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Influence of Surface Silanization on the Physicochemical Stability of Silver Nanocoatings: A Large Length-Scale Assessment Victor T. Noronha, Francisco Assis Sousa, Antonio Gomes Souza Filho, Cristiane Aparecida Silva, Francisco Afranio Cunha, Hyun Koo, Pierre B.A. Fechine, and Amauri Jardim Paula J. Phys. Chem. C, Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 4, 2017

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The Journal of Physical Chemistry

Influence of Surface Silanization on the Physicochemical Stability of Silver Nanocoatings: A Large Length-Scale Assessment Victor T. Noronha,a,‡ Francisco A. Sousa,a,‡ Antonio G. Souza Filho,b Cristiane A. Silva,c Francisco A. Cunha,d Hyun Koo,e,f Pierre B. A. Fechine,d Amauri J. Paulaa,*

a

Solid-Biological Interface Group (SolBIN), Departamento de Física, Universidade

Federal do Ceará, P.O. Box 6030, 60455-900, Fortaleza-CE, Brazil b

Laboratory of Raman Spectroscopy, Departamento de Física, Universidade Federal

do Ceará, Fortaleza-CE, Brazil c

Laboratório Nacional de Nanotecnologia (LNNano), Centro Nacional de Pesquisa em

Energia e Materiais - CNPEM, Campinas-SP, Brazil d

Group of Chemistry of Advanced Materials (GQMAT)- Departamento de Química

Analítica e Físico-Química, Universidade Federal do Ceará, Fortaleza-CE, Brazil e

Biofilm Research Lab, Levy Center for Oral Health, and fDepartment of

Orthodontics and Divisions of Pediatric Dentistry and Community Oral Health, School of Dental Medicine, University of Pennsylvania, Philadelphia-PA, United States ‡

Contributed equally.

Corresponding Author * Phone.: +55 85 3366 9270; email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT: We synthesized sub-100 nanometer “biogenic” protein-capped silver nanoparticles (bio-AgNPs) from a yeast-extract (Rhodotorula glutinis) and assessed the chemical stability of their coatings formed on various silane-modified substrates up to the millimeter length-scale. Large-field (LF) X-ray imaging was used for scanning AgNPs-coated substrates (5 x 5 mm) after they were immersed in physiological PBS for hours. Striking differences were found in the amount and the structural organization of bio-AgNPs in the coatings depending on the type of surface silanization. The relative amount of bio-AgNPs in the coatings increases due to the assembly of enlarged (8-30 µm2 in area) and chemically stable agglomerates of bioAgNPs formed on a multilayered aminosilane film (avg. 230 nm-thickness), which was generated with the use of 3-aminopropyltrimethoxysilane (APTMS). In contrast, substantially less bio-AgNPs was found on the hydrophobic silane film generated with the use of trimethoxyphenylsilane (TMPS), and on the films generated with 3trihydroxysilyl-propyl-methylphosphonate (THSMP) and 3-mercaptopropyl trimethoxysilane (MPTMS). As silver nanocoatings formed on APTMS films have a remarkable stability against nanoparticle lixiviation/detachment, this architecture has a great potential in several biotechnological applications that require resistant coatings of nanoparticle for acting in physiological medium/buffers and biological fluids that contain high ionic strength, especially for catalysis, sensors and as antimicrobials surfaces.


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INTRODUCTION Among the biogenic processes for the production of AgNPs, the fungi-mediated synthesis of AgNPs is simple, cost-effective, and present high yields.1,2 Fungi cultures, when compared to bacteria, have simple nutrition requirements, produce a large amount of biomass and are easily handled. In addition, most of fungi used for AgNPs production are non-pathogenic for humans.3 From these considerations, in a screening of soil fungi able to mediate the synthesis of metallic nanoparticles, we have discovered the potential of yeast Rhodotorula glutinis (Rg). This commercially important pigmented yeast (largely used in food industry) can be found and isolated from different natural sources such as soil, water, wood and fruits.4,5 Besides producing β-carothen, γ-carothen, torulen and lycopene,6 these yeasts have also a great potential in the biodiesel industry, with the capacity to convert the main biodiesel industry byproduct glycerin in carotenoids.7 Considering the physicochemical characteristics of the biogenic silver nanoparticles (bio-AgNPs) produced from R. glutinis, these nanoparticles could find many applications as films or coatings over solid surfaces such as metals, ceramic, glasses and polymers, in order to prevent bacterial and fungal contamination or to act as sensors.8–15 The advantages of their use might be related with the very nature of their production method. The bio-AgNPs nucleation→growth→colloidal stabilization steps are driven by interaction mechanisms largely ruled by proteins present in the bacterial/fungal extract or filtrate.9,16,17 However, the initial step of nucleation that involves a bio-reduction of Ag+ can be performed by biomacromolecules other than proteins, such as polysaccharides, amino acids and vitamins.18 At the end of the kinetic process, a protein corona phase that is stably adsorbed on the bio-AgNPs provides a long-term colloidal stability. This protein corona comprises a molecular


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capping made of biomacromolecules, including a myriad of proteins from the fungi extract.19,20 Furthermore, the chemical groups present in the protein corona may lead to the manifestation of several non-covalent chemical interactions (e.g. electrostatic, hydrophobic, hydrogen bonds and van der Waals) between the nanoparticles and solid surfaces.8,21–23 Even so, there are very few reports on the rational design of silver nanocoatings from the use of biogenic AgNPs.24–26 On the other hand, there are several studies in the literature on the formation of coatings from “synthetic” metallic nanoparticles (e.g. Ag, Au and TiO2) and their dependence on the chemical functionalization of solid surfaces upon which the nanoparticles are attached.27–32 Many report on the use of electrostatic interaction between the nanoparticles and surface (i.e. +/− or −/+) for increasing both the nanoparticle attachment and also the chemical stability of the coating/film formed. However, the characterization of these coatings/films in currently available studies is limited to small length scales (from nanometers to microns), commonly achieved through imaging techniques such as electron microscopy performed at high magnifications.8,9,11,33–41 Therefore, it is difficult to correlate the coating properties manifested at large length scales (e.g. antimicrobial, catalytic), which result from complex mechanisms of agglomeration, self-organization and physico-chemical stabilization of nanoparticles, with the critical design parameters manipulated at the nanoscale (i.e. size, shape, surface chemistry, flexibility/rigidity, architecture and elemental composition),42 which define the nanoparticles and the solid surface characteristics. Herein, the relationship between the surface microchemistry of silane-modified solid surfaces and the morphology/structure/dimensionality aspects of the resulting NPs coatings formed on SiO2/Si substrates was studied using a newly developed bio-


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AgNP produced from the extract of yeast Rhodotorula glutinis. The surface conjugation with silanes is a widely used method for the modification of oxide surfaces and is suitable for controlling the surface microchemical environment in terms of reactivity, charge and hydrophobicity.23,43–50 Consequently, silanization reactions have not only several technological applications but also provide a platform for fundamental studies in which the physicochemical features of the surface must be precisely engineered. Although there are very good perspectives for the use surface silanization to drive the formation of silver nanocoatings (from AgNPs), very few studies applied this approach by using a few types of organosilanes.51–53 In particular, the effect of the surface silanization on the physicochemical stability of silver nanocoatings has never been assessed either in small or large length-scales. In this context, the formation and stability of bio-AgNPs coatings were assessed on solid surfaces with positive and negative charges, and also with varied hydrophobicity and topography, by using large-field (LF) X-ray imaging in an scanning electron microscope (SEM) coupled to X-ray energy-dispersive spectroscopy (EDS). In this analytical approach, the sample surface is scanned at multiple length scales varying nanometers to millimeters.54 Information at this scale range can be provided with electron microscopy due to its high resolution at small length scales (i.e. below 1 nm). Furthermore, the X-ray originated from the electron beam-matter interaction is a very “clean” signal, with very few artifacts after sequential scanning of subjacent areas of a sample surface up to areas of centimeters, as recently shown by our group.54 In this sense, the coatings characteristics such as the coating homogeneity, amount of bio-AgNPs, and the nanoparticles-agglomerates distribution could be determined by computational analysis of the large-field images, i.e., assemblies of hundreds of adjacent electron micrographs and X-ray elemental


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maps acquired in the SEM. In addition, the silane films characteristics at the millimeter scale were also assessed. From this approach, we determined the optimum conditions for the formation of chemically stable silver nanocoatings, formed on thick silane films (> 100 nm) based on aminopropyl organosilanes. The resulting nanocoating containing a high amount of attached AgNPs is suitable for biotechnological applications that require catalytic, sensing and antimicrobial interfacial action in physiological medium/buffers and biological fluids that contain high ionic strength.

METHODS Synthesis of bio-AgNPs. We have screened several soil fungi for biogenic properties and have discovered the yeast Rhodotorula glutinis (R. glutinis) with the ability to mediate the synthesis of metallic nanoparticles. After isolation and identification from soil samples collected at the Federal University of Ceara (Pici campus: 3°44′20.832″S, 38°34′12.483″W), R. glutinis was grown in a 500 mL Erlenmeyer flasks with 200 mL of MGYP medium (malt extract, glucose, yeast extract and peptone) at 25 °C for 72 h. The samples were centrifuged at 4000 rcf for 10 min after fungal biomass growth, and washed three times to eliminate culture medium residues. A wet mass of 5.0 g of yeast was transferred into 100 mL of deionized water, and incubated at 25 °C for 48 h without stirring. The yeast suspension was then filtered with 0.45 µm polyvinylidene difluoride (PVDF) membranes. Finally, silver nitrate (AgNO3) was added to the filtrate in order to achieve a concentration of 1.0 mmol L–1 of Ag+, and the mixture was incubated at 25 °C for 168 h (7 days). Confirmation of AgNPs formation was attained with a characteristic plasmonic absorption band detected through UV-Vis


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spectrophotometry. Purification of the bio-AgNPs was performed by: (i) centrifuging the mixture at 10000 rcf for 20 min; (ii) resuspending the pellet, (iii) washing it three times with deionized water, and finally (iv) resuspending it in deionized water. The final concentration of the bio-AgNPs stock suspension was determined through inductively coupled plasma spectrometry (ICP) and then diluted to 0.05 mg mL–1. Production of bio-AgNPs Coatings. Precut 5 x 5 mm pieces of flat (root mean square roughness - RMS - of 0.26 nm) raw silicon substrates ( orientation; SiO2 layer < 5 nm; namely sample “Si”) were cleaned by sonication in acetone for 5 min at high frequency (40 kHz), and then thoroughly rinsed with deionized water produced by a Direct-Q® 3 UV system (Millipore-USA). After cleaning, the substrates were etched by immersion in 40 mL of a 1.0 mol L−1 KOH solution for 15 min at 60 ºC under magnetic stirring. Then, the substrates were thoroughly rinsed with deionized water, sonicated in 40 mL of deionized water for 5 min (40 kHz), and finally dried at room temperature. The resulting substrates (after etching with KOH, samples “Si-ETC”) had their topography, hydrophobicity and the surface chemistry slightly modified with the formation of craters and small pyramids (see Table 1 and Figure S1 in the Supporting Information). The surface silanol groups (Si–OH) were used as anchoring sites for the formation of an organosilane coating on the surface, in order to precisely modify the surface microchemical environment. In this sense, three coatings (TMPS, APTMS and THSPMP) with rather distinct physicochemical properties were generated. TMPS provides a surface with hydrophobic character; APTMS induces the formation of positively charged surfaces at neutral-acidic pH (due to the protonation of exposed primary amines); and THSPMP induces the formation of negatively charged surfaces due to the presence of deprotonated phosphonate groups (at neutral-alkaline


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pH).23,44,46,55,56 In order to form the organosilane films of APTMS and TMPS on the SiO2/Si substrates (Si-ETC), they were immersed in an upright position, held by tweezers, in 35 mL of anhydrous toluene containing 700 µL of each mentioned organosilane, and allowed to react for 60 min under magnetic stirring at 80 ºC. For generating the THSPMP film, sample Si-ETC was immersed in 35 mL of absolute ethanol containing 1.4 mL of the respective organosilane, at 60 ºC, for 60 min under stirring. Immediately after removing the substrates from the organosilane solution, a 10 µL drop of the silane solution was dispensed on each substrate and they were left to dry on a glass plate at 25 ºC, which was further heated in a rate of ~10 ºC min–1, up to 100 ºC, and kept at this temperature for 4 h. On the cleaned raw silicon substrate (sample Si), on the etched Si (sample SiETC), and on the organosilane-coated SiO2/Si substrates (samples Si-TMPS, SiAPTMS and Si-THSPMP), three sequential aliquots of 50 µL of a bio-AgNPs suspension were carefully dropped, and the substrate was left to dry on a Petri dish over a heating plate at 50 ºC between the sequential dropping procedures. The bioAgNPs suspension used for sample Si-APTMS was pH 4, for sample Si-THSPMP was pH 10, and for all other samples was pH 7 (Si, Si-ETC and Si-TMPS). The suspensions with varied pH were obtained by mixing 1.0 mL of the stock bio-AgNPs suspension (0.05 mg mL–1) with 200 µL of each of the following buffers: potassium biphthalate/sodium hydroxide for pH 10, disodium phosphate/monobasic potassium phosphate for pH 7, and boric acid/potassium chloride for pH 4. Different pHs were used in order to increase protonation/deprotonation of surface amine and phosphonate groups in samples Si-APTMS and Si-THSPMP, respectively. Finally, chemically stable bio-AgNPs coatings were obtained by immersing the substrates in an upright position into 5 mL of a 1x-diluted PBS solution (10 mmol L−1 of a phosphate buffer,


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2.7 mmol L−1 of potassium chloride and 137 mmol L−1 of sodium chloride; pH 7.4), and then kindly rinsed with 100 mL of ultrapure type 1 water in order to remove loosely bonded nanoparticles and salts. Most of the initial coating is lixiviated during this washing process, thus only tightly adhered bio-AgNPs remained on the various SiO2/Si substrates. Among several liquid media tested for this washing step, including those of physiological conditions (e.g. water, saline solution), the PBS solution was the most efficient for removing loosely bonded nanoparticles on the surface, due to its high ionic strength. Further information on the materials and reagents used is provided in the Supporting Information. LF X-Ray Imaging and Computational Analysis of LF Images. In the total area of the silicon substrate (5 x 5 mm), just a square area of 3.6 x 3.6 mm in the middle portion of the substrate was considered in the LF images. The substrate peripheral regions (a border with 0.7 mm from each side of the square silicon substrate) were excluded from the LF image due to artifacts introduced in both the silicon etching and the silane film formation. Furthermore, as the handling of the substrate was carried out with tweezers holding its borders, artifacts are introduced at the peripheral regions during these steps. In this way, the initial size of the raw LF images (i.e., elemental maps) was of approximately 10000 x 10000 pixels (96 pixels per inch). After binning and cropping them, the resulting images used for the quantitative assessment of the silane films and the AgNPs coatings had 2465 x 2465 pixels, with each pixel representing 1.4 x 1.4 µm of the substrate surface. The LF image processing was performed on Wolfram Mathematica with scripts generated by our group. More information on the conditions used for large-field scanning and also the SEM setup is provided in the Supporting Information.


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For localizing the AgNPs (distributed as agglomerates on the surface), LF Ag elemental maps were binarized, thus resulting in images where the pixel value “1” indicates the presence of the element silver (i.e. AgNPs) and “0” its absence. In the analysis of the relative amount of AgNPs over the substrate, all pixel values present in Ag elemental maps in grayscale (values varying from 0 to 1; 8-bits/channel; 2465 x 2465 pixels) were summed, thus resulting in the Intensity Counts values. All Ag elemental maps had their image contrast values normalized in regard to Si, which is the standardized element in all samples. The determination of the AgNPs agglomerates was performed for all elements identified in the binarized LF images, which sizes were equal to and larger than 1 pixel (~2 µm2), and their equivalent areas were determined. This determination was performed for at least three elemental maps acquired from at least three different samples, thus resulting in the mean and standard deviations. Precise quantitative morphological information at scales below 1.0 µm2 must be acquired at higher magnifications and by using longer beam dwell times in individual scans. This was not performed in this study since it was sought the morphological analysis of AgNPs agglomerates with sizes at the micrometermillimeter scale. However, it must be mentioned that in the calculation of the relative amount of bio-AgNPs (i.e. nanoparticle surface density), the EDS signal emitted from individual nanoparticles were accounted.

RESULTS AND DISCUSSION Characterization of the Silane Films at the Millimeter Length Scale. The formation of the silane films was assessed through EDS, acquired through a LF electron scanning (see Figure 1). The presence of well-resolved carbon (C Kα: 0.27


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keV), nitrogen (N Kα: 0.39 keV) and phosphorus (P Kα: 2.0 keV) peaks in the cumulative EDS spectra (see Figure 1, first column) indicate the presence of the respective silane films over the substrate: Si-TMPS, Si-APTMS and Si-THSPMP. The oxygen peak (O Kα: 0.52 keV) is related to surface silanol (Si–OH) groups present in sample Si-ETC, as well as the oxygen present in the polysiloxane chains (Si–O–Si) present in all samples. The silicon EDS peak (Si Kα: 1.7 keV) is also shown in spectra for sample Si-THSPMP.


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Figure 1. First column: cumulative large-field X-ray energy-dispersive spectroscopy (LF-EDS) of etched silicon surface (Si-ETC; with a modified topography compared to raw Si), and of silane films formed on the etched silicon surface (sample Si-ETC). Silane films comprise Si-TMPS, Si-APTMS, and Si-THSPMP. The cumulative spectra shown in the first column are the sum of hundreds of spectra individually obtained from a substrate area of ~2 µm2. Elemental maps extracted from LF-EDS are depicted from second to fourth columns, with an area of approximately 3.6 x 3.6 mm. Each pixel in the maps is represented with a colored contrast function (from 0 to 1), which is a normalized scale given as a function of the element peak area (in the EDS spectrum) associated with that pixel. Maps scales are represented in millimeters.

To maximize the cross-sections (and consequently to increase the X-ray signal used for identification of the silane films formed) for carbon, nitrogen and oxygen, the LF-scan was performed with 2 kV of accelerating voltage for samples Si-ETC, SiTMPS and Si-APTMS. For Si-THSPMP, the phosphorus cross-section was maximized using 7 kV. Both the resolution and sensitivity are largely increased in a large-field X-ray imaging since the resulting cumulative data contain signal from more than 300 individual spectra recorded along the scan (individual scans had horizontal and vertical fields of 0.274 mm and 0.188 mm, respectively; 512 x 352 pixels). The same occurs with the signal-to-noise ratio, which is largely increased along a large field scan (see Figure 1, first column). In this way, even trace elements can be identified on flat surfaces through this technique, as recently shown by our group.54 This aspect is evidenced in the LF elemental maps shown in Figure 1, which reveal how the films were formed on the silicon substrate, at the millimeter scale, even when the C, N and P concentrations are very low (

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