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Functional Nanostructured Materials (including low-D carbon)

One-pot green synthesis of ultra-bright N-doped fluorescent silicon nanoparticles for cellular imaging by using ethylenediaminetetraacetic acid disodium salt as an effective reductant Xin Geng, Zhaohui Li, Yalei Hu, Haifang Liu, Yuan-Qiang Sun, Hongmin Meng, Yingwen Wang, Ling-Bo Qu, and Yuehe Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09242 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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ACS Applied Materials & Interfaces

ABSTRACT: Due to excellent photoluminescence properties, robust chemical inertness and low cytotoxicity of silicon nanoparticles (Si NPs), exploration of their applications in bioimaging is of great interest. Up to date, method to synthesis Si NPs with high fluorescence quantum yield (QY) is still challenging. This situation limits the further applications of Si NPs. In this work, we report a mildly, simple and green one-pot method to synthesis N-doped fluorescent Si NPs with an ultra-high QY up to 62%, using ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) as an effective reductant. The obtained ultra-bright Si NPs has properties such as relative small size (about 2 nm), water dispersibility, robust stability, and biocompatibility. The as-prepared Si NPs were further applied for cellular imaging with satisfactory results, indicating their great potential in bioimaging applications.

KEYWORDS: N-doped silicon nanoparticles; ethylenediaminetetraacetic acid disodium salt; high fluorescence quantum yield; robust stability; cellular imaging.

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1. INTRODUCTION As the second most abundant element on the earth, silicon has been widely used in a myriad of fields covering electronics, energy and biology, which is owing to its intrinsic merits such as high inert, low-cost, low/non-toxicity, and favorable biocompatibility.1 In the past two decades, various types of silicon nanostructures have been reported.2-6 Among

them,

the

zero-dimension

silicon

nanostructure,

fluorescence

silicon

nanoparticles (Si NPs) have received significantly growing attentions and further accepted as alternatives to traditional organic dyes as well as semiconductor quantum dots in bioimaging or biomedical research fields.7-12 Some methods for Si NPs synthesis have been developed, such as solution-phase reductive strategy,13,14 microemulsion,15 laser ablation16-18 and so on, whereas most of them involved complicated multistep process,

strict

experimental

condition,

and

the

requirement

of

subsequent

functionalization. However, less study focus on the improvement of Si NPs fluorescence performance especially quantum yield (QY), which seriously limit the further applications of Si NPs in biomedical and biological studies. Recently, Si NPs have been synthesized through a plasma-based method with a high QY of 60%. However, these Si NPs can only disperse in nonpolar solvents and are not suitable for biological applications.19,20 Shao et al. synthesized Si NPs with the QY up to 75% via surfacemodified method, while the obtained Si NPs have a severe limitation to be further used in bioimaging because of their obvious biological toxicity.21 Overall, it is highly desirable to develop a simple and green method to synthesize water-dispersible Si NPs with high QY. As a high efficient heating technology, microwave irradiation has been widely used for materials synthesis.22-24 It offers super efficient energy, rapid volumetric heating,


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faster reaction time, strong reaction selectivity as well as outstanding product yield, which can proceed one-step synthesis of hydrophilic nanomaterials and provide a scalable platform for industrial applications.25-28 All these advantages make the microwave irradiation method more favorable for synthesizing water-dispersible bioapplicable Si NPs.29-31 However, most of the reported Si NPs synthesized through microwave irradiation have relatively low QY (about 20%). For the purpose of improving the fluorescence QY, some studies have been reported that heteroatom doping especially the nitrogen doping could improve the QY of nanomaterials effectively.32-34 Silicon source with higher nitrogen content has been approved an effective way of improving the optical properties.35 Besides the silicon source, the reduction reagent is also an important for the synthesis of high-quality Si NPs. Researchers usually utilize citrate and ascorbate as the common reduction reagents to produce Si NPs, whereas these reagents are not containing any nitrogen atoms.29,36,37 We then developed a new method to synthesis high QY of fluorescent Si NPs with nitrogen-containing reduction reagents. Herein in this work, we proposed a facile one-pot microwave irradiation method to synthesize water-dispersible Si NPs with ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) and N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO) as nitrogenrich reduction reagents as well as nitrogen-rich silicon source, respectively. Using nitrogen atom-containing EDTA-2Na as an effective starting reductant and the obtained Si NPs exhibited fluorescence with a much higher QY up to 62%, which is the highest QY for Si NPs synthesized under normal pressure up to now. Moreover, these Si NPs showed good water dispersibility, excellent chem/biostability, low toxicity, and favorable


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biocompatibility. Taking advantages of these robust performances, the as-prepared Si NPs were further utilized for cellular imaging. 2. EXPERIMENTAL 2.1. Materials. Ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) and glycerol were

obtained

from

J&K

Scientific

Ltd.

(Beijing,

China).

N-[3-

(trimethoxysilyl)propyl]ethylenediamine (DAMO, 95%) was obtained from Aladdin Chemistry Co. Ltd. (Shanghai, China). All amino acids were obtained from SigmaAldrich reagent Co. Ltd. (St. Louis, MO, USA). DMEM (High Glucose) was ordered from Thermo Scientific HyClone (MA, USA). Fetal bovine serum, streptomycin (30 mg/mL-1) and penicillin (100 KU/mL-1) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-d-phentltetrazolium bromide (MTT) was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Other reagents are analytical grade and obtained from J&K Scientific Ltd. (Beijing, China). Ultrapure water was used to prepare all solutions, which was acquired through a Millipore Milli-Q water purification system (Billerica, MA, USA) with an electric resistance ≥18.2 MΩ·cm. 2.2. Instruments. A SOP microwave reactor (CEM, USA) was used to synthesize the Si NPs. A TU-1810 spectrophotometer (Beijing, China) was used to record UV-visible (UV-vis) absorption spectrum. F-4600 spectrophotometer (Hitachi, Japan) was used to measure the fluorescence spectra. The value of absolute fluorescence quantum yield and the fluorescence lifetime decay curve were obtained through a FLS 980 fluorometer (Edinburgh, England). The IR spectra were collected on a Bruker Tensor 27 FT-IR spectrophotometer (Bruker, Germany) in the range of 4000-400 cm-1 with KBr pellets. Transmission electron microscopy (TEM) image was obtained by transmission electron


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microscope (FEI-Tecnai G2, USA) at an accelerating voltage of 200 kV. The X-ray photoelectron spectroscopy (XPS, Thermo ESCALAB 250Xi, USA) with Al Kα radiation was used to analyze the surface composition and chemical state of the product. Inductive coupled plasma emission spectrometer (ICP) (Thermo, USA) was used to measure the content of the silicon element in the as-prepared Si NPs. The TCS-SP8 confocal microscope (Leica, Germany) was used to capture the bright-field and fluorescence images of HeLa cells with a laser operated at 405 nm. 2.3. Preparation of the Silicon nanoparticles (Si NPs). The Si NPs were obtained by microwave irradiation method under normal pressure in one-step. Initially, during the process of stirring, put the Ethylenediaminetetraacetic acid disodium salt (EDTA-2Na, 0.1652 g) into Ar-saturated glycerol solution (3.2 mL). After 20 min, 0.8 mL of N-[3(trimethoxysilyl)propyl]ethylenediamine (DAMO) was added into the above solution dropwise under continuous stirring. After 10 min, the precursor solution was transferred to an exclusive vitreous vessel. The mixed solution was treated with the irradiation at 160 o

C for 10 min. When the temperature dropped below 60 oC, the resultant Si NPs were

removed. At last, the yellow solution was centrifuged at 2428 ×g for 10 min by an ultrafiltration device (MWCO: 3000 Da) to remove the excess DAMO and EDTA-2Na.38 The purified product was diluted 40 times with ultrapure water for further use (v/v, 0.88 mg/mL as Si, measured by ICP). 2.4. Fluorescence quantum yield (QY) measurement. The value of absolute fluorescence QY was directly measured and calculated via a FLS 980 fluorometer equipped with an integrating sphere attachment with 370 nm as the excitation wavelength.


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2.5. Cytotoxicity assays. The cytotoxicity assay for the Si NPs was carried out by a standard MTT assay. Human epithelial cervical cancer (HeLa) cells were first incubated in a 96-well plate with a density of 5×103 cells per well for 24 h. Then, the medium was replaced with 100 µL of fresh DMEM medium containing different concentrations of Si NPs (0, 50, 100, 150, 200, 300, 400, 500 µg/mL). After 24 h, the cells were incubated in 10 µL (0.5 mg/ mL) of MTT and 90 µL of fresh DMEM medium mixture for 4 h. At last, the supernatant was discarded and 100 µL of DMSO (dimethyl sulfoxide) was added to liberate the formed formazan. The cell viability was determined by Multiskan GO microplate reader (Thermo Scientific, Finland) at 490 nm. 2.6. Cellular imaging. To study the imaging capability of Si NPs in living cells, HeLa cells were first incubated in DMEM medium at 37 oC with 5% CO2 for overnight. After washed three times with PBS (0.1 M, pH=7.4), the cells were then incubated with a series of concentrations of Si NPs (50, 100, 150 and 200 µg/mL) in DMEM at 37 oC in 5% CO2, while the Si NPs-free cells were used as the control. Fluorescence imaging of cells were obtained under Leica laser scanning confocal microscope with a 405 nm laser. Cell images were collected with the wavelength range from 420 to 550 nm. 3. RESULTS AND DISCUSSION 3.1. The synthesis of the Si NPs. Hydrophilic Si NPs were directly synthesized in one pot via microwave irradiation, where DAMO acted as the silicon source and EDTA-2Na was regarded as the effective reduction reagent (Scheme 1). Because of glycerol solvent boiling point up to 290 oC, the synthesis process can be done under elevated temperature and normal pressure, which eliminates its potential safety problems.39 As can be seen from Figure S1, when the reaction system contains glycerol, the mixture of glycerol and


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EDTA-2Na, or the mixture of glycerol and DAMO, resulted products have no obvious fluorescence. Correspondingly, when EDTA-2Na, DAMO and glycerol all exist in the system together, a high fluorescence at 440 nm appears distinctly, indicating the successful synthesis of fluorescent Si NPs. Reaction condition such as temperature, reaction time and the molar ratio of reactants were optimized. As shown in Figure S2A-B, a noticeable increase of fluorescence intensity is observed when the temperature rises from 120 oC to 160 oC, which is due to the fast core growth and the reduction of surface defects.40 After 160 oC, the fluorescence intensity dropped down. Meanwhile, the color of Si NPs solution also changes from light yellow to brown under ambient light with the maximum emission wavelength shifts from 432 nm to 450 nm. As the reaction time increase, the fluorescence intensity of Si NPs increases significantly. After 10 min, the reaction reaches equilibrium (Figure S2C). Furthermore, a series of molar ratios between DAMO and EDTA-2Na were also investigated. The fluorescence intensity increases as the molar ratio of EDTA2Na increase and saturated at the molar ratio of 1:0.12 between DAMO and EDTA-2Na (Figure S2D). In conclusion, an optimized reaction condition: 160 oC, 10 min, and the molar ratio of 1:0.12 (DAMO: EDTA-2Na) were used for following experiments. 3.2. Characterization of Si NPs. The TEM image proves that Si NPs are monodispersed in water with an average diameter of 2.1 nm (Figure 1A). These nanoparticles with such small size can facilitate the renal clearance in vivo, indicating that the as-prepared Si NPs may possess good biocompatibility and little potential toxicity.41 The high-resolution TEM examination shows that Si NPs are amorphous structures due to difficulty observing lattice fringes (Figure S3). Additionally, a typical X-ray diffraction analysis confirms that


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the Si NPs are in the amorphous phase as well (Figure S4). Furthermore, XPS and FT-IR were performed to analyze the surface and element composition of the Si NPs. As illustrated in Figure 1B, five major peaks at 101.48, 153.08, 284.8, 399.0 and 531.8 eV corresponds to Si 2p, Si 1s, C 1s, N 1s and O 1s.42 The high resolution C 1s analysis reveals the four types of carbon atoms, originating from C-Si (284.00 eV), C-C (284.80 eV), C-N/C-O (285.66 eV) and C=O (286.96 eV) (Figure S6A).43 The O 1s spectrum manifests that the peaks at 531.00, 531.78 and 532.81 eV are for C=O, C-OH/C-O-C and Si-O (Figure S6B).44 The three fitted peaks at 100.80, 101.43 and 102.10 eV in Si 2p spectrum are assigned to Si-C, Si-N and Si-O groups (Figure 1C). 43, 45, 48 Consistent with Si 2p spectrum, the Si NPs exhibit a peak at 1043 cm-1 in the FT-IR spectrum, which assigns to the vibrational stretch of Si-O bonding (Figure S5). The N 1s signal can be described by three peaks at 398.48, 399.36 and 400.26 eV, which implies the presence of N-Si, C-N-C and N-H (Figure 1D).46 Similarly, the N-H bending vibration and the N-H stretching vibration are also observed at 1648 and 3334 cm-1. The nitrogen atoms have five valance electrons for easily binding with different atoms with an appropriate atom size, supporting the possibility of synthesizing N-doped materials. Moreover, it has been reported that nitrogen doping can result in a new surface state and effectively modulate the photic properties of the materials, finally resulting a higher QY.47 As for the prepared Si NPs here, the appearance of the N 1s peak and the high nitrogen content up to 12% indicate that the particles were successfully doped with nitrogen element by using the nitrogen containing starting source EDTA-2Na and DAMO, which contributes to the high QY of the obtained Si NPs successfully. Additionally, according to the FT-IR spectrum (Figure S5), the peak at 2933 cm-1 is assigned to the O-H bond, and the peak at 1648 and


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3334 cm-1 is due to the N-H bond, indicating that there are abundant hydrophilic groups on the surface such as hydroxyl and amino groups that originated from the starting materials.48 Consequently, ethyl acetate (the gravity lighter than water) and chloroform (the gravity heavier than water) were used to investigate the hydrophilicity of the Si NPs. This experiment clearly demonstrated the excellent hydrophilic property of the Si NPs (Figure 2). In comparison to the other methods that required complicated multistep process and subsequent functionalization to promote the water dispersibility, we used the hydrophilic silicon source DAMO and reductant source EDTA-2Na to synthesize Si NPs in one step without additional post-treatment, which is an efficient and green method to synthesize high fluorescence water-dispersible Si NPs suitable for bioimaging applications. 3.3. The optical properties of Si NPs. As illustrated in Figure 3A, the UV-PL spectra show that the Si NPs aqueous solution exhibits a strong absorption peak at 368 nm which could attribute to the n→π* transition. The existence of this peak might be due to the trapping of exited-state energy by the surface state, which leads to a strong fluorescence emission.49 Under the excitation of 370 nm, a bright blue light center at 440 nm can be obtained. Unlike most other reported Si NPs, the emission wavelength of the as-prepared Si NPs exhibits excitation-independent emission character.50,51 With the increase of excitation wavelength from 320 nm to 410 nm, the fluorescence peak at 440 nm does not shift. The maximum excitation wavelength is consistent with the UV-Vis absorption spectrum at 368 nm, suggesting the nonradiative transition has hardly occurred in the Si NPs.49 Figure 3B shows that the Si NPs only exhibits a single exponential decay function with the fluorescence lifetime of 8.32 ns. Such a short lifetime indicated that the radiative


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recombination of the surface defect states in Si NPs was responsible for the high fluorescence emissions.47 The absolute QY of the as-prepared Si NPs is 62%, which is much higher than most Si NPs reported, especially synthesized via microwave-assisted irradiation (Table S1). The high QY might attribute to the more defect emissive sites and most importantly the utilization of new reagent source with nitrogen-doping. It has been reported that the silicon source DAMO containing two nitrogen atoms could produce a higher QY than those synthesized by APTMS ((3-aminopropyl)trimethoxysilane) and APTES ((3aminopropyl)-triethoxysilane), indicating that increasing the content of nitrogen of reactants can greatly increase the fluorescence QY of Si NPs.35 Meanwhile, EDTA-2Na as a new adopted reduction material has rich nitrogen atom compared with the traditional reduction reagents such as citrate and ascorbate, which results in the further improvement of fluorescence QY of as-prepared Si NPs. Here, the amine groups that are contributed from DAMO and EDTA-2Na can increase the conjugation degree of Si NPs and the passivation degree of traps on the surfaces, which can decrease the non-radiation transition probability, and electrons return to the group states via radiative route, which plays an important role in the process of improving emission electron number, and further more improving the QY of Si NPs.32,47,49 Furthermore, synthesizing the nanoparticles in organic solvent such as glycerol can also decrease the surface defects, which further improve the fluorescence QY.52 Besides in the environment of normal buffer, we also detected the QY of Si NPs in biological fluids, such as DMEM medium with 10% FBS, and the value of QY is about 53%. As the factors mentioned above, the as-obtained Si


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NPs exhibited much higher fluorescence QY, making them to be robust candidates in bioimaging applications. 3.4. The stability of Si NPs. For the bioimaging application of developed Si NPs, the stability of the N-doped Si NPs was tested. After 120 min of irradiation, the fluorescence intensity of Si NPs still remains ~90%, indicating the high resistance to photo-bleaching (Figure 4A). With the change of pH value, as shown in Figure 4B-C, the fluorescence intensity of Si NPs does not change significantly, which is very conducive to their potential applications in bioimaging. Furthermore, as shown in Figure 4D, until in high concentration of NaCl solution up to 50 mM and 100 mM, the fluorescence intensity of the Si NPs only decreases to some extent. In addition, to test the practicality of Si NPs in complex biological environment, we studied their photo stability in cell culture medium. The Si NPs were incubated with the DMEM containing 10% fetal bovine serum for 6 h at 37 oC, and the fluorescence spectra were then recorded. As can be seen from Figure 4E, with nearly 109 mM NaCl in the DMEM medium, the fluorescence intensity of Si NPs remains similar, though slight decreased. Furthermore, when stored at 4 oC for more than 30 days, the Si NPs still maintain strong fluorescence with the robust storage stability (Figure 4F). In additional, it has been reported that EDTA as the chelating agent can easily chelated with metal ions in aqueous solution, and the heavy metal ions such as Hg2+ or Cu2+ ions might have the ability to quench the fluorescence of Si NPs.53 Therefore, we investigated different metal ions that may present in living cells as well as other heavy metal ions in nature. As shown in Figure S7A, only when add Mn2+ decrease of the fluorescence intensity, other metal ions did not influence the fluorescence intensity. Moreover, the impact of different amino acids with the concentration of 50 µM on the


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fluorescence intensity of Si NPs was further studied and showed no obvious interference (Figure S7B). All these results demonstrate that the Si NPs are stable enough to complicate and various environments, which facilitates their great potential application in biomedical and biological field. 3.5. Cytotoxicity assays. In order to use these Si NPs in bioimaging applications, the toxicity of the as-prepared Si NPs was verified by the MTT assay. HeLa cells were selected as a model in this study. HeLa cells were incubated with a series of concentrations of as-prepared Si NPs for 24 h. As shown in Figure 5, the cellular viability remains approximate 76% even with the concentration of the Si NPs up to 500 µg/mL. The result suggested that the Si NPs with good biocompatibility could be further used for cellular imaging. 3.6. Cellular imaging. Taking full advantages of these Si NPs including high fluorescence QY, robust stability, low toxicity and good biocompatibility, we further investigated the capability of the Si NPs for cellular imaging. HeLa cells were cultured for 6 h at 37 oC in different concentrations of Si NPs and subsequently analyzed under a laser scanning confocal microscope with the excitation of 405 nm. Figure 6 shows that the cells incubated with Si NPs have strong blue fluorescence and the intensity is getting stronger with the increasing of Si NPs concentrations, which were calculated by image J (Figure S8). As control, there is no obvious fluorescence signal for HeLa cells in the absence of Si NPs. The shape and morphology of HeLa cells did not show obvious changes after incubation with Si NPs for 6 h, which is in consistence with the result of MTT assay. Furthermore, HeLa cells treated with Si NPs exhibit strong and stable fluorescence signal even after 30 min irradiation (Figure S9-10), which is consistent with


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the spectrum results for Si NPs in aqueous solution and demonstrates that the Si NPs with strong photo-stability is suitable for long-term cell imaging. All these results indicate the potential application of Si NPs as a substitute for cellular imaging with some advantages such as high fluorescence QY, good photo/chemo stability, low toxicity and favorable biocompatibility. 4. CONCLUSION In this work, the synthesis of the N-doped Si NPs can be achieved by a one-pot and green strategy, where EDTA-2Na was used as nitrogen source and effective reduction material. Due to the N-doping, the fluorescence QY of the as-prepared Si NPs reached up to 62%, which is much higher than most of the reported Si NPs. This new Si NPs synthesis strategy not only provides an excellent way to increase the QY of Si NPs by heteroatom doping, but also confirms that EDTA-2Na can serve as the reductant in the preparation of nanoparticles. The cellular imaging results demonstrated that the N-doped Si NPs had highly chemical/photo stable fluorescence, favorable biocompatibility, low toxicity and excellent aqueous dispersibility, which promised the great potential of these Si NPs serving as high-performance fluorescence probes in biomedical and biological applications.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. The fluorescence emission spectra of products prepared using different starting raw source; the optimization of synthesis condition; the high-resolution TEM image of Si NPs;


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the XRD pattern of the Si NPs; the FT-IR spectrum of the Si NPs; XPS spectra of C 1s and O 1s peak of the Si NPs; effect of metal ions and amino acids on the fluorescence intensity of the Si NPs solution; photo-stability of fluorescence images of HeLa cells incubated with Si NPs; comparison of Si NPs prepared by different methods; the p value and relative percentage (F/F0) of the fluorescence intensity of Si NPs in different conditions. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported in part by the National Natural Science Foundation of China (21205108, 21605038), the Outstanding Young Talent Research Fund of Zhengzhou University (1421316038), and the Foundation for University Key Teacher by Henan Province (2017GGJS007). Thanks to the software Image J for the help on our experimental data analysis.

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Scheme 1. The schematic illustration of one-pot synthesis of high quantum yield Si NPs by using EDTA-2Na as an effective nitrogen-containing reductant.


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Figure 1. (A) TEM image of the Si NPs. The scale bar is 50 nm. The inset in (A) presents the size distribution of Si NPs measured by TEM. High resolution XPS spectra with full range (B), Si 2p peak (C) and N 1s peak (D) of the Si NPs.


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Figure 2. The photographs of the as-prepared Si NPs in the mixtures of water and chloroform (left), water and ethyl acetate (right) under ambient light and 365 nm UV irradiation, respectively.


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Figure 3. (A) The absorbance spectra and the excitation wavelength-independent emission spectra along with the excitation wavelength ranging from 320 to 410 nm at equal intervals. (B) The lifetime decay curves of as-prepared Si NPs.


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Figure 4. (A) The time-scan spectrum of Si NPs in water under 370 nm excitation. The stability of Si NPs in different pH of water (B), PBS (C), different concentration of NaCl (D), and DMEM medium (E). (F) Storage stability.


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1.0

Cell Activity (%)

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0.8 0.6 0.4 0.2 0.0 0

50

100

150

200

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400

500

Concentration of Si NPs (µg/mL) Figure 5. Cell viability of HeLa cells in the presence of different concentrations of the Si NPs.


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Figure 6. (A1-E1) The fluorescence images of HeLa cells incubated with different concentrations of Si NPs (0, 50, 100, 150, 200 µg/mL) at 37 oC for 6 h. (A2-E2) The bright-field images of HeLa cells corresponding with A1-E1. (A3-E3) The merged images. Scale bar, 20 µm.


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Table of Contents


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Due to excellent photoluminescence properties ... - ACS Publications

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