Colloidal silver nanoparticles: an effective nano-filler material to prevent fungal proliferation in bamboo

Abstract

Silver nanoparticles (Ag-NPs) have attracted attention as a promising nano-filler material for reinforcement and anti-bacterial effect in polymers and composites. In this work bamboo samples, Dendrocalamus Giganteus Munro, were impregnated using a colloidal solution of homemade Ag-NPs with the goal of improving its resistance to attacks by fungi. X-ray microtomography (μCT) was performed to investigate the 3D distribution of Ag-NPs within the biological matrix. 3D image reconstruction showed silver clusters distributed within the parenchymatic tissue. Quantitative information of the Ag-NPs agglomerate population was computed. Ag-NPs were characterized by UV-VIS, SERS, ICP-MS, TSEM, DLS and zeta potential analysis. The antimicrobial activity of homemade and commercial citrate-capped silver nanoparticles was evaluated against the Aspergillus niger fungus. Homemade nanoparticles (NP-01), presenting the smallest diameter (14.3 ± 3.6 nm), and the highest particle concentration (1.25 × 10¹¹ particles per mL) were able to inhibit 53% of Aspergillus niger growth in a concentration of 2.00 mg L⁻¹. Both engineered biocomposite material and untreated specimens were exposed to air and humidity. After five months the treated samples were free of fungal colonies, while colonization by the fungal hyphae was present on untreated bamboo specimens.

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Introduction

The use of bamboo has been investigated in lieu of raw materials in engineering, biomass production, power plant, furniture, textiles, paper, composite panels, decoration materials, building material and handcrafts.^1,2 Nevertheless, the application of natural bamboo has been restricted due to its degradation, which is affected by microbiological activity.^3–5 In this regard nanotechnology is focused on the use of silver nanoparticles against insects, bacteria and fungi, affecting the metabolism of these microorganisms, for industrial and biomedical applications.⁶

Among all the inorganic nanoparticles (NPs) based on gold (Au), silver (Ag), copper (Cu), zinc (Zn) and titanium (Ti), Ag-NPs are one of the most used engineered nanostructured materials for industrial applications and academic research. Inorganic nanoparticles, defined as nanofillers: “are reinforcement particles smaller than 100 nm in size which are usually blended with resins to create nanofilled resin composites. Incorporation of nanofillers into resin composites enables mechanical strengthening and excellent surface polishing”.⁷ Relatively easy to synthesize and characterize, Ag-NPs have good chemical stability when capped with proper organic stabilizer, high biocompatibility with human and plants, easy commercial availability and large atomic weight if compared to bamboo matrix, leading to significant image contrast in microtomography studies. Antifungal effect of silver nanoparticle (Ag-NPs) against plant pathogenic fungi has already been studied with success.^8,9 Ag-NPs can act as well as potential antimicrobial agents which mechanism inhibition of cell growth by silver ions (Ag⁺) has been established.¹⁰ Particle size, shape, type of ligand or stabilizer can influence its bactericidal properties.^11,12 There are already several applications in food packaging, food safety and as wound treatment agents.^13–15 Successful treatment of natural fibers with silver nanoparticles as antifungal protector, has also been reported.^16,17 Recently, a review shows a large number of nanomaterials used for dental care and dentistry which may improve mechanical reinforcement, aesthetic aspects, beyond inducing antimicrobial and therapeutic effects. Among them, Ag-NPs are already successfully used for dental care.^7,18 In the plant nanobiotechnology field, silver nanoparticles are used in plant tissues instead of classical antibiotics and chemotherapeutic agents to avoid phytotoxicity, retard plant tissue growth or risk of resistance to bacteria in case of extended exposure to conventional drugs.¹⁹ In nanoagriculture, engineered nanomaterials are used to increase the yield of plant for food and fuel.²⁰

Hamed et al., reported an in vitro study about the biodegradation of wood by the Aspergillus niger fungus.²¹ The work focus was to illustrate the morphological and structural wood decay induced by fungi. The concern was the lack of a conservation method or lifting procedure to preserve historically wooden artefacts.

The incorporation of silver, copper and zinc oxide nanoparticles into paints have been studied to inhibit molds growth in indoor environments and therefore to prevent air contamination. The best results were obtained incorporating silver nanoparticles with the smallest size (10 nm).²² Nowadays, two patents involving silver NPs as biocidal agents are already deposited: Horner et al., reported a practical application using nanosilver as an antimicrobial agent in building material for roofing, insulation, cladding, wall lining;²³ Kwon et al., developed an antibacterial paint containing silver nanoparticles.²⁴ A cheaper alternative to silver NPs is based on copper oxide nanoparticles. Meghana and Usman et al., reported its potential antimicrobial activity against bacteria.^25,26 In this early stage, no works are presented against fungus, or its potential hazard due to prolonged exposure of human beings and environment.^3,5

Bamboo is considered an important non-conventional building material for low income housing and sustainable urban architecture, but its response to microbiological activity is not yet established for biological decay.²⁷ It is a giant grass, and not a tree as commonly believed, belonging to the Bambusoideae family and found in large quantities in Asia, Africa and South America. Their culms are generally a cylinder shell, divided by transversal diaphragms at the nodes. The structure of bamboos is a composite material, mainly composed of multi-functional fibers of cellulose (55%), lignin (25%) and hemicellulose (20%).²⁸ Aligned cellulose fibers immersed in ligneous matrix are distributed anisotropically in a radial direction from the inner to outer section of the culm.²⁹ This structural composition, named sclerenchyma is responsible for bamboo's mechanical strength^30,31 and, at the same time, it supports the vascular bundles system to feed all the living cells of the parenchymal tissue. Due to its chemical, physical and mechanical properties, bamboo presents high versatility and viability to replace wood and steel.³² In addition, cellulose bamboo pulp can act as reinforcement in composites,³³ and in concrete to replace asbestos and glass fibers.³⁴

Within parenchymal tissue of the biological matrix there are starch granules and water, which are an energy source for the growth of microorganisms, which are prone to their attacks, especially by fungi and insects. In order to passive the bamboo from biological degradation, different methodologies have been explored, such as, immersion in boiling water, injection of water with positive pressure (Boucherie method),³⁵ and impregnation with polymeric resin.³⁶ To reduce the degradation of bamboo and to preserve the external aspect as well as all the mechanical properties, a treatment with antifungal chemical compounds, such as borax–boric acid mixture, ammonium compound or copper–chrome–arsenic, have been investigated, but not with total success.^4,37 Bamboo pulp³⁸ and bamboo rayon fabric¹⁷ were grafted with silver and copper nanoparticles, respectively. Jingpeng Li et al., have successfully deposited different nanostructured materials onto external bamboo surface creating: (1) a superhydrophobic bamboo timber based with TiO₂ anatase film for a self-cleaning and acid rain protection,^39,40 (2) Ag-NPs external deposition to construct a durable robust superhydrophobic surface with high conductivity,⁴¹ (3) ZnO nanocoating surface with improved mould-resistance.^42,43

In this work, for the first time, we propose to fill the bamboo matrix with silver colloids as nanofiller agents to verify its effectiveness against microbiological attacks, especially fungi.⁴⁴ Fungi is a kingdom containing a lot of micro-organisms divided into phylum, class, order, family, genus and species.⁴⁵ Many species are able to attack wood and bamboo, including the Aspergillus species.^21,46 Aspergillus niger is one of the most important species from this genus, for both industry and the environment.^47,48 These cells are able to produce lots of metabolites and enzymes involved in cellulose, hemicellulose and lignin degradation.⁴⁹ Aspergillus niger is easily cultivated, its morphology and metabolism are well understood and its cell growth is faster than for other fungus.⁴⁷ To save time and to avoid possible degradation of silver NPs in the culture medium during extended biological experiment, in this work, we decided to use Aspergillus niger. For all these mentioned reasons, we preferred to work with this fungi as a non-pathogenic fungus widely distributed in nature and to avoid opportunistic human pathogenic fungus.

The impregnation with an aqueous colloidal solution of Ag-NPs can take advantage from the hydrophilic surface properties of bamboo fibers.⁵⁰ In this regard to enhance the dispersion of the Ag-NPs the right choice of organic surfactant ligand is critical. X-ray microtomography (μCT) was chosen as the imaging technique to reveal the metal distribution within the bamboo matrix. μCT is a non-destructive technique that relies on the interaction and attenuation of X-rays when passing through a sample.^51,52 This technique is particularly suitable in this case, given the large atomic weight difference between the silver particles and the bamboo matrix, leading to significant image contrast. The technological advancements on X-ray tubes with smaller focal spot sizes and flat panel detectors made resolution at the micron scale possible. The result of μCT is a 3D image of the internal structure of the biocomposite sample, which allows a 3D analysis of parameters that are usually limited to a 2D evaluation.⁵³ Considering that μCT offers thousands of images and true 3D information, the statistics are far superior to any other available non-destructive technique. The advantages of the technique have been recognized in several areas of expertise such as orthodontics, biology, earth sciences, archaeology, and many others.^54–59

In this study, the nanofiller agent (Ag-NPs) was used to fill up the biological matrix of bamboo to obtain an engineered biocomposite. The silver deposition into bamboo matrix was analysed by means of μCT and quantitative information of the silver clusters was extracted using digital processing 3D images. Finally, optical microscope observation was performed to confirm the growth inhibition of fungal colonies on bamboo sample.

Experimental

Materials and methods

Silver nitrate (AgNO₃, >99.9% pure), sodium borohydride (NaBH₄, >99% pure), and trisodium citrate (Na₃Citrate, >99.0% pure) were purchased from Sigma-Aldrich and used as received. Water obtained from a Millipore Milli-Q purification system (resistivity ≥ 18.2 MΩ cm⁻¹) was used to make all the solutions for the desired reactions. For the synthesis of silver nanoparticles, in flow mode, two syringe pumps (Future Chemistry) and one glass microreactor (Micronit) were used. The syringe pumps are able to inject the solutions of silver precursor and organic ligand into the microchannel device to improve the mixing and the formation of the organic–metal complex in a shorter time when compared with the batch mode. PFA (perfluoroalkoxyalkane) tubes and PEEK connections (polyetheretherketone) (UpChurch) were used to inject the two solutions into the microreactor with 6 μL internal volume. Commercial PELCO Citrate NanoXact™ Silver Nanospheres with diameters about 20 nm (CO-20), 40 nm (CO-40) and 60 nm (CO-60) purchased from TedPella Inc were also used. The bamboo impregnation experiments were carried out using a four years old Dendrocalamus giganteus bamboo culm obtained from FZEA-USP, Pirassununga-SP, Brazil. Microbiological tests were performed with the Aspergillus niger fungus strain, grown in liquid Sabouroud, Kasvi (Slabor). The samples were cut with an automatic precision cut-off machine, Miniton from Struers, using a wafering blade made of diamond metal bonded, 3′′ × 0.006′′ × ½′′ (76 × 0.15 mm).

Synthesis procedures

Ag-NPs were synthesized employing NaBH₄ as reducing agent and Na₃Citrate as stabilizer organic ligand. In the first step of organic–metal complex formation, Na₃Citrate organic ligand with three carboxylate functional groups was able to coordinate silver ions (Ag⁺) into the microfluidic device. In the second step, during the chemical reduction of Ag⁺ to silver metal nanoparticles, Na₃Citrate acted as a capping agent, preventing the aggregation of Ag-NPs. The reduction process was conducted in continuous flow reaction, in which AgNO₃ solution (10⁻² mol L⁻¹) was mixed with sodium citrate organic ligand (10⁻² mol L⁻¹) in a glass of microreactor system.⁶⁰ The two solutions were injected by means of two syringe pumps at a flow rate of 0.25 mL min⁻¹. The Ag⁺ : ligand complex flowing out of the microreactor dropped directly into the fresh NaBH₄ solutions (10⁻² mol L⁻¹) under vigorous stirring, at room temperature. Colloidal solution NP-01 was used for the antimicrobial tests and impregnation of a bamboo specimen.

UV-VIS spectroscopy

The colloidal suspensions of Ag-NPs were characterized using UV-VIS spectrophotometer (Perkin-Elmer, model Lambda 950), with a spectral resolution of 1.0 nm and a spectral range between 300 and 500 nm. For the UV-VIS spectrophotometric analysis, all commercials nanoparticles were diluted with Millipore Milli-Q water in the ratio 1 : 4, with the exception of NP-01, which was diluted in the ratio 1 : 40.

RAMAN and surface-enhanced Raman scattering (SERS) spectroscopy

Raman and SERS spectroscopy were performed using a micro-Raman microscope (HORIBA, model XploRA), at the exciting wavelengths of 638 nm. The laser excitation beam was focused onto the liquid samples with an intensity of 25 Wmm⁻² by a 10× objective lens. The scans were performed using a fixed acquisition time of 5 s with 20 accumulations, with a range between 550 and 1800 cm⁻¹ and a spectral resolution of 4 cm⁻¹. Raman spectra of trisodium citrate aqueous solution (1.5 mol L⁻¹) was acquired and compared with the SERS spectra of citrate-capped Ag-NPs. In order to observe the SERS of citrate-absorbed Ag-NPs, sequentially, 3 drops of NPs suspensions were deposited on a silicon wafer to induce Ag-NPs aggregations by slow evaporation of the water solvent. Before the aggregation of NPs only background scattering was observed.

Morphological characterization by TSEM

For the morphological characterization of Ag-NPs using microscopy, a small drop (2.5 μL) of the nanoparticle solutions were placed on a carbon grid (holey carbon) and allowed to evaporate completely in the air before analysis. A field emission scanning electron microscope (FEG-SEM) (JEOL, JSM-6701F) operated in the transmission mode (TSEM) at 30 kV with a work distance of 6.0 mm using the bright-field detector was used. A longitudinal section of bamboo vascular bundle was also observed in scanning mode (SEM) at 1 kV with a work distance of 14.7 mm.

In order to obtain the size distribution of silver nanoparticles, an image analysis (IA) routine created in the FIJI/ImageJ program was applied. An intensity threshold was used for segmentation, followed by watershed separation to detect the individual silver nanoparticles. The particles' major and minor axes were measured, among other morphological attributes. Diameter distributions were inserted in the corresponding TSEM images with a mean value of 14.3 ± 3.6 nm for NP-01, 21.2 ± 3.2 nm for CO-20, 38.9 ± 7.3 nm for CO-40 and 60.3 ± 8.9 nm for CO-60. The results are in good agreement with the technical data sheet of the commercial nanoparticles purchased from TedPella Inc.

Element Ag detection by ICP-MS

The quantification of the concentration of colloids Ag-NPs was performed by inductively coupled plasma mass spectrometry (ICP-MS) to confirm the data of commercial nanoparticles (CO-20/40/60) and homemade NP-01. ICP-MS (Perkin-Elmer, ELAN Drc II Axial Field Technology) data acquisition was performed using 20 sweeps, in triplicate, and a dwell time of 50 ms. Default values were used for the rest of instrumental parameters. After acid digestion with concentrated HNO₃ in hot plate for 10 minutes, the diluted silver ionic solutions were direct injected and compared with the ionic silver standard solutions (5–30 ppm) into the ICP-MS system.

Size distributions and zeta potential analysis

Size distribution by dynamic light scattering (DLS) and zeta potential analysis for the colloid solutions were measured with a Nano-100SZ (Horiba instrument). NP-01 colloid solution was diluted to reach a citrate concentration of 2.0 × 10⁻³ mol L⁻¹, as indicated on the technical data sheet for the Ag-NPs commercialized by TedPella Inc. Before the analysis, the solutions were filtered through a 0.20 μm membrane, Cromafil® EXTRA PTFE-20/25. Measurement parameters were as follows: water viscosity medium of 0.894 mPa s⁻¹; refractive index dispersion medium of 1.333; detector scattering angle of 90°; temperature of 25 °C; particle refractive index for silver of 0.130–3.200i. For all samples, the count rate was between 500 and 1200 kCps. The polydispersity index (PDI) for the colloids silver nanoparticles was smaller than 0.58.

Bamboo impregnation

A 5 × 5 × 5 mm cubic section of Dendrocalamus Giganteus Munro bamboo was placed into a test tube with 0.50 mL of citrate-capped colloidal solution of silver nanoparticles (NP-01) for 2 hours. It was submitted to five impregnation cycles through a vacuum system. After each cycle, a fresh NP-01 solution was used. Then the impregnated bamboo specimen was analysed with X-ray microtomography.

X-ray microtomography (μCT)

A Zeiss Xradia Versa 510 microtomograph was employed. This system consists of an X-ray microfocus source, with up to 160 kV voltage and 10 W power and a detection system in which two-stage magnification scheme is used: (i) geometric magnification as with conventional μCT, (ii) a set of optical lens with scintillators converts X-rays to visible light, which is then optically magnified. Achievable true spatial resolution reaches 0.7 μm. The bamboo samples, before and after Ag-NPs impregnation, were scanned using the configurations presented in Table 1. A 3D digital video rendered from the μCT reconstructed images was used to visualize the different biological structures and the volumetric metal deposition within the engineered biocomposite material. This video is available through the following link https://www.youtube.com/watch?v=gbbyCVz3Pw0g.⁶¹

Table 1 X-ray microtomography (μCT) parameters

	Sample 1 pure bamboo	Sample 2 bamboo/Ag-NPs
Voltage (kV)	80	40
Power (W)	7	3
Pixel size (μm)	4	3.43
Exposure time (s)	0.2	1
Number of projections	1600	1600

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Image processing and analysis of μCT images

The μCT images of the impregnated bamboo specimen were processed and analyzed using FIJI/ImageJ free software.^62,63 The volume distribution and statistical information of Ag-NPs aggregates population in the bamboo matrix were obtained.

Microbiological tests

The antimicrobial activity of Ag-NPs was evaluated. For this purpose, Aspergillus niger was cultivated in Sabouroud liquid medium at 29 °C and 160 rpm in the presence and absence of Ag-NPs in the test tubes.⁴⁴ A volume of 1 mL of culture medium without Ag-NPs was used as a positive control for cell growth. Commercial and homemade colloidal solutions of Ag-NPs with original concentration as shown in Table 5 where introduced in 1 mL inoculated culture media with a final concentration of 2.00 mg L⁻¹ of each type of Ag-NPs. Microbial growth was followed during 24 and 48 hours. After 48 h, the tubes were vacuum filtered, and the pellets were dried in an oven at 60 °C. At different times, they were weighed with an analytical balance until constant dry weight. All the experiments were conducted in triplicate.

Results and discussion

Ag-NPs characterization

The Localized Surface Plasmon Resonance (LSPR) band of a diluted silver colloidal solution is presented in Fig. 1. The peak absorption of the LSPR band is centred at 393.1 nm for the homemade silver nanoparticles, NP-01. The plot also shows the spectra for commercial nanoparticles with 20 nm, 40 nm and 60 nm, with peak absorptions at 398.5 nm, 409.0 nm and 431.8 nm, respectively. According to the theory of surface plasmon resonance, the LSPR band of colloidal solutions is highly dependent on the diameter of silver nanoparticles.⁶⁴ Thus, the smallest wavelength for NP-01 indicates a colloidal solution contains smaller particles than the commercial ones.

Fig. 1 UV-VIS spectra of diluted colloidal solutions of citrate-capped Ag-NPs. Homemade (NP-01) and commercial colloidal solutions (CO-20, CO-40 and CO-60).

The Raman spectra of the trisodium citrate is reported in Fig. 2 and its scattering bands summarized in Table 2. The most intense single band Raman scattering is attributed to the carboxylate symmetric stretching band, ν_s(COO), at 1406 cm⁻¹. This band is broadened and shifted, from 1375 to 1383 cm⁻¹, in the SERS spectra of citrate absorbed on the surface of the commercial Ag-NPs (CO-20/40/60) (Fig. 3). This is possibly due to the interaction between the carboxylate groups and the silver metal surface. The broad band of the carboxylate symmetric stretching band indicates that the interactions with the metal surface are not equivalent, supposing that one of them might point out of the surface as confirmed by the zeta-potential measurements with a value up to −40 mV.⁶⁵

Fig. 2 Raman spectra of aqueous solution of trisodium citrate (1.5 mol L⁻¹) by excitation at 638 nm.

Table 2 Raman frequencies and band assignments for the aqueous solution of trisodium citrate (1.5 mol L⁻¹)^66–68

Frequency (cm^−1)	Assignment	Frequency (cm^−1)	Assignment
1567–1630	νa(COO)	943	ν(C–COO)
1406	νs(COO)	830	ν(C–C)
1257–1282	δ(COO)	667	δout-plane(COO)
1042–1082	ν(C–OH)

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Fig. 3 SERS spectra of citrate absorbed to nanoparticle surface obtained by excitation at 638 nm for CO-60 (red line), CO-40 (blue line), CO-20 (black line) and NP-01 (green line).

The weak double band Raman scattering at 1567–1630 cm⁻¹ is attributed to the carboxylate asymmetric stretching band, ν_a(COO), of pure citrate. These broadened bands are thin and centred at 1602 cm⁻¹ in the SERS spectra of the commercial Ag-NPs. This enhancement of the asymmetric vibration can corroborate the hypothesis of interactions between the carboxylate groups and the metal nanoparticle. An isolated SERS band, at 1721 cm⁻¹ for citrate-reduced silver colloids, was observed for the first time by Muoro et al.⁶⁸ It was attributed to the presence of decomposition products of citrate, such as, acetone dicarboxylic acid and acetoacetic acid. All these compounds with similar carboxylate groups can justify the broadening of the ν_s(COO) from 1314 to 1467 cm⁻¹.

For the commercial NPs we did not obtain disclosed information about the chemical preparation. For this reason it is difficult to justify and compare SERS spectra of homemade NPs with the commercial ones. Our homemade NPs are borohydride-reduced colloids what might justify the absence of the isolated SERS band at 1721 cm⁻¹. In Fig. 3, the SERS of homemade NPs confirms the presence of carboxylate symmetric stretching band, ν_s(COO), centred at 1393 cm⁻¹, but the carboxylate asymmetric stretching thin band, ν_a(COO), is broadened, weak and shifted to 1551 cm⁻¹. The bending vibration of carboxylate group, δ(COO), in the Raman spectra of pure citrate, appears as double bands from 1257 to 1282 cm⁻¹. SERS δ(COO) bands appeared at 1279 and 1296 cm⁻¹, respectively, for citrate absorbed on CO-60/40/20 and NP-01. All other SERS bands, summarized in Table 3 and 4, overlap with a small red shift that might be due to the different chemical nature of borohydride-reduced colloids suspensions (NP-01).^66,67

Table 3 SERS frequencies and band assignments for citrate absorbed on the metallic surface of commercial Ag-NPs aggregated from 3 drops of suspensions of CO-60, CO-40 and CO-20 on silicon wafer^66–68

Frequency [cm^−1]	Assignment	Frequency [cm^−1]	Assignment
1600–1603	νa(COO)	1112	ν(C–O)
1375–1382	νs(COO)	924	ν(C–COO)
1279	δ(COO)	824–846	ν(C–C)
1079	ν(C–OH)	791	δout-plane(COO)

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Table 4 SERS frequencies and band assignments for citrate absorbed at the surface of homemade Ag-NPs (NP-01) aggregated from 3 drops of suspension on silicon wafer^66–68

Frequency [cm^−1]	Assignment	Frequency [cm^−1]	Assignment
1551 very weak	νa(COO)	1121	ν(C–O)
1393	νs(COO)	939	ν(C–COO)
1296	δ(COO)	834	ν(C–C)
1084	ν(C–OH)	798	δout-plane(COO)

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ICP-MS analysis results of element (Ag) mass concentration are summarized in Table 5. The results for the commercial NPs from 17 to 22 mg L⁻¹ showed a good agreement with the technical data sheet of the commercial nanoparticles. Aliquots of the mother solutions were injected in 1 mL fungus medium with an equal final concentration of 2.00 mg L⁻¹. From the element (Ag) mass concentration and the size diameter distribution from TSEM images (Fig. S1†) we estimated the particle concentration of each colloid used for the microbiological tests. Homemade nanoparticles (NP-01) presenting the smallest diameter of 14.3 ± 3.6 nm show a particle concentration (1.25 × 10¹¹ particles per mL) 3.3 fold higher with respect to the smallest commercial nanoparticle CO-20 (3.82 × 10¹⁰ particles per mL). How will be shown in the microbiological test, the best biocidal activity of NP-01 is due to both reasons: the smaller size diameter, as reported previously,^69,70 and the higher number of NPs able to act against the fungus.

Table 5 Quantification of element (Ag) mass concentration and particles concentration for the colloids suspensions by direct injection of acid digested NPs solutions into ICP-MS

Sample	Mass concentration (Ag) [mg L^−1]	Mass concentration (Ag) in 1 mL media [mg L^−1]	Diameter from TSEM image [nm]	Particles concentration in 1 mL media [particles per mL]
NP-01	229.00 ± 0.31	2.00	14.3 ± 3.6	1.25 × 10^11
CO-20	16.66 ± 0.09	2.00	21.2 ± 3.2	3.82 × 10^10
CO-40	21.31 ± 0.10	2.00	38.9 ± 7.3	6.19 × 10^9
CO-60	21.28 ± 0.17	2.00	60.3 ± 8.9	1.66 × 10^9

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Colloid stability

Homemade NPs (NP-01) and commercial NPs (CO-20/40/60) were stored in the same conditions: at +5 °C and protected from the light. The DLS and zeta potential experiments were done with 5 month aged silver NPs. DLS size distribution shown by particle number is plotted in Fig. 4 and summarized in Table 6. Hydrodynamic diameter of all colloids suspensions confirms the size distribution obtained by TSEM analysis distribution (Fig. S1†). As well established in the literature,⁶⁵ the hydrodynamic diameter of NPs does not depend only on the metal core, but also on the organic ligand absorbed on the surface of the nanoparticle and the thickness of the electrical double layer (solvation shell). For these reasons the size distribution measured with DLS is higher in comparison to the TSEM analysis distribution.

Fig. 4 DLS size distribution shown by particle number for 5 months aged Ag-NPs colloids: NP-01 (17.2 nm), CO-20 (22.7 nm), CO-40 (44.7 nm) and CO-60 (62.4 nm).

Table 6 Summary results of size distribution of hydrodynamic radius and Zeta-potential for 5 months aged Ag-NPs colloids

Sample	Particle surface aqueous Na3Citrate [mol L^−1]	Hydrodynamic diameter (Z-average) [nm]	PDI	Zeta potential [mV]
a Data from the technical data sheet of TedPella Inc.
NP-01	2.0 × 10^−3	17.2	0.553	−41.4
CO-20	2.0 × 10^−3 ^a	22.7	0.587	−40.5
CO-40	2.0 × 10^−3 ^a	44.7	0.268	−43.3
CO-60	2.0 × 10^−3 ^a	62.4	0.296	−49.9

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Aged NPs with zeta potential of about −40 mV, confirms the stability of the NPs suspensions with pendant negative carboxylate groups of citrate absorbed on metal Ag-NPs.⁶⁵ NPs negatively charged by citrate ions are stabilized by electrostatic repulsions. Also, SERS spectra of aged NPs were acquired to confirm the presence of citrate ions absorbed on their surface (Fig. 3). We would like to highlight that all the biological and impregnation experiments were done with fresh synthetized NPs.

Microtomography study

Three dimensional (3D) μCT images of pure bamboo structures are presented in Fig. 5. The light grey colour shows the complex vascular bundles of bamboo with two large circular metaxylem vessels (100 μm), phloem and protoxylem wrapped or capped by fibers of sclerenchyma tissue (lignificated vegetable cells). The parenchyma shown in dark grey colour represents the living cells of the bamboo matrix. The parenchyma cells are vertically elongated in rectangular or shorter cube-like shapes, and alternate themself along the culm. The vascular bundles system varies along the bamboo culm length in size, distribution and shape. Meanwhile the tissue arrangements of the vascular bundles are strictly longitudinally oriented, as pillars.²⁸

Fig. 5 3D μCT image of pure bamboo specimen shows the complexity of vascular bundles constituted by: (A) metaxylem vessels (100 μm); (B) phloem; (C) protoxylem; (D) sclerenchyma fibers and (E) parenchyma tissue.

In Fig. 6 and 7, 2D and 3D μCT images present bamboo samples after Ag-NPs treatment with 5 cycles of impregnation. The images in Fig. 6 show a transversal (XY) and two longitudinal sections (YZ and XZ) of the biocomposite bamboo in which Ag-NPs aggregates are highlighted with red squares. It can be seen that the deposition was concentrated in the parenchyma only, leaving the metaxylem vessels free of Ag-NPs. The random deposition of the bright metal within the living cells of the bamboo matrix is evident in all represented sections. Moreover, the zoomed-in images reveal that metal deposits are vertically elongated in rectangular or shorter cube-like shapes, similar to the shape of the plant cells. In Fig. 7A and B, 3D μCT images, with front and lateral projections and pseudo colours, allow to identify silver aggregates inside the bamboo sample. In this case, red and yellow represent the bamboo matrix, and silver aggregates, respectively. In Fig. 7C and D the opacity of the bamboo parenchyma was reduced to highlight the silver aggregates within the whole bamboo volume.

Fig. 6 2D μCT images of bamboo after five impregnation cycles with citrate-capped Ag-NPs. In the transversal (top image) and longitudinal (bottom image) sections, red squares highlight silver aggregates in the parenchyma matrix of bamboo.

Fig. 7 3D μCT images of bamboo after five impregnation cycles with citrate-capped Ag-NPs. Red and yellow colors represent, respectively, the bamboo matrix and silver aggregates. (A–B) and (C–D) are front and lateral side projections.

The video available online shows the different physical parts of the biocomposite engineered material. One frame of the video is shown in Fig. 8. The orange colour represents the sclerenchyma and parenchyma tissues; purple represents the metaxylem, phloem and protoxylem vessels; finally, yellow represents the silver aggregates in the parenchyma matrix. Since μCT cannot show individual nanoparticles due to the limitation in the spatial resolution of this technique, this analysis focused on the study of the agglomerates deposited inside the bamboo matrix.

Fig. 8 One frame picture of the on-line video showing different physical parts of the biocomposite engineered material: sclerenchyma and parenchyma tissues (orange); metaxylem, phloem and protoxylem vessels (purple) and silver aggregates (yellow).

It is important to mention that only agglomerates with volume >160 μm³, corresponding to a diameter of approximately 5.4 μm, were analyzed. The histogram in Fig. 9 shows the volume distribution of these Ag-NPs aggregates. About 8900 of the total number of 9949 counted agglomerates are small particles with volume near the detection limit, between 160 and 2500 μm³. Higher presence of silver aggregates with volume between 160 and 190 000 μm³ was observed preferentially in the parenchyma tissue.

Fig. 9 Volume distribution of Ag-NPs aggregates in bamboo matrix.

Table 7 presents the main statistical parameters of the analyzed agglomerates. Half of the agglomerates have volume below 282 μm³ (median volume). It suggests the presence of a large number of undetected small aggregates with volume below 160 μm³.

Table 7 Volume statistical information of Ag-NPs aggregates population in bamboo matrix

Total number of particles	9449
Bamboo volume analyzed [μm^3]	121.0 × 10^9
Total volume occupied by Ag [μm^3]	1.86 × 10^7
Volume ratio	1.54 × 10^−4
Minimum volume of aggregate [μm^3]	161.41
Maximum volume of aggregate [μm^3]	180 057.79
Median volume [μm^3]	282.48
Mean volume [μm^3]	1967.71
Standard deviation [μm^3]	9437.77

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Finally, the microtomography analysis showed that the aggregates are concentrated in the parenchyma tissue inside the bamboo cells. This observation is still under investigation, but it is well known that the parenchyma, a tissue with living plant cells, is open and connected to the vessels by small apertures with diameter up to 5 μm, to allow inward and outward flows of water, mineral salts and other materials useful to sustain cell life.^1,28 As shown in Fig. 10, colloidal silver nanoparticles might flow through the vascular bundle into the living cell during the impregnation process.

Fig. 10 SEM image of the longitudinal section of a bamboo vascular bundle shows a metaxylem vessel with a diameter of 100 μm immersed in sclerenchyma fibers and holes with diameter up to 5 μm.

This observation supports the evidence of stronger deposition in the inner region of the parenchyma tissue. The negative charge of the citrate organic ligand, useful to stabilize the silver nanoparticles, might play an important role in the NPs distribution inside the living cells of the parenchyma tissue. In this regard other polyelectrolyte and neutral polymers capped Ag-NPs are under study to direct the silver deposition into other elements of the vascular bundle system.

Microbiology test

As shown in Fig. 11, comparing the dry-weight fungal colonies of the control test (without NPs) with the test in presence of NPs, approximately 53% of cell dry-weight decreased in the presence of NP-01, while 47% and 7% of cell growth inhibition were obtained with CO-20 and CO-40, respectively. According to Table 5, all silver NPs used in this work presented the same concentration in the culture media (2 mg mL⁻¹), but their diameter and also their concentration in particles per milliliter are different. Comparing only the physical characteristics of commercial NPs, we see that lower is the diameter (d), higher is the antimicrobial effect of Ag-NPs.

Fig. 11 Histogram of dry-weight fungal colonies after 48 hours of growth in the absence (control) and in the presence of different colloids Ag-NPs: homemade NP-01 and commercials CO-20/60.

In our experiments CO-20 (d = 21.2 nm) presented better antimicrobial effect when compared to CO-40 (d = 38.9 nm) and CO-60 (d = 60.3 nm). Moreover, NP-01 was more efficient compared to CO-20. On the other hand, evaluating particles concentration used in this work, we observed that higher is this parameter (particles per mL) higher is the inhibitory effect. At the same mass element (Ag) concentration of colloids, the solution with the smallest NPs presented the highest particle concentration (NP-01, d = 14.3 ± 3.6 nm and 1.25 × 10¹¹ particles per mL), conversely, the largest NPs diameter presented the lower particle concentration (CO-60, d = 60.3 ± 3.6 nm and 1.66 × 10⁹ particles per mL).

The antimicrobial tests revealed that homemade nanoparticles NP-01 in the media culture presented a particle concentration 3.3 fold higher (1.25 × 10¹¹ particles per mL) than the smallest commercial nanoparticle, CO-20, (3.82 × 10¹⁰ particles per mL). So, we can state that the highest inhibition of Aspergillus niger growth (53% with 2.00 ppm) is due to both reasons: the smaller size diameter and the higher number of NPs able to inhibit fungus growth. None of the reference papers mentioned particle concentration (particles per mL) to establish the effect of silver nanoparticles on microbial cell growth. All of them compare particles size, shape and concentration of mass element (Ag) concentration (mg mL⁻¹ or ppm).^8,69–71 The different behavior found for CO-60 related to an increase of cell growth, compared to the control test, can be attributed to the highest particles size diameter that contributes negatively to the inhibitory effect^11,72 and to the lowest number of particles per milliliter in the media culture. Thus, the number of particles with CO-60 was not enough to inhibit properly the cell growth.

Besides, in the absence of the inhibitory effect, citrate can be metabolized and used as additional carbon source for ATP production and additional cell growth.^73,74 It is worth mentioning that the mechanism of ATP production is the same in both bacteria and fungi. Literature reports mention that Ag-NPs effects on cells may involve different mechanisms: alteration of membrane-bound enzymes function, such as those in the respiratory chain,^73,74 loss of DNA replication ability that hinder the expression of many proteins and enzymes essential for energy production (ATP).^75,76 All these mechanisms are related to Ag⁺ release from Ag-NPs directly involved in metal ion–enzymes interactions, or in tiol-binding of tiolated protein to the metal silver surface. Both can modify the protein structure and consequently its biological function.

Finally, as NP-01 was the most effective, it was used for the impregnation bamboo test specimens. Pure bamboo and engineered biocomposite Ag-NPs/bamboo were exposed to air and ambient humidity (about 70–80%) during summer time in Rio de Janeiro, Brazil. All untreated samples showed the formation of fungal colonies, while those treated with Ag-NPs were preserved and protected from the fungi degradation. The optical microscope picture in Fig. 12 shows the proliferation of fungi on the bamboo surface without Ag-NPs treatment (A), while the specimen with Ag-NPs treatment (B) is free of fungal colonies.

Fig. 12 Optical microscope images of bamboo specimens: (A) without Ag-NPs treatment and (B) with Ag-NPs impregnation (NP-01). On left side the natural bamboo shows formation of fungal hyphae, while the engineered biocomposite of bamboo with Ag-NPs is free of fungi.

Conclusions

To our best knowledge, this is the first investigation in which X-ray microtomography analysis is used to study an engineered biocomposite metal-bamboo matrix (Ag-NP/Bamboo). Silver nanoparticles were synthesized, characterized and used to fill up bamboo specimen with the objective to protect it against fungi attack. Qualitative and quantitative analyses of Ag-NPs agglomerates population inside the bamboo matrix using 3D μCT images showed an important population of agglomerates with volumes between 160 and 190000 μm³, mainly deposited on the parenchyma tissue.

From the antimicrobial tests, we state that highest inhibition of Aspergillus niger growth, obtained with NP-01 (53% with 2 mg mL⁻¹), is due to both reasons: the smaller size diameter (14.3 ± 3.6 nm), and the higher number of NPs (1.25 × 10¹¹ particles per mL) able to inhibit fungus proliferation, when compared with the commercial NPs.

The results show that homemade silver colloidal solution is an effective nano-filler agent with antifungal activity able to improve the bamboo durability. The engineered biocomposite bamboo exposed to air and humidity during 5 months was free of fungal colonies, while, all untreated bamboo specimens presented a biological degradation. We believe that the use of NPs silver colloids will contribute to prevent fungal proliferation and microbiological activity for future industrial applications of engineered bamboo and wood. Our conservation method can be useful to preserve historical wooden artefact or handcraft made on bamboo.

Future studies will be focused on the selective distribution of polymer capped Ag-NPs into bamboo vascular bundles, as well as the variation of the metal volume deposition as a function of the number of impregnation cycles. Besides, the effect of different charged surfactant-capped Ag-NPs on their dispersion and their impact on bamboo degradation, will be evaluated. Pre-chemical treatments of bamboo fibers could directly interfere with adhesion of the nanoparticles and improve the impregnation of the Ag-NPs inside the biocomposite matrix.

Acknowledgements

The authors thank CNPQ for the approval of the Project: 458302/2013-9, CAPES and FAPERJ for their financial support and the scholarship of the under graduate student (E. L. L.), master student (R. S. M.) and post-doctoral researchers (S. M. L. K. and E. C. R.). The authors thank Rafael C. C. Rocha and Jefferson R. Souza for technical assistance during ICP-MS analysis.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: TSEM image of Ag-NPs. See DOI: 10.1039/c6ra12516f

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