Understanding the bactericidal mechanism of Cu(OH)2 nanorods in water through Mg-substitution: high production of toxic hydroxyl radicals by non-soluble particles

Abstract

To date, there is still a lack of definite knowledge regarding the toxicity of Cu(OH)₂ nanoparticles towards bacteria. This study was aimed at shedding light on the role played by released cupric ions in the toxicity of nanoparticles. To address this issue, the bactericidal activity of Cu(OH)₂ was at first evaluated in sterile water, a medium in which particles are not soluble. In parallel, an isovalent substitution of cupric ions by Mg²⁺ was attempted in the crystal structure of Cu(OH)₂ nanoparticles to increase their solubility and determine the impact on the bactericidal activity. For the first time, mixed Cu_1−xMg_x(OH)₂ nanorods (x ≤ 0.1) of about 15 nm in diameter and a few hundred nanometers in length were successfully prepared by a simple co-precipitation at room temperature in mixed alkaline (NaOH/Na₂CO₃) medium. For E. coli, 100% reduction of one million CFU per mL (6 log₁₀) occurs after only 180 min on contact with both Cu(OH)₂ and Cu_0.9Mg_0.1(OH)₂ nanorods. The entire initial inoculum of S. aureus is also killed by Cu(OH)₂ after 180 min (100% or 6 log₁₀ reduction), while 0.01% of these bacteria stay alive on contact with Cu_0.9Mg_0.1(OH)₂ (99.99% or 4 log₁₀ reduction). The bactericidal performances of Cu(OH)₂ and the magnesium-substituted counterparts (i.e. Cu_1−xMg_x(OH)₂) are not linked to cupric ions they release in water since their mass concentrations after 180 min are much lower than minimal concentrations inhibiting the growth of E. coli and S. aureus. Finally, an EPR spin trapping study reveals how these nanorods kill bacteria in water: only the presence of hydrogen peroxide, a by-product of the normal metabolism of oxygen in aerobic bacteria, allows the Cu(OH)₂ and its magnesium-substituted counterparts to produce a lethal amount of free radicals, the majority of which are the highly toxic HO˙.