PREPARATION OF SILVER-CHITOSAN NANOCOMPOSITES COLLOIDAL AND FILM AS ANTIBACTERIAL MATERIAL

Colloidal nanocomposites silver-chitosan have been made. Silver nanoparticles were produced by chemical reduction methods assisted microwave irradiation using chitosan from crab shells as a reducing agent and stabilizer, AgNO3 as a precursor and NaOH as an accelerator. This study investigated AgNO3 concentration toward localized surface plasmon resonance (LSPR) phenomenon of nanocomposites colloidal. The size and shape of the silver nanoparticles were confirmed by TEM. Furthermore, the stability of the storage was observed for twelve weeks. Colloidal and film nanocomposites silverchitosan have been made by casting method by drying at room temperature. After that, the film characterization was carried out, including swelling with gravimetry methods and surface morphology using scanning electron microscopy (SEM). Diffusion methods tested colloid antibacterial activity and silver-chitosan nanocomposite film’s against E. Coli and S. Aureus. The results showed that the formation of silver nanoparticles was identified by the LSPR absorption band's appearance at 413-419 nm. The increasing of AgNO3 concentration increased the intensity of the LSPR absorption band. Silver nanoparticles with sizes of about 3-9 nm are spherical. The silver nanoparticles were stable at 12 weeks of storage. The higher AgNO3 concentration tends to increase the swelling of the film. The surface of the silverchitosan nanocomposite film’s was rougher than that of the chitosan film. The higher the silver nanoparticle concentration, the higher the colloid and film antibacterial activity against E. Coli and S. Aureus.


INTRODUCTION
The medical world is a health sector that plays an essential role in human life so that everything continues to be developed.
One of the things developed in the medical world is a polymer. Currently, synthetic polymers are still widely used, such as polyurethane, polyethylene, polylactides, polyglycolide, and polyacrylonitrile, where there is a weakness, low biocompatibility. Therefore, biopolymers are continuously being developed to replace synthetic polymers. The requirements for biomedical polymers include, among others, that it must be non-toxic, not allergenic, easily sterilized, have adequate mechanical properties, strong, elastic, durability and biocompatibility.
Chitosan is one of the ingredients that is widely used as a basic material for making biofilms. Chitosan has many useful properties, including non-toxic, antibacterial, biodegradable and biocompatible [2]. Chitosan is a material that is widely used as a base for making biofilms. It has many useful properties, including non-toxic, antibacterial, biodegradable and biocompatible [2].
Chitosan is obtained from the isolation of fishery waste such as shrimp shells, small crab shells, crab shells, clamshells, and other shelled marine animals. Crab shells contain chitin and chitosan, the second-largest biopolymer compounds found in nature after cellulose [3]. Crab contains the highest percentage of chitin (70%) among crustaceans, insects, worms and fungi. This chitin is later deacetylated to become chitosan [4].
Chitosan is appropriate for use as a polymer in the medical field because it has an antibacterial and antifungal activity which can inhibit infection, reduce contractions, accelerate wound closure and healing [5]. Chitosan is a polymer compound in the form of a long chain of glucosamine with a chemical formula (2amino-2-deoxy-β-D-Glucose). Chitosan is also a multifunctional polymer because it contains an amine group and a hydroxyl group. The existence of this functional group causes chitosan to have high chemical reactivity [6].
Chitosan has antibacterial properties, but its antibacterial properties can still be improved by adding nanoparticles. One of the nanoparticles that are currently being developed is Ag (Silver) nanoparticles. The addition of silver nanoparticles to chitosan will form silver-chitosan nanocomposites biopolymers. Chitosan acts as a matrix or binding component, while silver nanoparticles act as a filler component [7]. Ag nanoparticles' anti-bacterial ability is influenced by nanomaterial physical characteristics, such as size, shape, and surface properties. Nanoparticles have many uses, such as detectors, catalysts, surface coating agents, and antibacterial agents. Among metal nanoparticles, silver nanoparticles have received much attention because of their physical and chemical properties. Silver has been used to treat medical ailments for more than 100 years because of its natural antibacterial and antifungal properties, and because it is nontoxic to humans [8].
Silver-chitosan nanocomposites biopolymers can be prepared by first synthesizing Ag nanoparticles. Chemical reduction methods can carry out the preparation process. Besides easy and quite effective, the costs required are also relatively affordable. There are 2 important components that must be involved in the synthesis process of silver nanoparticles using chemical reduction; 1) a reducing agent that will reduce Ag + to Ag 0 and 2) a stabilizer (stabilizing agent) which will provide stability to silver nanoparticles formed from oxidation, agglomeration and deposition processes [9], [10].
Besides acting as a matrix, chitosan can act as a stabilizer and a reducing agent in the synthesis of silver nanoparticles. [11][12] used chitosan as a stabilizer, while [13] used chitosan as a reducing agentmicrowave irradiation for 11 minutes with a break of 45 seconds every 1 minute. Thus, microwave irradiation assistance can provide time efficiency in the synthesis process of silver nanoparticles [13].
The synthesis of silver nanoparticles can also be added with an accelerator to increase the effectiveness and efficiency.
Based on research conducted by Susilowati [14], the use of NaOH as an accelerator can increase the effectiveness and efficiency of the silver nanoparticle synthesis process with glucose as the reducing agent. The use of NaOH as an accelerator can reduce the size of silver nanoparticles and increase their number.
This research was carried out on the preparation of colloid and film filled with silver nanoparticles (Ag) with a reduction method with chitosan from crabs shell as both reducing agent and stabilizing agent assisted by microwave irradiation became silverchitosan nanocomposite film's and colloidal to be used as an antibacterial material. NaOH is used as an accelerator to increase the effectiveness of silver nanoparticles producing.
Besides, chitosan also acts as a biopolymer matrix in the synthesis of silver-chitosan nanocomposites.

METHODS
The material used in this study was chitosan from crab shells produced by Biotech Surindo Indonesia. Acetic acid (CH3COOH), silver nitrate (AgNO3) and sodium hydroxide (NaOH) was produced by Merck. Aquades is produced by Laboratorium of Chemistry Education Sebelas Maret University, Indonesia.

Preparation of silver-chitosan nanocomposite colloidal (SCNC)
Initially, 1% chitosan solution was made in 1% (v / v) acetic acid solution. As much as 2.5 grams of chitosan from crab shells dissolved in 250 mL of 1% acetic acid, then stirred until homogeneous for 2 hours.
Then the chitosan solution was left to stand for 24 hours so that the chitosan was completely dissolved. Then a solution of AgNO3 0.12 g / ml and NaOH 2 M was also made.
The preparation of colloidal silverchitosan nanocomposite was started by taking 12.5 mL of 1% chitosan solution and then adding 0.12 g / ml AgNO3 as much as 0.25 ml stirring for 5 minutes. After that, 1.75 ml of 2 M NaOH was added and stirred vigorously for 5 minutes, and a white gel was formed. This gel was then irradiated with a microwave with a power of 100 watts for 4 minutes, and the gel changed colour to brown. This gel was then mixed with 47.5 chitosan and stirred until all the gel was dissolved and homogeneous to form colloids which in this study were called colloidal silverchitosan nanocomposites (SCNC1).
The same step was taken to variation the volume of AgNO3 0.12 g / ml, namely 0.50 ml; 0.75 ml; 1.00 ml; 1.25 ml and 1.5 ml with coded samples respectively are SCNC2, SCNC3, SCNC4, SCNC5 and SCNC6.

Preparation of silver-chitosan nanocompocite film
Nanocomposites silver-chitosan film was prepared by casting method.   These results follow Raghavendra's research [13] and Shah [12], which state that the SPR band silver nanoparticles on UV-Vis spectra can provide characteristic peak absorbance spectra wavelength range of 400-450 nm.
Based on UV-Vis spectra (Figure 2), the increase in the concentration of AgNO3 used is generally directly proportional to the increase in absorbance. Increased absorbance shows that the concentration of silver nanoparticles also formed increases. These results are following the research conducted by Susilowati [14].
Silver-chitosan nanocomposite film's was formed from silver-chitosan nanocomposite colloidal which was moulded and dried at room temperature to be further neutralized.
The film that is created has a darker colour variation, as shown in Figure 3.

Stability test of silver-chitosan nanocomposites colloidal
To determine the colloidal stability of silver nanoparticles by measuring the absorbance at various concentrations of AgNO3. Measurements were made every two weeks for 12 weeks in room temperature storage. Figure 4 shows the absorbance chart of silver-chitosan nanoparticles for 12 weeks. Based on the graph (Figure 4)

Swelling test of the silver-chitosan nanocomposite film's
The swelling test shows that the addition of AgNO3 affects the absorption of the film layer into water. The results of the swelling test are shown in Figure 5. The greater the addition of AgNO3, water absorption will increase, and the increase in the concentration of AgNO3 is directly proportional to the increase in water absorption. is also known to increase the silver-chitosan nanocomposite film's water absorption capacity [12].

TEM Analysis
The shape and size of silver nanoparticles in silver-chitosan nanocomposites colloidal were analyzed using TEM, the results of which can be seen in Figure 6. The samples analyzed were SCNC3 and SCNC6 only to compare the shape and size of colloids with low and high silver nanoparticles concentrations. Based on Figure 6, the resulting silver nanoparticles have a spherical shape with the size of about 3-9 nm. When the SCNC3 and SCNC6 samples were compared, it was seen that the SCNC6 samples had more silver nanoparticles than the SCNC3 samples. The spherical shape of silver nanoparticle in line with LSPR band about 400 nm [16]. The increase in the concentration of AgNO3 is directly proportional to the increase in the concentration of silver nanoparticles that formed that have proved from TEM image [16].

Morphological analysis of silver-chitosan nanocomposite film's
The morphology of the silver-chitosan nanocomposite film's was analyzed using SEM. The results of SEM analysis in Figure 7.
Sample SCNC0 is a pure sample of chitosan without the addition of Ag nanoparticles.
Based on the SEM results in Figure 7, the addition of AgNO3 causes the surface of the film to become uneven. This occurs because Ag nanoparticles' addition causes chitosan polymer chains to develop when interacting or binding with Ag nanoparticles.
Besides, there was clumping on the silverchitosan nanocomposite film's surface, which indicated that the silver nanoparticles were experiencing agglomeration. This is consistent with a study conducted by Susilowati [16] which stated that the higher the concentration of silver nanoparticles on the film, the higher the nanoparticles' tendency to experience agglomeration [16].   When compared, the diameter of the colloidal inhibition zone of silver-chitosan nanocomposites was slightly larger than the silver-chitosan nanocomposite film. According to a study conducted by Haryati [17], the good diffusion method will produce a larger diameter of the inhibition zone than the disk diffusion method [17]. This is because the well method occurs at a higher osmolarity process than the disk method. In the well diffusion method, the osmolarity that occurs is more thorough and more homogeneous, and the concentration is higher so that it is stronger to inhibit bacterial growth [17].
Dissolved chitosan and its derivatives are more effective at inhibiting bacterial growth.
Dissolved chitosan has a broad conformation, which allows the reaction to be effectively and efficiently, whereas the solid chitosan only comes into contact through its surface [18]. Coli and S. aureus bacteria.