316
C. Marquina
local damage at cell level. Moreover, as cyto- and phyto-toxicity are dose-dependent,
they cannot be discarded in case of treatments with more concentrated nanoparticle
suspensions.
13.3 Interaction of Silica Coated Magnetic Nanoparticles
with Pathogenic Fungi
In this section, the study of the interaction between silica coated iron oxide
(Fe 3 O 4 @SiO 2 ) nanoparticles and fungal cells is presented, paying attention to the
affinity and internalization of the particles by hyphal cells, and to their toxicity [48].
The core-shell nanoparticles were synthesized in two steps. First, the magnetite
core was synthesized by coprecipitation, as described in De Matteis et al. [75].
According to the X-ray diffraction pattern and HR-TEM images of the synthesized
powders, the nanoparticles were mainly composed by magnetite and only in some
batches a small fraction of maghemite was detected. The particles were subsequently
coated with an aminated silica shell and functionalized with protein G following
the protocols reported in [47]. The diameter of the silica-coated nanoparticles was
about 50–100 nm. The protein G on the surface was afterwards conjugated to AlexaFluor488, to visualize the nanoparticles by confocal microscopy when studying their
interaction with the Fusarium. In addition, when the nanoparticles were going to be
used for sensing purposes, the protein G would easily allow their further conjugation
with the biomolecule that specifically recognizes the pathogenic fungus [47]. All the
experiments described from here on were carried out on F. oxysporum forma specialis
lycopersici. The fungal strain was stored as microconidial suspension in 30% glycerol at −80 °C. For microconidia production, cultures were grown in potato dextrose
broth [48].
13.3.1 Internalization of Fe 3 O 4 @SiO 2 Nanoparticles
by Fungal Cells
First of all, the affinity between the Fe 3 O 4 @SiO 2 nanoparticles and the fungus was
studied. With this purpose, turbidity (or relative absorbance) measurements were
performed on conidial suspensions incubated with magnetic nanoparticles, after
different incubation times. In all the cases, 5 × 10
6 microconidia were grown for
16 h at 28 °C under agitation at 170 rpm in 1 mL of minimum medium (MM)
[76]. The measurements were performed after long-term and short-term incubation
experiments. In the first case the conidia were grown in MM supplemented with
nanoparticles at 200 μg ml
−1 . In short-term incubation experiments, conidia were
grown in the absence of nanoparticles. They were added to the MM after 16 h of
conidia growth and incubated there for 5 s, 15 min and 30 min. After incubation with
C. Marquina
local damage at cell level. Moreover, as cyto- and phyto-toxicity are dose-dependent,
they cannot be discarded in case of treatments with more concentrated nanoparticle
suspensions.
13.3 Interaction of Silica Coated Magnetic Nanoparticles
with Pathogenic Fungi
In this section, the study of the interaction between silica coated iron oxide
(Fe 3 O 4 @SiO 2 ) nanoparticles and fungal cells is presented, paying attention to the
affinity and internalization of the particles by hyphal cells, and to their toxicity [48].
The core-shell nanoparticles were synthesized in two steps. First, the magnetite
core was synthesized by coprecipitation, as described in De Matteis et al. [75].
According to the X-ray diffraction pattern and HR-TEM images of the synthesized
powders, the nanoparticles were mainly composed by magnetite and only in some
batches a small fraction of maghemite was detected. The particles were subsequently
coated with an aminated silica shell and functionalized with protein G following
the protocols reported in [47]. The diameter of the silica-coated nanoparticles was
about 50–100 nm. The protein G on the surface was afterwards conjugated to AlexaFluor488, to visualize the nanoparticles by confocal microscopy when studying their
interaction with the Fusarium. In addition, when the nanoparticles were going to be
used for sensing purposes, the protein G would easily allow their further conjugation
with the biomolecule that specifically recognizes the pathogenic fungus [47]. All the
experiments described from here on were carried out on F. oxysporum forma specialis
lycopersici. The fungal strain was stored as microconidial suspension in 30% glycerol at −80 °C. For microconidia production, cultures were grown in potato dextrose
broth [48].
13.3.1 Internalization of Fe 3 O 4 @SiO 2 Nanoparticles
by Fungal Cells
First of all, the affinity between the Fe 3 O 4 @SiO 2 nanoparticles and the fungus was
studied. With this purpose, turbidity (or relative absorbance) measurements were
performed on conidial suspensions incubated with magnetic nanoparticles, after
different incubation times. In all the cases, 5 × 10
6 microconidia were grown for
16 h at 28 °C under agitation at 170 rpm in 1 mL of minimum medium (MM)
[76]. The measurements were performed after long-term and short-term incubation
experiments. In the first case the conidia were grown in MM supplemented with
nanoparticles at 200 μg ml
−1 . In short-term incubation experiments, conidia were
grown in the absence of nanoparticles. They were added to the MM after 16 h of
conidia growth and incubated there for 5 s, 15 min and 30 min. After incubation with