an investigation of major factors affecting metal

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NANOTECHNOLOGY SCIENCE AND TECHNOLOGY

METAL NANOPARTICLES PROPERTIES, SYNTHESIS AND APPLICATIONS

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NANOTECHNOLOGY SCIENCE AND TECHNOLOGY

METAL NANOPARTICLES PROPERTIES, SYNTHESIS AND APPLICATIONS

YESENIA SAYLOR AND

VASILIKI IRBY EDITORS


Copyright © 2018 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website and locate the “Get Permission” button below the title description. This button is linked directly to the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search by title, ISBN, or ISSN. For further questions about using the service on copyright.com, please contact: Copyright Clearance Center Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: [email protected].

NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data ISBN:H%RRN

Published by Nova Science Publishers, Inc. † New York


CONTENTS Preface Chapter 1

Chapter 2

Chapter 3

Chapter 4

vii An Investigation of Major Factors Affecting Metal Nanoparticle Morphology in Island Films N. B. Leonov, V. A. Polishchuk and T. A. Vartanyan Silver Nanoparticles in Silicate Glasses: Synthesis, Modification and Destruction Nikolay Nikonorov and Alexander Sidorov Synthesis, Optical Properties and Surface Enhanced Raman Scattering Applications of Noble Metal Nanoparticles Embedded in Polymers Jai Prakash, Simona Badilescu, Rajeev Gupta, Hendrik Swart, Muthukumaran Packirisamy and Shuhui Sun Nanoparticles of Various Transition Metals and Their Applications as Antimicrobial Agents Armen Trchounian, Lilit Gabrielyan and Narine Mnatsakanyan


1

61

125

161

vi Chapter 5

Chapter 6

Chapter 7

Contents Metal Nanoparticles-Polymer Composites and Their Fuel Cell Applications Mohamed R. Berber and Inas H. Hafez

211

The Effect of Gold and Silver Nanoparticles on Plant Growth and Development Lev A. Dykman and Sergei Y. Shchyogolev

263

Application of Electrochemically Generated Platinum and Silver Nanoparticles on Conducting Polymers to the Determination of Antioxidant Capacity María Pilar Rivas, Rafael Estevez Brito and José Miguel Rodríguez Mellado

Index

301

327


PREFACE Metal nanoparticles that have already found numerous applications in science and technology may be obtained in different ways. In the opening study included in Metal Nanoparticles: Properties, Synthesis and Applications, several factors affecting metal nanoparticle morphology in island films are determined. The formation of islet metal films during their deposition in the process of thermal evaporation on a dielectric substrate and their spontaneous changes at room temperature are also described. Following this, the authors present their experimental results on synthesis, modification and destruction of silver nanoparticles in the bulk and surface of silicate glasses. Special attention is paid to the effects of laser and electron-beam irradiation on the above-mentioned processes. The authors go on to review the synthesis of embedded noble metal nanoparticles and their optical properties and potential applications in surface enhanced Raman scattering. The optical properties of NMNPs due to the localized surface plasmon resonance and the enhanced local electromagnetic field which govern their SERS activity will be discussed along with their fundamental mechanisms. The effects of zinc, titanium, copper and oxide thin films with nanostructured surfaces and iron oxide and silver nanoparticles on Enterococcus hirae and Escherichia coli growth and membrane activity are also presented and discussed. The authors suggest that different types of


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Yesenia Saylor and Vasiliki Irby

metal nanoparticles could be applied in medicine due to their antimicrobial activity, efficiency in anti-inflammatory effects and potential in anticancer therapy. Next, the development of platinum-metal nanoparticles and their coreshell structures is discussed. The authors analyze in detail the polymeric composite of the metal nanoparticles on the surface of different carbon support materials (e.g., carbon nanotubes, graphene, carbon-nanospheres, mesoporous carbon, and carbon nanofibers), showing the synergetic effects on the active surface area of platinum metal catalysts. In one review, the past decade’s data regarding the effects of nanoparticles of noble metals on higher plants are considered, as well as possible nanoparticle phytotoxicity. The review discusses the various effects that gold and silver nanoparticles can have on the state, growth, and productivity of plants. Published evidence, although incomplete and contradictory, indicates that metal nanoparticles can have both positive and negative effects on plants. Lastly, glassy carbon electrodes modified with polyphenazine conducting polymers and metallic nanoparticles were used to study the interaction between H2O2 and ascorbic acid. The antioxidant capacity of ascorbic acid measured from this interaction agreed with that reported using non-electrochemical techniques. Chapter 1 - Metal nanoparticles, which are now widely used in various scientific and technical areas, may be obtained in different ways. Physical vapor deposition is one of the most common ways for metal nanoparticle production. It is simple to implement and leads to the manifold of nanostructures depending on the control parameters, such as the substrate material and its temperature, deposition rate, and the amount of the deposited metal. Nevertheless, application of island films requires even more control over the properties of the nanoparticles arrays. To meet these demands, post processing of the prepared nanostructure may be successfully employed. In this study several factors affecting metal nanoparticle morphology in island films are determined. The equipment design allowed us to see changes in nanoparticle morphology both during the film deposition and upon their heating without breaking the vacuum conditions. Much more control over


Preface

ix

the size and shape distribution of silver nanoparticles may be performed with the help of intense laser illumination. In this case, instead of violent heating of the entire particles’ ensemble selective heating of those nanoparticles that possess plasmon excitations in resonance with the incident radiation, is achieved. The influence of organic solvents on the structure and optical properties of silver granular films was studied. It was found that irregularly shaped oblate silver grains transform into almost spherical nanoparticles. Simultaneously, optical extinction spectra shift to the short wavelength range and become narrower. It was noticed, that the shape of nanoparticles changes under the influence of these fluids, as in the process of thermal annealing. Finally, the specially prepared silver nanoparticle arrays are very sensitive to the applied voltage and change their conductivity under electrical bias. Both reversible and irreversible conductivity changes were observed under certain conditions. The authors address all the above mentioned tools to control the morphology of the metal nanoparticle arrays and, through the morphology, their optical and electrical properties. The authors describe the formation of islet metal films during their deposition in the process of thermal evaporation on a dielectric substrate and their spontaneous changes at room temperature. The morphology and properties of such films under the influence of various factors: heating, laser irradiation, the action of certain liquids, and the action of electric fields are also considered. The films were investigated with the optical spectroscopic and scanning electron microscopic techniques. The results obtained are interpreted within a model relating the film optical properties with the shape and size of the nanoparticles forming this film. Chapter 2 - Silver-containing glasses are widely used in optics and photonics. Non-organic glasses with silver nanoparticles are used as nonlinear optical materials, optical filters, media for holograms and optical information recording. By the external action of different types of radiation and thermal treatment the state of silver in glasses can be changed considerably: from ions and atoms to charged and neutral subnanosized molecular clusters and nanoparticles. Here the authors present their experimental results on synthesis, modification and destruction of silver nanoparticles in bulk and surface of silicate glasses, obtained in ITMO


x


University. The main attention is paid to the effects of laser and electronbeam irradiation on above-mentioned processes. Chapter 3 - Noble metal nanoparticles (NMNPs) exhibit unique physical and tunable optical properties that are applicable in a variety of fields, ranging from sensing, energy, and environment, to biomedical applications. However, these NMNPs suffer from the open environment (moisture, air) that leads to the degradation of their properties, thereby limiting their functional applications. Therefore, embedding them into a medium that allows to retain their properties with high reproducibility, could be very promising. In this chapter, the synthesis of embedded NMNPs, their optical properties and potential applications in surface enhanced Raman scattering (SERS) will be reviewed. The optical properties of NMNPs due to the localized surface plasmon resonance and the enhanced local electromagnetic field which govern their SERS activity will be discussed, together with their fundamental mechanisms. Particularly, the ultrasensitive detection of the organic and the bio-molecules will be discussed, with special emphasis on the reproducibility of results. Due to the tuning of the optical properties and to the structural stability of the NMNPs in the embedding media, the sensitivity of the SERS technique can be considerably increased. Recent developments in the SERS applications of the embedded NMNPs, including their synthesis and the effect of the embedding media on their optical properties and their SERS performance, will be discussed as well. Chapter 4 - Bacterial resistance to antibiotics results in a continuous need for new antimicrobial agents. Nanoparticles (NPs) of various transition metals can be used in this respect, as they have expressed antibacterial activity simultaneously being more reactive compared to their bulk material. The effects of zinc (II), titanium (IV), copper (II) and (I) oxides thin films with nanostructured surface and iron (III) oxide and silver NPs on Enterococcus hirae and Escherichia coli growth and membrane activity are presented and discussed. These results are of significance, since E. hirae and E. coli can be considered as model organisms for Gram-positive and Gramnegative bacteria, respectively; moreover, they are among pathogenic bacteria in nature. It was revealed that sapphire base plates with deposited ZnO, TiO2, CuO and Cu2O NPs had no effect either on E. hirae or E. coli


Preface

xi

growth, both on agar plates and in liquid medium. Fe3O4 NPs concentrationdependent inhibitory effect on E. coli was obtained. The reactive oxygen species together with superoxide radicals and singlet oxygen formed by Fe3O4 NPs could be the cause of inhibition. At the same time, Fe3O4 exhibited opposite effect on E. hirae: growth stimulation was observed. These results point out distinguishing antibacterial effects of Fe3O4 and Ag NPs on different bacteria; the difference between effects can be explained by specifics of bacterial membrane structure and properties. H+-transport and the FOF1-ATPase activity disturbance by NPs might be involved in antibacterial effects. Cellular mechanisms for antimicrobial activity of NPs are discussed. Distinguishing effects on different bacteria should be taken into consideration when applied. Metal NPs have also antifungal effects. Different types of metal NPs could be applied in medicine due to their antimicrobial activity, effeciency in anti-inflammatory effects, potential in anticancer therapy, drug delivery and others, developing a new era of nanomedicine; a further study is required. Chapter 5 - Polymer electrolyte fuel cells (PEFCs) are receiving much interest because they provide an efficient, a clean and an environmentally friendly power source with high energy density. The main roadblock that obstacle the wide spread application of PEFCs is the metal catalyst. The catalyst usually facilitates the reaction of the oxidant (oxygen or air) and the fuel (hydrogen or alcohol), in order to produce electricity with zero emission of carbon dioxide. The most efficient catalyst for PEFCs is platinum-based materials. Platinum (Pt) is currently the only efficient metal-catalyst that can withstand in the acidic environment of PEFCs. It is a very expensive material, in addition, its price is almost controlled by two countries, i.e., the republic of Russia and South-Africa. Accordingly, Pt-metal catalyst limits the wide spread application of PEFCs. In order to promote the application fuel cell technology, it is important to optimize the use-efficiency of Ptmetal through the nanoscience technology. In this chapter, the authors focus on the development of Pt-metal nanoparticles and their core-shell structures which are fabricated by different synthetic techniques. The authors discuss in details the polymeric composite of the metal nanoparticles on the surface of different carbon support materials (e.g., carbon nanotubes, graphene,


xii


carbon-nanospheres, mesoporous carbon, and carbon nanofibers), showing the synergetic effects on the active surface area of Pt metal catalyst. The authors also focus on the Pt nanosized controlling routes which are receiving a great deal of attention in the way of fabricating of highly active metal catalysts. In addition, the authors highlight the factors affecting the physicochemical properties of the Pt core-shell structure, and how these factors affect the fuel cell performance. Furthermore, the authors summarize the recent research results of some other metal-catalyst nanoparticles (e.g., Palladium, nickel, gold, and iron) used for the different fuel cell types (e.g., alkaline fuel cells, direct methanol fuel cells, and direct-ethanol fuel cells). Chapter 6 - This chapter considers the past decade’s data regarding the effects of nanoparticles of noble metals (gold and silver) on higher plants, as well as regarding possible nanoparticle phytotoxicity. It discusses the various effects that gold and silver nanoparticles can have on the state, growth, and productivity of plants. Research in this field is topical because (1) plant-nanoparticle interactions are caused by a diversity of natural and man-made factors and because (2) green chemistry now uses plants for the dedicated biotechnological synthesis of nanoparticles. Published evidence, although incomplete and contradictory, indicates that metal nanoparticles can have both positive and negative effects on plants and that intracellular penetration is determined mostly by the particles’ chemical nature, size, shape, surface charge, and dose. Thus, the need exists for a coordinated research program that can find correlations between particle characteristics, experimental design, and observed biological effects. Chapter 7 - Electrodes modified with conducting polymers and metal nanoparticles have been developed, characterized and optimized for the determination of antioxidant capacity of single components and complex food samples. Carbon materials as pure graphite and glassy carbon, and indium tin oxide (ITO) supports, covered either with polyaniline or polyphenazines, were prepared. After deposition of platinum or silver on the polymer surface, these electrodes were used to be used in the monitoring of the H2O2 reduction in the presence of antioxidants. The role of the conducting polymer in the electrochemical and electrocatalytic properties of the hybrid nanocomposite was explored by in situ electrochemical and


Preface

xiii

spectroscopic techniques. The reduction of H2 O2 in phosphate buffer solutions at neutral pH is catalyzed by the polarons of the nanocomposite, acting the polymer as a proton reservoir. Glassy carbon electrodes modified with polyphenazine conducting polymers and metal nanoparticles were used to study the interaction between H2O2 and ascorbic acid. The antioxidant capacity of ascorbic acid measured from this interaction agreed with that reported using non-electrochemical techniques. Electrodes based in platinum nanoparticles have higher durability, but silver nanoparticles modified electrodes are also a good selection because their lower cost. The exploration of the morphology of the nanoparticles-doped poly-phenazines on ITO, by SEM-EDX and Raman spectroscopy, showed that the polymers were electrodeposited in an inhomogeneous way. Pores nesting the nanoparticles are formed. At high deposition times, the formation of clusters implies the loss of electrocatalytic activity. Platinum nanoparticles (10-60 nm) cover the polymer agglomerates. After electrochemical measurements, nanoparticles and their aggregates decrease in size. An electrochemical cleaning results in the recovering of the sizes of aggregates, which justify the reproducibility of the measurements. For silver nanoparticles, formation of dendrites was observed by SEM-EDX. The glassy carbon electrode modified with poly-neutral red and Pt nanoparticles was used to explore the scavenging activity of extracts of teas, infusions and spices. The activity of individual antioxidants forming part of these foods was also investigated. The time and ease of measurement were improved with respect to other commonly used methods. The measurements were made in media close to physiological conditions, contrary to what happens with the Hg electrode. Antioxidants not showing activity in the DPPH radical scavenging assay can be studied. The advantages with respect to the CUPRAC assay are the lower measuring time involved and the absence of organic solvent, with the consequent closeness to physiological conditions. The sensitivity of the sensor is comparable to those of DPPH and CUPRAC.



In: Metal Nanoparticles Editors: Y. Saylor and V. Irby

ISBN: 978-1-53614-115-3 © 2018 Nova Science Publishers, Inc.

Chapter 1

AN INVESTIGATION OF MAJOR FACTORS AFFECTING METAL NANOPARTICLE MORPHOLOGY IN ISLAND FILMS N. B. Leonov, PhD, V. A. Polishchuk, PhD and T. A. Vartanyan, PhD ITMO University, St. Petersburg, Russian Federation

ABSTRACT Metal nanoparticles, which are now widely used in various scientific and technical areas, may be obtained in different ways. Physical vapor deposition is one of the most common ways for metal nanoparticle production. It is simple to implement and leads to the manifold of nanostructures depending on the control parameters, such as the substrate material and its temperature, deposition rate, and the amount of the deposited metal. Nevertheless, application of island films requires even more control over the properties of the nanoparticles arrays. To meet these demands, postprocessing of the prepared nanostructure may be successfully employed. 

Corresponding Author Email: [email protected].


2

N. B. Leonov, V. A. Polishchuk and T. A. Vartanayn In this study several factors affecting metal nanoparticle morphology in island films are determined. The equipment design allowed us to see changes in nanoparticle morphology both during the film deposition and upon their heating without breaking the vacuum conditions. Much more control over the size and shape distribution of silver nanoparticles may be performed with the help of intense laser illumination. In this case, instead of violent heating of the entire particles’ ensemble selective heating of those nanoparticles that possess plasmon excitations in resonance with the incident radiation, is achieved. The influence of organic solvents on the structure and optical properties of silver granular films was studied. It was found that irregularly shaped oblate silver grains transform into almost spherical nanoparticles. Simultaneously, optical extinction spectra shift to the short wavelength range and become narrower. It was noticed, that the shape of nanoparticles changes under the influence of these fluids, as in the process of thermal annealing. Finally, the specially prepared silver nanoparticle arrays are very sensitive to the applied voltage and change their conductivity under electrical bias. Both reversible and irreversible conductivity changes were observed under certain conditions. We address all the above mentioned tools to control the morphology of the metal nanoparticle arrays and, through the morphology, their optical and electrical properties. We describe the formation of islet metal films during their deposition in the process of thermal evaporation on a dielectric substrate and their spontaneous changes at room temperature. The morphology and properties of such films under the influence of various factors: heating, laser irradiation, the action of certain liquids, and the action of electric fields are also considered. The films were investigated with the optical spectroscopic and scanning electron microscopic techniques. The results obtained are interpreted within a model relating the film optical properties with the shape and size of the nanoparticles forming this film.

INTRODUCTION Nanoparticles have a number of unique properties due to their intermediate position between individual atoms and a massive solid body. One of the properties is the presence of a narrow band in the absorption spectrum due to the excitation of localized surface plasmons in metallic nanoparticles. Namely, this property is applied in different fields of modern science and technology. In some cases, to measure a minor amount of objects, localized surface plasmon resonance is used in medicine


An Investigation of Major Factors Affecting Metal Nanoparticle …

3

and biological sensors [1, 2], in surface–enhanced Raman scattering spectroscopy [3, 4], metal-enhanced fluorescence [5], and in improved photovoltaic devices [6]. The morphology of nanoparticles is very important in most applications. Plasmon frequencies depend on the shape of nanoparticles and, to a minor extent, on their size. Therefore, studying the shape of nanoparticles and the kinetics of their formation in vacuum deposition is of interest for various applications. Even more interesting is the influence of various factors on the shape of nanoparticles, for example, high temperature, light, the environment, into which nanoparticles are embedded, etc. Along with the well-known methods, such as electron, atomic force, and autoionization microscopies, optical methods proved to be very effective to study nanostructures. In these methods, optical characteristics of island films, extinction spectra in particular, are associated with the morphology of the particles forming the film. The comparison of numerous optical extinction spectra of different films with the corresponding electron micrographs of the islands composing these films shows obvious relationship of these spectra with the shape of the particles. In [7], the relation between changes in the extinction spectrum during heating of a thin gold film with a change in the shape of its 5-100 nm particles was clearly revealed. A similar study was carried out with a silver film [8]. While the extinction spectra are much easier to obtain than the electron microscopy images, they often provide more complete information. For example, extinction spectra are obtained from an area of several square millimeters, which allows smoothing and averaging of micro spottiness always present on the film. In this study, we show how the shape of islands varies under the influence of various factors, including exposure to light, heat, and electric field, and the presence of different liquids. Along with the proposition of using similar agents in various applications, we tried to understand some peculiarities of physical processes in nanometer-sized particles and differentiate them from the processes in bulk materials.


4

N. B. Leonov, V. A. Polishchuk and T. A. Vartanayn

We deliberately did not touch upon the topic of research into complexes - island metal film compounds with various substances: dyes, liquid crystals, quantum dots, etc., since this topic goes far beyond our work.

RESEARCH METHODS AND MATERIALS The films were prepared of various metals: silver, gold, copper, aluminum, and sodium. Most of the experiments were conducted with silver films, as they have a pronounced plasmon resonance in the visible spectral region and change more rapidly under the influence of external factors. Sodium films were also investigated, but they have a significant drawback their contact with atmospheric air is unacceptable. Therefore, sodium films were studied in special vacuum cells with sapphire or quartz windows at the ends. When one of the ends was heated, an islet film was formed on the opposite end [9]. All other metal films were formed via physical vapor deposition of thermally evaporated metals (purity 99.99%) onto the surface of ground sapphire substrates in a PVD 75 vacuum chamber (Kurt J. Lesker) at a residual pressure of ~10–7 Torr. A sample was mounted on a manipulator with a built-in furnace, which made it possible to vary the substrate temperature from room temperature to 300°C. The amount of deposited material was monitored using a quartz microbalance and expressed as an equivalent thickness of a homogeneous film. The deposition rate was varied from 0.01 to 0.1 nm/s. The design of the vacuum system and the use of a PMA-12 multichannel photon analyzer (Hamamatsu) allowed us to record the extinction spectra of the films deposited on the transparent substrates both during the deposition and upon the heating. The spectrophotometer made it possible to measure spectra with certain (specified) time intervals. We used steps from 30 s to 2 min. There was a possibility of depositing a film on a substrate at room temperature, with its subsequent heating, or depositing a film on a hot substrate. The extinction spectra were also obtained on a stationary



5

spectrophotometer in the wavelength range 200-1100 nm (in particular, it was used to study the spectra of sodium in vacuum cells). The film structure was analyzed with a scanning electron microscope (MERLIN, Carl Zeiss). Various methods of influencing the film will be described in the relevant sections.

EVOLUTION OF THE THIN METAL FILMS MORPHOLOGY DURING PHYSICAL VAPOR DEPOSITION Experimental Setup and Results of the Experiments Before proceeding to the study of the influence of various factors on the shapes of metal nanoparticles, one should become acquainted with the way they are formed during the deposition process and how this process is investigated using optical methods. The scheme of the experiment is shown in Figure 1. A film is deposited on a transparent substrate (sapphire or quartz) by vacuum evaporation. The substrate is illuminated by a light source (xenon lamp). After passing through the substrate, the light enters the detector of a spectrophotometer. On the monitor, we observe the extinction spectrum of the substrate together with the metal film deposited on it. The results of these studies are shown in Figure 2. As the film thickness grows, the optical absorption increases and the form of the extinction spectrum changes. As the deposition goes on, the spectral width of the absorption band increases, indicating that the islets acquire more and more irregular shape, far from spherical. The shift of the absorption band maximum to longer wavelengths indicates that the islets are becoming more flat. Figure 2 shows that, as the thickness of the film increases, a small peak at the wavelength of about 360 nm begins to grow. In 20-30 minutes after the completion of the deposition, this peak disappears, as is clearly seen in Figure 4.


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N. B. Leonov, V. A. Polishchuk and T. A. Vartanayn 4 6 1 8 2

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Figure 1. Experimental setup for studying metal films during deposition and heating. 1 – vacuum chamber, 2 – transparent substrate, 3,4 - quartz windows, 5 - light source, 6 – photodetector, 7 - crucible with metal, 8 – spectrophotometer. 1 - 0.1nm 2 - 0.2nm 3 - 0.3nm 4 - 0.4nm 5 - 0.5nm 6 - 0.6nm 7 - 0.7nm 8 - 0.8nm 9 - 0.9nm 10 - 10nm

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It can be assumed that along with large particles (several tens of nanometers in diameter) very small spherical particles, 2-3 nm in diameter, are formed. The calculated value of the plasmon resonance energy for particles of this shape corresponds to the wavelength of the short-lived extinction maximum. Due to the surface diffusion, particles of such a small size disappear quickly. The electron micrograph of a 4 nm-thick silver film deposited on sapphire at room temperature is shown in Figure 3. As seen, large fractal structures of various shape and size are present. The extinction spectra of such films are inhomogeneously broadened due to the formation of particles of different shapes. In addition, interaction between the particles contributes to the extinction spectra. The structure of the films and their extinction spectra depend weakly on the deposition rate. At larger deposition rates, with the growth of the film thickness, the plasmon band maximum shifts into the red region of the spectrum slightly faster than at lower rates.

Figure 3. SEM image of the 4-nm silver film on a sapphire surface.


8


The extinction spectra of silver films deposited on a substrate at room temperature spontaneously change in time (Figure 4). The maximum of the plasmon resonance shifts to the short-wavelength region of the spectrum, while the optical density decreases. This suggests that the islands change their structure in a short time and become more “compact” and less smeared over the substrate surface. Significant changes occurred mainly in the first 5-10 min after the end of the deposition, then the rate of the change decreased. Obviously, the changes occurred due to the diffusion of silver atoms over the surface of the substrate, as well as through self-diffusion. It should be noted, that in this respect, silver differs strongly from other metals. The copper and gold extinction spectra, obtained immediately after deposition, change much more slowly, which indicates very high mobility of silver atoms and, as a consequence, high rate of diffusion processes. 0,7

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Figure 5. Spectra of optical density D taken during the deposition of gold (a) and copper (b) films on a substrate of crystalline sapphire. Film thickness varies from 3 to 10.5nm for gold and from 0.5 to 11.5 nm for copper.

In a similar way, during the deposition process, the optical properties, and, consequently, the morphology of other metals, also change. Figure 5 shows the change in the extinction spectra during the deposition of gold and copper.


10


It should be noted that the gold atoms are much less mobile than the silver atoms and after the completion of the deposition the gold film spectrum stays practically unchanged. As was mentioned in the introduction, sodium films were produced in vacuum cells with flat sapphire or quartz windows. At first, sodium - by a small heating (up to 100-200°C) - was distilled on one side of the cell. Then, this side was heated to a temperature of 100°C and the metal was gradually deposited on the opposite window. The growth of an islet sodium film is shown in Figure 6. In this case, the effective thickness of the film cannot be measured, whereas it can be estimated only very approximately from the extinction spectrum.

Figure 6. Extinction spectrum for deposition of sodium on sapphire.

Sodium films, as well as silver films, spontaneously change in time. Especially noticeable is the change in the long-wave peak, it shifts toward short waves and becomes narrower. The nature of the changes is the same as for silver films. Sodium films obtained in cells with windows made of a polished fused quartz change much more rapidly than films obtained in the



11

cells with windows made of crystalline sapphire. This is apparently due to a decrease in the diffusion rate of metal atoms on surfaces whose roughness is comparable to the light wavelength (sapphire). The presence of two peaks with an energy of approximately 1.5 and 3.2 eV is amassing. The plasmon energy of a spherical sodium nanoparticle is calculated as 3.6 eV. At the beginning of the deposition, when the film is still very thin and the optical density of the peak with an energy of 1.5 eV reaches 0.12, a peak with an energy of 3.2 eV is absent (see Figure 6). However, as the thickness of the film grows, this peak begins to grow, where growth rate depends on the film thickness. All this allows us to assume the following model of sodium film growth: sodium islands formed on sapphire or quartz surface have a strongly flattened shape, which leads to a large shift of the plasmon resonance toward low energies. At the same time, a peak with an energy of about 3.2 eV, which appears when the film is already thick enough, can be explained by the presence of quadrupole resonance, which is the greater, the larger the island size [10].

Conclusion We see that in the processes of deposition of island (labyrinth) films and growth of their thickness, individual particles in the form of large fractal structures of various shape and size are formed. Along with such particles, spherical particles (1-3 nm in size) may be also formed to disappear within a few minutes after the deposition. After the deposition is complete, the shape of the fractal structures continues to change due to diffusion. But the rate of this phenomеnon is very different for different metals. For silver, these changes are significant for several tens of minutes. On the extinction spectra of sodium films, beginning with some thicknesses, a stable peak with energy of 1.5 eV appears. With increasing film thickness, it grows faster than the long-wave peak, which suggests that this peak corresponds to quadrupole plasmon resonance.


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CHANGES IN THE OPTICAL PROPERTIES AND STRUCTURE OF THE ISLAND FILMS DURING ANNEALING Experimental Results The simplest and most studied way of changing the shape of islands is heating. Since we are dealing with temperatures considerably lower than the melting point, the shape of the islands is changed due to diffusion of metal atoms on the substrate surface, as well as self-diffusion, that is, diffusion along the surface of the island itself. Annealing of films deposited on a cold substrate changes their structure radically. Figure 7 shows the extinction spectra of silver films recorded during their annealing at 70, 110, and 190°C. The heating time (in min) is indicated above the curves. 0,30

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1000

c

Figure 7. Annealing of silver films with a thickness of about 5 nm at temperatures of (a) 70, (b) 110, and (c) 190°C. The numbers near the curves show the time passed from the annealing onset.

It can be seen that the spectra first change rapidly but then the rate of their change decreases. Each temperature corresponds to a certain final state of the film and the composing particles. The narrowest extinction spectrum


14


is obtained for the film heated at 190°C for 35 min, which is very close to the extinction spectrum of the film obtained by deposition on a hot substrate. SEM images of these films (Fig. 8) suggest that the film particles are similar in shape to spheroids compressed to a certain extent. Similar modifications of silver nanoparticles shapes were reported in [11].

Figure 8. SEM image of an annealed silver film deposited on a sapphire substrate. The annealing temperature is 200°C and the film thickness, 4 nm.

The films annealed at lower temperatures exhibit wider final extinction spectra. This means that particles acquire the shape of spheroids compressed to certain extent at a given temperature; continuation of annealing at the same temperature does not change the shape of the extinction spectrum and, therefore, the particles’ shapes. The SEM images of the films obtained via deposition on a hot substrate are very close to the images of the annealed films. Figure 9 shows the extinction spectra obtained during the deposition on a hot substrate. At the beginning of the deposition on a hot substrate, the maximum of the extinction spectrum is at a wavelength of 400 nm. This position corresponds to the plasmon absorption band of a slightly compressed silver nanosphere in a homogeneous medium with a refractive index average between the refractive indices of the substrate and the vacuum. The width of the extinction spectrum at the beginning of the deposition process on a hot substrate is also close, although slightly exceeds the width of the plasmon



15

absorption band of the model nanospheroid. When deposited on a cold substrate, broadening, which can be related to the spread of particles by shape, is more significant. Subsequently, during deposition, the spectra of the resulting films broaden the stronger, the lower the substrate temperature.

0,8

D

0,6

0,4

0,2

0,0 400

500

600

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800

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,nm

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D

0,5 0,4 0,3 0,2 0,1 0,0 400

500

600

700

800

,nm

900

1000

1100

b

Figure 9. Optical density spectra taken during deposition on a heated substrate. The deposition rate is 0.01nm/s. The maximum thickness of the films is 10 nm. a) the substrate temperature is 200°C, b) the substrate temperature is 110°C.


16


At normal incidence of radiation, oscillations of electrons in the plane parallel to the plane of the substrate are excited in the films, which lead to intense absorption of light at the frequency of the surface plasmon localized in the nanoparticle. The frequency of the plasmon dipole mode depends on the particle shape [12]. The extinction spectra of films deposited on a hot substrate consist of one narrow absorption band, which indicates an insignificant dispersion of the particles in shapes.

Figure 10. Dependences of the optical density in the maximum of the extinction spectrum, plasmon-resonance position, and plasmon-resonance width on the amount of deposited silver.



17

The extinction spectra of the films deposited under different conditions were used to determine the dependences of the width of the plasmon resonance, its spectral position, and the optical density in the resonance peak on the film thickness. The results are presented in Figure 10. The optical density in the plasmon-resonance peak is independent of the film deposition conditions and depends almost linearly on the amount of the deposited material. Thus, one can state that the optical density maximum in extinction spectra is proportional to the amount of silver deposited. The equivalent film thickness is proportional to the amount of material adsorbed by quartz microbalance. The optical density depends not only on the amount of the deposited material, but also on its distribution over the substrate, which is presented in Figure 9. The optical density (at the same wavelength) is significantly different for films of equal thickness but deposited at different substrate temperatures. At the same time, the maximum in the extinction spectrum corresponds to a certain type of plasmon resonance. For example, the final spectrum in Figure 7c indicates a small spread of particles over shapes and sizes. Most islands are shaped as balls or spheroids. At the same time, in the case of inhomogeneous broadening, the maximum in the spectrum corresponds to the largest number of particles with identical shape. This number is proportional to the amount of the deposited material, which is confirmed by linear dependence in Figure 10. The wavelength corresponding to the maximum in the spectrum increases, while a spherical particle is transformed into a flattened spheroid [12]. The degree of particle flatness depends linearly on the particle size and, therefore, on the equivalent film thickness. It is noteworthy, that the plasmon-resonance FWHM also depends almost linearly on the film thickness. The plasmon-resonance width increases more slowly at an equivalent film thickness of 2–3 nm. Along with the increase in the size of islands and complication of their shapes, merging of islands into large clusters begins at these film thicknesses. The electric resistance of the films during their deposition becomes measurable ( Hg > Cu > Cd > Cr > Ni >Pb> Co > Zn > Fe > Ca. It turns out that Ag is the most efficient among the metals examined. All the metals studied were in the form of salts in combination with various anions (Martin, 1969). Despite this, there is strong evidence in the literature that the active component of any Ag compound is the Ag itself. The data in other works show that electrically generated Ag cations are more effective than Ag sulfadiazine or Ag nitrate (Berger et al. 1976).

ON THE CELLULAR MECHANISMS OF ANTIMICROBIAL ACTION OF TRANSITION METAL NANOPARTICLES Gram-negative bacteria E. coli and Gram-positive bacteria Enterococcus hirae are non-pathogenic microorganisms that can indicate the possible presence of pathogenic bacteria in the environment (Singh et al. 2016). E. coli are the most-studied and best-characterized Gram-negative bacteria; having high growth rate, metabolic pathways and physiological activity established more or less clearly, they are used in microbiological and biotechnological researches (Poladyan et al. 2013; Vardanyan et al. 2015; Trchounian et al. 2017). E. hirae can be considered as model organisms for Gram-positive bacteria (Gaechter et al. 2012; Arokiyaraj et al. 2014). E. hirae show antimicrobial activity against some bacteria (Arokiyaraj et al. 2014). Nowadays, they are widely used in food industry as bio-preservatives and in lactic acid production (Foulquié Moreno et al. 2006; Subramanian et al. 2015). Besides, E. hirae and E. coli have also pathogenic forms responsible for various diseases such as urinary tract and central nervous system infections (Foulquié Moreno et al. 2006; Clements et al. 2012; Vela et al. 2015). In this case, it is important to study the behavior of E. hirae and E. coli in the presence of metal NPs. The effects of NPs can be various for different bacteria, so these effects should be investigated for revealing the appropriate action mechanisms.


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Armen Trchounian, Lilit Gabrielyan and Narine Mnatsakanyan

Figure 3. Proposed schemes for metal NPs effect on Gram-negative E. coli and Grampositive E. hirae. NPs can directly or indirectly (via FHL complexes) affect the FOF1ATPase. In E. hirae’s membrane vesicles (protoplasts) the FoF1-ATPase is more susceptible to nanoparticles action than ATPase in E. coli’s membrane vesicles (spheroplasts) containing certain remains of the cell wall.

-1

Specific growth rate (h )

1.50

control (no addition) Fe3O4 (50 g/mL)

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Fe3O4 (100 g/mL) Fe3O4 (150 g/mL)

1.00

Fe3O4 (250 g/mL)

0.75 0.50 0.25 0.00

E. coli BW 25113

E. coli K-12

Figure 4. Specific growth rates of E. coli BW 25113 and E. coli K-12 in the presence of Fe3O4 NPs various concentrations. Control was without NPs addition.


Nanoparticles of Various Transition Metals …

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Ag NPs are suggested to affect not only the growth of both E. hirae ATCC 9790 and E. coli K-12, but also the H+-coupled membrane transport of bacteria (Vardanyan et al. 2015). The membrane-associated ATPase activity of E. coli and E. hirae and H+-coupled K+ transport are the result of the FOF1-ATPase interaction with K+uptake system (Trchounian & Kobayashi, 1998; Trchounian, 2004; Vardanyan et al. 2015). Ion fluxes have been shown to be increased even in the presence of the inhibitor N,N’dicyclohexylcarbodiimide (DCCD), indicating that Ag NPs affect the structure and permeability of bacterial membrane. These effects depended on bacterial species (Vardanyan et al. 2015). In E. coli the ratio of DCCDdependent H+–K+ exchange was fixed, whereas in the case of E. hirae Ag NPs changed the stoichiometry of H+–K+ exchange through membrane (Figure 3). The effect on FOF1-ATPase and disturbance of the interaction between FOF1-ATPase and K+uptake system in E. hirae (see Figure 3) can be responsible for the antibacterial effect (Vardanyan et al. 2015). Similar data were obtained in mammalian cells, where Ag NPs inhibited mitochondrial ATPase activity of rat liver cells (Chichova et al. 2014). Ag NPs change the permeability of bacterial membrane and inhibit cell respiration by penetrating via cell wall (Morones et al. 2005; Pal et al. 2007). These NPs affect the bacterial membranes, disrupting their vital activity and causing cell death (Lok et al. 2006). They are able to damage DNA and inactivate enzymes (Gibbins & Warner, 2005; Raffi et al. 2008). The researchers showed that the degree of antimicrobial activity of Ag NPs directly depends on its concentration, i.e., the higher the concentration of Ag NPs the more is its inhibitory effect towards microorganisms (Kim et al. 2007). Interestingly, Ag NPs are toxic for bacteria, and at low concentrations these NPs are non-toxic for human cells (Zhao & Stevens, 1998; Ral et al. 2009; Ansari et al. 2011). It has also been found that gold and iron oxide NPs affect the biofilm formation by various pathogens such as St. aureus and P. aeruginosa (Tran et al. 2010; Sathyanarayanan et al. 2013). The antimicrobial activity of metal NPs is a result of NPs interaction with bacterial membranes and their penetration into the bacterial cell, causing membrane damage and inactivation of bacteria (Seil & Webster, 2012;


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Arakha et al. 2015; Wang et al. 2017). Iron oxide NPs have been used as targeted drug carriers to treat various types of cancer in biomedicine because of their biocompatibility (Ficai et al. 2014; Mody et al. 2014; Assa et al. 2016; Stankic et al. 2016). Fe3O4 NPs are of a great interest for their super paramagnetic, high force, high magnetic susceptibility and other properties (Margabandhu et al. 2014; Assa et al. 2016). Lee with co-workers (2008) showed that zero-valent iron NPs could interact with intracellular oxygen, leading to oxidative stress and cell membrane disruption.

Lag phase duration (h)

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control (no addition) Fe3O4 (50 g/mL) Fe3O4 (100 g/mL) Fe3O4 (150 g/mL)

1.5

Fe3O4 (250 g/mL)

1.0

0.5

0.0

E. coli BW 25113

E. coli K-12

Figure 5. Lag phase duration of E. coli BW 25113 and E. coli K-12 in the presence of Fe3O4 NPs various concentrations. Control was without NPs addition.

The growth parameters of E. coli two wild type strains BW 25113 and K-12, grown under anaerobic conditions in the presence of colloidal Fe3O4 NPs (from 50 μg/mL to 250 μg/mL), have been investigated in our lab. Fe3O4 NPs show concentration dependent effect on E. coli growth (Figure 4). In the presence of 50 μg/mL Fe3O4 growth rate of bacteria was similar to the control. In the presence of 100–250 μg/mL Fe3O4, inhibition of E. coli growth was observed (Figure 4). The maximal inhibitory effect has been obtained at the concentration 250 μg/mL, which led to the decrease in



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bacterial specific growth rate by ~1.5-2.0-fold, indicating the bactericidal effect of the Fe3O4 NPs. The antibacterial effect of Fe3O4 could be due to several mechanisms. ROS together with superoxide radicals (O2-), hydroxide radical (OH-) and singlet oxygen (1O2) formed by Fe3O4 NPs could be the inhibition cause. The same results were obtained in the other studies that showed the antimicrobial activity of Fe3O4 NPs against E. coli (Chatterjee et al. 2011; Arakha et al. 2015). Latent (lag) growth phase duration was considerably increased (~2.0-fold in E. coli K-12) in the presence of Fe3O4 NPs in a concentration-dependent manner (Figure 5).

Figure 6. The number of viable colonies of E. coli BW 25113 grown in the absence and presence of 100 μg/mL Fe3O4 NPs as a function of initial bacterial suspension dilution.

Figures 6 and 7 display the relationship between bacterial suspension dilution and the number of colonies of E. coli grown in the absence and presence of 100 μg/mL NPs. The number of viable colonies of bacteria was decreased 1.4–2.0-fold in the presence of Fe3O4 NPs (Figures 6-8). At the


176


same time, Fe3O4 NPs have the opposite effect on E. hirae wild type strain ATCC 9790 (not shown). In the presence of Fe3O4, a stimulation of bacterial growth was observed. The 100 μg/mL Fe3O4 was the most effective: the specific growth rate was increased ~1.2-fold. The 500 μg/mL NPs led to the decrease in bacterial growth rate ~1.5-fold, indicating the bactericidal effect of this concentration.

Figure 7. The number of viable colonies of E. coli K-12 grown in the absence and presence of 100 μg/mL Fe3O4 NPs as a function of initial bacterial suspension dilution.

Thus, Fe3O4 NPs show significant antibacterial activity against Gramnegative but not Gram-positive bacteria. Different effects of Fe3O4 NPs on E. coli and E. hirae growth may be due to the differences in bacterial cell structure and components, and the metabolic peculiarities of these bacteria, as well as electrostatic interactions between bacterial cells and NPs (Vardanyan et al. 2015; Lu et al. 2017; Wang et al. 2017). Small size of NPs can contribute to their antibacterial activity (Lee et al. 2008; Sathyanarayanan et al. 2013; Vardanyan et al. 2015). NPs can interact closely with bacterial membranes and the inactivation of bacteria could be



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due to their penetration into the bacterial cell (Lee et al. 2008; Vardanyan et al. 2015).

Figure 8. The colonies of E. coli K-12 grown in the absence and presence of 100 μg/mL Fe3O4 NPs.

It is known that redox potential (Eh) is a very important factor of the environment, which can be defined as the ability of biological system to reduce or oxidize various compounds. The anaerobic growth of bacteria is coupled with a drop of Eh from positive to the low negative values, which determines the bacterial anaerobic growth (Vassilian & Trchounian, 2009; Poladyan et al. 2013). To reveal the action mechanisms of Fe3O4 NPs on E. coli and E. hirae the kinetics of Eh during bacterial growth has been studied. The anaerobic growth (6 h) of E. coli BW 25113 cells was accompanied by a drop in the Eh value from positive value (+150 ± 5 mV) at the beginning of growth lag phase to low negative value (–520 ± 15 mV) (Figure 9a). Such decrease indicates the enhancement of reduction processes, which characterizes bacterial metabolism under anaerobic conditions, and generation of H2 (Poladyan et al. 2013). Addition of the Fe3O4 NPs resulted in a delayed Eh drop. In the presence of 250 μg/mL and 500 μg/mL Fe3O4, the Eh values decreased up to (–460 ± 15 mV) and (–435 ± 10 mV), respectively (see Figure 9a). The inhibition of bacterial growth in the presence of Fe3O4 NPs can be coupled with Eh or with direct effect of Fe3O4 on bacterial membrane. In the case of E. hirae ATCC 9790 control cells the


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initial Eh value was +50 ± 5 mV, which decreased to negative value (–200 ± 5 mV) after 6 h growth (Figure 9b). Eh of E. hirae has not been changed much in the presence of NPs (see Figure 9b).

a Eh (mV)

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Figure 9. Changes of medium Eh during E. coli BW 25113 (a) and E. hirae ATCC 9790 (b) anaerobic growth in the presence of Fe3O4 NPs various concentrations. Control was without NPs addition.

A relationship between the decrease of Eh to low negative values and H2 generation was shown for various bacteria: the electron flow can be shifted toward the reduction of protons to H2 under strong reducing conditions (Poladyan et al. 2013; Gabrielyan et al. 2015). It is known, that E. coli



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produces H2 by the action of the membrane-associated formate hydrogen lyase (FHL) complexes, which split formate to H2 and CO2 (Trchounian et al. 2013, 2017).

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E. coli (control) Fe3O4 (50 g/mL)

Fe3O4 (100 g/mL)

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H2 yield (mmol/L)

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

3h

4h

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6h

Figure 10. The H2 yield in E. coli BW 25113 during anaerobic growth in the presence of Fe3O4 NPs’ various concentrations. Control was without NPs addition.

During glucose fermentation H2 production by E. coli control cells was observed after 3 h anaerobic growth and increased ~2-fold during the growth up to 5-6 h (Figure 10). The increase of H2 production might be coupled with the activity of membrane-associated H2-producing enzymes – hydrogenases, which are involved in H2 metabolism in E. coli (Blbulyan & Trchounian, 2015; Trchounian et al. 2017). Fe3O4 NPs have concentration dependent effect on H2 yield in E. coli (see Figure 10). H2 production by E. coli culture in the presence of 250 μg/mL Fe3O4, determined during 3 h anaerobic growth, was ~1.12-fold lower than H2 yield in control cells; whereas H2 yield in E. coli, determined during 5 h growth, was ~2.0-fold lower in cells, grown by addition of 250 μg/mL Fe3O4, than those of control cells (see Figure 10).


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H2 production by E. coli, grown in the medium with 500 μg/mL Fe3O4, was not observed during 3 h growth and decreased up to~2-fold after 5-6 h growth, in comparison with the control (see Figure 10). Table 1. The changes of H+-fluxes across the bacterial membranes of different bacteria in the presence of Fe3O4 and Ag nanoparticles Bacteria and conditions* E. coli BW 25113 (no addition) E. coli BW 25113 + Fe3O4 NPs E. coli K-12 (no addition) E. coli K-12 + Ag NPs E. hirae ATCC 9790 (no addition) E. hirae ATCC 9790 + Fe3O4 NPs E. hirae ATCC 9790 + Ag NPs

H+-fluxes (mmol H+/min/1010 cells) 2.68 ± 0.02*** 0.48 ± 0.01 2.50 ± 0.02 2.70 ± 0.02 1.32 ± 0.02

H+-fluxes** (mmol H+/min/1010 cells) 0.73 ± 0.01 0.45 ± 0.01 1.10 ± 0.02 1.25 ± 0.02 0.70 ± 0.02

0.90 ± 0.02

0.67 ± 0.02

2.65 ± 0.01

0.85 ± 0.01

*The

bacteria were washed and transferred into Tris-phosphate buffer; bacterial cells were treated with metal nanoparticles for 10 min. **The H+-fluxes in the presence of DCCD. ***Average values with standard errors from at least 3 independent experiments are shown.

Proton-coupled membrane transport has been determined in the cultures of E. coli and E. hirae in the absence and presence of colloidal Fe3O4, and data were compared with the effect of Ag NPs (Table 1). Bacteria were treated with metal NPs for 10 min. Indeed, Fe3O4 NPs suppressed energydependent H+-efflux by E. coli BW 25113 and E. hirae ATCC 9790 ~5.6and ~1.5-folds, respectively (see Table 1). H+-fluxes were also decreased in the presence of DCCD, inhibitor of the H+-translocating ATPases (see Table 1). The same results were obtained with E. coli K-12 (not shown). In comparison with Fe3O4, the presence of Ag NPs in the assay medium led to the increase in H+ fluxes (Vardanyan et al. 2015). More notable effect was observed with E. hirae: H+-flux was increased ~2.0-fold, whereas H+-flux in E. coli was increased too, but the effect of Ag NPs was weaker (see Table 1). At the same time, in the presence of Ag NPs the ion fluxes were increased



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even in the presence of DCCD, which indicates that Ag NPs affect bacterial membrane leading to changes in membrane structure and permeability (Vardanyan et al. 2015). 180

control (no addition) control + DCCD Fe3O4 (100 g/mL)

ATPase activity -1 -1 (nM Pin min g protein)

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120 100 80 60 40 20 0

E. coli

E. hirae

Figure 11. ATPase activity of E. coli BW 25113 and E. hirae ATCC 9790 membrane vesicles in the presence of Fe3O4 NPs and DCCD. Bacteria were grown in the presence of 100 μg/mL Fe3O4NPs; 0.2 mM DCCD was present in the assay medium, when indicated.

The membrane-bound H+-translocating FOF1-ATPase activities of E. coli and E. hirae membrane vesicles were analyzed in the presence of Fe3O4 NPs to reveal their effect on the ATPase. Note, during preparation of membrane vesicles, the protoplasts were obtained from Gram-positive E. hirae and the spheroplasts from Gram-negative E. coli. In mitochondria or aerobic bacteria, the FOF1-ATPase catalyzes the synthesis of ATP during oxidative phosphorylation. In anaerobic bacteria, like E. hirae with deficiency of respiratory chain, this enzyme catalyzes only ATP hydrolysis, which is coupled to the generation of a H+gradient (Shibata et al. 1992). In facultative anaerobic bacteria, such as E. coli, the FOF1-ATPase is reversible depending on bacterial growth conditions (Trchounian & Vassilian, 1994; Blbulyan & Trchounian, 2015). The FOF1-ATPase of the E. hirae is biochemically similar to the well-characterized ATPase of E. coli (Shibata


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et al. 1992; Trchounian & Vassilian, 1994; Capaldi et al. 2000). However, its function differs from other FOF1-ATPases: E. hirae ATPase can work as a regulator of cytoplasmic pH (Shibata et al. 1992). The obtained results indicate that the FOF1-ATPase might be a target for Fe3O4 in both types of bacteria. NPs resulted in ~1.4-fold increase of overall membrane-associated ATPase activity in membrane vesicles of E. coli and E. hirae, grown in the presence of 100 μg/mL Fe3O4 (Figure 11). DCCDsensitive ATPase activity of bacterial membrane vesicles has been increased ~1.4- and 3.7-folds in E. coli and E. hirae, respectively, in comparison with those of the control (see Figure 11). Indeed, DCCD-sensitive H+translocating ATPase activity of membrane vesicles has been increased more in E. hirae than in E. coli. These results suggest that FOF1-ATPase can be a sensitive target for Fe3O4 NPs. These NPs can directly affect FOF1ATPase, because ATPase activity was changed even in the absence of DCCD, the inhibitor of H+-translocating systems; or this effect can be intermediated by membrane-associated FHL complexes, which are responsible for H2 production in E. coli (see Figure 3). These studies confirm the fact, that the FOF1-ATPase, having a crucial role in cell metabolism, can be a target for various metals NPs. Differences in the effects of Fe3O4 on E. coli and E. hirae ATPase activity may be linked to differences in the structure of these protein complexes in the membranes as well as in bacterial cell wall structure (see Figure 3). In E. hirae’s protoplasts the FOF1-ATPase is more susceptible to Fe3O4 NPs action, than ATPase in E. coli’s spheroplasts containing certain remains of the cell wall (see Figure 3). These data obtained indicate that the membrane permeability may be changed during the bacterial growth in the presence of Fe3O4 NPs.

APPLICATION OF TRANSITION METAL NANOPARTICLES IN NANOMEDICINE NPs of transition metals of either simple or complex nature display unique, physical and chemical properties and represent an increasingly



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important material in the development of novel nanodevices which can be used in numerous physical, biological, biomedical and pharmaceutical applications (Martis et al. 2012; Nikalje, 2015). The antimicrobial nature of Ag NPs is the most exploited nature of AgNPs in the medical field. Ag NPs have demonstrated efficient inhibitory activity against viruses (Xiang et al. 2011), pathogenic fungi (Panácek et al. 2009) and bacteria (Jung et al. 2008). In addition to the study presented and analyzed above, dependence of the antibacterial activity of colloidal Ag NPs on their concentration and type of microorganisms has been determined using the disc diffusion method (Patel et al. 2014) and different test microorganisms including pathogenic bacteria and yeasts, namely Gram negative rod-shaped bacteria (E. coli and Pseudomonas aeruginosa), Gram positive cocci (St. aureus and E. faecalis) and yeast Candida albicans. It was shown that the same concentration of colloidal Ag affected different microorganisms in varying degrees. Meanwhile, different concentrations of colloidal Ag inhibited the growth of the same microorganism at different degrees – higher concentrations inhibited the growth of the microorganism more than the lower ones (Figure 12). Also, the minimum inhibitory concentration (MIC) of colloidal Ag towards the microorganisms was determined. MIC scores are important in diagnostic labs to confirm resistance of microorganisms to antimicrobial agents and also to monitor the activity of new antimicrobial agents. It was shown that MIC for E. coli and P. aeruginosa is 10 ppm of colloidal Ag, for St. aureus and E. faecalis – 20 ppm of colloidal Ag, for C. albicans – 40 ppm of colloidal Ag. This study confirms the findings that Ag inhibits the growth of microorganisms in varying degrees depending both on the concentration of colloidal Ag and the type of microorganism. Antifungal activity of Ag NPs was confirmed by many scientific works. Skin infections caused by fungi have become more common in recent years. In particular, fungal infections are more frequent in patients who are immunocompromised. The influence of Ag spherical NPs on the growth of dermatophytes, such as Candida species (14 strains) and Trichophytonmentagrophytes (30 strains) which are fungal pathogens of the skin was studied. Toward all fungal strains nano-Ag exhibited similar tivity with


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amphotericin B, but it was more potent activity than fluconazole (Kim et al. 2008). Amphotericin B is a fungicidal agent widely used in treating serious systemic infections, and fluconazole is used in the treatment of superficial skin infections caused by dermatophytes and Candida species. However, in contrast to antibacterial activity, the antifungal activity of Ag NPs against Candida biofilms doesn’t have size dependence. Also it was shown that Ag NPs inhibited the dimorphic transition of Candida albicans from yeast form to mycelial form, which is responsible for pathogenicity, with mycelial shapes being predominantly found during the invasion of host tissue (Monteiro et al. 2012). However, it should be considered that Ag NPs can penetrate through the skin in case of topical application. Results obtained in one of the studies demonstrated shape-dependent skin penetration of Ag NPs through intercellular pathway. Rod-shaped NPs demonstrated the highest penetration capabilities and accumulation in the dermal layer. Triangular NPs have shown slower penetration capabilities, compared to rod-shaped and spherical NPs (Yu et al. 2015). In this regard, follicular penetration pathways cannot discriminate skin penetration rates of differently shaped NPs, as the follicular opening diameter is too large (26 ± 1 μm) compared to the nanoscale of Ag. Thus, if the follicular penetration pathway plays a central role in mammalian skin absorption, the rates of skin penetration of all the differently shaped Ag NPs should have been similar. Considering the slow penetration capability and shape-dependent bactericidal activity of Ag NPs (the latter was mentioned above), triangular NPs can be considered as the most suitable for dermal applications, compared to the rod-shaped and spherical Ag NPs. Ag triangular NPs can reduce systemic toxicity and increase antibacterial efficacy at lower dose levels due to their slow penetration capabilities. Nevertheless, safety assessment issues should be considered. Overconsumption of Ag may lead to argyria, which results in permanent blue-grayish pigmentation of the skin, eyes and mucous membranes (U.S Public Health, 1990). The anti-inflammatory nature of Ag NPs is also considered. Nanocrystalline silver (NCS) has proven to be an important wound dressing particularly in chronic infected wounds. However, debate still rages around its use in the case of partially epithelialized wounds, particularly when these



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are non-infected. Gelatinase has been demonstrated to be pivotal in the reepithelialization of wounds. High levels of gelatinase promote increases in ongoing inflammation and chronicity. NCS has been demonstrated to decrease these undesirable high levels of gelatinase making it an ideal dressing for chronic infected wounds, acute inflamed wounds and burn wounds of all types. But in case of minimal inflammation the use of NCS may undesirably decrease the low levels of gelatinase and adversely affect epithelialization (Kirsner et al. 2001). These studies have proved that AgNPs are involved in the anti-inflammatory effects; however, the precise mechanism of action remains to be determined.

Figure 12. Antibacterial and antifungal activity of colloidal Ag against E. coli ATCC 25922, P. aeruginosa ATCC 9027, S. aureus ATCC 25923, E. faecalis ATCC 29212 and C. albicans isolated strain. The disk diffusion method was applied. After the incubation period, the growth inhibition zones of the test cultures were measured; average values are presented, standard errors are within the designations.

There are reports that suggest that AgNPs can cause adverse effects on humans, as well as on the environment. The adverse effects of Ag on humans include permanent bluish-gray discoloration of the skin (argyria) and exposure to soluble Ag compounds may produce toxic effects like liver and kidney damage; eye, skin, respiratory, and intestinal tract irritations; and untoward changes in blood cells (Panyala et al. 2008). Ag NP exposure


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could induce the changes of cell shape, reduce cell viability, and finally result in cell apoptosis and necrosis. Cytotoxicity is a direct outcome due to oxidation stress caused by Ag NPs and release of Ag ions (Zhang et al. 2014). Ag NPs are also associated with genotoxicity as a result of damage to DNA and chromosomes from oxidation stress (AshaRani et al. 2009). However, based on cytotoxicity and genotoxicity properties, Ag NPs have a potential for applications as anticancer agents (Abdel-Fattah & Ali, 2018). Noble metal NPs, especially Au NPs, have immense potential for cancer diagnosis and therapy on account of their surface plasmon resonance (SPR), enhanced light scattering and absorption. Conjugation of Au NPs to ligands specifically targeted to biomarkers on cancer cells allows molecular-specific imaging and detection of cancer. Additionally, Au NPs efficiently convert the strongly absorbed light into localized heat, which can be exploited for the selective laser photothermal therapy of cancer. By changing the shape or composition of Au NPs, the SPR can be tuned to the near-infrared region, allowing in vivo imaging and photothermal therapy of cancer (Jain et al. 2007). Recent developments in nanotechnology have led to the emergence of nanomedicine, a new ﬁeld which includes many diagnostic and therapeutic applications involving nanomaterials and nanodevices. Nanotechnology tools are used in process development and product development. Process development refers to both synthesis of drugs, drug intermediates, and to the development of analytical tools for diagnostics. One of the major applications of nanotechnology in relation to medicine is drug delivery. The problems with the new chemical entities such as insolubility, degradation, bioavailability, toxicologic effects, targeted drug delivery, and controlled drug release are solved by nanotechnology. For example, encapsulated drugs can be protected from degradation. Specific nanosized receptors present on the surface of the cell can recognize the drug and elicit appropriate response by delivering and releasing the therapy exactly wherever needed. Because of their small size and large surface area relative to their volume, NPs can readily interact with biomolecules (Estelrich et al. 2015a). NPs with targeting capability can deliver the therapeutic agents to specific tissues or cells and release the cargo in a



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sustained fashion, thus reducing their systematic toxicity (Zhang & Saltzman, 2013). The main aim of any new therapeutic application is to reduce the toxicity or any adverse action of the agents by making them more targets specific and hence reduce its dose. Magnetic resonance imaging (MRI) has become one of the most widely used and powerful tools for noninvasive clinical diagnosis owing to its high degree of soft tissue contrast, spatial resolution, and depth of penetration. Various NPs and complexes have been studied as MRI contrast agents, and several formulations have been approved for clinical use. These contrast agents are formed either from transition and lanthanide metals or from iron oxide NPs and more recently, ferrite NPs (Estelrich et al. 2015b). The in vitro labelling of cultured cells with iron oxide NPs is a frequent practice in biomedical research. To date, the potential cytotoxicity of these particles remains a matter of debate. The results of investigations highlight the importance of in-depth cytotoxic evaluation of cell labelling studies at nontoxic concentrations. From this point of view some particles are less suitable for the MR visualization of labelled cells (Soenen et al. 2011). The studies show that endosomal localization of different iron oxide particles results in cell degradation and in reduced MR contrast, the rate of degradation is governed mainly by the stability of the coating of iron oxide NPs. The release of ferric iron generates ROS, which greatly affects cell functionality. It was shown that lipid‐coated NPs display the highest stability and furthermore exhibit intracellular clustering, which significantly enhances their MR properties. Thereby, depending on the nature of the coating, particles can be rapidly degraded, completely annihilating their MR contrast to levels not detectable and greatly impeding cell functionality, thus hindering their application in functional in vivo studies (Soenen et al. 2010). One of the most commonly-used nanoscale materials are magnetic NPs (MNPs). MNPs are a class of NPs that can be manipulated using magnetic fields. Such particles commonly consist of two components, a magnetic core (Fe, Ni, Co) encapsulated in an organic or a polymeric coating. MNPs exhibit a variety of unique magnetic phenomena that are drastically different from those of their bulk counterparts; they are generating significant interest, since their properties can be utilized in a variety of applications in the


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biomedical sciences. Therefore, the nanoscaling laws of magnetic nanoparticles are important not only for understanding the behavior of existing materials, but also for developing novel nanomaterials with superior properties. MNPs can bind to drugs, proteins, enzymes, antibodies, or nucleotides and can be directed to an organ, tissue, or tumour using an external magnetic field. Since MNPs can be easily conjugated with biologically important constituents such as DNA, peptides, and antibodies, it is possible to construct versatile nano-bio hybrid particles, which simultaneously possess magnetic and biological functions for biomedical diagnostics and therapeutics (Jun et al. 2008). Besides the size, the surface properties of MNPs are essential for their applications. Coating MNPs with a layer of a different material is an interesting method for modifying their surface properties. Several groups of coating materials are used to modify the surface chemistry of MNPs: organic polymers (dextran, chitosan, polyethylene glycol, polysorbate, polyaniline); organic surfactants (sodium oleate, dodecylamine); inorganic metals (gold); inorganic (silica, carbon); bioactive molecules and structures (liposomes, peptides, ligands/receptors) (Shubayev et al. 2009). Coated NPs have major advantages over simple NPs due to their enhanced properties, such as less cytotoxicity; increased dispersibility and biocompatibility; better conjugation with other bioactive molecules; and increased thermal and chemical stability (Chatterjee et al. 2014). The scientific investigations and developments which have been carried out for improving the quality of magnetic particles, their size, shape, and surface coatings are very important (Gupta & Gupta, 2005; LaConte et al. 2007; Soenen et al. 2009). Various formulations of MNP have been developed for theragnostics applications. In particular, the use of MNPs, as drug carriers, has attracted enormous attention. However, in relation to magnetically-guided MNPs, efficient in vivo drug delivery is still elusive, with fundamental problems being the drop of magnetic field strength with distance inside the body and ever small targets, such as individual NPs, as well as the body’s physiological defense mechanisms against foreign agents. In contrast, the magnetically-guided approach has yielded clear benefits in gene



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transfection. Compared to all other physical methods, the major advantage of magneto-fection is that it is capable of combining simplicity, a modest cost, enhanced localization and efficiency of delivery and reductions in both incubation time and vector doses. Meanwhile, magnetically-sensitive MNPs constitute a platform that shows the highest diversity of innovations in the drug delivery field. Thus, NPs of different transition metals have great potential in nanomedicine mainly due to their antimicrobial activity, anti-inflammatory effects, potential in anticancer therapy and drug delivery; however, further studies are required to clarify their effective application.

ACKNOWLEDGMENTS The authors thank to Dr. V. Gevorgyan and Mrs. L. Rshtuni (RussianArmenian (Slavonic) University, Yerevan, Armenia), Mr. L. Hakobyan (“Tonus-Les” LLC, Armenia) for providing with different transition metals NPs used in our study.

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Yu, K. T., Sukdeb, P., Pravin, K. N., Sabarinathan, R. & Joon, M. S. (2015). Shape-dependent skin penetration of silver nanoparticles: does it really matter? Sci. Reports, 5, 16908. Zhang, T., Liming, W., Chen, Q. & Chen, C. (2014). Cytotoxic potential of silver nanoparticles. Yonsei Med. J., 55, 283–291. Zhang, J. & Saltzman, M. (2013). Engineering biodegradable nanoparticles for drug and gene delivery. Chem. Eng. Prog., 109, 25–30. Zhang, X. F., Liu, Z. G., Shen, W. & Gurunathan, S. (2016). Silver nanoparticles: synthesis, characterization, properties, applications and therapeutic approaches. Int. J. Mol. Sci., 17, 1534. Zhao, G. J. & Stevens, S. E. (1998). Multiple parameters for the comprehensive evaluation of the susceptibility of Escherichia coli to the silver ion. Biometals, 11, 27–32. Zhou, Y., Kong, Y., Kundu, S., Cirillo, J. D. & Liang, H. (2012). Antibacterial activities of gold and silver nanoparticles against Escherichia coli and bacillus Calmette-Guérin. J. Nanobiotechnol., 10, 19.

BIOGRAPHICAL SKETCH Armen Trchounian Affiliation: Yerevan State University, Yerevan; Russian-Armenian (Slavonic) University, Yerevan, Armenia Education: Yerevan State University, MSc. in Biophysics, PhD in Biophysics, DSc in Biophysics Business Address: 1 A. Manoukian Str., Yerevan 0025, Armenia Previous Positions: 

2011-2016: Head, Department of Microbiology and Plants and Microorganisms Biotechnology, Yerevan State University


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1993-2011: Professor (Full Professor); 1990-1994 Leading Researcher (Research Professor); 1981-1990 Senior, Junior Researcher, Department of Biophysics, Yerevan State University

Visiting Positions: 

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2016; 2013: Visiting Scholar, Institute of Biology/Microbiology, Martin Luther University of Halle-Wittenberg, Halle (Saale), Germany (c/o Prof. R. Gary Sawers) 2001; 1998-1999: Visiting Professor, School of Animal and Microbial Sciences, The University of Reading, Reading RG6 6AJ, UK (c/o Dr. Simon C. Andrews) 1999-2000: Visiting Fellow, Department of Molecular Physiology and Biological Physics, The University of Virginia, Charlottesville VA 22908, USA (c/o Prof. Dr. Robert K. Nakamoto) 1998: Invited Professor, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263, Japan (c/o Prof. Dr. Hiroshi Kobayashi)

Professional Appointments: Head & Professor, Department of Biochemistry, Microbiology and Biotechnology, Faculty of Biology, Yerevan State University, Yerevan; Supervisor & Leading Researcher, Laboratory of Microbiology, Bioenergetics and Biotechnology, Research Institute of Biology; Yerevan State University, Yerevan; Professor, Department of Medical Biochemistry and Biotechnology, RussianArmenian (Slavonic) State University, Yerevan Honors: Awards  

2003: Prize of the President of RA for the Achievement in Natural Sciences 2016: Akira Mitsui Award for Leadership in Biological Area of Hydrogen Energy of International Association of Hydrogen Energy


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Armen Trchounian, Lilit Gabrielyan and Narine Mnatsakanyan 

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2014: The outstanding service award for altruistic contribution to the cause of Hydrogen Economy of International Association of Hydrogen Energy 2011: The scientific award of excellence – 2011 of the American Biographical Institute (USA) in the field of Biophysics & Biotechnology 2010: Award of the National Academy of Sciences of RA, Ministry of Diaspora of RA, and Armenians Union of Russia for the best scientific paper in Natural Sciences

Orders and Medals     

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2004: Honor Cross Order of Merit of the European Academy of Natural Sciences (Germany) 2016: Gold Medal of the State Committee of Science, Ministry of Education and Science of RA 2004: Memorable Gold Medal of the Ministry of Education and Science of RA 2016: Medal of Prokhorov Academy of Engineering Sciences “For contribution to the development of engineering sciences” (Russia) 2011, 2008, 2003: Robert Koch Medal, Vladimir Negovsky Medal, Rudolf Virchow Medal, European Academy of Natural Sciences (Germany) 2010: Khachatur Abovyan Medal, Armenian Pedagogical State University, Yerevan 2008: Honorary Medal, Armenian Engineering Academy 2007: Academician N. M. Sissakian Medal, Russian Academy of Natural Sciences for the Achievement in Biochemistry and Space Biomedicine (Russia) 2006: Gold Medals of Yerevan State University and Yerevan State Architecture-Construction University, 2005: Gold Medal of Yerevan State Medical University, Memorable Medal of Yerevan State Linguistic University; Honor



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Deed of the Chairman of the National Assembly of RA for scientific achievements Publications from the Last 3 Years: Reviews Trchounian K., Sawers R. G., Trchounian A. (2017). Improving biohydrogen productivity by microbial dark- and photo-fermentations: novel data and future approaches. Renew. Sustain. Energy Rev. 80, 1201-1216. Soghomonyan D., Trchounian K., Trchounian A. (2016). Millimeter waves or extremely high frequency electromagnetic fields in the environment: what are their effects on bacteria? Appl. Microb. Biotechnol. 100, 47614771. Trchounian A. (2015). Mechanisms for hydrogen production by different bacteria during mixed-acid and photo-fermentation and perspectives of hydrogen production biotechnology. Crit. Rev. Biotechnol. 35, 103-113. Trchounian K., Trchounian A. (2015). Hydrogen production from glycerol by Escherichia coli and other bacteria: An overview and perspectives. Appl. Energy 156, 174-184. Original Papers Abrahamyan V., Poladyan A., Vassilian A., Trchounian A. (2015). Hydrogen production by Escherichia coli during glucose fermentation: effects of oxidative and reductive routes using the strain lacked of hydrogen oxidizing hydrogenases 1 (hya) and 2 (hyb). Int. J. Hydrogen Energy 40, 7459-7464. Aghajanyan A., Movsisyan Z., Trchounian A. (2017). Antihyperglycemic and antihyperlipidemic activity of hydroponic Stevia rebaudiana aqueous extract in hyperglycemia induced by immobilization stress in rabbits. BioMed Research Intern. 2017, ID 9251358, 6 p.


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Avakyan S. V., Kovalenok V. V., Savinykh V. P., Ivanchenkov A. S., Voronin N. A., Trchounian A., Baranova L. A. (2015). The role of a space patrol of solar X-ray radiation in the Provisioning of the safety of orbital and interplanetary manned space flights. Acta Astronautica 109, 194-202. Avetisyan A., Markosian A., Petrosyan M., Sahakyan N., Babayan A., Aloyan S., Trchounian A. (2017). Chemical composition and some biological activities of the essential oils from basil Ocimum different cultivars. BMC Compl. Altern. Med. 17, 60. Avtandilyan N., Javrusyan H., Petrosyan G., Trchounian A. (2018). The involvement of arginase and nitric oxide synthase in breast cancer development: arginase and no-synthase as therapeutic targets in Cancer. BioMed Research Intern. 2018, ID 8696923. 9 pages. Babayan A., Petrosyan M., Sahakyan N., Trchounian A. (2017). The radical scavenging activity of some species of Artemisia genus represented in Armenian flora. Biolog. J. Armenia 69(1), 96-99. Blbulyan S., Trchounian A. (2015). Impact of membrane-associated hydrogenases on the FoF1-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: atpase activity and its inhibition by N,N’-dicyclohexylcarbodiimide in the mutants lacking hydrogenases. Arch. Biochem. Biophys. 579, 67-72. Gabrielyan L., Hakobyan L., Trchounian A. (2016). Comparative effects of Ni(II) and Cu(II) ions and their combinations on redox potential and hydrogen photoproduction by Rhodobacter sphaeroides. J. Photochem. Photobiol. B: Biology 164, 271-275. Gabrielyan L., Hakobyan L., Trchounian A. (2017). Characterization of light-dependent hydrogen production by new green microalga Parachlorella kessleri in various conditions. J. Photochem. Photobiol. B: Biology 175, 207-210. Gabrielyan L., Sargsyan H., Trchounian A. (2015). Novel properties of photofermentative biohydrogen production by purple bacteria Rhodobacter sphaeroides: effects of protonophores and inhibitors of responsible enzymes. Microb. Cell Factories 14, 131.



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Gabrielyan L., Sargsyan H., Trchounian A. (2016). Biohydrogen production by purple non-sulfur bacteria: Effect of low-intensity electromagnetic irradiation. J. Photochem. Photobiol. B: Biology 162, 592-596. Ghazaryan A., Blbulyan S., Poladyan A., Trchounian A. (2015). Redox stress in geobacilli from geothermal springs: phenomenon and membrane-associated response mechanisms. Bioelectrochemistry 105, 1-6. Ginovyan M., Keryan A., Bazukyan I., Ghazaryan P., Trchounian A. (2015). The large scale antibacterial, antifungal and anti-phage efficiency of Petamcin-A: New multicomponent preparation for skin diseases treatment. Ann. Clin. Microbiol. Anrimicrob. 14, 28. Ginovyan M., Petrosyan M., Trchounian A. (2017). Antimicrobial activity of some plant materials used in Armenian traditional medicine. BMC Compl. Altern. Med. 17, 50. Ginovyan M., Trchounian A. (2017). Screening of some plant materials used in Armenian traditional medicine for their antimicrobial activity. Proc. Yerevan State Univ. Chem. Biology 51, 44-53. Griffin S., Alkhayer R., Mirzoyan S., Turabyan A., Zucca P., Sarfraz M. S., Nasim M.J., Trchounian A., Rescigno A., Keck C. M., Jacob C. (2017). Nanosizing Cynomorium: Thumbs up for potential antifungal applications. Inventions 2, 24. Hakobyan L., Gabrielyan L., Trchounian A. (2017). Bio-hydrogen production by Rhodobacter sphaeroides during mixed carbon fermentation. Biolog. J. Armenia 69(1), 110-113. Hakobyan L., Grigoryan K., Trchounian A. (2017). The content of sulfur dioxide in dried vine fruit realized in Armenia. Biolog. J. Armenia 69(1) 113-117. Hakobyan L., Harutyunyan K., Harutyunyan N., Melik-Andreasyan G., Trchounian A. (2016). Adhesive properties and acid forming activity of lactobacilli and streptococci under inhibitory substances, such as nitrates. Curr. Microbiol. 72, 776-782. Hakobyan L., Hatutyunyan N., Harutyunyan K., Trchounian A. (2016). Microbiological and adhesive properties of Armenian traditional


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fermented milk products, matsun and yogurt. Reports Natl. Acad. Sci Armenia 116, 154-167. Hakobyan L., Sargsyan H., Gabrielyan L., Trchounian A. (2016). The effect of Cu (I) and Cu (II) ions’ low concentrations on growth, biohydrogen production and the FoF1-ATPase activity of Rhodobacter sphaeroides. Int. J. Hydrogen Energy 41, 16807-16812. Hovhannisyan P., Turabyan A., Panosyan H., Trchounian A. (2016). Thermostable amylase producing bacilli isolated from Armenian geothermal springs. Biolog. J. Armenia 68 (Special Issue), 68-73. Hovnanyan K., Hovnanyan M., Trchounian A. (2017). Electron microscopic study of nanolike bacteria. Open Access Library J. 4(05), e3485. Hovnanyan K., Kalantaryan V., Trchounian A. (2017). The distinguishing effects of low intensity electromagnetic radiation of different extremely high frequencies on Enterococcus hirae: growth rate inhibition and scanning electron microscopy analysis. Lett. Appl. Microbiol. 65, 220225. Javrushyan H., Avtandilyan N., Mamikonyan A., Trchounian A. (2017). The change of polyamines and nitric oxide quantities in human blood serum of prostate and bladder cancer.Biolog. J. Armenia 69(1), 125-132. Karapetyan H., Barsegyan E., Trchounian A. (2017). Antioxidant properties of stevia (Stevia redaudiana Bertoni) during immobilization stress. Biolog. J. Armenia 69(1), 133-137. Keryan A., Bazukyan I., Trchounian A. (2017). Lactobacilli isolated from the Armenian fermented milk product matsoun: Growth properties, antibacterial and proteolytic activity and their dependence on pH. Int. J. Dairy Technol. 70, 289-398, Margaryan A., Badalyan H., Trchounian A. (2016). Comparative analysis of UV irradiation effects on Escherichia coli and Pseudomonas aeruginosa bacterial cells utilizing biological and computational approaches. Cell Biochem. Biophys. 74, 381-389. Mirzoyan S., Romero Parekha M. Coello M. D., Trchounian A., Trchounian K. (2017). Evidence for hydrogenase-4 catalyzed biohydrogen production in Escherichia coli. Int. J. Hydrogen Energy 42, 2169721703.



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Mirzoyan S., Vassilian A., Trchounian A., Trchounian K. (2018). Prolongation of H2 production during mixed carbon sources fermentation in E. coli batch cultures: new findings and role of different hydrogenases. Int. J. Hydrogen Energy 43, 8739-8746. Mnatsakanyan N., Trchounian A. (2016). Antibacterial activity of the nanocomposite filter based on porous mineral tuff and silver nanoparticles against gram-positive and gram-negative bacteria: the different effects and dependence on the concentration of silver. Rep. Natl. Acad. Sci. Armenia 116, 327-334. Mnatsakanyan N., Trchounian A. (2017). Preservation of sour-cream by colloidal silver. Food Processing Industry 2, 20-23. Mnatsakanyan N., Trchounian A. (2018). Nanocomposite filter made from porous mineral tuff with absorbed silver nanoparticles and its application for disinfection of water. J. Water Supply: Science and Technol. 67, 127-136. Navasardyan L., Marutyan S., Hovnanyan K., Trchounian A. (2017). Survival and changes in morphology, mitotic and metabolic activity of yeasts Candida guilliermondii exposed to X-irradiation. Ind. J. Biochem. Biophys. 54, 273-280 Panosyan H., Hakobyan A., Birkeland N.-K., Trchounian A. (2018). Bacilli community of saline-alkaline soils of Ararat plain (Armenia) assessed by molecular and culture-based methods. Syst. Appl. Microbiol. 41, 232240. Panosyan H., Margaryan A., Trchounian A. (2017). Denaturating gradient gel electrophoresis (DGGE) profiles of the partial 16S rRNA genes difenied bacterial population inhabitating in Armenian geothermal springs. Biolog. J. Armenia 69(3), 102-109. Petrosyan M., Sherbakovs Y., Sahakyan N., Vardanyan Z., Poladyan A., Popov Yu., Trchounian A. (2015). Alkanna orientalis (L.) Boiss. plant isolated cultures and antimicrobial activity of their extracts: Phenomenon, dependence on different factors and effects on some membrane-associated properties of bacteria. Plant Cell, Tissues and Organ Culture - J. Plant Biotechnol. 122, 727-738.


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Poladyan A., Trchounian K., Vassilian A., Trchounian A. (2018). Hydrogen production by Escherichia coli using brewery waste: optimal pretreatment of waste and role of different hydrogenases. Renew. Energy 115, 931-936. Sargsyan H., Gabrielyan L., Hakobyan L., Trchounian A. (2015). Light-dark duration alternation effects on Rhodobacter sphaeroides growth, membrane properties and bio-hydrogen production in batch culture. Int. J. Hydrogen Energy 40, 4084-4091. Sargsyan H., Gabrielyan L., Trchounian A. (2016). The distillers’ grains with solubles as a perspective substrate for obtaining biomass and producing bio-hydrogen by Rhodobacter sphaeroides. Biomass & Bioenergy 90, 90-94. Sargsyan H., Trchounian K., Gabrielyan L., Trchounian A. (2016). Novel approach of ethanol waste utilization: biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains. Int. J. Hydrogen Energy 41, 2377-2382. Semerjyan I., Petrosyan M., Sahakyan N., Trchounian A. (2017). Crupina vulgaris cass. in vitro culture obtaining. Biolog. J. Armenia 69(1), 163166. Shahinyan G., Margaryan A., Panosyan H., Trchounian A. (2017) Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs. BMC Microbiology 17, 103. Shahinyan G., Panosyan H., Trchounian A. (2015) Characterization of lipase producing thermophilic bacilli isolated from Armenian geothermal springs. Reports Natl. Acad. Sci. Armenia 115, 59-68. Sharifi Alghabpoor S. F., Trchounian A., Hosseinpoor M. (2015) Production of new thermostable and acid stable alpha-amylase from Bacillus sp. b1 and b2 under solid state fermentation. Eur. J. Biotechnol. Biosci. 3(7), 8-11. Soghomonyan D., Baghdasaryan M., Trchounian A. (2016). The combined effects of electromagnetic waves and sulphur dioxide on wine lactic acid bacteria. Biolog. J. Armenia 68 (special Issue), 110-113.



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Sogomonyan D., Margaryan A., Trchounian K., Ohanyan K., Badalyan H., Trchounian A. (2018). The Effects of low doses of gamma-radiation on growth and membrane activity of Pseudomonas aeruginosa GRP3 and Escherichia coli M17. Cell Biochem. Biophys. 76, 209-217. Toplaghaltsyan A., Bazukyan I., Trchounian A. (2017). The effects of different carbon sources on the antifungal activity by lactic acid bacteria. Curr. Microbiol. 74, 168-174. Torgomyan H., Trchounian A. (2015). The enhanced effects of antibiotics irradiated of extremely high frequency electromagnetic field on Escherichia coli growth properties. Cell Biochem. Biophys. 71, 419-424. Trchounian K., Abrahamyan V., Poladyan A., Trchounian A. (2015). Escherichia coli growth and hydrogen production in batch culture upon formate alone and with glycerol co-fermentation at different pHs. Int. J. Hydrogen Energy 40, 9935-9941. Trchounian K., Mirzoyan S., Poladyan A., Trchounian A. (2017). Hydrogen production by Escherichia coli growing in different nutrient media with glycerol: Effects of formate, pH, production kinetics and hydrogenases involved. Int. J. Hydrogen Energy 42, 24026-24034. Trchounian K., Muller N., Schink B., Trchounian A. (2017). Glycerol and mixture of carbon sources conversion to hydrogen by Clostridium beijerinckii DSM791 and effects of various heavy metals on hydrogenase activity // Int. J. Hydrogen Energy 42, 7875-7882. Trchounian K., Poladyan A., Trchounian A. (2016). Optimizing strategy for Escherichia coli growth and hydrogen production during glycerol fermentation in batch culture: effects of some heavy metal ions and their mixtures. Appl. Energy 177, 335-340. Trchounian K., Poladyan A., Trchounian A. (2017). Enhancement of Escherichia coli bacterial biomass and hydrogen production by some heavy metal ions and their mixtures during glycerol vs glucose fermentation at a relatively wide range of pH. Int. J. Hydrogen Energy 42, 6590-6597. Trchounian K., Sargsyan H., Trchounian A. (2015). H2 production by Escherichia coli batch cultures during utilization of acetate and mixture of glycerol and acetate. Int. J. Hydrogen Energy 40, 12187-12192.


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Trchounian K., Trchounian A. (2015). Escherichia coli hydrogen gas production from glycerol: effects of external formate. Renew. Energy 83, 345-351. Vardanyan Z., Gevorkyan V., Ananyan M., Vardapetyan H., Trchounian A. (2015). Effects of various heavy metal nanoparticles on Enterococcus hirae and Escherichia coli growth and proton-coupled membrane transport. J. Nanobiotechnology 13, 69. Vardanyan Z., Trchounian A. (2015). Cu(II), Fe(III) and Mn(II) combinations as environment stress factors have distinguishing effects on Enterococcus hirae. J. Environm. Sci. 28, 95-100.




Chapter 5

METAL NANOPARTICLES-POLYMER COMPOSITES AND THEIR FUEL CELL APPLICATIONS Mohamed R. Berber1,2,* and Inas H. Hafez3 1

International Institue for Carbon-Neutral Energy Research, Kyushu University, Fukuoka, Japan 2 Department of Chemistry, Faculty of Science, Tanta University, Tanta, Egypt 3 Department of Natural resources and Agricultural Engineering, Faculty of Agriculture, Damanhour University, Damanhour, Egypt

ABSTRACT Polymer electrolyte fuel cells (PEFCs) are receiving much interest because they provide an efficient, a clean and an environmentally friendly *

Corresponding Author Email: [email protected]; and [email protected].


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Mohamed R. Berber and Inas H. Hafez power source with high energy density. The main roadblock that obstacle the wide spread application of PEFCs is the metal catalyst. The catalyst usually facilitates the reaction of the oxidant (oxygen or air) and the fuel (hydrogen or alcohol), in order to produce electricity with zero emission of carbon dioxide. The most efficient catalyst for PEFCs is platinum-based materials. Platinum (Pt) is currently the only efficient metal-catalyst that can withstand in the acidic environment of PEFCs. It is a very expensive material, in addition, its price is almost controlled by two countries, i.e., the republic of Russia and South-Africa. Accordingly, Pt-metal catalyst limits the wide spread application of PEFCs. In order to promote the the application fuel cell technology, it is important to optimize the useefficiency of Pt-metal through the nanoscience technology. In this chapter, we focus on the development of Pt-metal nanoparticles and their core-shell structures which are fabricated by different synthetic techniques. We discuss in details the polymeric composite of the metal nanoparticles on the surface of different carbon support materials (e.g., carbon nanotubes, graphene, carbon-nanospheres, mesoporous carbon, and carbon nanofibers), showing the synergetic effects on the active surface area of Pt metal catalyst. We also focus on the Pt nanosized controlling routes which are receiving a great deal of attention in the way of fabricating of highly active metal catalysts. In addition, we highlight the factors affecting the physicochemical properties of the Pt core-shell structure, and how these factors affect the fuel cell performance. Furthermore, we summarize the recent research results of some other metal-catalyst nanoparticles (e.g., Palladium, nickel, gold, and iron) used for the different fuel cell types (e.g., alkaline fuel cells, direct methanol fuel cells, and direct-ethanol fuel cells).

1. FUEL CELLS Unlike the internal combustion engine that converts kinetic energy (produced from fuel burning) into electrical energy, a fuel cell is an electrochemical device that converts the chemical energy stored in some molecules into electrical energy. Also, unlike batteries, fuel cells do not need to be recharged. They only require a continuous source of fuel to produce electricity.


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2. TYPES OF FUEL CELLS There are mainly five types of fuel cells. Polymer electrolyte membrane fuel cells (PEMFCs), phosphoric acid fuel cells (PAFCs), alkaline based fuel cells (AFCs), molten carbonate fuel cells (MCFCs), and solid oxide fuel cells (SOFCs). All the five types of fuel cells are working under the same electrochemical principles in which the fuels and the oxidants are chemically combined together to produce electricity. The differences between these main five types of fuel cells are mainly found in the electrolyte type, the charge carrier, the operating temperature, and the catalyst as shown in Table 1. Table 1. The main five types of fuel cells

3. COMPONENTS OF FUEL CELLS As seen from Figure 1, PEFC compose of an anode electrode, an polymer electrolyte membrane, and a cathode electrode. The fuel is oxidized at the anode side, producing electrons and protons. The polymeric electrolyte membrane allows a smooth proton transfer process from the anode-side to cathode-side, while the electrons are passed through an external circuit, producing electricity. At the cathode side, the oxidant is reduced by reacting with the protons coming from the anode, and then water is formed.


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Hydrogen is the simplest fuel that can be used in PEMFCs. When methanol or ethanol are used as fuels, the cell is then named an alcoholbased fuel cell. When liquid phosphoric acid is used as an electrolyte, the cell is then named phosphoric acid fuel cell. When an alkaline water solution (e.g., potassium hydroxide solution or sodium hydroxide solution) or an alkaline-based membrane are used as electrolytes instead of the acid electrolyte membrane, the fuel cell is then named alkaline-based fuel cell. When the electrolyte is composed of a molten carbonate suspended in a chemically inert porous ceramic of lithium aluminium oxide matrix, the fuel cell is then named molten carbonate fuel cell. Finally, when a nonporous ceramic compound is used as an electrolyte, the cell is then named solid oxide fuel cell. When it comes to the catalyst side, it is important to say, this is the key material that facilitates the reaction of the fuel with the oxidant to produce the electricity. The catalysts used in fuel cells are usually made of noble metals. In this chapter, we will focus on the different types of metal catalysts, their properties and their nanoparticle synthetic techniques.

Figure 1. A schematic illustration of fuel cell components and its working process.



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4. METAL CATALYSTS FOR FUEL CELLS With no doubt the birth of nanoscience technology has brought a great change to the applications of metals catalysts. Metal-based electrodes have extremely progressed and the fuel cell market has grown very much since the metal catalyst has reached a nanosized structure. Thus, the story is behind the development of new synthetic techniques to improve the catalytic activity and the active site density of the catalysts. In addition, the nanostructure makes a cost reduction of the catalyst, and accordingly facilitates the mass production process of the fuel cell technology.

5. PLATINUM METAL CATALYST Platinum (Pt) is a precious metal with a noticeable chemical stability to corrosion and solubility even at harsh conditions. Pt is considered as an important catalyst for many chemical reactions. Because of its scarcity in Earth's crust and given the priority of uses, few tonnes are annually extracted. The current fuel cell technology is mainly depending on Pt as a catalyst where it efficiently catalyze the combination of hydrogen and oxygen to produce electricity. Typically, Pt accelerates the hydrogen splitting into protons and electrons and also promotes the splitting of oxygen through an oxygen reduction reaction. The following paragraphs will focus on the synthetic techniques of Pt nanoparticles (Pt-NPs) in order to use it as a nanocatalyst for fuel cell technology.

6. PT NANOPARTICLES (PT-NPS) Pt-NPs are platinum particles in a nanosized form (cubes, small rods or spheres) of liquid colloids (Figure 2). They can be synthesized by a reduction process of platinum ion precursors in a solution with a stabilizing agent to


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form colloidal nanoparticles, or by an impregnation technique on microporous carbon support materials. A reduction process of Pt-ion is a technique in which Pt-ion is reduced to elemental Pt using for example ethylene glycol as a reducing agent in a process called polyol. This technique is used to control the morphology of the Pt-NPs, providing a control of the Pt electronic, chemical, and magnetic properties. Figure 2 shows the TEM images of cubic Pt-NPs fabricated under argon and nitrogen gases. The grown Pt-NPs have showed a cubic morphology with a size of 7 to 10 nm [1]. Cubic Pt-NPs were also fabricated by a modified polyol technique, showing very sharp shapes of Pt cube. The Pt crystals have grown into cubic and octahedral shapes during a very short time. Large cubic and octahedral Pt-NPs of around 160 nm diameter were also recorded. The formation of a large size cubic Pt was due to an aggregation process of Pt clusters [2]. Cubic Pt-NPs were also fabricated with very high Pt surface areas for catalysis. In one study, the utilization efficiency of Pt has increased from 9.5 to 26.0% by reducing the edge-length of Pt-cube from 11.7 to 3.9 nm [3]. Despite the extensive use of this strategy, it was difficult to improve the specific activity of Pt-NPs by engineering their surface structure.

Figure 2. TEM images of cubic Pt-NPs when the polyol synthesis was conducted under (a) argon and (b) nitrogen protection. Reprinted with a permission from (Nano Lett., Vol. 4, No. 12, 2004). Copyright (2004) American Chemical Society [1].



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As known, the nanostructure of the electrode has a great influence on the fuel cells performance. Pt-nanorods for example have showed an enhanced catalytic activity in the case of aligned Pt-nanorods arrays for methanol fuel cells [4]. The improved fuel cell performance of this system has been ascribed to the highly ordered nanostructured-electrode, which has led to an increase in the active electrochemical surface area and the catalyst utilization efficiency, in addition to an enhancement of the oxygen mass transfer and water balance in the system. Pt metal dispersion on carbon supports is also an important parameter that determines the catalytic activity of the metal catalyst. The choice and the modification of the reagents and the carbon support material can strongly affect the catalyst behaviour during the fuel cell operation. Carbon black is widely used as a support for Pt metal catalyst. Specific treatments can be applied to increase or decrease the number of active sites available on the surface of carbon black. In other words, carbon black surface can be modified to introduce functional groups that act as binding sites for Pt immobilization. Besides the chemical nature of the surface of the carbon support, Pt-NP size is also influenced by the catalyst preparation technique and the handling process of the metal catalyst. In the following paragraphs, we show different carbon support materials that are functionalized with polymers in order to get homogenously distributed PtNPs with enhanced active surface area for fuel cell applications. Carbon nanotubes are currently receiving a great deal of attention for fuel cell applications due to their graphitized structure together with the relatively high electrical conductivity. In addition, its fibrous structure is advantageous for a smooth gas diffusion during the fuel cell operation [5]. Since pristine carbon nanotubes have a smooth surface with a defect-free structure. Thus, Pt-NPs aggregate during their immobilization process (see Figure 3). Accordingly, carbon nanotubes need to be functionalized before use as a metal-catalyst support [6]. Carbon nanotubes can be functionalized by chemical and physical techniques. To avoid the destructive functionalization techniques (chemical processes), various polymers, such as polyaniline [7] polyallylamine hydrochloride [8] polypyrrole [9] poly(diallyldimethylammonium chloride) [10] and chitosan


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[11] have been physically introduced to the surfaces of carbon nanotubes in order to bring binding sites for metal catalyst loading.

Figure 3. TEM image of Pt-NPs immobilized on the surface of pristine carbon nanotubes (unpublished data).

Berber el al., [12] have recently showed an advanced strategy of polymer wrapping of carbon nanotubes. In their work, a very thin polymer layer of polybenzimidazole has worked both as a binder for a highly dispersed Pt-NPs and as a proton conductor in the catalyst layer, facilitating the proton transfer into the membrane electrode assembly (Figure 4). The structure of the prepared Pt nanocomposite has been confirmed by X-ray photoelectron spectroscopy (Figure 5), showing metallic Pt-NPs and a very thin layer of the polymer around carbon nanotubes. This newly fabricated



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catalyst has showed both a remarkable fuel cell performance (Figure 6) and durability (Figure 7).

Figure 4. A TEM image of carbon nanotubes-polymer-Pt composites and their particle size histogram. Figure inset shows a schematic illustration of the polymer wrapped carbon nanotubes with Pt-NPs. Reprinted with permission from (Berber et al., J. Mater. Chem. A, 2014, 2, 19053-19059). Copyright (2014) Royal Society of Chemistry [12].

Figure 5. X-ray photoelectron spectroscopy (survey scan) of polymer-wrapped carbon nanotubes Pt-NPs. Figure insets show the narrow scans of Pt and N-atoms of the polymer layer. Reprinted with permission from (Berber et al., J. Mater. Chem. A, 2014, 2, 19053-19059). Copyright (2014) Royal Society of Chemistry.


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Figure 6. Fuel cell performance (potential curve at high temperature, 120º C) of the polymer-wrapped carbon nanotubes Pt-NPs. Reprinted with permission from (Berber et al., J. Mater. Chem. A, 2014, 2, 19053-19059). Copyright (2014) Royal Society of Chemistry.

Figure 7. Polarization curves of membrane electrode assembly of polymer-wrapped carbon nanotubes Pt-NPs composites at high temperature (120º C) during stress testing of the composite. Reprinted with permission from (Berber et al., J. Mater. Chem. A, 2014, 2, 19053-19059). Copyright (2014) Royal Society of Chemistry.



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Table 2. The PT-NPs particle size, electrochemical surface area, theoretical surface area and utilization efficiency for the CNT/PyPBI/Pt

Source: Reprinted with permission from (Sci Rep., Vol. 4, article no 6295, 2014). Copyright (2014), Nature.

Figure 8. A schematic illustration of the immobilization procedure of PT-NPs on the surface of polymer (polybenzimidazole) wrapped carbon nanotubes. Reprinted with permission from (Hafez et al., Sci Rep., Vol. 4, article no 6295, 2014). Copyright (2014), Nature [13].


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In a bottom up approach, Hafez et al., have showed an advanced technique of controlling Pt-NPs size (Figure 8) on a surface of polymer wrapped carbon nanotubes, providing a high surface area and an enhanced mass activity of Pt-NPs [13]. Typically, the Pt-NPs were immobilized on the surface of conducting polymer wrapped carbon nanotubes. The size of PTNPs has ranged from 4 nm to 2.5 nm (Figure 9).

Figure 9. TEM images Pt-NPs with different particle sizes prepared by polyol method. Reprinted with permission from (Hafez et al., Sci Rep., Vol. 4, article no 6295, 2014). Copyright (2014), Nature [13].



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As a result of Hafez et al. approach [13], a polymer electrolyte fuel cell employing the smallest Pt-NPs (2.3 nm) has exhibited 8 times higher mass activity in comparison to a cell with 3.7 nm Pt-NPs (see Table 2).

Figure 10. Cell voltage polymer coated carbon black and (grey curve) and non-coated carbon black (black curve) at 0.2 A/cm2. Reprinted with permission from (ACS Appl. Mater. Interfaces, 8 (23), 14494–14502, 2016). Copyright (2016), American Chemical Society.

Figure 11. A schematic illustration of the polymer coating of carbon black. Reprinted with permission from (ACS Appl. Mater. Interfaces, 8 (23), 14494–14502, 2016). Copyright (2016), American Chemical Society.


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In another approach, Fujigaya et al., have shown stable Pt-NPs with a remarkable durability on carbon black support (Figure 10). In their study, the polymer coating of carbon black has capped the gates of CB micropores, preventing the penetration of the electrolyte into the micropores. Figure 11 shows a schematic illustration of the polymer coating of carbon black. This process has avoided the oxidation of the interior of the carbon black micropore. Thus, it demolished the decomposition of carbon black [14]. Durability and stability of Pt-NPs are very important issues for fuel cell technology. Pt-NPs usually suffer from sintering and agglomeration during the fuel cell operation, leading to a low durable fuel cell. Berber et al., have showed the degradation mechanism of Pt-NPs supported on carbon materials [12]. For that purpose, they studied two different membrane electrode assemblies using pristine carbon nanotubes and commercial carbon black as carbon supports for Pt-NPs. The carbon supports were first wrapped by a polybenzimidazole polymer (Figure 12). Then, Pt-NPs were deposited on the surface of the polymer wrapped carbon. The fabricated catalysts were monitored by different spectroscopic techniques before and after durability testing. After 15,000 potential cycles of both membrane electrode assemblies (see Figure 13), it has been observed that the cell voltage of the carbon blackbased membrane electrode assembly drastically decreased compared to carbon nanotubes-based membrane electrode assembly which exhibited a gradual decrease upon cycling. This durability difference was related to the difference in the oxidation stability of the carbon supports.

Figure 12. The chemical structure of polybenzimidazole-based polymers. In the case of X=H, the polymer is named Benzene-based polybenzimidazole. In the case of X=N, the polymer is named pyridine-based polybenzimidazole.



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Figure 13. The cell voltage at 200 mA cm-2 as a function of the number of potential cycling for carbon black-based membrane electrode assembly (grey color) and carbon nanotubes-based membrane electrode assembly (black color) during durability testing. Reprinted with permission from (Berber et al., J. Mater. Chem. A, 2014, 2, 1905319059). Copyright (2014) Royal Society of Chemistry.

Figure 14. TEM images of the catalysts derived from carbon black-based membrane electrode assembly and carbon nanotubes-based membrane electrode assembly, and their particle size histogram distributions. Reprinted with permission from (Berber et al., J. Mater. Chem. A, 2014, 2, 19053-19059). Copyright (2014) Royal Society of Chemistry.


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The TEM analysis of two membrane electrode assemblies (Figure 14) has showed that upon potential cycling, an accelerated fusion process of the neighboring Pt-NPs occurs, leading to the formation of bigger Pt particles. The increase of the Pt particle size as seen from Figure 14 was more obvious in the case of the carbon black-based membrane electrode assembly where it reached 6.24 nm compared to 5.55 nm for the carbon nanotubesbased membrane electrode assembly. Such an increase in the Pt size of the carbon black-based membrane electrode assembly would be due to the faster corrosion process of the carbon black. The XRD pattern of the CB-based electrocatalyst (Figure 15) has showed an obvious broadening in the graphitic (002) peak after 12,000 potential cycles, confirming the fast corrosion process of the carbon blackbased catalyst. In a sharp contrast, such a broadening in the graphitic (002) peak was not observed in the carbon nanotubes-based catalyst after the same number of potential cycles. Accordingly, it was obvious that the corrosion of the carbon support has played a crucial role in the Pt aggregation. The aggregation was much higher in the case of carbon black support compared to carbon nanotube support. Also, it was clear that the polymer wrapping of carbon support demolish the Pt aggregation as shown in Berber et al., work [12].

Figure 15. X-ray patterns of the catalysts derived from carbon black-based membrane electrode assembly (a) and carbon nanotubes-based membrane electrode assembly (b). Reprinted with permission from (Berber et al., J. Mater. Chem. A, 2014, 2, 1905319059). Copyright (2014) Royal Society of Chemistry.



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Based on such studies [12], more advanced protective ways are still required to enhance the nanoparticles stability on the surface of carbon supports. In a bottom up synthetic protocol, Berber et al., [15] have showed highly stable carbon nanotubes and carbon black catalysts. In their study, PtNPs have been sandwiched between two conductive polymers (Figure 16). The carbon support material was first coated with pyridine-based polybenzimidazole polymer, then another thin layer of Nafion polymer was added on the surface of the composite. The interaction between the two polymers was then confirmed by FT-IR spectroscopy (Figure 17).

Figure 16. (a) The structure of polymer used to wrap the carbon support material (PyPBI). (b,c) Schematic illustrations for the preparation of carbon nanotubes and carbon black with two conductive polymers. Reprinted with permission from (Berber et al., Sci Rep., Vol. 5, article no 16711, 2015). Copyright (2015), Nature.


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Figure 17. Infrared spectra of Nafion polymer (faint grey), PyPBI polymer (grey), and Nafion-PyPBI composite (black). The inset shows the magnification of the low wave number region. Reprinted with permission from (Berber et al., Sci Rep., Vol. 5, article no 16711, 2015). Copyright (2015), Nature.

Figure 18. The durability result of double polymer coated carbon nanotubes-based and carbon black-based catalysts compared to the commercial carbon black-Pt catalyst. Reprinted with permission from (Berber et al., Sci Rep., Vol. 5, article no 16711, 2015). Copyright (2015), Nature.



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As a result of this double polymer coating technique, an improvement in durability was achieved (see Figure 18). The fuel cell performance of the double polymer wrapped carbon nanotubes-based membrane electrode assembly was slightly declined after 500000 potential cycles. After this huge number of potential-dynamic cycling, a 5% loss of the initial potential was observed. Additionally, the power density was decreased by only a 20% of the initial value. Further investigations of the double polymer wrapping effect on the stability of the fabricated catalysts was observed by Somaye Rasouli et al., [16]. The experimental investigations have showed the mechanism for the loss of electrochemical active surface area. The results have revealed that the electrochemical active surface area of the Pt particles of the double polymer wrapped catalyst has decreased because of the particle motion, followed by coalescence. In contrast, the Pt particles supported on carbon without polymers have suffered from a dissolution and/or a re-deposition process. This is confirmed by comparing the size of the spherical particles in the cathode of the membrane electrode assembly before and after 10,000 cycles. The results have showed no significant growth of Pt-NPs. On the other hand, the number of coalesced particles has increased considerably after the voltage cycling (see Figure 19). The coalescence mechanism of the Pt-NPs was shown in Figure 19. Figure 19a has showed two well separated nanoparticles on the left side of the carbon (square 1). As seen, the nanoparticles have moved until they contacted each other and coalesced via a neck growth. On the other hand (square 2 of Figure 19a and b), the change in the orientation of an individual nanoparticle has been observed after voltage cycling. As a result, a larger fraction of the surface area of Pt-NPs were covered by carbon, and thus become inaccessible to the fuel. Additionally, Figure 19c has showed six individual Pt-NPs (square 1) close to each other with a different orientation. During voltage cycling, the particles have moved toward each other and made a contact to lower the total energy of the grain boundaries, particle rotation or orientation alignment of the planes at the interface of the particles. This has led to the formation of a larger single crystal particle (Figure 19d).


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Figure 19. Aberration corrected TEM images of the Pt-NPs on double polymer wrapped carbon before and after 1000 voltage cycling. Reprinted with permission from (Microsc. Microanal. 21, Suppl 3, 2015). Copyright (2015), Cambridge Core.

The application of polymer supported Pt-NPs for other fuel cell types were also reported. Yang et al., [17] have showed a very high CO tolerance and a remarkable durability during the direct methanol oxidation (Figure 20) by using a double polymer-wrapped carbon black catalyst which is fabricated by applying a wrapping process with two conductive polymers



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(polyvinylphosphonic acid and polybenzimidazole). The methanol oxidation reaction (MOR) was measured before and after the durability test in N2-saturated 0.1 M HClO4 and 1 M CH3OH at room temperature. The double polymer wrapping technique of carbon black has exhibited an onset potential less positive than that for the catalyst without the polymer. This result has demonstrated a faster reaction kinetics for methanol oxidation reaction in the case of double polymer wrapped catalyst. The CO stripping results have also indicated that the CO tolerance of double polymer wrapped carbon black/Pt was the highest among the studied three catalysts, in a good agreement with the results of the methanol oxidation measurements.

Figure 20. Methanol oxidation before (solid line) and after (dotted line) the durability test for carbon black/Pt (a), single polymer wrapped carbon black/Pt (b), and double polymer wrapped carbon black/Pt (c). (d) Magnification of methanol oxidation profiles of the three catalysts from 0.27 to 0.7 V vs. RHE.


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7. PLATINUM ALLOYS FOR FUEL CELL APPLICATIONS Pt is currently the most efficient metal catalyst for fuel cell technology. It costs around 17% of the total price of the fuel cell unit. Since Pt is a rare metal with a limited amount on earth crust. Thus, it works as an obstacle for the widespread utilization of fuel cell technology. In order to commercialize the fuel cell technology, a cost reduction of Pt metal is required. A lot of research efforts are currently underway to reduce the contribution of Pt cost. One important strategy is to alloy Pt with other metal catalysts of lower cost, taking into account the catalyst stability and activity. The current research has showed that when Pt is alloyed with other metals like cobalt, nickel, iron, and chromium, an enhancement in the Pt catalytic activity is observed towards reduction and oxidation reactions of fuel cell. This improvement is attributed to a positive shift of onset potential for hydroxyl formation on the alloy [18]. During the synthesis of Pt alloy, a special care is devoted to the alloy morphology, composition, and shape. The understanding of such parameters is being critical in formulating highly active and stable Pt-alloys. Normally, synthesis of Pt-alloys is performed by a solution-based process. Thus, precursors type, capping agents, solvents, reducing agents, and supports are strongly affect the kinetics of the reactions. Hence, by controlling one or more of these parameters, the desired alloy product can be obtained [19]. One important issue of Pt-alloys is the composite stability in the acidic environment of PEMFCs. A dissolution process of the non-precious metal of the alloy composite occurs, leading to a collapse of the alloy structure, and accordingly, a decrease in the catalytic activity of the composite catalyst. The post treatment of the Pt-alloy has showed an improvement in the catalyst stability, fuel cell performance and durability. This improvement is attributed to a reduction of the dissolution rate of the non-noble metal catalysts. These post treatment includes, calcination of the alloys [20] to strength the bonding system of the unalloyed metal surface that accelerates the alloy collapse. Hence, it is important to say that alloying of Pt controls the crystalline structures and the morphologies of Pt-NPs, in particular Pt surface electronic structure and adsorption properties.



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Figure 21. X-Ray diffraction patterns of the Pt-Co alloys. Reprinted with permission from (J. Mater. Chem., 2004, 14, 1454-1460). Copyright (2004), Royal Society of Chemistry.


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Many carbon-supported Pt-alloy catalysts have been successfully synthesized. Mukerjee and Srinivasan [21] have showed an enhancement in the catalysis of oxygen reduction reaction on carbon supported alloys of Pt in acidic fuel cells. This study revealed an enhanced activity, lower activation energies and different reaction orders for the Pt-alloys compared to pure Pt. One important parameter during their synthesis process was the alloy composition (the ratio of Pt to the other metal). Most Pt-alloys, regardless of the ratio between the Pt metal and the non-noble metal have showed better stability compared to Pt itself. For example, Pt alloyed with chromium has showed much more stable performance than Pt alloys with nickel, resulting in a higher stability in an acidic medium of polymer electrolyte fuel cells. One possible explanation for such a difference is that chromium exhibits a higher degree of alloying with Pt than nickel do [22]. Xiong and Manthiram [23] have also synthesized different fuel cell catalyst alloys of Pt with cobalt metal by changing the ratio of cobalt using borohydride as a reducing agent followed by a calcination process at 900°C. Figure 21, shows the diffraction peaks of the Pt cobalt alloys. As seen, a shift to higher angles was observed for the Pt cobalt alloys compared to that of pure Pt. This result has reflected a lattice contraction arising from the substitution of the smaller Co atoms for the larger Pt atoms. As seen from Figure 21 and 22, Pt-Co alloys with better ordering exhibit better catalytic activity, and this was reflected on the fuel cell performance. Polymers are currently used as stabilizing agents to demolish the agglomeration behavior of Pt alloy nanoparticles supported on carbon materials. Poly(N-vinyl-2-pyrrolidone) has been used as a protecting agent to stabilize the Pt-alloy nanoparticles and prepare a series of carbonsupported Pt nickel alloys with different morphologies and particle size. It forms a complex with Pt metal. Thus, stabilize it during the fuel cell performance [24]. Namgee Jung et al., [25] have developed a highly active and a durable carbon-supported Pt-Co alloy using poly(N-isopropylacrylamide) functionalized-carbon as a support material to selectively modify the surface of the Co atoms with a N-containing polymer as shown in Figure 23.



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Figure 22. Comparison of the performances of the Pt-Co alloys in PEMFC cell: (a) Pt Cobalt alloys prepared by employing ammonium hydroxide, and (b) Pt-Co alloys prepared by employing sodium borohydride. Reprinted with permission from (J. Mater. Chem., 2004, 14, 1454-1460). Copyright (2004), Royal Society of Chemistry.

This composite structure was confirmed by X-ray photoelectron spectroscopy (Figure 24). The results showed the Co-N band as a result of interaction between Co and the polymer. This strategy has showed a highly active and a durable Pt-Co catalysts useful for the oxygen reduction reactions. The electronic structures of Pt and Co have dramatically changed by the selective interaction of poly(N-isopropylacrylamide) with Co.


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The enhanced performance of Pt-Co/N-containing polymer is thus attributed to the N-atoms attached to the Co sites on the Pt-Co nanoparticle surface, which prevented the further oxidation of the Co atoms.

Figure 23. A schematic illustration of the surface modified Pt-Co alloy using poly(Nisopropylacrylamide). Reprinted with permission from (NPG Asia Materials volume 8, page e237, 2016). Copyright (2016), Nature.

Figure 24. X-ray photoelectron spectroscopy (narrow scan of N 1s region). Reprinted with permission from (NPG Asia Materials volume 8, page e237, 2016). Copyright (2016), Nature.



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8. CORE-SHELLS OF PLATINUM METAL FOR FUEL CELL APPLICATIONS Core-shell technique was emerged as a new process of nanomaterial preparation for a cost reduction of Pt catalysts used in polymer electrolyte fuel cells. Pt Core-shell means a Pt monolayer (shell) is deposited on different metal core nanoparticles. This process is considered as one of the key technologies to improve the mass activity of Pt. The targets in this technique is to find a cost-effective and simple procedure for preparation of the Pt-NPs. Pt Core-shell nanoparticles can be prepared by different techniques. Poly(N‐vinyl‐2‐pyrolidone) was used to protect the core-shell structure of Pt/Au nanoparticles, which has been prepared by a multistep reduction process of HAuCl4 and H2PtCl6 alternatively by hydrogen adsorbed on platinum atom [26]. In a bottom up study, Pt/carbon black/polyaniline (PANI) core-shell catalyst was designed to address the durability of fuel cell catalysts. The catalyst was prepared through a direct polymerization of a thin layer of polyaniline on the carbon surface of the Pt/Carbon catalyst as shown in Figure 25. The aniline monomer was first selectively adsorbed onto the carbon surface via preferential π−π conjugation between the aniline and the carbon support and was then polymerized in-situ on the carbon surface via ammonium peroxodisulfate oxidation in acidic solution.

Figure 25. A schematic illustration of Pt/carbon black/polyaniline core-shell catalyst preparation. Reprinted with permission from (J. Am. Chem. Soc. 134, 13252−13255, 2012). Copyright (2012), American Chemical Society.


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Figure 26. I-V curves of a single fuel cell with cathodes fabricated from (a) Pt/Carbon/polyaniline (30%) and (b) Pt/Carbon black catalysts after the indicated numbers of CV cycles. Reprinted with permission from (J. Am. Chem. Soc. 134, 13252−13255, 2012). Copyright (2012), American Chemical Society.

Figure 27. (a) Normalized ECSAs of Pt/Ccarbon and Pt/C/Polyaniline (30%) catalysts as a function of CV cycles. (b−e) TEM images of (b) the uncycled Pt/Carbon catalyst, (c) the Pt/Carbon catalyst after 1500 CV cycles, (d) the uncycled Pt/C/PANI (30%) catalyst, and (e) the Pt/C/PANI (30%) catalyst after 1500 CV cycles. Reprinted with permission from (J. Am. Chem. Soc. 134, 13252−13255, 2012). Copyright (2012), American Chemical Society.



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The experimental results have showed a significant improvement in the stability of the catalyst after polymer treatment (Pt/Carbon/PANI) compared with the unmodified Pt/C catalyst (see Figure 26). In addition, a remarkably improved durability was observed as a result of polymer coating (see Figure 27). The TEM images have confirmed the improved stability in the case of polymer treated Pt/carbon core-shell.

9. GOLD METAL CATALYST Gold (Au) is a transition metal and one of the rarest element on earth crust. One important property of Au is its resistance to corrosion and surface oxidation. Normally, Au is a useless material for catalyst of chemical reactions because it doesn’t do much. When it comes to a nanoscale size, Au can act as a catalyst, bringing unprecedented catalytic activities for many reaction, in particular, carbon monoxide and methanol oxidations. Thus, the smaller the nanoparticle of Au, the larger the proportion of atoms at the surface, and the larger proportion of atoms at the corners of the crystal, and accordingly, the higher the catalytic activity. As a solution for reducing the cost of fuel cell technology, gold nanoparticles (Au-NPs) and their alloys have been recoginzed as an alternative solution for fuel cell catalysis because it is stable at harsh fuel cell conditions. In a recent study by Fujigaya et al., [27] Au-NPs have showed an excellent oxygen reduction activity, thanks to their preparation technique of Au-NPs at nanoscale. In this study, the Au-NPs were immobilized on the surface of polymer-coated graphene through a coordination bonding between Au-NPs and the polymer (see schematic illustration of Figure 28). The advantage of this technique is that the graphitic carbon surfaces of graphene were utilized without further oxidization. The X-ray photoelectron spectra (Figure 29) have revealed the coordination bonding of the polymer with the Au metal. The N1s peak and the Au4f peaks have revealed the successful coating process of graphene with the polymer and the good immobilization of Au metal on the surface of the polymer graphene


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composite. The transmittance electron micrographs with their particle size histograms (Figure 30) have showed the different nanosized structures of the Au-NPs, ranging from 4.5 to 1.6 nm.

Figure 28. A schematic illustration of preparation process of Au-NPs on the surface of polymer-coated graphene. Reprinted with permission from (Sci Rep vol. 6. Article no. 21314, 2016). Copyright (2016), Nature.

Figure 29. X-ray photoelectron spectra of Au-NPs supported on the surface of polymer-coated graphene. Reprinted with permission from (Sci Rep vol. 6. Article no. 21314, 2016). Copyright (2016), Nature.



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Figure 30. TEM images of Au-NPs supported on the surface of polymer-coated graphene, and their particle size histograms. Reprinted with permission from (Sci Rep vol. 6. Article no. 21314, 2016). Copyright (2016), Nature.

10. PALLADIUM NANOPARTICLES FOR FUEL CELLS Palladium is a material with an affinity to absorb large volumetric quantities of hydrogen at room temperature and atmospheric pressure, and subsequently forms a palladium hydride. On palladium surfaces, the dissociative adsorption of hydrogen molecules occurs. The superior dissociative properties of palladium enable it to serve as a catalyst, facilitating hydrogen absorption and desorption in other metal hydrides with a little or no activation energy barrier. Thus, palladium has a potential to play a major role in hydrogen economy, including hydrogen storage and fuel cells [28]. Carbon-supported palladium is currently used as an electrocatalyst in hydrogen based fuel cells that utilize hydrogen as a fuel.


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Thus, palladium is of great interest as a substitute metal catalyst for Pt, not only due to its chemical similarity to Pt, but also because of its significantly reduced cost (approximately 1/5 that of Pt) and 50 fold greater abundance [29]. For the oxygen reduction reaction, palladium-alloys have also demonstrated improved performance when compared to Pt, especially in the presence of methanol. The transition from Pt-based to palladiumbased catalysts has commenced and will likely result in the development of more cost effective materials with enhanced activity toward the commercialization of fuel cells [28]. In this context, Yang et al., [30] have prepared a novel Pd/polymer/ nanoporous carbon (NC) catalyst by a reduction process of palladium ions on the surface of polymer/carbon nanotube composites (see Figure 31). The polymer wrapping of carbon support has been reflected on the fuel cell performance of the prepared palladium catalysts. As seen from Figure 32, the power density of hybrid membrane electrode assembly fabricated from NC/PyPBI-doped phosphoric acid (PA)/ palladium (Pd) 0.1 with 0.45 mgPd cm−2 for anode and commercial carbon black/Pt (0.45 mgPt cm−2) for cathode reached 221 mW cm−2, which was somewhat higher compared to the MEA from NC/PyPBI-PA/Pd0.1 with 0.05 mgPd cm−2 for anode and commercial CB/Pt (0.45 mgPt cm−2). The hybrid membrane electrode assembly of palladium showed almost twice higher power density than Ptbased membrane electrode assembly due to the higher activity towards oxygen reduction reaction, suggesting that the homogeneous proton transfer (PA-polymer) in catalyst layer is essential for the enhancement in fuel cell performance.

Figure 31. Palladium-NPs supported on polymer functionalized Nanoporous Carbon. Reprinted with permission from (Sci Rep vol. 6. Article no. 36521, 2016). Copyright (2016), Nature.



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Figure 32. Potential and power density curves of Pd-NPs (different sizes) supported on polymer functionalized nanoporous carbon. Reprinted with permission from (Sci Rep vol. 6. Article no. 36521, 2016). Copyright (2016), Nature.

Figure 33. Preparation of the PANI/VrGO supporting material and the Pd/PANI/VrGO catalyst. Reprinted with permission from (ACS Appl. Mater. Interfaces, 2016, 8 (1), pp 169–176). Copyright (2015), American Chemical Society.

In another study, polyaniline (PANI) was used as a conducting polymer to enhance the electron transfer of the metal catalyst through the fabricated composite [31]. Typically, a vertically reduced graphene oxide (VrGO) was coupled with polyaniline through a simple one step electrodeposition technique. Then, a homogenous immobilization of Pd metal catalyst was performed through a spontaneous redox reaction (see Figure 33).


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The composite structure of the Pd/PANI/VrGO catalyst was confirmed by X-ray photoelectron spectroscopy. As seen, Figure 34 has showed the N1s signal at 400.0 eV which is attributed to PANI polymer. The narrow scan spectra of N1s region has further indicated four signals of binding energies centered at 400.72, 400.01, 399.50 398.85 eV being assigned to nitrogen cationic radical (N+•), benzenoid amine (-NH-), quinoid imine (=N-), and nitrogen atoms of PANI bonded with Pd nanoparticles, respectively. Additionally, the Pd3d XPS spectrum of Pd/PANI/VrGO was collected. Both the Pd3d5/2 (~337 eV) and the Pd3d3/2 (~343 eV) peaks, from the spin-orbital splitting of Pd3d, was deconvoluted into doublets assigned to Pd0 and Pd2+.

Figure 34. XPS spectra of Pd/PANI/VrGO (a), narrow scan of N1s spectrum of Pd/PANI/VrGO (b), and Pd3d spectra of Pd/PANI/VrGO (c) (upper panel) and Pd/VrGO (lower panel). Reprinted with permission from (ACS Appl. Mater. Interfaces, 2016, 8 (1), pp 169–176). Copyright (2015), American Chemical Society.

The presence of Pd2+ means a part of the Pd atoms are bonding with their support. Compared to the Pd/VrGO hybrid without PANI, Pd/PANI/VrGO has showed significantly positive shifts of the Pd peaks. Such chemical shifts



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towards higher binding energies has reflected an enhanced interaction of the Pd nanoparticles with the PANI/VrGO support in Pd/PANI/VrGO. The electrocatalytic activity of the prepared Pd/PANI/VrGO composite was evaluated by the methanol and ethanol oxidation reactions in alkaline media. The commercial Pd/C was used for comparison. Figure 35, has showed that the mass activity for the methanol oxidation reaction on Pd/PANI/VrGO is 375.6 A/g, which is 3.6 times than that of commercial Pd/C (105.2 A/g). In addition, the onset potential for the methanol oxidation reaction on Pd/PANI/VrGO is ca. –0.4 V, which is 100 mV more negative than the ca. –0.3 V observed on commercial Pd/C. The higher mass activity and the more negative onset potential has meant an enhanced electrocatalytic activity of Pd/PANI/VrGO for the the methanol oxidation reaction. Similarly, Pd/PANI/VrGO has exhibited a higher electrocatalytic activity towards the ethanol oxidation reaction compred to commercial Pd/carbon. It was also evidenced by the significantly higher anodic oxidation current and the lower onset potential. Moreover, in both cases, much higher If/Ib (If and Ib are the forward and the backward currents, respectively) ratios were observed for Pd/PANI/VrGO than commercial Pd/Carbon. This result indicates that alcohol molecules can be effectively oxidized by the Pd/PANI/VrGO catalyst with relatively less poisoning species yielded.

Figure 35. Cyclic voltammograms of Pd/PANI/VrGO and commercial Pd/Carbon for the methanol oxidation reaction (a) and ethanol oxidation reaction (b). Reprinted with permission from (ACS Appl. Mater. Interfaces, 2016, 8 (1), pp 169–176). Copyright (2015), American Chemical Society.


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Figure 36. Cyclic voltammograms of Pd/PANI@VrGO at the 5th, 25th, 50th, 250th and 500th cycles (a). (b) Plots of anodic peak current as a function of cycle number for Pd/PANI@VrGO, Pd/VrGO, and commercial Pd/Carbon. Reprinted with permission from (ACS Appl. Mater. Interfaces, 2016, 8 (1), pp 169–176). Copyright (2015), American Chemical Society.

The stability of Pd/PANI/VrGO was further evaluated using the CV technique. Figure 36 has showed the cyclic voltammograms of Pd/PANI/VrGO for the methanol oxidation reaction at the 5th, 25th, 50th, 250th and 500th cycles. The peak current initially increases till the 50th cycle, and afterwards, the peak current is almost constant, indicating a good durability of Pd/PANI/VrGO. In contrast, the prepared Pd/VrGO without PANI and commercial Pd/Carbon have showed obvious current deterioration during the CV scanning.

11. IRON METAL AS A NON-NOBLE METAL CATALYST FOR FUEL CELLS Iron is a superior metal catalyst towards oxygen reduction reactions because it is abundant and inexpensive. Thus, it can work as an alternative catalyst to precious metals. The use of N-containing polymers such as polyaniline (PANI), and polypyrrole as a source of both N and C was shown to be more facile than the conventional N-doping process, N-containing polymers also enable Ndoped sites to be distributed homogeneously. Wu et al. reported a PANIderived Fe-Co-doped carbon catalyst that has exhibited an outstanding



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performance and a durability in an acidic electrolyte [32]. Li et al. synthesized a Fe-N-CNT catalyst with a half-wave potential that was 30 mV more positive than Pt/Carbon catalysts in alkaline electrolytes [33]. Yong Yuan et al., [34] have shown an efficient iron phthalocyanine catalyst, using polyaniline/carbon black as an electrocatalyst support for oxygen reduction reactions in an air–cathode single-chamber direct methanol fuel cell. The iron/polyaniline/carbon black has showed a higher catalytic activity compared to the iron/carbon black catalyst, thanks to the polymer layer. A maximum power density of 650.5 mWm−2 was achieved from a direct methanol fuel cell with the iron/polyaniline/carbon black cathode compared to 336.6 mWm−2 for iron/carbon black (see Figure 37). Meanwhile, the power per cost of iron/polyaniline/carbon black was 7.5 times higher than that of Pt, demonstrating that the polyaniline/carbon black was an ideal supporting material for iron phthalocyanine. Thus, it offers a good alternative to Pt in direct methanol fuel cell practical applications. In a recent study by Yong Yuan et al., [35] a highly efficient nonprecious metal catalyst of iron-cobalt nanoparticle supported on polyaniline–multiwalled carbon nanotube composite is proposed for the oxygen evolution reaction (see Figure 38). This new catalyst is prepared through a novel and a simple in situ process under mild conditions. The introduction of polyaniline has improved the synergistic property between the iron-cobalt nanoparticle and the multiwalled carbon nanotube, promoting the stability and electrical conductivity of the catalyst. Meanwhile, polyaniline has provided more active sites to attach iron-cobalt nanoparticle uniformly and tightly. The electrochemical measurement have showed that the electrocatalyst displays excellent oxygen evolution reaction activities at a low overpotenial of 0.314 V for 10 mAcm−2 current density and a small Tafel slope of 0.03069 Vdec−1 in 1 M KOH. Furthermore, this catalyst has exhibited a remarkable durability which is evaluated by potential cycling for 1,000 cycles at 0.54 V (vs. Ag/AgCl) for almost 40 h. The obtained results have confirmed that the iron-coblt nanoparticle catalyst is an abundant and cheap fabricated anode catalyst for oxygen evolution reaction.


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Figure 37. Power density curves (A) and individual potentials of anode (open symbol) and cathode (filled symbol) (B). Reprinted with permission from (Journal of Power Sources 196, 1103–1106, 2011). Copyright (2011), Elsevier.

The composite structure of the prepared catalyst was confirmed by different spectroscopic techniques. For example, Figure 39 shows the TEM images of the composite materials that are fabricated during the synthetic route of the final catalyst. As seen, the TEM images have showed a successful covering of multiwalled carbon nanotube by polyaniline polymer. Also have showed the successful immobilization of the CoFe catalyst with homogenous distribution on the polymer coated carbon with a nanosized



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ranged from 28–10.59 nm with an average particle size of 6.22 nm. Additionally, the EDX spectra has confirmed the presence of the CoFe metals.

Figure 38. A schematic illustration of the process for the preparation of the iron-cobalt nanoparticle catalyst supported on polyaniline–multiwalled carbon nanotube composite and application in the oxygen evolution reaction. Reprinted with permission from (J. Mater. Chem. A, 2016, 4, 4472-4478). Copyright (2016), Royal Society of Chemistry.

In another recent study, a facile synthetic strategy for Fe-aniline metal catalysts was proposed for polymer electrolyte membrane fuel cells [36]. This catalyst was prepared by ultrasonicating a mixture containing an iron precursor, an aniline monomer, and a carbon black. In this study, it was supposed that ultrasound plays two kinds of roles. It could affect the crystallinity and structure of polyaniline which might have something to do with the active sites after a pyrolysis. In addition, it is able to reduce the polymerization time and cause an abrupt nucleation throughout the solution, which can help forming uniform and homogeneous distribution of active sites for oxygen reduction reaction (see Figure 40). The X-ray photoelectron spectroscopy (Figure 41) and TEM were used to characterize the prepared catalysts. X-ray photoelectron spectroscopy has showed the characteristic signals of the polymer and the metals deposited on the surface of the carbon support.


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Figure 39. 1 TEM images of multiwalled carbon nanotubes (A); polyaniline– multiwalled carbon nanotubes 1:20(B); polyaniline–multiwalled carbon nanotubes (40 wt%) (C); the iron-cobalt nanoparticle catalyst supported on polyaniline–multiwalled carbon nanotube 1:20 (40 wt%) (D–F); and the HR-TEM (G and H) images of the iron-cobalt nanoparticle catalyst supported on polyaniline–multiwalled carbon nanotubes 1:20 (40 wt%). The inset of TEM image (D) is size distribution histogram; and the inset of TEM image (F) is the EDX pattern. Reprinted with permission from (J. Mater. Chem. A, 2016,4, 4472-4478). Copyright (2016), Royal Society of Chemistry.



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Figure 40. The conceptual diagram of the facile synthetic method of Fe-aniline carbon black. Reprinted with permission from (Sci Rep vol. 7. Article no. 5396, 2017). Copyright (2016), Nature.

The fuel cell results of this study (see Figure 42) have showed that the optimum catalyst loading for Fe/PANI/C was 2 mg cm−2. The current density at 0.6 V was 139 mA cm−2 and the maximum power density of Fe/PANI/C was 157 mW cm−2, which was 83% of that of a membrane electrode assembly using commercial Pt/Carbon black as a cathode catalyst. The performance of the catalyst deteriorated at a loading of 3 mg cm−2 because of the high ohmic and the mass transfer resistance. The increased catalyst loading caused the catalyst layer thickness to increase from 30 μm to 96 μm, which hindered the transport of the reductant, product and hydroxide ions. In the same regard, a highly active iron-cathode catalyst, Fe/polyimide was successfully synthesized by the pyrolysis of a Fe-containing polyimide precursor (see Figure 43).


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Figure 41. X-ray photoelectron spectroscopy of the prepared composites, showing the different signals of the characteristic elements of the deposited materials. Reprinted with permission from (Sci Rep vol. 7. Article no. 5396, 2017). Copyright (2016), Nature.

Figure 42. Anion exchange membrane fuel cell performance evaluation of the new Febased catalyst. I–V curves of Fe/PANI/Carbon and Pt/Carbon cathodes. The loading of the Fe/PANI/carbon catalyst was changed from 1 mg cm−2 to 3 mg cm−2, the loading of the catalyst was 0.5 mg cm−2 for Pt/Carbon. Reprinted with permission from (Sci Rep vol. 7. Article no. 5396, 2017). Copyright (2016), Nature.



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This catalyst has demonstrated a remarkable fuel cell performance and a promising durability (see Figures 44 and 45), especially with air as the cathode gas. The improvement of performance by employing small size iron catalyst nanoparticles was due to the improvement of oxygen diffusion in the catalyst layer [37]. Figure 43 shows the synthesis route for the preparation of the polyimide nanoparticles and the SEM images of the asprepared and carbonized nanoparticles. The obtained Fe-containing polyimide nanoparticles were carbonized by a multistep pyrolysis process, where the Fe species catalyzes carbonization up to 600 °C and excess Fe species are removed before treatment at even higher temperatures to minimize the loss of nitrogen species at higher temperature [37]. To obtain smaller polyimide particles, the precursor and polymerization conditions were optimized.

Figure 43. (a) A synthetic route for the polyimide and SEM images of the polyimide particles (b) before and (c) after carbonization to produce Fe/polyimide size 100 nm. (d) Synthetic route for the polyimide and SEM images of the polyimide particles (e) before and (f) after carbonization to produce Fe/polyimide size 60 nm. Reprinted with permission from (Sci Rep vol. 6. Article no. 23276, 2016). Copyright (2016), Nature.


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Figure 44. The I-V performance curves (a) and the Tafel plots (b) of the I-V curves with the Fe/polyimide (100 nm) and Fe/polyimide (60 nm) cathode catalysts at pure or balanced O2 (humidified) at 80°C. The Electrolyte used was Nafion NR211. T = 80 °C. Reprinted with permission from (Sci Rep vol. 6. Article no. 23276, 2016). Copyright (2016), Nature.

Figure 45. Stability curves of the prepared catalysts at 0.2 A cm−2 with air as the cathode gas. Reprinted with permission from (Sci Rep vol. 6. Article no. 23276, 2016). Copyright (2016), Nature.



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As seen from Figure 44, the Fe/polyimide nanoparticle (size 60 nm) has showed a better fuel cell performance compared to the Fe/polyimide nanoparticle (size 100 nm). This difference was related to the mass transport diffusion rather than the kinetics. Also, as shown in Figure 45, the fuel cells were successfully operated for 600 h with stable performance.

12. NICKEL NANOPARTICLES AS A CATALYST FOR FUEL CELLS Nickel-containing materials have been also used as powerful catalysts for fuel cells, mainly alkaline fuel cells, direct alcohol fuel cells, and hydrogen oxidation reaction. Hydrogen production by the water electrolysis is also a great target to produce an economically and environmentally attractive renewable energy source. The drawbacks of this technology are mainly related to the high energy consumption, the low specific production rates of hydrogen, the low efficiency, and the large system volume. Hence, the improvement of the catalyst electrode material may enhance the utility of the hydrogen production technology from water [38]. Nickel-polymer composites were fabricated as efficient catalysts to serve this target. Polyaniline and polypyrrole have been used to support the homogenous synthesis and distribution of non-noble metal catalysts for hydrogen production by water electrolysis [39]. The current challenge in the synthesis of these catalysts is that the reduction potential of the metals is significantly more positive than the potentials commonly used for the electro-polymerization of monomers such as pyrrole and aniline, which can limit the process efficiency. In this context, Daniel Corte et al., [38] have showed a stable and an efficient nickel-polyaniline composite with enhanced catalytic activity towards hydrogen production, thanks to the preparation technique in which polyaniline particles are incorporated in the nickel during the electrodeposition process. In another study [39], polypyrrole has also showed a substantial decrease in the diameter of Ni. Accordingly, an


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increase in the surface area of electrodeposited nickel has been observed, offering a significant electrocatalytic activity. These results have demonstrated that the morphology of the polymer matrix has a great influence on the resulting morphology of the Ni-composite, and subsequently on the overall electrocatalytic activity of the catalyst [39]. In another recent study, Suparna Das et al., [40] have showed a more abundant and a less-expensive Ni-metal catalyst supported on Vulcan carbon-polyaniline (PANI) and partially sulfonated PANI (S-PANI) for the oxidation of methanol. The partially sulfonated polyaniline (S-PANI) was utilized as the aromatic conducting polymer (ACP). Additionally, the SO3H groups has been chemically incorporated into the aromatic conducting polymer structure, instead of using SO3H dopants. This step was added to produce better interaction between the catalyst particles and the support matrix (see Figure 46), accordingly providing a remarkable stability to the overall fabricated system. The fuel cell performance of this Ni-based catalyst (Figure 47) was comparable to that of the current market catalyst of PtRu/Carbon. Also, the durability testing (Figure 48) has showed a promising and stable catalyst for direct methanol fuel cells.

Figure 46. A schematic illustration of the S-PANI composite preparation and the deposition process of the catalyst. Reprinted with permission from (J. Mater. Chem. A, 2015, 3, 11349-11357). Copyright (2015), Royal Society of Chemistry.



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Figure 47. Direct methanol fuel cell performance of Ni/SPAni as anode catalysts at 60 °C in comparison to Pt–Ru/Carbon commercial catalyst. Reprinted with permission from (J. Mater. Chem. A, 2015, 3, 11349-11357). Copyright (2015), Royal Society of Chemistry.

Figure 48. The stability testing of the Ni/SPANI catalyst system in comparison to the commercial Pt–Ru/Carbon catalyst system. Reprinted with permission from (J. Mater. Chem. A, 2015, 3, 11349-11357). Copyright (2015), Royal Society of Chemistry.


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Chapter 6

THE EFFECT OF GOLD AND SILVER NANOPARTICLES ON PLANT GROWTH AND DEVELOPMENT Lev A. Dykman1, and Sergei Y. Shchyogolev1,2,† 1

Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, Saratov, Russia 2 Saratov State University, Saratov, Russia

ABSTRACT This review considers the past decade’s data regarding the effects of nanoparticles of noble metals (gold and silver) on higher plants, as well as regarding possible nanoparticle phytotoxicity. It discusses the various effects that gold and silver nanoparticles can have on the state, growth, and productivity of plants. Research in this field is topical because (1) plantnanoparticle interactions are caused by a diversity of natural and man-made factors and because (2) green chemistry now uses plants for the dedicated biotechnological synthesis of nanoparticles. Published evidence, although  †

Corresponding Author Email: [email protected]. Corresponding Author Email: [email protected].


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Lev A. Dykman and Sergei Y. Shchyogolev incomplete and contradictory, indicates that metal nanoparticles can have both positive and negative effects on plants and that intracellular penetration is determined mostly by the particles’ chemical nature, size, shape, surface charge, and dose. Thus, the need exists for a coordinated research program that can find correlations between particle characteristics, experimental design, and observed biological effects.

INTRODUCTION In the past few decades, scientists and the world community have paid much attention to nanotechnology, based on the use of sub-100 nm objects in the synthesis, assembly, and modification of substances, materials, and structures with unusual (and often unexpected) properties. This size area is characterized by its own phenomena and concepts owing to the specific characteristics of such materials and composites and owing to the use of specific methods for their preparation, study, and application [1]. Of separate interest are toxicological effects of metal and metal oxide nanoparticles on biological systems [2]. That is because the physicochemical, structural, and optical properties of nanoparticles differ greatly from those of microparticles and massive materials [3], as well as from those of molecular (ionic) forms of metals. Gold and silver nanoparticles have various uses in biomedicine, including uses as drug carriers, amplifiers and/or converters of the optical signal, and immunomarkers [4-6]. Industrial nanotechnologies make accidental effects of artificial nanoparticles on plants and animals increasingly probable; therefore, the consequences of such effects must be analyzed and predicted. Approximate estimates indicate that the annual production of silver nanoparticles (AgNPs) is 3-20 tons in the USA [7] and 5.5 tons in Europe [8]; about 800 tons is used globally [9]. Gold nanoparticles (AuNPs) are applied most widely for biomedical purposes [6]. Both types of nanoparticles inevitably enter the environment, causing difficult-to-predict (and sometimes unwanted) ecological effects [10]. The use of metal nanoparticles in medicine and biology entails questions about their biodistribution in organs and tissues, pharmacokinetics, and


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possible toxicity [11, 12]. But whereas the toxicity of nanoparticles to microbes and animals and their cellular penetration, transformation, and elimination from cells and entire organisms have been amply documented [11-16], nanoparticle interactions with plant cells is largely a terra incognita. The topicality of this review is attested to by the publication in 20162017 of several comprehensive reviews and monographs on the interactions of noble metal nanoparticles with plants [17-23]. Yet, very little is known about the positive or negative effect of nanoparticles on plants, and what data there are, are inconsistent [24-29]. A wide variety of nanosized particles are manufactured with which plants can come into contact under natural conditions. In addition, the physicochemical properties of man-made nanoparticles are often similar to those of natural ones. For example, recent findings indicate that in goldenriched geological deposits, climatic factors give rise to nanostructures (spheres and plates) whose sizes and shapes are similar to those of particles made in the laboratory [30]. Some approaches use plants and their metabolites for the biotechnological synthesis of nanoparticles (green chemistry) [31, 32]. Nanoparticle synthesis by plants growing on metalpolluted soils was suggested to be a detoxification means [33]. In particular, cyanobacteria (Synechocystis sp. PCC 6803), regarded as physiologically similar to eukaryotic algae, responded to Au ions with intracellular synthesis of AuNPs; this process was linked to changes in cellular metabolism (respiration and photosynthesis) [34]. Plants have developed a diversity of adaptive mechanisms to protect cellular metabolism against heavy metals in their surroundings. These mechanisms include (1) binding of heavy metals by the cell wall and by the cell-exuded substances, (2) reduction of the entry of heavy metals into the cell and their release from cytoplasm into apoplast, (3) chelation by peptides and proteins (phytochelatins and metallothioneins) in cytoplasm, (4) repair of damaged proteins, and (5) metal compartmentation in the vacuole through tonoplast transporters. One such protective mechanism could also be nanoparticle synthesis.


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This chapter summarizes the past few years’ data on the interaction of higher plants with the most widespread metallic nanomaterials – AuNPs and AgNPs.

ENTRY OF NANOPARTICLES INTO PLANT CELLS AND TISSUES Several recent reviews have dealt with the interaction of metal nanoparticles with higher plants [35-38] and with algae [39, 40]. It turned out that unicellular algae, specifically Dunaliella salina Teod., are a useful model for studying nanoparticle effects on living cells [41, 42]. Navarro et al. [43] were among the first to examine the penetration of nanoparticles into plant, fungal, and algal tissues and cells. They rightly pointed out that the cell wall – a structure typical of plants, fungi, and algae – is both a primary site for the interaction with nanoparticles and a barrier to their entry into cells. The cell wall allows the passage of small molecules or particles but limits the entry of large ones. The pore diameter (5-20 nm on average) restricts the size of nanoparticles able to overcome the cell wall. Nonetheless, nanoparticles themselves can modulate the pore size of the cell wall, thereby removing the rigid structural limitations that prevent them from reaching the plasmalemma [43]. Subsequently, nanoparticles may enter the cell interior through endocytosis, but this process is still understudied in plants. Some data suggest that AuNPs penetrate the tissues of Oryza sativa L. and Solanum lycopersicum L. through both clathrindependent and clathrin-independent endocytosis [44]. Moscatelli et al. [45] used negatively and positively charged AuNPs to investigate endocytosis in growing pollen tubes of tobacco (Nicotiana tabacum L.). With electron microscopy, they convincingly showed that owing to endocytosis, AuNPs are rapidly trapped and are delivered to membrane vesicles. The apical growth of pollen tubes is a fairly rapid process in which the cortical actin cytoskeleton and the plasmalemma are constantly renewed. Perhaps in such systems (and also in protoplasts),



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endocytosis is the way for nanoparticles to enter plant cells [46]. Other types of nanoparticles, such as gold nanostars [47], paramagnetic nanoparticles [48], nanoparticles of silicon oxide [49] and magnesium oxide [50], and carbon nanotubes [51], presumably enter plant tissues through endocytosis, too. Several studies [52-55] have pointed out that AuNPs are never found in the aerial parts of radish, pumpkin, barley, poplar, and wheat, unlike what is observed in tobacco, tomato, alfalfa, ryegrass, maize, bamboo, and rice. The efficacy of tissue penetration of AuNPs depends not only on the plant species used but also on the particles’ size and surface charge. Positively charged AuNPs are well absorbed only by plant roots, whereas negatively charged AuNPs also can effectively move from roots to stems and leaves [44, 56]. Some part in this process is played by the plant vascular system, as well as by plasmodesmata [52, 57]. When AuNPs are sprayed on seedlings of watermelon [Citrullus lanatus var. Lanatus (Thunb.) Matsum. & Nakai], they enter leaves via stomata and are translocated from leaves to roots by the phloem transport mechanism [58]. Small nanoparticles penetrate the aerial parts better than do large ones; in addition, they are more toxic. With AgNPs, this fact could be explained by the better solubility of small particles and by the toxicity of the metal ions [14, 59]. Intriguing data were obtained by mass spectrometry and X-ray fluorescence in a study of entry of 5-, 10, and 15-nm AuNPs into the tissues of Nicotiana tabacum L. cv. Xanthi [60]. AuNPs were found not only in tobacco leaves but also in the tissues of tobacco hornworm (Manduca sexta), which feeds on tobacco leaves. Using an artificial aquatic ecosystem, Ferry et al. [61] showed that gold nanorods penetrated the tissues of mollusks, shrimp, and fish better than they penetrated the tissues of the water plant Spartina alterniflora Loisel. Mask et al. [14] reported that AgNPs were toxic to algae and crustaceans at much lower concentrations than they were to mammalian cells (~0.1 mg Ag/l versus ~26 mg Ag/l). Seedlings of spring barley grown hydroponically for 2 weeks with 1-10 μg/ml of 10-nm AuNPs accumulated the nanoparticles both in leaves and in roots. A concentration-dependent effect was observed: the higher the


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nanoparticle concentration in the medium, the greater was the suppression of leaf and root growth. A hormetic effect was also found: with the minimal AuNP concentration (1 μg/ml), leaf growth was increased by an average of 10% and root growth was increased by an average of 60% [62].

BIOLOGICAL EFFECTS OF PLANT EXPOSURE TO METAL NANOPARTICLES As said above, much recent attention has been given to human-caused (technogenic) effects on plant development and performance. These effects include soil pollution by toxic heavy metals and nanoparticles. The phytotoxicity of nanoparticles has been covered in several thorough reviews [63-69], but the data on phytotoxicity mechanisms are scarce and contradictory. In a field study of AuNP effects on the growth and productivity of Brassica juncea L., Arora et al. [70] sprayed the plant with suspensions of various AuNP concentrations. The particles within plant tissues were detected by atomic absorption spectroscopy. The effects from AuNP application were positive, including increased stem length and diameter, increased numbers of leaves and shoots, and improved productivity. Similar results came from the germination of B. juncea seeds on a nutrient medium [71] and in the presence of AgNPs [72]. The addition of AuNPs to soil used for plant growth enhanced seed germination in Zea mays L. [73] and in Pennisetum glaucum L. R. Br. [74]. Using synchrotron-based X-ray microanalysis and high-resolution transmission electron microscopy, Sabo-Attwood [75] showed that 3.5-nm AuNPs entered Nicotiana xanthi through the roots and moved into the vascular system. Aggregates of 18-nm AuNPs were detected only in root cell cytoplasm. Exposure to small particles led to leaf necrosis after 14 days, but with large particles, no differences from the control were observed (Figure 1). At very high AuNP concentrations, no physiological effect or a



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slight such effect has been described in Glycine max L. [76] and in aquatic aquarium plants [77, 78].

Figure 1. Tobacco plants exposed to 3.5-nm AuNPs for 14 days have necrotic leaves (panel A, arrows), as compared to unexposed plants (panel B) [75].

When used early in the ontogenesis of Brassica napus L., AuNPs promoted substantial increases in the weight of roots and stems while slightly decreasing the energy and rate of seed germination [79]. However, treatment of seeds of Boswellia ovalifoliolata N. P. Balakr & A. N. Henry with AgNPs noticeably accelerated seed germination and seedling growth [80]. A similar effect was obtained from exposure of seeds of Asparagus officinalis L. to AgNPs [81]. In addition, seedlings treated with AgNPs contained more ascorbic acid and chlorophyll. The addition of AgNPs to the nutrient medium decreased seed germination, slowed down the formation of nodules (owing to decrease in the numbers of the symbiotic bacterium Rhizobium leguminosarum) and the growth of shoots, and reduced root length in Vicia faba L. [82]. In hydroponic culture, the seed germination in Solanum lycopersicum L. [83] and in Raphanus sativus L. [84] was not decreased by AgNPs, but root and shoot length did decrease and photosynthetic activity was slightly reduced. Exposure of the microalga Skeletonema costatum Grev. to AgNPs decreased cell viability and chlorophyll content because of an excess of reactive oxygen species present [85].


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The potential risk of negative effects of AgNPs on Lactuca sativa L. was very low: the Ag content in the edible parts of the plant was