Synthesis and Characterization of BSA–CuSe Nanosnakes
As shown in Fig. , we can clearly observe the BSA–CuSe nanosnakes with different lengths. We also observed that the cubic copper selenide nanostructures were firstly formed in Fig. . As the reaction time increased, the cubic copper selenide nanostructures gradually disappeared and became the nanosnakes. The resultant nanosnakes gradually grew longer and longer.
TEM images of BSA–CuSe nanosnakes (a) and the cubic copper selenide nanostructures (b)
Regarding the synthesis of one-dimensional BSA–CuSe nanostructures, sodium selenosulfate (Na2
) was used as Se source, which has been widely used to prepare nanocrystallite selenides such as CdSe [20
] and PbSe [21
]. Lakshmi et al. [22
] and Nair et al. [23
] have successfully prepared copper selenide (Cu2−x
Se and Cu3
) thin films by using Na2
as Se source. Na2
is much more active than Se powder, because it reacts easily with Cu2+
ions at room temperature, is also less toxic, and, therefore, is safer to use than Na2
Se or H2
]. Equations (1) and (2) describe the reaction processes:
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 [25
]. 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 [26
], such as Cu2+
, and AuCl4−
Potential Mechanism of BSA–CuSe Nanosnake Formation
In order to clarify the mechanism of synthesis of CuSe nanosnakes, we characterized the CuSe nanostructures by UV–vis spectroscopy. Figure shows the UV–vis absorption spectra of pure BSA, BSA–Cu2+, and BSA–CuSe. The pure BSA has a special absorption peak at 280 nm. The spectrum of BSA–Cu2+ complex did not display shift and enhancement of absorption peak at 280 nm, because the BSA protein can provide multiple binding sites for Cu2+. The spectrum of BSA–CuSe nanocomposites clearly showed the absorption peak shift from 280 nm to 228 nm after the Na2SeSO3 solution was added into the BSA–Cu2+ solution, indicating that the Se2− released from Na2SeSO3 and reacted with Cu2+ forming CuSe nanostructures. Figure clearly shows that the absorbance of BSA–CuSe nanostructures was markedly enhanced, because more and more BSA–CuSe nanosnakes were formed.
a UV–vis absorption spectrum of BSA, BSA–Cu2+, BSA–CuSe at 24 h, BSA–CuSe at 48 h, BSA–CuSe at 72 h, BSA–CuSe at 96 h; b The change of absorbance at 190 nm
The BSA–CuSe nanosnake was also characterized by using high-resolution transmission electron microscopy (HRTEM), selected-area electron diffraction (SAED), and energy dispersive spectroscopy (EDS). Figure – show representative TEM images of the BSA–CuSe nanosnakes at the different reaction time such as 24, 48, and 96 h, respectively. We can clearly observe that the well-dispersed nanostructures displayed different sizes, representing the different growth stages. Within the 24-h reaction time, BSA–CuSe nanostructures mainly exhibited cubic structure with average size of 30 nm. After 24 h, the BSA–CuSe nanosnakes formed gradually, their sizes were about 130 nm in length and 12 nm in width. After 48 h, the cubic nanostructures had little change, short rods appeared, and the nanosnakes grew wide and long (Fig. ). When the reaction time reached to 96 h, the cubic nanostructures almost completely disappeared, and the nanosnakes grew homogeneously up to about 200 nm in length, and 14 nm in width (Fig. , ). When the reaction time was over 96 h, the sizes of nanosnakes were almost unchanged. As shown in Fig. , the single nanosnake exhibits good crystalline and clear lattice fringes. The lattice fringe spacing was 0.172 nm, consistent with the interplanar spacing of the (113) plane of cubic berzelianite (Cu2−xSe) crystallites. Figure is the corresponding SAED pattern, revealing that the nanosnakes are crystalline and can be indexed to berzelianite Cu2−xSe.
Figure 3 TEM images of BSA–CuSe nanosnakes obtained after different aging time in the typical experiment: a 24 h, b 48 h, and c 96 h, respectively. d HRTEM image of an individual nanosnake. e SAED pattern in an area including many nanosnakes. f The histogram (more ...)
In order to investigate the typical growth stage of nanosnakes, we used HR-TEM to observe the samples at 48-h reaction time. Figure depicts the typical morphology of the cubic BSA–CuSe nanocomposites revealing that the peanut-like assemblies and shorter nanorods were generated. As shown in Fig. , the adjacent nanostructures attached without sharing a same crystallographic orientation. The experimental lattice fringe spacing, 0.146 nm, is consistent with the interplanar spacing in monoclinic Cu2
Se. The connected nanoparticles were rotated to find the common crystallographic orientation (indicated by the white arrow) [27
]. After the rotations were finished, they fused to form almost a perfect short nanorod (Fig. ). The lattice fringe spacing is 0.173 nm, which was in agreement with the interplanar spacing of the (113) plane of cubic berzelianite (Cu2−x
Figure 4 TEM images showing oriented attachment of cubic copper selenide in BSA solution for 48 h. a Low-magnification TEM image of sample. b HRTEM image of two primary crystallites forming “peanut” or “chain” via oriented attachment. (more ...)
To understand the growth mechanism of nanosnakes, the representative TEM images of the devourment of cubic nanoparticles were recorded in Fig. –, and the prepared nanosnakes also characterized by scanning electron microscope (SEM) (see Supplementary Fig. 2). When the cubic nanoparticles were captured by the nanosnakes, the square boundary gradually fuzzed with the reaction time and disappeared finally (shown by the white arrow in Fig. ). Figure , showed the capture transient and the devourment stage, respectively. Figure – recorded three different parts of an individual nanosnake, including the neck (D), the body (E), and the tail (F), whose lattice fringe spacing is respectively 0.266, 0.101, and 0.155 nm. The different parts have different crystallographic orientation and steadily existed in BSA solution. The EDS spectrum shows the presence of elements Cu and Se in the prepared nanosnakes (see Supplementary Fig. 3). The peaks of C and O element are due to the BSA. The Supplementary Table 1 documents the weight percentage and atomic percentage of silver and selenium elements of the measured area, which showed that the atomic ratio of Cu and Se does not match the stoichiometric molar ratio (Cu/Se) of copper selenide exactly. The main reason is that the amount of selenium in the BSA solution is excessive (see Supplementary Table 1). According to the above phenomena, it could be presumed that the nanosnakes are growing at the expense of the colloidal particles in the Ostwald ripening process, and BSA act as a stabilizing agent to modify the new generated nanosnakes surface.
Figure 5 TEM images showing oriented attachment of copper selenide nanosnakes in BSA solution for 48 h. a Low-magnification TEM image of sample. b, c TEM images of two different devour stages of copper selenide nanosnakes. HRTEM images of different parts of an (more ...)
To clarify the formation mechanism of BSA–CuSe nanosnakes, we also obtained the FT-IR spectra and Raman spectra of pure BSA, BSA–Cu2+, and BSA–CuSe powders. The FT-IR spectra and the data of the main peaks are shown in Fig. and supplementary Table 2. The IR peaks of pure BSA at 3,430, 3,062, 1,652, and 1,531 cm−1 are assigned to the stretching vibration of –OH, amide A (mainly—NH stretching vibration), amide I (mainly C=O stretching vibrations), and amide II (the coupling of bending vibrate of N–H and stretching vibrate of C–N) bands, respectively. The difference between the IR spectrum of pure BSA and that of BSA–Cu2+ is obvious. The characteristic peak of –NH groups disappeared, suggesting that there might be coordination interaction between Cu2+ and –NH groups of BSA, which may play an important role in the formation of CuSe nanosnakes. In addition, the new peaks of BSA–Cu2+ at 1,021 and 824 cm−1 might be contributed to the interaction of Cu2+ and BSA. The strong peak at 1,383 cm−1 in the BSA–Cu2+, and BSA–CuSe spectra is attribute to the absorption of NO3−1, which was introduced by the addition of Cu(NO3)2.Comparing the IR spectra of BSA–CuSe with those of pure BSA, the characteristic peak of –OH groups shifts to a high wavenumber of about 5 cm−1, and the characteristic peak of –NH groups disappears. The results indicate that the conjugate bonds existed between the CuSe nanosnakes and –OH groups and –NH groups of BSA.
a The FT-IR spectra of (a) pure BSA, (b) BSA–Cu2+, and (c) BSA–CuSe in BSA solution for 96 h. b Raman spectra (632.8 nm excitation) of pure BSA, BSA–Cu2+, BSA–CuSe in BSA solution for 96 h
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. , 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.
To further study the formation mechanism of the nanosnakes in the BSA aqueous solution, the conformation changes in the secondary structures of BSA in the reaction system were determined by CD spectroscopy, which is a valuable spectroscopic technique for studying protein and its complex. The CD spectra of pure BSA, BSA–Cu2+, and BSA–CuSe solutions are shown in Fig. . From the figure, it can be seen that the CD curve of BSA–Cu2+ solution is similar to that of the pure BSA solution, while the CD spectrum of the BSA–CuSe solution is different from that of pure BSA. According to the result, it can be seen that copper ions only induced the smaller deformation of the BSA molecules in the BSA–Cu2+ solution, whereas there were bigger changes in the BSA conformation in the BSA–CuSe nanosnake solution, resulting from the strong conjugate bonds between BSA and surfaces of the colloidal nanosnakes. With the growth of CuSe nanosnakes, more and more α-Helix were stretched and transformed into β-Sheets, which could be contribution to the impairment or break of hydrogen bonds.
The CD spectra of a pure BSA, b BSA–Cu2+, and c BSA–CuSe in BSA solution
According to the data mentioned above, we suggest one possible mechanism model of CuSe nanosnake formation based on use of BSA as soft-template, shown in Scheme . The basic principle is attributed to that whose structure decides whose function. BSA has reversible conformational isomerization in different pH condition, when pH value is lower than 4.7, BSA undergoes another expansion with a loss of the intra-domain helices (10) of domain I which is connected to helix (1) of domain II and that of helix (10) of domain II connected to helix (1) of domain III [27
] Then, BSA has three reversible forms: N forms, F forms, and E forms, which could bind with CuSe nanoparticles, finally result in the formation of different shapes of CuSe nanostructures, for example, CuSe nanoparticles bound with N forms formed the sphere nanostructures, CuSe nanoparticles bound with F forms formed the cubic nanostrctures, and CuSe nanoparticles bound with E forms formed the nanosnakes, final CuSe nanostrcutures strongly depend on the structures of BSA proteins under the reaction condition.
Schematic mechanism of synthesis of CuSe nanosnakes using BSA as soft-template