2.1 High-Entropy Alloy Preparation
The use of a powder mixture composed of pre-alloyed CoCrFeNi and Cu powders. Both the CoCrFeNi and Cu powders have a weight purity of 99.8% and particle sizes ranging from (15 − 53)µm as shown in Figure 1 (a-b). Table 1 displays a nearly equiatomic composition for the CoCrFeNi high entropy alloy. Moreover, the table illustrates that each element contributes roughly equivalent proportions to the overall composition, leading to a near-uniform distribution of these elements in the material. This distribution adheres to the principles of high entropy alloy design. Scanning Electron Microscopy (SEM) was employed to investigate and comprehend the variations in antibacterial and anti-biofilm efficacy among the samples prepared with optimized laser cladding parameters.
Table 1 Atomic percentage and weight checks using electron microscopy powder.
Element
|
Weight%
|
Weight% σ
|
Atomic
|
Chromium
|
27.708
|
1.007
|
29.877
|
Iron
|
23.954
|
1.099
|
24.048
|
Cobalt
|
23.828
|
1.273
|
22.669
|
Nickel
|
24.509
|
1.367
|
23.405
|
2.2 Preparation of CoCrCuFeNi Nano Particle
Lactobacillus acidophilus (LB), a Gram-positive bacterium, was cultured in the bacteriological laboratory at the College of Science, Mustansiriyah University in Baghdad, Iraq [26, 27]. To initial the procedure, 50 ml of MRS broth was inoculated with bacteria, followed by incubation at a temperature of 37o for 24 hours. Following the incubation, the pH of the bacterial culture was adjusted to 6.0. In meantime, a mixture of CoCrFeNi and Cu powders was prepared with a weight ratio of 78:22 (CoCrFeNi:Cu). Subsequently, 1.4 g of CoCrCuFeNi HEA was introduced into the flask containing the bacterial culture and heated in a water bath for 5-10 minutes at a temperature of (75 − 80)o C. Until a visible colored deposit emerged at the bottom of the flask, signifying the initiation of the transformation of CoCrCuFeNi HEA into CoCrCuFeNi NPs.
Then, the flask was cooled and left to incubate for 24 hours at 37o C to allow the particles to precipitate at the bottom of the flask. Finally, the residue left after centrifugation was washed twice with distilled water and then dried at 40o C for 2 hours [28]. The dimensions and morphology of the CoCrCuFeNi HEA nanoparticles were examined using Scanning Electron Microscopy (SEM). The specimen was prepared by depositing of the CoCrCuFeNi HEA stock onto Lactobacillus acidophilus. SEM was conducted to con-firm the formation of the CoCrCuFeNi HEA NPs, The SEM micrograph recorded from drop-coated films of the CoCrCuFeNi HEA nanoparticle clearly shows that the particle size ranges from 81.45 nm to 851.1 nm as depicted in Figure 2. The CoCrCuFeNi HEA NPs will be dissolved to create an LB supernatant stock solution using an ultrasonic cleaner for 30 minutes. The resulting CoCrCuFeNi NPs were utilized for subsequent experiments, as illustrated in Figure 3 (a-b).
2.3 Antibacterial and anti-biofilm activity of LB supernatant and CoCrCuFeNi HEA NPs
2.3.1 Antibacterial activity
The antibacterial assays were conducted in accordance with the procedures reported previously [22]. The agar well diffusion method was used to detect the effect of both CoCrCuFeNi HEA NPs and LB supernatant against Escherichia coli (E. coli) a Gram-negative bacterium, and Staphylococcus aureus (S. aureus) a Gram-positive bac-terium cultured, which obtained from Department of Biology, College of Science, Mustansiriyah university, located in Baghdad, Iraq as shown Figure 3 (a-d). To start the procedure, plates were prepared by spreading approximately 105 CFU/ml culture broth of each indicator bacterial isolates on the nutrient agar surface. Additionally, the nutrient agar sterilization process involved autoclaving at a temperature of 121o (with 15 lbs of pressure) for 15 minutes, followed by cooling to a temperature range of (45 − 50)o C. The agar plates were allowed to stand for 15 minute before aseptically dispensing 50µl of CoCrCuFeNi HEA NPs and LB supernatant into the pre-made agar holes. Subsequently, the plates were incubated at 37o C for 24 hours, as illustrated in Figure 6. The zones of inhibition were measured and recorded in millimeters, and one well was filled with sterilized D.W. and utilized as a control.
Co-culture technique, also was used to detected of antibacterial activity of CoCrCuFeNi HEA NPs + LB bacterial culture of pathogenic bacteria was grown in nutrient broth with a ratio 1:1 (CoCrCuFeNi HEA NPs solution and : nutrient broth) the control medium contained nutrient broth only. The mixture co-culture and control were then incubated at a temperature of 37o C for a duration of 24 h. After the incubation 1 ml of each cultures were serially diluted up to 10−1 to 10−8.
Then 0.1ml of 10−8 dilution sample was taken and spreaded on nutrient agar plates. The plates were incubated at 37o for 24 h. The colonies were counted and the inhibition activity was evaluated after 24 h and calculated percent reduction of bacterial using the following equation described as Ghosh et al [29].
R is the reduction rate, A is the number of bacterial colonies from control medium and B is the number of bacterial colonies from treated medium.
The second replicate of all the above treatments was tested with a scanning electron microscope for detection of biofilm formation on catheter.
2.3.2 Anti-biofilm activity
The process initiated with the inoculation of tubes with approximately 105 CFU/ml of each indicator bacterial isolate. The samples were prepared by cutting the catheters into three pieces, each approximately 1cm in length, and then placing them in tubes. Figure 4 (a) depicts the catheter piece with distilled water without bacterial, Figure 4 (b) illustrates the catheter piece with E.coli broth 105 CFU/ml, and Figure 4 (c) displays the catheter piece with E.coli and CoCrCuFeNi HEA NPs+LB supernatant followed by incubation at a temperature of 37o for 24 hours. The catheter pieces with CoCrCuFeNi HEA NPs and the control (non-coated catheter pieces) were placed on blotting paper to remove excess suspension and then left to dry at 50oC. Following the incubation period, all catheter pieces (treated and control) were stained with a 0.1% weight by volume solution of crystal violet for 30 minutes at room temperature. Subsequently, the catheters were washed three times with 95% ethanol at room temperature, and the resulting solution was analyzed using a spectrophotometer set to 620 nm. The percentage of biofilm inhibition was determined using the equation described by Namasivayam et al. [30].