PREAMBLE TO THE DESCRIPTION
THE FOLLOWING SPECIFICATIONS PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
DESCRIPTION
1. FIELD OF INVENTION
This present disclosure relates to the preparation of chitosan, graphene-silver, and tannic acid based polymeric nanocomposites, which enables sustained and controlled release of silver ions to an aqueous medium. The composite is highly antibacterial and effective against a wide spectrum of pathogenic organisms. The polymeric nanocomposite has wide possible applications including food packaging and disinfection of water.
2. PRIOR ART
Providing adequate access to safe drinking water is a challenging task and is now identified as one of the Sustainable Development Goals of United Nations. Addressing wide varieties of pollutants present in freshwater is vital to achieve the goal. Many freshwater resources across the country are polluted with diverse contaminants. According to the report of Global Analysis and Assessment of Sanitation and Drinking water, there are 2.4 million people who die due to lack of access to safe drinking water globally. More than 1.8 billion people around the world are consuming contaminated water with high microbial content [1]. Unfortunately, a significant portion of that is from developing world.
In rural areas of poor and developing countries, the infrastructure is either poorly developed or non-existent. Due to lack of infrastructure in place, water needs of the rural population are largely met by groundwater or untreated surface water.

Some parts of urban areas, especially urban slums, are also suffering from inadequate access to treated water. This is getting aggravated with time due to population expansion and migration of people from rural to urban areas. Among the various pollutants detected in water, pathogens need special attention due to its widespread occurrence and high potential to cause waterborne diseases.
Effective and affordable disinfection methods are indispensable to produce microbial safe drinking water. Chlorination is the most popular and best available technology for this purpose. However, the formation of toxic disinfection by¬products (DBPs) is an emerging concern in the chlorination process. More than 600 DBPs have been identified from various parts of the world. It is reported that deactivation of pathogens like Cryptosporidium parvum and Giardia lamblia are difficult with traditional disinfectants and hence requires an extremely high dosage of chlorine [2]. This may further lead to the excess formation of DBPs [3], Besides, chlorine is not user-friendly and requires special care during application. This is not practical in many cases, especially in rural areas or urban slums, when the situation demands a household (decentralized) water disinfection system.
Considering the challenges associated with conventional disinfectants, the solution based on the state-of-the-art technology holds the key to safe drinking water. In comparison to conventional chemical disinfectants, nanoscale materials are not strong oxidants and hence unlikely to produce harmful DBPs [4}. Several natural and engineered nanomaterials are available for the purpose of disinfection [5-8]. However, most of the nanotechnology-enabled disinfection systems are still far from the full-scale application. Among the nanotechnology-enabled disinfectants, silver-based systems are proved to be more effective in improving the microbial quality of water [9-11], However, these systems have not produced desired results in the field because they are available only as fine powders or as an aqueous dispersion. Besides, the presence of co-existing ions in water can cause ion induced aggregation of nanoparticles and thereby reduce the reactivity of the silver nanoparticles (AgNPs) [12], The said limitations can be overcome by

anchoring the nanoparticles (NPs) on a suitable matrix or by granulating NPs using appropriate binding agent. Several efforts have been made to immobilize the nanoparticles on various matrices. One such widely practiced approach includes immobilization of AgNPs on ceramic candles [13, 14]. Materials such as silver immobilized porous membranes [15], activated carbon [16], bactericidal paper [17], woven fabric [18] have also been developed [21-26].
However, such immobilization techniques may reduce the disinfection efficacy of the system due to interactions between NPs and support matrix. The interaction of AgNPs with the surrounding environment (for example, interaction with chloride and natural organic matter) can also reduce the antibacterial activity of the silver-based disinfection system significantly.
Notably, most of the silver-based disinfection systems developed over the years, have not produce desired results in the field due to the said limitations. Hence, an effective and affordable immobilization technique is the need of the hour. Moreover, it is also important to develop sustainable technologies with affordable cost. The said composite is eco-friendly and less embodied energy. The composite can be produced in a single reactor system at atmospheric pressure and temperature.
3. OBJECT OF INVENTION
The present invention describes a novel process for the preparation of a film forming composites that enables controlled and sustained release of an antibacterial agents or a combination of antibacterial agents to achieve disinfection of water. More specifically, the invention relates to the development of a film forming matrix that acts as a reservoir of AgNPs that enables controlled and sustained release of silver ions to an aqueous medium. The composite is hydraulically stable and can sustain in water more than a year without disintegrating. The composite can be used either as a standalone film or can be supported on a suitable solid surface to form a thin coating of the composite.

4. SUMMARY OF THE INVENTION:
According to one aspect of the invention, the present disclosure reveals a method
to provide controlled and sustained release of silver ions from a nanocomposite
film for the purpose of disinfection of drinking water.
In another aspect, the invention discloses a green and facile method for the
production of film-forming nanocomposite with enhanced mechanical strength
and antibacterial property.
In yet another aspect, the invention discloses an efficient immobilization
technique to protect the silver nanoparticles from the surrounding environment
and preserves the reactivity of the nanoparticles.
An important aspect of the invention is that the tannic acid in the nanocomposite
film helps in controlling the porosity, which governs the controlled and sustained
release of silver ions.
In another aspect, the film-forming ability of the said composite facilitates the
immobilization of the composite on the suitable solid surface easily. It also helps
in forming a standalone film that can act as independent disinfecting agent for the
point-of-use applications.
4. STATEMENT OF THE INVENTION IS GIVEN IN THE FOLLOWING EXAMPLES
The following examples are provided to illustrate the present invention. These examples will illustrate the skills in the art with the complete revelation and description of how the materials, compositions, and methods claimed herein are made and evaluated. The examples are intended to be purely exemplary of the invention and not intended to limit the scope of the invention, of what inventors regard as their invention, in any way.
Example 1:

This example describes the synthesis of Reduced Graphene Oxide (RGO) via photoreduction. The synthesis of Graphene Oxide (GO) was carried out using the modified Hummer's method [19]. In brief, the preparation procedure includes the pre-oxidation of powdered graphite to graphitic oxide, followed by the exfoliation of graphitic oxide to GO. The reduction of GO to Reduced Graphene Oxide (RGO) was carried out similar to a method reported by Mohandoss et al. [20].
Example 2:
This example describes the preparation of the silver decorated graphene sheets. In order to prepare the silver decorated graphene sheets, the following procedure was adopted. A desired concentration of AgN03 (0.01 mM to 10 mM) was added to 0.01% (w/v) RGO dispersion. The mixture was kept undisturbed at room temperature and atmospheric pressure for 12 to 24 h. A redox-like reaction between RGO and silver ions resulted in the formation of (AgNPs) decorated RGO sheets. The prepared composites were designated as RGO-Ag-0.01, RGO-Ag-0.5, RGO-Ag-1.25, RGO-Ag-2.5, RGO-Ag-5, and RGO-Ag-10. Where 0.0i, 0.5, 1.25 etc. represents the initial concentration of the silver precursor in the RGO dispersion.
Example 3:
This example describes the preparation of ,the said film-forming nanocomposite. The silver nanoparticles decorated RGO sheets were mixed thoroughly with 2% protonated chitosan solution at 1:1 (V/V) ratio along with 10-15 mg of tannic acid for 4h to form silver nanocomposite (AgNC). Then the resultant composite solution was poured onto Petri plate and dried at < 50 °C to form a stable and transparent nanocomposite film. The as-prepared nanocomposite film was cross-linked by 5 - 10% trisodium citrate. Based on the initial concentration of silver precursor, the prepared nanocomposite was designated as RGO-AgNC-0.5, RGO-AgNC-1.25, RGO-AgNC-2.5, RGO-AgNC-5,andRGO-AgNC-10.

Example 4:
This example describes the experimental studies performed to evaluate the antibacterial activity of the nanocomposite. Antibacterial activity of the samples was evaluated by disk diffusion assay [21]. In this assay, the Muller Hinton agar medium (38 g/L) was prepared and sterilized in an autoclave at 121 °C and 15 PSI for 15 min. The prepared medium was poured onto the sterilized petri plates and cooled. The Sterile cotton swab was dipped into the microbial inoculum and spread over the plates containing Muller Hinton agar medium. Sterilized Whatmann filter paper disks of 5 mm were placed onto Muller Hinton agar medium. 10 uX of RGO-Ag samples of different compositions were placed onto the paper disks placed on the Muller Hinton agar using sterile forceps. The plates were incubated overnight at 37 °C for 24 h. The clear zone present around the filter paper disks represents the zone of inhibition of RGO-Ag samples against the pathogens. The zone of inhibition was measured.
Example 5:
The experimental evidence supporting the sustained and controlled release of silver ions from the composite is demonstrated. The as-prepared nanocomposite films with different concentration of silver nanoparticles and different thicknesses were evaluated for the leaching of silver ions in deionized water (DIW). The nanocomposite film of radius 60 mm were placed in a batch reactor containing 100 mL of DIW in ambient conditions. Leaching studies were carried out by replacing water in every 30 min for first 15 cycles, followed by replacing water for every 24 h for the next 60 cycles and it was continued by replacing water for every 30 min for the remaining cycles. The collected water samples were digested by 2% HNO3 and analyzed for silver ions by Graphite Furnace Atomic Absorption Spectrophotometer. The release patterns of silver ions from the nanocomposite films were studied. One particular composition of the film, RGO-AgNC-10 was demonstrated for sustained and controlled releases of silver ions for more than 200 cycles.

Example 6:
This example narrates the details of the study conducted to evaluate the disinfection efficiency of the as-prepared nanocomposite film. The contaminated water was prepared by spiking 103 - 104 CFU/mL of E.coli and buffering agents K2HPO4, KH2P04, CioH1602N8 (EDTA) as per National Science Foundation (NSF) P231 protocol. The nanocomposite film was placed in 100 mL of the contaminated water. The reactor was left undisturbed. An aliquot of sample of 1.1 mL was withdrawn at predetermined intervals. From the collected sample, 0.1 mL of sample was separated and quenched with 2.64 uL of 6 g/L of sodium thiosulphate for 2 min at room temperature. Then it was plated onto Petri plates containing nutrient agar media and spread over by means of a sterile L-rod. The plates were incubated overnight at 37 °C and checked for the viable colonies after 24 h. The colonies were counted and reported as CFU/mL. The remaining 1 mL of aliquoted sample was digested with 2% HNO3 and analyzed for total silver ions. The reusability of the composite was demonstrated for multiple cycles. The bacterial culture was prepared daily to simulate the contaminated water.
Example 7:
The instrumentation employed for the characterization of the as-prepared nanocomposite films are described in this example. UV/Vis spectra were measured by Thermofisher Scientific (EVO 300 PC) UV/Vis spectrophotometer. The attenuated total reflectance infrared spectra (ATR-IR) in the range of 400 -4000 cm"1 were collected by Thermo Scientific NICOLET iSlO FT-IR Spectrometer. High-resolution transmission electron microscope (HRTEM) images of the samples were obtained with a UHR pole piece equipped with an EDAX (JEOL JEM3011™, 300 KV). X-Ray diffractograms of the samples were recorded by X-ray powder diffraction (XRD-SMART lab - Rikagu, JAPAN) using Cu-Ka radiation at X = 1.5418 A. A scan speed of 3° per minute and step width (29) of 0.02° was applied to record the patterns in the range from 10° to 80° (20). The concentration of silver ions release was analyzed by PerkinElmer Pinnacle 600 T Graphite Furnace Atomic absorption spectrophotometer.

5. DESCRIPTION WITH REFERENCE TO DRAWINGS AND TABULATED DATA
Figure. 1 illustrates the UV-Vis spectra and HR-TEM image of the nanocomposite.
Figure 1(A) shows the UV-Vis spectra of GO, RGO, AgN03, and RGO-Ag. GO peak shows strong absorbance at 230 nm that corresponds to the TC - TC* transition of the conjugate C-C bonds and shoulder at 301 nm attributing to the n -7t* transition of C = O bonds. Upon photoreduction, the peak from 230 nm is red-shifted to 264 nm indicating that the sp2 carbon structure is restored. The shoulder at 301 nm visible in GO spectrum is completely removed after 16 h exposure under sunlight. This shift of the peak from 230 nm to 264 nm confirms the reduction of GO to RGO. The silver nitrate shows the peak around 300 nm and is clearly shown in the inset of the figure. The UV-Vis spectra of RGO-Ag show the surface plasmon resonance (SPR) peak at 410 nm, indicating the formation of silver nanoparticles. The intensity of RGO peak decreases with a slight blue shift. This implies that the reduction of silver ions to AgNPs accompanies oxidation of RGO. A redox-like reaction between RGO and AgN03 leads to the formation of silver nanoparticles decorated RGO sheets.
The Figure 1(B) shows the UV-Vis spectra of tannic acid at 276 nm, indicating the presence of non- ionic polyphenolic groups. The protonated chitosan solution shows a sharp peak centered at 210 nm. There is no shift in the SPR peak, which is shown in Figure 1(B) of RGO-Ag nanocomposite indicates that there is no aggregation of AgNPs during the formation of the nanocomposite.
The Figure 1(C) show HRTEM images of RGO decorated AgNPs. The nanoparticles of size around 5 nm are uniformly distributed on RGO sheets. This is evident from Figure 1(C). From the Figure 1(D), it is seen that the silver nanoparticles are immobilized onto polymeric chitosan. The size of the nanoparticles is around 50 nm when the initial concentration of AgN03 is 10 mM. A direct correlation between the concentration of silver precursor and the size of the silver nanoparticles are observed. The higher the concentration of silver

precursor, the bigger the size of the nanoparticles formed are. This is attributed to the aggregation of the particles.
Figure. 2 illustrate ATR-IR spectra and X-Ray diffractograms for chitosan and the nanocomposite.
ATR-IR spectra of chitosan and the as-prepared nanocomposite are shown in the Figure 2(A). The broad peak at 3318 cm'1 shows the presence of-OH and -NH stretching vibrations. The sharp peak centered at 1632 cm"1 and 1062 cm"1 are ascribed to -OH bending and C-O stretching of saccharide structure of chitosan, respectively. The significant shift in the peak at 1551 cm"1 to 1582 cm4 indicates that there is an interaction between RGO-Ag sheets with the amine functional groups present on chitosan. Also, this shift is due to the reaction between amino groups in chitosan with the epoxy groups present in RGO by changing the primary amino (-NH2) groups to secondary (-NH) groups, resulting in nucleophilic addition reactions. The characteristic 29 peaks of chitosan at 9° and 20° in Figure 2(B) show the presence of hydrated crystalline structures and amorphous structure of chitosan. The presence of peaks at 38°, 44°, 65° in Figure 2(B) represents (111), (200), (220), (311) face centered cubic crystalline of silver (JCPDS No. 04-0783).
Figure. 3 depicts the antibacterial activity of the as-prepared RGO-Ag samples
The RGO-Ag samples showed a wide range of antibacterial activity against gram-positive (S.aureus, E.faecalis) and gram-negative bacteria {E.coli, K.pneumoniae, P.vulgaris, P.aeruginosa). The zone of inhibition is evident from the photographic images shown in the Figure 3. The clear zone around the Whatmann filter paper confirms the presence of antibacterial property of RGO-Ag against the gram-positive and gram-negative bacteria. The RGO-Ag samples containing high concentration of silver produces large diameter of the zone of inhibition than the other sample compositions. This is due to the higher availability of silver ions and their associated diffusion into the bacterial cells. The

zone of inhibition is found to be higher in case of gram-negative bacteria compared to gram-positive bacteria. This may be due to the presence of thick peptidoglycan layer in the gram-positive bacteria that resists the diffusion of silver ions.
Figure. 4 depicts the controlled and sustained release of the silver ions from the as-prepared composite.
Controlled leaching of silver ions from the composites was studied. The nanocomposite films with the different amount of silver nanoparticles (1.8 to 35 mg) per gram of the composites were evaluated. A direct relationship is observed -with the amount of AgNPs in the film and the concentration of silver ions' released. The leaching of silver ions from a film of thickness 0.09 mm is shown in. Figure 4. The concentration of the silver ions released with time is more or less constant for a particular film. The nanocomposite is hydraulically stable and can sustain in water even after prolonged exposure. The data shows that the system can act as an excellent reservoir of AgNPs, capable of releasing the silver ions at a sustained and controlled manner, indicating its suitability in real field application of disinfection of water.
Figure. 5 show the trend of prolonged release of silver ions from the nanocomposite.
The nanocomposite film of thickness 0.09 mm, containing 34.12 mg of AgNPs per gram of composite is demonstrated for its ability to release silver ions for a longer duration at sustained and controlled rate. The experiments were conducted for about 200 cycles to demonstrate the robustness of the film. No significant reduction in the leaching pattern was observed during the studies. The mass balance analysis also shows that only 36% percent of the silver is released and the system is capable of working for more number of cycles.
Figure. 6 illustrates the disinfection efficiency of the nanocomposite film.

The nanocomposite film containing 34.12 mg of AgNPs per gram of composite (0.09 mm thick) was evaluated for the disinfection efficiency. The disinfection follows the sigmoidal pattern, which is evident from the Figure 6. More than 70% of the disinfection is achieved in 30 min contact time and the complete (>99.99%) disinfection is achieved within 120 min of contact. The data also reveals that the reuse potential of the film is excellent and the system is capable of producing consistent performances with repeated use.

6. INDUSTRIAL APPLICABILITY: Yes/Ne
The said composite has wide possible industrial applications including disinfection of water and packaging of putrcsciblc goods. Being a film-forming composite with good mechanical strength, enhanced antibacterial property (6 log reduction), and hydraulic stability, the composite can be used as a standalone film. This film can also be supported on suitable solid surfaces for the said applications. The said composite film can be prepared through a simple, soft chemistry approach at atmospheric pressure and temperature without the need of special reactor setup. Facile single pot synthesis approach followed here supports the large-scale production of material at an affordable cost. The excellent ability of the said composite to retain AgNPs will help to improve the service life of the system. Excellent antibacterial property (>99.99%), ability to retain AgNPs for a prolonged period without compromising the reactivity, and its eco-friendly nature will improve the applicability of the material in the field.

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