nanomaterials

Article
Silver Nanoparticles on Cellulose Surfaces:
Quantitative Measurements
Ana Patrícia Carapeto 1, * , Ana Maria Ferraria 2, * and Ana Maria Botelho do Rego 2
1 BioISI—Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa,
1749-016 Lisboa, Portugal
2 Centro de Química-Física Molecular and IN-Institute for Nanosciences and Nanotechnologies and
IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa,
1049-001 Lisboa, Portugal; amrego@tecnico.ulisboa.pt
* Correspondence: apcarapeto@fc.ul.pt (A.P.C.); ana.ferraria@tecnico.ulisboa.pt (A.M.F.)




Received: 19 March 2019; Accepted: 19 May 2019; Published: 22 May 2019


Abstract: In this work, cellulose films pre-activated with carbonyldiimidazole (CDI) and grafted
with 1,6-hexanediamine, were decorated with silver nanoparticles (AgNPs). The generation of
AgNPs was followed by quartz crystal microbalance (QCM). The obtained films were characterized
by X-Ray Photoelectron Spectroscopy (XPS) and imaged by atomic force microscopy (AFM). XPS
confirmed the synthesis in situ of AgNPs on the film attesting their oxidation state. The results from
the three techniques were compared showing how sound the quantitative treatment of the results
issued from these techniques can be. The main objective of this work is exactly to show that the
quantitative exploration of the results of different characterization techniques can and should be
practiced systematically instead of just comparing them qualitatively.

Keywords: cellulose; silver; nanoparticles; thin films; AFM; QCM; XPS

1. Introduction
The use of a large battery of techniques to characterize a given system is, recently, a common
approach. Specifically, for the study of systems involving surface functionalization, several microscopies
(atomic force microscopy, AFM; scanning electron microscopy, SEM) as well as mass measuring tools
(quartz crystal microbalance, QCM) and surface-specific spectroscopic techniques such as X-ray
photoelectron spectroscopy (XPS) are frequently used together. However, their quantitative use to test
how their different probing range can enrich the obtained information is rare.
Here, an example dealing with the functionalization of cellulose films with metallic nanoparticles
will be used to exemplify how quantitative these studies can be.
Developing methods for immobilizing metallic nanoparticles (NPs) onto different surfaces is a
subject of fundamental interest, as nanometric structures enable the appearance of novel properties,
absent in bulk or even in micrometric dimensions. Silver NPs have been increasingly used in various
fields. These nanoparticles are being successfully used in cancer diagnosis and treatment [1,2].
Nano-silver systems also present several advantages that make them very interesting for use as
antimicrobial agents. They possess a very high activity against a broad range of microbes and
parasites [3,4] as well as biofilm inhibition in medical implants [5]. Furthermore, the incorporation of
releasable silver NPs into polymeric nanofibers could be a promising technique to provide effective
antibacterial action during wound healing [6].
Natural cotton is one of the purest cellulose sources and is the most significant commercial source
of natural fibers. As a material, cotton holds several attractive properties, including biocompatibility,
biodegradability, and both thermal and chemical stability. Silver NPs linked to cellulose reportedly

Nanomaterials 2019, 9, 780; doi:10.3390/nano9050780 www.mdpi.com/journal/nanomaterials

Nanomaterials
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cellulose reportedly demonstrate antimicrobial activity through the release of nanoscale silver [7].
demonstrate
Preceding antimicrobial
works published activity through
by our group on the
this release
subject,ofusing
nanoscale silver
cellulose [7].form
in the Preceding
of filmsworks
have
published by our group on this subject, using cellulose in the form of films
shown that the nucleation and growth of silver NPs on the surface of cellulose films, interacting have shown thatwith
the
anucleation and growth
suitable silver of silver
salt solution, NPs
can be on the surface
optimized by of cellulose
a prior films,activation
chemical interactingofwith a suitable
the alcohol silver
groups
salt solution, can be optimized by a prior chemical activation of the alcohol groups
on the cellulose surface followed by the grafting of aliphatic diamine molecules (Scheme 1). One of on the cellulose
surface
the aminefollowed
groups by willthe
begrafting
grafted of
onaliphatic diamine molecules
the film (specially (Scheme
to the cellulose 1). One
primary of the and
alcohol) amine thegroups
other
will will
one be grafted
complex on silver
the film (specially
ions. to the cellulose
The growth of NPs isprimary
achieved alcohol)
by theand the otherofone
interaction will complex
aqueous dilute
silver ions.
solutions ofThe growth
silver of (AgNO
nitrate NPs is achieved by modified
3) with this the interaction of aqueous
cellulose dilute [8–10],
film surface solutions
theofamine
silver group
nitrate
(AgNO3 ) withtothis
coordinating themodified
silver ioncellulose
and thefilm surfacealso
cellulose [8–10], the amine
having a role group
in the coordinating to theofsilver
partial reduction ion
further
and the cellulose also having a role in the partial reduction of further silver ions [11,12].
silver ions [11,12]. The procedure limits the generation of NPs only on the cellulose surface, keeping The procedure
limits
the the generation
dispersion medium of completely
NPs only onexemptthe cellulose surface,
of them. keeping NPs
The resulting the dispersion medium chemically
are, consequently, completely
exempt of them.
immobilized Thecellulose
on the resultingsurface.
NPs are, consequently, chemically immobilized on the cellulose surface.

Scheme 1.1.Activation
Scheme Activationof cellulose (Cell) with
of cellulose carbonyldiimidazole
(Cell) (CDI) followed
with carbonyldiimidazole by the
(CDI) functionalization
followed by the
with hexanediamine.
functionalization with hexanediamine.

The characterization
The characterization of of immobilized
immobilized NPs NPs atat surfaces,
surfaces, usually
usuallyincludes
includesthetheimaging
imagingby byAFM.
AFM.
However, when trying to quantify the total amount of silver in the form of
However, when trying to quantify the total amount of silver in the form of NPs, even if the imageNPs, even if the image
analysis allows
analysis allows forfor aa proper
proper evaluation
evaluation of of the
the NP
NP size
size distribution,
distribution, several
several assumptions
assumptions need need to
to be
be
made, namely,
made, namely,concerning
concerning thethe
shape of NPs.
shape of OnNPs.theOn
other hand,
the estimating
other the volume the
hand, estimating of nanoparticles
volume of
immobilized on a surface using the QCM requires other assumptions,
nanoparticles immobilized on a surface using the QCM requires other assumptions, namely namely that the metalthat
density
the
in thedensity
metal NPs is the same
in the NPsasisinthe
thesame
bulk,asand that
in the the silver
bulk, composing
and that the silverthe as-synthesized
composing NP is in the
the as-synthesized
metallic
NP is in form. Such chemical
the metallic form. Suchcharacterization can be done by
chemical characterization XPS.
can be Since
done these three
by XPS. complementary
Since these three
techniques require different assumptions, the complementarity of the data
complementary techniques require different assumptions, the complementarity of the data is very important to validate
is very
the obtained
important results. the obtained results.
to validate

2. Materials and Methods

2. Materials and Methods
Acetone (puriss.) was obtained from Fluka (Steinheim, Germany). Absolute ethanol was
Acetone (puriss.) was obtained from Fluka (Steinheim, Germany). Absolute ethanol was from
from Riedel-de Haën (Steinheim, Germany). Dimethylsulfoxide (DMSO), hydrochloric acid (HCl,
Riedel-de Haën (Steinheim, Germany). Dimethylsulfoxide (DMSO), hydrochloric acid (HCl, 37%),
37%), N,N0 -Carbonyldiimidazole (≥90%) (CDI), 1,6-hexanediamine (98%) and silver nitrate (AgNO ,
N,N′-Carbonyldiimidazole (≥90%) (CDI), 1,6-hexanediamine (98%) and silver nitrate (AgNO33,
99.9999%), were purchased from Aldrich (Steinheim, Germany). Deionized water (DIW) with a
99.9999%), were purchased from Aldrich (Steinheim, Germany). Deionized water (DIW) with a
resistivity of 18.2 MΩ cm was supplied by a MILLIPORE system (Darmstadt, Germany), fed with
resistivity of 18.2 MΩ cm was supplied by a MILLIPORE system (Darmstadt, Germany), fed with
distilled water.
distilled water.
QCM crystals (5 MHz Au/Cr, Stanford Research Systems, Sunnyvale, CA, USA) were degreased
QCM crystals (5 MHz Au/Cr, Stanford Research Systems, Sunnyvale, CA, USA) were degreased
with acetone and ethanol, and dried under an argon flux. The cellulose films were produced by
with acetone and ethanol, and dried under an argon flux. The cellulose films were produced by spin-
spin-coating (speed = 2000 rpm, t = 60 s, acceleration = 800 rpm/s) the clean crystal of Quartz/Au
coating (speed = 2000 rpm, t = 60 s, acceleration = 800 rpm/s) the clean crystal of Quartz/Au (electrode)
(electrode) with a trimethylsilylcellulose (TMSC) film from a solution in toluene (prepared as described
with a trimethylsilylcellulose (TMSC) film from a solution in toluene (prepared as described in [13])
in [13]) and exposing it immediately to a saturated HCl atmosphere to regenerate the cellulose. The film
and exposing it immediately to a saturated HCl atmosphere to regenerate the cellulose. The film
surface was then activated with CDI (QCM crystal/cellulose film was immersed in a CDI solution
surface−2was then activated with CDI (QCM crystal/cellulose film was immersed in a CDI solution 5 ×
5 × 10 M in DMSO, for 2 h), functionalized with 1,6-hexanediamine (QCM crystal/activated cellulose
10−2 M in DMSO, for 2 h), functionalized with 1,6-hexanediamine (QCM crystal/activated cellulose
Nanomaterials 2019, 9, 780 3 of 8

film was immersed in a diamine solution 1 × 10−2 M in DMSO, for 2 h) and thoroughly rinsed with
ethanol and water to remove any physisorbed amine.
The quartz crystal microbalance used in this work was the QCM 200 from Stanford Research
Systems. The QCM crystal coated with the cellulose film (grafted with 1,6-hexanediamine), was
mounted in the QCM and placed in interaction with AgNO3 (5 × 10−3 M aqueous solution) by injecting
in the reaction chamber, perpendicularly to the film surface, a flow of the silver salt solution for 15 min
and at room temperature. The variation in frequency (∆f ) was monitored as a function of the time.
The Sauerbrey equation:
∆f = −Cf ∆m, (1)

where ∆f is the frequency change, ∆m is the mass change per unit area and Cf is the sensitivity factor
for the crystal which is 56.6 Hz µg−1 cm2 , relates the mass change per unit area at the QCM electrode
surface to the observed change in the oscillation frequency of the crystal, provided that |∆f |/f 0 (where
f 0 = 5 MHz is the resonant frequency of the fundamental mode of the unloaded crystal) is less than 2%.
It was used to obtain the mass gain as the nanoparticles grew on the cellulose surface [14].
The resulting functionalized films were imaged by AFM (D3100 with a Nanoscope IIIa controller
from Digital Instruments, Santa Barbara, CA, USA) carried out in tapping mode under ambient
conditions with TESP tips (Material: 0.01–0.025 Ω cm Antimony (n) doped Si; R: 3.25–4.75 µm; L:
110–140 µm; W: 30–50 µm; f 0 : 279–389 kHz; k: 20–80 N/m; estimated nominal radius = 8 nm), Bruker
AFM Probes (Camarillo, CA, USA). The AFM images were analyzed with the software WSxM (5.0
Develop 6.5, Nanotec Electronica S.L., Madrid, Spain) [15]. However, the number and volume of
the nanoparticles could not be estimated by using the WSxM flooding tool, which allows to detect
different features of the probed area, like hills and/or holes. In this case, and due to the roughness
of the polymeric film and the dimensions of the nanoparticles, when detecting hills above a given
height, besides the nanoobjects identified as silver nanoparticles, it also detects cellulosic bundles
whose area and volume are also quantified by the software. To avoid misleading measurements,
a visual inspection of the images was done, counting and measuring the sizes of the nearly spherical
nanoparticles existing at the surface. The reliability of such simplistic data collection was analyzed by
comparing the results with other data obtained by a very different technique of analysis, the QCM.
The hybrid film was characterized by XPS, using a XSAM 800 dual anode spectrometer from
KRATOS (Manchester, UK). Non-monochromatic Al Kα X-rays (hν = 1486.6 eV) were used and the
take-off angle was 45◦ . The analyzed area in high magnification mode is approximately 1 mm × 3 mm.
Other operation and acquisition conditions and data treatment details were as described in [16].
No charge correction was needed. The quantification factors were 0.25, 0.66, 0.42, and 5.2 for C 1s, O
1s, N 1s, and Ag 3d, respectively.

3. Results
The growth of NPs was followed by QCM and the QCM crystal with the hybrid film was imaged
by AFM. The chemical characterization was performed by XPS.

3.1. QCM
The NPs growth was followed by QCM (Figure 1). From QCM measurements, |∆f | is less than
20 Hz, which for a crystal resonant frequency of 5 MHz, corresponds to a |∆f |/f 0 less than 4 × 10−4 %,
largely within the Sauerbrey Equation validity range (|∆f |/f 0 < 2%). The mass of nanoparticles formed
on the film surface is, therefore, computed from the Sauerbrey Equation (1), 0.22 ± 0.04 µg/cm2 . This
value was obtained based on three independent measurements (three samples) ranging from |∆f | = 11
to 14 Hz, yielding an average value of 12.5 ± 2.1 Hz. Assuming the same density for silver NPs as for
bulk silver (ρ = 10.5 g/cm3 ), the NPs should have a total volume of 210,331 ± 35,694 nm3 /µm2 . Even if
the assumption about the density is approximate, the order of magnitude should be this one.
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Figure 1. Quartz crystal microbalance (QCM) result: variation of the crystal frequency (∆f) after
Figure 1.1. Quartz
Figure Quartz crystal
crystal microbalance
microbalance (QCM)
(QCM) result:
result: variation
variation of
of the
the crystal
crystal frequency
frequency(∆f ) after
(∆f) after
interaction of the cellulose film (grafted with 1,6-hexanediamine) with AgNO3 monitored as a
interaction
interactionofofthethe
cellulose filmfilm
cellulose (grafted with with
(grafted 1,6-hexanediamine) with AgNO
1,6-hexanediamine) monitored
with 3AgNO as a function
3 monitored as a
function of the time.
of the time.
function of the time.

3.2.
3.2. AFM
AFM
3.2. AFM
Cellulose ultrathinhybrid
Cellulose ultrathin hybridfilm
film was
was imaged
imaged by AFM.
by AFM. EachEach measurement
measurement consisted
consisted of threeofimages
three
Cellulose ultrathin2 hybrid film was imaged by AFM. Each measurement consisted of three
images of the surface, 1 × 1
of the surface, 1 × 1 µm (or lower):µm 2 (or lower): height, amplitude, and phase images. The images
height, amplitude, and phase images. The images presented in
images of the surface, 1 × 1 µm2 (or lower): height, amplitude, and phase images. The images
presented in Figure to
Figure 2 correspond 2 correspond
one of seventodifferent
one of seven
pointsdifferent pointsTopographic
of the surface. of the surface.
andTopographic
phase imagesand are
presented in Figure 2 correspond to one of seven different points of the surface. Topographic and
phase
shownimages
in Figure are2a,b
shown in Figure Discrete
respectively. 2a,b respectively. Discreteafter
NP are observed NP the
are interaction
observed after
withthe
AgNOinteraction
3 . They
phase images are shown in Figure 2a,b respectively. Discrete NP are observed after the interaction
with AgNO
are better 3. They are
identified better
in the identified
phase in the
image than in phase image thanone.
the topographic in the topographic
Since one.
the cellulose Since
film the
surface
with AgNO3. They are better identified in the phase image than in the topographic one. Since the
cellulose film surface
is very rough, is verytool
no automatic rough, noas
(such automatic
WSxM) can toolbe
(such as WSxM)
applied can beto
to the image applied to the
count and image
measure
cellulose film surface is very rough, no automatic tool (such as WSxM) can be applied to the image
to
thecount and measure the nanoparticles.
nanoparticles.
to count and measure the nanoparticles.
31.30
31.30 nm nm
1 31.30
31.30 nm nm
1

0.8
0.8

0.6
0.6
Y[µm]
Y[µm]

0.4
0.4

0.2
0.2

0
0 0.00 nm
0 0.2 0.4 0.6 0.8 1 0.00 nm
0 0.2 0.4 0.6 0.8 1
X[µm]
X[µm]
(a) (b)
(a) (b)
Figure 2. Cont.
Nanomaterials 2019, 9, 780 5 of 8
Nanomaterials 2019, 9, x FOR PEER REVIEW 5 of 8

30
1
25
0.8
20

Height (nm)
Diameter
0.6 15 = 38.3 nm

10
0.4
5

0.2
0
0 200 400 600 800 1000
X (nm)
0
0 0.2 0.4 0.6 0.8 1
(c) (d)
2. (a) Atomic force microscopy (AFM) image of hybrid
Figure 2. hybrid cellulose
cellulose film
film (1000
(1000 nmnm ×× 1000 nm) in
topographic mode; (b) AFM image of hybrid cellulose film, the same as in (a) in phase mode; circles
were drawn around
around the
theundoubtedly
undoubtedlysilver
silvernanoparticles;
nanoparticles; (c)(c)
AFMAFM image
image of (a) superposed
of (a) withwith
superposed the
the
samesame circles
circles which
which were
were drawn
drawn in in
thethe phase
phase imageinin(b);
image (b);(d)
(d)Height
Heightprofile
profile for
for a horizontal line
crossing the
the white
white circle
circle center.
center.

To
To make
make anan estimation
estimation ofof the
the nanoparticle
nanoparticle volume,
volume, aa “manual”
“manual” method
method was
was adopted:
adopted: thethe NPs
NPs
were visually identified in the phase image and encircled (Figure
were visually identified in the phase image and encircled (Figure 2b). 2b).
This
This set
set of
of circles
circles was
was grouped
grouped andand paste
paste on
on the
the topographic
topographic image
image (Figure
(Figure 2c).
2c). Lines
Lines parallel
parallel to
to
the
the xxaxis
axiswere
weredrawn
drawn crossing thethe
crossing center of the
center of encircled particle
the encircled and the
particle andrespective profile profile
the respective was drawn
was
automatically by the software.
drawn automatically The full The
by the software. width at width
full half maximum of the profile
at half maximum feature
of the profilewas measured
feature was
and taken as
measured andthetaken
particle diameter
as the (see
particle Figure 2d
diameter forFigure
(see an example). Also
2d for an the heights
example). Alsowere
themeasured and
heights were
taken as the
measured radius
and takenofas
the
thesphere.
radius of the sphere.
i 4/3π(Di∑/2)
3 , was computed
P
With
With this
thisprocedure,
procedure, 32 NPs
32 NPscould be counted
could and theand
be counted totalthe
volume,
total volume, i 4/3π(Di /2) , was
3
3 3 3
P
to be 225,000 nm . Using the3 heights, the volume obtained, i 4/3πhi , was 199,000 nm . The associated
computed to be 225,000 nm . Using the heights, the volume obtained, ∑i 4/3πhi3, was 199,000 nm3. The
error is hard to estimate, given the intrinsic nature of the measurements in AFM and the made
associated error is hard to estimate, given the intrinsic nature of the measurements in AFM and the
assumptions. Anyway, the relative difference for the average value found by the QCM method, is less
made assumptions. Anyway, the relative difference for the average value found by the QCM method,
than 7%.
is less than 7%.
3.3. XPS
3.3. XPS
The chemical characterization was performed by XPS (Figure 3). XPS results attest undoubtedly
TheAg
that the chemical characterization
3d5/2 peak, centered atwas performed
368.1 ± 0.1 eV by XPS (Figure
corresponds to3). XPS results
metallic silverattest
(Ag0undoubtedly
) (Figure 3a).
that the Ag 3d 5/2 peak, centered at 368.1 ± 0.1 eV corresponds to metallic silver (Ag0) (Figure 3a). The
The silver oxidation state was deduced from the Auger parameters equal to 725.9 ± 0.1 eV and
silver±oxidation
719.9 0.1 eV forstate was deduced
the pairs [Bindingfrom the(BE)
Energy Auger(Agparameters equal
3d5/2 ), Kinetic to 725.9
Energy ± 0.1
(KE) (AgeV
M4and
N45 719.9
N45 )],±and
0.1
eV for the pairs [Binding Energy (BE) (Ag 3d 5/2), Kinetic Energy (KE) (Ag M4N45N45)], and [Ag 3d5/2,
[Ag 3d5/2 , Ag M5 N45 N45 ], respectively [17]. It is worth noting that for Ag 3d photoelectrons (kinetic
Ag M5N
energy N45],eV),
≈451118 respectively [17]. Itlength,
the attenuation is worth
λ, innoting that
silver is 1.6 for
nm Ag
[18]3d photoelectrons
which gives a probe(kinetic
depth, energy
d, taking≈
d = 5λ (99.32% of the signal comes from this depth), of ~8.0 nm. Therefore, for particles withdradii
1118 eV), the attenuation length, λ, in silver is 1.6 nm [18] which gives a probe depth, d, taking = 5λ
(99.32% of the signal comes from this depth), of ~8.0 nm. Therefore, for particles with
larger than 8 nm, this information does not come from the inner part of the particle but rather from an radii larger than
8 nm, shell
outer this information
8 nm thick. does not come from the inner part of the particle but rather from an outer shell
8 nm thick.
N 1s shows that no unreacted NO3−, from the silver precursor, remains in the film, since it would
be detected around 407 eV (Figure 3c). The nitrogen detected at 399.1 ± 0.2 eV comes from amine C–
NH2 groups and carbamate groups C–N=O, formed upon cellulose activation with CDI, and, at
higher binding energy (400.9 ± 0.2 eV), from protonated –NH3+ groups. Other diamine, cellulose and
gold (substrate) features are detected in C 1s, O 1s, and Au 4f XPS regions (not shown). The fact that
gold is detected confirms how thin the cellulose films here studied are. XPS analysis depth is of the
Nanomaterials 2019, 9, x FOR PEER REVIEW 6 of 8

order of 102019,
Nanomaterials nm.9,Therefore,
780 films are, in average, thinner than that value, confirming thicknesses
6 of 8
estimated earlier for similar films [16].

Ag 3d Ag MNN N 1s N-C
Ag 3d5/2 Ag M5N45N45
NH3+

Ag 3d3/2

Intensity (a.u.)
Intensity (a.u.)

Intensity (a.u.)
Ag M4N45N45

382 377 372 367 362 340 348 356 364 408 403 398 393
Binding Energy (eV) Kinetic Energy (eV) Binding Energy (eV)
(a) (b) (c)
Figure
Figure 3.
3. X-ray
X-rayphotoelectron
photoelectron spectroscopy
spectroscopy (XPS)
(XPS) regions:
regions: (a)
(a)Ag
Ag 3d;
3d; (b)
(b) Ag
Ag MNN;
MNN; and
and (c)
(c) N
N 1s.
1s.

N 1s shows
4. Discussion that
and no unreacted NO3 − , from the silver precursor, remains in the film, since it would
Conclusions
be detected around 407 eV (Figure 3c). The nitrogen detected at 399.1 ± 0.2 eV comes from amine
The values obtained with AFM and QCM are presented in Table 1. Given the rough
C–NH2 groups and carbamate groups C–N=O, formed upon cellulose activation with CDI, and, at
approximations used in both estimations, and the consequent associated error, the two values which
higher binding energy (400.9 ± 0.2 eV), from protonated –NH3 + groups. Other diamine, cellulose
differ from each other less than 7%, may be considered the same.
and gold (substrate) features are detected in C 1s, O 1s, and Au 4f XPS regions (not shown). The fact
This means that: i) the used approximations are not too crude and the method can be soundly
that gold is detected confirms how thin the cellulose films here studied are. XPS analysis depth is of
applied for other systems and ii) the nanoparticles were really formed at the film extreme surface as
the order of 10 nm. Therefore, films are, in average, thinner than that value, confirming thicknesses
expected given the low thickness of the films stated above and its functionalization with the diamines.
estimated earlier for similar films [16].
Table 1. Atomic force microscopy (AFM) and quartz crystal microbalance (QCM) values for the
4. Discussion and Conclusions
calculation of Ag nanoparticles volume.
The values obtained with AFM and QCM are presented in Table 1. Given the rough approximations
AFM QCM
used in both estimations, and the consequent associated error, the two values which differ from each
Number of NPs 32 Δf −12.5 ± 2.1 Hz
other less than 7%, may be considered the same.
Computed from Computed from
∆m 0.22 ± 0.04 µg/cm2
measured diameters measured heights
Table 1. Atomic force microscopy (AFM) and quartz crystal microbalance (QCM) values for the
Ag NPs
225,000 nm3/µm
calculation of Ag nanoparticles 2
volume. 199,000 nm3/µm2 210,331 ± 35,694 nm3/µm2
volume/Area
AFM QCM
One can, then, conclude that the AgNPs synthesis procedure here described limits the generation
Number of NPs 32 ∆f −12.5 ± 2.1 Hz
of NPs only to the cellulose surface, keeping the dispersion medium (solution) completely exempt of
Computed from Computed from
them. The resulting NPs are, consequently, chemically immobilized on∆m the cellulose ± 0.04 µg/cm2
surface.
0.22
measured diameters measured heights
The values obtained for the nanoparticles volume using AFM and QCM techniques are, within
Ag NPs
the experimental
volume/Area
error, exactly the3 /µm
225,000 nm same2
in spite of the
199,000 3 /µm2
nmdifferent sizes 210,331
of the explored 3 /µm2
sample
± 35,694 nm zones
(of the order of a few cm in QCM and of µm in AFM). XPS is not adequate for estimating the NP
2 2

volume but confirms that the silver oxidation state is Ag0. This information is indispensable to make
This meansbased
the estimations that: (i)
on the
AFMusedand approximations are not
QCM results. This work tooshows
crude and
that the
datamethod canthrough
obtained be soundly
the
applied for other systems and (ii) the nanoparticles were really formed at the film extreme
combined use of these analysis techniques allow for sound and reliable estimations of volume NPs, surface as
expected given the low thickness of the films stated above and its functionalization with
provided that they are essentially formed on the extreme surface of materials and should be used in the diamines.
Nanomaterials 2019, 9, 780 7 of 8

One can, then, conclude that the AgNPs synthesis procedure here described limits the generation
of NPs only to the cellulose surface, keeping the dispersion medium (solution) completely exempt of
them. The resulting NPs are, consequently, chemically immobilized on the cellulose surface.
The values obtained for the nanoparticles volume using AFM and QCM techniques are, within
the experimental error, exactly the same in spite of the different sizes of the explored sample zones
(of the order of a few cm2 in QCM and of µm2 in AFM). XPS is not adequate for estimating the NP
volume but confirms that the silver oxidation state is Ag0 . This information is indispensable to make
the estimations based on AFM and QCM results. This work shows that data obtained through the
combined use of these analysis techniques allow for sound and reliable estimations of volume NPs,
provided that they are essentially formed on the extreme surface of materials and should be used in
that way more often. This demonstration of the quantitative use of these techniques opens the way to
further exploration of QCM and AFM (or SEM, depending on the involved sizes) data when properly
combined with XPS.

Author Contributions: Experiments were performed by A.P.C.; Data were analyzed by A.P.C., A.M.F., and
A.M.B.d.R.; The original draft was prepared by A.P.C.; The final paper was written, edited and reviewed by A.M.F.
and A.M.B.d.R.
Funding: This research was funded by Fundação para a Ciência e a Tecnologia (FCT) projects references
UID/NAN/50024/2013 and UID/MULTI/04046/2013. APC and AMF were funded by FCT grants numbers
SFRH/BD/75734/2011 and SFRH/BPD/108338/2015, respectively.
Conflicts of Interest: The authors declare no conflict of interest.

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