Journal of Nanoparticle Research

, Volume 10, Issue 4, pp 559–566

Biomimetic synthesis of HgS nanoparticles in the bovine serum albumin solution

Authors

  • De Zhi Qin
    • College of Chemistry and Environmental ScienceHenan Normal University
    • College of Chemistry and Chemical EngineeringPingdingshan University
  • Xiao Ming Ma
    • College of Chemistry and Environmental ScienceHenan Normal University
    • College of Chemistry and Environmental ScienceHenan Normal University
  • Li Zhang
    • College of Chemistry and Chemical EngineeringPingdingshan University
  • Zhong Jun Ma
    • College of Chemistry and Environmental ScienceHenan Normal University
  • Jie Zhang
    • College of Chemistry and Environmental ScienceHenan Normal University
Research Paper

DOI: 10.1007/s11051-007-9284-9

Cite this article as:
Qin, D.Z., Ma, X.M., Yang, L. et al. J Nanopart Res (2008) 10: 559. doi:10.1007/s11051-007-9284-9

Abstract

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.

Keywords

Mercury(II) sulfideNanoparticlesBiomimetic synthesisBSAColloidsNanobiotechnology

Introduction

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.

Experimental

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.

Characterization

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.

To investigate the crystalline structure of the products, XRD measurements were carried out at room temperature. Figure 1 shows the XRD patterns of HgS samples. All the peaks can be indexed to cubic β-HgS with lattice constant comparable to the values of JCPDS 6–261. It is clear that the peaks are relatively broad, indicating that the nanoparticles have the small size. The EDS spectrum of the samples also showed the significant presence of only Hg and S with an atomic ratio (Hg/S) of nearly 1, in good agreement with the stoichiometric molar ratio of mercury(II) sulfide.
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-007-9284-9/MediaObjects/11051_2007_9284_Fig1_HTML.gif
Fig. 1

XRD pattern of the as-prepared HgS nanoparticles

Figure 2A shows a representative TEM image of the as-prepared HgS nanoparticles in the BSA solution (c = 2.0 mg/L) from the typical experiment. From the figure, these nanoparticles with average sizes 20–30 nm are obviously well dispersed. After the solutions were kept for 30 days, we also can get the same monodisperse nanoparticles as before. The result showed that the presence of BSA was a key factor in controlling and regulating the shape and size of the HgS nanoparticles. The related SAED pattern (Fig. 2B, inset of Fig. 2A) shows principally rings corresponded to the planets of cubic HgS of the zinc blende structure, which is consistent with the results of XRD measurement. The HRTEM image in Fig. 2C provides further insight into the structure of the products. The image exhibits lattice fringes with d spacing of 0.138 nm, which is consistent with the unique 0.134 nm separation between 331 planets in cubic HgS crystallites. Due to the sensitiveness of HgS to electron beams, we have not obtained clearly detailed images of Fig. 2 (B and C).
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-007-9284-9/MediaObjects/11051_2007_9284_Fig2_HTML.jpg
Fig. 2

(A) Typical TEM image of HgS nanoparticles in the BSA aqueous solution (c = 2.0 mg/L) (B) SAED pattern in an area including HgS nanoparticles (C) HRTEM image of HgS nanoparticles

Figure 3 shows the size distribution of the HgS nanoparticles synthesized in the BSA solution(c = 2.0 mg/L). The distribution is quite narrow with an average diameter of about 30 nm. This result demonstrates that HgS nanoparticles with monodisperse size can be synthesized in aqueous solution of BSA.
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-007-9284-9/MediaObjects/11051_2007_9284_Fig3_HTML.gif
Fig. 3

The size distribution of HgS nanoparticles in the BSA solution (c = 2.0 mg/L)

In order to observe the details of conjugation of BSA-HgS, the as-prepared nanoparticles were dyed by using phosphotungstic acid hematoxylin stain for TEM experiments. The picture of a single nanoparticle was magnified to look more closely at the BSA-nanoparticle conjugation in Fig. 4. The image shows HgS nanoparticle surrounded by soft materials whose width is 3–4 nm in size. Apparently, the HgS nanoparticles are uniformly coated with BSA like arrangement, with each nanoparticle seemingly immersed in a small pot of BSA. This result provides strong evidence for the conclusion that well-dispersed HgS nanoparticles directly conjugated with BSA.
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-007-9284-9/MediaObjects/11051_2007_9284_Fig4_HTML.jpg
Fig. 4

TEM image of a typical BSA-conjugated HgS nanoparticle

The UV-vis spectra of the samples (a, b, c) prepared in different concentrations of BSA aqueous solution are given in Fig. 5. On account of quantum size effect a broad absorption peak without maximum is observed. This optical spectra was used to calculate the band gap from
$$ \alpha {\left( v \right)} = A{\left( {hv/2 - E_{g} } \right)}^{{\raise0.7ex\hbox{$m$} \!\mathord{\left/ {\vphantom {m 2}}\right.\kern-\nulldelimiterspace} \!\lower0.7ex\hbox{$2$}}} $$
where α is the absorption coefficient and Eg is the band gap. For a direct transition m = 1 a plot of (αEphot)2 vs. Ephot was constructed and the value of Ephot extrapolated to α = 0 gives the band gap, Eg. The band gaps calculated for samples (a, b, c) are 2.74, 2.85 and 2.92 ev, while the corresponding concentrations of BSA solution were 1.0, 2.0 and 4.0 mg/L, respectively, which are higher than the band gap (2.0 ev) of bulk HgS due to a quantum size effect. The photoluminescence (PL) spectrum of the as-prepared HgS nanoparticles is shown in Fig. 6. The nanoparticles exhibit a well-defined absorption feature at 440 nm, which is considerably blue-shifted relative to the bulk HgS crystals, indicating a quantum size effect. These features suggested HgS nanoparticles have quantum-confined effects and size-tunable optical properties.
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-007-9284-9/MediaObjects/11051_2007_9284_Fig5_HTML.gif
Fig. 5

UV/vis absorption spectra of HgS-BSA nanocomposites with BSA solution concentrations (a) 1.0 mg/L, (b) 2.0 mg/L and (c) 4.0 mg/L

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Fig. 6

PL spectra of as-prepared HgS nanoparticles

To study the formation mechanism of the HgS nanoparticles in the BSA aqueous solution, the FT-IR spectra of pure BSA, BSA-Hg2+ and BSA-HgS solutions were determined. The FT-IR spectra and data of the main peaks are presented in Fig. 7 and Table 1. The IR peaks of pure BSA at 3,424, 3,071, 1,658 and 1,535 cm−1 are assigned to the stretching vibration of –OH, amide A′(mainly-NH stretching vibration), amide I, and amide II bands, respectively (Jackson et al. 1991). The amide I band is the characteristic band of the C=O stretching vibrations. The amide II is caused by the coupling of bending vibrate of N–H and stretching vibrate of C–N. The peak at ∼1385 cm−1 in the BSA-Hg2+ and BSA-HgS spectra is assigned to the absorption of nitrate ions, which was introduced by the addition of mercury nitrate. Comparing the IR spectrum of BSA-Hg2+ with those of pure BSA, there are negligible variations in the characteristic peaks of –OH groups, amide I and amide II bands, because that Hg2+ as a soft Lewis acid has a low affinity for the hard base. But the characteristic peak of amide A′ bands shifts to a high wavenumber of about 26 cm−1, suggesting that there might be coordination interaction between Hg2+ and –NH groups of BSA, which may play an important role in the formation of nanoparticles. Comparing the IR spectra of BSA-HgS with those of pure BSA, the characteristic peaks of -OH groups and amide A′ bands of the BSA-HgS shift to a high wavenumber of about 26 and 97 cm−1, respectively. The results showed that there might be the conjugate bonds between the HgS nanoparticles and –OH and –NH groups of BSA.
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-007-9284-9/MediaObjects/11051_2007_9284_Fig7_HTML.gif
Fig. 7

The FT-IR spectra of (a) pure BSA, (b) BSA-Hg2+ and (c) BSA-HgS nanoparticles

Table 1

The Main Peaks of Pure BSA, BSA-Hg2+ and BSA-HgS nanoparticles from the IR spectrum

Assignment

–OH (cm−1)

Amide A′(cm−1)

AmideI (cm−1)

AmideII (cm−1)

NO3 (cm−1)

Pure BSA

3,424

3,071

1,658

1,535

 

BSA-Hg2+

3,414

3,097

1,645

1,522

1,385

BSA-HgS

3,450

3,168

1,643

1,529

1,385

To further study the change in conformation of BSA, 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. Circular dichroism is observed when molecules absorb left and right circularly polarized light to different extent. The amide chromophore of the peptide bond in protein dominates their CD spectra below 250 nm. In a α-helix structure of protein, a negative band near 220 nm is observed due to the strong hydrogen-bonding environment of this conformation, a second transition at 190 nm is split into a negative band near 208 nm and a positive peak near 192 nm. The CD spectra of β-sheet display a negative band near 216 nm, a positive band between 195 and 200 nm, and a negative band near 175 nm. So CD spectroscopy was used in our experiments. The CD spectra of pure BSA, BSA-Hg2+ and BSA-HgS solutions are given in Fig. 8. From the figure, it can be seen the CD curve of pure BSA is different from that of BSA-Hg2+ and BSA-HgS. After the calculation by CDPro software, the experimental data of the reaction system are presented in Table 2. From Table 2, pure BSA contains 54.4% α-helices and 9.4% β-sheets, in good agreement with the previous reports (Gelamo et al. 2002). Comparing the CD results of BSA-Hg2+ and BSA-HgS with those of pure BSA, the content of α-helix of BSA decreased 33.7 and 38.5%, and the β-sheet content increased 18.2 and 23.6%, whereas the content of turns and random coils did not change much. The effect of formation of HgS on secondary structure of pure BSA is larger than that of Hg2+. In protein secondary structure, there are 3.6 amino acid residues in every ring of the α-helix segment. It is well known that the hydrogen bond formed between the oxygen atom of the (i) carboxylic group and the hydrogen atom of the (i + 4) amino group. And the β-sheet structure can be seen as a kind of special α-helix only with two amino acid residues through stretching, resulting from the break of the hydrogen bond. We deduce that mercury ions can react with –NH groups of BSA, so the hydrogen bonds break in α-helix structure of protein. The new hydrogen bonds develop between primary α-helices; which results in the formation of β-sheets structure. So there is a change in BSA secondary structure from α-helices to β-sheets resulted from the interaction of BSA and Hg2+. In addition, the BSA protein also involves 17 disulfide bonds with one free thiol in cysteine residues, which might also be attracted Hg2+ via thiolate linkages.
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-007-9284-9/MediaObjects/11051_2007_9284_Fig8_HTML.gif
Fig. 8

The CD spectra of (a) pure BSA, (b) BSA-Hg2+ and (c) BSA-HgS solutions

Table 2

The percentages of the secondary structures of pure BSA, BSA-Hg2+ and BSA-HgS

Assignment

α-helix%

β-sheet%

Turn%

Random%

Pure BSA

54.4

9.4

11.0

25.0

BSA-Hg2+

20.7

27.6

19.9

31.8

BSA-HgS

15.9

33.0

17.9

33.1

According to the discussion above, the scheme of the HgS nanoparticles formation in the BSA solution is illustrated in Fig. 9. Firstly, the Hg2+(yellow ball in Fig. 9) solution was added into BSA aqueous solution, there is an interaction between the mercury ions and binding sites of BSA. The concentration of the mercury ions around these sites is very high because of this interaction. When the TAA is added to the Hg2+-BSA solution, it releases S2- (red ball in Fig. 9) upon hydrolysis gradually. Then the S2- combines with Hg2+ to form HgS nanoparticles. During the formation of HgS nanoparticles, the nucleation and growth of the nanocrystals will be affected by the BSA through electrostatic matching, structural and stereochemical complementarity (interfacial molecular recognition).
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-007-9284-9/MediaObjects/11051_2007_9284_Fig9_HTML.gif
Fig. 9

Scheme of the HgS nanoparticles formation in the BSA solution

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.

Conclusions

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.

Acknowledgements

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.

Copyright information

© Springer Science+Business Media B.V. 2007