1. INTRODUCTION:

A variety of synthesis techniques have been developed including chemical reduction of silver ions in aqueous solutions, with or without stabilizing agents, thermal decomposition in organic solvents and chemical and photo reduction in reverse micelles. These methods are not only expensive and also involve toxic, hazardous chemicals which may pose potential environmental and biological risks. The use of leaf extracts, bacteria and fungi for the synthesis of silver nanoparticles offers numerous benefits as they are compatible for biological application with zero chemical toxicity on the application and environment. They are eco-friendly and well-suited for pharmaceutical and biomedical applications as they do not contain toxic chemicals in the synthesis process.

This kind of environmentally sustainable synthesis process has led to few bio-mimetic approaches which refer to applying biological principles in materials formation such as bio reduction. Numerous inorganic nanomaterials have been synthesized by bio reduction processes using various microorganisms like Pseudomonas stutzeri, Verticillum, Fusarium oxysporum, Aspergillus flavus etc. The usage of botanical materials for the synthesis of nanoparticles shall be advantageous over other biological processes since the tedious process of maintaining the microbial culture is avoided. Moreover this method is cost effective over chemical and physical methods and environment friendly. Since there is no need to use high pressure, energy, temperature and toxic chemicals, the technology is very simple. Neem leaves extract was chosen for the present study, because-

(i) Neem is a quite commonly available plant and abundant in nature

(ii) Excludes addition of external stabilizing agent during synthesis and

(iii) It offers synergistic effects to enhance the antimicrobial properties of the synthesized silver nanoparticles (one of the major end uses).

The UV spectroscopy analysis proved that the formed particles were silver nanoparticles in the wavelength ranging from 405 nm to 425 nm. The X-ray diffraction analysis confirmed the formation of crystalline silver nanoparticles. The Scanning and Transmission Electron Microscopy techniques revealed that the morphology and size of the nanoparticles were strongly dependent on the process parameters like reductant concentrations, reaction pH, mixing ratio of the reactants and interaction time of the reactants. Since water medium is the common breeding ground for many pathogens, the presence of bacteria is the main indication of water contamination. It is recommended that any water intended for drinking should not contain fecal contamination and the total coliform counts shall be reduced. The removal or inactivation of pathogenic microorganisms is the last step in the treatment of water. In conventional methods, chemical and physical agents, such as chlorine and its derivatives, AgNO3, ultraviolet light and radiation, are commonly used. The application of nanoparticles for water disinfection is relatively new. This will be a cutting edge technology because of the high reactivity due to the large surface to volume ratio of nanoparticles. This single aspect will play a crucial role in water purification as drinking water is an essential commodity for the well being and the survival of the society. An attempt is made through this study to establish the antibacterial properties of the polyurethane foam coated with synthesised silver nanoparticles. The common polyurethane (PU) foams can be coated with Silver nanoparticle by overnight exposure of the foams to nanoparticle solutions. Cyclic washing and air-drying yields uniformly coated PU foam. The coating of Nanoparticles is stable on the foam and is not leached by continuous water contact. These coated foams can be used as a water filter. E. coli, the most common bacteria in the drinking water was chosen in this study as indicators of fecal contamination. The input water had a bacterial load of 10⁵colony-forming units (CFU) per mL in which contact time of PU foam in water was of the order of 10 minute, the output count of Escherichia coli got significantly reduced. The low cost and high efficiency and the simplicity of this technology will be be of great attraction in developing countries for countering waterborne diseases and public health.

2. EXPERIMENTAL:

2.1. Synthesis of silver nanoparticles:

The Fresh Neem leaves were thoroughly washed and finely cut and again washed with distilled water. 25 g of the leaves were added to 100 mL of deionised water and boiled for one hour in a water bath. The mixture was filtered to obtain aqueous extract of 20 % concentration. The prepared neem leaf broth was interacted with 0.01 M AgNO₃ solution at 1:4 mixing ratio to make up 100 mL volume in 250 mL Erlenmeyer flask for synthesis of silver nanoparticles. Dilute Ammonium Hydroxide (NH₄OH) was used to maintain the pH of the reaction mixture in the range of 8. The flask was kept for 4 h in rotary shaker at 80 rpm to achieve homogenous reaction. The dry powders of the silver nanoparticles were obtained after 4 h of reaction period. The colour change from silver nitrate solution to reduced silver nanoparticles is indicated in Fig.1.. The broth containing silver nanoparticles was centrifuged at 7,000 rpm for 15 min. Thereafter, the particles were re-dispersed in sterile distilled water to get rid of any uncoordinated biological molecules. The same process was repeated three times to ensure better separation of free entities from the metal nanoparticles. The purified particles were dried using a hot air oven up to 70^oC. Solid silver nanoparticles obtained are shown in the Fig.2. A rough prediction of the reaction that takes place during the process has been given as Neem broth + AgNO₃ solution → Ag nanoparticles

2.2. Fabrication of silver-coated polyurethane foams:

Polyurethane (PU) foams of 10cm x 10cm x 5mm size was washed by deionised water, dried and then soaked in silver colloidal solutions for a day. In order to eliminate the adsorbed substances and impurities the sheets were washed repeatedly with water. It was found the coatings of nanoparticles were stable on the foam and not washed away by water on repeated washing. Also, the morphology of the foam was retained even after coating. The binding of nanoparticle was due to its interaction with the nitrogen atom of the PU. The following reaction takes place when PU foam is soaked in Ag nano solution:

- NH-(C=O)-O- + Ag → -N^-Ag⁺-(C=O)-O-

2.3. Microbiological Experimentation:

The contaminated water was analysed and found to contain following bacteria like E.coli, staphylococcus, clostridium, streptococcus species like dysnteriae, pyogenes, salmenela species, proteus species, pseudomonas species, and shigella species. E.coli, Staphylococcus aureus (gram positive bacterium) and Pseudomonas aeruginosa (gram negative bacterium) were selected as major indicators of bacterial contamination of water. Nutrient broth was used as the growing medium for the microorganisms. Bacteria were grown aerobically in nutrient broth at 30^oC for 15 h. The cultures were centrifuged, the cells were washed and suspended in distilled water, reaching a final concentration of 1 x 10⁵ to 1 x 10⁶ CFU/mL.

2.4. Test Tube Analysis:

For the test tube analysis, 20 mL of E.Coli, Staphylococcus aureus and Pseudomonas aeruginosa cells each suspended in sterile water was taken in sterilized falcon test tubes separately. 10cm x10cm x 5mm pieces of the foam was put into the tubes. These tubes were incubated in an orbital shaker at 30°C and 100 rpm. After 30 min, the foam samples were taken out from the tubes. The liquids were collected at the end of incubation; plating was done with this treated water by serial dilution method for 10⁰, 10^-3 and 10^-5 dilutions with trytpic soy agar (TSA). Plating was also done for the initial CFU count and with uncoated PU-treated solution. For every dilution, 10 µL of the solution were plated. Plating was done by the wet plate method. The bacterial colonies were counted after 48 hours of incubation at 30°C.

3. RESULTS AND DISCUSSIONS:

3.1. Characterization of silver nanoparticles:

a) XRD Analysis

X-Ray Diffraction analysis is the most useful method by which X-Rays of a known wavelength are passed through a sample to identify the crystalline structure. The X-Rays are diffracted by the lattice of crystal to give a unique pattern of peaks of 'reflections' at different angles and of different intensity, just as light can be diffracted by a grating of suitably spaced lines. The phase identification of the silver nanoparticles was also carried out by X-ray diffraction method. The sample was grounded using a mortar and pestle into powder. X-ray powder diffraction measurement was carried out by using Philips X`Pert MPD powder diffractometer with long fine focus Cu anode operated at 40 KV and 30 mA in Bragg-Brentano geometry.

Fig.3 XRD analysis of the silver nanoparticles

The X-Ray detector moves around the sample and measures the intensity of these peaks and the position of these peaks [diffraction angle 2θ]. The highest peak is defined as the 100% peak and the intensity of all the other peaks are measured as a percentage of the 100% peak. The powder XRD data were obtained in the 2q range from 10° to 80° in step-scan mode with 2q step of 0.02°. The X-ray diffractometer was calibrated by means of external silicon standard, SRM 640a. The diffraction pattern indicated that the sample is the silver nanoparticles. The conversion of silver nitrate to silver nanoparticle was greater than ninety percent and smaller peaks contributed to neem extract impurity. The XRD pattern of the mixture is shown in the Figure.3.

b) TEM Analysis

The silver nanoparticles synthesised from AgNO₃ solution and Neem leaf broth was analysed for its size in TEM. Mostly spherical and near spherical shapes of silver nanoparticles can be observed for interaction time 4 h in 20 nm size range. The image of the silver nanoparticles is shown in the Figure.4.

Fig.4 TEM images of the silver nanoparticles

c) SEM Analysis

To gain further insight into the features of the silver nanoparticles, analysis of the sample was performed using SEM method. The freeze-dried silver nanoparticles were mounted on specimen stubs with double-sided adhesive carbon tape, coated with Au/Pd alloy to make the surface conducting in a sputter coater (BAL-TEC SCD-005), and examined under a Philips XL-30 SEM at 12-16 kV with a tilt angle of 45^o. Scanning electron microscopy provided the morphology and size details of the silver nanoparticles. The experimental results showed that the diameter of prepared nanoparticles were in the range of 20 nm to 100 nm. The SEM image of the silver nanoparticles is given in Fig.5.

Fig.5 SEM image of a single silver nanoparticle

d) UV Analysis

Figure.6 shows the UV spectra of the silver colloid in the range 400 nm – 425 nm. UV absorption spectra has proved to be quite sensitive to the formation of silver colloids because silver nanoparticles exhibit an intense absorption peak due to the surface Plasmon (it describes the collective excitation of conduction electrons in a metal) excitation. The absorption band in visible light region (350 nm – 550 nm, Plasmon peak at 425 nm) is typical for silver nanoparticles. The Plasmon peak and the full-width of half-maximum (FWHM) depends on the extent of colloid aggregation. To monitor the stability of silver colloid, the absorption of the colloid was measured after different periods of time. There was no obvious change in peak position for a month, except for the increase of absorbance. As the particles increase in the size, the absorption peak usually shifts toward the red wavelengths. The stable position of absorbance peak indicates that newly formed particles do not aggregate.

Fig.6 UV Spectra of Silver Nanoparticles

There are two types of PUF material shown in Fig.7.. The first picture (Fig.7A.) shows pure polyurethane foam and it is white in colour. The second picture (Fig.7B.) is silver nanoparticles coated polyurethane foam and the colour changes from white to golden yellow.

(A) (B)

Fig.7. Pure polyurethane (PUF) (A) and PUF coated with silver nanoparticles (B).

4. Microbiological Results:

After a contact time of 30 min with silver nanoparticles coated PU, the bacterium quantity detected was zero in the treated water. For E. Coli, Staphylococcus aureus and Pseudomonas aeruginosa strains, the output count was nil for all the dilutions. The water treated with the Control sample (pure PU) showed substantial growth on the plates. Initial water sample (input) showed overgrowth in almost all the cases. The bacterium count was decreased in the output water after passing through the coated foam for E. coli, Staphylococcus aureus and Pseudomonas aeruginosa. This was checked for input loads of 1 x 10⁵and 1 x 10⁶CFU/mL. There was no growth below the PU coated with nanoparticles while growth was seen in case of pure PU, which again confirms the antibacterial property of PU coated with silver nanoparticles. Figure 8A shows the bacterial growth seen in water treated with pure PUF. Figure 8B and 8C show the immersion of Silver nanoparticles coated PUF and the growth of bacteria in that treated water.

Fig. 8: (A) Bacterial growth seen after plating treated water with pure PUF; (B) FALCON test tube containing silver nanoparticles coated PUF in contact with bacterial water; (C) Zero bacterial growth seen in treated water with silver coated PUF after plating

The following table gives an idea about the antibacterial effect of silver coated polyurethane foam on various kinds of bacteria

Table.1. Reduction in bacterial counts in PU foam coated with silver nanoparticles

S No	Bacterial strains	Polyurethane foam (PU)*
S No	Bacterial strains	Pure PU	Coated PU
1.	Staphylococcus aureus (Gram positive bacterium)	6.0 x 10⁶ CFU / ml	Nil
2.	Pseudmonas aeruginosa (Gram negative bacterium)	1.0 x 10⁷ CFU / ml	Nil
3.	Escherichia coli (Gram negative bacterium)	3.0 x 10⁶ CFU / ml	Nil

5. CONCLUSION:

The following conclusions were drawn after the completion of the experiments.

a) It was found that the shape and size of the nanoparticles produced through bio reduction by Neem leaves extract were strongly dependent on the process parameters like Neem broth concentration, mixing ratio of Neem extract to AgNO₃ solution, interaction time and pH of the solution.

b) It was determined that the minimum interaction time was about 4 hours to obtain the nanoparticles with nearly spherical shape of size below 50 nm.

c) It was confirmed from XRD analysis that there was full conversion of silver nitrate to silver nanoparticle and bio-organic components from Neem leaf broth acted as probable stabilizer for the silver nanoparticles.

d) With 24 hours of soaking, the silver nanoparticles coating was found to be stable with PU and did not get washed away after repeated washing and drying.

e) For a treatment of 30 minutes, the silver nanoparticles were found to have the stable binding with PU and did not get mixed with water.

f) With the input bacterial load of 1 x 10⁵ – 1 x 10⁶ CFU/mL, the performance of the material was checked for its antibacterial properties and efficient removal and the bacterium count was zero in the treated water.

g) It was found that the coated Pu can remove all types of bacteria i.e., gram positive and gram negative bacteria.

h) The life cycle of the coated PU was tested for 25 times and found to function with the same efficiency.

i) The method employed here is a biomimetic approach and the chemicals involved in the synthesis of nanoparticles are non-toxic, commonly available and cost effective.

As the chemistry involved in the preparation of nanoparticle-coated foams is very simple, it can be scaled up to industrial application without the involvement of high pressure or temperature. These qualities make this technology adaptable to underdeveloped nations where bacterial contamination is the principal issue in drinking water.

Received on 14.09.2013 Accepted on 03.10.2013

Asian J. Pharm. Tech. 2013; Vol. 3: Issue 4, Pg 170-174