Rapid and Sensitive Microplate Assay for Screening the Effect of Silver and Gold Nanoparticles on Bacteria

Rehab M Amin; Mona B Mohamed; Marwa A Ramadan; Thomas Verwanger; Barbara Krammer

Disclosures

Nanomedicine. 2009;4(6):637-643. 

In This Article

Results & Discussion

Metallic nanoparticles, such as silver and gold nanoparticles, were prepared chemically by the reduction of the metallic ions in the presence of biocompatible capping agents. Citrate ions are used as a capping material that is biologically safe and exhibits no apparent toxicity.[20] Figure 1 shows the absorption spectra and transmission-electron microscopy images of the prepared silver and gold spherical particles. Based on the absorption spectra it is clear that spherical metallic particles show only one absorption band owing to what is called surface plasmon resonance (SPR). The wavelength of the SPR of metal particles depends on the particle size and the dielectric constant of the metal itself. This strong absorption is due to the oscillation of the surface electron around the ionic core once it is exposed to visible light. Spherical gold particles have one absorption band at approximately 520 nm, depending on the particle size, and silver nanoparticles show only one SPR band at 400 nm.

Figure 1.

Characteristics of Silver and Gold Nanoparticles. (A) UV-Visible spectra of spherical silver nanoparticles; (B) UV-Visible spectra of spherical gold nanoparticles; (C) transmission-electron microscopy image of silver nanoclusters; and (D) transmission-electron microscopy image of gold nanoclusters.
Ag: Silver; Au: Gold.

The effects of silver nanoparticles on both Gram-negative and -positive bacteria are shown in Figure 2. Each point of the growth curve is the average of two OD measurements in four wells at any given time (Figures 2A & B). Bacterial growth curves are divided into four phases: lag phase, where the bacterial cells increase in size to start cell division; logarithmic phase, where cells and cell mass double at a constant rate; stationary phase, where some cells are dying and others still growing and dividing, thus there is no increase or decrease in cell number; and death phase, where the rate of decline becomes exponential within time. Since the doubling of the population (generation time) occurs at regular time intervals at the logarithmic phase, this phase is suitable for the assessment of the antimicrobial activity. Therefore, the microbial toxicity of metallic nanoparticles in the current study was assessed clearly through that phase.

Figure 2.

The Effect of Silver Nanoparticles on Both Gram-negative and -positive Bacteria. (A) Growth curves of Staphylococcus capitis using different concentrations of AgNCs; (B) growth curves of Escherichia coli using different concentrations of AgNCs; and (C) survival curves of both strains using AgNCs.
AgNC: Silver nanocluster; Cont.: Control; OD: Optical density.

Analysis of the data of the growth curve of S. capitis (Figure 2A) as an example of Gram-positive bacteria shows that with increasing concentration of silver nanoclusters, growth is increasingly reduced compared with the control values. Derived from that, the survival curve decreases until at a concentration of 150 μM up to 60% of bacterial cells are killed (Figure 2C). By contrast, the growth curve of E. coli (Figure 2B) shows that with increasing concentration, growth is accelerated until 30 μM and then increasingly reduced compared with the control. As a consequence, the survival curve increases up to a concentration of 30 μM and decreases thereafter until at a concentration of 150 μM up to 30% of bacterial cells are killed (Figure 2C).

Results indicate that an increase in the concentration of silver nanoparticles reduced the survival of bacteria. This finding is in agreement with that obtained by Pal et al. who mentioned that silver nanoparticles undergo shape-dependent interaction with Gram-negative E. coli and were being used as bactericidal agents,[21] and also agrees with Kim et al. and Choi et al., who investigated the inhibitory effect of silver nanoparticles on microorganisms.[22,23]

Effects of gold nanoparticles on both Gram-negative and -positive bacteria are shown in Figure 3. Analysis of the growth curve of S. capitis as an example of Gram-positive bacteria shows that with increasing concentration of gold nanoclusters the growth is increasingly reduced until 70 μM compared with the control values, where it then becomes relatively constant (Figure 3A). Derived from that, the survival curve decreases until at a concentration of 70 μM 20% of bacterial cells are killed (Figure 3C). By increasing the concentration up to 150 μM, the percentage of survival remains almost the same, in other words, the toxic effect of gold nanoclusters on the bacterial cells is no longer observed. The growth curve of E. coli using gold nanoclusters shows that with increasing concentration of gold nanoclusters, growth is moderately decreased until 70 μM compared with the control values, but increases thereafter until 130 μM (Figure 3B). As a consequence, the survival curve remains nearly the same with a slight decrease of only 10% up to a concentration of 70 μM. Between 70 and 130 μM, the survival curve is increased. At still higher concentrations the survival curve decreases again to almost 10% bacterial cell death (Figure 3C). This outcome is in agreement with the findings of Zharov et al. who did not observe significant killing of bacterial cells using gold nanoparticles.[24]

Figure 3.

The Effect of Gold Nanoparticles on Both Gram-negative and -positive Bacteria. (A) Growth curves of Staphylococcus capitis using different concentrations of AuNCs; (B) growth curves of Escherichia coli using different concentrations of AuNCs; and (C) survival curves of both strains using AuNCs.
AuNC: Gold nanocluster; Cont.: Control; OD: Optical density.

Although the examined nanoparticles were used at the same concentrations (10–150 μM) and the number of cells at the beginning of the experiment was the same (106 colony-forming unit/ml), it was demonstrated that the nanoparticles showed a higher killing effect on S. capitis than on E. coli owing to the Gram status. It is known that the difference between the outer wall of Gram-positive and -negative bacteria is given by the degree of permeability, with an exclusion limit for substances with a molecular weight of more than approximately 600 Da for the Gram-negative cells. The outer wall of Gram-negative bacteria acts as a permeability barrier due to the presence of a lipopolysaccharide layer that is able to exclude macromolecules and hydrophilic substances, thereby being responsible for the intrinsic resistance of Gram-negative bacteria.[25] Therefore, S. capitis is more susceptible to the lethal effects of our prepared nanoparticles than E. coli.

Results show that there is an increase in the percentage survival of E. coli above 100% either with silver nanoclusters at a concentration below 30 μM (Figure 2C) or with gold nanoclusters at concentrations between 70 and 130 μM (Figure 3C). This increase may be due to the catalytic activity of trace amounts of metallic nanoparticles. Our findings support the work of Limbach et al., who suggested that proactive development of nanomaterials should consider chemical and catalytic properties of nanomaterials beyond a mere focus on physical properties.[26] Furthermore, it is known that metal ions such as iron, magnesium, zinc, molybdenum, manganese and copper are often required in low quantities as trace elements functioning as cofactors/coenzymes or prosthetic groups of various enzymes.[27]

An increase in the survival percentage with gold and silver nanoparticles was demonstrated only in the Gram-negative bacteria; this could be due to the presence of the lipopolysaccharide layer, which is significant in membrane transport of Gram-negative bacteria[28] and may regulate the entrance of the nanoparticles in trace amounts to enhance the bacterial growth.

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