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Current Nanoscience, 2015, 11, 000-000
Microbicidal Tissue Paper Using Green Synthesized Silver Nanoparticles S.C.G. Kiruba Daniela, J. Abiramib, S. Kumaranb and M. Sivakumara,* a
Division of Nanoscience and Technology, Bharathidasan Institute of Technology, Anna University, Tiruchirappalli – 624 024 INDIA; bDepartment of Biotechnology, Periyar Maniyammai University, Thanjavur - 613 403 INDIA Abstract: Microbicidal tissue paper is made by a simple method of impregnating biosynthesized silver nanoparticles with the conventional tissue paper. A number of infections are being transmitted by conventional tissue paper especially through hospitals. Silver nanoparticles were synthesized by green synthetic route using the leaf extract of Eichornia crassipes, further characterized using UV vis spectroscopy exhibiting a SPR peak at 413nm and HRTEM having a size between 20 to 50 nm. We have developed a hybrid tissue paper for control of spreading of hospital infections. The hybrid tissue paper was characterized by X – Ray Diffraction and Field Emission Scanning Electron Microscopy in which the size of the nanoparticles present in the tissue paper was found to be ranging from 20 nm to 50 nm. Antimicrobial activity was evaluated against clinical pathogens - Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and release studies of nanoparticles from the hybrid tissue paper were carried out to ascertain the antimicrobial nature.
Keywords: Biosynthesized silver nanoparticles, Eichornia crassipes, microbicidal tissue paper, release studies. 1. INTRODUCTION Silver nanoparticles based nanoproducts are being developed for their effective antimicrobial activity . Silver based compounds are available for human use especially in Eastern medical systems like Siddha, Unani and Ayurveda. In medicine, silver nanoparticles were used as sensors [2, 3], antimicrobial membranes , and antimicrobial dressings . People in South India were using silver cups for drinking water, milk and other silver based utensils from time immemorial due to their potential ability to kill microorganisms. In our earlier studies we have reported biosynthesis of nanoparticles using weeds like Eichornia crassipes, Ipomea carnea and Dodonea viscose [4-6]. Toxicity of silver nanoparticles is much smaller at low concentrations  making them biocompatible to make antimicrobial products. ZnO nanorods have been found to have antimicrobial activity against common microbes S. aureus and E. coli . Silver nanoparticles impregnated paper exhibits antimicrobial activity [9, 10]. Antibacterial wall paper has been fabricated using ZnO nanoparticles for hospitals for better disinfection . ZnO nanoparticles based paper has been used for photocatalytic degradation of organic dyes . Carbon nanotube, gold nanoparticles and graphene coated papers have been used for simple, cheap biochips and antibacterial products [13-15]. Due to population explosion we are witnessing an unprecedented increase in diseases due to infections caused by pathogens. Among them hospital infections are leading to number of deaths and other serious medical conditions. Hospital infections are mainly spread through contaminated water, cloths, improper disposal of used tissue papers, cotton and other medical waste. Till now nobody has reported the *Address correspondence to this author at the Division of Nanoscience and Technology, Anna University-Tiruchirappalli, Tiruchirappalli-620024, India; Tel: +914312407959; Fax: +914312407999; E-mail: [email protected]
fabrication of biosynthesized silver nanoparticles based tissue paper. We have developed nanoparticles based antimicrobial tissue paper to immobilize pathogens when they come into contact with the tissue paper so as to prevent spread of harmful pathogens by conventional tissue paper. 2. METHODS Silver nanoparticles were synthesized using the leaf extract of Eichornia crassipes (Water hyacinth) as reported . Eichornia crassipes leaves are used to prepare plant leaf extract by taking 20 grams of thoroughly washed and finely cut leaves in a round bottom flask having 100 mL sterile distilled water. The mixture is boiled for 2h in reflux. The broth extract is filtered using filter paper and is kept at 4oC for further work. 10 mL of leaf broth is added to 90 mL of a 1mM solution of AgNO3 for reduction of silver ions and the reduction reaction temperature is optimized at 90oC. Synthesized silver nanoparticles were characterized using UV Vis absorption spectroscopy and High Resolution Transmission Electron Microscopy (HRTEM). HRTEM was done using FEI TECHNAI at 200 kV. For UV Vis absorption spectroscopy, samples were taken in a quartz cuvette and measured in a JASCO V 650 spectrophotometer containing double beam in identical compartments each for reference and test solution from 200 nm to 900 nm. Conventional tissue paper is dipped in silver nanoparticles solution and kept overnight. The silver nanoparticles impregnated tissue paper was dried in air and subjected to analysis. XRD analysis and FESEM – EDX (Field Emission Scanning Electron Microscopy-Energy Dispersive X-ray analysis) analysis of the silver nanoparticles impregnated tissue paper was taken. FESEM was done using FESEM Hitachi S - 3690. Antimicrobial activity of the silver nanoparticles impregnated tissue paper was evaluated against three clinical pathogens - Escherichia coli, Staphylococcus © 2015 Bentham Science Publishers
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aureus and Pseudomonas aeruginosa isolated and obtained from nearby hospital in Tiruchirappalli, India by Disk Diffusion Assay using antimicrobial and control tissue paper cut into standard diameter of 6 mm. Antimicrobial and control tissue paper was soaked in double distilled water and UV Vis absorption spectroscopic analysis was carried out at regular time intervals to check the release of metal nanoparticles from the tissue paper. 3. RESULTS AND DISCUSSION Eichornia crassipes leaves are used as a biosource for synthesis of nanoparticles instead of cash crops like Green Tea, Rose, since it is a notorious weed prevalent over South East Asia and other parts of the world. The plant requires very little care and can be produced in large amount in little time for up scaling the synthesis. Silver nanoparticles biosynthesized by using Eichornia crassipes leaf extract exhibites surface plasmon resonance peak at 413 nm (Fig. 1) which is well corroborated with an earlier report . The UV peak is narrow which describes the narrow size range of nanoparticles which is not the case if the peak is broad as
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observed by HRTEM. HRTEM imaging of the biosynthesized nanoparticles revealed the monodisperse and spherical nature of the nanoparticles (Fig. 1) with size of 20-40 nm. Silver nanoparticles impregnated tissue paper is slightly reddish brown in colour when compared to control tissue paper which is white in colour (Fig. 2A). In an earlier report  in which sonication was used as a tool to coat silver nanoparticles on the surface of paper to make it antimicrobial, a colour change from white to brown and grey was observed. XRD analysis revealed the presence of a peak at 34.74° corresponding to the  direction of silver nanoparticles in antimicrobial tissue paper which is absent in control tissue paper (Fig. 2B). Silver nanoparticles embedded in the tissue paper surface appear to be spherical in shape as seen in FESEM. The size of the silver nanoparticles with the tissue paper matrix as observed under FESEM (Fig. 3) was found to range from 20 nm to 50 nm. EDX analysis was done to ascertain the elemental composition of the nanoparticles. The weight percentage of silver is found to be 1.61% (Fig. 3). The silver nanoparticles concentration in the hybrid tissue paper is much smaller, so that it may be non toxic but kills microorganisms.
Fig. (1). UV vis absorption spectroscopy (A) and High Resolution Transmission Electron Microscopy images at 100 nm (B) and 2 nm (C) of silver nanoparticles biosynthesized using Eichornia crassipes leaf extract.
Fig. (2). A) Colour variation between control (a) and antimicrobial tissue paper (b) due to the impregnation of silver nanoparticles, B) XRD analysis of control tissue paper (a) and antimicrobial tissue paper (b) showing minor peak at 34.74 for the presence of silver nanoparticles which is absent in (b).
Microbicidal Tissue Paper Using Green Synthesized Silver Nanoparticles
Current Nanoscience, 2015, Vol. 11, No. 1
Fig. (3). Field Emission Scanning Electron microscopy images of antimicrobial tissue paper taken at 5X, 30X and 60X resolutions showing silver nanoparticles size of 20 – 50 nm. Energy Dispersive X – ray spectroscopy analysis of antimicrobial tissue paper revealing the presence of Ag having 1.61% weight percentage.
Fig. (4). Antimicrobial activity of bactericidal tissue paper (a - single layer and b - double layer) against pathogens S. aerous, P. aeruginosa and E. coli compared to control tissue paper.
A disk diffusion assay was carried out using control tissue paper and silver nanoparticles impregnated tissue paper against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa with single layer (Fig. 4a) and double layer (Fig. 4b) of tissue papers. Zone of inhibition of 18 mm for both single layer and double layer of microbicidal tissue paper is observed for Escherichia coli, Pseudomonas aeruginosa exhibited zones of inhibition of 23 mm for single layer and 30 mm for double layer of microbicidal tissue paper and for Staphylococcus aureus zones of inhibition are 16 mm for single layer and 26 mm for double
layer of microbicidal tissue paper. Two pathogens - Pseudomonas aeruginosa and Staphylococcus aureus have been more susceptible to the microbicidal tissue paper as layers increase while Escherichia coli have the same zone of inhibition. All the three pathogens have exhibited zones of inhibition which can be attributed to the microbicidal nature of the hybrid tissue paper. Control tissue paper (single and double layer) did not exhibit a zone of inhibition which may be due to the absence of silver nanoparticles. Release studies of the antimicrobial tissue paper were carried out to ascertain that silver nanoparticles are not released into the
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Fig. (5). Release studies of tissue paper and microbicidal tissue paper in Milli-Q water at different time intervals using UV Visible spectrophotometer.
environment from the tissue paper. The UV Vis absorption spectra did not show any peaks from 400 nm to 450 nm for silver nanoparticles but has peaks for organic components which are observed for both control and antimicrobial tissue paper (Fig. 5). The antimicrobial nature of the silver nanoparticles impregnated tissue paper is active when the pathogens come into contact with the tissue paper (contact inhibition) since nanoparticles as such are not released from the microbicidal tissue paper.
The silver nanoparticles impregnated tissue paper was made by a simple method and characterized using XRD and FESEM - EDX. The tissue paper embedded silver nanoparticles exhibited spherical shape. The silver nanoparticles present in tissue paper are of size 20 – 50 nm and weight fraction of 1.61 %. Zones of inhibition 18 mm, 16 mm, 23 mm for single layer and 18 mm, 26 mm and 30 mm for double layer have been observed for microbicidal tissue paper against clinical pathogens - Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa respectively. Further silver nanoparticles are not released back by the microbicidal tissue paper into water as observed by release studies. The microbicidal tissue paper can be used in place of conventional tissue paper for use in hospitals, lavatories where there is a high possibility of infections.
  
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of interest.
S.C.G. Kiruba Daniel would like to acknowledge that this work has been catalyzed and financially supported by the RFRS fellowship of Tamil Nadu State Council for Science and Technology, Government of Tamilnadu, India.
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Received: May 25, 2014
Revised: July 03, 2014
Accepted: July 21, 2014
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