FOS NBs were synthesized via a single-step, room-temperature method under an air atmosphere using aminopropyl silane as the organosilane source in the presence of L-AA and DI water. First, L-AA was dispersed in DI water, after which the brownish organosilane was added to the reaction solution to induce the formation of the FOS NBs. After the addition of the organosilane precursors, the color of the solution turned into a reddish color after 5 min. Ten minutes after the reaction initiation, the FOS NBs were collected via removing supernatant and adding extra ethanol into the reactors followed by centrifugation to obtain FOS NBs. Figure 1 shows the SEM images of the FOS NBs synthesized using APTES precursors. As shown in Fig. 1a, b, the FOS NBs exhibit spherical morphologies. The TEM images also reveal that the FOS NBs consist of amorphous structures without crystalline fringe patterns. Figure 1c shows the size histogram of the FOS NBs obtained via dynamic light scattering (DLS) measurement. The average size of the FOS NBs was approximately 426.8 nm with a narrow size distribution. In addition, although the FOS NBs were synthesized using different types of organosilane monomers (Additional file 1: Fig. S1), the type of organosilane monomer used for the reaction does not significantly influence on the size of the FOS NBs; however, the polydispersity index of the FOS NBs synthesized with APTES was smaller than those synthesized with APTMS. In addition, the size and size distribution were not significantly varied by changing the reaction temperature (25 °C/40 °C) and reaction time (20 min/24 h/48 h). Furthermore, although the surface of SiO2 NBs is negatively charged owing to the silanol group on the surface, ζ-potential measurements revealed the presence of positive charges on the surface of the FOS NBs (Fig. 1d). The positive charges on the surface of the FOS NBs could be attributed to the presence of an amine group in the organosilane monomers, which formed positively charged ammonium moiety on the surface of the FOS NBs. The ammonium groups on the surfaces of the FOS NBs enabled the colloidal stability of the FOS NBs in DI water owing to the electrostatic interaction between the ammonium groups. Therefore, the as-synthesized FOS NBs exhibited excellent colloidal stability without precipitation in DI water.
The chemical structure of the FOS NBs was characterized using FTIR measurements. Figure 2a shows the FTIR spectra of the FOS NBs. A sharp and strong peak was observed at 1031 cm−1, which is attributed to the characteristic Si–O stretching. In addition, a sharp absorbance peak was observed at 1652 cm−1, which corresponded to the asymmetric –NH3+ deformation mode [35], indicating the presence of amine group in the NBs. Furthermore, two additional peaks were observed at 1516 cm−1 (N–H bending) and 3340 cm−1 (N–H stretching), indicating the presence of –NH2 groups in the FOS NBs. The absorbance peaks from 690 to 755 cm−1 indicated that free water was adsorbed on the surface of the NBs via hydrogen bonding [36]. Furthermore, the peak observed at 2931 cm−1 corresponded to the C–H vibration of aminopropyl groups. In addition, the shoulder was observed at 919 cm−1, which corresponded to the existence of silanol groups (Si–OH) in the FOS NBs. These results indicate that the FOS NBs mainly consisted of aminopropyl silane groups. In addition, a broad peak with a Bragg angle at 2θ = 21.7° was observed in the XRD pattern, confirming the amorphous structure of the FOS NBs (Fig. 2b).
For the elemental analysis of the as-synthesized FOS NBs, XPS measurement was performed on the FOS NBs. Five peaks were observed in the full-range XPS spectra (Additional file 1: Fig. S2) of the FOS NBs at 102.5, 152.7, 285.9, 400.4, and 532.4 eV, which were attributed to Si 2p, Si 2s, C 1s, N 1s, and O 1s, respectively. In addition, the high-resolution XPS spectrum of Si 2p (Fig. 2c) revealed that the silicon existed in two different chemical environments originating from Si–O (102.3 eV) and Si–C (102.2 eV) [37], which corresponds to the aminopropyl silane group. The three fitted peaks at 533.4, 532.2, and 530.2 eV in the O 1s spectrum (Fig. 2d) were assigned to Si–O, Si–OH, and O2− groups, respectively. In addition, four peaks were observed in the C 1s spectrum (Fig. 2e) at 287.7, 285.5, 284.6, and 283.6 eV, which could be attributed to C=O, C–N/C–O, C–C/C=C, and C–Si [30], respectively, indicating the presence of aminopropyl group in the FOS NBs. Furthermore, the peaks at 400.81 and 399.1 eV in the N 1s spectrum (Fig. 2f) could be attributed to the presence of –NH3+ and –NH2 [38], indicating the presence of positively charged ammonium functional group in the FOS NBs, which is consistent with the corresponding FTIR spectrum. The structural analysis of the FOS NBs by FTIR, XRD, and XPS confirmed that the as-synthesized FOS NBs were amorphous organosilica with positively charged ammonium functional groups on their surfaces.
The formation of FOS NBs occurred via the acid-catalyzed reaction of silane monomers in the silane/acid/DI water ternary phase [39, 40]. Synthesis of spherical SiO2 NBs with a diameter range of hundreds of nanometers to micrometers has been reported for various types of organic (e.g., acetic, tartaric) or inorganic (hydrochloric, nitric) acids. It is reported that the particle size was controlled by varying the relative molar ratio of the silane monomers and acids [41,42,43]. In our system, L-AA induces hydrolysis and condensation reactions of silane monomers to form FOS NBs with a diameter of several hundred nanometers. After a 10 min reaction time, the precipitated sols sank to the bottom of the flask, leaving a clear, reddish supernatant liquid. These precipitated sols may have been the heavy liquid phases of the reaction intermediates, for example, polysilicic acid [42]. There were no FOS NBs present in the supernatant after centrifugation, therefore, the supernatant was discarded, leaving the reaction precipitates in the reaction flask. After adding an excess amount of EtOH to the flask, the solution became cloudy, indicating the separation and dispersion of FOS NBs in EtOH.
L-AA contributed not only to the reaction of silane monomers but also to the incorporation of luminescent properties into the FOS NBs. Figure 3a shows the photograph of the colloidal dispersion of the FOS NBs under white light and UV irradiation of 365 nm. The FOS NBs formed a milky dispersion in DI water, which could be attributed to the scattering by several hundred nanosized NBs. In addition, the FOS NBs exhibited blue PL under UV irradiation without high-temperature calcination. Figure 3b shows the excitation and PL spectra of the FOS NBs. The maximum PL emission peak of the FOS NBs was observed at 485 nm (Fig. 3b) and the PL spectra was independent of the excitation wavelength (Additional file 1: Fig. S3). In addition, the PL spectra of the FOS NBs synthesized for 10 min, 12 h, and 24 h were measured. The PL and PL excitation (PLE) spectra of all the samples were almost identical properties of the FOS NBs (Additional file 1: Fig. S4). The absolute photoluminescence quantum yield (PLQY) of the FOS NBs measured using an integrating sphere was approximately 2.4%. The long-term stability of PL properties under storage was observed under ambient conditions. The FOS NBs dispersed in DI water were stored for 30 d in an atmospheric environment, and their PL properties were investigated. The results revealed that long-term storage did not significantly deteriorate the PL properties of FOS NBs (Additional file 1: Fig. S5).
Thus far, several studies have reported the synthesis of Si-based NPs by the reaction between amine-containing organosilanes and organic reductants, such as L-AA and citric acid in aqueous medium [32,33,34, 44, 45]. Early literature argues that the luminescence properties of these Si-based NBs could be attributed to the presence of reduced crystalline Si owing to the reduction of the siloxane monomers by the organic reductants. However, the structure and origin of the luminescent properties of the Si-based NPs synthesized in aqueous medium are still unclear because nano-sized Si is susceptible to oxidation in aqueous medium [46, 47]. In this study, we confirmed that the FOS NBs were composed of the organosilica group. Furthermore, no strong evidence of the presence of zerovalent Si in the NBs was observed as corroborated by the FTIR, XPS, and XRD results. In addition, recent studies have reported that the products formed via the reaction of alkylamine and organic reductants exhibit similar luminescence properties as Si-based NBs [46]. Therefore, the results of this study suggest that the silane groups were not reduced to zerovalent Si, but only acted as nano-sized organosilica templates, indicating that the luminescence properties of the FOS NBs may have originated from the reaction between amine group and L-AA. This could be related to the formation of luminescence carbon-containing particles, such as carbon dots [48,49,50,51]. Although previous studies have reported that luminescent carbon-containing particles are formed under high-temperature thermal decomposition conditions that induce carbonization, our result revealed that luminescent particles can also be formed under room-temperature conditions within a short reaction time (10 min).
In addition, several literatures have reported that the reaction between organosilane and organic reductants induces the formation of nanoparticles of less than tens of nanometers in size [32,33,34, 44, 45]. However, we observed that the reaction products were mainly NBs of several hundreds of nanometers in size. As previously discussed, the color of the reaction solution changed to a reddish color. After 10 min of the reaction, the solution retained its red color, as shown in Additional file 1: Fig. S6a; however, white precipitates were observed at the bottom of the reaction flask. Subsequently, the supernatants were selectively discarded, and extra ethanol was added to collect the white precipitates in the flask. After adding ethanol, the precipitates were redispersed in ethanol to form a milky solution (Additional file 1: Fig. S6b), which is the obtained colloidal dispersion of FOS NBs in ethanol. The PL properties of the supernatant and precipitates were measured under UV irradiation of 365 nm. The results revealed that the luminescence properties were mainly observed in the precipitates (Additional file 1: Fig. S7). This result confirmed that the PL properties, which originated from the carbon-based luminescent centers, is attributed to the organosilica NBs.
To investigate the potential utilization of the FOS NBs in bioimaging application, the cytotoxicity of the FOS NBs was investigated by CCK-8 assay (Fig. 4). The cytotoxicity of the FOS NBs was measured during 24 h and 48 h incubation periods using triton X-100 as the positive control. Triton X-100 exhibited complete toxicity to the hASCs in terms of viability loss. The FOS NBs were injected into the hASCs at concentrations of 50, 100, and 500 µg mL−1. At a concentration of 500 µg mL−1, the FOS NBs exhibited slight cytotoxicity toward the hASCs, which may be due to the presence of positively charged ammonium groups on their surface known to induce toxicity. This is accounted for more ability of positively charged NBs easily enter to cells due to the electrostatic attraction between the negatively charged cell membrane glycoproteins and positively charged NBs and being slightly toxic [52]. However, the cells preserved over 80% viability, demonstrating the good biocompatibility of the FOS NBs.
Finally, we investigated the potential of FOS NBs for cellular imaging applications owing to their biocompatibility and luminescence properties. The cellular uptake of FOS NBs into the hASCs was evaluated by examining the intrinsic fluorescence of the NPs by confocal laser scanning microscope (LSM). The hASCs were incubated with the FOS NBs (50 µg/mL) for 24 h, and cell nuclei were selectively stained with a nucleus-selective dye (DAPI) to located cell location. As shown in Fig. 5, the greenish emission is observed in label-free FOS NBs. This is attributed to the fact that FOS NBs exhibit the relatively broad emission from blue to green wavelength and blue emission from FOS NBs were removed from blue filter of confocal LSM. It is observed that FOS NPs were effectively internalized into cells and selectively distributed in the lysosomes around nuclei stained using DAPI. In addition, no fluorescence signal of FOS NBs was observed in the nuclei, indicating that the NPs could not penetrate the nuclear membrane. This result indicates the potential of the label-free FOS NBs for effective drug delivery applications in future studies.