The Effect of Nanogold - Nanosilver Injection on Increasing the Immunity of Community Affected by Covid-19

: This study aims to determine the best concentration of Nanogold and the effect of Nanogold - Nanosilver injection on increasing the immune system of people affected by Covid-19. The method used is quantitative and quantitative descriptive by observing the progress of giving injections once a month (May – November 2021) regarding the complaints experienced. The best concentration of Nanogold for injection observed on the antioxidant activity and λ maximum stability of Nanogold solution 5, 10, 15, 20, 25, 30 ppm after synthesis and after being stored for 7 days using UV-Vis Spectrophotometry instruments. Produce antioxidant activity 44,21%; 47,88%; 51,37%; 52,32%; 56,16%; 61,57%, with an IC50 of 14 ppm and a λ maximum stability of 5 20 ppm is stable; 25 - 30 ppm shifted by 0,50 nm. So, the best concentration of Nanogold was 20 ppm which was then characterized using the TEM instrument, resulting in an average size of 22,67 nm. A mixture of Nanogold - Nanosilver was injected into the respondents and obtained interview data after each injection. Respondents acknowledged that there was a significant difference, as evidenced by the percentage of development of body condition getting better. Thus, Nanogold and Nanosilver materials can increase immunity during the Covid-19 pandemic.


Synthesis of Gold Nanoparticles (Nanogold)
Nanogold was synthesized from the basic material of HAuCl4 1000 ppm which is yellow. Then heated 100 mL of distilled water in a beaker glass until boiling, then added 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, and 3 mL of 1000 ppm HAuCl4 in each beaker containing boiling distilled water. so that it becomes a Nanogold solution with concentrations of 5, 10, 15, 20, 25, and 30 ppm. After that, 2 grams of sodium citrate were added to each solution. Stir and leave until the color changes from yellow to burgundy (red Nanogold) [17].

Synthesis of Silver Nanoparticles (Nanosilver)
Nanosilver was synthesized from colorless 1000 ppm AgNO3 as the base material. Heated 100 mL of distilled water in a beaker until it boiled, then added 2 mL of 1000 ppm AgNO3 so that it became a Nanosilver solution with a concentration of 20 ppm. After that added 2 grams of sodium citrate. Stir and leave until the solution color changes to yellow [18]. Nanogold Antioxidant Activity Test 0.002 grams of DPPH crystals were dissolved in a 50 ml volumetric flask using ethanol to obtain a 0.004% DPPH solution. 2 ml of Nanogold sample was added with 1 ml of 0.004% DPPH solution and the incubation process was carried out for 30 minutes at room temperature. Then read the absorbance using UV-Vis spectrophotometry at the maximum wavelength of the DPPH solution (517 nm) [19].

Nanogold characterization using UV-Vis Spectrophotometry and TEM
The characterization of the stability of the Nanogold solution was observed through the difference in maximum (wavelength) between the initial synthesis and after being stored for 7 days using UV-Vis Spectrophotometry. While the characterization of the shape and size of 20 ppm Nanogold was observed using a TEM instrument with a magnification scale of up to 20 nm [20].

Immune Test
The type of solution injected in this immune test is a mixture of 20 ppm Nanogold and 20 ppm Nanosilver with a 9:1 ratio of 3 cc per injection [21]. This test is carried out in the Gununganyar area of Surabaya once a month for 7 months, starting from May -November 2021. The injection is given to people of all ages who are willing to be respondents. The indicator is the improvement of public health conditions for the better [22].

Synthesis of Gold Nanoparticles (Nanogold)
Nanogold is one type of nanoparticle that is easily synthesized [7]. There are many methods in the synthesis of nanoparticles, which generally use physical, chemical, and biological methods [12]. This synthesis uses a chemical method which is the most popular method that is still used today. The method was discovered by Turkevich in 1951 [23].
This method involves three main components, namely 1000 ppm HauCl4 as a metal precursor (basic material) and sodium citrate as a reducing agent as well as a stabilizer so that the Nanogold solution is not easily aggregated [24]. Sodium citrate acts as a reducing agent by reducing the gold metal ion (Au 3+ ) HauCl4 to an uncharged gold atom (Au 0 ). Au 0 will combine to form clusters that continue to experience nano-sized cluster enlargement [25]. The strength of the reducing agent plays an important role in controlling the reaction rate, thereby influencing the morphology obtained [23].
When gold ions (Au 3+ ) are reduced by sodium citrate to form neutral gold atoms (Au 0 ), the atoms will be closer to each other through intermolecular bonds. The longer the cluster will grow and form a nanometer size [24].  The higher the concentration of Nanogold, the smaller the distance between the clusters and the more Nanogold clusters formed. So that the resulting color will be more concentrated or stronger [24]. The Nanogold synthesis process changes its color to burgundy as shown in Figure 2. This is following the theory that Nanogold has a characteristic burgundy color [6]. To determine the best concentration of Nanogold, it can be seen based on the differences that occur after being stored for 7 days. This is appropriate, where the Nanogold solution with a concentration above 20 ppm if left for several hours or even a few days will form a precipitate, which means the Nanogold solution is not homogeneous and undergoes aggregation to form a nonnano particle [26].

Synthesis of Silver Nanoparticles (Nanosilver)
The Nanosilver synthesis process also uses the same method as the Nanogold synthesis, namely the chemical method. Where there are three main components, namely, AgNO3 1000 ppm as a metal precursor (basic material), sodium citrate as a reducing agent as well as a stabilizer. Sodium citrate will reduce silver metal ions (Ag + ) to uncharged silver atoms (Ag 0 ). In addition, citrate ions will also react with silver ions to form a complex [(Ag)2] + ....(citrate + )] so that it will coat and protect the formed Nanosilver so that agglomeration does not occur. The reaction that occurs is 4Ag + C6H5O7Na3 + 2H2O → 4Ag 0 + C6H5O7H3 + 3Na + + H + + O2↑ [18]. Reducing agents and stabilizing agents can affect the size and stability of nanoparticles [27].  The Nanosilver synthesis process occurs when the color changes from a colorless AgNO3 base solution to a yellow color. The advantages of synthesizing nanoparticles using chemical methods are easy and inexpensive [12].

Nanogold Antioxidant Activity Test
The antioxidant activity of Nanogold was tested using the DPPH test method. The instrument used in this method is a UV-Vis Spectrophotometer. The DPPH concentration used was 0.004% with a maximum wavelength of 517.00 nm and an absorbance of 0.7041 as shown in Figure 5. The maximum wavelength is used to read the absorbance of the sample which reduces free radicals. While the absorbance value obtained, will be used as absorbance blank in the calculation of % inhibition.
The antioxidant activity of Nanogold was tested by mixing 2 mL of Nanogold samples with concentrations of 5, 10, 15, 20, 25, 30 ppm with 1 mL of 0.004% DPPH solution. The mixture was shaken until homogeneous and incubated for 30 minutes at room temperature so that the DPPH could react with the Nanogold sample. Then the absorbance was read using a UV-Vis spectrophotometer at the maximum wavelength of DPPH, which is 517 nm.
Nanogold is a synthetic antioxidant compound that does not have a carcinogenic effect and has strong, long-lasting activity, and is a very effective antioxidant to reduce free radicals. The higher the concentration of Nanogold, the percentage of attenuation against free radicals will increase [25]. The reactions that occur between DPPH and Nanogold solution are: The ability of a compound to scavenge or scavenge DPPH free radicals can be expressed as a percentage of inhibition or a percentage of reduction [28]. From the curve, a straight line equation is obtained which is then used to calculate the IC50 value, by calculating the x value where the y value = 50. The IC50 value is the concentration of a compound needed to inhibit 50% of DPPH free radicals [29]. The IC50 value produced from Nanogold is 14.0028 ppm. So, it can be seen that nanogold is a very strong antioxidant. Where <50 is very strong, 50-100 is strong, 100-150 is currently, 150-200 is weak, and >200 is very weak [38]. The antioxidant activity of Nanogold produced is 4 times better than the antioxidant activity of Vitamin E, which is 57.45 ppm [30].

Nanogold characterization using UV-Vis Spectrophotometry and TEM
The characterization of Nanogold can be observed using a UV-Vis Spectrophotometer instrument. This characterization aims to determine the stability of the observed Nanogold solution through the maximum (wavelength).
There is a slight difference in the λ maximum of the nanogold solution between the initial synthesis and after being stored for 7 days, as shown in Table 2. Nanogold solutions with concentrations of 5, 10, 15, and 20 ppm had a maximum that was stable and did not shift. Meanwhile, the maximum at concentrations of 25 and 30 ppm shifted by 0.50 nm.
The Nanogold characterization process can also be observed through the shape and size of TEM analysis. TEM can see the size of a particle to nano size with a magnification of up to 150,000,000 times [20].
Based on the stability and antioxidant activity produced, it is known that the best concentration of Nanogold solution is 20 ppm. Where at a concentration of 20 ppm, it has a stability that is seen based on the maximum that does not shift and the percentage of inhibition is 52.32%. Thus, to determine the size of the nanoparticles, it is enough to look at a concentration of 20 ppm.   Figure 6, it can be seen that there are 14 nanogold clusters of various sizes shaped like a circle, with an average size of 22.6701 nm. The results obtained are following the theory, where the nanoparticle size ranges from 1-100 nm and the nanogold size is around 20.68 nm [17].

Immune Test
The type of solution injected in this immune test is a mixture of 20 ppm Nanogold and 20 ppm Nanosilver with a 9:1 ratio of 3 cc per injection [21]. Only 1 part of the Nanosilver is used, because Nanosilver acts as a stabilizer and preservative which has stable properties and has potential applications in various fields, including as a catalyst and antibacterial agent [31].
This procedure is carried out with observations regarding immunity body of respondents conducted in the Gununganyar area of Surabaya once a month for 7 months, starting from May -November 2021, following relevant laws and institutional guidelines, and respondents have obtained informed consent.

Figure 7. Nanogold-Nanosilver Injection Activities
Based on interview data, there were several complaints experienced by respondents before being given a Nanogold-Nanosilver injection. The largest percentage of complaints experienced by respondents is about body fitness or commonly said to have low body resistance as many as 39 people (46.9%). Where, respondents feel more easily sleepy, tired, weak, and not feeling well. Meanwhile, the smallest percentage was exposed to Covid-19, tremor, vertigo, and sneezing as many as 1 person each (1.8%).
After being given the injection 7 times, observations were made again by interviewing respondents about the development of complaints experienced as written in table 4. Description: Indicator 0-24%: progress is very little improved (still relapses often) Indicator 25-49%: progress slightly improved (reduced complaints and do not recur frequently) Indicator 50-74%: progress is improving (rarely relapse or pain) Indicator 75-99%: very good progress (complaints disappear) Based on the data in table 4, proves that injection of Nanogold Nanosilver is a material that can increase immunity in people affected by Covid-19, where body conditions are getting better. This is because Nanogold materials work to increase the immune system at the cellular level by building complexes with glutathione in cells [9]. Glutathione is a natural antioxidant that is in cells and plays a role in the process, formation, division, proliferation, and defense of T lymphocyte cells to fight pathogens [22].