Biomimetic Synthesis of Gelatin Polypeptide-Assisted Noble-Metal Nanoparticles and Their Interaction Study
© Liu et al. 2010
Received: 6 July 2010
Accepted: 12 August 2010
Published: 2 September 2010
Herein, the generation of gold, silver, and silver–gold (Ag–Au) bimetallic nanoparticles was carried out in collagen (gelatin) solution. It first showed that the major ingredient in gelatin polypeptide, glutamic acid, acted as reducing agent to biomimetically synthesize noble metal nanoparticles at 80°C. The size of nanoparticles can be controlled not only by the mass ratio of gelatin to gold ion but also by pH of gelatin solution. Interaction between noble-metal nanoparticles and polypeptide has been investigated by TEM, UV–visible, fluorescence spectroscopy, and HNMR. This study testified that the degradation of gelatin protein could not alter the morphology of nanoparticles, but it made nanoparticles aggregated clusters array (opposing three-dimensional α-helix folding structure) into isolated nanoparticles stabilized by gelatin residues. This is a promising merit of gelatin to apply in the synthesis of nanoparticles. Therefore, gelatin protein is an excellent template for biomimetic synthesis of noble metal/bimetallic nanoparticle growth to form nanometer-sized device.
Noble-metal nanocrystals with small uniform size have attracted more attentions for use in binding biomolecules including proteins, enzymes, DNA due to their huge accessible surface area. Great efforts have been devoted to the development of various synthesis methods for preparing monodisperse nanocrystals. For example, Huang et al.  reported a general synthesis strategy for metallic nanocrystals in a two-phase liquid–liquid system, which involves a quite fast nucleation stage overlapped with the growth stage within 10 min. Ideal nanometer-sized devices will combine features of nature's own nanodevices (proteins) such as specific recognition, energy transduction, and cooperativity with the electronic, magnetic, and optical properties of nanomaterials [2–5]. As a result of these, biologic molecules such as proteins and DNA are an ideal type of matrix which acts as biotemplates in the direct chemical synthesis of nanometer-sized devices. For instance, peptides with high affinities and specificities has been used to control the interparticle spacing of aggregated gold colloid through the folding of a helix-loop-helix forming polypeptide on the particle's surface . A number of research groups have investigated the ability of amino acids to act as reducing and stabilizing agent for the synthesis of gold nanoparticles [7–9]. Aspartic acid , lysine , tyrosine , and tryptophan [12, 13] have been found to initiate and control the synthesis of gold nanostructures at room temperature. Mandal and colleagues [11, 13] have found that tyrosine and tryptophan residues reduced gold and silver ions in solution, resulting in the formation of nanoparticles at room temperature. While peptides lacking tyrosine residues were used to modify the shape and size of gold nanoparticles produced with the aid of Sodium Borohydride, NaH, Potassium Borohydride, etc. , polypeptides containing a higher number of tandem peptide repeats were found to be more effective in controlling nanoparticle growth. More recently, CuSe Nanosnakes have been fabricated by using bovine serum albumin polypeptides as biotemplates at room temperature .
Gelatin is a soluble polypeptide derived from insoluble collagen, the most abundant protein in animal skin and bone, and has been extensively used for food, pharmaceutical, and medical applications . Gelatin is a material that gels and melts reversibly below the normal human body temperature (37°C). Intensive research has been done on α-helix folding of gelatin from random coils to its conformation in the biologically active form. At elevated temperature, gelatin polypeptide chains exist predominantly in the form of flexible, unfold coils in solution and can be partially renatured to the ordered α-helix upon cooling to room temperature. Due to the presence of functional groups including suspended double bond, –NH2, –SH and –COOH, gelatin can be further modified with other bio-molecules for different purposes. Therefore, gelatin is an ideal natural protein for biotemplating nanoparticles into ideal engineering nanometer-sized devices. The biologic impacts of nanoparticles are affected by the nature of the adsorbed protein layer or the protein (bimolecular) corona. Nanoparticles possess more surface area which increases non-specific interactions of amino acid side chains of the gelatin protein with the nanoparticle surface. Nevertheless, little attention has been paid to the interaction between gelatin and noble-metal nanoparticles. This limits the wide use of gelatin polypeptide–mediated noble-metal nanoparticles, particularly in biologic applications. Zhang et al.  simply reported that biocompatible gold nanoparticles could be successfully prepared in one step by using gelatin polypeptide upon thermal reduction. In this paper, we researched on the generation of gold, silver and gold–silver bimetallic nanoparticles using gelatin as a reducing and stabilizing agent, and the interaction between nanoparticles and gelatin. In addition, the shape control of gold nanoparticles, against NaCl salt-induced and pH-induced aggregation was discussed. The potential use of nanoparticles in biologic applications and the increasing importance of the emerging field of nanotoxicology, which aims to address the safety of engineered nanoparticles, are well known. Therefore, this paper will be an ignited clue for gelatin polypeptide application in engineering nanometer-sized devices.
Materials and Methods
Gelatin (type B, extracted from bovine skin) was purchased from Arcos Organics. HAuCl4 · 4H2O (Au% > 48%) and AgNO3 were from Shanghai Chemical Reagent Co. (Shanghai, China). All chemicals were used without further purification.
Morphology analyses of samples were carried out on a JEOL TEM operating at 200 kV. X-ray diffraction (XRD) patterns of the samples were recorded on a Bruker D8 using CuKα radiation (λ = 1.5418 Å) in the range 10–70°. The film sample was prepared by the metal nanoparticle colloid drying at room temperature under vacuum. The UV–vis absorption spectra were carried out with a Beijing Eraic UV-1201 spectrometer. Fluorolog-1040 (JobinYvoh Horiba) was used for Fluorescence measurements. 1HNMR Watergate water suppression spectra of gelatin solution and gelatin-AuNPs colloid were recorded on a Bruker 400 MHz spectrometer in H2O.
Generation of Gold Nanoparticles
Gold nanoparticles (AuNPs) with various diameters were prepared using gelatin as reducing/stabilizing reagent. The aqueous gelatin solution with different solid concentrations (100 mL) was heated to 80°C with vigorous stirring, then 2.0 mL of HAuCl4 solution (0.4 wt%) was added rapidly. The mixed solution was stirred continually for 4 h at 80°C, after which a red gelatin-AuNPs solution was obtained.
To obtain the information on AuNPs stabilized by gelatin, the gelatin backbone was removed by hydrolyzing gelatin-AuNPs in 2 M HCl for 24 h under reflux. Then the solution was characterized with UV–vis and TEM.
Generation of Silver Nanoparticles
Silver nanoparticles (AgNPs) with various diameters were prepared using gelatin as reducing/stabilizing reagent. The aqueous gelatin solution with different solid concentrations (100 mL) was heated to 80°C. A volume of 2.0 mL of the AgNO3 solution (0.4 wt%) was added rapidly with vigorous stirring. After silver nitrate was added into gelatin solution, the color of the solution changed from faint yellow to white at once. This is due to the formation of complex ion of gelatin and silver (I) . The reaction mixture is under the dark environment to avoid the light. The mixed solution was stirred vigorously for 8 h at 80°C, at which a gelatin-AgNPs solution was obtained.
Generation of Non-Spherical Gold Nanoparticles
The aqueous gelatin solution with different solid concentrations (100 mL) was heated to 80°C. The 2 mL silver seeds (Synthesis according to the procedure in "Generation of Silver Nanoparticles") was added into the gelatin solution. After 10 min, 2.0 mL of the HAuCl4 solution (0.4 wt%) was added rapidly under vigorous stirring. The mixed solution was stirred vigorously for 4 h at 80°C, after which a purplish red gelatin-AuNPs solution was obtained.
Generation of Silver–Gold Bimetallic Nanoparticles (Ag–AuNPs)
Silver–Gold nanoparticles (Ag–AuNPs) with various diameters were prepared using gelatin as reducing/stabilizing reagent. The aqueous gelatin solution with different solid concentrations (100 mL) was heated to 80°C. When stirred vigorously, AgNO3 solution (0.4 wt%) was added rapidly. The mixed solution was stirred vigorously for 8 h at 80°C. And then, HAuCl4 solution (0.4 wt%) was added under vigorous stirring. The mixed solution turned colorless at once. The colorless solution was stirred vigorously for 4 h at 80°C, after which a purple gelatin-Ag–AuNPs solution was obtained.
Results and Discussion
Generation of Gelatin-Directed Gold Nanoparticles (AuNPs) and Their Interaction Study
Generation of Gelatin-Silver Nanoparticle (AgNPs), Non-Spherical AuNPs and Ag–Au Bimetallic Nanoparticles
In this paper, we reported the generation of Au, Ag and Ag–Au bimetallic nanoparticles by using gelatin as reducing and stabilizing agent, the interaction between nanoparticles and gelatin polypeptides, and shape control of gold nanoparticles, against salt-induced (NaCl) and pH-induced aggregation. This study reveals that metal nanoparticles are not only stabilized by gelatin polypeptides chains but also joined in the self-assembling activities of gelatin polypeptides upon cooling to room temperature. It first showed that the major ingredient in gelatin polypeptide, glutamic acid, acted as reducing agent to biomimetically synthesize noble metal nanoparticles at 80°C. The destruction of the three-dimensional α-helix folding structure by gelatin polypeptide acid degradation could not alter the morphology of nanoparticles, but it made nanoparticles aggregated clusters array (opposing three-dimensional α-helix folding structure) into isolated nanoparticles stabilized by gelatin residues. This is a promising merit of gelatin to apply in the synthesis of nanoparticles. Therefore, gelatin protein is an excellent template to regulate the nanoparticle growth. Gelatin-noble-metal colloids can be also used as an excellent engineering nanometer-sized device in bio-detection, optical devices and so on.
This work was supported by the Natural Science Foundation of China (No. 50772048) and the Natural Science Foundation of China and China Engineering Physics (No. 10776014). The authors thank Dr. Masood Akhtar, School of Chemistry, University of Manchester, United Kingdom, for his helpful discussion.
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