Functionalised Anodised Aluminium Oxide as a Biocidal Agent

Abstract

In this article, we describe the antimicrobial properties of a new composite based on anodic aluminium oxide (AAO) membranes containing propyl-copper-phosphonate units arranged at a predetermined density inside the AAO channels. The samples were prepared with four concentrations of copper ions and tested as antimicrobial drug on four different strains of Escherichia coli (K12, R2, R3 and R4). For comparison, the same strains were tested with three types of antibiotics using the minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests. Moreover, DNA was isolated from the analysed bacteria which was additionally digested with formamidopyrimidine-DNA glycosylase (Fpg) protein from the group of repair glycosases. These enzymes are markers of modified oxidised bases in nucleic acids produced during oxidative stress in cells. Preliminary cellular studies, MIC and MBC tests and digestion with Fpg protein after modification of bacterial DNA suggest that these compounds may have greater potential as antibacterial agents than antibiotics such as ciprofloxacin, bleomycin and cloxacillin. The described composites are highly specific for the analysed model Escherichia coli strains and may be used in the future as new substitutes for commonly used antibiotics in clinical and nosocomial infections in the progressing pandemic era. The results show much stronger antibacterial properties of the functionalised membranes on the action of bacterial membranes in comparison to the antibiotics in the Fpg digestion experiment. This is most likely due to the strong induction of oxidative stress in the cell through the breakdown of the analysed bacterial DNA. We have also observed that the intermolecular distances between the functional units play an important role for the antimicrobial properties of the used material. Hence, we utilised the idea of the 2D solvent to tailor them.

Keywords: Fpg glycosylase; anodic aluminium oxide; antibiotics; bacterial E. coli strains; oxidative stress; surface functionalization.

Figure 1

The schematic representation of the investigated material. The density of functional units is determined by the statistical number of spacer units (N) between functional molecules.

Figure 2

The SEM and TEM micrographs of the anodic aluminium oxide membranes: (a) a planar view, (b) a cross-section, (c) a transmission micrograph of a fragment of a cross-section of an AAO membrane.

Figure 3

The pore size distribution of the AAO pores.

Figure 4

Differential pulse anodic stripping voltammetry curves recorded after 1-min electrolysis at −0.25 V in 0.1 M NaNO${}_3$ electrolyte solution using AAO membranes and flat aluminium oxide (AO) containing various amount of copper ions. DPASV curves are presented with the offset of 0.5 μA.

Figure 5

Examples of MIC and MBC on microplates with different concentrations of studied compounds (μg/cm${}^3$). The numbers correspond to sample names after “Cu” (“4”—AAO Cu 4). Resazurin was added as an indicator of microbial growth with K12, R2, R3, and R4 strains with the four tested compounds as described in Table 1. The third row shows examples of MIC with different strains K12, R2, R3, and R4 of the studied antibiotics: ciprofloxacin (ci), bleomycin (b) and cloxacillin (cl).

Figure 6

The antibacterial properties of the functionalised AAO membranes: (a) minimal inhibitory concentration of the AAO Cu in model bacterial strains, (b) minimal bactericidal concentration of the amidoximes in model bacterial strains, (c) MBC/MIC of the coumarin derivatives. Each experiment was performed independently in three replications (n = 3).

Figure 7

The percentage of bacterial DNA recognised by Fpg enzyme in model bacterial strains after ciprofloxacin, bleomycin, and cloxacillin treatment. The compounds were statistically significant at p < 0.05. Each experiment was performed independently in three replications (n = 3).

Figure 8

Examples of MIC with model bacterial strains K12, R2, R3, and R4 with ciprofloxacin, bleomycin, and cloxacillin. Each experiment was performed independently in three replications ($n=3$).

Figure 9

Percentage of plasmid DNA recognised by Fpg enzyme (y-axis) with model bacterial, K12, and R2–R4 strains (x-axis). Each experiment was performed independently in three replications ($n=3$).