Omic strategies in the evaluation of transgenic and non-transgenic soybean plants after cultivation with silver nanoparticles

Orientador: Marco Aurelio Zezzi ArrudaTese (doutorado) - Universidade Estadual de Campinas, Instituto de QuímicaResumo: Inicialmente, todo um estudo acerca da caracterização e quantificação das nanopartículas de prata (AgNP) foi realizado a fim de se avaliar a concentração de prata (Ag) tanto nas AgNP após a síntese, quanto nas suas frações (AgNP-res e Ag-sob). Com esta abordagem, observou-se que aproximadamente 50% da Ag está na forma de nanopartículas (NPs) na solução de AgNP após a síntese, enquanto os outros 50% da Ag está na forma livre iônica. Tais resultados sugerem que, para estudos sobre a aplicação das AgNPs, é necessária a purificação das mesmas via centrifugação e remoção do sobrenadante (Ag-sob), para que apenas a fração AgNP-res, que contém as NPs, seja empregada. Se essa estratégia não for considerada e a solução após a síntese for diretamente utilizada, possivelmente, os efeitos observados em um estudo serão sinérgicos devido à presença das NPs e da Ag livre na solução após a síntese. Após a avaliação das AgNP a serem empregadas neste estudo, foi realizado um cultivo-teste com uma concentração final de 250 mg kg-1 de Ag no substrato. Nestas condições, ambas as plantas não-transgênicas e transgênicas não sobreviveram ao cultivo com o nitrato de Ag (AgNO3), indicando que as plantas interagem de maneira distinta com as diferentes formas de Ag. Por isso, todos os demais cultivos foram realizados com uma concentração final de 50 mg kg-1 de Ag no substrato. Então, um estudo proteômico por 2-D DIGE foi realizado e diversos spots diferenciais foram encontrados entre as plantas de soja NT e T dos grupos controle e as plantas cultivadas com as AgNP e AgNO3. As plantas de soja NT e T apresentaram um comportamento distinto, em termos de spots diferenciais, uma vez que as plantas NT foram mais afetadas pelo cultivo com o AgNO3, enquanto que as plantas T foram mais sensíveis ao tratamento com as AgNPs, inclusive com um incremento significativo (teste de Tukey - p < 0,05) na quantidade total de proteínas nas folhas de soja. Após os resultados obtidos pela técnica de 2-D DIGE, os estudos proteômicos foram finalizados com a identificação, por espectrometria de massas, daquelas proteínas presentes nos spots diferenciais entre os grupos controle e os cultivados com as AgNP e AgNO3. De maneira geral, independente da forma de Ag empregada no cultivo, ambas as variedades de soja, NT e T, estão sob condição de estresse, uma vez que algumas proteínas relacionadas à tal condição foram identificadas, dentre elas a ribulose. Além da ribulose, espécies como a superóxido dismutase, ATP synthase, triosephosphate isomerase, entre outras, foram identificadas dentre os grupos de soja NT e T tratadas, sugerindo que os cultivos com AgNO3 e AgNP, possivelmente, resultaram em uma condição de estresse oxidativo. Adicionalmente, foi realizado um estudo enzimático com a finalidade de ampliar o entendimento do estresse oxidativo causado pelos cultivos com AgNO3 e AgNP observado nos estudos proteômicos. Enzimas como a catalase (CAT) e a superóxido dismutase (SOD), bem como o conteúdo de H2O2 e malondialdeído (MDA) foram avaliados, confirmando que as plantas de soja NT e T cultivadas com as AgNP e AgNO3 estão sob condição de estresse. Todos os resultados obtidos por meio de uma abordagem proteômica e avaliação enzimática sugerem que, de fato, a resposta de uma planta, em termos de ROS, conteúdo e atividade das enzimas antioxidantes, sob condições de estresse oxidativo, é dependente de vários fatores, dentre eles a espécie de planta estudada (no nosso caso, da variedade) e do tipo de metal/metalóide ou NPs empregados durante o cultivo. Com relação à absorção e assimilação de Ag, de maneira geral, as plantas de soja T translocaram uma maior quantidade de Ag em comparação com a soja NT. Considerando os diferentes tratamentos, independente da variedade de soja, o cultivo com AgNP resultou em uma maior taxa de translocação. Considerando o estudo ionômico, diferenças significativas (p ? 0,05) em termos de macro e micronutrientes foram encontradas em todos os compartimentos, principalmente nas raízes. Sendo assim, a presença de Ag, elemento não essencial para a bioquímica das plantas, em suas distintas formas (AgNP e AgNO3) interfere na absorção de alguns nutrientes. Tal fato pode estar relacionado com condições de estresse e consequente formação de clorose nas folhas das plantas, como observado em nosso estudo. Adicionalmente, foi observada a formação de necrose na parte basal do caule das plantas NT e T expostas às AgNP ou AgNO3. Por fim, o emprego de tampões extratores brandos e estratégias hifenadas baseadas em cromatografia líquida acoplada a ambas as técnicas ICP-MS e ESI-Orbitrap foram consideradas na avaliação de complexos metálicos que possam estar alterados nas folhas de soja NT e T devido aos cultivos com AgNP ou AgNO3. Após a obtenção de perfis de SEC-ICP-MS, diferenças em termos de intensidade foram observadas para Mn, Fe, Zn e Cu entre os grupos controle e tratados. Então, das quatro frações encontradas em SEC-ICP-MS, a fração três (F3) foi considerada e analisada por HILIC-ICP-MS em que perfis para Fe, Zn e Cu, previamente monitorados por SEC-ICP-MS, foram também obtidos. Então, a mesma fração F3 foi analisada por HILIC-ESI-Orbitrap e alguns íons com padrões isotópicos para Cu e Zn foram encontrados. Tais íons foram selecionados para futuros experimentos de fragmentação por HILIC-ESI-Orbitrap no modo tandem. Com tais resultados, espera-se identificar as espécies metálicas e, consequentemente, as possíveis vias que possam estar alteradas nas plantas de soja NT e T devido aos cultivos com AgNP e AgNO3Abstract: Initially, a study concerning characterization and quantification of silver nanoparticles (AgNP) was performed to evaluate silver (Ag) concentration in AgNP after the synthesis, as well as in its fractions (AgNP-res and Ag-sup ). According to results, approximately 50% of Ag is in the form of nanoparticles (NPs) in the AgNP solution after the synthesis, while the other 50% of Ag is in the ionic free form. These results suggest that, for studies related to AgNP application, is necessary to purify them by centrifugation and removal of the supernatant (Ag-sup), so that only the AgNP-res fraction containing NPs will be used. If this strategy is not considered and the solution after the synthesis is directly employed, possibly the effects observed in a study will be synergistic due to the presence of both NPs and free Ag in the solution after the synthesis. After the evaluation of AgNP to be used in this study, a cultivation test was first carried out with a final concentration of 250 mg kg-1 of Ag in the substrate. Under these conditions, both non-transgenic (NT) and transgenic (T) soybean plants did not survive to the cultivation with Ag (AgNO3) nitrate, indicating that the plants interacted differently with the different forms of Ag. Then, for a comparative evaluation, cultivations with final concentration of 50 mg kg-1 Ag on the substrate were carried out. Then, a proteomic study by 2-D DIGE was performed and several differentially abundant spots were found between NT and T soybeans from control groups and those plants which grown in the presence of AgNP or AgNO3. NT and T plants showed a different behavior in terms of differential spots, since NT soybeans were more affected by the cultivation with AgNO3, whereas T plants were more sensitive to the treatment with AgNP, including a significant increase (Tukey test - p < 0.05) in the total amount of proteins in soybean leaves. Then, the proteomic evaluation was finalized with the identification, by mass spectrometry, of those proteins present in the differential spots between the control groups and those which were exposed to AgNP or AgNO3. In general, regardless of the Ag form used in the cultivation, both soybean varieties are under stress condition, since some proteins related to such condition have been identified, among them ribulose. In addition to ribulose, species such as superoxide dismutase, ATP synthase, triosephosphate isomerase, among others, were identified in treated NT and T soybean groups, suggesting that the cultivation with AgNP and AgNO3 possibly resulted in an oxidative stress condition. Additionally, an enzymatic study was performed to increase the understanding regarding oxidative stress caused by AgNP and AgNO3 cultivations, previously observed in the proteomic evaluation. Enzymes such as catalase (CAT) and superoxide dismutase (SOD), as well as the content of H2O2 and malondialdehyde (MDA) were assessed, confirming that NT and T plants exposed to AgNP and AgNO3 are under stress conditions. All the results obtained through a proteomic and enzymatic approaches suggest that, in fact, the response of a plant, in terms of reactive oxygen species, content and activity of antioxidant enzymes, under oxidative stress conditions, is dependent on several factors, among them the plant species studied (in our case the variety) and the type of metal/metalloid or NPs used during cultivation. Regarding the uptake and assimilation of Ag, in general, T soybeans translocated a greater amount of Ag when compared to NT plants. Considering the different treatments, regardless of the soybean variety, the cultivation with AgNP resulted in a higher translocation rate. Through ionomic evaluation, significant differences (p ? 0.05) in terms of macro and micronutrients were found in all compartments, especially in the roots. Therefore, the presence of Ag, a non-essential element for the plant biochemistry, in its different forms (AgNP and AgNO3) interferes with the absorption of some nutrients. This fact may be related to stress conditions and consequent formation of chlorosis in the leaves of those treated plants, as observed in our study. Additionally, necrosis formation in the basal part of stems was observed for NT and T plants exposed to AgNP or AgNO3. Finally, the utilization of mild extractor buffers and hyphenated strategies based on liquid chromatography coupled to both ICP-MS and ESI-Orbitrap techniques were considered in the evaluation of metal species that may be altered in NT and T soybean leaves due to the cultivation with AgNP or AgNO3. After obtaining SEC-ICP-MS profiles, changes in the signal intensity were observed for Mn, Fe, Zn and Cu between control and treated groups. Then, from the four fractions found in SEC-ICP-MS, fraction three (F3) was only considered and analyzed by HILIC-ICP-MS. Profiles for Fe, Zn and Cu, previously monitored by SEC-ICP-MS, were also obtained. Then, the same F3 fraction was evaluated by HILIC-ESI-Orbitrap and some ions with isotopic patterns, mainly Cu and Zn, were found. These ions were selected for future HILIC-ESI-Orbitrap fragmentation experiments in tandem mode. With such results, it is expected to identify metal species and, consequently, provide additional information regarding possible pathways that may be altered in NT and T soybean plants due to AgNP and AgNO3 cultivationsDoutoradoQuimica AnaliticaDoutor em Ciências2013/15646-3, 2016/18373-6FAPES

Evaluation of two bioadsorbents for removing paracetamol from aqueous media

The frequent contamination of water resources with drugs comprises one the most important environmental problems. In order to avoid serious disturbances for aquatic life, efficient and economically viable procedures should be developed for removing common pollutants, as paracetamol. From these considerations, the present work evaluated the efficiency of sugar cane bagasse (SCB) and vegetable sponge (VS), two natural adsorbents commonly found in Brazil, for retaining paracetamol molecules dispersed in aqueous solutions. Thus, systems composed of glass columns and peristaltic pumps were optimized and, for pH, the best value was 7.0. After optimisation, adsorption isotherms were built and it was possible to calculate the MACF values for SCB (120.5 \ub5g/g) and VS (37.5 \ub5g/g). Additionally, real matrices of pretreated water, from a municipal plant for water catchment, were enriched with paracetamol at 5 \ub5M and passed through glass columns packed with SCB, VS and activated carbon (AC). The results showed that SCB was more attractive than AC in terms of price and efficiency (60% against 45% adsorption, respectively), while VS was responsible for removing 40% of paracetamol dissolved in the enriched water samples. Thus, the proposed natural adsorbents can be classified as viable materials to remove paracetamol from water used for human consumption

Employment of a metallic furnace hydride generation atomic absorption spectrometry (MF-HG-AAS) method for tin determination : analytical and morphological parameters analysis of metallic atomizer

Orientador: Marco Aurélio Zezzi ArrudaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de QuímicaResumo: Nessa dissertação, otimizou-se um método para a determinação de estanho (Sn) em amostras biológicas empregando a técnica de Espectrometria de Absorção Atômica por Geração de Hidretos e Forno Metálico (MF-HG-AAS, do inglês Metallic Furnace Hydride Generation Atomic Absorption Spectrometry). Foram avaliadas algumas variáveis químicas como o tipo e concentração do carregador/diluente da solução padrão, concentração do redutor tetraidridoborato (-1) de sódio (THB) e concentração de hidróxido de sódio, bem como variáveis físicas do sistema dentre elas a vazão de carregador, proporção de acetileno:ar na chama, volume de solução injetado, vazão de argônio como gás de arraste, vazão de água no nebulizador e área total de furos no tubo metalico Inconel600®. Foi realizado um estudo do efeito de memória observado em condições de chama oxidante nas vazões de 1,5:9; 1,4:9; 1,3:9; 1,4:10; 1,4:8 e 1,5:11 L min de acetileno:ar, respectivamente. Em todas essas proporções de chama diferentes da otimizada (1,5:10 L min acetileno:ar) há efeito de memória, o que é extremamente indesejável. Após a otimização do sistema, foram realizados testes de exatidão e precisão do mesmo com os materiais PACS-2 (sedimento) e SRM 1643e (amostra de água) em que, em ambos, foi possível recuperar o Sn adicionado obtendo limites de detecção (LD) de 7,1 mg kg e 7,6 mg L, respectivamente. Um estudo com concomitantes foi realizado para avaliar se algum dos elementos em questão poderia interferir na determinação do Sn. Foram estudados como possíveis concomitantes o cobre, chumbo e zinco em três níveis diferentes. Somente o cobre, nas razões de 1:10 e 1:20 (Sn:concomitante), interferiu na detecção de Sn. Por fim, a morfologia do atomizador foi avaliada por meio da técnica de Microscopia Eletrônica de Varredura (SEM, do inglês Scanning Electron Microscopy). Mesmo após a realização de todos os experimentos envolvendo a otimização do sistema e análise das amostras, os principais constituintes do tubo metálico Inconel600® (ferro, níquel e cromo) permaneceram homogeneamente distribuídos, indicando que esses metais podem não participar da rota de atomização do Sn. Além disso, houve formação de óxidos no atomizador que, juntamente com o uso de uma chama oxidante, sugerem uma rota de atomização do Sn via formação de óxidos. Considerando os LD, bem como a exatidão e precisão por meio da recuperação de Sn obtida nas amostras, constata-se a potencialidade da MF-HG-AAS frente a outras técnicas empregadas na determinação de SnAbstract: In this work, a Metallic Furnace Hydride Generation Atomic Absorption Spectrometry (MF-HG-AAS) was optimized for tin (Sn) determination in biological samples. Chemical variables, such as the type and carrier concentration, the sodium tetrahydrideborate (-1) (THB), and the sodium hydroxide concentration, as well as physical variables, such as carrier flow-rate, ratio of acetylene and air in the flame, injection volume, argon flow-rate as carrier of stannane, water flow-rate in nebulizer and total hole area of a Inconel600® metallic furnace were evaluated. A study of a memory effect observed in oxidant flame conditions such as 1.5:9; 1.4:9; 1.3:9; 1.4:10; 1.4:8 e 1.5:11 L minof acetylene:air, respectively, was realized. In any other flame flow-rate, which the optimized flame ratio (1.5:10 L min acetylene:air) was observed a memory effect, which is very undesirable. After optimizing of the system, test of accuracy and precision were realized with the PACS-2 (sediment) and SRM 1643e (water sample) materials wherein, in both, a recovery of the Sn added to the samples was possible with limits of detection (LOD) as 7,1 mg kg and 7,6 mg L, respectively. A concomitant study was carried out for checking the interferences in the Sn determination. Copper, lead and zinc at three different levels were studied as possible concomitants. Only copper, at 1:10 and 1:20 (Sn:concomitant) ratios interfered in the determination of Sn. Finally, the morphology of the atomizer employed was evaluated through Scanning Electron Microscopy (SEM) technique. Even after carrying out all experiments involved in the optimization of the system and in the sample analysis, the main constituents of Inconel600® metallic furnace (iron, nickel and chromium) remained homogeneously distributed, indicating which these elements may not participate of the Sn atomization route. Moreover, the oxide formation in the atomizer was detected, which, together with an oxidant flame used, suggests the Sn atomization route via oxides formation. Considering the LOD, as well the accuracy and precision through the Sn recovery in the samples, the MF-HG-AAS potentially is well pointed out when it is compared to other techniques employed for Sn determinationMestradoQuimica AnaliticaMestre em Químic

Optimization Of A Hydride Generation Metallic Furnace Atomic Absorption Spectrometry (hg-mf-aas) Method For Tin Determination: Analytical And Morphological Parameters Of A Metallic Atomizer.

The present work describes a metallic tube as hydride atomizer for atomic absorption spectrometry. Its performance is evaluated through tin determination, and the accuracy of the method assessed through the analysis of sediment and water samples. Some chemical parameters (referring to the generation of the hydride) such as acid, NaOH and THB concentrations, as well as physical parameters (referring to the transport of the hydride) such as carrier, acetylene, air flow-rates, flame composition, coil length, tube hole area, among others, are evaluated for optimization of the method. Scanning electron microscopy is used for evaluating morphological parameters in both new and used (after 150 h) tube atomizers. The method presents linear Sn concentration from 50 to 1000 µg L(-1) (r>0.9995; n=3) and the analytical frequency of ca. 40 h(-1). The limit of detection (LOD) is 7.1 µg L(-1) and the precision, expressed as RSD is less than 4% (200 µg L(-1); n=10). The accuracy is evaluated through reference materials, and the results are similar at 95% confidence level according to the t-test.117543-

The Importance Of Evaluating The Real Metal Concentration In Nanoparticles Post-synthesis For Their Applications: A Case-study Using Silver Nanoparticles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)To determine whether the effect observed in a study is related to the nanoparticle only or to their synergic effect with the "free" metal ions, the real concentration of silver (104 +/- 8 and 100 +/- 2 mg L-1) after AgNP synthesis is obtained through ICP-MS and ICP OES in the solution after the AgNP synthesis and in different fractions after centrifugation (at 8100g for 40 min). From the resuspension of the AgNP contained in the solution (AgNP-total) after synthesis (AgNP-res), concentrations of 49 +/- 3 and 51 3 mg L-1 are found and concentrations of 50 +/- 7 and 47 +/- 2 mg L-1 in the supernatant (Ag-sup) are found using ICP-MS and ICP OES respectively. The characterization of AgNP-total, AgNP-res and Ag-sup is performed by HRTEM and UV-vis, corroborating the results in terms of Ag determination, and indicates that half of the total silver concentration is in the AgNP form and that the other half is in the "free" silver form. The results of the stability test of the NPs indicate a 7% decrease in Ag as NP three months after its synthesis. (C) 2015 Elsevier B.V. All rights reserved.146795800Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Bipolar Disorder: Recent Advances And Future Trends In Bioanalytical Developments For Biomarker Discovery.

In this manuscript we briefly describe bipolar disorder (a depressive and manic mental disease), its classification, its effects on the patient, which sometimes include suicidal tendencies, and the drugs used for treatment. We also address the status quo with regard to diagnosis of bipolar disorder and recent advances in bioanalytical approaches for biomarker discovery. These approaches focus on blood samples (serum and plasma) and proteins as the main biomarker targets, and use various strategies for protein depletion. Strategies include use of commercially available kits or other homemade strategies and use of classical proteomics methods for protein identification based on bottom-up or top-down approaches, which used SELDI, ESI, or MALDI as sources for mass spectrometry, and up-to-date mass analyzers, for example Orbitrap. We also discuss some future objectives for treatment of this disorder and possible directions for the correct diagnosis of this still-unclear mental illness.407661-

Evaluation of two bioadsorbents for removing paracetamol from aqueous media

The frequent contamination of water resources with drugs comprises one the most important environmental problems. In order to avoid serious disturbances for aquatic life, efficient and economically viable procedures should be developed for removing common pollutants, as paracetamol. From these considerations, the present work evaluated the efficiency of sugar cane bagasse (SCB) and vegetable sponge (VS), two natural adsorbents commonly found in Brazil, for retaining paracetamol molecules dispersed in aqueous solutions. Thus, systems composed of glass columns and peristaltic pumps were optimized and, for pH, the best value was 7.0. After optimisation, adsorption isotherms were built and it was possible to calculate the MACF values for SCB (120.5 µg/g) and VS (37.5 µg/g). Additionally, real matrices of pretreated water, from a municipal plant for water catchment, were enriched with paracetamol at 5 µM and passed through glass columns packed with SCB, VS and activated carbon (AC). The results showed that SCB was more attractive than AC in terms of price and efficiency (60% against 45% adsorption, respectively), while VS was responsible for removing 40% of paracetamol dissolved in the enriched water samples. Thus, the proposed natural adsorbents can be classified as viable materials to remove paracetamol from water used for human consumption

Evaluation of some effects on plant metabolism through proteins and enzymes in transgenic and non-transgenic soybeans after cultivation with silver nanoparticles

To evaluate the effects of silver nanoparticles (AgNP) exposition, transgenic (through gene cp4EPSPS) and non-isogenic non-transgenic soybeans were cultivated in the presence or absence of AgNP or silver nitrate (AgNO3) at 50 mg/kg of silver. Physiological aspects of the plants including mass production and development of roots, proteomics such as protein amount and differential proteins, enzymes and lipid peroxidation were determined after exposition. The mass production of non-transgenic plants treated with AgNP or AgNO3 was decreased by 25 and 19%, respectively, on their mass based, while for transgenic soybean this effect was observed for AgNP cultivation only. Fifty-nine proteins were identified from the differentially abundant spots by two-dimensional difference gel electrophoresis and nano-electrospray ionization liquid chromatography coupled tandem mass spectrometry. Identified species as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), ATP synthase, superoxide dismutase (SOD), related to plant metabolism were less abundant for the cultivation with either AgNP and AgNO3 than the control. Finally, this work demonstrated significant correlation as evidenced by changes in lipid peroxidation content and catalase activity, which were a result of exposure to either AgNP or AgNO3 cultivations. Further, necrotic areas in the basal part of the stems and damage or chlorotic areas were found in the leaves. Significance: Once nanoparticles have been employed for several applications in recent years and they can be released in the environmental matrices, this study highlights proteomic and enzymatic alterations in transgenic and non-transgenic soybeans, an important crop, after cultivation with silver nanoparticles. Such strategy employing proteomic and enzymatic approaches to evaluate soybeans exposed to silver nanoparticles has not yet been reported. Therefore, the results obtained in this study can expand the information concerning the effects of silver nanoparticles in soybean plants19188106CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP401170/2016-0sem informação2016/07384-7; 2013/15646-

The importance of evaluating the real metal concentration in nanoparticles post-synthesis for their applications: A case-study using silver nanoparticles

To determine whether the effect observed in a study is related to the nanoparticle only or to their synergic effect with the "free" metal ions, the real concentration of silver (104 +/- 8 and 100 +/- 2 mg L-1) after AgNP synthesis is obtained through ICP-MS and ICP OES in the solution after the AgNP synthesis and in different fractions after centrifugation (at 8100g for 40 min). From the resuspension of the AgNP contained in the solution (AgNP-total) after synthesis (AgNP-res), concentrations of 49 +/- 3 and 51 3 mg L-1 are found and concentrations of 50 +/- 7 and 47 +/- 2 mg L-1 in the supernatant (Ag-sup) are found using ICP-MS and ICP OES respectively. The characterization of AgNP-total, AgNP-res and Ag-sup is performed by HRTEM and UV-vis, corroborating the results in terms of Ag determination, and indicates that half of the total silver concentration is in the AgNP form and that the other half is in the "free" silver form. The results of the stability test of the NPs indicate a 7% decrease in Ag as NP three months after its synthesis. (C) 2015 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Spectrometry, Sample Preparation and Mechanization Group, Institute of Chemistry, University of Campinas – Unicamp, P.O. Box 6154, Campinas, SP 13083-970, BrazilNational Institute of Science and Technology for Bioanalytics, Institute of Chemistry, University of Campinas – Unicamp, P.O. Box 6154, Campinas, SP 13083-970, BrazilInstitute of Science and Technology, Federal University of São Paulo, 12231-280 São José dos Campos-SP, BrazilFunctional Materials Laboratory, Institute of Chemistry, University of Campinas – Unicamp, P.O. Box 6154, Campinas, SP 13083-970, BrazilInstitute of Science and Technology, Federal University of São Paulo, 12231-280 São José dos Campos-SP, BrazilWeb of Scienc