Biomimetic synthesis of HgS nanoparticles in the bovine serum albumin solution
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- Qin, D.Z., Ma, X.M., Yang, L. et al. J Nanopart Res (2008) 10: 559. doi:10.1007/s11051-007-9284-9
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HgS nanocrystals conjugated with protein were synthesized in aqueous solution of Bovine Serum Albumin (BSA) at room temperature. The obtained HgS nanoparticles with average diameter about 20–40 nm were characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), selected-area electron diffraction (SAED) and high-resolution transmission electron microscopy (HRTEM). The quantum-confined effect of the HgS nanoparticles is confirmed by the ultraviolet-visible (UV-vis) and photoluminescence (PL) spectrum. The rescults indicate that the BSA not only induce the nucleation, but inhibit the further growth of HgS nanoparticles. The effect of Hg2+ on BSA and the change of BSA conformation were studied through Fourier transform infrared (FTIR) spectroscopy and Circular dichroism (CD) spectroscopy. The possible mechanism of HgS nanoparticles growth in the BSA solution was also discussed.
KeywordsMercury(II) sulfideNanoparticlesBiomimetic synthesisBSAColloidsNanobiotechnology
There is a keen interest in the synthesis of semiconductor nanomaterials in recent years. Among these materials, semiconductor sulfide nanocrystals have been most studied due to their quantum size effects and size-tunable optical properties (Fan et al. 2006; Liu et al. 2004;Yang et al. 2006). In the quantum-confined regime it is possible to tune the band gap by the control of the particle size and in principle to achieve targeted optical properties (Higginson et al. 2002). Various synthetic schemes have been developed for the size- and shape-controlled synthesis of metal sulfide nanocrystals, using organometallic precursors (Ghezelbash et al. 2005; Ma et al. 2003), coordinating solvent (Tran et al. 2002; Lee et al. 2002), reverse micelles (Zhang et al. 2002; Liu et al. 2000), microemulsions (Gao et al. 2003) and polymer films (Gao et al. 1996, 1998). Whatever the method of preparing metal sulfide nanocrystals, it requires organic solvent or ligand as template which can control the size, shape and monodispersity of nanoparticles. The development of simple, mild and effective methods of preparing nanocrystals is of importance to nanotechnology and remains a key research challenge. Thus, suitable water-soluble bio-macromolecules can be chosen as matrix to synthesis inorganic materials because of their non-toxicity and bio-compatibility.
Biological macromolecules are capable of controlling inorganic crystals nucleation and growth to a remarkable degree through biomineralization (Berman et al. 1993). Thereupon, it is a logical approach to use biomolecules to grow monodisperse nanocrystals via biomimetic methods. Mann et al. have prepared CdS nanocrystals using self-assembled bacterial S-layers (Shenton et al. 1997). We have synthesized Ag2S nanorods in the Bovine serum albumin (BSA) solution and CdS nanocrystals in the pepsin solution (Yang et al. 2005). Many other researchers also have synthesized biocompatible inorganic minerals in bio-macromolecules matrices (Meziani and Sun 2003; Coffer et al. 1996; Storhoff et al. 1999; Mamedova et al. 2001). All these results suggest a possible biomimetic role for these bio-macromolecules as “soft templates” in materials chemistry. Formation of crystalline materials in biological environments is largely dominated by macromolecules, such as their functional groups, electric property and advanced structure (Addadi et al. 2001; Izhaky and Addadi 1998; Falini et al. 1996; Jimenez-Lopez et al. 2003). On the other hand, the formation of inorganic crystals has important influence on the configuration of bio-macromolecules matrices, but reports on this effect are lacking. Proteins are important bio-macromolecules in bodies and play crucial roles in many biological activities. Serum albumin lies in animal blood plasma, 50% of the total amount of plasma protein consists of it (Rees et al. 2004). BSA is a globular protein with a average molecular weight of 68,000 Da, consisting of 582 amino acid residues, with 17 disulfide bridges and one free thiol group, which is quite similar to human serum albumin (HSA) (Guharay et al. 2001).
Among these semiconductor sulfide nanocrystals, cadmium sulfide and zinc sulfide have attracted considerable attention because of its relatively easy synthesis and distinct particle size dependent on optical properties (Bao et al. 2004; Wang et al. 2005; Zhang et al. 2003). There have been very few studies on HgS nanocrystals due to difficulties associated with its synthesis and the toxicity of mercury. Mercury sulfide is a useful material and it can be widely used in ultrasonic transducers, electrostatic image materials and photoelectric conversion devices. It also has application in the field of infrared sense because of its narrow band gap (Pal et al. 2003). This paper reports an effective method for the synthesis of HgS nanoparticles in BSA aqueous solution and the influence of the formation of HgS on the conformation of BSA.
BSA was of electrophoretic purity, purchased from Xiamen Sanland Chemicals Company Limited, China. The average molecular weight of it is about 68,000 Da. In a typical process, three parallel 50 mL of 25 mM mercury nitrate (≥99.8%, Mw = 333.62 A.R.) aqueous solution were prepared and transferred into 250 mL round-bottom flask, then three 100 mL BSA aqueous solutions were added, while the corresponding concentrations of BSA solution were 1.0, 2.0, 4.0 mg/L respectively. The mixed solutions of BSA-Hg2+ emulsion were kept static under N2 atmosphere for 8 h at room temperature. Then three parallel 50 mL of 25 mM TAA (≥99%, Mw = 75.13 A.R.) solutions were added into as-prepared solutions. After TAA addition, the color of solution changed to yellow immediately. Gradually, the color changed from yellow to orange yellow and finally to dark brown. The mixed reaction solution was again maintain under a static condition for 72 h, then separated by high speed centrifuging at 15,000 rpm. The collected solid-state product was washed with double distilled water and ethanol then dried in a vacuum at room temperature for 24 h. To investigate the influence of BSA on the formation of HgS nanoparticles, control experiment was carried out in the aqueous solution without BSA.
Powder X-ray diffraction (XRD) patterns were recorded on a Bruker D&Advance X-ray powder diffractometer with graphite monochromatized Cu Kα (λ = 0.15406 nm). A scanning rate of 0.05 deg/s was applied to record the pattern in the 2θ range of 10–70°. Transmission electron microscopy (TEM), accompanied by selected-area electron diffraction (SAED) was carried out on a JEOL JEL−100CX-II Transmission electron microscope. High-resolution transmission electron microscopy (HRTEM) investigations were conducted on a JEOL JEL−2010 transmission electron microscope. To prepare the TEM and HRTEM samples, a 5 μL droplet of dilute alcohol solution was dripped onto a carbon coated copper grid, then dried at room temperature. A Perkin-Elmer Lambda 20 ultraviolet-visible (UV-vis) spectrophotometer was used to carry out optical measurements; samples were placed in quartz cuvettes (1 cm path length). A Shimadzu RF−540 PC instrument was used to record the photoluminescence (PL) spectrum. Infrared spectra were taken on a Bio-Rad FTS−40 Fourier transform infrared spectrograph in the wavenumber range of 4,000–500 cm−1, the spectra were collected at 2 cm−1 resolution with 128 scans by preparing KBr pellets with a 3:100 “sample-to-KBr” ratio. The Circular dichroism (CD) spectra of reaction systems were measured at 10 ± 1 °C with a Jasco−810 spectropolarimeter. The same samples were repeated three times. This instrument had been calibrated previously for wavelength with benzene vapor, and for optical rotation with d10-camphorsulfonic acid. A cell with a path length of 0.1 cm was used. The parameters used were as follows: bandwidth, 1 nm; step resolution, 0.1 nm; scan speed, 50 nm/min; response time, 0.25 s. Each spectrum was obtained after an average of six scans. Quantitative estimations of the secondary-structure content were made with the CDpro software package, which includes the programs CDSSTR, CONTIN, and SELCON3. We used these three programs to analyze our CD spectrum.
Results and discussion
The synthesis of HgS nanoparticles was performed by two-step procedure. The first step was the generation of the Hg(II)-BSA complex by mixing of the mercury nitrate and BSA solutions. The second step was the formation of HgS nanoparticles by adding TAA into the above mixing solution at ambient temperature. TAA was comparatively unstable and slowly hydrolyzed to release S2− into the reaction solution. We once tried to use Na2S instead of TAA, but the HgS nanoparticles could not be formed, indicating that the slow release of sulfide ions was essential for the formation of the nanoparticles. To investigate the influence of BSA on the formation of HgS nanoparticles, we carried out control experiment in the absence of BSA. The products which were obtained from the reaction solution without BSA were badly aggregated. In order to study the influence of concentration of BSA on HgS crystallization, three experiments were performed, while the corresponding concentrations of BSA solution were 1.0, 2.0, 4.0 mg/L respectively. It is becoming clear that the smaller nanoparticles associated with greater amounts of BSA. So we can easily control size-tunable optical properties of nanoparticles through altering concentration of BSA.
The Main Peaks of Pure BSA, BSA-Hg2+ and BSA-HgS nanoparticles from the IR spectrum
The percentages of the secondary structures of pure BSA, BSA-Hg2+ and BSA-HgS
On the basis of these results, possible formation mechanism was proposed. As a soft Lewis acid, Hg2+ has a high affinity for the soft donor sulfur atom and moderate affinity for nitrogen donors. In addition, the negative charged oxygen atoms contained in deprotonated carboxylate also interacts with Hg2+. Therefore the bio-macromolecule BSA can provide multiple binding sites for Hg2+, mercury ions can react with BSA and the binding sites may include the –NH, –SH and –OH groups. After the mercury nitrate solution was added into the BSA aqueous solution, mercury ions coordinated with groups of BSA, resulting in the relatively high mercury ion concentration around these groups. Then, the S2- released from TAA reacted with Hg2+ ions to form HgS nuclei surrounding some special sites of BSA. With aging, the growth of HgS nanoparticles started on them. As a soft template, BSA is anti-aggregation agents and has great effect on the monodisperse of nanoparticles. As a result, the as-prepared HgS nanoparticles were obtained. On the other hand, the interaction between Hg2+ and BSA causes the BSA conformation change from α-helix to β-sheet, and β-sheet secondary structure forms a suitable conformation for the oriented growth of HgS nanoparticles which result in the HgS nanoparticles are uniformly coated with BSA.
In summary, cubic HgS nanoparticles conjugated with protein were synthesized by sequentially adding mercury nitrate and TAA into the BSA aqueous solution. The synthesis process is very simple, mild and controllable and the bio-macromolecules we selected as matrix are non-toxicity and bio-compatibility. The obtained HgS nanoparticles have a high degree of crystallinity and good photoluminescence. In addition, the nanocrystals exhibit quantum size effects and size-tunable optical properties. The influence of formation of HgS nanoparticles on conformation of BSA was also studied; the process induces the change from α-helix to β-sheet in BSA secondary structure.
This study was supported by the National Basic Research Program of China (Grant No. 2005CB724306) and the National Science Foundation of China (Grant No. 20371016). We are grateful to the Lab for Special Functional Materials of Henan University for the help with HRTEM measurements. We also thank the referees for helpful comments.