Structural and Optical Properties of Palladium , Silver, Core–Shell Nanoparticles Prepared using Pla Technique for Antibacterial Activation

INTRODUCTION
The process of Laser ablation to generate nanoparticles in liquids is a widely used process at the present time, although some of the resulting particles are not clearly known, such as the concentration of particles and their size distribution, and studies are continuing in this regard [1]. 
The process of laser ablation in a liquid has many applications [2]. It can be entered in the electronic industries and biotechnology, and most importantly the production of nanoparticles for many metals such as (gold-silver-palladium-lead-copper) and others [3]. The laser ablation mechanism is generally when the laser beam is absorbed by the surface of the target (used metal) that is immersed in a liquid such as (deionized water-ethanol-acetone) and others [4], taking into account the laser parameters of wavelength, energy, number of laser pulses, frequency, amount of liquid and its height For the metal piece that is removed after that, the surface of the material is melted or it evaporates and nanoparticles are generated [5], and by changing the laser parameters mentioned, the size and concentration of the resulting particles can be controlled [6]
Metal nanoparticles are used in a wide range of applications as they are used in biomedicine due to their small size and selectivity for bacteria, as well as their anti-disease activity as they have a non-specific microbial toxicity mechanism [7,8].
The nanoparticles resulting from laser ablation of metal alloys in a liquid have many interesting properties that can be used in technology and scientific research and lies in their use in sensing and stimulation systems, energy conversion and storage and in the field of Nano medicine [9].

MATERIALS AND METHODS
(pd-Ag core-shell Nps) were prepared using (PLA) technique by (Nd-YAG) laser with a wavelength of (532nm) in (10mL) deionized water with different number of laser pulses as shown in the Fig. 1 and Table 1.
Then the structural properties were studied (XRD, SEM, EDX). The nanoparticles were diluted on glass bases at room temperature, as shown in the Fig. 2.'

RESULTS AND DISCUSSION
XRD (X-ray diffraction)
examinations of the [Pd-Ag core-shell NPS] samples shown in Fig. 3 showed that the intensity increased slightly with increasing the number of laser pulses and that the D rate calculated from the Spark equation increased with increasing the number of laser pulses [9], as in the Table 2.
The purpose of measuring X-ray diffraction of nanoparticles is to know the crystalline nature of these particles and determine their crystal size. This examination also reveals the pattern of cubic structures. The basic information of the peaks that appeared in the laboratory results of (XRD) for all prepared samples was analyzed and the (Origin Pro 8) program was used to draw and give the final results. As we notice in Fig. 1 which shows the X-ray diffraction measurements of palladium and silver nanoparticles (Pd-Ag) NPs that Fabricated by pulsed laser ablation in deionized water (PLAL) using the second harmonic of the wavelength (532 nm) and with different pulse numbers (250@500 pulse) and When using laser energy (500mJ) notice the appearance of two distinct peaks at (39.6, 46.2 = 𝜃2) respectively and they have the highest intensity at Miller coefficients (*) (°) respectively and according to what is mentioned in the numbered card (JCPDS NO. 01-087-0638) Ag: (JCPDS card No. 04-0783) at issued by (Joint committee on powder diffraction standards [10], which is the joint committee on the standard diffraction card for powder materials. Through the drawing, we can also notice the appearance of a third peak but it is less intense than the previous two peaks It appears at (73.1=𝜃2) and has the highest intensity at Miller coefficients (*) according to the above-mentioned card. Under the same previous working conditions, a laser energy of (500 mJ) and a number of pulses (350@750 pulse) were used. X-ray diffraction measurements were observed for palladium and silver nanoparticles Produced by laser ablation in deionized water. We also notice the appearance of two distinct peaks at and) 40.6, 41.7 = 𝜃2) respectively, which have the highest intensity at Miller coefficients (°) (*) respectively. A third peak was also observed, but it was less intense than the previous two peaks and its shape was irregular and random at (75.9=𝜃2) and has the highest intensity at Miller coefficients (*). The crystal size was also calculated for all these values ​​using the (Scherer) equation. From this, we can conclude that the X-ray diffraction intensity is high when we used a laser energy of (500 mJ) for a wavelength of (532 nm). In addition, the crystal size was calculated (Crystal size) for these
peaks appearing using the (Scherer) equation as shown in Table 1 which shows the values ​​of the crystalline levels and the average grain size of the palladium and silver nanoparticles as well as the maximum width at the middle of the peak (FWHM) and the distance between the levels (d-spacing). As for the laser energy (500mJ) for the same wavelength (532nm) and the number of pulses (pulse500@1000) that were prepared under the same previous working conditions, we find that the analysis of the X-ray diffraction data for these nanoparticles also shows two prominent peaks at) 39.5 ֯ 45.7, =𝜃2) respectively, and they have the highest intensity at Miller coefficients (°) (*) respectively. In addition, a third peak appears that is less intense than the previous two peaks at (֯ 74.4 =𝜃2) and has the highest intensity at Miller coefficients (*). As for the laser energy (500mJ) the same wavelength (532nm) and the number of pulses (1250@675 pulse) that were prepared under the same previous working conditions, we find that the analysis of the X-ray diffraction data for these nanoparticles also shows two prominent peaks at (39.7, 45.9= 𝜃2) respectively, which have an intensity at Miller coefficients (°) (*) respectively. In addition, a third value appears that is less intense than the previous two peaks at (75.06 =𝜃2) and has the highest intensity at Miller coefficients (*).
Also, as shown in Table 2, we find that the solution that was prepared is characterized by high purity. Looking at the average particle size of the particles, we find that there is a difference between the sizes from energy to and the number of other different pulses, as we notice that the more pulses used in the ablation process, the smaller the particle size, and this indicates the quality of the production of nanoparticles by the laser ablation process in a liquid when using a different laser and number of pulse [11].
The size of the crystals of these particles was calculated using the Debye-Scherrer equation, Eq. 1 [12]:

D = particle size, 𝐾 = phase constant, 𝜆 = wavelength, 𝛽 = FWHM,
𝜃 = Braak angle
By observing the peaks that appear in the (XRD) analysis, we find that these peaks are sharp and clear, which indicates that the resulting palladium and silver (Pd-Ag) NPs nanoparticles are well crystallized and can be polycrystalline.
The distance between crystal planes (d-spacing) was also calculated by Bragg’s diffraction law, Eq. 2, and these values ​​show good comparison with the standard values ​​for palladium and silver nanoparticles [12].

d is the distance between crystal planes, ϴ is the angle of incidence of X-rays, n is an integer representing the order of interference.
It was also found that palladium and silver nanoparticles have cubic crystal phase and also the high range of peaks appearing indicates an increase in crystal size.
This result is consistent with the standard results. In addition, this increase in crystal structure can be interpreted as an enhancement of film crystallization by reducing crystal defects, i.e. crystals grow in one direction. In conclusion, all the results presented above are good and almost consistent with the standard results [13].
Scanning Electron Microscopy (SEM)
Samples for SEM experiments were prepared by depositing a drop of solution containing Pd@Ag nanoparticles on silicon grids and leaving them to dry completely at room temperature. The liquid medium has a significant effect on the structure of PLAL nanoparticles. SEM images of Pd@Ag nanoparticles produced by the PLAL process in two distinct surrounding liquids (polymer and distilled water) are shown in Fig. 4. The nanoparticles are spherical or almost spherical, as shown in the SEM images in the figures. The SEM images in the figures also show that there are many spherical core-shell nanoparticles in liquids, especially in liquid environments (compared to other environments). The results reveal that the average size and size distribution of nanoparticles in a given liquid environment vary. The optical properties of the target materials, such as absorption coefficient, and their physical properties, such as thermal conductivity, melting and boiling temperatures, electron-phonon coupling constant, and surface energy, contribute to this variation. These target characteristics were found to affect distinct stages of nanoparticle evolution. 
Compared to ablation at lower wavelengths, nanoparticles generated at higher wavelengths have larger particle size [14,15] .
The number of different ablation energy pulses increases with decreasing laser wavelength. Laser atomization in liquids (LFL) results in smaller particle size, which reduces the number of energy pulses required for ablation despite higher ablation at shorter wavelengths.
This figure also observes agglomeration and clustering of the samples.
Increasing the laser flux leads to the formation of small droplets of palladium and silver which fragment as a result of their interaction with the incident laser beam, followed by rapid quenching, thus forming larger nanoparticles. When the laser flux is increased with increasing wavelength, pure hexagonal nanoparticles are observed as shown in the figures, which is in good agreement with the XRD results [16].
Increasing the laser flux rate results in the formation of larger hexagonal particles and some rod-like structures. The effect of laser flux rate on the morphology of palladium-silver and palladium-silver nanoparticles can be explained based on the properties of the plasma generated on the surface target. The van der Waals attraction potential increases with increasing laser flux rate, leading to an increase in the density of agglomerated and clustered particles. Irregular particles were observed when the laser wavelength was increased to 532 nm. This result can be explained on the basis that increasing the laser flux
rate results in an increase in pressure and temperature, which in turn collapses the cavitation bubbles and thus gives irregular shaped particles [12].

Energy dispersive spectroscopy (EDS) 
The test confirmed the chemical properties of colloidal solutions of metallic palladium and silver prepared in a laboratory using laser ablation in deionized water. had high purity for some samples, but other samples contained some impurities that were probably generated during the precipitation process., Different ratios of elements like (oxygen-carbon-sodium-magnesium) were observed but not taken into account. The Fig. 5 proves the presence of palladium and silver by plotting the positions The first signal was observed in the Edx spectrum point profiles. reflects palladium and silver, the second signal reflects oxygen, and the rest reflect atoms of other elements. [17].
The edx test results indicate that all samples, including palladium and silver, remained unchanged during preparation, as indicated by the intensity of the fall and the energy of the incident electron.

Optical linear properties
Absorbance
Fig. 6 shows the absorbance (Pd-Ag core-shell nanopavticont). We note from the figure that with the increase in the number of laser pulses, the absorbance increased due to the increase in the density of the generated nanoparticles and thus the increase in interactions between photons and nanoparticles, which led to an increase in the absorbance [18].

Coefficient of Absorption
 Fig. 7 shows the absorption coefficient and we notice an increase in the absorption coefficient with the increase in the number of laser pulses. The same reason that led to the increase in absorbance is because the absorption coefficient is directly proportional to the absorbance according to the relationship [14].

Transmittance
Permeability. Fig. 8 shows the permeability and we notice a decrease in permeability with the increase in the number of laser pulses. The same reason that led to the decrease in permeability is because permeability is inversely proportional to absorbance according to the following relationship [15]:

Antibacterial efficacy of palladium and silver nanoparticle solutions
After testing Laser ablation prepared palladium silver colloid solution on two Common bacteria, such as Staphylococcus aureus and Escherichia coli,
 as seen in Figs. 9 and 10, which display distinct inhibition zones on the Mueller Hinton agar surface that contain both. Analysis of the When the colloidal solutions were given to bacteria, it was demonstrated that they could inhibit the growth of “Escherichia coli” and “Staphylococcus aureus.”
Comparing the effects of palladium and silver nanoparticle solutions prepared using one wavelength (532 nm), we found that the greater the number of strokes used, the larger the inhibition zone diameters for Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), as shown in Table 4.
We found similar inhibition values for both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. This implies that palladium’s antibacterial action is what caused the bacterial reaction. and there are no notable differences between these two kinds of bacteria in terms of silver nanoparticles. The inhibitory zone is formed in part by the bacterial cell wall. The Gram-positive bacterial cell wall (S. aureus) consists of a deep layer of a membrane The nanoparticles consist of linear polysaccharide chains, but in Gram-negative bacteria (Escherichia coli), their wall consists of a thin film layer. This suggests that the nanoparticles have a large enough surface area to interact with each other, thereby enhancing the antibacterial effect.
Different inhibition values for different pulse numbers when producing palladium and silver nanoparticles have completely opposite effects on bacteria. It can be concluded that using different pulse both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. This suggests that the bacterial response was brought on by palladium’s antibacterial properties. ... the silver nanoparticle content of these two types of bacteria does not significantly differ from one another. The bacterial cell wall contributes to the formation of the inhibitory zone. The Gram
The inhibition value is very high compared to other metal nanoparticles. This confirms the superiority of palladium and silver nanoparticles in antibacterial activity [16].
Effective → (5 mm – 10 mm)
Good effect → (10 mm-20 mm)
High efficiency → (20 mm and above)
The inhibitory impact of the palladium and silver nanoparticle sizes on the two employed microorganisms is evident as the surface area of ​​these particles has a greater antimicrobial effect compared to their small diameterThese particles are rich in reactive oxygen species (ROS), including (OH), (H2O2), and (O2,) which, when penetrated and infiltrated the bacterial cell wall, lead to high reactivity, causing significant damage to the proteins and DNA of the bacterial cells, thus destroying the cell membrane, allowing its contents to seep out. They have the ability to enter the microbial cells from within, and when adhered to the cell membrane, interact with the cell structure and important components such as enzymes, lipids and DNA, thus reducing the respiration of the bacterial cells, thus destroying and killing them.
Studies have shown that Both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria are highly sensitive to antibiotics and have poor resistance. solutions of palladium and silver nanoparticles. We discovered by merely looking at the inhibition zone that the highest inhibition
effect was the sample with a wavelength of 532 nm prepared with different pulse numbers and energy (500 mJ). The inhibition effect was 10 µm for E. coli and 10 µm for S. aureus.bacteria is (15 mm). The second sample comes immediately after it with an inhibition amount for (E.coli) bacteria (12 mm) and for (S. aureus) bacteria (14 mm), where these samples have shown good effectiveness. The diameter of inhibition for the bacteria is at which the third sample was prepared, the inhibition amount for (Ecbolic) bacteria is (14 mm) and for (S.aureus) bacteria is (16 mm). The fourth sample was prepared with an inhibition amount for (E.coli) bacteria (15 mm) and for (S.aureus) bacteria (13 mm).
We conclude from the above that palladium-silver nanoparticle (Pd-Ag)NPs solution has high antibacterial efficacy against both microorganisms that are both Gram-positive and Gram-negative. This trait can be found in samples. with different energy values ​​and pulse numbers due to increasing palladium concentrations. and silver nanoparticles. However, we observed a higher bacterial growth inhibition rate in samples with a higher pulse number. This is due to the small particle size of the nanoparticles, which allows them to penetrate the cell wall quickly and easily. Another reason is the presence The primary cause of bacterial mortality and destruction is reactive oxygen species (ROS). [17].
 Both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria demonstrated minimal resistance and high susceptibility to colloidal palladium solution. with Escherichia coli showing a stronger response to the antibacterial effect of palladium and silver nanoparticles than S. aureus.

Antioxidant activity of Pd-Ag NPs nanoparticles
The effect of palladium and silver nanoparticles prepared using the pulsed laser ablation method in distilled water at a wavelength of )532 nm( and a different pulse frequency, using a laser energy of )500 mJ(. DPPH (1-Diphenyl- 2-picrylhydrazyl). A free radical scavenging experiment was conducted to determine the antioxidant capacity of palladium and silver nanoparticles in vitro by reducing DPPH free radicals. Thirty minutes after introducing nanoparticles at concentrations of) 0.12, 0.25, 0.5, and 1 μg/ml( to the DPPH solution, the absorbance at 517 nm was determined. The ability of the nanoparticles to scavenge DPPH free radicals was demonstrated by measuring color changes [18]. DPPH, which reduces the effect of nanoparticles, increased with increasing concentrations of bioactive palladium and silver nanoparticles.
It was 59.3% at 1 μg/ml, 52.9% at 0.5 μg/ml, 51.5% at 0.25 μg/ml, and 42.5% at 0.12 μg/ml for palladium and silver nanoparticles, as shown in Fig. 11.
1-Diphenyl-2-picrylhydrazyl, which reduces the activity of the nanoparticles, increases with increasing concentration of palladium and silver bio-nanoparticles. 7.71% at 1 µg/ml, 54.5% at 0.5 µg/ml, 48.1% at 0.25 µg/ml, and 38.6% at 0.12 µg/ml palladium and silver nanoparticles, as shown in Fig. 12.
1- Diphenyl-2-picrylhydrazyl, which reduces the activity of the nanoparticles, increases with increasing concentration of palladium and silver bio-nanoparticles: 81.4% at 1 µg/ml, 61.1% at 0.5 µg/ml, 46.4% at 0.25 µg/ml, and 31.0% at 0.12 µg/ml palladium and silver nanoparticles, as shown in Fig. 13.
Diphenyl-2-picrylhydrazyl, which reduces the activity of the nanoparticles, increases with increasing concentration of palladium and silver bio-nanoparticles. 91.4% at 1 μg/ml, 85.6% at 0.5 μg/ml, 74.5% at 0.25 μg/ml, and 49.9% at 0.12 μg/ml palladium and silver nanoparticles, as shown in Fig. 14.
A single compound, 1-diphenyl-2-picrylhydrazyl (DPPH), has been commonly used to assess the antioxidant free radical scavenging capabilities in vitro. DPPH appears to be a more stable and well-known free radical that relies on the reduction of palladium and silver nanoparticles. By observing the color change, the antioxidant potential of DPPH was tested. The DPPH scavenging activity of the nanoparticles increased with increasing concentration, indicating that the inhibition ratio of DPPH increased with increasing concentration of palladium and silver nanoparticles, suggesting that DPPH exhibited greater inhibition due to increased donation in the nanoparticles. Antioxidants function not only by scavenging free radicals but also by inhibiting the formation of free radicals [19].
Free radicals have a powerful bactericidal effect on oxidative stress. Not only was the membrane damaged, but major biological molecules such as proteins, lipids, DNA enzymes, and DNA that promote cell death also caused damage [20].

Effect of palladium and silver nanoparticles on hemolysis
Hemolysis is a condition in which red blood cells rupture and release their components, leading to anemia, jaundice, and kidney failure. Because all substances entering the blood come into contact with red blood cells, it is crucial to assess the hemolytic potential of the substances [21].
Hemolysis was determined using Triton X-100 as a positive control. Sterile phosphate-buffered saline was used as a negative control, allowing the base solution to be stored at room temperature. Palladium and silver NPS particles at all concentrations (1, 0.5, 0.25, and 12 µg/ml) did not induce hemolysis in the whole blood samples examined, as shown in Table 5, and . This finding is consistent with [22], who found that hemolysis is not caused by the nanoparticles or the solvents present in the nanoparticles or polymer.

CONCLUSION 
Palladium silver (Pd-Ag) NPs nanoparticles were prepared using the laser-in-liquid (LI) technique. The liquid used was deionized water (DIW). A wavelength of 532 nm, an energy of 500 mJ, and a different pulse count were used for each sample. Tests were performed on the samples obtained, including UV-Vis testing, to determine the absorbance, absorptivity, and transmittance of the Pd-Ag nanoparticles. It was observed that the absorbance value and the absorption coefficient of the nanoparticles varied with the number of pulses. the more laser pulses that are used to create the Pd-Ag) NPs, the higher the absorbance and absorption coefficient values, and the lower the transmittance value. XRD and SEM tests were performed. Scanning electron microscope (SEM) analysis showed the that resulting particles are spherical in shape and aggregate to form various shapes and cubic crystalline structures of the Pd-Ag nanoparticles. (Edx) to ascertain the chemical makeup and purity of the samples utilized in biological testing. Palladium-silver nanoparticles’ efficacy as colloidal solutions was onfirmed as antibacterial, antioxidant, and hemolytic, whether negative or positive. We also concluded The palladium sample with the highest inhibition was found to be the one with a wavelength of 532 nm. for bacteria (Escherichia coli).

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.