Herein we firstly reported a simple, environment-friendly, controllable synthetic method of CuSe nanosnakes at room temperature using copper salts and sodium selenosulfate as the reactants, and bovine serum albumin (BSA) as foaming agent. As the amounts of selenide ions (Se2−) released from Na2SeSO3 in the solution increased, the cubic and snake-like CuSe nanostructures were formed gradually, the cubic nanostructures were captured by the CuSe nanosnakes, the CuSe nanosnakes grew wider and longer as the reaction time increased. Finally, the cubic CuSe nanostructures were completely replaced by BSA–CuSe nanosnakes. The prepared BSA–CuSe nanosnakes exhibited enhanced biocompatibility than the CuSe nanocrystals, which highly suggest that as-prepared BSA–CuSe nanosnakes have great potentials in applications such as biomedical engineering.
KeywordsCopper selenide Nanosnakes Bovine serum albumin Synthesis Characterization Mechanism Biocompatibility
Copper selenides (CuSe) are well-known p-type semiconductors having potential applications in solar cells, optical filters, nanoswitches, thermoelectric and photoelectric transformers, and superconductors . A lot of efforts have been devoted to the synthesis of copper selenides micro- and nanocrystallites with various morphologies, such as particles , tubes , cages , and flake-like structures . There have been a few reports on the synthesis of copper selenide 1D nanomaterials. For example, Cu2−x Se nanowires with lengths of several micrometers and diameters of 30–50 nm have been prepared by employing selenium-bridged copper cluster as precursor in a chemical vapor deposition (CVD) process . Also synthesized are arrays of copper selenide nanowires of mixed compositions of Cu3Se2/Cu2−x Se or Cu2−x Se/Cu in various proportions with lengths of several micrometers and diameters of 13–17 nm by using porous anodic aluminum oxide film as template . However, to our knowledge, few reports are closely associated with the environmental-friendly controllable synthesis of 1D snake-like morphological CuSe nanomaterials based on biomolecule-assisted synthesis. For example, Muñoz-Rojas et al.  synthesized Ag@PPy nanomaterials that had snake-like shape and showed the properties of bending and folding under hydrothermal conditions while retaining the crystallographic coherence of the silver core, which were highly suggested that snake-like 1D nanomaterials might have some unique properties and potential application.
In recent years, biomimetic synthesis has become a hotspot . For example, Yang et al.  reported biomimetic synthesis of Ag2S , HgS , and PbS , etc. in the bovine serum albumin (BSA) solution. These synthesized 1D nanomaterials have unique electrical, optoelectronic, biological, and mechanical properties with fundamental significance and great potential in applications such as electrochemical storage cells, solar cells, solid-state electrochemical sensors, semiconductive optical devices, catalyst, superionic materials, and biomedical engineering [13–16] and have attracted tremendous attentions from researchers in the field of materials, micro-electronics, and nanotechnology in recent years. However, how to fully use the advantage of bionanomaterials such as DNA, RNA and proteins, and metal nanomaterials as assistant media to fabricate 1D nanocomposites with controllable shapes and unique properties is still a great challenge. Up to date, few reports are associated with application of CuSe nanomaterials in biomedical engineering.
Herein, we selected one-dimensional copper selenide nanocrystals (CuSe) as research target, chose BSA as assistant reagent, developed a simple, nontoxic, room temperature, environmentally friendly method to synthesize controllably 1D BSA-wrapped copper selenide snake-like nanocomposites, and investigated these as-prepared products’ properties by UV–vis spectroscopy, high-resolution transmission electron microscopy, selected-area electron diffraction, energy dispersive spectroscopy, Raman spectroscopy, and MTT method. We found that as-prepared CuSe nanosnakes own some unique properties and enhanced biocompatibility, the possible formation mechanism of CuSe nanosnakes is also explored. Our primary results show that BSA–CuSe nanosnakes have great potential applications in biomedical engineering.
All the reagents, including Cu(NO3)2, Na2SO3, and Se powder, were from Sinopharm Company, China. BSA with average molecular weight of about 68 KD was from Xiamen Sanland Chemicals Company Limited, China. All other reagents were from Sigma Inc. Human fibroblast cell line was obtained from the American Type Collection Company. RPMI 1640 medium containing 10% fetal calf serum was from Gibco Company. Agarose was from Sigma (St. Louis, United States). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Dojin Laboratories (Kumamoto, Japan).
Synthesis of CuSe Nanosnakes
The Na2SeSO3 solution was prepared by refluxing selenium powder (5 mmol) and Na2SO3(5 mmol) in distilled water (200 ml) under nitrogen atmosphere for 24 h. In a typical synthesis process, 5 ml of 25 mM copper nitrate aqueous solution and 10 ml of 3 mg/ml BSA aqueous solution were mixed under vigorous stirring at room temperature (25°C). The mixed solution of the BSA–Cu2+ emulsion was kept static under nitrogen protection for 2 h. Then, 5 ml of 25 mM Na2SeSO3 solution was added. The color of the mixed solution rapidly changed to black. The mixed reaction solution was kept static under ambient conditions for 96 h, and then was separated by centrifugation at 15,000 rpm. The collected black solid-state products were washed with double distilled water and ethanol for three times and dried in a vacuum at room temperature for 24 h. During the process of nanosnakes growth, four replicas of the same experiment were run in parallel. Each replica was terminated at different times such as 24, 48, 72, and 96 h. To investigate the influence of BSA on the formation of copper selenide nanosnakes, a control experiment was carried out, copper selenide was prepared in the aqueous solution without BSA, and other conditions and procedures were the same as in a typical experiment.
Characterization of Synthesized BSA–CuSe Nanosnakes
These synthesized BSA–CuSe nanosnakes were characterized by a UNICAM UV300 spectrophotometer (Thermo Spectronic, USA), high-resolution transmission electron microscopy(HR-TEM, Hitachi H-700H, Hitachi, Japan), selected-area electron diffraction, energy dispersive spectroscopy, a PerkinElmer LS55 spectrofluorimeter, Laser Raman spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy (an FTS135 infrared spectrometer from BIO-RAD, United States).
Cell Culture and MTT Analysis
Human fibroblast cell line was cultured in RPMI 1640, containing 1 × 105 mU/ml of penicillin and 0.1 mg/ml of streptomycin supplemented with 10% (v/v) FCS, at 37°C in a humidified 5% CO2 and 95% air atmosphere for 48 h. These cells were collected and added into 24-well plates at the concentration of 5,000 cells/well and continued to culture for 24 h. Then, the 100 μl CuSe nanocrystals (20 μg/ml) and 100 μl BSA–CuSe nanocrystals(20 μg/ml) were added into the 24-well plates, not added into the control wells, and continued to culture for 3 days. MTT (5 mg/ml) was prepared in PBS, and 20 μl was added to each well, and the cells were incubated for 4 h at 37°C, then the medium was removed, 200 μl dimethyl sulfoxide was added to each well, and optical density (OD) was read at 515 nm. The cell viability was calculated by the following formula: cell viability (%) = OD (optical density) of the treated cells/OD of the nontreated cells. The percentage of cell growth was calculated as a ratio of numbers of CuSe or BSA–CuSe nanosnakes-treated cells and control cells with 0.5% DMSO vehicle [17–19].
Each experiment was repeated three times in duplicate. The results were presented as mean ± SD. Statistical significance was accepted at a level of P < 0.05.
Results and Discussion
Synthesis and Characterization of BSA–CuSe Nanosnakes
In the course of synthesis of 1D BSA–CuSe nanostructures, BSA was used as the soft-template to control the nucleation and growth of the nanocrystals, and also the dispersion and stabilization of the nanocrystals in solvents. As well known, BSA possesses a zwitterionic character at the isoelectric point (pI 4.7), displayed reversible conformational isomerization as the pH value changing . BSA can bind with different sites of a variety of cationic and anionic groups, which makes possible utilization of BSA-decorated nanomaterials in a variety of supramolecular assemblies. For example, any conformational BSA can form covalent adduct with various metal ions , such as Cu2+, Ni2+, Hg2+, Ag+, and AuCl4−.
Potential Mechanism of BSA–CuSe Nanosnake Formation
Raman spectroscopy is used to investigate the changes in the electronic properties of nanomaterials through the special electron–phonon coupling that occurs under strong resonant conditions. Therefore, Raman spectra are very powerful to detect of the new chemical bonds. As shown in Fig. 6b, the difference between the Raman spectrum of pure BSA and that of BSA–Cu2+ is obvious. The bands C–H of BSA at 2,926 cm−1 disappeared, suggesting that there might be coordination interaction between Cu2+ and BSA. Comparing the Raman spectra of BSA–CuSe with those of pure BSA and BSA–Cu2+, the characteristic peak of Cu–Se bonds at 250 cm−1 was found, which is consistent with the standard Raman spectra of cubic berzelianite (Cu2−xSe) crystallites(RRUFF ID: R060260.2). The above facts highly suggested that the Cu2−xSe nanosnakes were successfully synthesized in the BSA solution.
Biocompatibility of CuSe Nanocrystals and BSA–CuSe Nanosnakes
In conclusion, CuSe nanosnakes were successfully synthesized at room temperature using BSA as soft-template. Regarding the potential mechanism of the phenomena, we suggested a possible model: at first, the cationic Cu2+ions were covalently adducted to BSA, as the amounts of selenide ions (Se2−) released from Na2SeSO3 in the solution increased, the cubic and snake-like CuSe nanostructures were formed gradually. Secondly, the cubic nanostructures were captured by the CuSe nanosnakes, the CuSe nanosnakes grew wider and longer as the reaction time increased. Finally, the cubic CuSe nanostructures were completely replaced by BSA–CuSe nanosnakes. The prepared BSA–CuSe nanosnakes exhibited enhanced biocompatibility than the CuSe nanocrystals, which highly suggest that as-prepared BSA–CuSe nanosnakes have great potentials in applications such as biomedical engineering.
(See supplementary material 1)
This work was supported by the National Natural Science Foundation of China (No.20803040 and No.20471599), Chinese 973 Project (2010CB933901), 863 Key Project (2007AA022004), New Century Excellent Talent of Ministry of Education of China (NCET-08-0350), Special Infection Diseases Key Project of China (2009ZX10004-311), Shanghai Science and Technology Fund (10XD1406100). The authors thank the Instrumental Analysis Center of Shanghai Jiao Tong University for the Materials Characterization.
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