What Color Is Positive Silver Or Copper
Due to the concern to notice an alternative to reduce the colonization (microfouling and macrofouling) or the biocorrosion of surfaces submerged for long periods in water, we evaluated the antifouling activity of a commercial paint added with silver nanoparticles (AgNP's) and copper nanoparticles (CuNP's), beside copper-soybean chelate, by electrolytic synthesis, using them in depression concentrations (vi.94E − 04 mg Ag g−one pigment, nine.07Due east − 03 mg Cu g−1 paint, and 1.fourteenE − 02 mg Cu thousand−1 pigment, respectively). The exam for pigment samples was carried out by JIS Z2801-ISO 22196 for periods of initial time, 6 months, and 12 months, against three bacterial strains of marine origin, Bacillus subtilis, Bacillus pumilus, and Bacillus altitudinis. Information technology was possible to demonstrate, according to the standard, that the sample with the greatest antimicrobial activeness was the copper-soybean chelate against two of the iii strains studied (B. pumilus with R = two.11 and B. subtilis with R = two.41), which represents more than 99% of bacterial inhibition. Therefore, we considered a novel choice for inhibiting bacterial growth with nanoparticles every bit antifouling additives.
ane. Introduction
Nanotechnology, being an emerging field where other sciences contribute, generates a large number of new applications based on the novel physicochemical backdrop acquired by resizing a material to the gild of nanometers. Working at the nanoscale, seek to solve current problems in various areas, including materials science, where for example, in the expanse of paints. Some authors propose the incorporation of unlike types of additives, including nanomaterials, to requite them new characteristics, as self-cleaning, antibacterial, scratch-resistant, fire-retardant, UV-protected, woods preservation, anticorrosive [1], and antifouling paints. Using antifouling paints forestall damage to gunkhole hulls acquired past marine microorganisms [2]. Existing products whose purpose is to prevent the growth and settlement of bacteria, fungi, and algae on ships, the presence of those implies adverse effects for maritime navigation, such every bit a reduction in speed caused by greater resistance to friction, that leads to an increase in fuel consumption up to xl%. It tin can also provoke high corrosion rates in the send'southward hull, and consequently, the generation of toxic waste and the introduction of exotic species in its wake [3]. In the heart of the 19th century began to formulate antifouling paints. These had copper oxide, arsenic, and mercury oxide in resin binders and showed expert effectiveness [four]. In the 1950s, tributyltin (TBT), an organometallic chemical compound, began to be used and for approximately two decades was constitute in a large percentage of ships effectually the world [five], but which, despite representing economic benefits, demonstrated at low concentrations, a negative impact on marine organisms [half-dozen]. Thus began the search for alternatives to the use of TBT. Some biocides that accept replaced information technology are copper-based compounds such as cuprous thiocyanate and cuprous oxide [7], the latter being the most used in formulations for antifouling paints. In recent inquiry, it has been proposed to use nanomaterials because it would not only help to reduce the concentration of the biocide used but also forestall harmful effects to other marine species and the resistance of some microorganisms to antifoulants that are already on the market. Owing to the antibacterial properties of argent and copper nanoparticles, they are incorporated into some materials to solve current bug [8] such equally bacterial contamination [nine].
In general, nanoparticles as antifouling agents have been obtained by biosynthesis or chemical reduction methods. According to the study carried out past Inbakandan et al., AgNP's biosynthesis using Acanthella elongata produced particles less than 40 nm in diameter, which were evaluated against 16 strains that form marine biofilms. Their results show that the inhibition chapters depends both on the species to be evaluated and on the concentration of nanoparticles used, and they ended that they obtained a nanomaterial with great bactericidal capacity and antifouling potential [10]. An important finding was presented by Ramasubburayan et al. who constitute minimum values of minimum inhibitory and bactericidal concentration for their silver nanoparticles, obtained from a biosynthesis mediated by Bacillus vallismortis, confronting encrusting strains, showing promising antibiofilm activities [xi].
Copper, due to its properties confronting microorganisms, including marine organisms, has been used for many years in antifouling paints; therefore, all its structures and compounds have been investigated. Abiraman and Balasubramanian reported a green synthesis of copper nanoparticles busy with chitosan, with an average size less than ii nm and whose antifouling activeness against marine and green algae showed an antifouling efficacy of between lxxx and 95% [12]. Nowadays, there are materials that accept attracted interest to investigate them due to their composition and their possible uses in unlike fields, such is the case of a study conducted past DeAlba-Montero et al. where they demonstrated that there are more options of nanometric materials that have antimicrobial properties, such as copper-soybean chelate. They written report growth inhibition against E. coli, S. aureus, and E. faecalis strains at low chelate concentrations [13]. These results generate the concern to utilise and investigate copper-soybean chelate as a new antifouling condiment selection.
In addition to green synthesis or biosynthesis for obtaining nanomaterials as proposed by several authors, some other suggested method that likewise presents advantages such as nigh zero contagion is the electrochemical method. In this type of synthesis, the nanomaterial can be easily isolated from the precipitate, and information technology is an easy to replicate method in which the particle size is usually very well divers. Reetz et al. pioneered the electrochemical synthesis of metallic nanocrystals [14]. His method consists of half-dozen elementary steps: the oxidative dissolution of the anode, the migration of metallic ions to the cathode, the reduction of the ions to a state of naught-valent, the germination of particles by nucleation and growth, the abort of growth by stabilizing agents, and particle precipitation [15]. One of the master advantages of this method is its bang-up reproducibility and the possibility of modifying different variants in the process. The electrochemical synthesis method for obtaining copper and argent nanomaterials, among others, has been published in the literature; however, they utilise very long reaction times, high temperatures, more than ii or three reagents to activate the solution that acts as electrolytic solution and in some cases, a high voltage [sixteen–21]. In this research, we were able to optimize the synthesis method in such a way that nanoparticles were obtained in a reduced time, at room temperature, with a certain voltage and without the inclusion of a big number of reactives, thus proposing a highly reproducible method for both silver and copper, with a calculated reaction efficiency higher than 90%.
The Organisation for Economic Co-functioning and Development (OECD) has emphasized that at that place are different protocols and relevant standards at a global level that allow the evaluation of articles and products that contain antibacterial additives [22], among which can be listed every bit follows: (i) ASTM D5590-94: Standard Examination Method for Determining the Resistance of Paint Films and Related Coatings to Fungal Defacement by Accelerated Four-Week Agar Plate Assay (2) Singapore standard SS 345:2015: Specification for algae resistant emulsion paint for decorative purposes (iii) TESHSA NSI method: A nonsuspended inoculum method for determining the antibacterial activity of coated surfaces
These standards are only some of those that can be used to evaluate antimicrobial products. The vast majority of evaluations are based on qualitative tests, and we can notation that these methods determine the action of fungi and algae, with the exception of the TESHSA NSI method that allows the evaluation with bacteria and being a method that does non belong to any international organization for the standardization of evaluation methods and maybe is the reason why the Paint Research Association (PRA) in the United Kingdom recommends using the Japanese standard JIS Z2801. Every day more than companies use information technology to validate their products since information technology is a method supported at an industrial level past pigment manufacturing companies [23–25].
We propose the employ of silver and copper nanoparticles and copper-soybean chelate every bit additives to provide antifouling properties to a commercial paint. Information technology is relevant to the fact that these nanomaterials are easily synthesized due to our optimized and simplified synthesis method as well as the preparation of copper-soybean chelate, being a circuitous containing an organic part, is of great interest for its use in antifouling applications. We advise that the possible antifouling action will be due to the action that have the nanostructured compounds added to the paint, and this volition exist determined past performing an evaluation according to the Japanese Industrial Standard JIS Z2801-ISO22196 [26], as this is a quantitative and standardized method, which provides reliable and accurate results that improve surface evaluations; however, due to its complexity, it is rarely used. The analyses carried out volition allow us to get-go decide the characteristics of the materials that we are using as antifouling additives, to later observe their beliefs equally additives in paints and to establish whether the pigment prepared with these solutions tin be considered as antifouling and/or antimicrobial.
2. Materials and Methods
ii.1. Electrolytic Synthesis of Nanoparticles
ii.1.one. Silver Nanoparticles
The reactive materials employed for this synthesis were gallic acid (Sigma Aldrich Co.), nitric acrid (HNOthree 64–66%), and ammonium hydroxide (NH4OH) from Fermont Co. and used without farther purification. The electrodes used (silver sheet as anode and graphite every bit cathode) were placed vertically confront-to-face up inside the electrolysis chalice. 0.01 g of gallic acid was diluted and added to 150 ml of deionized water and started stirring; later, 0.5 ml of nitric acid and 0.5 ml of ammonium hydroxide were added to the previous solution. A potential was applied past 30 V for iii minutes, and the resulting solution corresponds to silver nanoparticles (AgNP'south).
two.1.2. Copper Nanoparticles
All the chemicals used in this projection were of reactive grade: sodium borohydride (NaBHiv ≥98%) and sulphuric acrid (H2SO4 95–98%) were purchased from Sigma Aldrich Co. and Fermont Co., respectively, and used without further purification. The experiments were carried out in an electrolysis beaker containing a sacrificial copper sheet every bit anode and graphite equally cathode. These electrodes were placed vertically face-to-face inside the cell, 150 ml of deionized water was added, and kept under stirring. Then, 0.5 ml of H2SO4 was added. Earlier each experiment, the electrodes were polished and done with deionized water. The electrodes were activated by a potential of 30 5 for iii minutes. After this time, 0.1 g NaBHiv was diluted in 5 ml of deionized water and added to the solution. The precipitate obtained was filtered and done with a pocket-sized amount of acetone to prevent the oxidation of the copper nanoparticles (CuNP's). In Effigy 1, we can discover a schematic representation of the suggested process for the electrochemical synthesis of nanoparticles [27]. Additionally, we propose the possible chemical reactions involved in the synthesis, which are presented in Figure one for copper and silver nanoparticles.
2.1.3. Copper-Soybean Extract Chelate
The solution was prepared co-ordinate to the method previously reported by Guajardo-Pacheco et al. [28], where 1 chiliad of CuSO4⋅5HiiO was added to a soybean extract solution; information technology was kept under magnetic stirring for 15 minutes, and the pH was adjusted to 7 using a 1 G sodium hydroxide solution.
two.2. Characterization
Copper and silver nanoparticles formation were confirmed by different structural characterization techniques. Optical absorption spectra were obtained with an Ocean Optics S2000-UV-Vis arrangement. The average particle size, polydispersion index (PDI), and Z potential were measured with a dynamic calorie-free handful (DLS) Zetasizer Nano ZS. Manual electron microscopy (TEM) measurements were performed on a JEOL JEM-1230, working at 100 kV, obtaining images respective to the shape and size. XRD analysis of copper nanoparticles was conducted using an X-ray diffractometer Firmament Alpha 1 of Malvern Panalytical., with Cu-Thouα X-rays of wavelength (λ) = i.54056 Å, and infrared spectroscopy (FTIR) was performed with an IR Affinity-ane for the Copper-Soybean Chelate sample.
ii.three. Antimicrobial Exam of Nanoparticles
The following reactive materials were used: phosphate buffer Na2HPO4 and KH2PO4 (Fermont), Mueller-Hinton goop (BD Difco), and sodium chloride (NaCl, CTR Scientific). We analyzed the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by the standard microdilution method (CLSI M100-S25 January 2015) [29], to determine the antimicrobial activity of the nanoparticles synthesized. The strains exposed to nanoparticles as the exam control were Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212); after, three strains of marine origin were used, Bacillus subtilis, Bacillus pumilus, and Bacillus altitudinis. The bacterial concentration was standardized using the McFarland scale, and information technology was realized to an optical density of 0.2 at 568 nm (approximately 1 × 108 CFU ml−1). The nanoparticles concentration employed against the strains was ii.five mg·ml−ane for copper nanoparticles and 1.54E − 01 mg ml−ane for silver nanoparticles. Once the nanoparticles were dispersed, they were diluted with 50μl of Mueller–Hinton broth and 50μ50 of phosphate buffer previously inoculated with the tested strains at a concentration of one × 10five CFU·ml−1. Equally the last footstep, the plate was incubated for 24 h at 36 ± 1°C.
2.4. Paint Preparation
For the grooming of antifouling paint, a commercial vinyl-acrylic paint Comex® Pro chiliad plus was used, which was prepared co-ordinate to the method reported by Dominguez-Wong et al. [30]. 15% five/5 of the Ag and Cu nanoparticles and copper-soybean chelate in solution was added to the paint. The concentration of the solutions added to the paint was 2.5 mg·ml−1 for copper nanoparticles and copper-soybean chelate and 1.54Due east − 01 mg·ml−1 for silverish nanoparticles. This concentration is the aforementioned as that used for the antimicrobial analysis of nanoparticles in solution. The paint without additives (command paint) was prepared according to the manufacturer'southward instructions, adding 15% water. To ensure proficient dispersion of the nanoparticles in the paint, each sample was homogenized using ultrasonic liquid processors (Sonics Vibra cell model CV33). The samples were processed for 10 minutes. An enough quantity of paint with additives was prepared for further evaluation over one year (initial fourth dimension, 6 months, and 12 months). The glass samples used had a measure of 5 × 5 × 0.6 cmiii, which were covered with 3 layers of paint for later on evaluation. All the samples were evaluated in triplicate.
ii.v. Evaluation of Color Variation in Paint Samples using the CIELAB76 Model
Colorimetric tests were carried out on the paints to define the color departure using the CIELAB 1976 standard, which is one of the about widely used systems proposed past the Commission Internationale de l'Eclairage [31]. This system is defined by three coordinates, L∗ indicates the lightness and a∗ and b∗ are the chromatic coordinates [32]. Fifty∗ = lightness a∗ = ruby-red/green coordinates (+a indicates reddish, -a indicates dark-green) b∗ = yellow/blue coordinates (+b indicates yellow, -b indicates bluish)
The color difference or color variation is defined as the numerical comparison of a sample with a standard and is represented as delta (ΔEastward∗), and this color difference is determined according to Equation 1.
Color is an important property in the coatings and paints industry. The CIELAB76 calculation method is still the most commonly used. The tolerance or color acceptance is the maximum color difference admitted in products in relation to a standard, and in the coatings and paints industry, it is established that this variation must be in the range of 0.0 to 5.0 ΔE ∗units. This divergence is industrially interpreted equally follows [33–35]: ΔE ∗< ane Imperceptible ΔDue east ∗ < 2 Minimum ΔEast ∗ < 3 Adequate ΔEastward ∗ < 5 Almost unacceptable ΔE ∗ = 5 Unacceptable
The color coordinates of the paints (CIEL∗a∗b∗) were determined with the OceanOptics SpectraSuite® spectrometer software, and subsequently the colour variation was calculated.
2.half dozen. Conclusion of Cu and Ag Species Concentration in Pigment
This exam was performed to make up one's mind the existent concentration of silver and copper species, one time added to the pigment, and correlate it with its initial concentration. The concentration of the Cu and Ag species contained in each pigment sample was adamant in an inductively coupled plasma atomic emission spectroscopy (ICP-OES iCAP 7000 serial, Mod. ICAP 7400 Duo, Thermo Scientific). For this analysis, 25 ml of the acid mixture for digestion was added to one k of paint, it was heated for 2 hours at 200°C, and the recovered supernatant was fabricated up to 25 ml with deionized water.
two.7. Antimicrobial Examination of Paint
two.seven.one. Qualitative Examination
The qualitative evaluation of the pigment samples was carried out by adapting the Standard Exercise for Determining Resistance of Plastics to Bacteria [36]. Different samples of Mueller–Hinton agar were inoculated at 45°C with iii strains of marine origin, B. subtilis, B. pumilus, and B. altitudinis, with a final concentration of 5 × xv cells·ml−1. One time the agar was inoculated, 15 Petri dishes were filled for each bacterial strain, each sample was tested in triplicate (control, command paint, paint + AgNP's, pigment + CuNP's, and paint + copper-soybean chelate). Before the full solidification of the Mueller–Hinton agar, the painted glasses were placed on the surface of the agar. Finally, they were incubated at a temperature of 36°C ± 1°C for 24 h. The result of this test is through visual appreciation later on incubation.
2.seven.two. Quantitative Test
For quantitative evaluation, the pigment was evaluated based on the Japanese Industrial Standard JIS Z2801-ISO 22196, Antibacterial products—Test for antibacterial activity and efficacy [26]. According to the standard, the test was performed in triplicate for each paint sample, and the R value was calculated according to equation (2). Established equally the antibacterial activity of an evaluated product, it tin also be expressed every bit the decrease in bacterial growth in orders of magnitude. The JIS Z2801-ISO 22196 standard indicates that for a production to be classified as antimicrobial, the R value must be ≥two. The glass samples painted with the previously prepared paint were employed and untreated glasses were used as a positive control. The inoculum was prepared from pure cultures of the iii strains of marine origin, B. subtilis, B. pumilus, and B. altitudinis, obtaining a concentration of 5 × 105 cells·ml−ane. The samples were inoculated with 200μ50 of the inoculum, and a 4 × iv cm2 polyethylene film was placed on summit of each one. Afterwards, serial dilutions of tenfold were fabricated, and the method of pouring into a plate with Mueller–Hinton agar was carried out, for the recovery and counting of the colony forming units (CFU). Petri dishes were incubated at a temperature of 36°C ± one°C. where R is the value of antimicrobial action. A is the average of the number of viable cells recovered immediately (time t = 0 h) after inoculation of the sample without additive. B is the average of the number of viable cells recovered later on 24 hours (fourth dimension t = 24 h) after inoculation of the sample without additive. C is the boilerplate of the number of viable cells recovered after 24 hours (time t = 24 h) after inoculation of the sample with additive.
All solutions used in this exam were used at the appropriate salinity level and the principal components to simulate seawater conditions [37]. The purpose of this test is to simulate the outset stage of colonization of a surface, which is characterized by the presence of algae, invertebrates, and bacteria followed by protozoa and diatoms. This flick of organisms is chosen primary picture or microfouling which is formed during the offset hours of contact. The character of the resulting customs is the one that gives way to macrofouling, upwards to development the biofilm [38, 39]. Therefore, if the first colonization tin can be inhibited, information technology is very probable that the formation of the biofilm will exist prevented. Figure two is a schematic representation of the possible antifouling mechanism past which paints tin can act against microorganisms.
three. Results
3.1. Label of Nanoparticles Solutions
iii.1.i. UV-Vis Assay
Effigy iii shows a grouping of UV-Vis spectra of the different electrolytic solutions. Figure three(a) shows a typical absorption spectrum of the silver nanoparticles, and a narrow absorption band centered at 415 nm is shown [19, 40]. Effigy iii(b) shows a divers absorption spectrum which proves the formation of the copper nanoparticles in the solution. The sample display a peak at effectually 543 nm [41, 42]. These peaks are due to the surface plasmon bands for the nanoparticles. Figure 3(c) corresponds to the sample of the copper-soybean chelate, where information technology is observed that the band associated with copper fades due to the formation of the soybean extract-copper complex [xiii]. The exact position of the resulting bands in the UV-Vis spectrum tin alter depending on the backdrop of the individual particles.
3.1.2. DLS Assay
The nanoparticle solutions were analyzed by size using dynamic light scattering (DLS) also as the polydispersion index (PDI) which is a measure of the size ranges present in the solution. The scale varies from 0 to 1, values close to zero point that the sample is monodisperse and values close to unity indicate that the sample has a cracking variety of sizes. Z potential was a measure out too. This indicates if the surface charge of the nanoparticles is high enough to ensure the electrostatic stability of the suspension in the long term and avoid aggregation. The particle size distribution of colloidal solutions is shown in Figure 4. Figure 4(b) corresponds to the measurement of argent nanoparticles where it shows an boilerplate size of seven nm, and it is observed that the histogram has a narrow size distribution. The PDI obtained is 0.109 which determines the monodispersion of the sample, and the value obtained for the Z potential of the silver nanoparticles was −32.7 ± 11.5 mV indicates that the surface accuse of the nanoparticles is high enough to ensure the electrostatic stability of their break, which prevents aggregation and contributes to the stabilization in long term. In Figure iv(d), it can exist observed that the average particle size is 155 nm, obtained from the measurement of the copper nanoparticle sample. The PDI is 0.450, and the Z potential was −19.8 ± 12.4 mV, demonstrating the low stability causing the assemblage, which can be corroborated in the TEM images [42, 43].
3.one.iii. TEM Analysis
Figure iv shows the TEM images of the nanoparticles prepared by electrolytic synthesis. Figure iv(a) shows the silver nanoparticles, where information technology is shown the formation of the well-dispersed nanoparticles with spherical shape and uniform size, and the presence of particle aggregation is not observed [forty]. Effigy 4(c) corresponds to copper nanoparticles where the distribution is non compatible and have an irregular shape; in this example, the particles are larger and the presence of dumbo agglomerates or the trend to cluster is observed.
three.1.4. XRD Analysis
Figure 5 corresponds to 10-ray analysis of the nanoparticles. In Figure 5(a), there are four strong reflection peaks at 2θ values of 38.23°, 44.25°, 64.72°, and 77.45° respective to lattice planes of silver (111), (200), (220), and (311), respectively. All reflections correspond to FCC silver, and the lattice constant was determined at 4.07 Å. This effect has highly matched with the standard powder diffraction card (JCPDS no. 04-0783) of Joint Committee on Powder Diffraction Standards. The XRD pattern of copper nanoparticles are shown in Figure 5(b)) with reflections at iiθ = 43.22°, 50.34°, and 73.95°, which corresponds to the crystalline plane (111), (200), and (220), respectively, of the face-centered-cubic construction (FCC), and the network parameter was iii.62 Å. It has been reported that for FCC materials, the reflection corresponding to plane (111) is the one with the highest intensity, which agrees with the measurements of the nanoparticles samples.
(a)
(b)
3.i.5. Infrared Spectroscopy (FTIR)
Figure six shows the infrared assimilation spectrum of the copper-soybean chelate solution that presents bands in the range of 400 to 500 cm−1 which correspond to the germination of chelate, specifically the band at 460 cm−1 is associated to symmetrical stretching vibrations of Cu-N and COtwo bend wagging and the bands at 618 cm−one and 775 cm−1 can exist attributed to rocking vibrations of COii and NH2 due to the complex information technology forms with the amino acids [44]. At 1097 cm−ane, the vibrations correspond to C-N, and the peaks at 1633 cm−1 are attributed to bend scissoring vibrations of NH2. After the 3000 cm−i range (3290 to 3390 cm−i), the bands correspond to O-H and N-H vibrations.
3.two. Antimicrobial Assay of Nanoparticles
Table 1 shows the results for the antimicrobial exam of the copper-soybean chelate, copper nanoparticles, and silver nanoparticles. The copper-soybean chelate compared to copper nanoparticles has a greater antibacterial activity when it was tested against B. altitudinis and E. faecalis, which is in accordance reported in the literature [13]. Apropos silver nanoparticles, the best antibacterial effect was observed against S. aureus, B. pumilus, and B. altitudinis, obtaining the same MIC value for the iii strains, and in this particular analysis, a higher efficacy was demonstrated for silver nanoparticles approximately ten times more effective compared to copper nanoparticles and copper-soybean chelate.
| | |||
| MIC nanoparticles (mg·ml−one) | |||
| Bacterial strains | Sample | ||
| Copper-soybean chelate | Copper nanoparticles | Silverish nanoparticles | |
| (2.5 mg·ml−i) | (1.54Due east − 01 mg·ml−1) | ||
| | |||
| E. coli ATCC 25922 | 0.62 | ane.25 | nine.viiE − 03 |
| South. aureus ATCC 29213 | 0.62 | 2.five | 4.8E − 03 |
| E. faecalis ATCC 29212 | 0.31 | i.25 | 3.9E − 02 |
| B. subtilis | ii.five | 1.25 | 9.7E − 03 |
| B. pumilus | 2.v | one.25 | 4.eightE − 03 |
| B. altitudinis | 0.62 | i.25 | 4.8E − 03 |
| | |||
3.iii. Color Variation of Paints using the CIELAB76 Model
The CIELAB organization allows to define the color and the formula of color variation or color difference, and it manages to quantify the homogeneity of the color compared to a standard or reference co-ordinate to the analyzed measurements. The values obtained from the paint samples to analyze the color variation are presented in Table two, such as lightness (Fifty∗), chromatic coordinates (a∗, b∗), and total color difference (ΔE ∗). This test was reported in triplicate for each sample. The ΔDue east ∗ levels calculated were less than i for the paint with copper nanoparticles and the pigment with copper-soybean chelate and less than 2 for the paint with silver nanoparticles. These represent an imperceptible and minimum degree of color variation, respectively. The variation of the parameters in each coordinate with respect to the reference, and the ΔE ∗ values bespeak a good colour homogeneity. The similarity betwixt the samples is consequent with the use of the same paint additive.
| | |||||
| Color variation of paints using the CIELAB76 model | |||||
| Sample | CIE Fifty∗ | CIE a∗ | CIE b∗ | ΔE ∗ | Color acceptance |
| | |||||
| Control paint reference | 99.81 | 0.3 | 0.two | ||
| Paint + copper-soybean chelate | |||||
| 1 | 99.41 | 0.two | 0.viii | 0.73 | Imperceptible |
| 2 | 99.41 | 0.1 | 0.eight | 0.75 | |
| 3 | 99.60 | 0.2 | 0.ix | 0.74 | |
| Paint + CuNP's | |||||
| one | 98.95 | −0.1 | 0.5 | 0.99 | Imperceptible |
| 2 | 99.15 | −0.one | 0.five | 0.83 | |
| iii | 99.01 | 0.one | 0.iv | 0.85 | |
| Paint + AgNP'due south | |||||
| 1 | 99.6 | −0.1 | 1.six | 1.47 | Minimum |
| 2 | 99.8 | −0.2 | 1.v | 1.39 | |
| 3 | 99.8 | −0.2 | 1.four | ane.3 | |
| | |||||
3.iv. Cu and Ag Species Concentration in Paint
The analysis of the paint using ICP-OES allowed usa to decide the existent concentration of the Cu and Ag species independent in each sample, and these results are shown in Table three. For the command paint, a negative concentration calculation was obtained, which indicates that the paint used equally the control does not comprise Cu and Ag species in its initial composition. These values let u.s. to compare the percentage of Cu and Ag species present in the pigment. If we compare them confronting the initial concentration of the added solutions (2.5 mg·ml−1 for copper nanoparticles and copper-soybean chelate and 1.54E − 01 mg·ml−ane for silverish nanoparticles), the concentration adamant by ICP-OES represents less than 1% of the initial concentration. In spite of low concentrations of Cu and Ag species, they can be correlated with a good antimicrobial activity, presented in the later sections.
| ||||||||||||||||||||||||
| aNo testify of Cu or Ag species was found in these samples. | ||||||||||||||||||||||||
iii.5. Antimicrobial Assay of Paint
3.v.1. Qualitative Assay
The samples evaluated by the Standard Practise for Determining Resistance of Plastics to Bacteria (control paint, paint + AgNP'southward, pigment + CuNP's, and paint + copper-soybean chelate) presented the same caste of inhibition when in contact with the surface for all samples; however, they did not show an inhibition halo in the culture medium.
3.v.2. Quantitative Assay—JIS Z2801-ISO 22196
The boilerplate of the count of recovered bacterial cells is presented in data of log10 CFU·ml−1 after ane year of evaluation in Table 4, with test periods (initial time, 6 months, and 12 months). After evaluation, the control paint sample did not show any antibacterial activeness, and the pigment samples added with nanoparticles were compared in the percentage of activity and logx CFU·ml−one against the control pigment. At that place is a difference of approximately 2 orders of magnitude (≈ii logten CFU·ml−1) between all the paints with additive and the control pigment for the 3 strains, which is equivalent to more than 99% inhibition in bacterial growth. Throughout the year of evaluation, there was an increase in the bacterial count (logten CFU·ml−i) that indicates a slight subtract in the per centum of antibacterial activity of the paints.
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| Paint + CS-Ch = paint + copper-soybean Chelate. Bold values are the outstanding results of the samples subsequently being evaluated in triplicate. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
4. Discussion
Later on the respective evaluations, we determined that the antifouling activity of the evaluated paint is attributed to the addition of the synthesized compounds.
TEM assay results for copper nanoparticles (Figure 4(c)) showed the presence of agglomerates of particles. This is probably due to the solubility of copper nanoparticles in h2o, and this is one of the principal disadvantages of these particles equally well as their rapid oxidation [45–47]. The chosen method for efficient measurement of the nanoparticles was to disperse the powder by sonication just before depositing it on the TEM grid to avoid precipitation. XRD design (Effigy v(b)) showed boosted peaks that are assigned to CutwoO, the network parameters are similar to those reported in the literature, and the germination of Cu2O indicates the partial oxidation of the nanoparticles. It can exist indicated that the result obtained from the XRD pattern is as expected, contrasting to what has been reported by different authors for copper nanoparticles synthesized past unlike methods [41, 42, 46–48].
The deviation in the results of the antimicrobial activeness of the samples tested against the strains may exist due to the construction of each bacterium: the cell wall is wider for Gram-positive strains, adhesion structures, virulence factors or pili type as well every bit the presence or absence of mobility structures or flagella [49]. Even so in the case of argent nanoparticles, we observed that despite being 2 different genders of bacteria, Bacillus genus and Staphylococcus genus, the same MIC values were obtained in these strains, which tin can be explained by previous studies where they reported that through an atomic force microscope (AFM), the surface characteristics of Gram-negative and Gram-positive leaner were studied and observed that species of the genus Staphylococcus showed a smooth surface practically equal to that of the surface area of species of the genus Bacillus [50]. Regarding paint, we can mention that the visible backdrop of the paint such as gloss and color were not modified when adding the nanoparticle solutions and the copper-soybean chelate. The qualitative analysis of the paint shows the absence of an inhibition halo in the medium, which could imply a stiff adherence of the Cu and Ag species to the paint, that explains the absenteeism of leaching of the biocidal species and only their degree of inhibition on contact with the surface.
According to the Japanese Industrial Standard JIS Z2801-ISO 22196, a product is antimicrobial when the value of R ≥ 2. Effigy seven shows the antibacterial activeness (R) calculated in the 3 test times, where it is possible to capeesh the decrease of the R value and the behavior that each sample presented afterwards one year of evaluation, and the graphics evidence that the paints with copper species (paint + copper-soybean chelate and paint + CuNP's) have a similar antibacterial action in the initial fourth dimension (R = 2.78, ii.69). Later 12 months, the paint + copper-soybean chelate maintains an R value > 2 for B. pumilus and B. subtilis which is equivalent to 99.22% and 99.61% inhibition of bacterial growth, respectively; for B. altitudinis, the R value decreased (R = 1.90) with this paint. In the example of B. altitudinis, information technology is observed that later 6 months of evaluation for paint + CuNP's and pigment + AgNP's, antimicrobial activity decreases and after 12 months, the 3 paint samples with additive take a value of R < 2 (R = ane.ninety, 1.79, and ane.74). For B. pumilus, the only sample that decreased its antimicrobial activity was paint + AgNP's at 12 months of evaluation (R = 1.91), and this sample is the one that showed the lowest antimicrobial activeness for the 3 strains. The three paint samples with condiment had a better antimicrobial activity when they were tested with B. subtilis, and in that location was a slight subtract throughout the year of evaluation; however, the R value remained at >2 (R = 2.41, 2.14, and 2.01) which is equivalent to more 99% bacterial inhibition.
Correlating the results of the surface evaluation (JIS Z2801-ISO 22196) with the effective concentration of silvery and copper species in the pigment, obtained by ICP-OES, we tin aspect, as mentioned to a higher place, the antimicrobial action of the paints to the activity of the added biocidal species. What is important to highlight at this betoken is the high inhibitory capacity of the paints despite containing a very low concentration of Cu and Ag species compared to paints that have been used for antifouling purposes [51–53]. This tin be translated into a bully and novel option, getting effective antifouling additives at low concentrations, equally well as the ease of admission to these by ways of a reproducible synthesis method.
The JIS Z2801-ISO22196 exam show an important inhibition of bacterial growth, highlighting the results obtained for the paint added with copper-soybean chelate. We suggest that the presence of the organic role (amino acids) provided past the soybean excerpt in the chelate composition is the one that interferes in the furnishings of a high bacterial inhibition [thirteen]. Biocidal species in ionic form accept a specific interaction with amino acids due to the content of costless sugar in the bacteria and the amino sugars present in the peptidoglycan chains [54] for that the copper-soybean chelate has the ability to attract microorganisms and crusade their prison cell death because the ions it contains has a major affinity than the nanoparticles and it is easier for them to penetrate the cell membrane of the bacteria. For this reason, despite having a different concentration than the copper and silver nanoparticles in the paint, it tin resemble and even improve its power to inhibit bacteria. It is of import to mention that this piece of work is the showtime phase of the study, and the results obtained will let us to continue with the investigation of the sample that produced the best results (copper-soybean chelate), to carry out the growth of a bacterial biofilm and the corresponding study of the interactions between this and the paint sample, in order to establish whether the second stage of colonization of a surface can be inhibited besides as to determine the concentration of ions released from the additive afterward interacting with the biofilm.
Nowadays, the use of compounds and nanostructures of different materials to determine their antimicrobial, antifungal, antifouling, and other properties is increasing; however, the biochemical process or the mechanism by which they act on microorganisms has not been completely elucidated, and dissimilar routes of action have been proposed for these biocidal species to crusade jail cell or bacterial death.
Talking about the ionic form of silvery or argent nanoparticles, the biochemical mechanisms involved can be several, such equally the impairment of the prison cell wall produced by silver likewise equally the accumulation of argent in the bacterial membrane. It is believed that Ag breaks the permeability of the outer membrane of the bacteria and affects their peptidoglycan chains, causing the leakage of cellular materials [55]. This leads to the inhibition of respiratory chain dehydrogenases, while some proteins and phospholipids induce membrane collapse and subsequently jail cell decease [54].
It is well known that transition metals are toxic to leaner, and although copper is an essential trace element for bacterial cells because information technology is involved in the synthesis of metalloproteins, such as electron-transport proteins, its ionic form is considered toxic at higher concentrations. A low molecular-weight compound can bind to bachelor copper ions and thus modify their biological function [56]. The probable biochemical process involved in the antibacterial activity of copper species begins with the absorption of Cu ions past the bacteria, which adhere to the bacterial cell wall that is negatively charged and crusade the breakdown, followed by a cascade of events for the reduction of these ions. Subsequently, these particles are released from the cell using the bacterial efflux organisation, that causes poly peptide denaturation and prison cell death [57, 58]. One time copper enters the bacterial cell, information technology can bind to deoxyribonucleic acid molecules causing rapid Dna degradation, followed by a reduction in bacterial respiration as well equally the inhibition of bacterial membrane cytochromes. Absorption of copper ions by bacterial cells as well disrupts important biochemical processes [57, 59, sixty].
5. Conclusions
This work conferred a significant improvement to the paint samples employing nanostructured compounds as additives and studying their antifouling capacity, which showed a loftier percentage of bacterial inhibition against Bacillus species, in which copper-soybean chelate presented the best antifouling action. Our results suggest that using copper species and AgNP'due south can increase the resistance to biofouling of surfaces.
Data Availability
The data that support the results of this study are available on request from the corresponding writer.
Conflicts of Interest
The authors declare that they have no conflicts of involvement regarding the publication of this commodity.
Acknowledgments
The authors are grateful to Consejo Nacional de Ciencia y Tecnología (CONACYT) for the partial support to this project. Thou. Thousand. Loredo-Becerra, A. Durán-Almendárez, and A.K. Calvillo-Anguiano would similar to give thanks CONACYT for scholarship nos. 465639, 733623, and 733766, respectively. The authors appreciate the contribution of PhD María Elena García Arreola, PhD Marcos Loredo Tovías, and Geochemistry Laboratory of the Geology Found UASLP regarding the ICP-OES analysis of the paint.
Copyright
Copyright © 2022 G. 1000. Loredo-Becerra et al. This is an open up admission article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original piece of work is properly cited.
What Color Is Positive Silver Or Copper,
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