Efficiency of Cu, Ag, and Fe Nanoparticles As Detergents Preservatives Against E. coli and S. aureus

Document Type: Research Paper


1 Department of Chemistry, Tarbiat Modares University, Tehran, Iran

2 Industrial paints of Iran Co., Isfahan, Iran


In this study, Cu, Ag, and Fe nanoparticles (NPs) are used in shampoo, hand washing liquid (HWL) and dish washing liquid (DWL) instead of the conventional synthetic preservatives such as isothiazolinones; since the latter often act as potent sensitizers that leads to development of allergic contact dermatitis. The above NPs are considerably effective against Escherichia coli and Staphylococcus aureus. Our metal NPs are deliberately made durable and pure through arc fabrication. They appear in spherical morphology as indicated by XRD and SEM. This study clearly demonstrates the advantages of using rather low concentrations of Ag, Cu and Fe NPs as preservatives instead of carcinogenic Kathone CG, which is commonly used in detergent products.
The shampoo formulation with 0.1 g/L Cu NPs and HWL and DWL with 0.1 g/L Ag NPs exhibit the best antibacterial activity against tested E. coli and S. aureus. Generally, the order of antibacterial activity of these preservatives is: Cu NPs>Ag NPs> Fe NPs.



A detergent, as a kind of synthetic surfactant, is engineered to perform under desired conditions [1] and composed of water, primary surfactant (cleansing agents), foam boaster, thickeners, modifiers, and desired additives [2-6]. While, the sterility of detergents is not often necessary, contamination with pathogenic microorganisms and microbial contaminants, especially in high numbers, are not desired [7]. In addition, transmission of bacteria on the skin can cause infections [1]. Microorganisms such as E. coliS. aureus, and P. aeruginosa easily grow in places not completely dried up (bathtubs, shower areas, sinks, etc.) [8]. In such places, microbes can easily be transmitted to shampoo products, which are generally based on sodium lauryl sulphate surfactants that separate or discolor upon proliferation of Pseudomonas species [9]. Of special concern is detection of the latter pathogen that has spoilage potentials and is the most common microorganism associated with recall of many cosmetic formulations in the United States and Europe [4, 10,11].

Hence, antimicrobial ingredients are employed as antiseptics and (in lower concentrations) as preservatives for detergents [12]. Although the efficacy of a detergent is influenced by the formulation, the type and concentration of active ingredients are of particular importance[13,14]. A large number of chemical compounds have the ability to kill or inhibit the growth and metabolism of microorganisms [1]. Despite their rather large number, each antimicrobial has its own physical, chemical or toxicological limitations. So, the interest in development of new and better ones remains high [15]. One of the most efficient preservatives with powerful biocide effects is methylchloroisothiazolinone which is used in conjunction with methylisothiazolinone (MCI/MI) (Kathone CG) [16]. It is used in the manufacture of cosmetics, detergents, paint, glue, and synthetic rubber and for disinfecting cooling systems. Kathone CG is known to be a potent sensitizer that may lead to the development of allergic contact dermatitis among cosmetics users and personnel working in industries where the substance is commonly used [17].

The emergence of nano-science and nanotechnology in the last decade has provided opportunities for exploring the bactericidal effects of metal nanoparticles [15,18]. Such effects have been attributed to their small size, and high surface to volume ratio, allowing them to interact closely with microbial membranes, coupled with the release of metal ions in solutions [19]. The antifungal and bacteriostatic properties of Ag NPs as well as the antimicrobial activity of Cu NPs, and Fe NPs have been reported [19-26]. Unlike antibiotics, NPs adaptation does not occur for microorganisms. These properties of Ag, Cu, Au, Fe, Zn, etc. nanoparticles can make them applicable in various fields; i.e., medical instruments and devices, water treatment and food processing [18-22,27].

This study investigates bactericidal efficacy of Ag, Cu, and Fe NPs against Escherichia coli (Gram negative), and Staphylococcus aureus (Gram positive) bacteria and potential application of the NPs as preservative in detergents including shampoo, hand washing liquid (HWL) and dish washing liquid (DWL).


Preparation of Metal Nanoparticles

Metal NPs are prepared by DC-arc discharge method optimized in our group [28-30].

Silver electrodes, with an 80o angle, are exposed to pulses of 5-10 A/cm2 in 10% glycerin/distilled water.

Pulses of 50 A/cm2 are passed through Cu as well as Fe rods, with a 45o angle, in distilled water.

The resulting NPs are separated upon centrifuging and drying at 70 oC for 24 h. The XRD (Philips X’pert MPD, CoKα irradiation, λ = 1.78897 Å) at a scanning speed of 2o/min from, 20o to 80o (2θ)) SEM (KYKY EM3200- 25 KV), and TEM (ZEISS, EM10C, 80 KV), are used for depictions of crystalline structures, morphology and size of the metal NPs.

Preparation of Detergents/Metal NPs

The shampoo samples were prepared using distilled water, sodium luryl ether sulfate (SLES) as surfactant, betain, glycerin, and coconut fatty acid. After mixing, pH was adjusted to neutral (6.5-7).

The HWL samples were formulated using four ingredients such as sodium luryl ether sulfate (SLES), betain, glycerin, coconut fatty acid and distilled water as vehicle. The pH of mixed solution was adjusted by adding sufficient quantity of citric acid solution (pH=6).

To formulate a basic DWL, definite amount of sulfonic acid and coconut fatty acid were added to distilled water. After mixing of the formulated DWL, the pH (6.5-7) was adjusted by adding diethanolamine.

Three samples of Ag NPs/detergent (0.1, 1.0, 10.0 g/L) were prepared by addition of 5, 50 and 500 mg of Ag NPs to three vials, containing 50 mL of detergent. Similarly, samples of Cu NPs as well as Fe NPs were prepared. Accordingly, twenty-seven different metal NPs/detergent vials were obtained (nine for any detergents). Each sample vial was placed in ultrasonic bath for 10 min until NPs are thoroughly dispersed. In order to compare the results, a commercial concentration of Kathone CG /detergent (50 mL) was also prepared (three vials).

Antibacterial Assay of Detergents/Metal NPs

Preservative capacity of the detergent samples was probed against detergent samples containing only Kathone CG. All of these thirty samples were contaminated with E. coli (ATCC 25922), and S. aureus (ATCC 25923) separately. Specifically, each of the above mentioned bacterial strains was cultured overnight on Muller-Hilton agar (ATCC, WDCM1). A suspension of each was prepared in 5 mL normal saline containing 10cells/mL. These suspensions (100 μL) were mixed and homogenized with 500 μL detergent samples (60 samples). After a specific time, 5 mL sterile normal saline was added to these mixtures and vortexed for one minute. Dilutions of each tube were cultured on Muller-Hilton agar. The plates were maintained at 37 oC for 18 h. Finally, the number of colonies per sample was counted.


Size, morphology, and durability of metal NPs highly depend on the method of synthesis. Following our quest for stable nanomaterials [28-32] and our recent interest in reaching for molded detergents with preservative and antibacterial applications, here we take up fabrication of durable metal nanoparticles, including Ag, Cu, and Fe NPs, through arc discharge. They are mixed (in 3 concentrations) with shampoo, HWL and DWL formulations.

Fabrication of Durable Metal NPs Through Arc Discharge

Our DC arc discharge technique involves explosion of metal rods by a pulse current. Fairly pure metal electrodes (99%) with diameters of 2 mm and lengths of 40 mm were used as anode and cathode. Explosion of silver, copper and iron rods occur at a pulse current of 5-10, 50 and 50 A/cm2, respectively. Average particle size for Ag, Cu and Fe NPs appears as small as 8, 20 and 37nm based on their TEM images respectively (see supplementary file).

Antibacterial Assay of Shampoo, HWL and DWL Formulations/Metal Nanoparticles

Preservative capacities of Ag, Cu, Fe NPs are probed for twenty-seven samples with three different concentrations of metal nanoparticles and compared to those of three samples containing only Kathone CG. All the above samples were separately tested against deliberate contamination with E. coli, and S. aureus. Specifically, each of the above mentioned bacterial strains was cultured overnight on Muller-Hilton agar. A suspension of each was prepared in 5 mL normal saline containing 10cells/mL. Then, 100 μL of each suspension of bacteria was separately added to 500 μL of each of the thirty detergent samples. Consequently, the above samples were homogenized via a shaker. After 24 hours, 5 mL sterile normal saline was added to these mixtures and vortexed for one minute. Three dilutions of each sample (1, 0.1, and 0.01 v/v) were cultured on Muller-Hilton agar. The 90 plates were maintained at 37oC for 18 h, and then the number of colonies per sample was counted. The overnight vicinity (24 h) shampoo, HWL and DWL samples extirpated all of the bacteria. Hence, vicinage time was gradually decreased and experiments were repeated down to 12 and then 6 h, where all the bacteria were similarly extirpated. So, the time was decreased again. After 1 h and in dilution of 0.01 v/v,alterations were observable and results were recorded (Table 1).

Quantitatively, these results are based on the percent bacterial growth (X), shown by X= (1-((B-T)/B)) ×100. Here, B is the number of colonies in a bacterial assay used as the “Control” (Sample B). “Control” is attributed to every one of the two strains of bacteria directly cultured on agar, without being subjected to the shampoo, HWL and DWL formulations. T is the number of colonies found in either one of the thirty detergent samples.

9 detergent samples containing three concentrations of Ag NPs (0.1, 1.0 and 10.0 g/L) were investigated. The bactericidal efficiency of these products (samples 1-9) was studied against E. coli and S. aureus. Based on the results, Ag NPs with either of its three concentrations in shampoo and DWL samples show an efficient antibacterial activity against S. aureus (Fig. 1a and 1c), while in HWL in concentration of 10 g/L it does not show a good efficiency (Fig. 1b).

The results for E. coli are completely different especially in shampoo samples. X values are really large in all shampoo samples (Fig. 1a). The results of assessment of antibacterial potency (Fig. 1b) revealed that Ag NPs (0.1, 1.0 g/L) in HWL and (0.1, 10 g/L) in DWL have potent antibacterial activities against E. coli cells. This efficacy at concentration of 10.0 g/L HWL and 1.0 g/L DWL decreased. Here, S. aureus is more sensitive than E. coli to the Ag NPs, suggesting that E. coli is more resistant to Ag NPs. Outer membrane of Gram- negative bacteria is mostly formed from lipopolysaccharide molecules causing a resistance barrier against nanoparticles [33]. It is known that possible free silver ion (Ag+), and those Ag+ and Ag0 can be released by Ag NPs (eq. 1) which are highly toxic to bacteria [34-36]. On the other hand, bacterial cell wall has a negative charge because of teichoic acids (in Gram positive bacteria) linked to either the peptidoglycan or to the underlying plasma membrane and outer covering of phospholipids and Lipopolysaccharides (in Gram negative) [37]. This negative charge (9.57 r/e (-)) in S. aureus is really more than that in E. coli (1.33 r/e (-)) [38]. So, released Ag+ ion can easily connect to S. aureus cells wall and may affect them through deactivation of cellular enzymes and DNA by reacting with electron-donating groups such as thiol (–SH) groups and generates ROS ultimately leading to cell lysis and death [39-44]. Although, the antimicrobial mechanistic action of Ag NPs is not clearly understood, previous research has demonstrated that Ag NPs attach the surface of cell membrane, causing the change of membrane permeability, dissipation of the ATP pool and proton motive force, bringing about the final cell death [44-47]. Some studies suggest that E. coli cells may have stronger oxidizing/reducing power and interact with Ag species to form cell-particle aggregates [39,45,48]. To study the effect of Ag+ on E. coli and S. aureus, Ag+ (released from Ag NPs during 6 months) in three concentrations in studied detergents was used (Table 2).

The results of antibacterial assay in shampoo and DWL samples show that with increasing Ag+ concentration, growth of E. coli increases, but in the HWL samples E. coli growth falls due to raising the Ag+ concentration (Fig. 2).

The results show Ag+ ions, even at very low concentration, eliminate all S. aureus cells and show that this bacterium is really sensitive to Ag+ ions even in a very low concentration.


In Cu NPs study, 9 formulated detergents involving three different concentrations of Cu NPs (0.1, 1.0 and 10.0 g/L shampoo, HWL and DWL formulations) were investigated. The bactericidal effects of these products (Table 1, samples10-18) were studied against E. coli and S. aureus. Fortunately, all bacteria were extirpated in all shampoo samples (Fig. 3a). Based on the results of antibacterial efficacy tests of HWL (Fig. 3b), it can be perceived that presence of Cu NPs (1.0, and 10.0 g/L HWL formulation) completely inhibited growth of both E. coli and S. aureus. The antibacterial tests of DWL conducted using Cu NPs proved that the Cu NPs have high antibacterial efficiency against S. aureus. However, maximum efficiency of 100 % against E. coli strain was observed in sample 18 with 10.0g/L Cu NPs in DWL formulation (Fig. 3c).

Hence, Cu NPs could be considered as a potential antibacterial agent against E. coli strain. The elimination percentage of this bacterium increased with raising quantity of Cu NPs. Among various metal nanoparticles, Cu NPs is said to have attracted more attention because of its antifungal/antibacterial properties [19,23,24,49-51]. The bactericidal effect of Cu NPs is attributed to their small size, and high surface to volume ratio, enabling them to interact closely with microbial membranes [19,46]. Bacteria exhibit differential sensitivities to different Cu species. Gram positive bacteria are more sensitive than Gram negative bacteria [52]. The exact mechanism of antimicrobial action of Cu NPs is not clearly reported and the general view seems to be a combination of several factors including: released Cu2+ ions in the solution from Cu NPs and consequent changes in pH and conductivity, direct penetration and disruption of cell membrane by Cu2+ ions, DNA damage and disruption of biochemical pathway by chelating cellular enzymes, etc. The above have been reported to occur simultaneously during interactions with microbial cells, where Cu2+ exerts antimicrobial effect on the freely moving bacteria [18,24,53-56].

To study Fe NPs in detergents, 9 formulated detergents including shampoo, HWL and DWL formulations with three different concentrations of Fe NPs (0.1, 1.0 and 10.0 g/L) were investigated. The bactericidal effects of these products (Table 1, samples 19-27) were studied against E. coli and S. aureus(Fig.4). The antibacterial activity of Fe NPs in shampoo samples is frustrating at 1 h vicinage time especially against E. coli (Fig. 4a). Even in HWL, the results for E. coli are not

reasonable, while the X values of S. aureus are acceptable (Fig. 4b). In DWL samples, E. coli cells growth is high but not as much as shampoo and HWL samples (Fig. 4c).

The best results for Fe NPs antibacterial activity is against S. aureus in DWL samples which all cells were extirpated in all concentrations studied. As to the mechanism involved, the released ions by Fe NPs may attach to the negatively charged bacterial cell wall and rupture it, thereby leading to protein denaturation and cell death [57,58]. Also, Fe2+ may react with intracellular oxygen or hydrogen peroxide and produce reactive oxygen species which may have induced oxidative stress and inflammatory responses [59-61]. Moreover, such cellular processes may lead to cell death via cell necrosis or apoptosis [58]. In a short time, releasing Fe2+ or Fe3+ ions from Fe NPs is more difficult because of the redox potential, so, antibacterial activity of Fe NPs is less than Ag and Cu NPs in this study.

Although all of the studied metallic NPs exhibit excellent antibacterial efficiency in the time of more than 1 h, in short vicinage time, growth of cells especially in E. coli increased and there is not any special order for that. Since the detergents contain one or more surfactants, they help growth of bacteria in short time after contamination [62,63]. Comparing the antibacterial assay of NPs in time tested (Fig. 5) shows Cu NPs have an average best activity against E. coli and S. aureus in shampoo, HWL, and DWL samples and the order of antibacterial activity of these preservatives is:

Cu NPs>Ag NPs> Fe NPs.

Based on using minimum amount of NPs, shampoo samples with 0.1 g/L Cu NPs and HWL and DWL with 0.1 g/L Ag NPs indicate outstanding results against considered E. coli and S. aureus strains. Among different detergents, S. aureus cells approximately were extirpated in all DWL samples that may be due to chemical make-up of DWL formulation.


This study clearly demonstrates the advantages of using rather low concentrations of Ag, Cu and Fe NPs as preservatives

instead of carcinogenic Kathone CG, which is commonly used in detergent products. The shampoo formulation with 0.1 g/L

Cu NPs and HWL and DWL with 0.1 g/L Ag NPs exhibit the best antibacterial activity against tested E. coli and S. aureus. Conclusively, the order of antibacterial activity on these preservatives was: Cu NPs>Ag NPs> Fe NPs.


The authors appreciate the financial support of this work by the Iran National Science Foundation (INSF) and Tarbiat Modares University. We are indebted to Dr. Mobarez group especially Dr. Khoramabadi for his generous cooperation.


This study was funded by the Iran National Science Foundation (INSF).

This article does not contain any studies with human participants or animals performed by any of the authors.



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