13 Magnetic Nanoparticles for Life Sciences Applications
317
the nanoparticles, the suspensions were filtered to remove nanoparticles not internalized/not attached in/to the conidia, and the filtrate was resuspended in new fresh MM.
This suspension was introduced in a magnetic separator, and kept under the action of
the magnetic field for 1, 3 and 5 min. At these times, an aliquot of the medium was
taken for turbidity measurements. All those fungi attached to the magnetic nanoparticles were going to be trapped by the magnet, decreasing the conidia concentration
in the aliquot, and consequently, decreasing its turbidity. The optical density of this
aliquot was measured at 600 nm (A600), and compared with the absorbance of a
control sample (a sample not introduced in the magnetic separator). The reduction in
absorbance was correlated with the nanoparticle attachment/internalization in/to the
fungus. After long-term incubation with magnetic nanoparticles, the conidia were
attracted by the magnet as early as 1 min after being introduced in the magnetic
separator, and the turbidity of the medium was reduced dramatically, confirming the
attachment of the nanoparticles to the hyphae. The long-term incubation experiments
were also performed with the Fe 3 O 4 @SiO 2 nanoparticles after their functionalization with protein G, to verify whether the protein influenced the fungus-nanoparticle
interaction. In fact, the functionalization of the nanoparticle surface delayed the
particle movement towards the magnet, although a turbidity decrease similar to that
observed when working with the non-functionalized nanoparticles was detected after
5 min. Short-term incubation experiments were also performed incubating the grown
conidia with functionalized and non-functionalized Fe 3 O 4 @SiO 2 nanoparticles. The
turbidity measurements were carried out following the protocol described above for
long term experiments. A drastic reduction of the turbidity was observed after incubating fungal conidia with both functionalized and non-functionalized nanoparticles
for as short as 5 s. These results evidence the high affinity of the functionalized and
non-functionalized Fe 3 O 4 @SiO 2 nanoparticles for the fungal hyphae.
In order to investigate whether the nanoparticles were able to penetrate the fungal
hypha or remained attached to its surface, visible, confocal and transmission electron
(TEM) microscopies on conidia cultures after long-term and short-term incubation
with Fe 3 O 4 @SiO 2 nanoparticles were carried out. For these studies, 5 × 10
6 conidia
were grown for 16 h at 28 °C under agitation at 170 rpm in 1 ml of MM. For long-term
studies, 200 μg ml
−1 of nanoparticle suspension was added at the time of fungus
inoculation, while for short-term studies the nanoparticles were added to MM after
16 h growth and incubated for either 10 min or 3 h before visualization. First of all,
confocal images of thin time-course confocal optical sections (~1 μm thick) were
acquired. Details about the microscope and the instrumental conditions can be found
in [48]. The results are displayed in Fig. 13.8. After 10 min of incubation, fluorescent Fe 3 O 4 @SiO 2 nanoparticle aggregates attached to the fungal hyphal surfaces
were clearly seen (pointed by an arrow head at visible field and stack projection in
Fig. 13.8a). However, theses aggregates did not penetrate the fungal hyphae since
3D optical sections showed a clear signal only for the largest aggregates attached
to the hypha. Although the penetration of non-aggregated nanoparticles could not
be inferred from the confocal images, it cannot be discarded. After 16 h of incubation, nanoparticle aggregates were still visible and attached on the hyphal surface,
although they were smaller than those attached after 10 min incubation, and no signal
317
the nanoparticles, the suspensions were filtered to remove nanoparticles not internalized/not attached in/to the conidia, and the filtrate was resuspended in new fresh MM.
This suspension was introduced in a magnetic separator, and kept under the action of
the magnetic field for 1, 3 and 5 min. At these times, an aliquot of the medium was
taken for turbidity measurements. All those fungi attached to the magnetic nanoparticles were going to be trapped by the magnet, decreasing the conidia concentration
in the aliquot, and consequently, decreasing its turbidity. The optical density of this
aliquot was measured at 600 nm (A600), and compared with the absorbance of a
control sample (a sample not introduced in the magnetic separator). The reduction in
absorbance was correlated with the nanoparticle attachment/internalization in/to the
fungus. After long-term incubation with magnetic nanoparticles, the conidia were
attracted by the magnet as early as 1 min after being introduced in the magnetic
separator, and the turbidity of the medium was reduced dramatically, confirming the
attachment of the nanoparticles to the hyphae. The long-term incubation experiments
were also performed with the Fe 3 O 4 @SiO 2 nanoparticles after their functionalization with protein G, to verify whether the protein influenced the fungus-nanoparticle
interaction. In fact, the functionalization of the nanoparticle surface delayed the
particle movement towards the magnet, although a turbidity decrease similar to that
observed when working with the non-functionalized nanoparticles was detected after
5 min. Short-term incubation experiments were also performed incubating the grown
conidia with functionalized and non-functionalized Fe 3 O 4 @SiO 2 nanoparticles. The
turbidity measurements were carried out following the protocol described above for
long term experiments. A drastic reduction of the turbidity was observed after incubating fungal conidia with both functionalized and non-functionalized nanoparticles
for as short as 5 s. These results evidence the high affinity of the functionalized and
non-functionalized Fe 3 O 4 @SiO 2 nanoparticles for the fungal hyphae.
In order to investigate whether the nanoparticles were able to penetrate the fungal
hypha or remained attached to its surface, visible, confocal and transmission electron
(TEM) microscopies on conidia cultures after long-term and short-term incubation
with Fe 3 O 4 @SiO 2 nanoparticles were carried out. For these studies, 5 × 10
6 conidia
were grown for 16 h at 28 °C under agitation at 170 rpm in 1 ml of MM. For long-term
studies, 200 μg ml
−1 of nanoparticle suspension was added at the time of fungus
inoculation, while for short-term studies the nanoparticles were added to MM after
16 h growth and incubated for either 10 min or 3 h before visualization. First of all,
confocal images of thin time-course confocal optical sections (~1 μm thick) were
acquired. Details about the microscope and the instrumental conditions can be found
in [48]. The results are displayed in Fig. 13.8. After 10 min of incubation, fluorescent Fe 3 O 4 @SiO 2 nanoparticle aggregates attached to the fungal hyphal surfaces
were clearly seen (pointed by an arrow head at visible field and stack projection in
Fig. 13.8a). However, theses aggregates did not penetrate the fungal hyphae since
3D optical sections showed a clear signal only for the largest aggregates attached
to the hypha. Although the penetration of non-aggregated nanoparticles could not
be inferred from the confocal images, it cannot be discarded. After 16 h of incubation, nanoparticle aggregates were still visible and attached on the hyphal surface,
although they were smaller than those attached after 10 min incubation, and no signal