Synthesis of Gold Nanoparticles by using Batch Method and Study its Antibacterial Activity

The research involves preparing gold nanoparticles (AuNPs) and studying the factors that influence the shape, sizes and distribution ratio of the prepared particles according to Turkevich method. These factors include (reaction temperature, initial heating, concentration of gold ions, concentration and quantity of added citrate, reaction time and order of reactant addition). Gold nanoparticles prepared were characterized by the following measurements: UV-Visible spectroscopy, X-ray diffraction and scanning electron microscopy. The average size of gold nanoparticles was formed in the range (20 -35) nm. The amount of added citrate was changed and studied. In addition, the concentration of added gold ions was changed and the calibration curve was calculated used to estimate Au ions in different samples. The effectiveness of gold nanoparticles prepared according to Turkevich method was studied as antibacterial agents against E. coli bacteria. The minimum inhibition concentration of gold nanoparticles that inhibit bacterial growth was calculated using the broth dilution method, which is based on several dilutions to determine the inhibition concentration.

Then followed by slow growth by reducing. Finally, a rapid reduction of gold ions loaded on seeds to form nanoparticles.
Nanotechnology-specific scientific studies confirm the effectiveness of nanoparticles as antimicrobial agents. Furthermore, gold nanoparticle affects Staphylococcus aureus cells and leads to biological changes such as cellular membrane decay, damage DNA, and negatively affecting cellular functions such as protein synthesis and metabolism (12), finally inhibits cell growth. Nanoparticle is a powerful and highly effective agent for a range of positive and negative bacteria (E. coli, Enterobacter cloacae, P. aeruginosa) which cause urinary tract diseases and resistance to many antibiotics (13).
The aim of the research is to prepare gold nanoparticles by chemical methods and study the factors affecting the shapes and sizes of the nanoparticles formed and study the antibacterial activity of AuNPs against Escherichia coli bacteria and to determine the minimum inhibitory concentration (MIC) of Au 3+ that used to inhibit bacterial growth using broth method.

Materials and Methods:
Chemical solutions The solution of trisodium citrate One gm of the trisodium citrate was dissolved in (100 mL) of distilled water to produce percentage of concentration of citrate 1% (34 mmol/L). The solution of HAuCl 4 .3H 2 O One gm of chloroauric acid was dissolved in (100 mL) of distilled water to prepare (25 mmol / L) as a stock solution. The required concentrations could be prepared by carrying out successive dilution processes.

AuNPs preparation method (Standard preparation
Solution A (HAuCl 4 .3H 2 O) was prepared after dilution with distilled water to different concentrations. After dilution by using distilled water, (50 mL) of solution was taken and heated at 95 o C by a magnetic hot plate with continuous stirring for 10 minutes. (5 mL) of solution B was added to (trisodium citrate Na 3 C 6 H 5 O 7 ). The solution was heated and stirred continuously for 20 minutes. Then, the colour of the solution changed to red. The solution then was cooled at room temperature and maintained in the refrigerator at 4 o C.

Antibacterial activity of AuNPs
The method of Broth dilution was used in order to study the antibacterial activity of AuNPs (14). This method involves using several tubes and doing a series of dilution to find the minimum inhibitory concentration (MIC). The process can be described with the following points: 1-Adding (2 mL) of Lauryl Tryptose Broth (L.T.B) to a series of tubes. (L.T.B is a medium used to detect coliform bacteria in sanitary materials and wastewater). 2-Adding (2 mL) of AuNPs into the first tube and mix with the material L.T.B. 3-Transfering (2 mL) of the first tube and adding to the second tube and mixing with the material L.T.B, the process continues with the third tube until reaching the last tube. 4-Adding (2 mL) of material Lauryl Tryptose Broth (L.T.B) to two tubes considered as a negative control and positive control. 5-All tubes except the negative control are fertilized with (0.2 mL) of bacterial growth (concentration 1.5 ×10 8 CFU). Shake the tubes and store them in an incubator at 37 ° C for 24 hour.

Results and Discussion:
Parameters influence on particle growth, shapes and the final size of AuNPs prepared.

-Au 3+ concentration
Many solutions of gold nanoparticles were prepared by using Turkevich methods and changing the concentration of gold ions. The lowest concentration of Au 3+ was (10 mg/L). Otherwise, the concentration is insufficient to prepare the gold nanoparticle (gold particles does not enough to form the seeds).

Spectral characterization -UV-Visible Spectroscopy
Through the UV-Visible spectrum, much information can be obtained to characterize the prepared samples and study the effect of changing the concentration of the reactants as shown in Fig.1.

Figure 1. Absorbance for different concentrations of Au 3+
From Fig.1, it is noted that increasing the concentration of the gold ions leads to changes in color, absorbance, and max wavelengths (wavelength is decreasing and suffer a blue shifting (15)). In addition, according to the seed growth mechanism (16) (The high concentration of Au 3+ will provide a greater probability to reduction more amounts of gold ions which will provides more monomers of Au 0 . These monomers form the seeds; these seeds determine the final size of the particles).
Producing more seeds means a greater distribution of residual gold ions on these seeds which lead to small final size as shown in Fig.2

Figure 2. The effect Au 3+ concentration According to theory seed growth mechanism A) Concentration of Au 3+ = 20 (mg/L), B) concentration of Au 3+ =100 (mg/L)
Kimling J, et al (17) explained that the increased concentration of HAuCl 4 will reduce the particle sizes of the gold nanoparticles formed. Results showed that when increasing the concentration of HAuCl 4 from 0.125 M to 0.5 M, the sizes of the formed nanoparticles decrease from 14 nm to 5 nm. The reason for the decrease in the particle sizes of gold nanoparticles is because the value of Chloroauric acid will determine the ratio of [AuCl 4 ] -, and increasing the amount [AuCl 4 ]means to produce more seeds, which leads to forming smaller particles.
Polte J, et al (18) proved by using the method of analysis that depend of X-ray absorption near-edge spectroscopy (XANES) that changing the concentration of HAuCl 4 from 0.25 mmol to 0.375 mmol (stabilizing other conditions, temperature 75 o C, concentration of citrate 2.5 mmol) will leads to a decrease in the particles sizes in addition to significantly accelerating the formation of nanoparticles as it leads to increase the reaction rate.

X-ray diffraction (XRD)
X-Ray diffraction patterns of the sample are shown in Fig Fig.5, it is noted that the absorption value is very converged. Increasing the volume of citrate shows its effect on the gold nanoparticle over time. Increasing the amount of citrate reduces the stability of the nanoparticles, then increases the size of the nanoparticle by the aggregation process (19). This is noticed when using 5 mL of citrate. The colour of the solution changes to purple over time as a result of the aggregation process. It was found that the effect of using different volumes of citrate is negligible 1, 2, 3, 4 solutions.
Islam Z A, et al (19) studied the effect of increasing the amount of citrate from 0.5 to 3 mL on the gold nanoparticles formed. Results showed that the absorbance peaks were significantly close, even though the values of SPR are similar (520.8 and 520.93) nm respectively. Additionally, the effect of increasing the amount of citrate on the resulting particle sizes is small (65.1 and 70.44) nm respectively and the only effect is that it affects the mono dispersity. Add more citrate means to get less mono dispersity.
Kumar S, et al (20) found that the concentration of the citrate has no effect on the final size of the particles as long as its buffer ability guarantees a neutral pH value when it reacts with HAuCl 4 . The results proved that changing the concentration of citrate from 2.5 mmol to 3.75 mmol will change the final size of the particles very slightly (13.8 and 14.2) nm respectively.

Reaction time
Over time, the radius of particles increases as explained in the theory of seed growth. As shown in Table 1 and Fig.6, the absorption value will increase over time until reaching the end of the reaction. It can be observed in Table 1 the required time for converting Au 3+ to the nanoscale at different concentrations (100 and 20) mg/L.
When using different concentrations of gold ions, the reaction time will vary. At high concentrations of gold ions, the time needed to reach the nanoscale is less. The change in reaction time can be understood through seed formation theory. Increasing the concentration leads to an increase in the number of seeds formed and thus increases the whole surface area of the seed surface that attracted or attached by Au ions. Therefore, the kinetics of steps 3, 4 (from seed growth mechanism) are increased.   Hendel T, et al (21) studied the effect of reaction time on gold nanoparticles formed and proved that although absorbance changed over time, the final size of the particles is determined in the seed formation step (the second step of seed growth mechanism). The results assumed that if the standard method to produce gold nanoparticles requires 17 min, the seed formation step will take place during the first minutes which consumes 5% of the gold ions concentration. Therefore, the determination of the particle size will be within a short time. The only effect that changes over time is poly dispersity, which decreases over time.

Effect of temperature
The composition of the nanoparticles and the sizes formed are related to the reaction temperature. Using high temperatures usually produces smaller particle sizes. On the other side, at room temperature, the particles have large diameters and poly dispersity greater than 25%. These diameters decrease with increasing temperature until reaching the ideal temperature (22). It should be noted that increasing the temperature would reduce the required time for converting Au 3+ to the nanoscale as shown in Fig.7. Polte J, et al (18) studied the effect of changing the reaction temperature on the gold nanoparticles formed. The results showed that raising the reaction temperature from 75 o C to 85 o C leads to a rising in the absorbance peaks. A higher temperature will lead to the faster formation of the nanoparticles. Likewise, the increase in temperature leads to an increase the reaction rate, and that is mean to get smaller particles (assuming that there is no interference between the particles at high temperatures)

Initial heating
Without preheating of HAuCl 4 . 3H 2 O, the nucleation process is not fast enough and we get a very broad size distribution. Therefore, the key to this method is that nucleation occurs fast practically exhausting the supply of gold ions as shown in Fig.8.

Inverse method of preparing AuNPs
The inverse method could apply by adding a solution of chloroauric acid (high concentration) into a large volume of preheated citrate solution. It was found that using this method the final particle sizes and reaction time decreases. Changing the order of addition of reactants affects the final size of the particles as shown in Fig. 9. The concentration of gold ions is usually about (1000 -10000) mg/L, and the concentration of the citrate range from (0.5-1) mmol.
Sivaraman S, et al (23) explained that adopting the inverse method for producing gold nanoparticles leads to the formation of particles with sizes ranging from (5-10) nm (unlike the standard method that produces particles greater than 10 nm). The results demonstrated also that the order of addition process has a role in producing monodisperse nanoparticles. Using inverse methods will make the timescale of synthesis shorter and synthesis of nanoparticle will be completed in less than 3 min compared with the standard method that lasts 10 min.

Application of Batch method in Au 3+ estimation
Using the increasing concentrations of gold ions, the calibration curve of AuNPs solutions is shown in Fig. 10, the linearity of the results were (10 -100) mg /L, R 2 0.9988, correlation coefficient 0.9993 and the detection limit 5 mg /L.

Antibacterial activity of AuNPs
In order to study the antibacterial activity of gold nanoparticles, the efficacy of gold nanoparticles was tested against Escherichia coli bacteria. The method used is the Broth dilution method. The broth dilution method expresses results in (mg / L) or (μg/mL) to determine the MIC, which is considered the lowest concentration to prevent bacterial growth (24). The concentration of Au 3+ used in the synthesis of AuNPs was 100 mg /L.
After the preparation and incubation of tubes at 37 o C for 24 hours, the absorbance of the prepared tubes is measured at (630 nm) and compared with the positive and negative tubes absorption as showed in Table.3 and Fig .11.  Figure 11. The samples prepared to study MIC It can be seen that the bacteria effect depends on the concentration of the nanoparticles. It shows us that the concentration of 50 mg /L is the minimum concentration used to inhibit bacterial growth. To ensure the effectiveness of this concentration, we performed a second culture on a nutrient agar plate and incubated this plate at 37 0 C for 24 hour as showed in Fig.11 B. No bacterial growth on the plate proves the antibacterial activity of concentration 50 mg/L. Rai A, et al (25) studied the preparation of AuNPs and cover it with cefaclor (a second-generation antibiotic).
The rusltes showed that AuNPs prepared from this method have activity against gram-negative for E. coli. MICs of gold nanoparticles were 100 mg mL -1 for (E. coli).
Odai N, et al (26) studied the effect of AuNPs as an antibacterial against species bacterial such as E.coli. Au nanoparticles were prepared by pulsed laser ablation in liquid (PLAL). The results showed that the AuNPs were less active against gram-positive than the gram-negative. This status could be attributed to the variation in the composition of bacterial cell membranes. Inhibition zone (mm) of AuNPs was (14 for E.Coli).

Conclusions:
This paper includes the preparation of gold nanoparticle in the standard method and the inverse method. By using the standard method, gold nanoparticles' solutions are prepared with average sizes ranging from (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) nm. Changing the parameters that influence the Turkevich method (concentration of gold ions, the temperature, the amount of citrate, the initial heating, the reaction time and change order of reactant addition) show up differences in colors, absorbances, and wavelengths of the prepared AuNPs solutions. These factors according to the theory of seed growth mechanism mean changes in the sizes and poly dispersity of the prepared particles. By changing the concentrations of gold ions, it is possible to draw a calibration curve used to determine the concentrations of gold ions in aqueous solutions and Gold Jewellery after converting them into gold nanoparticles. The results show good recovery and a small relative error. The biological effectiveness of the nanoparticles is studied and the minimum inhibition concentration is calculated using the Broth dilution method, where AuNPs are used to inhibit the bacterial growth of Escherichia coli.