Antibacterial properties of Doped Nanoparticles: A Review Proma Bhattacharya, Sudarsan Neogi* Department of Chemical Engineering, Indian Institute of Technology Kharagpur West Bengal 721302, India email id:
[email protected],
[email protected]
*corresponding author
Abstract: Nanoparticles have high potential as antibacterial agents owing to its ability to produce reactive oxygen species (ROS). Recent studies have indicated that this ROS generation is highly affected by the modification of band structure by introduction of various dopant materials into them. Thus, doped nanoparticles have been extensively studied in recent literature. The types of dopants, synthesis techniques, and experimental parameters have been found to affect the overall electronic structure of the material, leading to varied antibacterial efficiency. This review summarizes some of the prominent dopant nanomaterials, various methods of synthesizing doped nanoparticles used against bacterial cells and the main factors involved in it. Despite the extensive research on the mechanism of the antibacterial action it is still poorly understood, mainly due to the inherent complexities and dynamics in cell membranes. Some of the major proposed mechanisms of action of each kind of dopant nanomaterial have also been reported in this work, focussing on the bacterial cell structure. Keywords: bacterial cell, doping, antibacterial mechanism, sol-gel, nanoparticles, reactive oxygen species
1. Introduction: 1.1 Bacterial Cell Structure Bacteria are basically a group of single celled, prokaryotic organisms which are the first life forms that appeared on earth surface. It was first observed by Antonie van Leeuwenhoek (Chimileski and Kolter, 2017) in 1676. They can inhabit any condition, ranging from soil, acidic springs, deep sea vents to below earth crust. Bacteria consists of a well-defined cell structure which is responsible for their different characteristics and toxicity. Figure 1 illustrates the cell structure of a typical bacterial cell. They have a wide range of shapes, with some typical examples like coccus (spherical shaped), bacillus (rod-like), spiral (DNA shaped), filamentlike (elongated) etc. The plasma membrane and cell wall constitutes the cell envelope, which provides structural integrity to the bacterial cell. This cell wall, located just outside the cytoplasmic membrane, is made up of peptidoglycan which is responsible for the cell wall rigidity. Some also contains an additional thin layer made of peptidoglycan next to the cytoplasmic membrane. The cytoplasmic membrane is basically a bilayer of phospholipids, and acts as a permeable barrier for various molecules (Neidhardt et al., 1990).
Figure 1 Schematic diagram of a typical prokaryotic bacterial cell
Chromosomes and ribosomes are the only easily observable intracellular organelle present in all bacteria (Hammond et al., 2017) The bacterial DNA, which is responsible for the transport of information via translation, replication and transcription, is found unbounded in the bacterial cytoplasm. Ribosomes, most numerous intracellular structure, are the sites for protein synthesis in cells. Other organelles like gas vacuoles are found which are membrane-bound, spindleshaped vesicles, that gives buoyancy to cells by reducing the cell density. Endospores are also found in some bacteria, which are bacterial survival structures that are highly resistant to chemical and environmental conditions. Some research proposes that the formation of endospore has allowed for the survival of a class of bacteria for millions of years (Priest, 1993). The types of bacteria found on earth are extremely numerous, but in order to comprehend and classify, Cohn in 1872 has divided them in several groups (showed in table 1) based on their morphological and behavioural factors (Lowy, 2009). Table 1 Comprehensive classification and description of bacteria on different basis Classification Basis Shape
Nutrition
Cell Wall
Name and description 1. Cocci: Unicellular, spherical or elliptical shaped, e.g. Micrococcus flavus, Diplococcus pneumonia, Staphylococcus aureus. 2. Bacilli: Rod or cylindrical shaped, e.g. Bacillus cereus. 3. Vibrio: Curved or comma shaped, e.g. Vibro cholerae 4. Spirilla: Spiral or spring-like along with curvature and terminal flagella, e.g. Spirillum volutans. 1. Autotrophic: Capable of self-sustenance 1.1 Photoautotrophs: contains bacterioviridin and bacteriochlorophyll, derives nutrition from sunlight, e.g. green sulphur bacteria. 1.2 Chemoautotrophs: Derives nutrition from inorganic substrances like hydrogen sulfide, elemental sulfur, ferrous iron, molecular hydrogen, and ammonia, e.g. Thiobacillus Ferrooxidans (iron oxidising bacteria), Thiobacillus Thiooxidans (sulphur bacteria). 2. Heterotrophic: Derives food by ingesting or absorbing organic carbon, e.g. heliobacteria. 1. Gram positive: Absorbs the crystal violet stain used in the test, and then appears purple through a microscope, e.g. Bacillus subtilis.
Temperature Response
Number of Flagella
2. Gram negative: Unable to retain the crystal violet stain when washed with alcohol and acetone, have to be counterstained with safranin, e.g. Escherichia coli. 1. Psychrophilic: Able to grow in cold, about 10˚C, e.g Arthrobacter sp., Pseudomonas syringae. 2. Mesophilic: Grows best in moderate temperatures, typically between 20 and 45 °C, e.g. Listeria monocytogenes, Thiobacillus novellus. 3. Thermophilic: Thrives at extreme temperatures, between 41 and 122 °C, e.g. Sulfolobus solfataricus, Thermococcus litoralis 1. Atrichos: No flagella, e.g. Lactobacillus acidophilus, Pasteurella multocida 2. Monotrichous: Only one flagellum which is present at one end, e.g. Vibrio cholerae 3. Lophotrichous: Two flagella, one at each end, e.g. Pseudomonas fluorescens 4. Amphitrichous: One flagellum is present at each end, e.g. Rhodospirillum rubrum 5. Peritrichous: Evenly distributed flagella distributed all over the cell surface, e.g. Bacillus thuringiensis.
These different kinds of bacteria are closely related to different functions of human, plants or animals. About 99% of the bacteria are useful, rest are associated with negative effects. Some of the most common helpful and harmful bacteria are as follows: Helpful bacteria: Lactobacillus/ Doderlein’s bacillus: Lactobacilli species, present in milk and some fermented foods, are able to produce lactic acid and are important for processes like fermentation, pickling, digestion etc. Prominent examples include L. acidophilus, L. reuteri, L. plantarum. Bifidobacterium: Present in human gastrointestinal tract, these bacteria prevent growth of other harmful bacteria. Escherichia coli: Aids digestion and produces vitamin K and biotin.
Streptomyces: Decomposes organic matter that are present in soil, and possesses the ability of production of bioactive secondary metabolites, such as such as antifungals, antivirals, immune-suppressants, antibiotics and anti-hypertensives. Typical examples are Chloramphenicol from S. venezuelae, Fosfomycin from S. fradiae, Streptomycin from S. griseus etc. Rhizobia: Known for fixing atmospheric nitrogen and help in its absorption by the plants. E.g. Rhizobium etli, Bradyrhizobium spp., Azorhizobium spp. Cyanobacteria/ Cyanophyta: Essential for fixing nitrogen in aquatic habitats, and maintaining ecological balance in coral reef. Harmful bacteria: Xanthomonas: Causes bacterial spots and blights of leaves, stems, and fruits etc. Prominent examples are X. albilineans, X. citri, X. oryzae etc. Mycobacteria: Pathogens of different human diseases, like M. tuberculosis and M. leprae cause tuberculosis and leprosy, respectively. M. ulcerans causes ulcers in the skin, M. bovis leads to cattle tuberculosis. Bacillus anthracis: Causative agent of anthrax in sheep, goats, cattle. Helicobacter pylori: Causes gastritis and peptic ulcers. Clostridium tetani: Causes tetanus. Yersinia pestis: Causes bubonic and pneumonic plague Vibrio cholera: Causes cholera, a fatal disease. Salmonella typhi: Leads to typhoid. 1.2 Conventional antibacterial agents
An antibacterial agent is anything that is responsible for the inhibition or suppression of microbial growth. There are three types of proposed mechanisms how they work, which are the following: (1) Inhibition of cell wall synthesis; (2) Inhibition of protein synthesis; (3) Inhibition of bacterial nucleic acid synthesis. The discovery, development and clinical use of antibacterial materials have been a major thrust to biomedical fields because of their extensive applications for arresting bacterial infections. In medieval ages, people used moulds, soil and different plants to treat bacterial infections. The concept of roasting the raw meat was derived from the idea of disinfection. In 1864, Lewis Pasteur invented a method of heating liquids below their boiling points to destroy microbes. Thus food borne pathogens were treated but human infections could only be treated by antibiotics (Gualerzi et al., 2013). This era began in 1907 with discovery of synthetic antibiotic derived from arsenic (Williams, 2009) by Alfred Bertheim and Ehrlich, and Ehrlich was awarded Nobel Prize in Physiology or Medicine for this. The modern day anti-bacterials can be classified on the basis of their mode of action(McIlwain, 1943) into two broad divisions; (a) Non-Residue-Producing Ones: The ones which act rapidly and then evaporate, leaving no traces or residue behind. Eg. Alcohols (ethanol, isopropanol), aldehydes (glutaraldehyde, formaldehyde), halogen-releasing compounds (chlorine compounds, iodine compounds), peroxides (hydrogen peroxide, ozone, peracetic acid) etc. (b) Residue-Producing Ones: Compunds that leave long-acting residues on the surface to be disinfected and thus have a prolonged action, like triclocarban, chlorhexidine, triclosan, silver compounds, mercury compounds, phenol, cresols etc.
But, due to wide spread and overuse of these agents, there has been an accelerated emergence of antibiotic-resistant pathogens. These resistant bacteria are generally referred to as superbugs and they are responsible for some diseases which are hard to cure or even detect. This resistance can be of two types depending on the source of the resistant genes, intrinsic resistance (caused by uninhibited mutation of existing or foreign genes) and acquired resistance (which is caused by acquisition of resistance genes from another organism). The emergence of multidrug resistance (MDR) is a result of the acquirement of different types of drug resistant genes by the same bacterial cell. This problem demands development of newer kinds of materials as antibacterial agents. In this effort, nanoparticles have shown great promise and is much researched, as discussed in the following section. 1.3 Nanoparticles as Antimicrobial Agents There have been advances in synthesis and functionalization of nanoparticles which have brought a considerable increase in their biomedical applications. This includes drug delivery (Zhang et al., 2010), biomedical imaging (Nune et al., 2009), blood purification (Herrmann et al., 2010), as biosensors (Luo et al., 2006) , bio detection of pathogens (Wang and Irudayaraj, 2010), tissue engineering (Ito and Kamihira, 2011)(Okamoto and John, 2013) and in fabrication of various medical nano-devices. Through control over material size, morphology, and chemical structure, nanomaterials can be tailored to achieve exceptional mechanical, chemical, optical, electrical and magnetic properties. Various mechanisms have been proposed on the pathway of nanoparticle-cell interaction. The most accepted theories being (a) Physical route, viz. disruption of membrane potential and integrity and (b) Chemical routes, viz. production of reactive oxygen species (ROS), also known as oxygen-free radicals or direct electron transfer. Membrane rupture occurs when the nanoparticles attaches electrostatically to the cell membrane. This results in modification of membrane potential, depolarization, and loss of integrity, which leads to an transport imbalance, disturbed
respiration, cell lysis, and subsequent cell death (Pelgrift and Friedman, 2013). ROS, on the other hand, are promoted either by breakage of respiratory chain or by the nanoparticles (Pelgrift and Friedman, 2013). In cells, molecular oxygen gets reduced to water through a series of proton-electron transfer reactions, and ATP (Adenosine triphosphate) is synthesized. During this procedure, the oxygen which is not reduced completely results in the formation of superoxide anion and other oxygen-containing radicals. So, ROS are by-products of cellular oxidation, maximum part of which occurs in the mitochondria of the cells. ROS leads to oxidative stress, lipid peroxidation, protein alteration, variation of inflammatory responses through signal transduction, impediment to formation of enzymes, modulation of gene expression by activation of various transcription factors, and RNA and DNA damage (Choudhury et al., 2017, 2017; Zhao et al., 2016). The cells fail to maintain normal physiological redox-regulated function due to this oxidative stress. Researchers propose different mechanisms like direct inhibition of some essential enzymes, induction of nitrogen reactive species (NRS) (Pelgrift and Friedman, 2013) and programmed cell death. Profuse research has been going on how to externally induce the ROS formation for inducing bacterial death, and surface functionalization or the technique of doping is a much convenient route for this, as doping leads to lattice defects which are in turn, responsible for ROS generation. Detailed discussion on how different dopants help in ROS induction are done in the next sections. Extensive research is going on different kinds of doping in nanoparticles and optimisation of their synthesis parameters. The following review mainly focuses on the current status of the doped nanoparticles and their activity as antimicrobial agents. 2. Doped nanoparticles: novel antibacterial agents Doping refers to the method of intentional introduction of some foreign elements in the vacant crystal lattice of another element with the intention of modifying their properties. In
nanoparticles, the addition of a few defect atoms can make the particle highly doped. They exhibit improved properties and can be employed for various purposes as tabulated in table 2. Table 2 Different kinds of doped nano-particles with their widely varied applications Type of doped nanoparticle
Applications
Reference
Mg-Doped ZnO
Sunlight-Driven Photo catalysis
(Etacheri et al., 2012)
Al doped ZnO
As electrical conductor
(Akdağ et al., 2016)
Co-doped ZnO
Spintronic material
(Wojnarowicz et al., 2015)
La doped nanoparticles
Diagnostic Sensing and brain (Lee et al., 2017) (Portioli et al., penetration 2016)
Mg doped ZnO
Optoelectronics
(Shayesteh and Dizgah, 2012)
Li doped ZnO
Inducing ferromagnetism
(Ullah Awan et al., 2012)
Ag-doped ZnO
Photoluminescence and (Sánchez Zeferino et al., 2011) enhanced ethanol gas sensing (Blanc et al., 2012)
Zndoped calcium Orthopaedic applications pyrophosphate dihydrate
(Vasant and Joshi, 2011)
Erbium-doped nanoparticles
Optical fibres
(Blanc et al., 2012)
Sn doped ZnO
Photocatalysis
(Yurddaskal et al., 2017)
W-Doped TiO2
Photocatalytic Treatment Landfill Leachate
La-doped NaLaF4
Luminescence
(Ladol et al., 2016)
Cu-Doped Fe3O4
Photocatalysis
(Mohanraj and Sivakumar, 2017)
of (Azadi et al., 2017)
As evident from table 2, doped nanoparticles have wide and variety of applications ranging from photo catalysis to biomedicine. In this review paper, we have focused on the antibacterial applications of the doped nanoparticles. These particles can exhibit properties like improved cellular internalization ability, non-cytotoxicity, and improved binding capacity. Majority of
researchers have concluded that the main reason for enhanced antibacterial activities of doped nanoparticles is their ability to generate reactive oxygen species. ROS are nothing but chemically reactive chemical species which contains oxygen. The main example of ROS are:
Singlet Oxygen: These high energetic forms of oxygen are far more reactive towards
organic compounds than its more prevalent triplet ground state of O 2 . It is denoted as 1O 2.
Superoxides: A superoxide is a chemical compound containing the superoxide anion
having the chemical formula •O2- . This is responsible for the formation of most other kinds of ROS. O2 +e− → •O 2-………………. (1) Dismutation of superoxides lead to the formation of hydrogen peroxide(H 2 O2) , which in turn partially reduces to hydroxyl radical ( •OH) (Burello and Worth, 2011). 2H+ + •O2- + •O2 - → H2O2 + O2…………………. (2) H2O2 + e− → HO − + •OH ……………………… (3)
Hydroxyl Radicals: These are the neutral form of hydroxide ion and are highly
reactive.
Peroxides: Peroxide is a compound with structural formulae R-O-O-R. different types
of peroxides include peroxide ion, organic peroxide, organic hydroperoxide, peracids etc. These different types of ROS impart certain toxicological stress to cells according to various mechanisms as described before. ROS is mainly generated by electron transfer by nanoparticle surfaces, specially controlled by the positions of the energy bands (Gajewicz et al., 2015). The electron bands are separated by a band gap, which is defined as an energy difference (measured in eV) between the topmost part of the valence band and the lowermost level of conduction band. An important parameter in this band theory is the fermi level, which is basically the top of the total collection of energy levels at absolute zero temperature. This fermi
level is an imaginary energy level of an electron, which would have a 50% probability of being filled at any given instance at thermodynamic equilibrium. The positioning of this Fermi level is a major determining factor in modulating the electrical properties of the element, and it is further regulated by imperfections at nano-level or insertion of a dopant material. The location of the fermi level is driven various factors like temperature, concentration of free electrons and holes, and their effective masses. When a dopant is inserted in the valence band, the fermi level moves towards one of the energy bands depending on the type of the dopant. If the dopants are of donor type (which add electrons to the system), the fermi level moves towards the conduction band in order to maintain the neutrality equation (which states that the electrical neutrality has to be fulfilled, i.e., the number of negative charges are to be exactly counterpoised by same number of positive charges). If the impurities inserted are of acceptor type (which trap electrons from the system, thus giving rise to an excessive amount of holes), the fermi level moves towards the valence band. Thus the band gap is altered when a dopant material is introduced in the lattice of a pure material. When the nanoparticles are activated by an external source (UV radiation or otherwise), the electrons from the valence band jumps to the conduction band (Ec), resulting in excess amount of electrons in conduction band and positive holes in the valence band(Ev). These excess conduction band electrons interact with oxygen molecules to give rise to superoxide anions (O 2.-), while the holes in the valence band, which have high oxidising property, lead to generation of hydroxyl radicals ( . OH) from water or hydroxyl anions (Burello and Worth, 2011).
Figure 2 : (a) Undoped nanoparticle showing its energy bands; conduction band distant from cellular reduction potential (CRP) and (b) the change in the position of electron band to with the CRP in doped nanoparticle via doping-induced band bending (Saleh et al)
This generation of ROS by introduction of a dopant leads to a variation in the cellular redox potential of the cell (Saleh et al., 2016), which leads to differences in cell responses and ultimate cell apoptosis, which is described in figure 2. The variation in synthesis method is also an important factor, as it can alter the crystal vacancies and dopant percentage. Some of the major dopant materials are described in the following section along with their proposed mechanism and synthesis methods. 2.1 Silver doping Silver, the metallic element with atomic number 47, with electronic configuration of [Ar] 3d10 4s2 4p6 4d10 5s1, finds a very prominent place in the field of doped nanoparticles in biomedical field. It is a transition metal, which has properties like the highest electrical conductivity and reflectivity of any metal. The antibacterial properties of silver have been long established. The ancient Phoenicians was wise enough so that they used to keep water, wine and vinegar in vessels made of silver to discourage contamination. In late 1800’s, it was very much in vogue to administer silver nitrate into the eyes of a newborn baby to prevent infection from mother during childbirth. The mechanism by which silver exhibits its antimicrobial nature
is widely studied to get an insight. Klueh et al (2000) suggested that Ag+2 ions enter the cells and bind with bacterial DNA. This binding causes denaturation by displacement of hydrogen bonds between adjacent purines and pyrimidines. Jansen et al (Jansen et al., 1994) came up with a theory that silver ions blocks the respiratory chain of bacteria present in the cytochrome oxidase and NADH-succinate-dehydrogenase. All these led to an increasing use of silver in various medicines, antibacterial bandages etc. But some recent studies show that silver can be toxic when administered in dosages beyond a certified level (Stensberg et al., 2011). This is one of the main reasons that silver is widely used as dopants to inert nanoparticles like silica or zinc oxide to enhance their bactericidal properties. In early 2005, Ping Li et al (2005) explored the synergetic antimicrobial effects of silver combined with amoxicillin, a very prominent β-lactam antibiotic against E.coli. It was found that the Ag-amoxicillin composite exhibited better results than silver or the antibiotic alone. In 2007, Thiel et al (2007) synthesized silver doped TiO2 nanoparticles via sol gel method and examined its antibacterial efficiency against E.coli bacteria. After that, wide research have been done to assess the antibacterial tests of Ag doped titania nanoparticles (Garcidueñas-Piña et al., 2016; Gupta et al., 2013; Imran et al., 2016), which showed its use as potential as antibacterial agents. Most recently, silver doped titanium dioxide nanoparticles find its place as antimicrobial additives to dental polymers (Chambers et al., 2017). It was found that even small quantities of Ag–TiO2 nanoparticles (>2% wt) were able to produce a significant bactericidal effect when in contact with S. mutans under visible light. Moreover, it was found that the use of silver doping led to a measurable band gap shift towards the visible spectrum which is necessary for TiO2 to show antibacterial activities. Zinc oxide is widely renowned for its antibacterial uses. The main reason for this is the free electrons and positive holes which are generated when zinc oxide, having a high band gap, is bombarded with an energy equal or higher than its band gap energy. But these free electrons
and holes recombine quite fast, not allowing them enough time to take part in any chemical reactions. So, the electrons and the holes should be captured by any material which exist on the surface (ions, atoms, molecules, etc.), or by some surface traps (Morales-Flores et al., 2011). The doping comes as a solution to this problem, and thus several researches have been done to enhance its activity by doping it with silver, another prime candidate in this area of research. Silver on zinc oxide surfaces acts as a sink for the free electrons, aids in the interfacial transfer of charge between the silver and ZnO, and it effectively inhibits the recombination of electrons and holes. Following that, the electrons can be captured by the soluble oxygen and the holes can be captured by the surface hydroxyl, both resulting in the formation of hydroxyl radical (OH.) (Morales-Flores et al., 2011). Yang et al (2011) formulated the following scheme for the formation of silver-zinc oxide hetero-structure: Zn2+ + 4OH− → Zn(OH)42− …………. … (4) Ag+ + 2OH− → Ag(OH)2 −………………… (5) Zn(OH)4 2− + 2Ag(OH)2 − → Ag2O/ZnO + 2H2O + 4OH− ………………… (6) Ag2O/ZnO Ag/ZnO + O2 (reactions occurs under light)………………… (7) The mechanism of silver doping on zinc oxide substrate is attributed to two factors. The lattice and symmetry match between ZnO and Ag in the corresponding planes is considered the foremost factor, while the direct interfacing of the zinc layer with silver is the next important parameter affecting the growth mechanism (Fan et al., 2009). Wang et al ( 2004) found out that Ag doped ZnO nanocrystallites have increased surface area and change in surface properties such as oxygen vacancies and crystal deficiencies. Various methods of synthesis have been adopted by scientists for doping silver onto zinc oxide nanoparticles, viz., sonication of the zinc salt solution at about (37 ± 3 kHz, 100 W) (Karunakaran et al., 2010), sol gel (Karunakaran
et al., 2011), dip coating (Thongsuriwong et al., 2012), combustion method where zinc nitrate and glycine (with silver nitrate) were employed as reactant cum oxidant and fuel leading to the formation of pale pink Ag doped ZnO nanoparticles (Karunakaran et al., 2011b), microwave assisted techniques using 2.45 × 109 Hz working frequency, at an on-off cycle of 30 seconds keeping the power at 800W (Karunakaran et al., 2011c). Sol- gel have been the most widely method owing to its ease of operation, purity of products, etc. A simplistic diagram of the solgel process is shown in figure 3.
Figure 3 Step by step Schematic diagram of Sol-Gel synthesis method of nanoparticles
Silver doped zinc oxide nanoparticles have showed great antibacterial efficiency tested against various strains of gram positive and gram negative bacteria. There is a wide variety in terms of dosage, applications and substrate material used for silver doping. Table 3 enlists some of the promising publications on silver doping on different substrates, and it has been observed that silver doped particles have found its use in various fields, starting from textile treatment to photo catalysis to biomedical fields.
Table 3 Different silver doped nano particles with their major findings Substrate material
Zinc oxide
Key findings
Particle Size
Reference
When modified by a di-block copolymer, AgZnO showed enhanced antibacterial and photocatalytic effects. The size of the particles decreased with increasing silver content, the pinning effect of the Ag at grain boundary can be the probable reason. MIC for 1.0 mole % Ag-ZnO: 256 µg/ml for S. aureus 512 µg/ml for E. coli Mechanochemical synthesis of silver doped ZnO in a ball mill. Decrease in size and increase in antibacterial efficiency was observed with increasing silver content. S. aureus was found to be more resistant than E. coli. MIC for 0.10 % Ag-ZnO: 100 /ml for S. aureus 40 µg/ml for E. coli Sonochemical (100W, 37 kHz) synthesis of silver doped ZnO. Silver doping sharpens band gap absorption and promotes charge transfer resistance. 0.5 at % Ag-ZnO showed promising bactericidal effect, tested against E. coli Photocatalytic activities of ZnO and Ag-ZnO are found to be substrate specific.
~ 40 nm
Amornpitoksuk et. al(Amornpitoksuk et al., 2012)
16-30 nm
Talari et. al ( 2012)
Increase in doping reduces agglomeration. Stable solutions of ZnO and Ag doped ZnO nanoparticles were applied as liquid form coating agent for textile treatment. Bacterial susceptibility against M. luteus and E. coli increased with increasing silver content. Zinc oxide Fast and reliable microwave assisted technique of supported synthesis. by Bentonite only exhibited antibacterial properties after bentonite acid treatment. clay Silver doping enhanced antimicrobial nature of clay by several folds. Titanium Presence of small Ag nanoparticles around TiO2 in dioxide SEM images confirm surface doping.
87 nm Karunakaran et. al for (2011d) doped ZnO 58 nm for undoped ZnO 13-30 nm
Mariana (2014)
et.
al
9-30 nm Motshekga et. al ( for Ag- 2013) ZnO
~ 20 ± Prakash et. al ( 10 nm 2016)
With increasing doping, enhancement in scattering signals of methyl orange molecules were observed, as observed by Raman spectroscopy measurements. Doped nanoparticles showed promising cell annihilation properties, unlike the undoped ones. Calcium phosphate nanoparticles are doped with silver and stabilised by carboxymethyl cellulose. The nanoparticles are bactericidal against E. coli and S. aureus and their sensitivity are comparable to silver acetate. Tests with mammalian cell lines indicate that the toxicity is due to the silver ion release and the rapid Calcium uptake of these ions by the cell lines is enhancing the Phosphate effect. Bone The changes in the properties of the silver doped Cement ceramic nanopowders were investigated. Antibacterial tests against E. coli, P. aeruginosa, S. aureus, and C. albicans proved these nanopowders to be promising bactericidal. The cytotoxicity tests on three different fibroblast cells and human umbilical vein endothelial cells (HUVECs) revealed that these stimulated proliferations of these cell lines. The angiogenic activity of ABT was investigated by tube formation assay in enthothelial cells. Apoptosis and wound healing effects were examined on fibroblasts and it was seen that ABT powders didn’t induce collapse of membrane potential of mitochondria. Cellulose Electrospun nanofibers were generated with silver Acetate nanoparticles on its surface. It exhibited strong antibacterial activity. Bioactive Alkali mediated sol-gel method of synthesis is used Glass Highly porous nanoparticles were obtained Zone of inhibition assay revealed its promise in antibacterial use, owing to the release of the silver ions. Poly(Vinyl Electrospun PVA nanofibers are suitable for wound Alcohol) healing bandages Heat treatment leads to accumulation of silver ions of the fibre surfaces. S. aureus and K. pneumonia exhibited high cell viability with these doped fibres.
2.2 Copper doping
143 ± 4 nm
Peetsch et. al ( 2013)
40-60 nm
Bostancıoğlu al ( 2015)
~ 600 nm
Son et. al ( 2006)
< 100 nm
Kady et. al ( 2012)
~ 6 nm
Hong et. al (2006)
et.
Copper, an element with atomic number 29, has electronic configuration of [Ar]3d104s1 . It is a soft, malleable, and ductile metal with very high thermal and electrical conductivity. It has been known for its antimicrobial nature since ancient times. It is said to exhibit oligodynamic effect, i.e. biocidal effect even in low concentrations. As reported by Sreshtha et. al ( 2010), the colony counts of E. coli reduced significantly within 4 hours of holding time with copper and brass, while it took about 24 hours for brass. The exact mechanism of this effect is still being researched, but some probable theories suggest that metal ions inactivate the reactive groups by binding to the proteins of the target cells. Various mechanisms are being proposed (Grass et al., 2011; Raffi et al., 2010), the most prominent being the generation of oxidative stress and hydrogen peroxide by the presence of elevated copper levels in cells, giving rise to the Fenton reaction. Basically, copper ions react with cysteine or glutathione leading to the formation of hydrogen peroxide, which in turn helps generating reactive hydroxyl radicals, which is proven to be destroy cellular molecules. 2Cu2+ +2RSH 2Cu2+ + RSSR + 2H + …….. (8) 2Cu2+ + 2H+ + O 2 2Cu2+ + H2O2 ……… (9) .
Cu2+ + H2O2 Cu2+ + OH- + OH …………. (10)
Some researchers are of the opinion that copper mainly kills by contact technique, which completely degrades the plasmid DNA post cell death, and it is enhanced by the fact that cells cannot multiply when in touch with copper surfaces. Thus copper prevents the cells from acquiring resistance (Warnes et al., 2010). Others are of the opinion that excess copper results in loss in the membrane integrity of microbes which leads to leakage of essential cell nutrients, such as potassium and glutamate. This leads to desiccation and subsequent cell death. Disruption of osmotic pressure of the cell and lipid peroxidation remains as other popular proposed mechanisms. Lee et. al., (2016) demonstrated that copper nanoparticles induce
systemic toxicity with various functional and morphological changes in spleen, liver and kidneys. It was observed that the liver is mainly responsible for the metabolism of the copper ions, and thus it gets excreted by the bile. These nanoparticles react with the acidic gastric juice and transform into ionic states. The unabsorbed nanoparticles are excreted via the stool, and a small amount is eliminated through urinary tract (Chen et al., 2006). That is why, doping a small amount of copper on some benign substrate is always preferred, and researched widely. Table 4 summarises some of the studied copper doped nanoparticles along with its significant results and applications. Table 4 Different types of copper doped nanoparticles along with their prominent results Substrate Material
Titanium dioxide
Silica
Key findings Cu doped TiO2 were found to be ferromagnetic at room temperature, activated by the created oxygen vacancies. The saturation magnetization of the nanorods was modulated by the aspect ratio and the amount of dopant. Cu doping enhances the optical absorbance of TiO2 to the visible spectrum. Simple sol-gel synthesis. The nanoparticles only showed bactericidal effect under the effect of UV irradiation. Coumarin based fluorescence probe method confirms that generation of hydroxyl radicals which is responsible for the oxidative damage caused to the bacterial cells. Cu-doped TiO2 NPs inhibited the M. smegmatis growth rate, but did not affect S. oneidensis growth. Addition of EDTA decreased the efficiency of the nanoparticles. The main reason of the antibacterial effect is considered to the Cu ion release, which interfered with the enzyme formation. Presence of chelating agents can enhance the bactericidal nature of the NPs. 2(Si-O-H) + Cu2+ (Si-O)2Cu + 2H+ This has been proposed as the route of copper doping on silica nanoparticles.
Size
Reference
3±1 nm You et. al ( diameter 2010) 30±12 nm length 8-12 nm
Yadav et. al ( 2014)
~20 nm
Wu et. (2010)
~ 88 nm
Leyland et. al ( 2016)
al
Zinc oxide
It showed promising antimicrobial activity against activities were detected against bacterial cells like S. aureus, E. cloacae, E. coli, and P. citrinum, fungi and yeast. Inclusion of catalyst enhanced the binding energy due to the formation of nucleophiles. Cu doping decreases crystallite size of ZnO nanoparticles. Band gap was found to decrease after copper doping. Strain inclusion has an significant effect on the grain size of the NPs. Sol-gel technique using gelatine and nitrate precursors were used. Energy band gap increased and absorption spectra is blue shifted due to Cu doping. Cell viability of E. coli was found to be concentration dependant of doped particles. Proposed use of these Cu doped ZnO in dermatological medicines. Zinc oxide is doped by Cu and its polythiophene (Pth) composites are prepared by sol-gel method. The amount of Cu2+ present was determined by iodometric method. PTh coating on doped particles results in a core– shell structure with copper doped zinc as core and PTh as shell. Two main mechanisms have been proposed: Generation of ROS by the Cu doped Zno and the Pth acts as stabilizing agent which prolongs the time taken by the ROS for cell killing. Thus a synergistic effect is obtained between Pth and Cu-ZnO. The Cu ions released from the nanoparticles can get adsorbed to the bacteria, destroying the charge balance leading to bacteriolysis. Modified neutralization method is used to dope Cu onto Hap. C. albicans count was reduced significantly by the effect of doped nanoparticles The mechanism of cell-particle interaction is proposed to be the presence of metal ions on the surface of the crystals, which form strong bonds with different functional groups present in the cell membrane proteins, leading to structural changes.
25-50 nm
Thakur et. al (2014)
30-52 nm
Samavati et. al (2016)
~ 20 nm
Ma et. (2014)
15-25 nm
Stanic et. al (2010)
al
Hydroxyapatite Hydrothermal method can be used for doping Cu2+ on HAp. The doped particles provided consistent antimicrobial efficiency against good antimicrobial activity against S. aureus, E. coli, P. aeruginosa and C. albicans. MTT assay revealed the non-cytotoxic nature of the particles, proving its use as scaffolds for dental fillings. Silver Sulfide Doping with copper increased the photocatalytic effectiveness of the silver sulfide nanoparticles. Imaging of nanoparticle treated E. faecalis and S. aureus proved the excellent antibacterial ability of the particles. The main mechanism proposed was the electronhole pair generated by the metal ion, which also acts as a charge carrier traps. Cadmium Doping by copper is confirmed by the band shift. Sulfide Both star shaped and spherical doped particles were formed. Star shaped particles exhibited better antimicrobial efficacy than spherical ones against Serratia marcescens. Cobalt Ferrite Inclusion of dopant decreases crystallite size, specific saturation magnetization, remanence and coercivity. Increase in copper concentration increases the ROS generation and this is the prime mechanism of action of the particles on bacterial cells. Substitution of Cu with Co leads to the preferential excitation of electrons from valence band to conduction band, which leads to superoxide formation.
< 100 nm
Radovanović et. al (2014)
30 nm
Fakhri et. al (2015)
30-40 nm
Sundaria et. al (2015)
20-30nm
Samavati et. al (2016)
From table 4, this can be interpreted that in most cases, generation of reactive oxygen species has been proposed to the predominant mechanism behind the bactericidal nature of copper ions and that is why, the doping of copper is so much prevailing in this field. The variation of shapes and sizes also contribute to this property of the nanoparticles. 2.3 Cobalt doping
Cobalt, with symbol Co and atomic number 27, belongs to group 9, period 4 of the modern periodic table, has an electronic configuration of [Ar]3d 74s2. Cobalt is used from as early as 1950s, when cobalt-60 was used to generate a beam of gamma rays to kill tumour tissues. Till today, cobalt treatment still has a useful role to play in certain applications and is still in widespread use worldwide, since the machinery is relatively reliable and simple to maintain. This element has further more uses in medical treatment. Co(III) is used in pernicious anemia because it enhances the formation of red blood cells (erythropoiesis), and is prescribed in fatigue, digestive and neuro-muscular problems as it is a part of vitamin B-12. Cobalt also has a significant role in the biotin-dependent Krebs-cycle, the process by which the sugar is broken to energy in human body. Researchers have found cobalt to be antibacterial (Chang et al., 2010). It is used as a dopant in various nanoparticles like ZnO (Vanaja and Rao, 2016), TiO2 (Bagheri et al., 2014; Barakat et al., 2005; Choudhury and Choudhury, 2012; Mugundan et al., 2015; Yano et al., 2013), CdS (T Lavanya, K Satheesh, 2015), SnO 2 (Entradas et al., 2014) etc by using various methods of synthesis like sol-gel (Lima et al., 2014), chemical precipitation (T Lavanya, K Satheesh, 2015) or microwave synthesis (Sheik Muhideen Badhusha, 2016). Substantial work has been reported on the enhancement of antibacterial efficacy due to cobalt doping (Anandan et al., 2016; Nair et al., 2011; Oves et al., 2015). Sood et. al., reported a mechano-chemical synthesis method of Co/Fe co doped ZnO which exhibited significant antibacterial activity and acceptable cytotoxicity (Sood and Sharma, 2016). However, regardless of these pros, cobalt is not without its cons. It can create toxicity in the liver, kidney, heart, pancreas, and even in skeletal muscles. It has been found to generate tumours in animals and is a human carcinogen as well in certain above-the-limit dosages. 3.4 Other novel doping materials: Doping is a widely accepted method for the alteration of the antibacterial properties of nanoparticles. The defect-related properties and the oxygen
vacancies affect these properties. Various other dopants are being investigated worldwide as described in table 5. Table 5 Comprehensive tabulation of different kinds of antibacterial doped nano particles with the most probable mechanism proposed by the literature Substrate
Dopant
Mechanism proposed
Less than 5% doping of The Fe3+ eluted Fe does not enhance from the the antimicrobial nanoparticles get property of the absorbed on the nanoparticles. cell membrane, imparting damage and leaking the cell material. Neodymium The pure and doped The oxygen particles was of rod vacancies created and flower shaped by the dopant respectively result in the 800 µg/ml of doped formation of ROS, particles resulted in disintegrating the cell membrane. 100% cell death. Tantalum The antimicrobial The synergistic efficiency of the effect of increased particles is more under surface bioactivity visible light. and enhanced 5% doping is electrostatic force considered to be because of doping is considered as the optimum. main reason behind the enhancement of activity. Yttrium Lattice parameters and Decrease of the unit cell volume band gap due to confirms successful Y doping helps in doping. generation of Doped particles has more ROS. increased activity against E. coli, B. subtilis, S. typhi, unlike S. aureus. Iron
Zinc oxide
Key findings
Size
Reference
25-45 nm
Khatir et. al.( 2016)
~ 33 nm
Hameed et. al. ( 2016)
~ 30 nm
Hameed al.(2015)
16-30 nm
Mote et. al. (2015)
et.
Manganese Mn doping results in Production of blue shift of ZnO. ROS and elution of metal ions both 10% Mn-ZnO showed promising antibacterial acted activity against E. simultaneously coli, K. pneumonia, P. for the rupture of the cell aeruginosa, S. membrane, aureus, S. leading to cell typhimurium and S. lysis. agalactiae. Nickel ---- Ni is incorporated on ZnO substrate by Hydrothermal decomposition method. Doped particles exhibited antimicrobial properties against P aeruginosa and B subtilis Selenium Antibacterial activity The reduction in decreased after doping antibacterial with Selenium. efficiency was due to two opposing factors like ROS generation, which is countered by sustained growth of bacteria due to the presence of micronutrients like Se in culture medium. Tin ------ Doping with tin enhanced the inhibitory activity of the zinc oxide particles. Tests proved the nanoparticles to be very bio-safe and biocompatible towards SH-SY5Y cells
~ 20 nm
Dhanalakshmi et. al. (2016)
~ 28 nm
Vimala (2015)
10.2±3.4 nm
Dutta et. al. (2014)
24-36 nm
Jan et. (2013)
al.
Titanium dioxide
Cerium Oxide
Copper oxide
Silver and Hydrolysis -------nitrogen precipitation method is used for synthesis. The doped particles exhibited higher efficacy under fluorescent light irradiation. The absorption band of TiO2 co doped by Ag and N had red shift. Indium and Use of acetic acid as The main Iron solvent affected the mechanism is the size and the binding of the bactericidal nature of nanoparticles to TiO2. the cell membrane in Iron doping fail to resulting of enhance the hindrance antimicrobial effect as active transport to it is an essential leading of micronutrient for inhibition RNA, DNA and bacterial sustainment. protein synthesis. Tungsten Increased doping The trioxide decreased the photogenerated crystallite size. hydroxyl radicals superoxide Doped particles was and powerful coated on charcoal and are oxidising agents, treated with E. coli resulting in lipid under UV irradiation. 1% doping showed peroxidation. promising antibacterial effect. Gadolinium Cubic shaped Penetration of the morphologies was decreased sized shown by doped particles through particles the cell membrane The cell killing leads to leakage of potential of cytoplasmic material. CeO2 considerably increased by doping. Zinc Doping is done by a The main green sono-chemical mechanism is said method. to be the piercing Zn-Cuo exhibited process on the cell brilliant cell membrane leading accelerated annihilation abilities to
20-40 nm
Yuan et. al. (2010)
8-12 nm
Castroalarcón et. al. (2016)
13-16 nm
Sangchay (2017)
~ 58 nm
Khadar et. al. (2017)
--
Wu et. (2016)
al.
against strains.
bacterial
cytoplasmic material leakage.
4. Conclusion With the emergence of antibiotic resistant bacterial strains, more and more attention is given to research involving novel materials. The number of new publications and patents in the field of nanoparticle doping has been exponentially increasing for the last decade. Change in energy levels, band gap narrowing and oxygen vacancies develop due to doping of a nanoparticle, are the main reason of their altered behaviour towards bacterial cells. Novel dopant materials are patented and discussed widely. Mainly, it has been proven that by varying synthesis parameters, dopant concentration, and by surface modification, very powerful antimicrobials can be obtained. This current review aims at summarising the extensive ongoing research on antimicrobial applications of doped nanoparticles for setting a well-built reference for scientists in their future endeavours. The main prominent area of future research would be the synthesis and development of novel dopant materials, advanced methods of dopant incorporation into nanoparticles as well as new modes of application for medical technology. Much more attention is required to the chemistry of the dopant material so as to develop a perfect antimicrobial with tailor made properties. The mechanism of action of the doped nanoparticles against bacterial cells have not been elucidated properly, much more insight is still in need of research. Further in-vivo studies are necessary to completely understand the effect of various dopants keeping their bio compatibility in consideration. With further optimisation, luminescence behaviour of doped particles can be used as non-toxic bio imaging agents. References:
Anandan M, Dinesh S, Krishnakumar N, Balamurugan K. Influence of Co doping on combined photocatalytic and antibacterial activity of ZnO nanoparticles. Mater Res Express 2016;3:115009. doi:10.1088/2053-1591/3/11/115009. Bagheri S, Chekin F, Hamid SBA. Cobalt doped titanium dioxide nanoparticles: Synthesis, characterization and electrocatalytic study. J Chinese Chem Soc 2014;61:702–6. doi:10.1002/jccs.201300486. Barakat MA, Hayes G, Shah SI. Effect of Cobalt Doping on the Phase Transformation of TiO 2 Nanoparticles. J Nanosci Nanotechnol 2005;X:1–7. doi:10.1166/jnn.2005.087. Burello E, Worth AP. A theoretical framework for predicting the oxidative stress potential of oxide nanoparticles. Nanotoxicology 2011;5:228–35. doi:10.3109/17435390.2010.502980. Chambers C, Stewart SB, Su B, Jenkinson HF, Sandy JR, Ireland AJ. Silver doped titanium dioxide nanoparticles as antimicrobial additives to dental polymers. Dent Mater 2017;33:e115– 23. doi:10.1016/j.dental.2016.11.008. Chang EL, Simmers C, Knight DA. Cobalt complexes as antiviral and antibacterial agents. Pharmaceuticals 2010;3:1711–28. doi:10.3390/ph3061711. Chen Z, Meng H, Xing G, Chen C, Zhao Y, Jia G, et al. Acute toxicological effects of copper nanoparticles in vivo. Toxicol Lett 2006;163:109–20. doi:10.1016/j.toxlet.2005.10.003. Chimileski S, Kolter R. Life at the Edge of Sight: A Photographic Exploration of the Microbial World. 2017. Choudhury B, Choudhury A. Luminescence characteristics of cobalt doped TiO2 nanoparticles. J Lumin 2012;132:178–84. doi:10.1016/j.jlumin.2011.08.020. Choudhury SR, Ordaz J, Lo CL, Damayanti NP, Zhou F, Irudayaraj J. Zinc oxide
nanoparticles-induced reactive oxygen species promotes multimodal cyto- and epigenetic toxicity. Toxicol Sci 2017a;156:261–74. doi:10.1093/toxsci/kfw252. Choudhury SR, Ordaz J, Lo CL, Damayanti NP, Zhou F, Irudayaraj J. Zinc oxide nanoparticles-induced reactive oxygen species promotes multimodal cyto- and epigenetic toxicity. Toxicol Sci 2017b;156:261–74. doi:10.1093/toxsci/kfw252. Entradas T, Cabrita JF, Dalui S, Nunes MR, Monteiro OC, Silvestre AJ. Synthesis of sub-5 nm Co-doped SnO2 nanoparticles and their structural, microstructural, optical and photocatalytic properties. Mater Chem Phys 2014;147:563–71. doi:10.1016/j.matchemphys.2014.05.032. Fan FR, Ding Y, Liu DY, Tian ZQ, Zhong LW. Facet-selective epitaxial growth of heterogeneous nanostructures of semiconductor and metal: ZnO nanorods on ag nanocrystals. J Am Chem Soc 2009;131:12036–7. doi:10.1021/ja9036324. Gajewicz A, Schaeublin N, Rasulev B, Hussain S, Leszczynska D, Puzyn T, et al. Towards understanding mechanisms governing cytotoxicity of metal oxides nanoparticles: Hints from nano-QSAR studies. Nanotoxicology 2015;9:313–25. doi:10.3109/17435390.2014.930195. Garcidueñas-Piña C, Medina-Ramírez IE, Guzmán P, Rico-Martínez R, Morales-Domínguez JF, Rubio-Franchini I. Evaluation of the Antimicrobial Activity of Nanostructured Materials of Titanium Dioxide Doped with Silver and/or Copper and Their Effects on Arabidopsis thaliana. Int J Photoenergy 2016;2016:1–14. doi:10.1155/2016/8060847. Grass G, Rensing C, Solioz M. Metallic copper as an antimicrobial surface. Appl Environ Microbiol 2011;77:1541–7. doi:10.1128/AEM.02766-10. Gualerzi CO, Brandi L, Fabbretti A, Pon CL. Antibiotics : Targets, Mechanisms and Resistance. Wiley; 2013.
Gupta K, Singh RP, Pandey A, Pandey A. Photocatalytic antibacterial performance of TiO 2 and Ag-doped TiO 2 against S. aureus . P. aeruginosa and E. coli. Beilstein J Nanotechnol 2013;4:345–51. doi:10.3762/bjnano.4.40. Hammond CM, Strømme CB, Huang H, Patel DJ, Groth A. Histone chaperone networks shaping
chromatin
function.
Nat
Rev
Mol
Cell
Biol
2017;18:141–58.
doi:10.1038/nrm.2016.159. Herrmann IK, Urner M, Koehler FM, Hasler M, Roth-Z’graggen B, Grass RN, et al. Blood purification
using
functionalized
core/shell
nanomagnets.
Small
2010;6:1388–92.
doi:10.1002/smll.201000438. Imran M, Muazzam AG, Habib A, Matin A. Synthesis, characterization and amoebicidal potential of locally synthesized TiO2 nanoparticles against pathogenic Acanthamoeba trophozoites
in
vitro.
J
Photochem
Photobiol
B
Biol
2016;159:125–32.
doi:10.1016/j.jphotobiol.2016.03.014. Ito A, Kamihira M. Tissue engineering using magnetite nanoparticles. Prog Mol Biol Transl Sci 2011;104:355–95. doi:10.1016/B978-0-12-416020-0.00009-7. Jansen B, Rinck M, Wolbring P, Strohmeier A, Jahns T. In vitro Evaluation of the Antimicrobial Efficacy and Biocompatibility of a Silver-Coated Central Venous Catheter. J Biomater Appl 1994;9:55–70. doi:10.1177/088532829400900103. Karunakaran C, Rajeswari V, Gomathisankar P. Enhanced photocatalytic and antibacterial activities of sol–gel synthesized ZnO and Ag-ZnO. Mater Sci Semicond Process 2011a;14:133–8. doi:10.1016/j.mssp.2011.01.017. Karunakaran C, Rajeswari V, Gomathisankar P. Combustion synthesis of ZnO and Ag-doped ZnO and their bactericidal and photocatalytic activities. Superlattices Microstruct
2011b;50:234–41. doi:10.1016/j.spmi.2011.06.005. Karunakaran C, Rajeswari V, Gomathisankar P. Optical, electrical, photocatalytic, and bactericidal properties of microwave synthesized nanocrystalline Ag-ZnO and ZnO. Solid State Sci 2011c;13:923–8. doi:10.1016/j.solidstatesciences.2011.02.016. Karunakaran C, Rajeswari V, Gomathisankar P. Antibacterial and photocatalytic activities of sonochemically prepared ZnO and Ag–ZnO. J Alloys Compd 2010;508:587–91. doi:10.1016/j.jallcom.2010.08.128. Klueh U, Wagner V, Kelly S, Johnson A, Bryers JD. Efficacy of silver-coated fabric to prevent bacterial colonization and subsequent device-based biofilm formation. J Biomed Mater Res 2000;53:621–31. doi:10.1002/1097-4636(2000)53:63.0.CO;2-Q. Lee IC, Ko JW, Park SH, Lim JO, Shin IS, Moon C, et al. Comparative toxicity and biodistribution of copper nanoparticles and cupric ions in rats. Int J Nanomedicine 2016;11:2883–900. doi:10.2147/IJN.S106346. Li P, Li J, Wu C, Wu Q, Li J. Synergistic antibacterial effects of β-lactam antibiotic combined with silver nanoparticles. Nanotechnology 2005;16:1912–7. doi:10.1088/0957-4484/16/9/082. Lima MK, Fernandes DM, Silva MF, Baesso ML, Neto AM, de Morais GR, et al. Co-doped ZnO nanoparticles synthesized by an adapted sol???gel method: Effects on the structural, optical, photocatalytic and antibacterial properties. J Sol-Gel Sci Technol 2014;72:301–9. doi:10.1007/s10971-014-3310-z. Lowy F. Bacterial Classification , Structure and Function. Columbia Univ 2009:1–6. Luo X, Morrin A, Killard AJ, Smyth MR. Application of nanoparticles in electrochemical sensors and biosensors. Electroanalysis 2006;18:319–26. doi:10.1002/elan.200503415.
McIlwain
H.
Biochemistry
and
chemotherapy.
Nature
1943;151:270–3.
doi:10.1038/151270a0. Morales-Flores N, Pal U, Sánchez Mora E. Photocatalytic behavior of ZnO and Pt-incorporated ZnO nanoparticles in phenol degradation. Appl Catal A Gen 2011;394:269–75. doi:10.1016/j.apcata.2011.01.011. Mugundan S, Rajamannan B, Viruthagiri G, Shanmugam N, Gobi R, Praveen P. Synthesis and characterization of undoped and cobalt-doped TiO2 nanoparticles via sol–gel technique. Appl Nanosci 2015;5:449–56. doi:10.1007/s13204-014-0337-y. Nair MG, Nirmala M, Rekha K, Anukaliani A. Structural, optical, photo catalytic and antibacterial activity of ZnO and Co doped ZnO nanoparticles. Mater Lett 2011;65:1797–800. doi:10.1016/j.matlet.2011.03.079. Neidhardt F., Ingraham J., Schaechter M. Physiology of the bacterial cell — A molecular approach. vol. 20. 1990. doi:10.1016/0168-9525(91)90427-R. Nune SK, Gunda P, Thallapally PK, Lin Y, Laird M, Berkland CJ. Nanoparticles for biomedical
imaging.
Expert
Opin
Drug
Deliv
2009;6:1175–94.
doi:10.1517/17425240903229031.Nanoparticles. Okamoto M, John B. Synthetic biopolymer nanocomposites for tissue engineering scaffolds. Prog Polym Sci 2013;38:1487–503. doi:10.1016/j.progpolymsci.2013.06.001. Oves M, Arshad M, Khan MS, Ahmed AS, Azam A, Ismail IMI. Anti-microbial activity of cobalt doped zinc oxide nanoparticles: Targeting water borne bacteria. J Saudi Chem Soc 2015;19:581–8. doi:10.1016/j.jscs.2015.05.003. Pelgrift RY, Friedman AJ. Nanotechnology as a therapeutic tool to combat microbial
resistance. Adv Drug Deliv Rev 2013;65:1803–15. doi:10.1016/j.addr.2013.07.011. Priest FG. Systematics and Ecology of Bacillus. Bacillus subtilis Other Gram-Positive Bact., American Society of Microbiology; 1993, p. 3–16. doi:10.1128/9781555818388.ch1. Raffi M, Mehrwan S, Bhatti TM, Akhter JI, Hameed A, Yawar W, et al. Investigations into the antibacterial behavior of copper nanoparticles against Escherichia coli. Ann Microbiol 2010;60:75–80. doi:10.1007/s13213-010-0015-6. Saleh NB, Milliron DJ, Aich N, Katz LE, Liljestrand HM, Kirisits MJ. Importance of doping, dopant distribution, and defects on electronic band structure alteration of metal oxide nanoparticles: Implications for reactive oxygen species. Sci Total Environ 2016;568:926–32. doi:10.1016/j.scitotenv.2016.06.145. Sheik Muhideen Badhusha M. Microwave assisted synthesis of ZnO and Co doped ZnO nanoparticles and their antibacterial activity. Pharma Chem 2016;8:78–84. Shrestha R, Joshi DR, Gopali J, Piya S. Oligodynamic Action of Silver, Copper and Brass on Enteric Bacteria Isolated from Water of Kathmandu Valley. Nepal J Sci Technol 2010;10:189– 93. doi:10.3126/njst.v10i0.2959. Sood S, Sharma N. Antibacterial Activity and Cytotoxicity of Co/Fe Doped ZnO Nanoparticles. Adv Sci Eng Med 2016;8:468–76. doi:10.1166/asem.2016.1880. Stensberg MC, Wei Q, McLamore ES, Porterfield DM, Wei A, Sepúlveda MS. Toxicological studies on silver nanoparticles: challenges and opportunities in assessment, monitoring and imaging. Nanomedicine (Lond) 2011;6:879–98. doi:10.2217/nnm.11.78. T Lavanya, K Satheesh NVJ. Ferromagnetic behaviour at room temperature of Co doped CdS nanoparticles by chemical precipitation method: Synthesis, optical and electrical properties.
IOP Conf Ser Mater Sci Eng 2015;73:012114. doi:1088/1757-899X/73/1/012114. Thiel J, Pakstis L, Buzby S, Raffi M, Ni C, Pochan DJ, et al. Antibacterial Properties of SilverDoped Titania. Small 2007;3:799–803. doi:10.1002/smll.200600481. Thongsuriwong K, Amornpitoksuk P, Suwanboon S. Photocatalytic and antibacterial activities of Ag-doped ZnO thin films prepared by a sol–gel dip-coating method. J Sol-Gel Sci Technol 2012;62:304–12. doi:10.1007/s10971-012-2725-7. Vanaja A, Rao KS. Effect of Co Doping on Structural and Optical Properties of Zinc Oxide Nanoparticles
Synthesized
by
Sol-Gel
Method.
Adv
Nanoparticles
2016;5:83–9.
doi:10.4236/anp.2016.51010. Wang C, Irudayaraj J. Multifunctional magnetic-optical nanoparticle probes for simultaneous detection, separation, and thermal ablation of multiple pathogens. Small 2010;6:283–9. doi:10.1002/smll.200901596. Wang R, Xin JH, Yang Y, Liu H, Xu L, Hu J. The characteristics and photocatalytic activities of
silver
doped
ZnO
nanocrystallites.
Appl
Surf
Sci
2004;227:312–7.
doi:10.1016/j.apsusc.2003.12.012. Warnes SL, Green SM, Michels HT, Keevil CW. Biocidal efficacy of copper alloys against pathogenic enterococci involves degradation of genomic and plasmid DNAs. Appl Environ Microbiol 2010;76:5390–401. doi:10.1128/AEM.03050-09. Williams KJ. The introduction of “chemotherapy” using arsphenamine - The first magic bullet. J R Soc Med 2009;102:343–8. doi:10.1258/jrsm.2009.09k036. Yang Z, Zhang P, Ding Y, Jiang Y, Long Z, Dai W. Facile synthesis of Ag/ZnO heterostructures assisted by UV irradiation: Highly photocatalytic property and enhanced
photostability. Mater Res Bull 2011;46:1625–31. doi:10.1016/j.materresbull.2011.06.016. Yano S, Kurokawa A, Takeuchi H, Yanoh T, Onuma K, Kondo T, et al. Synthesis and study of magnetic properties of Co-doped anatase TiO2 nanoparticles. J Phys Conf Ser 2013;433:012019/1-012019/7, 7 pp. doi:10.1088/1742-6596/433/1/012019. Zhang L, Pornpattananangkul D, Hu C-MJ, Huang C-M. Development of Nanoparticles for Antimicrobial
Drug
Delivery.
Curr
Med
Chem
2010;17:585–94.
doi:10.2174/092986710790416290. Zhao X, Ren X, Zhu R, Luo Z, Ren B. Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria-mediated apoptosis in zebrafish embryos. Aquat Toxicol 2016;180:56–70. doi:10.1016/j.aquatox.2016.09.013.
