Fabrication of ZnS nanoparticle chains on a protein template
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- Padalkar, S., Hulleman, J., Kim, S.M. et al. J Nanopart Res (2009) 11: 2031. doi:10.1007/s11051-009-9689-8
In the present study, we have exploited the properties of a fibrillar protein for the template synthesis of zinc sulfide (ZnS) nanoparticle chains. The diameter of the ZnS nanoparticle chains was tuned in range of ~30 to ~165 nm by varying the process variables. The nanoparticle chains were characterized by field emission scanning electron microscopy, UV–Visible spectroscopy, transmission electron microscopy, electron energy loss spectroscopy, and high-resolution transmission electron microscopy. The effect of incubation temperature on the morphology of the nanoparticle chains was also studied.
KeywordsNanoparticle chainsTemplateSynthesisMorphologyOne-dimensional nanostructure
One-dimensional structures (1D) such as nanowires, nanorods, nanoparticle chains, and nanotubes have attracted much attention in recent years (Cui et al. 2001; Diehl et al. 2002; Huang et al. 2001; Bachtold et al. 2001; Collins et al. 2001; Murray et al. 2000; Kimberly et al. 2002; Johnson et al. 2002; Alivisatos 1996). Their growing importance is due to the unique properties they exhibit. The 1D structures show future promise in a variety of fields such as electronics, optoelectronics, catalysis, and biosensing. Although the advances in the field of nanotechnology are promising, there are few obstacles that need to be overcome. The synthesis of 1D nanostructures by solvothermal process (Chen et al. 2003), thermal evaporation (Meng et al. 2003; Wang et al. 2002), liquid crystal template (Jiang et al. 2001), and electrodeposition (Xu et al. 2006) in porous anodic alumina templates require high temperatures or pressures and the precise control of the process variables. Moreover, after synthesis it is generally difficult to manipulate and position the nanostructures in devices. In turn, biological molecules have the chemical recognition capacity that is promising in allowing for a higher degree of flexibility in their positioning in specific places in nanoelectronic devices. In addition, certain peptides and proteins can self-assemble into chemically reactive readymade shapes that can serve as templates for further growth of inorganic nanostructures. Biological systems possess a high degree of organization from molecular building blocks (peptides, amino acids, proteins, and nucleic acids) and are perfect models for bottom–up strategies for controlled material synthesis. Their molecular recognition capabilities, combined with the specificity toward certain ions and molecules, can be used to precisely control the fundamental processes involved in materials synthesis and processing, such as phase stability, nucleation and growth, pattern formation, and assembly. The electroless deposition, especially on DNA molecules and viruses, has lead to the fabrication of several different 1D structures (Klein et al. 1997; Keren et al. 2003; Claridge et al. 2005; Flynn et al. 2003; Yan et al. 2003; Monson and Woolley 2003; Deng and Mao 2003; Richter et al. 2001; Dong et al. 2007; Mao et al. 2004; Huang et al. 2005; Nam et al. 2006). However, peptides and proteins, with the ability to self-assemble into ordered fibrils have been much less investigated. While several metallic nanowires, as well as CdS nanoparticle chains have been synthesized via protein-directed nucleation and growth in our laboratory (Padalkar et al. 2007, 2008), to our knowledge, there are no reports in literature regarding the use of fibrillar proteins for the template synthesis of zinc sulfide (ZnS) nanowires or nanoparticle chains. ZnS is an II–VI semiconducting material having a band gap of 3.7 eV. It is a particularly interesting material due to its wide range of potential applications. It shows promise in several fields and has applications in electronics and photonics. ZnS has semiconducting, photoluminescent, and field emission properties. These properties have been exploited in many applications such as light converting electrodes, ultraviolet light-emitting diodes, phosphors in cathode ray tubes, flat panel displays, injection lasers, and infrared windows (Kar et al. 2003; Xu et al. 2006; Lu et al. 2007). Several ZnS 1D structures such as nanorods, nanowires, nanobelts, and nanotubes (Zhang et al. 2002; Yin et al. 2005; Ma et al. 2003) have be fabricated. All these structures have been synthesized at high temperatures and require long reaction times. However, the fabrication process described here was carried out at atmospheric conditions and requires a very short synthesis time typically not more than 15 min for the completion of the entire experiment.
Here, we report the synthesis of ZnS nanoparticle chains on a fibrillar protein (α-synuclein) template. Synuclein is a 14.4 kDa amyloidogenic protein, which is found in the human brain (Spillantini et al. 1997). This protein has the ability to self-assemble into fibrillar structures having an approximate diameter of 8 nm and a length between 500 nm and 1 μm (Conway et al. 2000; Hoyer et al. 2002). The presence of protein fibrils in the human brain can lead to different pathologies (Serio et al. 2000; Scheibel and Lindquist 2001; Scheibel et al. 2003; DePace and Weissman 2002). However, when the α-synuclein protein self-assembles into fibrils in vitro, its properties can be potentially useful for the synthesis of inorganic nanostructures. The structure of amyloidogenic fibrillar proteins, such as α-synuclein, is mainly composed of adjacent β-sheets assembled into a twisted fibrillar structure by hydrogen bonding (Vilar et al. 2008; Serpell et al. 2000; Nelson and Eisenberg 2006; Rochet 2007). The charge on the β-sheets can be manipulated during the self-assembly process to obtain fibrillar structures with different charge arrangements, thus making them ideal structural templates for the fabrication of 1D nanostructures.
Self-assembly of α-synuclein fibrils
The expression and purification of α-synuclein were carried out as previously described (Conway et al. 2000; Rochet et al. 2000). The E46K mutant of α-synuclein, was used because it has the ability to rapidly self-assemble into fibrils. The lyophilized protein was dissolved in phosphate-buffered saline (PBS) with pH 7.4, 0.02% (w/v) NaN3 and dialyzed against the same buffer at 4 °C, for 24 h. The protein solution was filtered through a 0.22 μm nylon spin filter followed by a Microcon-100 spin filter, yielding a stock solution depleted of aggregates. The final concentration of protein in PBS was of 100–300 μM [determined by bicinchoninic acid (BCA) assay]. The protein was incubated at 37 °C for 12–96 h in a tissue culture rolling drum to generate fibrils.
Synthesis of ZnS nanoparticle chains
The synthesis of ZnS nanoparticle chains was carried out by using zinc chloride (ZnCl2; 2 mM) as the salt solution, and hydrogen sulfide (H2S) gas as the sulfur source. A stock solution of ZnCl2 was prepared and its pH value was adjusted to be in the acidic regime by the addition of concentrated hydrochloric acid. This was done to avoid precipitation of zinc hydroxide in solution. For the synthesis of ZnS nanoparticle chains a p-type silicon (Si) (111) wafer was used as a substrate to prepare a field emission scanning electron microscopy (FESEM) sample. The same synthesis procedure was performed on a 3-mm-diameter carbon-coated gold grid as a substrate to obtain a transmission electron microscopy (TEM) sample. A volume of 10 μL of α-synuclein fibrils suspended in the PBS buffer was pipetted onto the, Si wafer, substrate and dried in a desiccator. The ZnCl2 solution (10 μL) was deposited on to the dried protein solution, followed by an incubation time of 5 min. The substrate with the protein and the ZnCl2 solution was then exposed to H2S gas for 5 min. Later, the substrate was rinsed using deionized water and dried under a jet of air. A similar procedure was carried out for the preparation of a TEM sample. A similar, ZnS nanoparticle chain, TEM sample was prepared on a carbon-coated gold grid.
Characterization of α-synuclein fibril and ZnS nanoparticle chains
The diameter and morphology of the α-synuclein fibril were studied, with TEM, by using the Philips CM-10 operated under 80 kV accelerating voltage. A carbon-coated copper TEM grid was used as the substrate. The protein solution (3 μL) was pipetted out on to the TEM grid and was stained using 2% uranyl acetate solution for 1 min. The excess solution on the grid was then blotted and the sample was used for imaging.
The average diameter and morphology of the ZnS nanoparticle chains were analyzed, with FESEM, by using a Hitachi S4800 field emission scanning electron microscope and, with TEM, by using an FEI Titan 80/300 transmission electron microscope. High-resolution transmission electron microscopy (HRTEM) images were registered to investigate the crystalline nature of the sample. Further, an electron diffraction pattern was obtained to study the crystal structure of the sample. Electron energy loss spectra (EELS) were obtained from the ZnS nanoparticle chains to verify the presence of zinc (Zn) and sulfur (S) in the samples. Further, elemental mapping of Zn and S were also obtained to study the distribution of Zn and S in the samples. Finally, UV–visible (UV–Vis) absorption spectra were obtained from the ZnS sample and from the ZnS colloidal sample having a particle size of 10 μm, purchased from Sigma Aldrich to compare the absorption peaks. The FESEM analyses were performed on a Hitachi S4800. TEM imaging was performed on either Philips CM-10 operating at 80 kV or on an FEI Titan 80/300 transmission electron microscope having a Gatan Imaging Filter (GIF) and a 2 k CCD, which operated at 300 kV. EELS and HRTEM images were registered on the FEI Titan. The UV–Vis absorption spectra of the ZnS nanoparticle chains and colloidal ZnS were recorded with a molecular device microplate reader.
Results and discussion
The α-synuclein fibril formation
Synthesis and characterization of ZnS nanoparticle chains
Controlling the size and packing density of nanocrystals on a biological scaffold can be an effective way of tuning the electrical properties of the nanostructures. To achieve controlled growth kinetics of nanocrystals on the self-assembled polypeptide scaffold, it is important to understand the parameters that influence their formation. The reduction of ionic silver to a metallic form in the presence of proteins and DNA was first described by Merril et al. (1981) and Merril (1990). The method is widely applied for the detection of proteins and nucleic acids and is based on the differences between the redox potentials of the biomolecules and those of the matrix. A similar chemical mechanism, in which the metal ions are selectively reduced to a metallic form in the presence of biomolecules was previously used in our laboratory for the fabrication of inorganic nanoparticle chains on biological scaffolds, both metallic and semiconducting (Padalkar et al. 2007, 2008). During the synthesis process, the cations from a salt source [e.g., AgNO3, CdCl2, aned Pb(NO3)2] that reacts with the negatively charged aminoacyl side chains of the protein, at basic pH (Merril et al. 1981; Merril 1990). When these cations are subsequently reduced to the elemental state, metallic nanoparticle chains grow on the protein template. The negatively charged C-terminal domain of α-synuclein contains five aspartate and ten glutamate negatively charged side chains and therefore has the potential of forming complexes with metal cations and subsequently nucleating nanocrystals on the fiber surface. A number of studies have revealed that these negatively charged side chains into the fibril is not known with precision, based on our results that show formation of ZnS nanoparticle chains on the protein fiber scaffold, it can be speculated that some of these negatively charged aminoacyl side chains are exposed at the fiber’s surface rather than buried within the fiber (Qin et al. 2007; Chen et al. 2007; Heise et al. 2005; Murray et al. 2003). For semiconductor compounds, such as ZnS, the metal cations bind to the same negatively charged aminoacyl side chains of α-synuclein. After the introduction of the sulfide anion, semiconductor nanoparticles are expected to nucleate on the protein fiber surface. It could be speculated that there are several regions along the protein fiber where the protein side groups have a significant affinity for the semiconductor nanoparticles, leading to their stabilization.
The same generic mechanism could be expanded to other polypeptide scaffolds and can therefore be of significant potential importance for the field of designing bottom–up strategies for nanomaterials fabrication on biomolecular templates. Our results prove the biomineralization capacities of the α-synuclein protein, and can be extended to other fibrillar proteins or polypeptides.
The changes in the process variables help in varying the size of the nanoparticle chains and also help in varying the connectivity between the nanoparticles, thus making the nanoparticle chains more smoothly connected.
In summary, we report the use of α-synuclein fibrils as biological templates for the synthesis of ZnS nanoparticle chains. The size of the nanoparticle chains can be controlled by varying the process variables. This result was confirmed by TEM imaging carried out on the ZnS samples. The nanoparticles are composed of several nanocrystals having a dimension of ~2 nm. The diffraction pattern reveals the zinc blende structure of ZnS. The EELS confirm the presence of Zn and S in the ZnS nanoparticle chains. Further, elemental mapping of Zn and S shows uniform distribution of both the elements on the nanoparticle chains.