J. Coat. Technol. Res.
https://doi.org/10.1007/s11998-018-00175-1
A regioselective coating onto microarray channels of bamboo
with chitosan-based silver nanoparticles
Omar Ginoble Pandoli , Raquel S. Martins, Karen L. G. De Toni, Sidnei Paciornik,
Marcos H. P. Maurı́cio, Renan M. C. Lima, Nikolas B. Padilha, Sonia Letichevsky,
Roberto R. Avillez, Elton J. R. Rodrigues, Khosrow Ghavami
 American Coatings Association 2019
Abstract In this investigation, bamboo (Dendrocala-
mus giganteus Munro) timbers were coated selectively
into vascular vessel bundles with a potential antimi-
crobial colloidal solution of silver nanoparticles (Ag-
NPs). Electric charge and size of Ag-NPs, with differ-
ent charged organic ligands (trisodium citrate and
chitosan), affect their self-sorting in different anatom-
ical structures of bamboo when submitted up to 20
impregnation cycles through a vacuum system. Physic-
ochemical characterization of Ag-NPs was performed
by spectroscopic techniques and electron microscopy.
Confocal laser scanning microscopy and scanning
electron microscopy were employed to characterize
natural bamboo. Qualitative and quantitative determi-
nation of the metal coating in bamboo specimens was
performed with X-ray microtomography (lCT), en-
ergy-dispersive X-ray spectroscopy, and X-ray diffrac-
tion. lCT revealed a gradient deposition of citrate-
capped Ag-NPs into the parenchyma tissue with the
higher concentration at the outer part of the bamboo.
On the other hand, the chitosan-capped Ag-NPs were
deposited mainly in the vessel bundles.
O. Ginoble Pandoli (&), R. S. Martins,
E. J. R. Rodrigues
Chemistry Department, PUC-RIO, Rio de Janeiro, Brazil
e-mail: omarpandoli@puc-rio.br
K. L. G. De Toni
Rio de Janeiro Botanical Garden, Rio de Janeiro, Brazil
S. Paciornik,
MarcosH. P. Maurı́cio, R. M. C. Lima, N.
B. Padilha, S. Letichevsky,
RobertoR. Avillez
Chemical and Materials Engineering Department, PUC-
Rio, Rio de Janeiro, Brazil
K. Ghavami
Civil Engineering Department, PUC-Rio, Rio de Janeiro,
Brazil
Keywords Bamboo, Silver nanofiller, Nanocoating,
Bionanocomposite, Regioselective coating, Hybrid
bio-based composite
Introduction
A polymeric nanocomposite is a polymer matrix with
reinforced nanometric filler, such as nanoparticles
(NPs) or fibers. According to the nanofiller (NF)
material, it is possible to enhance or add new properties
to the matrix polymer, such as thermal stability, weather
resistance, self-cleaning, fire resistance, antibacterial,
and catalytic activities.1–3 The bamboo species (D.
giganteus) grows very fast, up to 70 cm day 1. It is a
low-cost, energy-saving raw material employed in engi-
neering, furniture, textiles, paper, composite panels,
decoration, food, and building materials.4,5 Because of
its rapid growth and biomass production, the bamboo
plant is useful for storing carbon dioxide from the
atmosphere6,7 and for soil remediation, removing met-
als from contaminated soil and water.8 Bamboo, as well
as wood, is a natural nanostructured biopolymer com-
posed of polysaccharides—mainly cellulose, lignin, and
hemicellulose. At the microlevel structure, all the
constituent polymers are organized into microfibrils
that are self-organized in macrofibrils around straight
microsized channels.4 The microarray of lignin–cellu-
lose parallel channels constitutes the system of internal
vascular bundles of bamboo with an internal diameter
ranging from 50 to 200 lm, while isodiametric, honey-
comb-like cells with curved cell walls and with variable
volume of 1000–2000 lm3 constitute the parenchyma.
The fibers constitute the sclerenchymatous tissue. This
tissue is composed of slender fiber cells with polylamel-
late secondary walls. This lamellation consists of alter-
nation of lignin, cellulose, and hemicellulose. An
irregular distribution of vascular bundles (atactostele)
is organized with a radial gradient, from the inner to the
J. Coat. Technol. Res.
outer region of the stem.9 A recent article in Nature’s
Scientific Reports showed a theoretical study about the
molecular origin of the strength and stiffness of bamboo
fiber based on the large presence of hydrogen-bonded
and van der Waals interactions between lignin mole-
cules and cellulose microfibrils, named lignin carbohy-
drate complex (LCC).10 This LCC nanostructured
composite is responsible for the intrinsic mechanical
strength and supports the vascular bundles system
(phloem and metaxylem) to transport fluid and nutri-
ents (Fig. 1, left side).11 SEM images (Fig. 1, right side)
show the metaxylem vascular bundle with several
surface pits through which the fluids reach the scle-
renchyma fibers and parenchymatic living cells. Sus-
tainable and renewable wood-derived materials are
already used for green electronics, biological device and
energy applications.12 If the metal deposition in the
bamboo matrix is fully controlled, this new material can
be employed for bio-nanotechnology applications, such
as supercapacitors,13 catalytic biomass-derived porous
carbon materials,14 lignocellulose-based analytical de-
vices15 and bio-microfluidic devices.16
A disadvantage of bamboo is its low biostability and
ensuing low durability over time. The high concentra-
tion of starch allows natural biodegradation by
microorganisms such as fungi, molds, and bacteria.17–19
Nanostructured materials have been used for wood
surface coating with different purposes and are already
on the market: water proofing (clay, SiO2, TiO2); UV
protection (SiO2, TiO2, ZnO, Fe2O3); biotic decay
protection (Ag, Cu, and ZnO); fire resistance (SiO2,
TiO2, and clay); self-cleaning (TiO2, ZnO); antiscratch-
ing (Al2O3, SiO2, TiO2).20–22 Jin et al. have used
several nanoparticles for an external coating of bam-
boo timber to add new functional properties: superhy-
drophobicity and self-cleaning (TiO2)23 (ZnO)24; flame
Inner wall
Outer wall
Internode Culm
Node
Phloem
Parenchyma
Fibers 200 μm
Fig. 1: Left: Culm morphology representation of Dendrocalamus giganteus with an optical image of the vascular tissue.
Right: SEM images with a detailed metaxylem vascular bundle showing the pits tissue in the internal wall
retardancy (TiO2)25; conductivity (Ag)26; and mold
resistance (ZnO).27 Different compositions of organic
and inorganic UV light absorbers have been used to
increase the photostability of bamboo surfaces.28 The
potential use and benefits of nanotechnology in agri-
culture and in some areas of materials science are still
uncertain.29 The public opinion and the political
regulation are still far from a common position.
Nanotoxicology implications of nanofertilizers and
nanopesticides, studies on life cycle assessment of
nanocomposites, occupational risks, bio-safety, and
biological activities of said materials, once dispersed
into the environment, require more research before
considering the introduction of nanotech products in
the market.30–33
An antibacterial coating of chitosan-Ag-NPs on
different biomaterials has already been described
elsewhere.34–36 The feasibility of a new functional
bionanocomposite based on internally coated bamboo
with antimicrobial citrate-Ag-NPs was reported for the
first time by our research group.37,38 The antibacterial
efficacy controlled by the diameter of Ag-NPs is well
established.39 Thus, we want to incorporate two
antimicrobial agents, chitosan surfactant and smaller
silver nanoparticles, for chitosan-based silver nanopar-
ticles used as regioselective coating into microarray
channels of bamboo, with a potential resistance
improvement to fungi attacks. On the other hand, if
the parenchyma behaves like a foam, as described in
reference (40), the cell walls deform primarily by
bending, and if the sclerenchyma structures around
the vessel bundles are mainly responsible for the
strength of the bamboo, then the present work repre-
sents an attempt to control the internal coating of the
natural microchannel array of bamboo by Ag-NPs with
antifungal properties, increasing the biostability of the
Outer part
Fibers
2000 μm
Inner part
Metaxylem
Xylem
Protoxylem
 J. Coat. Technol. Res.

material and, consequently, improving the mechanical Synthesis of Ag-NFs

properties of the biomaterial. Nonetheless, the possi-
bility to cover the internal wall of the microarray of
channels with catalytic polysaccharide metal nanopar-
ticles opens up a new biofabrication process to design
and prototype lignocellulose-based microfluidic de-
vices for catalyzed organic reactions.16,41 In this
perspective, a lignocellulose-based analytical device
was already obtained from bamboo specimens impreg-
nated with colorimetric indicators for complete urinal-
ysis.15
X-ray computed microtomography has been em-
ployed as a noninvasive approach to visualize and
analyze the biological structure of bamboo, such as the
complex vascular system in bamboo,42 deformation
mechanism following chemical treatment43 and
microstructural characterization.44,45 In particular,
Palombini et al.45 used a high-resolution X-ray micro-
tomography (lCT) for the reconstruction of a 3D
bamboo model to evaluate the strength of complex
parenchyma and sclerenchyma structures with nonlin-
ear finite element analysis (FEA). Dixon et al.40 used
lCT images of moso bamboo (Phyllostachys pubes-
cens) to rebuild a physical 3D structure of parenchyma
by stereolithography printing, with the aim to charac-
terize its mechanical properties and structure–property
relationships. Other research groups used synchrotron-
based microCT (SyncMicroCT) to investigate the plant
hydraulics in opaque xylem vessels of vascular plants,
and gold nanoparticles (Au-NPs) have been used as
tracers for the physiological mechanism of plants.46
Moreover, high-resolution X-ray tomography is a
powerful tool for coating research.47,48 lCT can be
used to evaluate the thickness, the penetration depth,
and the dispersion of the internal coating of Ag-NPs.
To the best of our knowledge, the characterization of
regioselective coating in bamboo timber with chitosan-
Silver nanofiller colloidal solutions were synthesized
with a glass chip microreactor of 100 lL internal
volume, a FlowStart B-200 unit to control the reaction
temperature and two syringe pumps (Fig. 2).49 Aqu-
eous stock solutions of AgNO3 (10 2 mol L 1) and
Na3Citrate (10 2 mol L 1) were prepared by dissolving
the salts in ultrapure water. A chitosan solution (10 g
L 1) in ultrapure water and acetic acid (pH 5) was
prepared by dissolving the polymer gradually at 60C.
NaBH4 reducing agent solution (10 2 mol L 1), related
to the silver solution in a 1.5:1 proportion, was used to
promote the chemical reduction of silver ions to Ag-
NPs. In continuous flow, silver nitrate and organic
ligand solutions were introduced into the microreactor
at the same flow rate of 0.25 mL min 1. The complex
Ag+-ligand from the outlet channel of the microreactor
was dropped directly into the flask containing NaBH4
(10 3 mol L 1) under vigorous magnetic stirring, in
which they reacted to form the corresponding silver
nanoparticles. The final colloidal solutions, protected
from light at 4C, were characterized by UV–Vis
spectrophotometry (Perkin-Elmer, model Lambda
950), dynamic light scattering (DLS), and zeta poten-
tial (Horiba, Nanoparticle Analyzer, SZ-100). For
UV–Vis spectra, DLS, and zeta potential analyses,
the solutions were diluted 1:10 with ultrapure water.
AgNO3
Glass
Two microreactor
syringe
pumps
Ligands
NaNBH4

based silver nanoparticles has never been published.

Chip Stirrer
holder
HOH2C HOH2C
O OH O
Materials and methods ... O O
O O ...
O O HO HO

Silver nitrate (AgNO3 > 99.9% pure), sodium borohy- O O 3 Na NHCCH3 NH2

dride (NaBH4 > 99% pure), chitosan with degree of Trisodium O

Chitosan
citrate
deacetylation (75%), and trisodium citrate (Na3Ci- polymer
trate > 99.0% pure) were acquired from Sigma-Al-
drich and used as received. All solutions were prepared
using ultrapure water (resistivity of 18.2 MX cm 1)
obtained from a water purifier Milli-Q Gradient Citrate-Ag-NFs Chitosan-Ag-NFs
System A10, Millipore. Four-year-old Dendrocalamus (negatively charged) (positively charged)
giganteus bamboo culms were obtained from FZEA-
USP, Pirassununga-SP, Brazil. Internode number 7, Fig. 2: Schematic representation of the flow chemical
about 2 m from the ground, with a thickness of 12 mm synthesis setup of citrate-Ag-NFs and chitosan-Ag-NFs.
was selected, and eight specimens of 18 9 12 9 5 mm Two syringe pumps operating at the same flow rate injected
(height, thickness, width) were cut with an automatic AgNO3 and organic ligand solutions into the microreactor
precision saw, Struers Miniton, using a metal-bonded channel, and thereafter, the final solution was dropped into
diamond wafering blade (76 9 0.15 mm). the reducing agent solution under vigorous stirring
J. Coat. Technol. Res.
Morphological characterization of Ag-NFs
and bamboo specimens
A field-emission scanning electron microscope (FEG-
SEM) (JEOL, JSM-6701F) operating in transmission
mode (STEM) at 30 keV with a working distance of
6.0 mm and a bright-field detector was used to char-
acterize the Ag-NFs. A scanning electron microscope
(SEM) (JEOL, JSM-6510LV) operating at 20 kV,
using a backscattered detector and EDS system, was
also employed to analyze a longitudinal section of the
bamboo vascular bundle and chitosan-Ag-NFs-coated
bamboo. High-resolution transmission electron micro-
scopy of citrate-Ag-NFs was conducted in a JEOL
JEM 2100F equipment, operated at 200 kV, by drop-
casting 20 lL of the colloidal solution on a copper grid.
High-resolution optical images of the bamboo sections
previously described were obtained with a confocal
laser scanning microscope (Leica TCS SPE). Laser
channels at 488 and 405 nm were used to excite the
fluorochromes auramine O and calcofluor white, emit-
ting at 540–656 nm and 480–500 nm, respectively, to
detect lignin and cellulose, respectively. The images
were captured by direct acquisition with a 72 lm Z
step, generating 50–60 optical sections in the LAS AF
Lite 2.6.0 software (Leica Microsystems).
Bamboo impregnation
We treated four bamboo samples (18 9 12 9 5 mm)
with each colloidal solution (citrate-Ag-NPs and chi-
tosan-Ag-NPs): two of them with 10 impregnation
cycles and the other two with 20 impregnation cycles
(total of eight samples). As depicted in Fig. 3, bamboo
specimens were placed in test tube with 4 mL of
colloidal solution of silver nanoparticles. Each one was
submitted to 10 or 20 impregnation cycles of 1 h each,
under negative pressure through a vacuum system and
then 30 min drying in an oven at 60C (Fig. 3). For
each treatment cycle, a fresh colloidal solution was
used.
Wide
5mm
Height 18 mm
Inner
wall Thickness
12 mm
Inner
wall
Citrate- Chitosan-
Ag-NFs Ag-NFs
Impregnation cycles ×10 or ×20
Fig. 3: Schematic illustration of bamboo
(18 3 12 3 5 mm) impregnation methodology
X-ray microtomography (lCT)
A Zeiss Xradia 510 Versa microtomograph was
employed to visualize and quantify the 3D structure
of Ag-NFs-coated bamboo. The microtomography
operational parameters are compiled in Table 1. This
system allows sample imaging as a conventional lCT,
with the source–sample–detector distances controlling
the geometric magnification and also achieves higher
resolution using a set of lenses with scintillators that
convert X-rays to light. Note that the projection images
are obtained with the rotation of the sample around a
vertical axis. Thus, in order to verify the homogeneity
of the colloidal solution diffusion along the longitudi-
nal fiber direction with higher magnification, Fig. 4
shows the edge and central portions of the bamboo
specimen analyzed with the 49 objective lens. With the
different lCT fields of view, it was possible to analyze
different regions of interest (ROIs: edge and central
part with 4 9 12 9 4 mm dimension) or the whole
sample (18 9 12 9 5 mm) as indicated in Table 1. This
equipment setup allows us to split the acquisition
routines and assemble the projection images during
image post-processing. On the other hand, the whole
volume of the sample was scanned at low resolution
with the 0.49 macro-lens with a longer distance from
the objective lens. Considering the high cost of lCT
analysis and the time-consuming computational proce-
dures involved in the image analysis, quantitative
results were obtained for two different ROIs of two
bamboo samples treated with 20 impregnation cycles
with each colloidal solution. However, ray diffraction
analyses were performed for the eight bamboo sam-
ples.
Image processing and analysis of lCT images
The lCT images were processed using the FIJI/
ImageJ50 free software enabling the identification of
Ag-NF aggregates within the bamboo structure with a
Objective lens 4.0x:
Citrate-Ag-NF and chitosan-Ag-NFs
Vaccum
pump Edge Central
0.4 cm 0.4 cm 1.2 cm
1h Exterior
0.5 cm
Interior
1.8 cm
Oven
30min, 60°C
Objective Lens 0.4×
Fig. 4: Regions of interest scanned with higher resolution
with a 43 objective lens for both citrate-Ag-NF- and
chitosan-Ag-NF-coated bamboos, and the whole bamboo
sample volume (18 3 12 3 5 mm) scanned with a 0.43 macro-
objective lens for bamboo treated with chitosan-Ag-NF

Table 1: X-ray microtomography (lCT) operational parameters
Citrate-Ag-NFs
Objective lens 94
Voxel size (lm) 4 4
Voltage (kV) 50
Power (W) 4 4
Exposure time (s) 2 2
Number of projections 1601
Field of view 4 9 12 9 4 mm
Fig. 5: Confocal laser scanning microscopy (CLSM)
images of a pure bamboo transversal section with two
fluorochromes: calcofluor for cellulose (blue–cyan) and
auramine O for lignin (green–yellow)
manual threshold and post-processing techniques nec-
essary to reduce the noise before the quantification of
the particles. The 3D model was reconstructed using
the DragonFly software (Version 3.1, Object Research
Systems, 2017), and the 3D images were then analyzed
layer by layer to provide data from different axes:
radial (from the internal to the external wall) and
longitudinal (along the fiber direction). These two axes
were selected to identify how the diffusion of the
colloidal solutions can be affected along the vascular
bundles if it is possible to selectively reach the
parenchymatic living cells.
XRD of pure and coated bamboo
X-ray diffraction analyses of microslices of in natura
and Ag-NFs-coated bamboo were performed with a
D8-Discover diffractometer from Bruker, using a
Bragg–Brentano geometry (coupled h–2h), copper tube
(k = 1.5418 Å) operating at 40 kV and 40 mA and a
J. Coat. Technol. Res.
Bamboo specimens
Chitosan-Ag-NFs
94 9 0.4
19
50 50
4
2
1601 1601
4 9 12 9 4 mm 18 9 12 9 5 mm
LynxEye detector. The scan was performed from 5 to
90 with 0.02 2h step. The original diffraction pattern
showed very strong air scattering at lower angles, so a
diffraction pattern of the empty sample holder was
obtained and smoothed by a fast Fourier procedure
with 15 points. The smoothed empty sample holder
pattern was scaled and subtracted from the sample
pattern. The scale was chosen by trial and error to
provide a final pattern with positive data and accept-
able background. The processed pattern was fitted by
the Rietveld method with fundamental parameters
using the TOPAS program (Version 4.2; Bruker, 2009).
Results and discussion
As shown by the SEM image in Fig. 1 (right side), the
pits in the internal wall channels allow the feeding of
the living cells of vegetal tissues. These results were
confirmed by the confocal laser scanning microscopy
(CLSM) images (Figs. 5 and 6). In Fig. 5, colored
images of pure bamboo with two selective fluo-
rochromes are shown: calcofluor for cellulose (blue–
cyan) and auramine O for lignin (green–yellow). The
white arrows inside the vascular bundles indicate the
presence of cellulose on the internal walls. This
transportation channel system is one of the targets
for the coating with Ag-NFs, which will help to protect
the bamboo matrix from the biotic decay. In the
zoomed images of Fig. 5B and C, a transversal section
of parenchymatic living cells presents rich areas of
cellulose, while rich areas of lignin polymers are
observed in sclerenchyma tissues (fibers). Neverthe-
less, the two polymers are present in both tissues. In
Fig. 6, which presents longitudinal CLSM images of
fibers (a) and living cells (b), it is possible to see the
micron-sized (1.4–1.8 lm) and submicron-sized (500–
600 nm) pits in sclerenchyma and parenchyma tissues,
respectively. This information confirms the presence of
a communication network between the main holed
vascular bundles and the parenchymatic living cells.
The network of communication channels between
micron-sized pits in the vessels and submicron-sized
J. Coat. Technol. Res.

Fig. 6: Confocal laser scanning microscopy images of longitudinal section of sclerenchyma tissue (a) and parenchyma
tissue (b) of pure bamboo showing micro- and nanosized pits

(a) (b)

100 nm 100 nm
100 100 100 100

N: 207 N: 207 N: 547 N: 547

Mean: 10.40 nm Mean: 7.93 nm Mean: 4.24 nm
Relative frequency (%)

Relative frequency (%)

Relative frequency (%)

Relative frequency (%)

80 80 80 Mean: 5.41 nm 80
SD: 3.69 nm SD: 3.04 nm SD: 1.64 nm SD: 1.50 nm
Median: 10.30 nm Median: 7.95 nm Median: 5.37 nm Median: 4.10 nm

60 60 60 60

40 40 40 40

20 20 20 20

0 0 0 0
5 10 15 20 5 10 15 5 10 5 10
Feret (nm) Min Feret (nm) Feret (nm) Min Feret (nm)

Fig. 7: STEM images for (a) citrate-Ag-NFs and (b) chitosan-Ag-NFs and the corresponding maximum and minimum
projection distributions

pits allows the nutrients to flow from the ground to the imum (MinFeret) projections were measured, as shown
whole bamboo matrix. in the plots of Fig. 7. The equivalent diameter was
The Ag-NFs were characterized using STEM obtained from the average of the two projections.
microscopy, and the corresponding diameter distribu- Citrate-Ag-NFs and chitosan-Ag-NFs colloidal solu-
tions are shown in Fig. 7. As the particles are not tions present an average equivalent diameter of 9.17
perfectly round, both the maximum (Feret) and min- and 4.83 nm, respectively.
 J. Coat. Technol. Res.

1.0 Unlike STEM analysis that provides a metal particle

Ag-NPs-citrate size measurement, UV–Vis spectroscopy and DLS
Ag-NPs-chitosan
analyses provide information about the surface plas-
394 nm
mon resonance (SPR) and hydrodynamic particle
diameter, respectively. UV–Vis and DLS suggest that
the positively charged chitosan polymer might stabilize
Absorbance

0.5 the clusters of the Ag-NPs in the polymer matrix as

402 nm illustrated in Fig. 8 and Table 2. This aggregation of
Ag-NPs driven by the chitosan polymer explains the
redshift of the SPR band at 402 nm, the higher
hydrodynamic diameter of 120 nm and the higher zeta
potential value of + 62.5 mV compared to citrate-Ag-
0.0 NFs (Table 2).
300 400 500 600 The Ag-NFs depositions into the bamboo matrix,
Wavelength (nm)
after 10 and 20 impregnation cycles, were analyzed by
lCT, and quantitative information of the silver clusters
Fig. 8: UV–Vis spectroscopy analyses of citrate-Ag-NFs
and chitosan-Ag-NFs colloidal solutions. SPR band shift of was obtained from 3D image analysis.38 Due to the low
chitosan-Ag-NFs at 402 nm is the result of the Ag-NPs resolution of the equipment setup, it was not possible
clusters stabilized inside the chitosan polymer matrix to detect silver aggregates in the samples with 10
impregnation cycles. The samples submitted to 20
Table 2: Dynamic light scattering (DLS) and zeta impregnation cycles showed a higher amount of metal
potential measurements for citrate-Ag-NFs and deposition. High-resolution lCT images (Figs. 9 and
chitosan-Ag-NFs colloidal solution 10), with the 4.09 objective lens, allowed analyzing a
small portion of the bamboo specimen
Ag-NFs Hydrodynamic particle Zeta potential (» 4 9 4 9 12 mm). The selection of those parts, as
diameter (nm) (mV) depicted in Fig. 4, is necessary due to the restricted
lCT equipment field of view and to analyze the Ag-
Citrate-Ag-NFs 17.2 40.5
NPs concentrations in different regions of the sample
Chitosan-Ag-NFs 120 + 62.5
(the edge and the central part). A qualitative repre-
sentation of all volumetric distributions of silver
aggregates after 20 impregnation cycles is shown in

Fig. 9: Comparison of the volumetric distribution of Ag-NFs aggregates revealed by lCT, (a) edge citrate; (b) central citrate;
(c) edge chitosan; (d) central chitosan (sample 4 3 12 3 4 mm)
J. Coat. Technol. Res.

Fig. 10: Central chitosan sample’s 3D lCT visualization (4 3 12 3 4 mm) shows how the metaxylem tubes are coated after
20 impregnation cycles

Table 3: 3D lCT image analyses of silver aggregates in bamboo for different regions along different axes
ROI Along radial axis Along longitudinal axis
2
Aggregates/cm Area fraction (%) Aggregates/cm2 Area fraction (%)

Citrate-Ag-NFs Edge 1492 0.15 1651 0.15

Central 1657 0.17 1653 0.17
Chitosan-Ag-NFs Edge 331 0.08 526 0.09
Central 809 0.23 984 0.25

1.2 2.0
Citrate-Ag-NFs (edge) Citrate-Ag-NFs (central)
1.8
1.0
1.6

0.8 1.4

1.2
Area (%)
Area (%)

0.6 1.0

0.8
0.4
0.6

0.2 0.4

0.2
0.0
0.0

0 2000 4000 6000 8000 10000 12000 0 2000 4000 6000 8000 10000 12000
Distance (μm) Distance (μm)

Fig. 11: Silver concentration (% area) along the radial axis of edge and central citrate sample

Fig. 9. The polymer bamboo matrix was suppressed to
simplify the interpretation of the 3D lCT images,
which shows only the silver aggregates (in yellow).
Figures 9a and 9b show the edge and central bamboo
specimens treated with citrate-Ag-NFs, with a higher
concentration of metal deposition on the parenchy-
matic tissue and mainly on the external wall of the
sample. Figures 9c and 9d show the edge and the
central bamboo specimens treated with chitosan-Ag-
NFs, revealing a high concentration of metal deposi-
tion into the microchannel array of bamboo.
Figure 10 presents a high-resolution lCT image of
the central part of the bamboo specimen impregnated
with the chitosan-Ag-NF colloidal solution. From left
to right, as the vegetal matrix is digitally removed, the
location of silver deposits in the bamboo matrix
becomes clear. The deposition of positively charged
Ag-NFs is mostly concentrated in the internal wall of
the microchannel array creating silver-coated xylem
tubes.
Table 3 summarizes the data analyses from the lCT
3D high-resolution images along different axes: radial
(from outer to inner bamboo wall) and longitudinal
(along the fiber and the vascular vessels). The results
are shown in detail in Figs. 11, 12, and 13.
The area fraction of bamboo volume occupied by
silver aggregates, along the two different axes, is
approximately the same, which is enough to confirm
 J. Coat. Technol. Res.

1.25 3.0
Chitosan-Ag-NFs (edge) Chitosan-Ag-NFs (central)
2.5
1.00

2.0
0.75
Area (%)

Area (%)
1.5
0.50
1.0

0.25
0.5

0.00
0.0

0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
Distance (μm) Distance (μm)

Fig. 12: Silver concentration (% area) along the radial axis of edge and central chitosan sample

(a) 0.25 (b) 0.5

Edge
Edge
Central
Central
0.4
Citrate-Ag-NFs Chitosan-Ag-NFs
0.20
0.3
Area (%)
Area (%)

0.2
0.15

0.1

0.10 0.0
0 1000 2000 3000 4000 5000 6000 0 1000 2000 4000 5000 6000 7000
Distance (μm) Distance (μm)

Fig. 13: Citrate (a) and chitosan-Ag-NFs (b) aggregates concentration (area %) through the xylem tube’s axis of bamboo
specimens impregnated with silver colloidal solutions

the reliability of the quantitative analysis. The aggre-
gates size differences explain the slight distinction
between their numbers. When the axis is changed,
some aggregates that seem to be independent appear
as a single large unit. Bamboo treated with citrate-Ag-
NFs showed a higher number of aggregates with
smaller dimension compared to the bamboo treated
with chitosan-Ag-NFs.
Analysis of the data related to the metal deposition
along the radial axis for citrate-Ag-NFs-coated bam-
boo is shown in Fig. 11. The results show a higher
concentration of silver deposition on the exterior part
of the sample, which suggests a different coating
process from the outer to the inner part of the
bamboo matrix. The silver concentration in the
exterior region, between 0.2% and 0.8%, can be
visualized in Figs. 9a and 9b for both portions of the
sample, edge and central parts. Along the radial
direction, toward the interior region of the sample,
from 4000 to 12,000 lm, metal deposition decreases,
which represents an irregular coating mechanism in
the parenchymatic tissue.
In comparison, the bamboo treated with chitosan-
Ag-NFs, as shown in Figs. 9c and 9d, is characterized
by the deposition of metal aggregates only in the
internal wall of microchannel arrays. The data in
Fig. 12 show a different profile from citrate-Ag-NFs
(Fig. 11). The sharp peaks in the plot of Fig. 12 have a
distance compatible with the microchannels’ diameter
for all the portions (edge and central) analyzed. An
overlap between the radial axis image (Figs. 9c and 9d)
and the plot in Fig. 12 allows matching of the position
and the width of metaxylem tubes coated by silver with
the corresponding analyzed peak. While Lee et al.46
used gold nanoparticles to trace the physiological
mechanism of a vegetal plant, we show that Ag-NFs
can be used as a contrast element to reveal the vegetal
structure of bamboo.
From the analysis of the citrate-Ag-NFs metal
aggregates through the tube’s xylem axis, along the
overall bamboo sample (edge and central part), a
percentage of occupied area around 0.16% was
obtained (Fig. 13a). Note that there is a gap between
2500 lm and 4000 lm, which represents the gap
J. Coat. Technol. Res.

between the edge and the central part of the sample
analyzed. Otherwise, the chitosan-Ag-NFs metal
aggregates in the bamboo matrix present an increasing
distribution from the edge to the central portion of the
sample, from 0.08% to 0.25%, respectively (Fig. 13b).
Some assumptions have been made to explain these
different behaviors. The negatively charged citrate-Ag-
NFs colloidal solution goes through the vascular
bundles and penetrates the bamboo matrix until it fills
up some parenchymatic living cells. A 2D lCT image,
Fig. 14: 2D lCT images with citrate-Ag-NFs aggregates
deposited into the parenchymatic living cells
in Fig. 14, presents citrate-Ag-NFs aggregates with a
random distribution into the parenchyma tissue, a
morphology corresponding to the honeycomb-like cells
of the bamboo living cell as revealed by the CLSM
images (Fig. 6). This behavior is uniform for all the
portions analyzed along the longitudinal axis, from the
edge to the central section of the bamboo (Fig. 13a).
The diffusion of chitosan-Ag-NFs colloidal solution
into the vascular bundles is not uniform, and a higher
coating of internal channels’ walls in the central
portion of the bamboo specimen was observed.
As shown in Fig. 15, lower resolution 2D and 3D
lCT images, as well as SEM images and respective
EDS spectra of a bamboo specimen, confirm the
abundant presence of metal deposition in the straight
vessel channels. In Figs. 15a and 15b, decreasing the
resolution of lCT images with a macro-objective lens
of 0.49, we can visualize the whole sample
(5 9 12 9 18 mm), where vascular bamboo vessels
are a natural template for the formation of silver
metal tubes. The positively charged chitosan-Ag-NF
might have a higher affinity with the cellulose–lignin
polymer constituent of the internal wall of vessels.
After a first deposition, the chitosan-Ag-NFs with a
hydrodynamic diameter of 120 nm (larger than the
citrate-Ag-NFs) might have blocked the pits of internal
bamboo walls, thus rendering those surfaces imperme-
able and halting the eventual penetration of the Ag-
NFs into the parenchymatic tissue. The higher concen-
tration of nanoparticles in the central portion of the
bamboo can be linked to the evaporation process of
the colloidal solution that drags Ag-NFs from the
external to the internal portion of the tubes. This

Fig. 15: (a) 2D lCT low-resolution images with chitosan-Ag-NFs aggregates intercalated into the metaxylem vessel
probably deposited on the cleavage plate of the biological structure (5 3 12 3 18 mm); (b) 3D lCT images of chitosan-Ag-
NFs in the bamboo matrix. To simplify the interpretation of the image, the biological matrix of bamboo was excluded during
the image processing (5 3 12 3 18 mm); (c) SEM image and EDS analysis of longitudinal bamboo specimen coated with
chitosan-Ag-NFs

Table 4: Mean crystallite size for different preparation methods with an estimated error range from 10% to 20%
Surface slab, 10 cycles
Citrate-Ag-NP 8.1
Chitosan-Ag-NP 14.0
Intensity/a.u.
10 20 30 40 50 60 70
2θ/°
Fig. 16: XRD patterns of (a) natural bamboo and coated
bamboo with silver nanofiller using (b) chitosan and (c)
citrate as stabilizers
treatment, repeated during the impregnation process
(20 cycles), might have created this kind of cylindrical
metal deposition inside the microchannel array. The
selective distribution of chitosan-Ag-NFs is possibly
mediated via the positive charge, the higher hydrody-
namic diameter and the interfacial compatibility
between the similar macromolecular structures of
chitosan and cellulose. For negatively charged citrate-
Ag-NFs, with lower hydrodynamic diameter and small
organic surfactant, the abundant metal aggregates were
not observed in the microchannel array. The deeper
penetration into the parenchymatic cells may arise
from electrostatic attraction, permeation through inter-
nal vascular bundles and chemical affinity with the
internal wall of the cells.
After the nondestructive lCT analysis, the Ag-NFs-
treated bamboos were sliced with a microtome along the
bamboo longitudinal axis, and the cross sections were
analyzed to detect X-ray diffraction pattern of crys-
talline silver nanoparticles. Chitosan- and citrate-Ag-
NF-coated bamboo samples prepared by 10 and 20
impregnation cycles were analyzed. The Ag crystallite
mean sizes are presented in Table 4 and do not show any
significant change from 10 to 20 treatment cycles.
Figure 16 shows XRD patterns of the crystalline cellu-
J. Coat. Technol. Res.
Mean crystallite size (nm)
Surface slab, 10 cycles Surface slab, 20 cycles Surface slab, 20 cycles
7.8 8.3 9.8
19.9 16.8 17.7
lose of bamboo in natura and Ag-NFs-coated bamboos.
Ag In addition to cellulose peaks (16.2 and 22.5), both
chitosan-Ag-NFs and citrate-Ag-NFs showed peaks
related to silver at 38.1, 44.2, 64.4 and 77.3 assigned
to the planes (1 1 1), (2 0 0), (2 2 0), and (3 1 1),
respectively. As expected, the XRD experiments con-
c firmed the presence of the silver aggregates for both
impregnation cycles setup, while lCT provided false
negative results for the 10 impregnation cycles sample
due to the lack of spatial resolution.
b The mean crystallite sizes determined from Ag
diffraction peaks are slightly smaller (8–10 nm) for the
citrate-Ag-NFs aggregates in the bamboo than for the
citrate-Ag-NFs observed by STEM (Fig. 7). This is
consistent with citrate-Ag-NFs formed by more than
one crystallite, which is the region of coherent diffrac-
a
tion. Figure 17 depicts an HR-TEM image of citrate-
Ag-NF with different crystallites and confirms the data
80 90
obtained by XRD. The chitosan-Ag-NP aggregates
show crystallite sizes (14–20 nm) larger than the grain
size determined by STEM for the freestanding chi-
tosan-Ag-NFs. These larger crystallite sizes point to
some coalescence process. Indeed, lCT analysis estab-
lished that the chitosan-Ag-NFs have a very distinct
penetration behavior, even causing the clogging of
bamboo vessels. Such clogging would favor the aggre-
gation and eventually the coalescence of the nanopar-
ticles, a process already observed at room temperature
for citrate functionalized silver nanoparticles.51 Fur-
thermore, the 60C heat treatment performed after the
impregnation process is reported as enough to degrade
the chitosan,52 allowing contact between the silver
nanoparticles.
Conclusions
Ag-NPs colloidal solutions, capped with different
anionic and cationic organic ligands, were selectively
used to coat the internal microenvironment bamboo
matrix. Ag-NFs were synthesized with a microfluidic
system operating in continuous flow and stabilized with
negatively (sodium citrate) and positively (chitosan)
charged organic ligands, with hydrodynamic diameters
of 17 and 120 nm, respectively. The results suggest that
the self-sorting filling process of hybrid metal biocom-
posite bamboo was driven by the electric charge, size of
the organic ligands-capped Ag-NFs and chemical affin-
ity between organic capping agent and bamboo matrix.
J. Coat. Technol. Res.
Fig. 17: Citrate-Ag-NP HR-TEM with different crystallite structure of citrate-Ag nanoparticles
In this work, it was found that the most important
parameters that have to be considered and further
investigated to accomplish a precise nanocoating of
bamboo vascular bundles’ internal walls are (1) interfa-
cial compatibility, based on self-assembling interactions,
between the capped-NFs surface and the lignin-cellulose
walls of bamboo vessels; (2) prevention of Ag-NFs
agglomeration inside the bamboo’s microchannels; (3)
control of colloidal solution permeability into the matrix
related to wetting parameters of bamboo and rheology
properties of Ag-NFs solution. Further research is being
focused on the use of other polysaccharide-capped metal
nanoparticles with different physical and chemical
characteristics to realize bamboo-derived biocomposite
materials for specific electrochemical and microfluidics
applications. Further experiments should be considered
to study a potential enhancement of long-term durability
and mechanical properties of different bamboo species
depending on the regioselective coating of specific
biological microstructures.
Acknowledgments This work was supported by
Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnologico-Brasil (CNPq) (Grant Number 458302/
2013-9) and the Serrapilheira Institute (Grant Number
Serra-1709-17482) and Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel Superior - Brasil
(CAPES) - Finance Code 001. O.P. thank FAPERJ-
Brazil for the JCNE fellowship (E-26/203.281/2016).
PhD student R. S. M, Post-Doc E.J.R.R. are grateful to
CNPq for scholar fellowships. We thank Dr. Julio C.
Spadotto and Prof. Guillermo Solorzano for the HR-
TEM images and Centro Brasileiro de Pesquisa Fisica
(CBPF) for the use of their facilities.
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