Printable nanocomposites of polymers and silver nanoparticles for antibacterial devices produced by DoD technology
Printable nanocomposites of polymers and silver nanoparticles for antibacterial devices produced by DoD technology
Nicole Barrera 0 1
Lizeth Guerrero 0 1
Alexis Debut 0 1
Petrus Santa-Cruz 1
☯ These authors contributed equally to this work. 1
0 Universidad de las Fuerzas Armadas ESPE, SangolquÂõ, Ecuador, 2 Departamento de QuÂõmica Fundamental, Universidade Federal de Pernambuco - UFPE , Recife, Pernambuco , Brazil
1 Editor: Yogendra Kumar Mishra, Institute of Materials Science , GERMANY
Silver nanoparticles (Ag-NPs) are known for their efficient bactericidal activity and are widely used in industry. This study aims to produce printable antibacterial devices by drop-ondemand (DoD) inkjet technology, using Ag-NPs as the active part in complex printable fluids. The synthesis of this active part is described using two methods to obtain monodisperse NPs: chemical and microwave irradiation. The synthesized NPs were characterized by UVVIS, STEM, TEM, DLS and XRD. Two printable fluids were produced based: one with AgNPs and a second one, a polymeric nanocomposite, using silver nanoparticles and polyvinyl butyral (Ag-NPs/PVB). Cellulose acetate was used as a flexible substrate. The ecotoxicity analysis of fluids and substrate was performed with Artemia franciscana nauplii. Optimized electric pulse waveforms for drop formation of the functional fluids were obtained for the piezoelectric-based DoD printing. Activity of printed antibacterial devices was evaluated using the Kirby-Bauer method with Staphylococcus aureus and Escherichia coli. The results show that the printed device with Ag-NP fluids evidenced a bacterial inhibition. An important advantage in using the DoD process is the possibility of printing, layer by layer or side by side, more than one active principle, allowing an interleaved or simultaneous release of silver NP and other molecules of interest as for example with a second functional fluid to ensure effectiveness of Ag activity.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was funded by the "LaboratoÂrio
de Arquitetura de Nanodispositivos FotoÃnicos e
Bioinspiradosº LANDFOTON-UFPE (Recife-Brazil)
Group, supported by the Brazilian Research
Agencies FINEP (CT-HIDRO/NAMICRO), CNPq,
FACEPE, and CAPES (Nanobiotec-Brasil Network
36), for the financing of the project carried out, the
CETENE Research Center (MCTIC) and the
Silver has been used for thousands of years as an antimicrobial and antibacterial agent, up to
the antibiotic era which started 90 years ago [1±2]. Nowadays, the dramatic rise of
antibioticresistant bacteria has led to revisiting the antibacterial activity of silver [3±4]. In the last two
decades, nanotechnology has allowed to obtain silver nanoparticles (Ag-NPs) of controlled
size and morphology [
]. Silver nanoparticles present a strong broad-spectrum bactericidal
activity, and develop little or no bacterial resistance [
]. Also, they have the potential to be
used alone or in combination with polymers [
]. Other materials are also of interest for such
CENCINAT ªCentro de Nanociencia y
NanotecnologÂõaº (Quito-Ecuador), for their
research support in the characterization of the
cases. For example, a comparative study of ZnO, CuO and Fe2O3 nanoparticles activity against
Gram-positive and Gram-negative pathogenic bacteria demonstrated that the order of
antibacterial activity was ZnO>CuO>Fe2O3 [
]. A comparison of cytotoxicity of Zn-containing
structures showed lower cytotoxic potency of ZnO tetrapods (ZnO-T) than that of ZnO NP,
and several parameters were revealed to the assessment of ZnO-T toxicity in cell cultures [
The multi-functionality of ZnO is highlighted by Mishra and Adelung (2018) [
both bactericidal activities and biosafe material properties. The low toxicity of ZnO-T,
combined with its interconnected porous network present ZnO-T as smart nanocomposites for
biomaterials coatings [
]. Regardless nanostructures recently described several pathogenic
bacteria have developed resistance against antibiotics and metallic silver, in the form of
AgNP, remains as an important option as a new generation of antimicrobials. Silver binds to
bacterial cell wall and cell membrane and inhibit the respiration process, very likely by the metal
interaction with thiol group compounds found in the respiratory enzymes of bacterial cells. In
case of E. coli, silver may act by inhibiting the uptake of phosphate [
]. The large
surface/volume ratio of NPs provides better contact, allowing attachment to cell wall, penetration in the
bacteria cell, thus, preferably attacking the respiratory chain and leading to cell death. Also, as
NP releases silver ions in the bacterial cells, this can enhance their bactericidal activity, by
DNA interfering DNA replication after Ag+ contact [
]. The presence of harmful bacteria in
water, food and medical equipment has caused the industry to have the need to generate
solutions that ensure sterility of products and the prevention of infection [
]. In this field,
nanotechnology has been successfully implemented in the industry with the development of
processes and products containing nanomaterials, such as the production of Ag-NPs obtained
on a large-scale and polymer films, combined or not, to counteract microbial growth [15±16].
There has been an increase in the variety of physical and chemical methods for obtaining
silver nanoparticles, leading to new technological applications arising in different areas [17±
19], including bioinspired materials as antibacterial devices based on silver nanoparticles [
Among the different methods of preparation of Ag-NPs [21±23], we can mention the chemical
method that uses the addition of sodium borohydride or sodium citrate and the microwave
irradiation method. Silver nitrate is then reduced until obtaining metallic nanoparticles [24±
25]. In order to maintain the size and morphology in time of nanoparticles, it is necessary to
use stabilizing agents, which immediately wrap them to prevent their continuous growth and
To apply these new functional materials by controlled deposition, one of the most
innovative techniques to produce template-based devices is the drop-on-demand (DoD), allowing a
layer-by-layer production. Inkjet printer system using piezoelectric (PZT) driven printheads
may deliver 1 pL drops through an array of nozzles as small as 9 μm. The PZT actuators used
in this soft direct-write process do not heat the fluid, avoiding a thermal evolution (thermal
reduction, for instance) during the printing process that includes nanomaterials such as
AgNPs or Ag-NPs polymeric composites. It allows to develop and create new devices that can be
applied in different areas such as medicine and food [27±28]. The device properties may also
be exploited as a function of the number of printed layers, as for example in ªintelligent paper
], or other nanodevices [30±32].
DoD offers several advantages such as high precision and resolution (ten times higher than
the best 3D conventional printer), low sample and reagent consumption, leading to a cost and
analysis time reduction [
]. The control of print parameters may be assisted by build-in
stroboscopic cameras, and electric pulse waveforms for the drop formation may be obtained for
each specific fluid. The final device can be replicated and evaluated. The printing process of
nanomaterials encompasses a series of requirements that may be tuned as a function of its
composition, allowing a wide range of nanomaterials and even soft materials [
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As several pathogenic bacteria have developed resistance against antibiotics, bacterial
resistance to silver nanoparticles has been discussed in the literature. PanaÂček et al. reported
Escherichia coli and Pseudomonas aeruginosa resistance to silver nanoparticles after repeated and
long exposure, due to the production of an adhesive flagellum protein flagellin, which triggers
the NPs aggregation, thus eliminate their antibacterial activity. However, this process can be
suppressed in the presence of pomegranate rind extract, which inhibited the production of
flagellin protein [
]. One of the advantages of the present proposed system is the novelty of
printing more than one active principle, layer by layer or side by side, thanks to the DoD
process. In a well-defined architecture template, this would allow an interleaved or simultaneous
release of silver and inhibitors of proteins produced by Gram-negative bacteria, that can result
in resistance to the antibiotic activity of silver NP.
Each part of the device that will be printed, including functional fluids and substrates, must
be evaluated by bioassays to confirm the possible toxicity it may generate on an ecosystem
]. The effects that are generally evaluated in bioassay are mortality, immobility, behavioral
alteration, among others; these allow to observe whether the chemicals or used materials could
generate environmental impacts [
]. One of the most common indicator organisms in
ecotoxicity tests is Artemia franciscana [38±39]. This is a small crustacean that lives in aquatic,
high salinity environments. Artemia franciscana has been used as a biomodel in preliminary
stages of research for new products since 1982 [40±41], and is recommended by FAO [
The ability to feed through non-selective filtration makes this micro-crustacean an efficient
biosensor for detecting toxicity by filtering large quantities of materials, such as polymeric
materials in water [
]. According to the bioassay protocols, to avoid the influence of other
factors on their evaluation, A. franciscana nauplii in their first larval state must remain in
optimum conditions of hatching such as temperature, salinity, pH, oxygenation and illumination
Hereafter, the synthesis and characterization of Ag-NPs obtained by chemical method and
microwave irradiation, for the creation of two antibacterial devices printed with the DoD
technique is described. The first device created uses only Ag-NPs and the second is a polymer
nanocomposite made from a mixture of Ag-NPs and polyvinyl butyral. The two devices were
printed on acetate cellulose substrate (AC). The ecotoxic evaluation of the two polymers used
in the printed device, polyvinyl butyral (in the fluid) and acetate cellulose (substrate) is carried
out by studying the mortality of Artemia franciscana.
To verify the potential application in a medical environment, antibacterial effects of the
devices were evaluated in Escherichia coli and Staphylococcus aureus, the most prevalent
human-associated species of gram-negative and gram-positive bacteria, respectively.
Materials and methods
Silver nitrate (AgNO3, 99.0%), sodium borohydride (NaBH4, 98%) and sodium citrate
(C6H5Na3O7, 99.0%), were purchased from (Sigma-Aldrich, BR). Polyvinyl pyrrolidone
(PVP, K90), N,N-dimethylformamide (DMF) and isopropyl alcohol (IA, 99.5%) were
purchased from (Vetec, BR). Polyvinyl butyral (PVB) was supplied by (Solutia). Acetate cellulose
(AC) and Ethanol (Et, 96%) were supplied by (DinaÃmica, BR). Mueller-Hinton (M-H) and
agar media were provided by (Difco, BR).
The active parts of the functionalized fluids to produce the printable antibacterial devices was
synthesized using two different process. Chemical synthesis of silver nanoparticles (Ag-NPs
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(Q)) was performed using the following process: NaBH4 (3 mM, 25 mL) was placed in an
Erlenmeyer flask under constant stirring, to which AgNO3 (1 mM, 10 ml) was added at a rate
of one drop per second. C6H5Na3O7 (0.05 M, 5 mL) and PVP (3% w/v, 200 μL) were then
added at the same speed to stabilize the solution. Microwave radiation synthesis of silver
nanoparticles (Ag-NPs (M)) was performed using the following process: DMF (5 mL), AgNO3 (4
mM, 1 mL) and PVP (3% w/v, 200 μL) were placed in a test tube, which was placed in the
Discover single-mode reactor microwave, CEM brand, at 90ÊC, 150 W and 250 Psi for 30 seconds
with constant magnetic agitation.
The formation of the silver nanoparticles was monitored using UV-Vis spectroscopy. The
optical absorption spectra of the Ag-NPs in solution was measured through a spectrophotometer
(Analytik Jena SPECORD S 600, DE, using the WinASPECT software). To determine the
hydrodynamic diameter distribution of the Ag-NPs, a Dynamic Light Scattering (DLS)
analysis was carried out with the filtered samples (0.2 μm filter), using the LB-550 DLS Nanoparticle
Size Analyzer (HORIBA, JP). All DLS measurements were performed at a 25 ÊC fixed
temperature. X-ray diffraction (XRD) patterns were collected on an EMPYREAN diffractometer
(PANalytical, NL) in Bragg-Brentano configuration at 40 kV and 45 A and monochromatic
Xrays of Cu K-α wavelength (λ = 1.541 Å). The analysis was performed from filtered samples,
which were previously centrifuged 10 minutes at 10000 rpm, and letting them dry on a
microscope slide on a hot plate at 60ÊC. To analyze the size and morphology of the synthesized
AgNPs of the filtered samples, images were taken by Scanning Transmission Electron Microscope
(STEM), MIRA 3 Field-Emission Gun SEM (Tescan CZ) and Transmission Electron
Microscope (TEM), Tecnai G20 Spirit Twin (FEI, NL). The images were analyzed by a software
published in [
Ecotoxic analysis of PVB and AC
The ecotoxicity of PVB as a fluid component and AC, as printing support substrate, were
analyzed by exposure with Artemia franciscana. For the hatching of A. franciscana cysts, a 35%
saline water solution was prepared at pH 8,9. In an Artemio JBL incubator set, 500 mL of the
solution was placed with 50 mg of Artemia franciscana cysts, maintaining a constant
temperature (26ÊC) and illuminance (1000 lux) for 48 hours, monitored with a luxmeter (WHDZ LX
1010B). After hatching, the artemias were placed on a 24-well microtitre plate (5 individuals
per well) and each well with 1 mL of the previously prepared saline solution.
AC was taken in a layer disc form of 6 mm diameter. PVB was prepared by evaporation, for
which 6 mg of PVB was placed in 10 mL of Et and the mixture was done using an
ultrasonicator (UNIQUE) for 30 minutes at 50ÊC. The mixture was then placed on silicone supports in an
oven (Technical Novel 512) at 100ÊC to obtain the polymeric film.
With a metal perforating punch, PVB and AC discs were made. 1, 2 and 3 discs were
distributed in different wells, 5 repetitions each. The assay was controlled by two targets in which
five individuals were placed. The survival and mortality of Artemia franciscana was evaluated
at 24 hours, as described in [
Printing procedure for antibacterial printing devices
The antibacterial devices were printed on a Dimatix Materials Printer DMP-2831 (FUJIFILM
Dimatix Inc.) using DMC-11610 cartridges (10 picoliters, 16 nozzles) and the jettable fluids
were Ag-NPs (Q) and Ag-NPs (M) / PVB (1:1) as a polymer nanocomposite (PNC). In both
cases, the printing support was AC.
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For each fluid, the drop formation, which is function of the viscosity and surface tension,
was optimized varying the electric pulse waveform of the printer. These waveforms, very
important mainly for Non-Newtonian fluids, allows the droplets to be ejected properly from
the nozzle, carrying the Ag-NPs. The voltage variation (Vxt) to be applied in each PZT
actuator was obtained for each fluid with the help of embedded drop watcher and fiducial cameras.
DMC-11610 and DMC-11601 are user-fillable 1.5 ml disposable cartridges, that integrates
printheads based on PZT silicon single-crystals in micro-electro-mechanical systems (MEMS).
They have 16-nozzles of 21 μm or 9 μm diameter respectively, linearly spaced, with drop
volume of 10 pL or 1 pL, jetting the fluid in a rate of 5 kHz. Using the Waveform Editor tool
embedded in the printer software and the voltage and frequency parameters obtained
empirically from a standard fluid, modifications were made to the waveform shape, which influences
the formation, shape, and volume of the produced droplets. In order to determine the
parameters, the following steps were performed:
1. The DDP applied to the nozzles was scanned between 10±40 V, to determine the minimum
DDP for the drop ejection without causing nozzle obstruction and the maximum without
2. The maximum frequency of the jetting cycle (kHz) was then determined avoiding
obstruction, generation of satellite drops or jet drift;
3. The Adjustment of the constant DDP plateau was done without fluid flow;
4. The Adjustment of the plateau and filling slopes of the ejection antechamber was done,
followed by the same adjustment to increase the DDP applied to the beginning of the ejection
of the fluid, and finally for the controlled decrease of the DDP, to guarantee the formation
of the drop with the minimum turbulence.
The waveform changes were tracked with the DropWatcher tool camera, while the print
parameters were determined from the Drop Spacing tool: to adjust the distance between the
center of two adjacent drops, the angle of the print head was adjusted, which influences the
resolution of the print.
Although it's possible to print complex templates, for a Proof of Concept (PoC) purpose
device, a basic print pattern (1x1 cm squares) was established to print layer-by-layer assembled
devices as a function of the number of layers: 5, 15 or 30, in the present case. The drying time
between layer was set to 5 min. Devices were produced with different number of layers for
Analysis of the bactericidal effect with Escherichia coli and Staphylococcus
Pure strains of Escherichia coli (UFPEDA 224) and Staphylococcus aureus (UFPEDA 02) were
used for bacteriological analysis. The inocula were adjusted to 0.5 on the McFarland scale. A
6.5% w/v solution of the M-H medium with constant agitation at 60ÊC was prepared for the
culture medium and 20 g of agar was then added. The mixture was autoclaved and placed in
Petri boxes. The bacteria were sown in the Petri dish boxes with a swab over the entire surface,
on which the discs of printed devices (5,15 and 30 layers) were placed. The discs were prepared
for this assay using a standard metal perforating punch. As target control, sterile AC discs
were used. The boxes were incubated at 37ÊC for 24 hours and the inhibition zone was then
measured with a digital caliper (Mitutoyo). To insert the Ag-NPs, a well has been made in the
center of the Petri dish, drilling the middle and removing it with the help of a cork drill. The
diffusion coefficient of Ag-NPs in the M-H-agar medium was calculated by measuring the
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apparent dynamic viscosity of the culture medium in the following concentrations: M-H
(0.50% w/v) and agar (0.15% w/v), M-H (1.00% w/v) and agar (0.31% w/v), M-H (1.50% w/v)
and agar (0.46% w/v). The data were then extrapolated to determine the apparent viscosity
at the concentration M-H (6.50% w/v) and agar (2.00% w/v) used in the bactericidal effect
analysis. The diffusion coefficients (D) were determined with the Stokes-Einstein equation:
H (herein nm2/s), where k is the Boltzmann constant, T the absolute temperature, η
the dynamic viscosity and d(H) the hydrodynamic diameter. Finally, the measured inhibition
zone was related to the minimum inhibition zone Amin expected in relation to the diffusion
coefficient calculated in 24 h of bacterial incubation, as indicated in the following relation:
Amin D 24h (herein mm2).
Survival of Artemia franciscana: statistical analysis was performed using an ANOVA variance
analysis of a 2x3 factorial design (6 treatments) to evaluate the combination of two types of
levels: material type (PVB and AC) and concentrations of each (0.4,0.8,1.2 mg/mL for PVB
and 1,2.3 mg/mL for AC) using the IBM SPSS statistical program. A p value 0.05 was
Bacterial inhibition: Two analyses of variance (ANOVA) were performed for a factorial 2x3
(6 treatments) design. One for the Ag-NPs (Q) printed device and one for the Ag-NPs (M) /
PVB printed device. In both analyses the combination of bacterial type (E. coli and S. aureus)
and number of printing layers (5,15 and 30) was evaluated by the IBM SPSS statistical
program. A p value 0.05 was considered. For each experimental measurement, average value
and standard deviation were calculated for the inhibition zone using a millimeter ruler.
Results and discussion
The presence of silver nanoparticles was confirmed both in the chemical synthesis method with
a maximum absorption peak at 425 nm and in the microwave irradiation method with a peak
at 421 nm (see Fig 1A). This indicates an approximate size for spherical silver nanoparticles
Fig 1. UV-VIS spectrum. (A) and DLS measurements for silver nanoparticles produced by microwave (B) and chemical (C) synthesis.
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between 35 to 50 nm [
]. This conclusion is based on the size dependence of the optical
property of the plasmon resonance of silver nanoparticles [
]. The frequency of the surface plasmon
resonance over the silver nanoparticles depends on the nanoparticle shape and size, as well as
the dielectric functions of the host matrix and nanoparticle. Also, the formation of
nanoparticles aggregates or clusters may displace the absorption band up to the infrared, not observed in
the present work [
Dynamic Light Scattering
The hydrodynamic diameters of the nanoparticles were determined using DLS. In cases of
polydisperse size distribution of nanoparticles, the greater the dispersity of the sample, the
greater the probability of unsuccessful data from DLS technique. The average diameter was
found to be 59.59 ± 15.8 nm for Ag-NPs (Q), and 64.29 ± 8.62 nm for Ag-NPs (M), as shown
in Fig 1B and 1C.
From Fig 2, diffracted peaks were observed at 32.12Ê and 38.07Ê for Ag-NPs (Q). They
correspond to the set of planes (101) and (111) of the crystalline structure of silver (FCC). For
AgNPs (M), the diffraction angles 32.12Ê and 38.06Ê correspond to the (101) and (111) planes,
respectively [50±52]. As shown in the diffractogram of Fig 2, one may notice that, regardless of
the nanoparticle preparation technique (microwave or chemical), the ratio between the Bragg
peaks intensities from the metallic silver and the scattering band associated to the amorphous
part of the material, do not changes, although the intensity ratio between the peaks related to
Fig 2. XRD patterns from Ag-NPs produced by microwave and chemical preparation.
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Fig 3. TEM and STEM micrographs of silver nanoparticles. Prepared from microwave synthesis (A) and (B), and from chemical synthesis (C) and
(D). The white bar scale is 50 nm.
(101) and (111) planes have its ratio intensities changed, probably due to a possible preferential
orientation of the powders during the preparation of the samples.
Scanning Transmission Electron Microscope (STEM) and Transmission
Electron Microscope (TEM)
From Fig 3, one can observe that Ag-NPs (Q) and Ag-NPs (M) are spherical. Using an analysis
software developed in our group [
], an average diameter of 18.9 ± 12.3 nm was found for
Ag-NPs (Q), taking 132 nanoparticles into consideration. For Ag-NPs (M) an average
diameter of 17.83 ± 7.3 nm was found measuring an amount of 113 nanoparticles. This result is
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different from the DLS measurements, as the refraction index of the formed complex is
unknown and due to the fact that the sample distribution is not perfectly monodisperse [
Ecotoxic analysis of PVB and AC
In this research a range of 0 to 10% of A. franciscana mortality was used to determine the
nontoxicity of the polymer, based on the results obtained by Chinnasamy et al. [
]. In addition,
an experimental error rate of 5% was established. To verify this experimental error rate, A.
franciscana nauplii were exposed to PVB. A mortality of 4% against exposure of 0.4 mg/ml and
8% against exposure of 0.8 and 1.2 mg/ml was obtained, lower than the established
experimental error. For individuals exposed to AC, an 8% mortality was observed at exposure of 1 mg/ml
AC and a 12% mortality at exposure of 2 and 3 mg/ml AC, lower than the considered
experimental error. Therefore, comparing the survival rate of control individuals with respect to
individuals exposed to PVB and AC, we conclude that there is no influence of these polymers
on the survival of A. franciscana.
Printed devices and analysis of the bactericidal effect with Escherichia coli and Staphylococcus aureus
To produce the two printed devices, the fluids droplets ejected through the nozzles were
optimized applying a specific electric pulse waveform to the PZT actuators. Table 1 and Fig 4 (left)
Fig 4. Waveform curves (Vxt). Vxt optimized for Ag-NPs (M) / PVB (A) and Ag-NPs (Q) (B) -based fluids (left) and the corresponding quick shot
images of the jetted droplets showing six drops (of 16) from the DoD printer at final stages of drop formation (right).
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Fig 5. Bactericidal effect on E. coli. (A) Ag-NPs (Q), (B) Ag-NPs (M) and on S. Aureus (C) Ag-NPs (Q), (D) Ag-NPs (M).
show the four-segment control settings and the corresponding waveform curves (Vxt) for
printing Ag-NPs (M) / PVB and Ag-NPs (Q), and the corresponding quick shot images of the
jetted droplets is shown beside (Fig 4, right). A detailed protocol can be found in [
In order to evaluate the efficacy of the printed antibacterial devices, initially the bactericidal
effect of silver nanoparticles without PVB and before printing was observed for Ag-NPs (M).
The effect was compared between Ag-NPs (Q) and Ag-NPs (M) after incubation of E. coli,
shown in Fig 5A(a) and 5B, respectively, and with S. aureus, shown in Fig 5C and 5D,
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Fig 6. Bactericidal effect on E. coli. (A) Ag-NPs (Q) on AC and 5 layers (5L), (B) Ag-NPs (Q) on AC and 15 and 30 layers (15L and 30L), (C) Ag-NPs
(M)/PVB on 5 layers (5L), (D) Ag-NPs (M)/PVB on 15 and 30 layers (15L and 30L).
After the production of the printed devices, the bactericidal effect was analyzed using the
method of diffusion in agar with KirbyÐBauer discs, where the inhibition zone generated by
the devices of Ag-NPs (Q) and Ag-NPs (M) in both bacteria was evidenced. The results for the
AC (substrate) and the PVB polymer printed without Ag-NPs (called PC5, PC15 and PC30 in
the figures) were used as controls. There were compared with the nanoparticle-printed devices,
where a higher inhibition zone was observed in the Ag-NPs (M) / PVB devices, as shown in
Fig 6. Measurements were registered (Table 2) for the inhibition zones produced by devices
printed with Ag-NPs (Q) with 5 (Fig 6A), 15 and 30 (Fig 6B) printed layers, and devices with
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Ag-NPs (M) with 5 (Fig 6C), 15 and 30 (Fig 6D) printed layers, on E. coli. The same process of
measurements was performed for S. aureus (Fig 7). All the inhibition zones measurements are
summarized in Table 2.
By the previous results, one can conclude that the cell wall thickness of the Gram-positive
bacteria affects the action of the devices, showing less effect in S. aureus with respect to E. coli.
This is also confirmed by other research groups, as published by Malegowd et al. [
Diffusion coefficients were determined for Ag-NPs (Q) and Ag-NPs (M), see Table 3.
Furthermore, it was measured an apparent minimum inhibition zone for Ag-NPs (Q) of
0.041 mm and 0.039 mm for Ag-NPs (M) / PVB. These experiments show clearly the
antibacterial effect of the obtained devices. However, the antibacterial mechanism of Ag-NPs is
not clearly defined. There are several theories in the current literature. Ag-NPs can anchor
to the cell membrane, causing damage and leakage of intracellular material. The theory
mentions that the formation of free radicals by Ag-NPs is one of the causes of cell death
since they have the ability to generate pores in the membrane [
]. Another theory
considers that the release of silver ions from nanoparticles damages the integrity and permeability
of the membrane, and that these ions can react with functional protein molecules and DNA,
interfering with DNA metabolism and replication, originating the cell death [
Considering these various theories, we consider the schematic diagram for explaining the
antibacterial mechanism presented here in the nanocomposite system, see Fig 8. The bacteria interact
with the composite and in presence of water a process of creating silver ion is generated,
thus leading to the antibacterial effect. The mechanism presented in Fig 8 is related to
metallic pure silver nanoparticles, that releases ionic silver in aqueous solution, as proposed here,
but, using the same procedure proposed in the present paper, it is also possible to print new
hybrid structures, as those recently proposed by Assis et Al. [
]. These hybrid nanoparticles
act by a different mechanism, in which a semiconductor attracts bacterial agents and Ag
nanoparticles neutralize them, in α-Ag2WO4 nanoparticles, produced by femtosecond laser
irradiation. The new class of spherical hybrid nanoparticle presents a 32-fold improvement
of antibacterial performance and may be fully compatible with the presented DoD printing
The result of the ANOVA, carried out in the survival of Artemia franciscana, showed that
there is no influence of the polymer type (user in fluid and substrate), since the p value
obtained for the type and the concentration of each polymer is 0.865 and 0.523, respectively.
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Fig 7. Bactericidal effect on S. aureus. (A) Ag-NPs (Q) on AC and 5 layers (5L), (B) Ag-NPs (Q) on 15 and 30 layers (15L and 30L), (C) Ag-NPs (M)/
PVB on AC and 5 layers (5L), (D) Ag-NPs (M)/PVB on 15 and 30 layers (15L and 30L).
DLS average diameter of Ag-NPs (nm)
k = 1.38064852 x 10−23 J.K-1
T = 310.15 K
η = 2.926x10-28 J.s/nm3
Diffusion coefficients (nm2/s)
D = 19177
D = 17677
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Fig 8. Schematic diagram of antibacterial mechanism of the device impregnated of AgNPs.
Moreover, these values are greater than 0.05. So, with a 95% confidence interval, we
determined that there is no statistically significant influence of this parameter against the
microcrustaceans taken as a biomodel. Regarding the ANOVA analysis on bacterial inhibition,
maintaining a 95% confidence interval (p_value 0.05), we determined that the bacterial type
has a statistically significant influence on this parameter, since the p_value resulting from the
analysis was 0.028. However, no significant influence related to the number of printing layers
on bacterial inhibition was found, since the calculated p_value was 0.15.
Two antibacterial devices printed using DoD technology were obtained. The first, with silver
nanoparticles obtained by chemical process and the second one, with silver nanoparticles
produced under microwave irradiation, in a PVB composite. We show that both devices have a
bactericidal effect against S. aureus and E. coli. The substrate (acetate cellulose) and the
polymer used in the fluid (PVB) were subjected to an ecotoxic test, in which it was determined
that both polymers do not generate any type of toxicity, according to the biomodel used. The
results indicate that the best antibacterial characteristics of the printed device is obtained for
the polymer compound with silver nanoparticles synthesized by microwave irradiation and
considering the highest number of printing layers. In addition, the inclusion of silver
nanoparticles into the polymer matrix avoid any aggregation and so the possibility of a reduction of
its antibacterial activity. To ensure the effect on a long time, an interleaved or simultaneous
release of silver and inhibitors of proteins produced by Gram-negative bacteria could be
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produced in the next future. Therefore, this kind of device could be used for industrial
applications as a printable antibacterial device produced by drop-on-demand inkjet technology.
S1 Fig. Original print screen of waveform curves (Vxt). Vxt optimized for Ag-NPs (M) /
S2 Fig. Original print screen of waveform curves (Vxt). Vxt optimized for Ag-NPs (Q).
S1 Video. Droplet video obtained during the printing process using parameters defined in S1 Fig.
S2 Video. Droplet video obtained during the printing process using parameters defined in S2 Fig.
The authors thank the ªLaboratoÂrio de Arquitetura de Nanodispositivos FotoÃnicos e
Bioinspiradosº LANDFOTON-UFPE (Recife-Brazil) Group, supported by the Brazilian Research
Agencies FINEP (CT-HIDRO/NAMICRO), CNPq, FACEPE, and CAPES
(NanobiotecBrasil Network 36), for the financing of the project carried out, the CETENE Research Center
(MCTIC) and the CENCINAT ªCentro de Nanociencia y NanotecnologÂõaº (Quito-Ecuador),
for their research support in the characterization of the nanoparticles. The authors are
especially grateful to Prof. Norma Gusmão (Departamento de Antibioticos, UFPE) for the support
for antibacterial assays.
Conceptualization: Lizeth Guerrero, Petrus Santa-Cruz.
Data curation: Alexis Debut.
Funding acquisition: Petrus Santa-Cruz.
Investigation: Nicole Barrera, Lizeth Guerrero, Alexis Debut, Petrus Santa-Cruz.
Methodology: Nicole Barrera, Lizeth Guerrero, Petrus Santa-Cruz.
Project administration: Petrus Santa-Cruz.
Supervision: Alexis Debut.
Writing ± original draft: Nicole Barrera, Lizeth Guerrero, Alexis Debut, Petrus Santa-Cruz.
Writing ± review & editing: Lizeth Guerrero, Alexis Debut, Petrus Santa-Cruz.
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