Synthesis of Magnetic Nanoparticles with Bacteriophages

Synthesis was carried out using the modified protocol of Thanh et al.⁵⁵ The following solutions were prepared: 2130 mg (13.1 mmol) of anhydrous iron(iii) chloride (FeCl₃, Sigma-Aldrich) dissolved in 200 mL of deionized water (ddH₂O); 800 mg (4 mmol) of tetrahydrate iron(ii) chloride (FeCl₂·4H₂O, Sigma-Aldrich) dissolved in 100 mL of ddH₂O. For synthesis, the following solutions were used: 8 mL of FeCl₃, 4 mL of FeCl₂ and 1 mL of bacteriophage suspension (1 × 10¹⁰ virions per mL). For the control sample, instead of the bacteriophage solution, 1 mL of 0.9% NaCl solution was added. The solutions were placed in a beaker, stirred mechanically (600 rpm), heated up, and after reaching 40 °C, 1 mL of 25% NH₃(aq) was added dropwise until pH 11. The solution turned dark brown and was continuously stirred for 5 min. Subsequently, the nanoparticles were separated using a magnet (0.25 T), and the separated nanoparticles were rinsed 10 times with ddH₂O to remove the non-magnetic part, reagent residues and reach neutral pH. The following sample identification was used: (i) NP – magnetic nanoparticles, (ii) NP/P1 – magnetic nanoparticles with P1 bacteriophages, (iii) NP/Φ6 – magnetic nanoparticles with Φ6 bacteriophages. The nanoparticles obtained were stained with SybrGold® and observed under fluorescence microscopy (DM500 filter with a bandpass 460–490 nm excitation filter). Scanning Electron Microscopy (SEM) was conducted using a Carl Zeiss AURIGA (Carl Zeiss Microscopy GmbH). The samples were placed on a glass slide and coated with 20 nm layer of gold by a vacuum coater (Quorum 150T ES). Analysis was done with a 20 kV acceleration voltage.