high stability, strong luminescence, easy surface functionalization, and being friendly to environment and human body have been designed for new applications, especially for biomedical fluorescence labelling [1][2][3][4][5][6][7][8]. Most nanoparticles reported in past immunoassays are smaller than 200 nm in diameter for the biomolecular [9,10]. The size of these particles is proven to provide obvious prolonged equilibration time and enhanced nonspecific adsorption [11][12][13]. Therefore, the kinetic properties of smaller nanoparticles can be improved, and the nonspecific adsorption decreases. On the other hand, nanoparticles should be controlled to be big enough in size to bind with several proteins and cells on the surface, which is expected to increase the immunological affinity [14]. Among nanostructured materials containing rare-earth elements, YVO 4 :Eu 3+ nanomaterials have received a great deal of interest because of biologically appropriate emission in the visible region and biocompatibility. It is surveyed from the literature that yttrium(III) orthovanadate, YVO 4 , is one of the most commonly used host lattices containing rare earth ions to prepare efficient luminescent materials with different color emittings because of its high luminescence quantum yields of f-f transitions [15]. Accordingly, when being doped with Eu 3+ ions, YVO 4 :Eu 3+ nanomaterials with high quantum efficiency have strong fluorescence emission at 618 nm. The luminescent 4f -4f transitions of europium(III) ion and the effective energy transfer from ligands to europium(III) ion lead to a strong emission of red light that makes them widely employed in detection, biomedical imaging, and luminescent labels [16,17].
The functionalization of nanostructured materials is a key step toward biomedical applications. The applications of nanostructured materials require preliminary grafting at the nanophosphor's surface by organic or bio-organic functional groups. Different approaches are used such as encapsulation with functional polymers or direct grafting of biofunctional ligands. The 3-aminopropyl triethoxysilane (APTES) solution is well-known as a functionalized bio-compatible agent, because its ligands can create ethoxy groups on an inorganic surface. Meanwhile, glutaraldehyde (GDA) is one of the most popular bis-aldehyde homobifunctional crosslinkers that can be incorporated into nanostructured materials containing rare earth elements for many biomedical applications, including labelling, biosensing, drug delivery, and other therapies [18][19][20]. However, the structure of GDA is complicated, and its reaction mechanism is not fully understood. The reactions of GDA on proteins and other amine-containing molecules through the formation of a Schiff base that is lacking and still in progress [21]. In this work, we reported the results of the synthesis and in vitro testing of YVO 4 :Eu 3+ @silica-NH-GDA-IgG bio-nanocomplexes for labelling cancer cells. Figure 1a,b show the scanning electron microscopy (SEM) images of the YVO 4 :Eu 3+ and YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles, respectively. It indicated that YVO 4 :Eu 3+ nanomaterials were nearly spherical with diameters ranging from 10 to 20 nm (Figure 1a). After being covered with silica and functionalized with APTES and IgG, the spherical nanoparticles with diameters increasing up to 20-25 nm were observed ( Figure 1b). yttrium(III) orthovanadate, YVO4, is one of the most commonly used host lattices containing rare earth ions to prepare efficient luminescent materials with different color emittings because of its high luminescence quantum yields of f−f transitions [15]. Accordingly, when being doped with Eu 3+ ions, YVO4:Eu 3+ nanomaterials with high quantum efficiency have strong fluorescence emission at 618 nm. The luminescent 4f−4f transitions of europium(III) ion and the effective energy transfer from ligands to europium(III) ion lead to a strong emission of red light that makes them widely employed in detection, biomedical imaging, and luminescent labels [16,17].

Morphological Characterization
The functionalization of nanostructured materials is a key step toward biomedical applications. The applications of nanostructured materials require preliminary grafting at the nanophosphor's surface by organic or bio-organic functional groups. Different approaches are used such as encapsulation with functional polymers or direct grafting of biofunctional ligands. The 3-aminopropyl triethoxysilane (APTES) solution is well-known as a functionalized bio-compatible agent, because its ligands can create ethoxy groups on an inorganic surface. Meanwhile, glutaraldehyde (GDA) is one of the most popular bis-aldehyde homobifunctional crosslinkers that can be incorporated into nanostructured materials containing rare earth elements for many biomedical applications, including labelling, biosensing, drug delivery, and other therapies [18][19][20]. However, the structure of GDA is complicated, and its reaction mechanism is not fully understood. The reactions of GDA on proteins and other amine-containing molecules through the formation of a Schiff base that is lacking and still in progress [21]. In this work, we reported the results of the synthesis and in vitro testing of YVO4:Eu 3+ @silica-NH-GDA-IgG bio-nanocomplexes for labelling cancer cells. Figure 1a,b show the scanning electron microscopy (SEM) images of the YVO4:Eu 3+ and YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles, respectively. It indicated that YVO4:Eu 3+ nanomaterials were nearly spherical with diameters ranging from 10 to 20 nm ( Figure 1a). After being covered with silica and functionalized with APTES and IgG, the spherical nanoparticles with diameters increasing up to 20-25 nm were observed ( Figure  1b).  Figure 2a shows the X-ray diffraction (XRD) pattern of the YVO4:Eu 3+ sample. One can realize that all diffraction peaks of the sample coincided well with the standard data of the tetragonal-phase YVO4:Eu 3+ structure (JCPDS No. 17-0341). Furthermore, no impurity peaks were detected, indicating that the dopant Eu 3+ ions were well inserted into the host lattice of YVO4.  Figure 2a shows the X-ray diffraction (XRD) pattern of the YVO 4 :Eu 3+ sample. One can realize that all diffraction peaks of the sample coincided well with the standard data of the tetragonal-phase YVO 4 :Eu 3+ structure (JCPDS No. 17-0341). Furthermore, no impurity peaks were detected, indicating that the dopant Eu 3+ ions were well inserted into the host lattice of YVO 4 . nanocomplex [20]. Compared to the case of the unconjugated YVO4:Eu 3+ sample ( Figure  2c) in addition to the strong peaks belonging to Y, V, and O elements, there were additionally weaker characteristic peaks of Eu and other peaks of Si, N, and C in the energy-dispersive X-ray (EDX) spectrum of the YVO4:Eu 3+ @silica-NH-GDA-IgG sample ( Figure 2d). It confirmed that the silica shell and the amine group were successfully coated on the surface of the YVO4:Eu 3+ core.  Figure 3a shows the photoluminescence (PL) spectra of the YVO4:Eu 3+ , YVO4:Eu 3+ @silica−NH2, and YVO4:Eu 3+ @silica-NH-GDA-IgG samples at a 355 nm excitation wavelength. The PL spectra consisted of a narrow band corresponding to the well-known Eu 3+ emission from intra 4f transitions ( 5 D0− 7 F1, 5 D0− 7 F2, 5 D0− 7 F3, and 5 D0− 7 F4). The strongest emission peaks were yielded by the 5 D0− 7 F2 transition at around 618 nm (red light). Specially, the positions of PL peaks of the YVO4:Eu 3+ @silica-NH-GDA-IgG The characteristic chemical bonds of the YVO 4 :Eu 3+ and YVO 4 :Eu 3+ @silica-NH-GDA-IgG samples were analyzed via Fourier-transform infrared (FTIR) spectra as shown in Figure 2b. For both cases, the observed absorption peaks at low frequencies of vibration such as at 644 and 790 cm −1 corresponded to the characteristic Y-O and Eu-O bonds, respectively. We also observed oscillations of the O-H bond at around 1638 and 3347 cm −1 [22]. In the case of YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles, the band around 2880 cm −1 was attributed to C-H stretching vibration of alkanes. The N-H stretching vibration and the O-H bond were around 3347 cm −1 . The characteristic band of the Si-O∓R bond was observed at around 1011 cm −1 . Furthermore, an intense peak at 2360 cm −1 was associated with C=N stretching vibration that was formed by the reaction between the glutaraldehyde with amine-NH 2 groups of functionalized YVO 4 :Eu 3+ @silica-NH-GDA-IgG antibodies. It indicated that the conjugation between luminescent nanoparticles and IgG was formed in the YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanocomplex [20]. Compared to the case of the unconjugated YVO 4 :Eu 3+ sample ( Figure 2c) in addition to the strong peaks belonging to Y, V, and O elements, there were additionally weaker characteristic peaks of Eu and other peaks of Si, N, and C in the energy-dispersive X-ray (EDX) spectrum of the YVO 4 :Eu 3+ @silica-NH-GDA-IgG sample ( Figure 2d). It confirmed that the silica shell and the amine group were successfully coated on the surface of the YVO 4 :Eu 3+ core. Figure 3a shows the photoluminescence (PL) spectra of the YVO 4 :Eu 3+ , YVO 4 :Eu 3+ @silica-NH 2 , and YVO 4 :Eu 3+ @silica-NH-GDA-IgG samples at a 355 nm excitation wavelength. The PL spectra consisted of a narrow band corresponding to the well-known Eu 3+ emission from intra 4f transitions ( 5 D 0 -7 F 1 , 5 D 0 -7 F 2 , 5 D 0 -7 F 3 , and 5 D 0 -7 F 4 ). The strongest emission peaks were yielded by the 5 D 0 -7 F 2 transition at around 618 nm (red light). Specially, the positions of PL peaks of the YVO 4 :Eu 3+ @silica-NH-GDA-IgG sample were almost the same as those of the YVO 4 :Eu 3+ sample and the YVO 4 :Eu 3+ @silica-NH 2 sample. It implies that the PL property of the YVO 4 :Eu 3+ @silica-NH-GDA-IgG sample was unchanged after function-alization and biological conjugation. Moreover, the PL intensity of the YVO 4 :Eu 3+ @silica-NH-GDA-IgG sample was enhanced. This result can be explained by substitution of the O-H luminescence quenching with O=C and C-H in the case of surface modification. These fluorescence properties of samples containing YVO 4 :Eu 3+ nanoparticles have attracted a great deal of attention in biology and medicine. It can be indicated that the surface modification of the YVO 4 :Eu 3+ nanoparticles not only improved their bio-compatibility, but also increased their PL intensity. Furthermore, the YVO 4 :Eu 3+ @silica-NH-GDA-IgG sample remained stable for quite a long time. The PL intensity of the YVO 4 :Eu 3+ @silica-NH-GDA-IgG sample had insignificant changes after three and six months of the synthesis as shown in Figure 3b. Thus, these nanoparticles can be used for bio-labelling applications. sample were almost the same as those of the YVO4:Eu 3+ sample and the YVO4:Eu 3+ @silica−NH2 sample. It implies that the PL property of the YVO4:Eu 3+ @silica-NH-GDA-IgG sample was unchanged after functionalization and biological conjugation. Moreover, the PL intensity of the YVO4:Eu 3+ @silica-NH-GDA-IgG sample was enhanced. This result can be explained by substitution of the O−H luminescence quenching with O=C and C−H in the case of surface modification. These fluorescence properties of samples containing YVO4:Eu 3+ nanoparticles have attracted a great deal of attention in biology and medicine. It can be indicated that the surface modification of the YVO4:Eu 3+ nanoparticles not only improved their bio-compatibility, but also increased their PL intensity. Furthermore, the YVO4:Eu 3+ @silica-NH-GDA-IgG sample remained stable for quite a long time. The PL intensity of the YVO4:Eu 3+ @silica-NH-GDA-IgG sample had insignificant changes after three and six months of the synthesis as shown in Figure 3b. Thus, these nanoparticles can be used for bio-labelling applications.

In Vitro Cellular Imaging
We used fluorescence microscopy to evaluate the linking ability between YVO4:Eu 3+ @silica-NH-GDA-IgG conjugates and MCF-7 breast cancer cells after the incubation process. Figure 4a-c show the fluorescent images for three cases of MCF-7 breast cancer cells (negative control), incubated MCF-7 breast cancer cells with YVO4:Eu 3+ @silica−NH2 nanoparticles, and incubated MCF-7 breast cancer cells with YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles, respectively.

In Vitro Cellular Imaging
We used fluorescence microscopy to evaluate the linking ability between YVO 4 :Eu 3+ @silica-NH-GDA-IgG conjugates and MCF-7 breast cancer cells after the incubation process. Figure 4a-c show the fluorescent images for three cases of MCF-7 breast cancer cells (negative control), incubated MCF-7 breast cancer cells with YVO 4 :Eu 3+ @silica-NH 2 nanoparticles, and incubated MCF-7 breast cancer cells with YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles, respectively.
sample were almost the same as those of the YVO4:Eu 3+ sample and the YVO4:Eu 3+ @silica−NH2 sample. It implies that the PL property of the YVO4:Eu 3+ @silica-NH-GDA-IgG sample was unchanged after functionalization and biological conjugation. Moreover, the PL intensity of the YVO4:Eu 3+ @silica-NH-GDA-IgG sample was enhanced. This result can be explained by substitution of the O−H luminescence quenching with O=C and C−H in the case of surface modification. These fluorescence properties of samples containing YVO4:Eu 3+ nanoparticles have attracted a great deal of attention in biology and medicine. It can be indicated that the surface modification of the YVO4:Eu 3+ nanoparticles not only improved their bio-compatibility, but also increased their PL intensity. Furthermore, the YVO4:Eu 3+ @silica-NH-GDA-IgG sample remained stable for quite a long time. The PL intensity of the YVO4:Eu 3+ @silica-NH-GDA-IgG sample had insignificant changes after three and six months of the synthesis as shown in Figure 3b. Thus, these nanoparticles can be used for bio-labelling applications.

In Vitro Cellular Imaging
We used fluorescence microscopy to evaluate the linking ability between YVO4:Eu 3+ @silica-NH-GDA-IgG conjugates and MCF-7 breast cancer cells after the incubation process. Figure 4a-c show the fluorescent images for three cases of MCF-7 breast cancer cells (negative control), incubated MCF-7 breast cancer cells with YVO4:Eu 3+ @silica−NH2 nanoparticles, and incubated MCF-7 breast cancer cells with YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles, respectively.  For the first case, we did not observe PL emission in the reference sample (Figure 4a). In the second case, the incubated MCF-7 breast cancer cells with the YVO 4 :Eu 3+ @silica-NH 2 sample showed a blur and tiny PL intensity (Figure 4b). This can be explained by the existence of the weak bonds between YVO 4 :Eu 3+ @silica-NH 2 nanoparticles and the cancer cells. As reported, muscarinic acetylcholine receptors (mAChR) belong to the Gprotein-coupled receptor family and are extensively expressed in human breast tumor cells. In addition, immunoglobulin G (IgG) has been described that the presence of IgG in tumor cells establishes correlations between high antibody levels and promotion of cancer cell proliferation, invasion, and poor clinical prognosis for tumor patients. Blocking tumor-cellderived IgG inhibits tumor cells. Tumor-cell-derived IgG might impede antigen-dependent cellular cytotoxicity by binding antigens such as mAChR while, at the same time, lacking the capacity for complement activation [23]. However, we can observe bright red pixels in the case of YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles in Figure 4c. This evidence demonstrated a strong coupling between YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles and MCF-7 breast cancer cells due to biological conjugation.
Furthermore, it can be seen that YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles were localized within the cell cytoplasm. The high GDA concentration allowed the ligand binding between cells and luminescent labelling particles. After that, the YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles were internalized into the cell via the invagination process. Therefore, YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles could be used as a potential bio-label for MCF-7 breast cancer cells. In comparison to the other techniques, the use of YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles is expected to be more advantageous by providing a facile method without requirements of any complex apparatus as well as processing such as spectral equipment and data analysis [24,25]. Additionally, it is a visual tool that is needed for some specific studies in biology.
In addition, the nanocomplex exhibited much fewer probing activities on HEK-293A human embryonic kidney cells, which were non-cancerous cell lines. Figure 5a-c show the fluorescence images of the HEK-293A cells with YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles with different detection modes: (a)-negative control, (b)-Dark field, (c)-Merge, respectively. The experimental conditions were the same as those for the MCF-7 breast cancer cells, and the images were taken in the cases of the bright field, dark field, and merged modes. Moreover, the YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles could not probe healthy cells of HEK-293A. 4a). In the second case, the incubated MCF-7 breast cancer cells with the YVO4:Eu 3+ @silica−NH2 sample showed a blur and tiny PL intensity (Figure 4b). This can be explained by the existence of the weak bonds between YVO4:Eu 3+ @silica−NH2 nanoparticles and the cancer cells. As reported, muscarinic acetylcholine receptors (mAChR) belong to the G-protein-coupled receptor family and are extensively expressed in human breast tumor cells. In addition, immunoglobulin G (IgG) has been described that the presence of IgG in tumor cells establishes correlations between high antibody levels and promotion of cancer cell proliferation, invasion, and poor clinical prognosis for tumor patients. Blocking tumor-cell-derived IgG inhibits tumor cells. Tumor-cell-derived IgG might impede antigen-dependent cellular cytotoxicity by binding antigens such as mAChR while, at the same time, lacking the capacity for complement activation [23]. However, we can observe bright red pixels in the case of YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles in Figure 4c. This evidence demonstrated a strong coupling between YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles and MCF-7 breast cancer cells due to biological conjugation.
Furthermore, it can be seen that YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles were localized within the cell cytoplasm. The high GDA concentration allowed the ligand binding between cells and luminescent labelling particles. After that, the YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles were internalized into the cell via the invagination process. Therefore, YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles could be used as a potential bio-label for MCF-7 breast cancer cells. In comparison to the other techniques, the use of YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles is expected to be more advantageous by providing a facile method without requirements of any complex apparatus as well as processing such as spectral equipment and data analysis [24,25]. Additionally, it is a visual tool that is needed for some specific studies in biology.
In addition, the nanocomplex exhibited much fewer probing activities on HEK-293A human embryonic kidney cells, which were non-cancerous cell lines. Figure 5a-c show the fluorescence images of the HEK-293A cells with YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles with different detection modes: (a)-negative control, (b)-Dark field, (c)-Merge, respectively. The experimental conditions were the same as those for the MCF-7 breast cancer cells, and the images were taken in the cases of the bright field, dark field, and merged modes. Moreover, the YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles could not probe healthy cells of HEK-293A. The flowcytometry results also provided the percentage of probed cells using the YVO4:Eu 3+ @silica-NH-GDA-IgG nanocomplex. As shown in Figure  6, YVO4:Eu 3+ @silica-NH-GDA-IgG nanocomplexes were found in about 82.11% of MCF-7 cells when stained with YVO4:Eu 3+ @silica-NH-GDA-IgG nanocomplexes. A similar result The flowcytometry results also provided the percentage of probed cells using the YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanocomplex. As shown in Figure 6, YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanocomplexes were found in about 82.11% of MCF-7 cells when stained with YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanocomplexes. A similar result (1.55%) was found in MCF-7 cells that were incubated with YVO 4 :Eu 3+ @silica-NH 2 nanoparticles. The percentage of MCF-7 cells was only 0.57% for MCF-7 cells incubated with unconjugated YVO 4 :Eu 3+ nanoparticles. Thus, the detection percentage of MCF-7 breast cancer cells increased pronouncedly from 0.57% for the case of unconjugated YVO 4 :Eu 3+ nanoparticles to 1.55% for the case of YVO 4 :Eu 3+ @silica-NH 2 nanoparticles and achieved a highest value of 82.11% with YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles, indicating the crucial role of the functionalization and conjugation of YVO 4 :Eu 3+ nanoparticles.
noparticles. The percentage of MCF-7 cells was only 0.57% for MCF-7 cells incubated with unconjugated YVO4:Eu 3+ nanoparticles. Thus, the detection percentage of MCF-7 breast cancer cells increased pronouncedly from 0.57% for the case of unconjugated YVO4:Eu 3+ nanoparticles to 1.55% for the case of YVO4:Eu 3+ @silica−NH2 nanoparticles and achieved a highest value of 82.11% with YVO4:Eu 3+ @silica-NH-GDA-IgG nanoparticles, indicating the crucial role of the functionalization and conjugation of YVO4:Eu 3+ nanoparticles.
By using APTES, a reagent contains a short organic 3-amino propyl group, which terminates in a primary amine, and ethoxy groups are not reactive enough to couple spontaneously with OH groups on an inorganic surface without prior hydrolysis to make silanol. This protocol can be used to modify the surface of particles with this reagent as described in Figure 7. Firstly, the reaction involves the hydrolysis of the alkoxysilane group to create highly reactive silanol that undergoes hydrogen bonding with other silanol groups in solutions and on the particle surface, resulting in the associated organosilane derivatives. Then, a condensation reaction takes place to form a polymerized coating of the organosilane on the particle surface [21]. Secondly, APTES is coated on the YVO4:Eu 3+ @silica surface to create a covalent shell containing a primary amine group. The reaction occurs in a partially aqueous environment, because ethoxy groups are unreactive enough to substrate OH groups without prior hydrolysis. This is typically performed in 5% water in ethanol that is acidified with acetic acid to pH values of 4.5-5.5. The pro-
By using APTES, a reagent contains a short organic 3-amino propyl group, which terminates in a primary amine, and ethoxy groups are not reactive enough to couple spontaneously with OH groups on an inorganic surface without prior hydrolysis to make silanol. This protocol can be used to modify the surface of particles with this reagent as described in Figure 7. Firstly, the reaction involves the hydrolysis of the alkoxysilane group to create highly reactive silanol that undergoes hydrogen bonding with other silanol groups in solutions and on the particle surface, resulting in the associated organosilane derivatives. Then, a condensation reaction takes place to form a polymerized coating of the organosilane on the particle surface [21]. Secondly, APTES is coated on the YVO 4 :Eu 3+ @silica surface to create a covalent shell containing a primary amine group. The reaction occurs in a partially aqueous environment, because ethoxy groups are unreactive enough to substrate OH groups without prior hydrolysis. This is typically performed in 5% water in ethanol that is acidified with acetic acid to pH values of 4.5-5.5. The process results in a layer containing about 3-8 organosilanes in thickness and masks the inorganic substrate with aminopropyl groups. The advantage of this process is providing a thin and controllable silane layer that can be created a monolayer of the aminopropyl group on the surface. 4 :Eu 3+ @silica-NH-GDA-IgG Bio-Nanocomplexes  The YVO 4 :Eu 3+ @silica-NH-GDA-IgG products were collected by centrifugation (5900 rpm) with water for three times and stored at 4 • C in a closing box. The reaction mechanism can be explained via a Schiff base linkage with amine on proteins [21]. Proteins can be coupled to the -NH 2 groups via an amine-reactive linker-glutaraldehyde-to form activated derivatives that enable to make crosslink with other proteins. As shown in Figure 8, the amine groups react with the aldehyde groups to form a Schiff base, resulting in a polymeric coating that contains both aldehydes and double bonds for further coupling with amine-containing molecules. cess results in a layer containing about 3-8 organosilanes in thickness and masks the inorganic substrate with aminopropyl groups. The advantage of this process is providing a thin and controllable silane layer that can be created a monolayer of the aminopropyl group on the surface.  Figure 8 describes the functionalization of the YVO4:Eu 3+ @silica−NH nanoparticles with glutaraldehyde (GDA) and immunoglobulin G (IgG). The YVO4:Eu 3+ @silica−NH2 nanoparticles and GDA were dispersed in vanadate-buffered saline (PBS, 0.1 M, pH 5) with a concentration of 5 gL −1 . Then, this compound was added to different concentrations of IgG. These reaction mixtures were incubated with glycerol at room temperature for 4 h. The YVO4:Eu 3+ @silica-NH-GDA-IgG products were collected by centrifugation (5900 rpm) with water for three times and stored at 4 °C in a closing box. The reaction mechanism can be explained via a Schiff base linkage with amine on proteins [21]. Proteins can be coupled to the −NH2 groups via an amine-reactive linker-glutaraldehyde-to form activated derivatives that enable to make crosslink with other proteins. As shown in Figure 8, the amine groups react with the aldehyde groups to form a Schiff base, resulting in a polymeric coating that contains both aldehydes and double bonds for further coupling with amine-containing molecules.   HTB-22). The results were then compared with HEK-293A Figure 8. Functionalization of the YVO 4 :Eu 3+ @silica-NH nanoparticles with GDA and IgG. This conjugation strategy has been used to associate biomolecules containing amine groups with aminated YVO 4 :Eu 3+ @silica-NH 2 nanomaterials, usually utilizing glutaraldehyde, a molecule containing two aldehyde moieties. One aldehyde group forms a Schiff base with the amine groups, while the other binds to the amino groups of the biomolecules. The reactions with glutaraldehyde are favored in alkaline media, being more efficient at high pH values [21].

MCF-7 Breast Cancer Cell and HEK-293A Cell Culture and Fluorescence Imaging of Cells
In this study, the experiments were implemented on MCF-7 breast cancer cells (MCF-7 was kindly presented by Prof. Chi-Ying Huang, National Yangming University, Taiwan-MCF7 (ATCC # HTB-22). The results were then compared with HEK-293A healthy cells (these cells were kindly provided from Prof. Young-Pil Kim, HanYang University, Korea-HEK-293A (Invitrogen # P/N 51-0036)) that were maintained in a cultured medium-Dulbecco's Modified Eagle Medium (DMEM) with fetal bovine serum (10%) (Sigma) and gentamicin (50 µg/mL) at 37 • C and 5% CO 2 in a humidified atmosphere [29,30]. The cells were seeded at a density of 5.10 4 cells/mL. To study the uptake capacity of the YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles, the MCF-7 breast cancer cells and HEK-293A cells (10 6 cells/mL) at the log phase were seeded in 24-well plates and then incubated for 24 h. The YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles (with a concentration of 20 µg/mL) were then added to the cell-seeded wells for 3 h. After the assigned time, the cultured medium was discarded. The cells were then washed three times with phosphatebuffered saline. At the end of the process, phosphate-buffered saline was added to the wells, before and after MCF-7 breast cancer cells and HEK-293A cells were incubated with YVO 4 :Eu 3+ @silica-NH-GDA-IgG bio-nanocomplexes. The cell images were obtained using an Olympus ScanR 100X fluorescent microscope.

Cellular Surface Labelling Analysis Using Flowcytometry
MCF-7 cancer cells, a cultured medium (DMEM), a fetal bovine serum (FBS), trypsin-EDTA, and bovine insulin were obtained from Invitrogen (Carlsbad, CA, USA). MCF-7 cells (5 × 10 4 cells/mL) were cultured in 6-well plates at 37 • C and 5% CO 2 for 24 h. Then, the cells were detached with 0.05% trypsin-EDTA and centrifuged at 1000 rpm for 5 min to obtain the cell pellets. The cells were fixed with 4% formaldehyde for 24 h at 4 • C and washed with cold phosphate-buffered saline (PBS). To access cellular surface labeling, the YVO 4 :Eu 3+ -NH 2 and YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles were employed and incubated with fixed cells for 2 h. The labelled cells were washed with cold PBS twice before resuspending in PBS for analyzing with a flowcytometry Novocyte system (ACEA Bioscience inc.) and NovoExpress software. The cells were requested for light protection.

Characterization Techniques
The X-ray diffraction (XRD) analysis of the samples was carried out on a Siemens D5000 diffractometer with λ = 1.5406 Å. The morphologies and the energy-dispersive X-ray spectra were observed and measured by field emission scanning electron microscopy (S-4800; Hitachi) attached with an energy-dispersive X-ray spectrometer. The infrared absorption spectra were performed by employing a Fourier-transform infrared spectrometer (FTIR NEXUS 670). The photoluminescence (PL) spectra of the samples were studied by using an iHR550 photoluminescence system (Horiba). The cells were observed under an Olympus ScanR fluorescence microscope (Olympus Europa SE & Co.KG, Hamburg, DE).

Conclusions
YVO 4 :Eu 3+ nanoparticles with a uniform size of 10-25 nm were synthesized via a hydrothermal process, followed by further functionalizations to form the YVO 4 :Eu 3+ @silica-NH-GDA-IgG bio-nanocomplexes. The surface modification of the YVO 4 :Eu 3+ nanoparticles not only improved the bio-compatible media, but also increased their PL intensity due to enhancement of chemical stability. The experimental evidence indicated that the YVO 4 :Eu 3+ @silica-NH-GDA-IgG nanoparticles could selectively detect MCF-7 breast cancer cells while it could not probe HEK-293A healthy cells for in vitro tests. The YVO 4 :Eu 3+ @silica-NH-GDA-IgG bio-nanocomplexes with a significant bio-compatible capability exhibited a strong, enhanced red emission, which provides a visual tool for bio-labelling applications.