biosynthesis of gold nanoparticles using peptides · 2020. 5. 20. · fig.2.representative tem...

GSJ: Volume 8, Issue 5, May 2020, Online: ISSN 2320-9186

www.globalscientificjournal.com BIOSYNTHESIS OF GOLD NANOPARTICLES USING PEPTIDES NevkaaDooshima Ngulianga1, Carole Perry2

1: Department of Microbiology, University of Agriculture Makurdi Nigeria email: [email protected] 2: Interdisciplinary biomedical research laboratory, Nottingham Trent University United Kingdom KeyWords Gold, nanoparticles,Peptides,surface plasmon resonance,synthesistransmission electron microscopy, Uv-vis spectroscopy,

ABSTRACT The applications of gold nanoparticles in pharmaceutical and biomedical areas is important, as seen in their use in antibacterial fields, The synthesis of Noble Metal nanoparticles and gold in particular has proven to be useful as molecular-specific probes, bio imaging, cancer di-agnostics and therapeutics in the biomedical field. Several methods exist for the synthesis of gold nanoparticles but these methods involve harsh chemicals and are not environmentally friendly. This study presents a simple environmentally friendly method for synthesis of gold nanoparticles using peptides in aqueous media and room temperature. The morphology and size of the nanoparticles is determined by Transmission electron microscopy and range from 7 to 50nm which corresponds with the surface plasmon resonance bands as characterized by Uv-vis spectroscopy. The binding properties of peptides to gold nanoparticles and surface characterization was determined by Fourier transform- infra red and thermogravimetric analysis. All peptides synthesized gold nanoparticles due to the presence of tyrosine and lysine amino acids. This simple one pot method may be adopted for synthesis of gold nanoparticles for biological applications. Gold Nanoparticles (Au NPs) study is well established in the nano and biotechnological fields, this is because they are biocompatible, chemically stable, and possess optical and electronic properties [1, 2]. Among the well-studied nanoparticles, gold is inert and less toxic which facilitates its use as drug delivery [3,4,5]. The wide application of gold nanoparticles is largely due to the unique surface plasmon resonance (SPR) properties, a feature that can vary with the particle shape and size [6,7].The synthesis of Noble Metal na-noparticles and gold in particular has proven to be useful as molecular-specific probes, bio imaging, cancer diagnostics and therapeu-tics in the biomedical field [8]. The applications of gold nanoparticles in pharmaceutical and biomedical areas is important, as seen in their use in antibacterial fields [9, 10]. There are also several studies on the non-covalent binding of gold nanoparticles with antibio-tics which have shown enhanced antibacterial activities,[9-14] all aimed at combating the challenge of antibiotic drug resistance by bacteria.The Various modifications on Au NPs using several biomolecules such as peptides, proteins, and nucleic acids, combined with the original properties of gold provide unique biological functions [15] Monodisperse gold nanoparticles can be synthesised with sizes ranging from 1-100 nm, different methods are employed for synthe-sis of monodisperse gold nanoparticles with different sizes. GNPs are fabricated by the reduction of a gold salt with a stabilizing agent that further prevents aggregation of the particles. Two common methods of gold nanoparticles synthesis are reduction of the gold chloride (HAuCl4) salt using sodium citrate in an aqueous medium where the citrate acts as the capping agent and stabilization agent [16]. Another method is by reduction of gold chloride with sodium borohydride in the presence of thiol capping agent [17].The so-dium borohydride method involves the use of harsh chemicals such as, cetyltrimethylammonium bromide, CTAB or tetraoctylammo-nium bromide OTAB) and at times toluene as an organic solvent to synthesise spherical gold nanoparticles. An alkane thiol is ex-changed with the added OTAB which then keeps the nanoparticles of 5-6nm in diameter stabilized [18, 19]. The use of biomolecules to assist the synthesis and stabilization of gold nanoparticles is proving to have high potential applications in a variety of research areas [20, 21]. Among the earliest research group to work on the potential use of peptides for GNPs fabrication was Brown [22, 23]. Their research formed a method of cell surface display (CSD) to identify and screen for peptides on E.coli grown

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in gold salt solution in the presence of sodium ascorbate as a reducing agent [24, 25]. They identified three peptides binding to E.coli surface by this method.Studies have been carried out on some already identified peptide sequences with known affinity for gold sur-faces. A3 (AYSSGAPPMPPF) identified by phage display from the research work of [15] and found to bind to silver nanoparticles, has been extensively studied for its gold binding capacity as well [15]. A3 peptide was able to reduce the gold nanoparticles and further stabilize them forming an average of 12.5 nm diameter size. Yu et al., 2011 studied the binding activity of the peptide (AYSS-GAPPMPPF) and other closely related peptides on two different types of Gold surfaces, their results gave reasonable insight on de-sign of new peptides for synthesis of gold nanoparticles with new morphologies [25]. It is possible to synthesise Gold nanoparticles by the reduction of chloro auric acid in the presence of peptides, where the formation of chloroaurate complexes by the amino acids such as tyrosine enhances reduction of gold [26]. Tan et al., 2010 in their study formulated design rules for shape and size controlled synthesis of gold nanoparticles by peptides from properties of the 20 natural amino acids for the ability to reduce and bind to gold in aqueous solution. This study revealed that the ability of peptide sequences to reduce gold depends on the presence of certain reduc-ing amino acids residues with tryptophan being the fastest reducing amino acid, the net charge of the peptides can as well have ef-fect on the nucleation and growth of gold nanoparticles and neighbouring amino acid residues [27]. Developing approaches to coat gold nanoparticles with peptide will increase the efficiency of their biomedical applications [2]. The use of peptides as biocompatible molecules therefore becomes important for synthesis of metal nanoparticles, because then the synthesis can occur at neutral pH conditions, in aqueous media and at room temperature [27]. Another advantage will be the vast majority of identified peptide sequences available to explore for potential applications [27] Table 1: Peptides used in the study and their general properties as determined byinnovagen technology, all amino acids are L- amino acids. Name if available

Sequence Molecular Weight (g/mol)

Net charge pH7

Iso electric Point (pH)

Water solubility

Extinction coefficient M-1cm-1

A3 PMB

AYSSGAPPMPPF KTKKKFLKKT AYKTKKK

1221.4 1249.61 866.07

0 6 4

5.93 11.21 10.63

Poor Good Good

1280 0 1280

Materials and methods Gold nanoparticles synthesis A simple synthesis method for gold nanoparticles at room temperature and near neutral pH was adopted for this study,a synthesis method was adopted based on a modified previous method by Slocik et al., 2005 [27].10 µL of a 10 mg/ml peptide sample dissolved in ddH20 was added to 500 µL of 0.1 M of HEPES buffer (pH 7.2) in a 1.5 mL microfuge tube. A 2.5 µL of a 0.1M HAuCl4 is further added to the solution and stirred for 30 min . All reactions were carried out at room temperature, while the concentration of HEPES buffer and Chloroauric acid remained same, the peptide concentration was varied for A3 (8.18 mM), AYKTKK (23 mM) and PMB (8 mM). Gold ions reduction and nanoparticles formation was monitored by UV/Vis spectroscopy using a Varian caryBio50 UV-vis spec-trophotometer at a wavelength of 200 to 800 nm, the wavelength of SPR bands of gold nanoparticles is expected within this range. Size and morphology characterization of nanoparticles The size of the gold nanoparticles synthesized by peptides where measured by Dynamic Light Scattering (DLS) with a Malvern Zeta-sizer (Nano-S). The particle morphology and size where measured on a Joel 2010 Transmission Electron Microscopy (TEM) operated at an accelerating voltage of 200 kV. The samples for TEM were prepared by pipetting drops of gold nanoparticle solution onto a 3 mm carbon-coated TEM copper grid and air drying at room temperature for 15 min. The extra solution was removed from the grid by blotting with a tissue paper. A minimum of 50 particles were measured with an image J software for analysis of the particle size dis-tribution, mean diameter and standard deviation.



Characterisation of the surface properties of Gold nanoparticles The binding of peptide to the gold surface was measured by FTIR spectra using a Nicolet 6700 Fourier Transform infrared (FTIR) Thermo scientific at 4 cm-1 with 128 scans. The FTIR sample was prepared by weighing out 198 mg of KBr and 2 mg of the freeze

dried peptide/gold nanopar-ticles. Thermogravimetric (TGA) analysis of peptide capped gold nanoparticles and peptide powder was per-formed using a Mettler Tole-do (TGA/SDTA851e) over a temperature range of 30 to 700oC at a heating of 10oC min-1 in the presence of air. Results and discussion UV- vis Spectroscopy characterisation The formation of gold nanoparticles was moni-tored by UV-vis spectros-copy. Fig. 1 shows the UV-vis spectra of the gold

nanoparticles synthesised at room temperature showing the characteristic surface plasmon resonance (SPR) bands. Fig. 1 Representative UV-vis spectra of gold nanoparticles synthesised at room temperature in the pres-ence of (curve a) polymyxin B peptide after 30 min and completion of synthesis, SPR band is seen at 610 nm, (curve b) polymyxin B after 15 min of synthesis (SPR at 550 nm) (curve c) A3 peptide with SPR band at 534 nm (curve d) AYKTKKK 525 nm, and (curve e) HEPES buffer having a SPR band at 668 nm. Inset shows the digital photographs of colloidal nanoparticles solution for the different peptides and HEPES buf-fer.



Fig.2. Representative TEM images of gold nanoparticles synthesised in the presence of peptides at room tem-perature (A) A3 has a mean diameter of 10.68 and std. dev. Of 1.390, N= 50 (B) Polymyxin B with mean diame-ter around 28.30 nm, N=54, std. Dev.=11.03 (C) Polymyxin B after 30 min of gold nanoparticles synthesis showing aggregated clusters (D) AYKTKKK with mean diameter of 7.665, std. Dec = 0.8404, N=52 fig. 6 insets are particle size distribution of gold nanoparticles presented as a percentage of the total number of nanopar-ticles counted

Results for the surface characterisation of gold nanoparticles

Thermogravimetric analysis (TGA) was performed to quantify the amount of peptide bound to the gold nano-particles. Figure 3 presents the TGA analysis of peptide without gold and peptide capped gold nanoparticles. To further analyse the binding of peptide to the surface of gold nanoparticles, Fourier transform infrared (FTIR) spectroscopy was performed fig. 3b



Fig. 3(A) TGA analysis of peptide is shown in (curve 1), gold nanoparticles synthesised with Polymyxin B (curve 2), A3 (curve 3) and AYKTKKK curve 4. (B) FTIR spectra of peptide synthesised gold nanoparticles curve 1 represents Polymyxin B, AYKTKKK AuNPs (curve 2) and A3 AuNPs (curve 3).

Conclusion In conclusion, this work has reported a simple biological approach for the synthesis of gold nanoparticles in aqueous media at neutral pH. And room temperature using three peptides. All peptides are able to synthesise gold nanoparticles that range from 7 to 50 nm. The spr bands of the nanoparticles range from 534 nm to 668nm either as disperse or aggregated particles. The ability of the pep-tides to form nanoparticles comes from the presence of tyrosine and lysine amino acids. Acknowledgment The authors wish to thank Tertiary education trust fund Nigeria for providing funding

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