Synthesis of Fluorescent and Water-Dispersed Germanium

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Synthesis of fluorescent and water dispersed germanium nanoparticles and their cellular imaging application Jiali Hu, Qiujun Lu, Cuiyan Wu, Meiling Liu, Haitao Li, Youyu Zhang, and Shouzhuo Yao Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01543 • Publication Date (Web): 07 Jul 2018 Downloaded from http://pubs.acs.org on July 15, 2018

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Synthesis of fluorescent and water dispersed germanium nanoparticles and their cellular imaging application Jiali Hu, Qiujun Lu, Cuiyan Wu, Meiling Liu, Haitao Li, Youyu Zhang* and Shouzhuo Yao

Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education) College of Chemistry and Chemical Engineering Hunan Normal University, Changsha 410081 (P. R. China)

KEYWORDS: germanium nanoparticles, one-step approach, organogermanes, fluorescence probe, cellular imaging

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ABSTRACT

In recent years, Ge nanomaterials have aroused a great deal of attention due to their unique physical and chemical properties. However, the current synthesis methods bear some disadvantages, such as high reaction temperature, dangerous reagents and inert atmospheres. In this paper, we developed a facile one-step route for preparing fluorescent and water dispersed germanium nanoparticles (Ge NPs) by utilizing organogermanes as the precursor, operated at mild reactive conditions. The as-synthesized Ge NPs have an average diameter of 2.6 ± 0.5 nm and intense blue-green fluorescence. Furthermore, the as-synthesized Ge NPs show remarkable water dispersibility, favorable biocompatibility, outstanding photostability, excellent storage stability and low cytotoxicity. More importantly, these Ge NPs can act as satisfactory fluorescence probe and successfully be applied to cellular imaging of HeLa. The present study offers a simple and moderate strategy for preparation of Ge NPs and expedites Ge NPs for bioimaging applications.

Introduction

Group IV nanomaterials have been widely studied in the optoelectronic technologies and biomedical applications field,1 owing to their intrinsic advantages, such as relatively low toxicity, excellent compatibility, favorable stability and uniquely optical properties.2 In the past


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decades, the synthesis of group IV (Si, Ge) semiconductor nanomaterials has attracted significant attention because they can take the place of the toxic heavy-metal-containing II-VI semiconductor nanomaterials in the field of biological fluorescence imaging and electronic devices.3 Indeed, Si nanoparticles have been extensively researched in the above areas.4 Compared with Si, Ge possesses not only a larger excitonic Bohr radius (24.3 nm)3e, 5 but also the smaller bandgap (0.67 eV),6 implying Ge NPs have more prominent quantum confinement effects1b, 7 and stronger visible photoluminescence.5 Based on the above characteristics, Ge NPs are emerging as promising nanomaterials for potential application related to solar cells,8 photodetectors,9 photothermal therapy,10 charge storage11 and bioimaging,12 etc.

Up to now, a diverse range of strategies have been developed for the synthesis of Ge NPs. These methods can be classified into two groups: top-down and bottom-up methods. The top-down preparation methods generally use bulk germanium materials as precursor, including chemical liquid deposition (CLD),13 molecular beam epitaxy (MBE),14 magnetron sputtering,15 femtosecond laser pulses,16 ball milling technique17 and so on. Nevertheless, these strategies not only require expensive equipments, complex procedures or high temperature, but also are difficult to control the size of the particles. Bottom-up synthetic approaches can better regulate the size of Ge NPs, generally utilizing the redox reaction of some chemical reagents containing germanium. For instance, McMillan’s group obtained Ge NPs by the metathesis reaction between GeCl4 and Mg2Ge in diglyme.18 Kim et al. prepared Ge NPs in toluene solvent by using


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germanium iodides as the precursor and LiAlH4 as the reducing agent.19 Zaitseva group reported the preparation of Ge NPs by thermal decomposition of three organogermanes precursors.20 Although these methods can be used to synthesize Ge NPs, they exist with some disadvantages: they usually need rigorous conditions without water and oxygen; the reducing agents, such as LiAlH4 and sodium anphthalide, are highly dangerous; generally require the coverage of organic ligands or surfactants to avoid aggregation of Ge NPs. Hence, a facile method to produce watersoluble Ge NPs under normal pressure is urgently required.

Herein, we present a one-step approach for the synthesis of Ge NPs under aqueous-solution by adopting organogermanes as the precursors and NaBH4 as the reducing agent. Compared with traditional methods, this synthesis method can operate at an ambient atmosphere and lower reaction temperature, and does not require dangerous reagents and further surface modification process. The as-synthesized Ge NPs have diameters of 2.6 ± 0.5 nm and excellent waterdispersibility. They also exhibit blue-green fluorescence and excitation-dependent fluorescence behaviour. In addition, the as-synthesized Ge NPs demonstrate robust photostability, negligible toxicity and superior biocompatibility. As a satisfying biological probe, the as-synthesized Ge NPs have been further successfully applied in the cellular imaging of HeLa.

Experimental Section

Synthesis of Ge NPs: The Ge NPs were synthesized by using diverse organogermanes under different reaction conditions. Typically, 0.1 mL Tetrabutoxygermane was added to 6 mL


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aqueous solution while stirring. Then, 0.016 g NaBH4 was added to the above mixture. The total mixture was put into an oil bath at 80 oC for 12 h. At last, a pale-yellow solution was formed and then dialyzed (Mw 500) against water for two days. By changing the reactants to 0.1 mL Tetrabutylgermanium and 0.03 g NaBH4, the pale-yellow solution was obtained similarly. In addition, the Ge NPs could also be prepared from Diphenylgermanium dichloride by hydrothermal treatment. Detailedly, 0.1 mL Diphenylgermanium dichloride and 20 mg NaBH4 were added to 10 mL aqueous solution, then transferred into 25 mL Teflon-lined autoclave and heated at 180 oC for a period of 30 h. At last, the colorless solution was generated and then dialyzed (Mw 500) against water for two days. Cytotoxity assays and cellular imaging: The HeLa cells were incubated in DMEM (Dulbecco’s Modified Eagle’s Medium) containing 10% (v/v) FBS (fetal bovine serum) at 37 °C in 5% CO2 atmosphere. The as-synthesized Ge NPs were modified with PEI according previously reported method.21 The cytotoxicity of Ge NPs was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Briefly, Hela cells were first cultured in a 96-well plate overnight. Then, the cells were washed three times with phosphate buffered saline (PBS), and incubated in DMEM containing Ge NPs with various concentrations (0, 0.05, 0.10, 0.15, 0.20 and 0.25 mg mL-1) for 24 h. Next, the cultured solution was poured out, and MTT (0.5 mg mL-1) solution was added. After 4 h, MTT solution was removed and DMSO was put into each well. Finally, the absorbance at 570 nm of each well was monitored in a spectrophotometer after being shaked for 10 min. In the cell imaging experiments, the cells were incubated with Ge NPs


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(0.2 mg mL-1) in DMEM (3 mL) for 8 h at 37 °C, and the extracellular Ge NPs were eliminated by washing with PBS three times. Next, the cell fluorescence images were captured with a confocal laser scanning fluorescence microscope. Results and Discussion The Ge NPs were prepared under aqueous-solution by adopting tetrabutoxygermane as the precursor and NaBH4 as the reducing agent. At the beginning of the reaction, the mixture quickly turned into brown-red precipitates (defined as intermediate-1), then gradually formed black precipitates (defined as intermediate-2) and generated the pale-yellow solution (as-synthesized Ge NPs) finally. Figure 1A shows the transmission electron microscopy (TEM) image of the as-synthesized Ge NPs, which exhibits spherical particles with good dispersity. The high resolution transmission electron microscopy (HRTEM) image (the inset of Figure 1A) clearly manifests that the assynthesized Ge NPs possess high crystallinity with a distinct lattice spacing of 0.2 nm corresponding to the (220) plane of germanium, which is consistent with the reported literature and indicates Ge NPs have been synthesized successfully.19 Figure S1A shows the size distribution of the Ge NPs with an average diameter of 2.6 ± 0.5 nm, as determined by analyzing 300 randomly chosen particles from the TEM images. The corresponding dynamic light scattering (DLS) result confirms the small size of the as-synthesized Ge NPs with a hydrodynamic diameter of ～6.02 nm (Figure S1B). The different diameters measured by TEM and DLS are due to different surface states of the sample under the different measurement


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conditions. Specifically, the aqueous Ge NPs samples are directly measured by DLS, while water in the Ge NPs samples must be strictly removed for TEM characterization, resulting in a larger hydrodynamic diameter than the TEM measurements.22 The energy dispersive X-ray (EDX) spectroscopy has displayed the element content in Ge NPs (Figure S1C), and it can find that the Ge NPs contain C, O, and Ge elements.

Figure 1. (A) TEM image of as-synthesized Ge NPs; inset: the HRTEM image of single Ge NPs. (B) FT-IR spectrum of as-synthesized Ge NPs.

To identify the functional groups of the as-synthesized Ge NPs, Fourier transform infrared spectrum (FT-IR) was carried out (Figure 1B). Typically, the obvious signal at 1450 cm-1 is attributed to bending vibration of the Ge-C bond.23 The broad absorbance between 3000 and 3600 cm-1 corresponds to the O-H stretching vibration. The absorbance peaks in the range of 2850-3000 cm-1 are assigned to the C-H symmetric and asymmetric stretching vibrations.


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Additionally, the band at 800-900 cm-1 is assigned to the stretching vibration of Ge-O,24 implying the surface of the Ge NPs were partially oxidized. We speculated that the abundant functional groups of Ge NPs were responsible for the good water-solubility and dispersibility of the Ge NPs. As shown in Figure S1D, the zeta potential of the as-synthesized Ge NPs in aqueous solution is about －29 mV, illustrating that these Ge NPs have strong negative charges on surfaces which further demonstrates that the nanoparticles surface functionalized with oxygencontaining functional groups. Owing to the negative charges, the particles exclude each other and well dispersed in the aqueous solution. The UV-Vis absorption spectrum and fluorescence spectra of the as-synthesized Ge NPs solution are shown in Figure 2. The as-synthesized Ge NPs possess a strong absorption band in the UV region (red line in Figure 2A) and display a maximum emission peak at 505 nm when excited at 382 nm. As shown in the inset in Figure 2A, the as-synthesized Ge NPs solution exhibits paleyellow under ambient light and bright blue-green fluorescence under 365 nm UV lamp irradiation. The fluorescence quantum yield (QY) is about 5.6%, estimated by using quinine sulfate as reference (quantum yield of 54% at 0.1 M H2SO4), which compared with other literatures in Table S1. We speculated that the difference of QY may be due to the size, surface states of Ge NPs synthesized by different methods, which is similar to the C, Si nanomaterials.25 In addition, an excitation-dependent fluorescence behavior of the as-synthesized Ge NPs is observed in Figure 2B, which is similar to the other group IV nanomaterials. 21, 26


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Figure 2. (A) UV-vis and fluorescence excitation and emission spectra of as-synthesized Ge NPs; inset: Photographs captured without (left) and with (right) 365 nm UV lamp irradiation. (B) Corresponding fluorescence spectra at different excitation wavelengths ranging from 320 nm to 440 nm.

The possible synthesis mechanism based on reaction phenomenon and the results of the characterization experiments was proposed. To explore the mechanism of the synthesis process, FTIR and X-ray photoelectron spectra (XPS) were carried out. As shown in Figure 3A, the strong signals at 810 cm-1 are attributed to the stretching vibration of Ge-O bond, indicating that the intermediate-1 (brown-red precipitates, a line) and intermediate-2 (black precipitates, b line) are mainly germanium oxides. However, the stretching vibration of Ge-O bond of the assynthesized Ge NPs (c line) is not obvious, manifesting the content of germanium oxides is lower. It can be inferred that germanium oxides were gradually reduced to Ge0 as the reaction was prolonged. In addition, the XPS spectra of Ge3d further determined the chemical


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compositions of the above three substances. As shown in Figure 3B and 3C, the intermediate-1 and intermediate-2 exhibit two broad peaks at 33.2 eV and 31.0 eV, which associating with Ge4+ and Gex+ (0

Synthesis of Fluorescent and Water-Dispersed Germanium

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