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Synthesis and Antibacterial Activity of gold and silver Nanoparticles against Pathogen species, E. coli and S.aureus
Synthesis and Antibacterial Activity of gold and silver Nanoparticles against Pathogen species, E. coli and S.aureus
Odai N.Salman*,, Duha S. Ahmed,a*, Mohammed O. Dawood
This work involved preparation Gold and Silver nanoparticles by pulsed laser ablation (PLAL) using laser operated at 532 nm wavelength at energy density about 17 J/cm2 for pure gold and 10 J/cm2 for silver metal plate immersed in Deionize Water DW and another aqueous solvent of 5mM Sodium dodecyl sulfate (SDS). The morphology, size and crystalline nature of nanoparticles were estimated by Transmission electron microscopy (TEM) techniques. Absorption spectrum of forming Au and Ag nanoparticles colloidal was calculated using UV-Vis Spectrophotometer. The Zeta potential of synthesized AgNPs and AuNPs was determined at 25°C using Zeta plus and deionized water DW was used as dispersant. Finally, the antibacterial activity of Au and Ag nanoparticles against bacterial species E. coli and S. aureus demonstrate that Ag and Au NPs were more active against Gram negative bacteria than Gram positive bacteria and this was attributed to change in the bacterial cell membranes composition.
Keywords: Gold and Silver nanoparticles, Surface Plasmon Resonance (SPR), surfactants, thiol and sulfhydryl groups, Laser ablation method
Noble metal nanoparticles like silver and gold nanoparticles are represented a resource of great attention because of their good electrical, optical, physical, chemical and magnetic features because of their surface plasmon resonance (SPR) associated features that are basically practical in biology filed [1,2].Pulsed laser ablation in liquids (PLAL) represented a base technique in manufacture of NPs with controlling shape and size. Ablation of noble metals in water environment is consider as a different process to usual chemical reduction processes for obtain colloids of nanoparticles, since this approach represented environmentally friend green procedure with products that commonly without stabilized molecules or additional chemicals. PLAL process can be apply in clean Deioniz Water (DW) or in biological aqueous solvent and can be joined with good establish protocols to improve the sensitivity of classic vibration spectroscope like in the case of surface enhanced raman (SER) phenomena [3,4]
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Controlling of noble metals NPs size that synthesis via Laser Ablation could be achieve by addition particular molecules to aqueous manufacture solution, which physically or chemically interaction with the surfaces of form particles, limiting their increase. Ionic surfactants,  like Cyclodextrins and Sodium Chloride were effectively used to decrease the particle size of noble metals. As well as, the accurate method reducing the increase of the particles remain uncertain [6,7].
Besides, sodium dodecyl sulfate SDS, represented the most qualified surfactants to limit average sizing of AuNPs and AgNPs down to 5nm through laser ablation of NPs. Recently, laser ablation technique permit to synthesis NPs with no difficulty and with no Contaminate by a reduced agent, but coagulation development of atoms leads to broaden size distribution of the NPs and it’s hard to be controlled . Alternative, a perfect metal nanoparticles solutions can be efficiently create, when laser ablation take place in a liquid environmental .Since, noble metals have been used for centuries as bactericidal agents, can be represented by silver, gold and zinc, each with various properties, activity spectrum and their main antibacterial activity were due to to their cytotoxicity against a number of bacteria. Besides, AgNPs and AuNPs can interaction with the functional groups on the cell membrane of bacteria which leads to inactive pathogen .The main characteristics of metallic NPs are large surface energies, specific electronic structure, plasmon excitation and quantum confinement .For AgNPs, it probable consider that the high specific area and high fraction of surface atoms of AgNPs result in high antimicrobial activity when compare with bulk Ag metals. The antimicrobial effects of AgNPs have been used generally in various medical applications for years. Using AgNPs in bone ravels good quality of antimicrobial activity with no existence of cytotoxicity as studies in vitro assessment filed. AuNPs is one of significant applications in the medical filed because of they are easily tacking up by membrane of bacteria. The toxicity of AuNPs against various cell kinds depends on their sizing. Furthermore, the capability of pathogenic bacteria in resist antimicrobial agents is a fact that has emerging in latest years and represents a main health trouble [11-14]. Therefore, in this study we evaluate the synthesis and bactericidal activity of AgNPs and AuNPs against Escherichia coli and Staphylococcus aureus produced by using pulsed laser ablation in liquids, PLAL. Optical spectra and morphology of colloidal of AgNPs and AuNPs prepare by pulsed laser ablation in liquids, PLAL were explored.
Materials and methods
Preparation of silver and gold nanoparticles using PLAL (Pulse Laser Ablation in Liquid)
In this part, silver and gold nanoparticles are synthesized by irradiation the metal target plate by pulsed laser ablation in liquid (PLAL) and a thickness of 1 mm located on the underneath of quartz container contain deionized water DW and additional aqueous solvent of surfactants like (SDS) of 5mM concentration at ambient temperature. Laser ablation can be achieving used focusing production of pulse laser Nd: YAG (Huafei model) working with a repetition rate of 5 Hz and pulse width of 9 ns. Laser ablation was take place with laser operate at 532 nm wavelengths at energy density set in 17 J/cm2 for Au and10 J/cm2 for Ag with a positive lens containing focal length of 10 cm. The spot sizing of the laser beam on surface of metallic plate was 2 mm in width by the distance between the focus lens and the metallic plate at 7 cm. Laser is fixed by a holder on the target at the bottom of container as shown in Fig (1). Each of gold and silver plate was fixing in a container full with 5 ml DW water.
Size and sizing distribution of AuNPs and AgNPs are confirmed by Transmission Electron Microscope (TEM,Carl Zeiss – Germany – EM10 C- 100 KV, Daypetronic co., Iran)that produced by PLAL technique. Absorption spectrum of AuNPs and AgNPs colloidal are determined using UV-Vis Spectrophotometer (SHIMADZU UV-1800) with wavelength range 190 Ì´ 1100 nm and the speed of the wavelength scan is up to 2500nm/min. The Zeta potential of synthesized AgNPs and AuNPs was determined at 25°C using Zeta plus(Malvern Instruments Ltd., UK)with the range between -250 mV to 250 mV(sample dependent)and Deionized water (DW) was used as dispersant.
Antimicrobial susceptibility testing had done with quality control strains by using S. aureus and E.coli for appropriate antimicrobials. The antibacterial test is performing by using Disk diffusion method. The sterility paper discs (8mm) soaking with AuNPs and AgNPs are suspend in pure Deionized Water (DW) and let drying at 37°C for 24h in a clean circumstance. Suspension of bacteria is provide by preparing the saline solutions of isolating colonies choose from nutrient agar plate, then agar plates are grow for 18h. 5ml of sterilized nutrient broth is inoculation with a loop full of test organism and incubation in 24h. Then, 0.2 ml from overnight culture of organism are distribute into 9ml of sterility nutrient broth and incubation for 3h to make standard McFarland turbidity using the Spectrophotometer of 600nm anf production the culture with concentration 106 CFU/ml. Surface of (MHA) agar was absolutely inoculate using a clean cotton swab in all directions and rotating the plate. The soaking discs are then placed on the inoculation agar and incubating at 37°C for 24 h. After incubation, the diameter of the growth inhibition zone is measuring by width of the inhibition.
Results and Discussion
Zeta potential analysis
Electric charge on the surface of AuNPs and AgNPs is measuring using Zeta potential. Zeta potential was found to be −28.13 mV for AgNPs and -40.31 mV for AuNPs as shown in Fig (2 a, b). The negative zeta potential confirms the negative charge on the surface of colloidal nanoparticles. The columbic repulsion forces induced by surface negative charge decrease the aggregation and thus contribute to the stability of the green synthesized nanoparticles. The attractive and repulsive of NPs can be evaluated using zeta potential. If the particles get a high positive or negative value of zeta potential, they repel each other and result a stable suspending. Since, the point at which zeta potential reads 0 mV is called zero electric point. Zeta analysis is essential in industries such as ceramics, minerals processing, water treatment, pharmaceuticals etc .
Optical properties of gold and silver nanoparticles by PLAL technique
The unique optical phenomenon of metal nanoparticles, in the UV region enables their easy detection. This property occurs due to surface plasmon oscillation of free electrons. Fig (3) shows the absorption spectra of AgNPs and AuNPs colloidal solutions synthesized by laser ablation technique with wavelength 532 nm and using 5mM SDS and Deionized Water (DW).
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The results reveal the absorption peaks of AuNPs (blue line) and AgNPs (red line) depending on SDS concentration 5mM. In Fig (3), peaks of absorbance tend to increase by addition of SDS, 5mM. Moreover, depending on results the surfactant SDS, 5 mM fabricate high quantity of AuNPs and AgNPs depending on the experiment conditions. Obviously, the surfactant (SDS) playing a significant role in determine stable and size of the AuNPs and AgNPs, because of the extinction of NPs growing is controlling by the distribution and attaching rates of SDS on the nanoparticles. The samples set in (SDS) liquid, the particles were enveloped with surfactant. Since, using 5mM of (SDS) surfactants make distributed width become thin. Extremely negative charge nanoparticles are repelling each other more efficiently, that allow SDS surfactants covering them before connection take place. Since, the kinetics consideration is predictable to bound coalescence of the form clusters, resulting to lesser particles. The surfactants that contact with nanoparticles through the condensation avoid NPs from more coalescence and agglomerated and successfully stabilizing and reducing size of Au and Ag nanoparticles.
Determination of bactericidal activity of silver and gold nanoparticles
The antibacterial assay of AuNPs and AgNPs are evaluated against standards of the E.coli and S. aureusby using Disc Diffusion test with muller hinton agar (MHA) to determine the inhibition zone. The zone of inhibition was measured by the diameter of the inhibition circle as shown in Table 1.
Table 1: inhibition Zones diameters in (mm) of AgNPs and AuNPs impregnated against S. aureus and E.coli bacteria
inhibition Zones diameters in (mm) of AgNPs
inhibition Zones diameters in (mm) of AuNPs
The results in Table 1 show the bacteria are susceptible to the AgNPs and AuNPs. AgNPs illustrate further activity on test pathogens comparison to AuNPs. Besides, AgNPs and AuNPs colloidal reveal good antibacterial activity against tested pathogens as shown in Fig (4a,b). These results showed that AgNPs have higher antibacterial activity than AuNPs due to the relatively inert chemical nature of gold. In Fig (4 a), AgNPs were further activity against Gram negative bacteria than Gram positive bacteria and this was attributed to change in the bacterial cell membranes composition. AuNPs are less efficient against the tested pathogens as reveal in Fig (4a,b). The bactericidal mechanism of AgNPs may consist of variation of thiol or sulfhydryl groups including biomolecules like proteins and electrochemical collapse that penetrate across the cell membranes of bacteria . It is assumed that heavy metals like silver and gold release ions reacting with thiol or sulfhydryl groups (-SH) of proteins and inactivates them. Ionic silver and gold efficiently interact with thiol group of vital enzymes and inactivate bacteria. Since, ionic silver, Ag+ replaced ionc hydrogen H+ of thiol groups that deactivates the proteins by decreasing the permeability which leads to cell dye . It is normally to state that attaching of AgNPs or AuNPs to the bacteria cell depends on the surface area presented for interaction. Nanoparticles have larger surface area presented for interactions, which improve bactericidal effect than the large size particles; therefore they convey cytotoxicity to the microorganisms 
In present, mechanisms of penetrating bacteria using nanoparticles is still now not complete, but propose metals are cytotoxicity and reacting with protein, as a result the metals connect proteins molecules, metals efficiently interact with thiol group of vital enzymes and inactivate bacteria. Moreover, it is suppose that AgNPs and AuNPs binding to functiona groups of protein resultant in proteins inactivation, so that cellular metabolism is inhibiting causing inactivation of microorganisms [17, 18].
TEM analysis confirmed the nanosize of AgNPs and AuNPs prepared by laser ablation in liquids (PLAL) with 5 Mm SDS surfactant solutions. The spherical shape of the Ag and Au nanoparticles is shown in TEM images in Fig (5 a,b), respectively. Besides, in TEM analysis, both AgNPs and AuNPs are dispersed and mostly spherical in shape with obvious morphology of Ag and Au nanoparticles and no aggregation. The average diameter AuNPs range of 20- 30 nm while the average diameters AgNPs range of 40-60nm, respectively.
To summarize, a high purity of silver and gold nanoparticles have been prepared via using pulse Nd: YAG laser operating at 532 nm wavelengths with a repeating rate of 5Hz and pulse length of 9ns in SDS solution. Obviously, the results exhibited, the surfactants (SDS) play a significant role in determination the stabile and sizing of AuNPs and AgNPs as shown in optical properties that affected by laser fluence and wavelength. The negative zeta potential confirms the negative charges on exterior of colloidal nanoparticles because of addition surfactant like SDS solution. Besides, AgNPs and AuNPs are shifting to a lesser size because of surfactant SDS solution. Since, the synthesized Ag and Au nanoparticles contain spherical shape and size distributed is almost Gaussian. Finally, AgNPs and AuNPs colloidal reveal good antibacterial activity against tested pathogens and becomes more active against Gram negative bacteria than Gram positive bacteria and this was attributed to change in the bacterial cell membranes composition. Since, complete bacterial inhibition depends the effect of laser ablation conditions in synthesized AgNPs and AuNPs colloidal and on number of bacteria cells; and concludes that silver and gold nanoparticles contain high bactericidal and potential effects in reduced bacterial growing in practical applications.
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