Thermo-chemical induced production of silver nanoparticles (Ag-NPs) and their antimicrobial activity towards human pathogens

 

S. Krishnakumar*, R. Divya, N.R. Kanchana Devi, G. Keerthana, A. Ancy Judi

School of Bio and Chemical Engineering, Department of Biomedical Engineering, Sathyabama University, Chennai 600 119, Tamilnadu, India.

 

ABSTRACT:

Nanotechnology is a fast growing and emerging new technology in recent years due to their extensive application. In current scenario, chemically induced synthesis of silver nanoparticles has played a significant role. In this study silver nanoparticles were produced in de-ionized water and dimethyl sulphoxide (DMSO) using D-galactose as reducing agent at 80^oC. Silver nitrate (AgNO₃) was employed as the metal precursor for the synthesis of silver nanoparticles (Ag-NPs). D-galactose acted as reducing agent for the conversion of silver salts into silver ions under experimental condition. The formation of nanoparticles and Plasmon resonance kinetic energy was determined by UV – visible spectroscopy. The de-ionized water solution of D-galactose was better reductive activity in 400nm whereas DMSO solution of D-galactose in 350nm. The produced nanoparticles were subjected to perform antimicrobial efficacy against selected pathogens. D-galactose induced silver nanoparticles produced in de-ionized water exhibited higher antimicrobial activity against bacterial pathogens. Among the pathogens tested Pseudomonas aeruginosa and Salmonella paratyphi A showed maximum antibacterial activity. No activity was observed towards yeast Candida albicans. In conclusion D-galactose mediated silver nanoparticles using de-ionized water could be cost effective and eco-friendly technique for bulk production. The characterization studies are under progress to understand the size, shape and topography of the nanoparticles. Further biocompatibility and cytotoxicity studies are essential for pharmaceutical and biomedical applications.

 

 

 

1.0 INTRODUCTION:

Nanotechnology is an emerging field of modern nano-science dealing with synthesis and manipulation of particles ranging from 1-100 nm. Now a days nanotechnology have involved in various sector such as health care industry, cosmetics and  food, environmental, biomedical sciences, chemical industries, drug delivery and  catalysis applications.^1-3  Nanobiotechnology is a new branch of nanotechnology that integrates principles of biology with physical and chemical protocols to produce small particles (nano-sized) with specific functions.⁴ 

 

Nanobiotechnology is an important area of current research by the researchers for the synthesis of NPs with different sources of biological, chemical and physical methods to produce different size, shape and morphology.^5-7 There are different methods have been developed for the production of metallic nanoparticles especially silver nanoparticles. The most important current method is crystallization of nanoparticles in micro-emulsions using a variety of chemicals as precursors and surfactants as stabilizing agents.⁸ In recent years there is a growing need to develop cheap and eco-friendly processes, which do not involve any type of toxic chemicals in the synthesis procedures. Water is an eco-friendly universal solvent and polysaccharides as capping/reducing agents for the production of silver nanoparticles. Synthesis of starch silver nanoparticles (SSNPs) was carried out in a gently heated system with starch as capping agent and β-D-glucose as reducing agent.⁹

 

Although biosynthetic pathway of nanoparticle was considered to be cost effective and eco- friendly but, relatively time consuming process.¹⁰ Chemical or physical methods of synthesis of nanoparticles are normally used because they are readily available in large quantities and can be used to synthesize in large amounts with relatively minimum time. Chemical reduction technique is the most frequently adopted method for the preparation of silver nanoparticle as a stable, colloidal dispersion in water or other organic solvents. The reduction of silver ions (Ag⁺) with aqueous solution usually yields colloidal silver with the particle diameter size of several nanometers.¹¹ In this context the present research has been initiated for the production of silver nanoparticles using D-galactose dissolved in deionized water and DMSO. D-galactose acts as reducing agent and stabilizing agent for the transition of silver nitrate into silver ions. The formation of silver nanoparticles was confirmed by ultraviolet-visible (UV-Vis) spectroscopy.  The thermo-chemically produced silver nanoparticles were subjected to perform antimicrobial assay against selected pathogens.

 

2.0 MATERIALS AND METHODS:

2.1 Chemicals:

All chemicals and Hi-media were procured from Merck India Private Ltd and Hi-media Private Ltd., Mumbai, India.

 

2.2 Test pathogens:

The produced silver nanoparticles were subjected to antimicrobial assay against selected pathogens. Bacterial pathogens such as, Escherichia coli, Pseudomonas aeruginosa, Salmonella paratyphi A, Bacillus subtilis, S. aureus, and yeast C. albicans were used for the antimicrobial assay. The tested pathogens were maintained and stored as pure culture in our Microbiology laboratory.

 

2.3 Thermochemical induced production of silver nanoparticles (Ag-NPs) by deionized water:

In this technique, D-galactose has been used as a reducing and stabilizing agent in de-ionized water for the production of silver nanoparticles. Analytical grade of silver nitrate (AgNO₃) as a starting materials and D-galactose as a reducing material was used for the production of silver nanoparticles under high temperature. Fifty milligrams of D-galactose have been taken in 100 ml of de-ionized water in 250ml of Erlenmeyer flask by continuous stirring using clean glass rod to dissolve completely. Then 17 mg of silver nitrate was accurately weighed and transferred into the reaction flask. The reaction mixture was continuously heated to 80^oC using thermo-regulated heating mantle until the colour change was observed from clourless to yellowish brown indicate the synthesis of nanoparticles. The formation of silver nanoparticles and Plasmon resonance absorption was measured by following UV-visible spectroscopy analysis.

 

2.4 Thermochemical induced production of silver nanoparticles (Ag-NPs) by DMSO:

In this method, D-galactose has been used as a reducing and stabilizing agent in DMSO for the production of silver nanoparticles. Analytical grade of silver nitrate (AgNO₃) as a starting materials and D-galactose as a reducing material was used for the production of silver nanoparticles under high temperature. Fifty milligrams of D-galactose have been taken in 100 ml of de-ionized water in 250ml of Erlenmeyer flask by continuous stirring using clean glass rod to dissolve completely. Then 17 mg of silver nitrate was accurately weighed and transferred into the reaction flask. The reaction mixture was continuously heated to 80^oC using thermo-regulated heating mantle until the colour change was observed from clourless to yellowish brown indicate the synthesis of nanoparticles. The formation of silver nanoparticles and Plasmon resonance absorption was measured and confirmed by following UV-visible spectroscopy analysis.

 

2.5 UV-Visible spectroscopy analysis:

The thermochemical reduction of silver ions was monitored by measuring the UV-Vis spectrum of the reaction mixture. UV-Vis spectral analysis was carried out using Systronics type, model -118, UV-Vis spectrophotometer.

 

2.6 Antimicrobial assay:

The antimicrobial susceptibility pattern of silver nanoparticles was evaluated by standard disc diffusion assay method. Commercially available sterile empty discs (Hi-media) with the size of 6mm diameter were impregnated with different concentrations (10 µl, 20 µl, 30 µl, 40 µl and 50 µl per disc) of silver nanoparticles for antimicrobial assay. Sterile Muller Hinton agar (MHA) plates was prepared and seeded over night broth cultures of each test pathogens (10⁸ cells) separately under aseptic condition. The nanoparticle impregnated discs were placed aseptically at the center to center pattern of the Petri plate each other. Triplicates were maintained for each concentration of the nanoparticles towards all the test pathogens to obtain mean zone of inhibition. Negative control was maintained by the disc impregnated with D-galactose solution alone (25µl/disc) to compare the antimicrobial efficiency. The plates were incubated at 35±2^oC for 16 to 18 hours. The different levels of zone of inhibition was measured transversely the disc and recorded in mm in diameter for each tested pathogen.

 

2.7 Statistical analysis:

The antibacterial assay against tested pathogens using silver nanoparticle produced by two different techniques was calculated as mean diameter of zone of inhibition in mm ± standard deviation (mean ± SD).

 

3.0 RESULTS AND DISCUSSION:

Nanobiotechnology is the most important emerging discipline in the field of both nanotechnology and biological science. In the present investigation thermo-chemical induced silver nanoparticles were produced using de-ionized water and DMSO. The reaction mixture was heated at high temperature for the production of nanoparticles. The reaction mixture containing D-galactose induced the colloidal solution which was turned reddish brown for the solvent de-ionized water indicating that the formation of silver nanoparticles is depicted in fig.1. The reaction mixture of colloidal solution was turned greyish brown for the solvent DMSO indicating that the formation of silver nanoparticles is portrayed in fig. 2. The produced nanoparticles were adopted to analyse UV-visible spectroscopy. The surface plasmon resonance of nanoparticles with optical density of colloids prepared using D-galactose by de-ionized water and DMSO is presented in fig. 3 and 4 respectively. The reaction mixture showed a surface plasmon resonance absorption band with a maximum peak of 400nm and 350nm respectively. The result shows that the silver nanoparticles were formed under experimental condition over a period of reaction time at high temperature. The produced nanoparticles were stored in screw capped tube under room temperature for further studies.

 

Fig. 1  D-galactose induced production of sivernanoparticles using de-ionized water; A) Before; B)  After

 

Fig. 2  D-galactose induced production of sivernanoparticles using DMSO; A) Before; B) After

 

Fig. 3 UV-visible spectroscopy analysis of Ag-NPs produced by D-galactose in de-ionized water

 

Fig. 4 UV-visible spectroscopy analysis of Ag-NPs produced by D-galactose in DMSO

Table 1 Antimicrobial activity of silver nanoparticles produced by D-galactose with de-ionized water

Values are the average of triplicates; NA – No activity

 

Table 2 Antimicrobial activity of silver nanoparticles produced by D-galactose with DMSO

Values are the average of triplicates; NA – No activity

 

Plate 1 Antimibacterial activity of silver nanoparticles produced by D-galactose with deionized water; A) Salmonella paratyphi A; B) Staphylococcus aureus

 

Antibacterial properties of silver are predicted since 1000 B.C., when silver vessels were used to preserve water. The first scientific investigation describing the medical use of silver reported that the prevention of eye infection in neonates in late 18^th century. Later during 1901 it was reported for antisepsis for internal organs. In older days silver has been used against various diseases and found to use as antiseptic and antibacterial agents.¹² The actual mechanisms of antibacterial activity of silver nanoparticles are not clearly understood and are yet to be evaluated on specific microbes. Early research on silver and their leads reported that silver strongly inhibit bacterial growth by the suppression of respiratory enzymes and electron transport components through intervention with DNA functions.¹³ Silver nanoparticle exhibit antimicrobial activity against human pathogens¹⁴ and plant pathogens.¹⁵ Silver nanoparticles are rendering to interact physically with the cell surface of various Gram positive and Gram negative bacteria. This is predominantly in the case of Gram negative bacteria where the adhesion and accumulation of AgNPs to the bacterial surface. This is confirmed by the present study where the Gram negative bacteria are more susceptible than the Gram positive bacteria and yeast pathogens studied. Early studies have reported that the AgNPs can damage cell membranes leading to structural changes and provide bacteria more permeable.¹⁶

 

Antimicrobial activity of D-galactose induced silver nanoparticle synthesized  using deionized water and  DMSO is portrayed in table 1 and2 respectively. Plate 1 depicted antibacterial activity of synthesized silver nanoparticles. Among the pathogens tested bacterial pathogens are more susceptible than the fungal pathogen. The produced silver nanoparticles showed highest antibacterial activity against Gram negative bacteria namely Pseudomonas aeruginosa  and Salmonella paratyphi A  with the inhibition zone of 16mm for nanoparticle produced using D-galactose as a reducing agent by deionized water and DMSO. No activity was reported by silver nanoparticles produced by both deionized water and  DMSO against yeast Candida albicans. Whereas Krishnakumar et al¹⁷ reported that starch mediated production of  silver nanoparticles exhibit highest antimicrobial activity against Candida albicans than the bacterial pathogens.

 

4.0 CONCLUSION:

Thermo-chemical induced production of silver nanoparticles was performed by the reduction of silver salts into silver ions using D-galactose. Silver nanoparticles were produced by deionized water and DMSO under high temperature using silver nitrate as precursor and D-galactose as a reducing and stabilizing agent. The formation of nanoparticles was determined by UV–visible spectroscopy where surface plasmon absorption maxima can be observed at 400 nm. These NPs have significant inhibitory effects against selected Gram negative bacterial pathogens than the Gram positive and yeast pathogen. This study could be suggested that silver nanoparticles produced by deionized water using D-galactose as cost effective and safe antibacterial agents in a diverse range of pharmaceutical ingredients as nanomedicine to treat bacterial diseases.

 

5.0 ACKNOWLEDGMENT:

The authors are grateful to management of Sathyabama University, Department of Biomedical Engineering, School of Bio and Chemical Engineering, Chennai, Tamil Nadu, India for providing all facilities to complete this research successfully.

 

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Accepted on 25.03.2017           © RJPT All right reserved