1.1 Introduction to Nanoscience and Nanotechnology
Nanotechnology is the study of phenomena and fine tuning of materials at atomic, molecular and macromolecular scales, where properties differ significantly from those at a larger scale. The term “Nano” refers to the metric prefix 10-9. It means one billionth of something. “Nano” can be ascribed to any unit of measure, for example, a very small mass in nanograms or the amount of liquid in one cell in terms of nanoliters.
Nanoscale structures have existed in nature long before scientists began studying in laboratories. A single strand of DNA, the building block of all living things, is about 3 nanometers wide. The scales on a morpho butterfly’s wings contain nano structures that change the way light wave interact with each other, giving the wings brilliant metallic blue and green hues. Peacock feathers and soap bubbles also get their iridescent coloration from light interacting with structures just tens of nanometers thick. Scientists have even created nano structures in the laboratory that mimic some of nature’s amazing nanostructures. The foundations of nanotechnology have emerged over many decades of research in many different fields. Computer circuits have been getting smaller. Chemicals have been getting more complex. Biochemists have learned more about how to study and control the molecular basis of organisms. Mechanical engineering has been getting more precise. In 1959, the great physicist Richard Feynman (Figure 1.1) suggested that it should be possible to build machine small enough to manufacture object with atomic precision. His talk, “There’s Plenty of Room at the Bottom”, is widely considered to be the foreshadowing of nanotechnology. Among other things, he predicted that information could be stored with amazing density. In the late 1970?s, Eric Drexler (Figure 1.2) began to invent what would become molecular manufacturing. He quickly realized that molecular machine could control the chemical manufacture of complex products, including additional manufacturing systems which would be a very powerful technology. In 1986 he introduced the term “Nanotechnology” in his book Engines of Creation. 1.1
Nanoscience and nanotechnology involve the ability to see and to control individual atoms and molecules. Everything on Earth is made up of atoms the food we eat, the clothes we wear, the buildings and houses we live in, and our own bodies. But something as small as an atom is impossible to see with the naked eye. In fact, it’s impossible to see with the microscopes typically used in a high school science classes. The microscopes needed to see things at the nanoscale were invented relatively recently about 30 years ago. Today’s scientists and engineers are finding a wide variety of ways to deliberately make materials at the nanoscale to take advantage of their enhanced properties such as higher strength, lighter weight, increased control of light spectrum, and greater chemical reactivity than their larger-scale counterparts. 1.2
1.1.1 Fundamental concepts in Nanoscience and Nanotechnology
The fundamental properties of matter change at the nanoscale. The properties of atoms and molecules are not governed by the same physical laws as larger objects, but by “quantum mechanics”. The physical and chemical properties of nanoparticles can be quite different from those of larger particles of the same substance. Altered properties can include but are not limited to colour, solubility, material strength, electrical conductivity, magnetic behavior, mobility (within the environment and within the human body), chemical reactivity and biological activity. Medieval stained glass windows and Lycurgus cup (Figure 1.3) are the examples of how nanotechnology was used in the pre-modern era. It’s hard to imagine just how small nanotechnology is. One nanometer is a billionth of a meter, or 10-9 of a meter.
Here are a few illustrative examples:
There are 25,400,000 nanometers in an inch
A sheet of newspaper is about 100,000 nanometers thick
On a comparative scale, if a marble were a nanometer, then one meter would be the size of the Earth.
Nanoscience and nanotechnology involve the ability to see and to control individual atoms and molecules. Everything on Earth is made up of atoms the food we eat, the clothes we wear, the buildings and houses we live in, and our own bodies.
But something as small as an atom is impossible to see with the naked eye. In fact, it’s impossible to see with the microscopes typically used in a high school science classes. The microscopes needed to see things at the nanoscale were invented relatively recently about 30 years ago.
Once scientists had the right tools, such as the scanning tunneling microscope (STM) and the atomic force microscope (AFM), the age of nanotechnology was born. Although modern nanoscience and nanotechnology are quite new, nanoscale materials were used for centuries. Alternate-sized gold and silver particles created colors in the stained glass windows of medieval churches hundreds of years ago. The artists back then just didn’t know that the process they used to create these beautiful works of art actually led to changes in the composition of the materials they were working with.
Today’s scientists and engineers are finding a wide variety of ways to deliberately make materials at the nanoscale to take advantage of their enhanced properties such as higher strength, lighter weight, increased control of light spectrum, and greater chemical reactivity than their larger-scale counterparts. 1.2
A nanoparticle is the most fundamental component in the fabrication of a nanostructure, and is far smaller than the world of everyday objects that are described by Newton’s Laws of Motion, but bigger than an atom or a simple molecule that are governed by Quantum Mechanics. Nanoparticles are between 1 and 100 nanometers in size. In nanotechnology, a particle is defined as a small object that behaves as a whole unit with respect to its transport and properties. Particles are further classified according to diameter. Ultrafine particles are the same as nanoparticles and between 1 and 100 nanometers in size, fine particles are sized between 100 and 2,500 nanometers, and coarse particles cover a range between 2,500 and 10,000 nanometers. Scientific research on nanoparticles is intense as they have many potential applications in medicine, optics, and electronics. The term nanoparticle is not usually applied to individual molecules, and usually refers to inorganic materials.
The reason for the synonymous definition of nanoparticles and ultrafine particles is that, during the 1970-80s, when the first thorough fundamental studies with “nanoparticles” were underway in the USA and Japan, they were called “ultrafine particles” (UFP). However, during the 1990s before the National Nanotechnology Initiative was launched in the USA, the new name, “nanoparticle,” had become fashionable (see, for example the same senior author’s paper 20 years later addressing the same issue, lognormal distribution of sizes. Nanoparticles can exhibit size-related properties significantly different from those of either fine particles or bulk materials. 1.3
1.2.1 Properties of Nanoparticles
Physical and chemical characteristics of nanomaterial as compare to conventional bulk particles nanomaterials exhibits some unique physical properties including electrical, catalytic, magnetic, mechanical, thermal, or imaging features that make the nanomaterials a relevant topic in medical pharmaceutical possesses some remarkable and specific peculiar properties which may be significantly distinctive from the physical properties of bulk materials.
18.104.22.168 Optical Properties
Optical properties exhibited by nanomaterials are quite different from their bulk counterpart. The reason behind this change in property is mainly due to the effect of the Surface Plasmon Resonance. There are few examples where the materials show the different colour when they are converted to nanoparticles. As per example when the gold materials are converted to nanomaterials they turn into red color at 25 nm (Figure1.3). Gold nanoparticle interaction with light is strongly governed by the particle size (~2-150 nm) have high surface electron densities which are called as surface plasmons undergo a collective oscillation when they are excited by light at specific wavelengths. This oscillation is described as a surface plasmon resonance (SPR). For small (~30 nm) monodisperse gold nanoparticles the surface plasmon resonance phenomena is responsible for an absorption the blue-green portion of the spectrum (~450 nm) while red light (`700nm) is reflected, producing a rich red color.
22.214.171.124 Thermal Properties
As the melting point drastically falls, the particle size of the material approaches to the nanoscale ranges. This phenomenon related to melting point depression is very prominent in nanoscale materials which melt at temperatures hundreds of degrees lower than bulk materials. Melting point depression is most evident in nanowires, nanotubes and nanoparticles, which all melt at lower temperatures than bulk form of the same material. Changes in melting point occur because nanoscale materials have a much larger surface to volume ratio than bulk materials, drastically altering their thermodynamic and thermal properties.
126.96.36.199 Mechanical Properties
All the nanomaterial possesses high mechanical strength as compared to their conventional counterparts. The mechanical strength of nanomaterials may be one or two times higher in magnitude than that of single crystals in the bulk form. Defects in the form of atomic vacancies can lower the tensile strength of the materials by up to 85%. Conversion of materials in to nanoscale increases crystal perfection or reduction of defects, which would result the enhancement in mechanical strength.
188.8.131.52 Electrical Properties
This is quite complex phenomenon. Reduction in materials dimensions would have two different contrasting effects on electrical conductivity. By its property nanoparticle product enhance the crystal perfection and as well as it reduce the defects. As a result electron scattering phenomenon due to crystal defects are also reduced and a reduction in resistivity is experienced.
184.108.40.206 Chemical Properties
The chemical properties are also changed when bulk particles converts to nano range. Due to increase of exposed surface area of the nanoparticles as compared with conventional bulk objects, reactivity of those particles increases enormously.
1.2.2 Classification of Nanoparticles
There are many types of intentionally produced nanomaterials, and the varieties of others are expected to appear in the future.
There are various approaches for the classification of Nanoparticles. They are:
220.127.116.11 On the Basis of Origin
Nanomaterials which are formed originally in nature are called Natural nanomaterials. Examples include Virus, Protein molecules and Antibodies. Besides these, minerals such as clay, natural colloids such as milk and blood, mineralized natural materials such as shells, volcanic ash, ocean spray etc. are naturally occurring materials of nano dimension.
Artificial nanomaterials are those which are prepared deliberately through a well-defined mechanical and fabricated process. Examples include Carbon nanotubes, Semiconductor nanoparticles like Quantum dots etc.
18.104.22.168 On the Basis of Dimension
Zero dimensional (0-D)
These nanomaterials have nanodimensions in all the three directions. Metallic nanoparticles including gold and silver nanoparticles and semiconductor such as quantum dots are the perfect example of this kind of nanoparticles. Most of these nanoparticles are spherical in size and the diameter of these particles will be in the 1-50 nm range. Cubes and polygons shapes are also found for this kind of nanomaterials.
One dimensional (1-D)
In these nanostructures, one dimension of the nanostructure will be outside the nanometer range. These include nanowires, nanorods and nanotubes. These materials are long (several micrometer in length), but with diameter of only a few nanometer. Nanowires and nanotubes of metals, oxides and other materials are few examples of this kind of materials.
Two dimensional (2-D)
In this type of nanomaterials, two dimensions are outside the nanometer range. These include different kind of nano films such as coating and thin-film-multilayers, nano sheets or Nano-walls. The area of the nano films can be large (several square micrometer), but the thickness is always in nano scale range.
Three dimensional (3-D)
All dimensions of these are outside the nanometer range. These include bulk materials composed of the individual blocks which are in the nanometer scale (1-100 nm)
22.214.171.124 On the Basis of Structural Configuration
Carbon based material
These nanomaterials are composed mostly of carbon, most commonly taking the form of a hollow spheres, ellipsoids, or tubes. Spherical and ellipsoidal carbon nanomaterials are referred to as fullerenes, while cylindrical ones are called nanotubes. These particles have many potential applications including improved films and coatings, stronger and lighter materials, and applications in electronics.
Metal based materials
These nanomaterials include quantum dots, nanogold, nanosilver, and metal oxides, such as titanium dioxide. A quantum dot is a closely packed semiconductor crystal comprised of hundreds or thousands of atoms, and whose size is on the order of a few nanometers to a few hundred nanometers. Changing the size of quantum dots changes their optical properties.
These nanomaterials are nanosized polymers built from branched units. The surface of a dendrimer has numerous chain ends, which can be tailored to perform specific chemical functions. This property could also be useful for catalysis. Also, because three dimensional dendrimers contain interior cavities into which other molecules could be placed, they may be useful for drug delivery.
Composites combine nanoparticles with other nanoparticles or with larger, bulk type material. Nanoparticles, such as nanosized clays are already being added to products ranging from auto parts to packaging materials, to enhance mechanical, thermal, barrier and flame-retardant properties. 1.1
1.2.3 Traditional Methods of Synthesis
Basically there are two approaches for nanoparticle for nanoparticle synthesis namely the ‘Top-down’ approach and ‘Bottom-up’ approach.
In the Top-down approach, nano objects are constructed from larger entities without atomic level and control. These seek to create smaller devices by using larger ones to direct their assembly. The method refers to a set of fabrication technologies which fabricate by removing certain parts from a bulk material substrate. The removing methods can be mechanical, chemical, electrochemical and etc., depending on the material of the base substrate and requirement of the feature sizes. The formed structures usually share the same material with the base substrate. There are a couple of manufacturing technologies in the conventional scale which can be categorized Top-down. Milling is a representative example. In the milling process, material is selectively removed from the substrate, usually a metal sheet, forming a cavity with certain geometries. The dimensions of the cavity depend on the travel path of the mill, which can be precisely controlled with the help of computer assisted numerical systems. The milling technique, along with similar methods such as drilling and grinding, is the most widely used technique in conventional manufacturing industry. People have attempted to extend Top-down method into nanometer domain and supplemented the mechanical removing methods with chemical and electrochemical methods. Many technologies that descended from conventional solid-state silicon methods for fabricating microprocessors are now capable of creating features smaller than 100 nm. Solid-state techniques can also be used to create devices known as Nanoelectromechanical systems (NEMS).
In the “Bottom-up” approach, materials and devices are built from molecular components which assemble themselves chemically by principles of molecular recognition. These seek to arrange smaller components into more complex assemblies. The methods refer to a set of technologies which fabricate by stacking materials on top of a base substrate. These methods are similar in principle to welding and riveting at the conventional scale, in which a different type of material is attached to the base component melted solder or physical fitting. In welding and riveting, attention is mainly paid to the strength of the contact area in order to maintain the construct as a reliable component for high load application. Similarly, in bottom-up nanofabrication, the adhesion of the surface layer to the base substrate is also an important concern 1.4. There is extensive research on the surfactants to enhance adherence and avoid cracks during the subsequent processing. Research has also focused on autonomous patterning of the surface layer into nanometer scale features since manipulation of nanoscale components is not ever an easy task as compared to that at the conventional scale. DNA nanotechnology utilizes the specificity of Watson-crick base pairing to construct well defined structures out of DNA and other nucleic acids. Approaches from the field of classical chemical synthesis also aim at designing molecules with well-defined shape.
Traditionally nanoparticles were produced only by physical and chemical methods. Some of the commonly used physical and chemical methods are ion sputtering, solvothermal synthesis, reduction and sol-gel technique 1.5. An overview of these methods is given below.
It is a wet chemical technique used for the fabrication of metal oxides from a chemical solution which acts a precursor for integrated network (gel) of discrete particles or polymers. The precursor sol can be either deposited on the substrate to form a film, cast into a suitable container with desired shape or used to synthesize powders.
This is a versatile low temperature route in which polar solvents under pressure and at low temperatures above their boiling points are used. Under solvothermal conditions, the solubility of reactants increases significantly, enabling reaction to take place at lower temperature.
It is the reduction of an ionic salt in an appropriate medium in the presence of surfactant using reducing agents. Some of the commonly used reducing agents are sodium borohydride, hydrazine hydrate and sodium citrate.
It is a process of removing material from a solid surface by irradiating with a laser beam. At lower laser flux, the material is heated by absorbed laser energy and evaporates or sublimates. At higher flux, the material is converted to plasma. The depth over which laser energy is absorbed and the amount of material removed by single laser pulse depends on the optical properties of the material and the laser wavelength. Carbon nanotubes can be produced by this method.
Inert gas condensation:
Here different metals are evaporated in separate crucibles inside an ultra-high vacuum chamber filled with helium or argon gas at typical pressure of a few 100 Pascal. As a result of inter atomic collisions with gas atoms in chamber, the evaporated metal atoms lose their kinetic energy and condense in the form of small crystals which accumulate on liquid nitrogen filled cold finger. For example, gold nanoparticles can be synthesized from gold wires using this technique 1.6.
1.3 Synthesis of nanoparticles using Plants and Bio-organism
The physical and chemical syntheses of nanoparticles are not eco-friendly because of many drawbacks such as the presence of toxic organic solvents, production of hazardous by-products and intermediary compounds and high energy consumption. A greener approach in the synthesis of nanomaterials is therefore needed 1.5. This approach, called Green synthesis mainly concerns the elimination of hazardous wastes and the utilization of sustainable processes, implementation of environmental friendly chemicals, solvents and renewable materials. In the early researches on greener methods, scientists used microorganisms and then plant extracts.
It has long been known that plants are able to reduce metal ions both on their surface and in various organs and tissues remote from the ion penetration site. Due to this, plants have been used for extracting precious metals from land which would be economically unjustifiable to mine. This approach is known as phytomining 1.7. The metal accumulated by the plants can be recovered after harvesting through sintering and smelting methods. Interestingly, study of the bioaccumulation process in plants has revealed that metals are usually deposited in the form of nanoparticles. The advantage of using plants for the synthesis of nanoparticles is that they are easily available, safe to handle and possess a broad variability of metabolites that may add in reduction. The most important bioreductants in the synthesis of metal nanoparticles are plant extracts.
Synthesis using bio-organisms is compatible with the green chemistry principles as the bio-organism, the reducing agent employed and the capping agent in the reaction are eco-friendly. Initially bacteria used to synthesize nanoparticles and this was later succeeded with the use of fungi because they are known to secrete much higher amounts of proteins, thus might have significantly higher productivity of nanoparticles in biosynthetic approach. Bacteria are known to produce inorganic materials either intracellularly or extracellularly. Some well-known examples of bacteria synthesizing inorganic materials include magnetotactic bacteria (synthesizing magnetic nanoparticles) and S layer bacteria which produce gypsum and calcium carbonate layers. Many bacteria produce nanostructured mineral crystals and metallic nanoparticles with properties similar to chemically synthesized materials, while exercising strict control over size, shape and composition of the particles. Examples include the formation of magnetic nanoparticles by magnetotactic bacteria, the production of silver nanoparticles within the periplasmic space of Pseudomonas stutzeri and the formation of palladium nanoparticles using sulphate reducing bacteria in the presence of an exogenous electron donor. In the case of bacteria, most metal ions are toxic and therefore the reduction of ions or the formation of water insoluble complexes is a defense mechanism developed by the bacteria to overcome such toxicity.
Focus on actinomycetes (microorganisms originally designated as ray fungi) has primarily centered on their exceptional ability to produce secondary metabolites such as antibiotics. It has been observed that a novel alkalothermophilic actinomycete, Thermomonospora sp. synthesized gold nanoparticles extracellularly when exposed to gold ions under alkaline conditions. Extreme biological conditions such as alkalinity and slightly elevated temperature were favorable for the formation of monodisperse gold nanoparticles by Thermomonospora sp. Alkalotolerant actinomycete Rhodococcus sp. has also been used for the intracellular synthesis of gold nanoparticles.
Fungi have been widely used for the biosynthesis of nanoparticles and the mechanistic aspects governing the nanoparticle formation have also been documented for a few of them. In addition to monodispersity, nanoparticles with well-defined dimensions can be obtained using fungi. Compared to bacteria, fungi could be used as a source for the production of large amount of nanoparticles. This is due to the fact that fungi secrete more amounts of proteins which directly translate to higher productivity of nanoparticle formation. Yeast, belonging to the class ascomycetes of fungi has shown to have good potential for the synthesis of nanoparticles. Gold nanoparticles have been synthesized intracellularly using the fungi V.luteoalbum. Cladosporium cladosporioides was used to synthesize silver nanoparticles extracellularly.
Microbiological methods generate nanoparticles at a much slower rate than that observed when plant extracts are used 1.6. This is one of the major drawbacks of biological synthesis of nanoparticles using microbes.
Factors affecting Bioreduction
The reduction process of metal ions leading to the formation of nanoparticles is affected by a large number of factors. Besides the nature of the plant extract providing active biomolecules in different combinations and concentrations, the other factors include the reaction mixture PH, incubation temperature, reaction time, concentration, and electrochemical potential of the metal ion.
PH value of the plant extract
The PH value of a plant extract exerts great influence on the formation of nanoparticles. A change in PH results in a charge change in the natural phytochemicals contained in an extract, which affects their ability to bind and reduce cations and anions in the course of nanoparticle synthesis. This in turn may affect the shape, size, and yield of nanoparticles. For example, in the Avena sativa (common oat) extract more numerous small-sized gold nanoparticles were formed at PH 3.0 and 4.0, whereas more aggregate particles were observed at PH 2.0. Therefore, it has been suggested that nanoparticle aggregation is dominant over the process of reduction and primary nucleation of reduced atoms at very acidic PH values. At PH 2.0 the most accessible metal ions are apparently involved in a smaller number of nucleation events, which leads to agglomeration of the metal. However there are cases in which the particles are formed under alkaline PH also. In the case of silver ions and the tuber powder of Curcuma longa (turmeric), a substantially larger number of silver nanoparticles are synthesized at alkaline PHs, at which extracts may contain more negatively charged functional groups capable of efficient binding and reduction of silver ions.
Temperature is another important factor affecting the formation of nanoparticles in plant extracts. In general, temperature elevation increases the reaction rate and efficiency of nanoparticle synthesis. It was found that in Alfalfa plants, triangular silver nanoparticles are formed only at temperatures above 30? .Furthermore, experiments on the synthesis of silver nanoparticles in lemon verbena extracts (Aloysia citrodora) demonstrated that increasing the reaction temperature is accompanied by an increase in the efficiency of the silver ion reduction. Moreover, crystal particles are formed much more frequently at high temperatures than at room temperature. In experiments with Cassia fistula (golden shower tree) extracts, it was found that temperature may also affect the structural form of the synthesized nanoparticles. For instance, silver nanoribbons are mainly formed at room temperature, whereas spherical nanoparticles predominate at temperatures above 60?. In this case it is believed that higher temperatures alter the interaction of phytochemicals with the nanoparticle surface, thereby inhibiting incorporation of adjacent nanoparticles into the structure of nanoribbons.
Electrochemical potential of the metal ion
Due to the limited ability of plants to reduce metal ions, the efficiency of metal nanoparticle synthesis also depends on the electrochemical potential of the ion. The ability of a plant extract to effectively reduce metal ions may be significantly higher in the case of ions having a large positive electrochemical potential (for example, Ag+) than in the case of ions with a low electrochemical potential such as Ag(S2O3)23-. 1.8
Applications of nano materials
1.4.1 Applications of nanomaterials in electronics
The use of nanotechnology on electronic components is referred to as nano electronics. The important applications include:
The use of carbon nanotubes in semiconductor chips.
The use of a variety of nanomaterials in lighting technologies
(Light emitting diodes or LEDs and organic light emitting diodes or OLEDs).
Nanomaterials are used in laser technology (use of quantum dots in lasers).
A variety of nanomaterials (such as LiBi02) are used in lithium-ion batteries.
Nanoradios have been developed structural around carbon nanotubes.
Research is ongoing to use nanowires and other nano structured materials with the hope to create cheaper and more efficient solar cells than are possible with conventional planar silicon solar cells.
There is also research into energy production devices called bio-nano generators. A bio- nano generator is a nanoscale electrochemical device like a fuel cell or galvanic cell, but drawing power from blood glucose in a living body, much the same as how the body generates energy from food. The electricity generated by such a device could power devices embedded in the body (such as pacemakers) or sugar fed nanorobots.
Applications of nanomaterials in vehicles
A series of nanomaterials, including metal nanoclusters, metal nanocolloids, metal nanopowders, metal nanoparticles and magnetic fluids are now available with many applications in the automotive market. Potential applications are:
Use of catalytic converters has significantly reduced emissions of hazardous air pollutants from automobiles. The catalytic reactivity of platinum nanoparticles has significantly enhanced over existing catalysts due to the fact that a much greater surface area of the metal is exposed.
Shock absorbers provide comfortable ride. Nanotechnology specifically magnetic nanoparticles have advancing shock absorber capabilities further than ever before. This application is due to the fact that the viscosity of the magnetic fluids, comprised of magnetic nanoparticles in a fluid suspension, can be controlled dynamically.
Nanoparticles when added to heat transfer fluids increase their performance. The solid nanoparticles conduct heat better than the liquid. Nanoparticles work best because they stay suspended in liquids longer than larger particles. They also have a much greater surface area.
Applications of nanomaterials in robotics
Nanorobotics is an emerging technology field of creating machines or robots whose components are at or close to the microscopic scale of a nanometer. More specifically nanorobotics refers to the nanotechnology engineering discipline of designing and building nanorobots, with devices ranging in size from 0.1-10 micrometers and constructed of nanoscale or molecular Components. The names nanobots, nanoids, nanites, nanomachines or nanomites have also been used to describe these devices.
Nubot or nucleic acid robots are synthetic robotic devices at nanoscale. Potential applications for nanorobotics in medicine include early diagnosis and targeted drug delivery for cancer, biomedical instrument surgery, pharmacokinetics, monitoring of diabetes and heath care. In such plans, future medical nanotechnology is expected to employ nanorobots injected into the patient to perform work at cellular level. Nanorobots once introduced into the body can detect or repair damages and infections.
Application of nanomaterials in computers
Nanomaterials and nanoelectronics holds the promise of making computer processors more powerful than are possible with conventional semiconductor fabrication techniques. A number of approaches are currently being researched, including new forms of nanolithography, as well as the use of nanomaterials such as nanowires or small molecules in place of traditional CMOS components. Field effect transistors have been made by using both semiconducting carbon nanotubes and with heterostructured semiconductor nanowires.
Applications of nanomaterials in sensors
Nanosensors are any biological, chemical or surgical sensory points used to convey information about nanoparticles to the macroscopic world. Their use mainly include various medicinal purposes and as gateways for building other nanoproducts such as computer chips that work at the nanoscale and nanorobots. Nanosensors exist in the biological world as natural receptors of outside simulation. For instance, sense of smell, especially in animals in which it is particularly strong, such as dogs, functions using receptors that sense nanosized molecules. Certain plants, too use nanosensors to detect sunlight and various fishes use nanosensors to detect small vibrations in the surrounding water. Chemical sensors, too have been built using nanotubes to detect various properties of gaseous molecules. Carbon nanotubues have been used to sense ionization of gaseous, molecules while nanotubes made out of titanium have been employed to detect atmospheric concentrations of hydrogen at the molecular level. Nanoparticles cast either separately as free stranding films or incorporated into film forming polymeric matrices and gels, are also being developed as sensors for gas molecules. Semiconducting metal oxide nanoparticles are being widely researched as chemoresistive gas sensors. Single gas detection, leakage detectors, fire detectors and humidity sensors fall under the category of gas sensors. The operating principle of these electronic noses is that of variation in the resistance when exposed to certain gases.
Applications of nanomaterials in medicine
Nanomedicine is the medical application of nanotechnology. It seeks to deliver a valuable set of research tools and clinically useful devices in the near future. Medical use of nanomaterials are:
Nanomedical approaches to drug delivery center on developing nanoscale particles or molecules to improve drug bioavailability. Bioavailability refers to the presence of drug molecules where they are needed in the body where they will do the most good. Drug delivery focuses on maximizing bioavailability both at specific places in the body and over a period of time. This can potentially be achieved by molecular targeting by nanoengineered devices.
Protein and peptide delivery
Proteins and peptides exert multiple biological actions in human body and they have been identified as showing great premise for treatment of various diseases and disorders. These macromolecules are called biopharmaceuticals. Targeted and or controlled delivery of these biopharmaceuticals using nanomaterials like nanoparticles and dendrimers is an emerging field called nanobiopharmceutics.
Molecular imaging and therapy
The small size of nanoparticles endows them with properties that can be very useful in oncology, particularly in imaging. Quantum dots (nanoparticles with quantum confinement properties such as size tunable light emission), when used in conjunction with MRI (magnetic resonance imaging) can produce exceptional images of tumor sites. Nanoparticles of cadmium selenide (quantum dots) glow when exposed to ultraviolet light. When injected, they seep into cancer tumors. The surgeon can see the glowing tumor and use it as a guide for more accurate tumor removal.
At Rice University, a flesh welder is used to fuse two pieces of chicken meat into a single piece. The two pieces of chicken are placed together touching. A greenish liquid containing gold coated nanoshells is dribbled along the seam. An infrared laser is traced along the seam, causing the two sides to weld together. This could solve the difficulties and blood leaks caused when the surgeon tries to restich the arteries that have been cut during a kidney or heart transplant. The flesh welder could weld the artery perfectly.
Various kinds of nanosystems in use
Nanoshells represent a unique class of medically prominent nanoparticles. These are made of drug coated metal nanospheres or dielectric metal nanospheres. When these nanoshells are irradiated with a laser of known intensity, it causes release the drug coat present on the nanoparticle surface.
Nanopores are essentially nanoparticles whose surface contains pores, which can be used for containing drugs.
Dendrimers are branched tree shaped nanoparticles, which have an immense potential for use in clinical diagnostics and therapeutics. Multicomponent nanodevices can be formed by attaching different types of dendrimers with each other though their branches forming tectodendrimers.
1.4.7 Application of nanomaterials in mobile electronic devices
Highly integrated flash memory, a major non-volatile memory, is in high demand for portable devices such as mobile phones. A floating gate stores the charge in the flash memory and these floating gates are now being replaced by a number of nanodots. These nanodots are non-continuous film and work smoothly even when film contains conventional semiconductor memory. 1.9
Introduction to silver nanoparticle
Silver nanoparticles are nanoparticles of silver which are in the range of 1 and 100 nm in size. Silver nanoparticles have unique properties which help in molecular diagnostics, in therapies, as well as in devices that are used in several medical procedures. The major methods used for silver nanoparticle synthesis are the physical and chemical methods. The problem with the chemical and physical methods is that the synthesis is expensive and can also have toxic substances absorbed onto them. To overcome this, the biological method provides a feasible alternative. The major biological systems involved in this are bacteria, fungi, and plant extracts. There are many ways depicted in various literatures to synthesize silver nanoparticles. These include physical, chemical, and biological methods. The physical and chemical methods are numerous in number, and many of these methods are expensive or use toxic substances which are major factors that make them ‘not so favored’ methods of synthesis. An alternate, feasible method to synthesize silver nanoparticles is to employ biological methods of using microbes and plants.
The production of nanoparticles majorly involves physical and chemical processes. Silver nanomaterials can be obtained by both the so called ‘topdown’ and ‘bottomup’ methods. The topdown method involves the mechanical grinding of bulk metals and subsequent stabilization of the resulting nanosized metal particles by the addition of colloidal protecting agents. The bottomup methods, on the other hand, include reduction of metals, electrochemical methods, and sonodecomposition.
In the case of biological method there are three major sources of synthesizing silver nanoparticles: bacteria, fungi, and plant extracts. Biosynthesis of silver nanoparticles is a bottomup approach that mostly involves reduction/oxidation reactions. It is majorly the microbial enzymes or the plant phytochemicals with antioxidant or reducing properties that act on the respective compounds and give the desired nanoparticles. The three major components involved in the preparation of nanoparticles using biological methods are the solvent medium for synthesis, the environmentally friendly reducing agent, and a nontoxic stabilizing agent The mechanism of silver nanoparticle production by fungi is said to follow the following steps: trapping of Ag ions at the surface of the fungal cells and the subsequent reduction of the silver ions by the enzymes present in the fungal system. The extracellular enzymes like naphthoquinones and anthraquinones are said to facilitate the reduction. A major drawback of using microbes to synthesize silver nanoparticles is that it is a very slow process when in comparison with plant extracts. 1.10
1.5.1 Characterization of silver nanoparticles
Characterization of nanoparticles is important to understand and control nanoparticles synthesis and applications. Characterization is performed using a variety of different techniques such as transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR), and UV–Vis spectroscopy. These techniques are used for determination of different parameters such as particle size, shape, crystallinity, fractal dimensions, pore size and surface area. Moreover, orientation, for instance, the morphology and particle size could be determined by TEM, SEM and AFM. The advantage of AFM over traditional microscopes such as SEM and TEM is that AFM measures three dimensional images so that particle height and volume can be calculated. Furthermore, dynamic light scattering is used for determination of particles size distribution. Moreover, X-ray diffraction is used for the determination of crystallinity, while UV–Vis spectroscopy is used to confirm sample formation by showing the plasmon resonance. 1.11
Dynamic light scattering (DLS)
DLS is widely used to determine the size of Brownian nanoparticles in colloidal suspensions in the nano and submicron ranges. Shining monochromatic light (laser) onto a solution of spherical particles in Brownian motion causes a Doppler shift when the light hits the moving particle, changing the wavelength of the incoming light. This change is related to the size of the particle. It is possible to extract the size distribution and give a description of the particle’s motion in the medium, measuring the diffusion coefficient of the particle and using the autocorrelation function.
Scanning Electron microscopy
Scanning electron microscopy (SEM) is giving morphological examination with direct visualization. The techniques based on electron microscopy offer several advantages in morphological and sizing analysis; however, they provide limited information about the size distribution and true population average. For SEM characterization, nanoparticles solution should be first converted into a dry powder, which is then mounted on a sample holder followed by coating with a conductive metal, such as gold, using a sputter coater. The sample is then scanned with a focused fine beam of electrons (Jores et al., 2004). The surface characteristics of the sample are obtained from the secondary electrons emitted from the sample surface.
Transmission electron microscope
TEM operates on different principle than SEM, yet it often brings same type of data. The sample preparation for TEM is complex and time consuming because of its requirement to be ultra-thin for the electron transmittance. The surface characteristics of the sample are obtained when a beam of electrons is transmitted through an ultra-thin sample, interacting with the sample as it passes through.
Atomic force microscopy
Atomic force microscopy (AFM) offers ultra-high resolution in particle size measurement and is based on a physical scanning of samples at sub-micron level using a probe tip of atomic scale (Muhlen et al., 1996). Instrument provides a topographical map of sample based on forces between the tip and the sample surface. Samples are usually scanned in contact or noncontact mode depending on their properties. 1.12
X-ray Diffraction studies
X-ray Diffraction (XRD) is a popular analytical technique, which has been used for the analysis of both molecular and crystal structures, qualitative identification of various compounds, quantitative resolution of chemical species, measuring the degree of crystallinity, isomorphous substitutions, stacking faults, polymorphisms, phase transitions, particle sizes etc. 1.18. When X-ray light reflects on any crystal, it leads to the formation of many diffraction patterns and the patterns reflect the physico-chemical characteristics of the crystal structures. In powder specimens, the diffracted beams coming from the sample reveal its structural and physic-chemical features. Thus XRD technique can analyze structural features with other ambiguities of a wide range of materials such as inorganic catalysts, superconductors, biomolecules, glasses, polymers and so on. Analysis of these materials largely depends on forming diffraction patterns. Each material has its unique diffraction beam, which can define and identify the material by comparing the diffracted beams with reference database in JCPDS (Joint Committee on Powder Diffraction Standards) Library. The diffraction patterns also explain whether the sample materials are pure or contain impurities. Therefore, XRD have long been used to define and identify both bulk materials and nanomaterials, forensic specimens, industry and geochemical sample materials.
UV-Visible Spectroscopy refers to the absorption or reflectance spectroscopy in the UV-Visible spectral region. It uses light in the visible and adjacent (near –UV and near infrared) regions. The instruments used to study or measure the absorption or emission of electromagnetic radiation in the UV-Visible range as a function of wavelength is called UV-Visible spectrophotometers. UV-Visible Spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as highly conjugated organic compounds, transition metal ions and biological macromolecules.
UV-Visible absorption spectroscopy is an important technique to monitor the formation and stability of metal nanoparticles in aqueous solution. Nanoparticles have optical properties that are sensitive to size, shape, concentration, agglomeration state and refractive index near the nanoparticle surface, which makes UV-Visible Spectroscopy a valuable tool for identifying, characterizing and studying these materials. Nanoparticles made from certain metals, such as gold and silver, strongly interact with specific wavelengths of light and the unique optical properties of these materials is the foundation for the field of plasmonics. 1.13
In vibrational spectroscopy we look at the changes in vibrational motion of atoms in a molecule, which are greatly influenced by the masses of atoms, their geometrical arrangement and strength of their chemical bonds. IR involve transition between quantized vibrational states and provide a complementary image of molecular vibrations. Interaction of IR radiation with vibrating molecule is only possible if the molecular dipole moment is modulated by the vibration (IR active).
IR encompasses a spectral region from red end of visible spectrum electromagnetic spectrum and is conveniently divided into near IR (12500 to 4000 cm-1), mid IR (4000 to 400 cm-1) and far IR (400 to 10 cm-1). The main significance of this division is that most fundamental molecular vibrations occur in mid-IR making this region richest in chemical information while overtones and combination of fundamental vibrations especially those involving hydrogen atoms appear in the near IR and far IR contains vibrations involving heavy atoms, lattice modes of solids and some rotational absorption of small molecules. Fourier transform spectrometers are superior to the dispersive IR spectrometers. FTIR spectrometers are based upon Michelson interferometer. The powder sample is mixed with nujol to form a thick paste and held between salt plates or thoroughly mixed with potassium bromide (KBr) and pressed using a hydraulic press to form pellets and then placed in the sample holder of the instrument. 1.14
1.5.2 Antimicrobial activity of silver nanoparticle
Silver nanoparticles are well known potent antimicrobial agents. One important parameter of the antimicrobial activity of silver nanoparticles is the surface area of the nanomaterial. The highest concentration of released silver ions was observed in the case of the highest surface area of silver nanoparticles. The lowest concentration of silver ions released was noted for silver nanoparticles with the lowest surface area, resulting in weak antimicrobial properties. Silver nanoparticles have the ability to anchor to the bacterial cell wall and subsequently penetrate it, thereby causing structural changes in the cell membrane like the permeability of the cell membrane and death of the cell. There is formation of ‘pits’ on the cell surface, and there is accumulation of the nanoparticles on the cell surface. The formation of free radicals by the silver nanoparticles may be considered to be another mechanism by which the cells die. There have been electron spin resonance spectroscopy studies that suggested that there is formation of free radicals by the silver nanoparticles when in contact with the bacteria, and these free radicals have the ability to damage the cell membrane and make it porous which can ultimately lead to cell death. 1.15
1.5.3 Applications of silver nanoparticle
The use of silver nanoparticle (AgNP) in various fields is receiving attention among researchers in recent years. The physical, chemical and mechanical properties of AgNP are unique and useful in the field of electronics and electrical, biotechnology and Bioengineering, textile engineering, environmental and pharmaceuticals.
The optical properties of the silver nanoparticle are owing to the interaction between incoming light and free conduction electrons. When the wavelength of the incident light matches with the oscillating frequency of the conduction electron, a surface plasmon resonance occurs, this gives rise to the absorption band in the visible region. This surface plasmon resonance peak depends on the particle size, shape, surface charge, separation between the particle and the nature of the environment. This leads to the formation of different colors of the metal nano-sol. The surface structure of the nanoparticles determines the charge of the nanoparticles. The silver nanoparticle are used in solar cells for trapping solar energy. In conventional solar cells the coating layer was made up of silicon. So the system was not efficient because silicon is a poor absorber of light. Hence the light trapping efficiency of solar cells was enhanced by undercoating the silver nanoparticle layer along with silicon layer. In optical fiber analyzers the Ag-doped silica nano composite is coated as a membrane on the surface of an optical fiber along with bent silica. The improved optical sensor fiber useful to trace ammonia in a gas sample. The plasmonic and photonic characteristics of silver nanoparticle and the silver nanoparticle loaded compound such as silver loaded poly vinyl alcohol (Ag-PVA) (Figure 1.8) are used for light guiding and optical sensing applications.
Electrical and electronic applications
Silver nanoparticle is successfully employed in the field of microelectronic materials. The melting point of smaller sized silver nanoparticle drastically reduced and with increased surface energy. This property of silver nanoparticle is useful in electronics and used as conductive fillers in electronically conductive adhesives (ECAs). The electrical conductors fabricated with a thick film of silver nanoparticle reduces the electrical loss. The lower electrical losses at higher frequency is attributed to the lower surface roughness that give better packing and used to fabricate antennas. The electro reflectance (ER) effect of silver nanoparticle is important in the field of electro-optical devices and sensors. Silver nanoparticles are being used in numerous technologies and incorporated in to a wide array of consumer products that take advantage of their desirable optical, conductive and antibacterial properties.
Bioengineering and Biomedical applications
Nanoparticle in medicine are used for therapeutic, imaging, and diagnostics of cancer and other diseases leading an entrapped or bound therapeutic or diagnostic target material in the area of interest, e.g., a tumor. The destination of targeted delivery may be found by physical forces (magnetic) or with surface bound antibodies. Carbohydrate modified AgNP may provide new tools that can be used in phototherapy for killing cancerous cells and diagnosis. In electrochemical DNA sensors, silver nanoparticle are used as an indicator that bounded with the oligonucleotide probes that able to paired with the sample DNA sequence for disease detection application such as disease diagnosis, drug screening, epidemic prevention and environmental protection.
The redox property of silver nanoparticle is useful for the successful preparation of DNA sensors and the electrochemical polymerization of conducting polymer-pyroll that coated with silver nanoparticle possesses good sensitivity and stability. Silver nanoparticle conjugated to oligonucleotides have recently emerged as powerful tools for the detection of target DNA sequences, and are used in the design of colorimetric assays based on aggregation induced by sequence-specific hybridization.
The figure 1.9 showed the site targeted identification of the desired probe using the detector DNA probe tagged with silver nanoparticle. This DNA-sensing concept used to detect the targeted DNA that can be captured in a sandwich DNA hybrid assay. The emerging field of nano medicine seeks to exploit the novel properties of engineered nano-materials for diagnostics and therapeutic applications. Silver nanoparticle are being used increasingly in wound dressings, catheters, and various household products due to their antimicrobial activity. The surgical meshes that made of silver nanoparticle coated polypropylene are used as an ideal candidate for surgical meshes due to its antimicrobial, anti-inflammatory properties.
Waterborne pathogens in drinking water makes health risk to the human beings as well as ecosystems. For instance, Ag and Ag-containing compound nanomaterial, mostly in the form of nanoparticles and nanocomposites, are intensively used for diverse bio-related applications. Silver has also been impregnated in filters in point-of-use tap water purification devices in order to retard the growth of microorganisms. So, it can be deduced that the water purification filter which possesses Ag-containing nanomaterial can be a more cost-effective way because of its semi-permanent characteristics than chemical method. The inhibitory effect of Ag+ ions on bacterial growth is due to their adsorption to negatively charged bacterial cell walls, inactivation of enzymes, disruption of membrane permeability, and, ultimately, cell death. Moreover, silver nanoparticle was reported as stable, biocompatible and non-toxic at lower concentration, the silver nanoparticle was not influencing the human epidermal keratinocytes. The lower concentration of silver nanoparticle from 2 ppm -4 ppm was reported as nontoxic to human cells (HEK 293) and toxic to bacterial cells.
Textile industrial application
Silver nanoparticle deposited onto the surface of different fabrics (nylon, polyester and cotton) by ultrasound irradiation, possess the antibacterial activity. The coated fabrics have potential application in sports socks to control odour, dirt, microbes, etc., and used in medical bandages and bed linings as shown in figure 1.10.The intensified interest in polymer nanocomposites with silver nanoparticle is due to the high antimicrobial effect of nanosilver. The silver nanoparticle are readily form bonds with the fibers and produce the nano engineered coating of the material. The high surface area relative to the volume of particles increases their chemical reactivity, allowing them to stick to materials more permanently. The nano engineered fabrics that kill bacteria, eliminate moisture, odour, and prevent static electricity. Polymer nanofiber coatings applied to textiles that make a bond to the fabric material at one end of the polymer fabrics. These polymer nanofiber coated on the surface of the materials prevent liquid contact or act as a waterproof to the textile materials. Nanofabrics with dirt-proof, stain-proof, and super hydrophobic properties are possible as a result of the layer formed by polymer nanofibers. The Ag–fabric composite were prepared and experimented against Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive) cultures, showed strong antibacterial activity. The electrospun nanocomposites of Poly vinyl alcohol (PVA) and Poly vinyl pyrrolidone (PVP) prepared by immobilizing the silver nanoparticle because of the strong affinity with a pyridyl group of metals and its ability to undergo hydrogen bonding with polar species. 1.16
K.K. Chattopahyay,A.N.Banerjee, Introduction to Nano Science and Nano Technology, Prentice Hall of India (2009)
National nanotechnology initiative available at http://www.nano.gov
Taylor, Robert; Coulombe, Sylvain; Otanicar, Todd; Phelan, Patrick; Gunawan, Andrey; Lv,Wei; Rosengarten, Gary; Prasher, Ravi; Tyagi, Himanshu (2013). “Small particles, big impacts: A review of the diverse applications of nanofluids”.
Manoj Singh, Manikandan, S. and Kumaraguru, A.K. (2010), Nanoparticles: A New Technology with wide Applications, Research Journal of Nanoscience and Nanotechnology, ISSN 1996-5044/ DOI: 10.3923/rjnn.2010.
Mohanpuria, P., Rana, K.N. and Yadav, S.K. (2008), Biosynthesis of Nanoparticles: Technological concepts and future applications, Journal of Nanoparticle Research, vol.10, 507-517.
Prathna, T.C., Lazar Mathew, Chandrasekharan, N., Ashok M. Raichur and Amitava Mukherjee (2000), Biomimetic synthesis of Nanoparticles: Science, Technology & Applicability, Biomimetics learning from Nature, Amitava Mukherjee (Ed.), ISBN: 978-953-307-025-4, In Tech, Available from: www.intechopen.com, Accessed 21 May 2016
Makarov, V.V., Love, A.J., Sinitsyna, O.V., Makarova, S.S., Yaminsky, I.V., Talinsky, M.E. and Kalinina, N.O. (2014), “Green” Nanotechnologies: Synthesis of Metal Nanoparticles Using Plants, Acta Naturae, 6(1), 35-44, PMC ID: PMC 3999464, Available from: PubMed Central®, Accessed 28 May 2016
Makarov, V.V., Love, A.J., Sinitsyna, O.V., Makarova, S.S., Yaminsky, I.V.,Talinsky, M.E. and Kalinina, N.O. (2014), “Green” Nanotechnologies: Synthesis of Metal Nanoparticles Using Plants, Acta Naturae, 6(1), 35-44, PMC ID: PMC 3999464,Available from: PubMed Central®, Accessed 28 May 2016
Dr.M.Viswanathan, Principles of Physical and Inorganic Chemistry
Sukumaran Prabhu, Eldho K Poulose, Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects, December 2012, 2:32
Kholoud M.M. Abou ElNour Ala’a Eftaiha, Abdulrhman AlWarthan , Reda A.A. Ammar, Synthesis and applications of silver nanoparticles, Arabian Journal of Chemistry, Volume 3, Issue 3, July 2010, Pages 1351401.
Sovan Lal Pal, Utpal Jana, P. K. Manna, G. P. Mohanta, R. Manavalan , Nanoparticle: An overview of preparation and Characterization ,Journal of Applied Pharmaceutical Science 01 (06); 2011: 228-234.
Rasel Das, Md. Eaqub Ali and Sharifah Bee Abd. Hamid (2014), Current Applications of X-Ray Powder Diffraction- A Review, Rev. Adv. Mater. Sci, 38, 45-109, Accessed 28 May 2016
K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds Part A: Theory and Applications in Inorganic Chemistry (5th edn.), Wiley- Interscience, New York, (1997)
Sukumaran Prabhu, Eldho K Poulose, Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects, December 2012, 2:32
V.Thamilselvi and K.V.Radha, A Review On The Diverse Application Of Silver Nanoparticle, IOSR Journal Of Pharmacy, (e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219, Volume 7, Issue 1 Version. 1 (Jan 2017), PP. 21-27
MATERIALS AND METHODS
This chapter deals with the synthesis methods, materials used, characterization techniques and other methods of measurement for analyzing the sample.
2.1 Synthesis technique
Fungi and Precursor salt used
Fungi were collected form 10 days old bread surface. It is a widely distributed threadlike mucoralean mold, it takes food and nutrients from the bread and causes damage to the surface where it lives.
Scientific name and Common name
Black Bread Mold
General description of the organism
Black Bread Mold is a threadlike mold and a heterotrophic species; it is dependant on sugar or starch for its source of carbon substances for food. It uses food matter, generally breads or soft fruits, like grapes or strawberries, as a food source for growth nutrition and reproduction. Black Bread Mold is a mass of mycelium, the vegetative filaments of the fungus, and a fruiting structure. Most of the mycelium is composed of multi-nucleate, rapidly growing hyphae. When the mold’s spores are released they produce more mycelium through germination. As the mold matures it begins to turn black. It is an agent of plant disease; it breaks down organic matter through decomposition. When kept in a moist environment, such as a piece of bread, the parasite can quickly spread within a few days. Its spores are commonly found in the air. The spores grow most rapidly at temperatures between 15°C and 30°C where they are able to germinate to their full potential. Black Bread Mold commercial use in the manufacturing of alcohol and organic acids.
Importance of the organism to the environment
The Black Bread Mold causes rotting of fruits and, in some cases, infections to humans. They grow inside food and use absorption to take in nutrients and dissolve the substrate with extracellular enzymes. Rhizopus stolonifer play a key role in the carbon cycle because they work as decomposers in soil, dung and in many foods.
Common or endangered?
Black Bread Mold is one of the most common and fastest growing species in the Zygomycota. It often grows within a few days in moist and humid conditions because its spores are quite common in the air. The species is a common mold and is therefore not endangered at all; it grows on common foods and spreads very quickly. 2.2
The precursor salt, AgNO3 (Figure 2.3) was purchased from Nice Chemicals Private Limited, Edappally Kochi.
Preparation of fungal extract
The Black Bread Mold (Rhizopus stolonifera) was plucked carefully from 10 days old bread surface using blade and plucker (Figure 2.4) during May 2017. Around 6 g of mold were weighed using an electronic balance and was put into a beaker, which was washed properly using de-ionized water. Then 100 mL of distilled water was added to it and stirred well. Then the beaker was kept in ‘heating mantle’ and heated for almost one hour at 600C and stirred out at regular intervals. The beaker was covered with tissue paper and left for overnight. The next day the solutions left in the beaker were filtered firstly through ordinary filter paper and then by whatmann filter paper (Figure 2.5). The amount of filtered solution obtained in the measuring jar was noted. From this solution, 75mL was taken for the remaining process.
Synthesis of Silver nanoparticles
For the Synthesis we use 0.05 Molar AgNO3 solution (Figure 2.6) therefore about 0.4246 g of AgNO3 (precursor salt) were measured using the Magnetic Analytical balance in a dark room and the collected precursor salt was dissolved in 50 ml of distilled water (Figure 2.6) and covered the solution using aluminium foil paper for reducing the presence of light then stirred it well using a Magnetic stirrer. Then the filtered extract solution was placed over the Magnetic stirrer at room temperature with 120 rpm and it was also covered with aluminium foil paper then 15 ml of salt solution was added drop wise into it and stirred well for almost 3hr and heated frequently at about 600C. The pH and colour of solution were also noted frequently. Initially pH was below 4. Then 80% saturated ammonium sulphate ((NH4)2SO4) solution was added drop wise until pH paper became blue which indicate pH reached above 7. Thus a brown solution was obtained (Figure 2.7). This means Silver nanoparticles are formed .Then gel like accumulation will be noted and keeping moment for 3hr with constant heating & stirring. It is necessary to keep pH between 2 to 6 in order to obtain better precipitation of Silver nanoparticles. The solution in the magnetic stirrer was fully covered with aluminium foil paper and the xero-gel obtained will be kept an overnight to settle down and the residue obtained was taken in a petri dish and dried using heating mantle then crushed it for half an hour using a granite mortar to obtain fine and labelled. The collected sample was then sent for various analytical studies.
2.2 Characterization Techniques
There are various techniques for detecting, measuring and characterizing nanoparticles. A particular method cannot be selected as the “best” because the selectivity of a method depends upon the type of the sample, the information required, time constraints and the cost of the analysis. A straight forward technique may simply detect the presence of nanoparticles, others may give the quantity, the size distribution or the surface area of the nanoparticles. The characterization techniques for nanoparticles assess the chemical contents of a nanoparticle sample, the reactions on the surface of the nanoparticles or the interactions with other chemical species present. Here we discuss techniques such as X-ray Diffraction (XRD), UV-Visible Spectrophotometry,Fourier Transform Infrared Spectroscopy (FTIR) and Antimicrobial study.
2.2.1 X-ray Diffractometry (XRD)
For a solid crystalline material, it is well known that the atoms are arranged in a regular pattern in three dimensions, where a certain basic repeating unit of structure having minimum volume called the unit cell is the building block. A unit cell may be decided by the lengths of its sides (a, b and c) called as lattice parameters. In a crystal lattice there are several sets of possible parallel planes. Each set of parallel planes is uniquely described by three integral co-ordinates (h k l) called as Miller indices. The examples of such parallel planes in case of cubic structure are as (111), (200), (311), (220), (222), (400), (440), (333), (444), etc.
When the wavelength of X-rays incident on a crystal is of the order of the inter-atomic distance in the crystal, the X-rays are diffracted. Diffraction is essentially due to the existence of certain phase relations between two or more waves. The diffraction from a crystal is depicted in figure 2.9.
Incident and reflected rays are shown for two neighboring planes. ? is the scattering angle and ? is the wave length of the X-ray beam. Consider a wave incident on a crystal. If the different planes of crystal (A, B and C) are d distance apart then the path difference between the waves scattered from two consecutive planes would be 2dsin?, ?- being the incident angle.
In order that the scattered rays be completely in phase with each other their path difference should be integer number n of wavelengths.
2dsin? = n? 2.1
This is known as Bragg’s law. Diffraction takes place from the crystal only when Bragg condition is fulfilled 2.3-2.4. This condition can be achieved by continuously changing either ? or during the experiment. There are mainly three experimental methods for measuring X-ray diffraction.
Figure 2.9: Bragg reflection from a particular family of lattice planes separated by a
Laue method variable fixed
Rotating crystal method fixed variable
Debye-Scherrer powder method fixed variable
In the Debye-Scherrer powder method the powder to be examined is placed in the path of a beam of monochromatic X-rays. In the crystalline powder specimen various reciprocal lattice vectors are randomly oriented with respect to the incident beam and Bragg diffraction occurs over a set of cones, known as Debye scherrer cones. The incident monochromatic radiation strikes on a fine powdered or a fine grained polycrystalline specimen contained in a sample holder. Diffracted beams goes out from the individual crystallites that happen to be oriented with planes making an incident angle ? with the beam satisfying the Bragg equation 2dsin? = n?. Diffracted rays leave the specimen along the generators of the cones concentric with original beam. The generators make an angle of 2? with the direction of the original beam, where ? is the Bragg angle. Powder specimen is rotated in order to increase the number of planes contributing to each reflection. The sample is rotated by an angle ? and the detector by 2? continuously in such a way that only the crystals whose planes are parallel to the sample surface take part in the direction and result in constructive interference. The advantages of this method are: (1) small amount of powder is required, (2) practically complete coverage of all thee reflections produced by the specimen, (3) relative simplicity of the apparatus. 2.4-2.5
Indexing and determination of lattice constant
In the powder diffraction measurements, the observed data comes in the form of the intensity of diffracted rays as a function of angle 2?. The exact 2? angle at which Bragg’s peaks are observed are carefully noted down and then with the help of Bragg’s law the corresponding d values are calculated. If one has knowledge of the crystal structure, then with an initial guess of cell constants, d values for various (hkl) reflections can be calculated using the following relations.
For cubic systems 2.2(a)
For tetragonal system 2.2(b)
For Hexagonal systems 2.2(c)
For orthorhombic systems 2.2(d)
The calculated values are compared with those experimentally observed and thus the observed Bragg reflections are assigned hkl values. The success of the indexing procedure depends on the accuracy of the experimental values and hence also on the purity of the examined sample. Presence of any low intensity peaks that cannot be indexed could be indicative of the existence of some impurity phase in the sample. When the whole diffraction pattern has been indexed, then by using the above standard formulae (and the values of hkl and the corresponding d values) the actual cell parameters of the sample are calculated. The cell parameters are refined through a least square refinement. A least square method based computer program can be used to do the above calculations for determining the crystal structure of a specimen. 2.3-2.6
Determination of particle size
It has been indicated that, in order for a crystal to diffract at all, the reflecting plane must meet the incident X-ray beam at one of a set of specified angles. This is necessary in order that the X-rays reflected from different points on these planes reach the detector in phase, i.e. their path lengths differ by integral multiples of one wave length. When the diffracting crystal is large, containing thousands of parallel planes, this condition is satisfied very precisely and when it happens, the diffraction maxima are sharp. However, as the crystallites become smaller, this condition is somewhat relaxed due to smaller number of co-operating planes. Finally, when the crystallites are so small that they contain only a few planes in phase, diffraction by these planes is no longer capable of producing sharp diffraction minima.
Now we discuss how crystalline size affects the broadening. If the path difference between rays scattered by the first two planes differs only slightly from an integral multiple of wavelength then the planes scattering a ray exactly out of phase with the ray from the first plane will lie deep within the crystal. If the crystal is so small that this plane does not exist, then complete cancellation of all the scattered rays will not exist. It follows there is a connection between the amount of ‘out of phaseness’ that can be tolerated and the size of crystal. Thus broadening of the peaks depends upon the particle size which can be calculated. 2.4-2.6
Suppose that the crystal has thickness’t’ measured in a direction perpendicular to a particular set of reflecting planes. Let there be (m+1) planes. Bragg angle ? will be regarded as variable and let ?B be the angle which exactly satisfies the Bragg’s law
2dsin? = n?
In figure 2.10 A, D & M makes angle ?B with reflecting planes. Thus D’ and A’ have path difference ? and M’ and A’ have that of m ?. So rays A’, D’, M’ etc. those are in phase give a beam of maximum intensity. But if glancing angle is slightly larger than ?B i.e. ?1, such that ray L’ is
Figure 2.10: Effect of crystal size on diffraction
(m+1) ? out of phase with B’. This means that midway in the crystal there is a plane scattering array which is an integer plus ½ wavelengths out of phase with ray B’ giving rise to destructive interference between similar pairs having ?/2 path difference. Similarly if glancing angle is slightly less than ?B i.e. ?2 as in figure for ray C and N such that the diffracted C’ and N’ have path difference (m-1) ?. Then also it will give rise to the destructive interference between similar pairs having path difference ?/2. Thus beams diffracted at 2?1 and 2?2 have zero intensity and intensity at angles between 2?1 and 2?2 is not zero but immediate between zero and maxima (at ?B). The form of pattern is shown in Figure 2.10(a) in contrast to Figure 2.10(b) which illustrate the hypothetical case of diffraction occurring only at ?B. The width B is measured in radiance at an intensity equal to half the maximum intensity. As a rough measure of B, we can take half the difference between the two extreme angles where intensity is zero, so
B=½(2 ?1-2?2) = ?1-?2 2.3
Therefore the path difference equation for these two angles related to the entire thickness of the crystal:
2t sin ?1= (m+1) ?
2t sin ?2= (m-1) ?
So t (sin ?1- sin ?2) = ?
If ?1+ ?2 ~2 ?B and sin (?1-?2)/2~ (?1-?2)/2
Then, 2t (?1-?2)/2 cos ?B = ?
More exact treatment of the problem gives
This is known as Scherrer formula.
Now the problem is shifted to determination of broadening B. It is essentially zero when particle size exceeds 1000Å. All diffraction lines have a measurable breadth BM even when the crystal size exceeds 1000Å due to such causes as divergence of incident beams and size of sample, width of X-ray source. Of the many methods proposed for measuring B, Warren’s is the simplest, given by following formula,
Where BS is the measured breadth at half maximum intensity of the line from the standard (in our case Si) samples of bulk sized particles (>1000 Å). 2.7-2.8
The X-ray Diffractometer used was XPERT-PRO Diffractometer (Figure 2.11) system having type 0000000083005153 with continuous scan mode of step size, 2?°= 0.0170 in the gonio axis in the 2? range 10° to 89.9°, installed at National Centre for Earth Science Studies (NCESS), Thiruvananthapuram.
The experimental set up of the X-ray diffractometer consists of a goniometer driven by a stepper motor and a solid state detector (all enclosed in a dust proof cover), a high voltage generator and an X-ray tube, an electronic unit to control the system, a chart recorder, collimators, the automatic divergence slit and the sample holder . The optical arrangement of the setup is shown in Figure 2.13. A description of the different units of the setup is given in the following sections. 2.9
Figure 2.13: Optical arrangement of the X-ray diffractometer set up.
The X-ray tube has an iron target mounted in the tube tower. We have used iron target giving Cu-K? radiation of wave length ?=1.54 A°. The high tension between source and target can be varied between 10kV and 50kV and the filament current is adjustable between 5 mA and 60 mA. In most of the runs we have operated 45 kV and 40 mA.
The goniometer is mounted on the X-ray tube tower of the diffractometer and is of a Bragg-Brentano design. The goniometer is driven by a four phase hybrid stopper motor which rotates in 1.8° steps. The principle of ?-2? transmission is an endless belt wound from the 2? drive disc around two guide rollers to rotate the ? axis (sample) at half the angular rotation of the 2? axis (detector). The angular range for 2? is 0-120°.
Automatic divergence slit
For avoiding high background with unwanted diffraction lines from the sample holder material and excessive broadening of diffraction lines there is an automatic divergence slit which is placed between the X-ray tube line source and the collimator. This provides a constant illuminated sample area independent of angle of incidence.
As far the X-ray beam falling on the sample is perfectly parallel rays and if we could rotate the sample, it is possible to get diffraction pattern from low angle to high angle. For the purpose of obtaining parallel beam of x-rays two collimators are provided, one for incident X-ray beam and the other for diffracted X-ray beam.
The sample holder is an aluminum plate with a rectangular opening having an area of 15mm x 20mm. Powder sample is filled in this rectangular space and compacted by pressing the surfaces with flat plate.
Diffractometer control unit
This is the main electronic unit of the instrument and it is microprocessor based. This unit controls the movements of stepper motor and detector. It contains a key board for setting different parameters like speed, angle regions, step range, chart speed and slit.
A standard silicon sample has been used for calibration of the powder diffractometer. It is a circular plate of silicon of high purity. Recorded peak positions are compared with those corresponding to standard values of lattice parameter of silicon. 2.10
2.2.2 UV-Visible Spectrophotometry
UV spectroscopy is type of absorption spectroscopy in which light of ultra-violet region (200-400 nm.) is absorbed by the molecule. Absorption of the ultra-violet radiations results in the excitation of the electrons from the ground state to higher energy state. The energy of the ultra-violet radiation that are absorbed is equal to the energy difference between the ground state and higher energy states (delta E = h f). Generally, the most favoured transition is from the highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO). For most of the molecules, the lowest energy occupied molecular orbitals are s orbital, which correspond to sigma bonds. The p orbitals are at somewhat higher energy levels, the orbitals (nonbonding orbitals) with unshared paired of electrons lie at higher energy levels. The unoccupied or antibonding orbitals (pie and sigma) are the highest energy occupied orbitals. In all the compounds (other than alkanes), the electrons undergo various transitions. Some of the important transitions with increasing energies are: nonbonding to pie, nonbonding to sigma, pie to pie, sigma to pie and sigma to sigma.
Principle of UV spectroscopy
UV spectroscopy obeys the Beer-Lambert law, which states that: when a beam of monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation with thickness of the absorbing solution is proportional to the incident radiation as well as the concentration of the solution.
The expression of Beer-Lambert law is
A = log (I0 /I) = Ecl
Where, A = absorbance
I0= intensity of light incident upon sample cell
I = intensity of light leaving sample cell
C = molar concentration of solute
L = length of sample cell (cm.)
E = molar absorptivity
From the Beer-Lambert law it is clear that greater the number of molecules capable of absorbing light of a given wavelength, the greater the extent of light absorption. This is the basic principle of UV spectroscopy.
Concept of Chromophore and Auxochrome in the UV spectroscopy
Chromophore- Chromophore is defined as any isolated covalently bonded group that shows a characteristic absorption in the ultraviolet or visible region (200-800 nm). Chromophores can be divided into two groups
Chromophores which contain p electrons and which undergo pie to pie transitions. Ethylenes and acetylenes are the example of such chromophores.
Chromophores which contain both p and nonbonding electrons. They undergo two types of transitions; pie to pie and nonbonding to pie. Carbonyl, nitriles, azo compounds, nitro compounds are the example of such chromophores.
Auxochromes: An auxochrome can be defined as any group which does not itself act as a chromophore but whose presence brings about a shift of the absorption band towards the longer wavelength of the spectrum. –OH,-OR,-NH,-NHR, -SH etc. are the examples of auxochromic groups.
Absorption and intensity shifts in the UV spectroscopy
There are four types of shifts observed in the UV spectroscopy
Bathochromic effect- This type of shift is also known as red shift. Bathochromic shift is an effect by virtue of which the absorption maximum is shifted towards the longer wavelength due to the presence of an auxochrome or change in solvents. The nonbonding to pie transition of carbonyl compounds observes bathochromic or red shift.
Hypsochromic shift- This effect is also known as blue shift. Hypsochromic shift is an effect by virtue of which absorption maximum is shifted towards the shorter wavelength. Generally it is caused due to the removal of conjugation or by changing the polarity of the solvents.
Hyperchromic effect- Hyperchromic shift is an effect by virtue of which absorption maximum increases. The introduction of an auxochrome in the compound generally results in the hyperchromic effect.
Hypochromic effect- Hyperchromic effect is defined as the effect by virtue of intensity of absorption maximum decreases. Hyperchromic effect occurs due to the distortion of the geometry of the molecule with an introduction of new group.
Applications of UV spectroscopy
Detection of functional groups- UV spectroscopy is used to detect the presence or absence of chromophore in the compound. This is technique is not useful for the detection of chromophore in complex compounds. The absence of a band at a particular band can be seen as an evidence for the absence of a particular group. If the spectrum of a compound comes out to be transparent above 200 nm than it confirms the absence of a) Conjugation b) A carbonyl group c) Benzene or aromatic compound d) Bromo or iodo atoms.
Detection of extent of conjugation- The extent of conjugation in the polyenes can be detected with the help of UV spectroscopy. With the increase in double bonds the absorption shifts towards the longer wavelength. If the double bond is increased by 8 in the polyenes then that polyene appears visible to the human eye as the absorption comes in the visible region.
Identification of an unknown compound- An unknown compound can be identified with the help of UV spectroscopy. The spectrum of unknown compound is compared with the spectrum of a reference compound and if both the spectrums coincide then it confirms the identification of the unknown substance.
Determination of configurations of geometrical isomers- It is observed that cis-alkenes absorb at different wavelength than the trans-alkenes. The two isomers can be distinguished with each other when one of the isomers has non-coplanar structure due to steric hindrances. The cis-isomer suffers distortion and absorbs at lower wavelength as compared to trans-isomer.
Determination of the purity of a substance- Purity of a substance can also be determined with the help of UV spectroscopy. The absorption of the sample solution is compared with the absorption of the reference solution. The intensity of the absorption can be used for the relative calculation of the purity of the sample substance.
Most of the modern UV spectrometers consist of the following parts
Light Source: Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region. Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm.
Monochromator: Monochromators generally composed of prisms and slits. The most of the spectrophotometers are double beam spectrophotometers. The radiation emitted from the primary source is dispersed with the help of rotating prisms. The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose. The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism.
Sample and reference cells: One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution. Both sample and reference solution are contained in the cells. These cells are made of either silica or quartz. Glass can’t be used for the cells as it also absorbs light in the UV region.
Detector: Generally two photocells serve the purpose of detector in UV spectroscopy. One of the photocell receives the beam from sample cell and second detector receives the beam from the reference. The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells.
Amplifier: The alternating current generated in the photocells is transferred to the amplifier. The amplifier is coupled to a small servometer. Generally current generated in the photocells is of very low intensity, the main purpose of amplifier is to amplify the signals many times so we can get clear and recordable signals.
Recording devices: Most of the time amplifier is coupled to a pen recorder which is connected to the computer. Computer stores all the data generated and produces the spectrum of the desired compound.2.11
The UV-Visible Spectrophotometer used was JASCO V-650 (Figure 2.18) installed at Department of Physics, Sreenarayana College, Kollam. The V-650 is a double-beam spectrophotometer with a photomultiplier tube detector. The high sensitivity of the photomultiplier tube detector enables accurate measurements of low concentration samples. By controlling the high voltage applied to the PM tube, the dynode feedback circuit allows a wider dynamic range. It also enables the use of such solid sample handling accessories as integrating spheres to collect diffuse light transmitted or reflected by the sample. The advanced optical design results in high optical throughput and allows the bandwidth to be set as low as 0.1 nm for high resolution work such as gas and vapor phase spectroscopy. Low stray light slit settings provide excellent linearity of up to 4 absorbance units. Two graphical user interfaces are available including a newly redesigned intelligent remote module (iRM) with a color LCD touch screen and Spectra Manager™ II software, the latest version of JASCO’s innovative cross-platform spectroscopy software. Both of these interfaces allow full system control and advanced data processing. Spectra Manager CFR is a 21 CFR part 11 compliant version of software and is available as an option.
IQ Accessory and IQ Start
User-friendly features include the IQ Accessory function for automatic accessory recognition and IQ Start for immediate start of registered control programs when conducting routine measurements.
Excellent Optical Performance
Recent advances in optical technology have been utilized to provide excellent reliability and assure highly accurate results. High throughput optics combined with a modern electronic design provide excellent sensitivity and stability.
Full Line of Accessories
A full complement of accessories is available to optimize the V-650 for particular applications. Options include a wide variety of liquid cell holders, micro cell holders, flow cell units and accessories for solid samples. Advanced accessories such as automated cell changers, sippers and programmable temperature control systems allow full control by the iRM or Spectra Manager II.
Hardware specifications JASCO V-650 UV-Visible Spectrophotometer is shown in the table. 2.12
Single monochromator,1200 lines/mm plane grating, Czerny-Turner mount, Double-beam
Deuterium lamp: 190 to 350 nm
Halogen lamp: 330 to 900 nm
Light source exchange wavelength
User-selectable in the range of 330 to 350 nm
190 to 900 nm
±0.2 nm (using a spectral bandwidth of 0.5 nm;
wavelength: 656.1 nm; stabilized room temperature)
0.1, 0.2, 0.5, 1, 2, 5, 10 nm
L2, L5, L10 nm (low stray-light mode)
M1, M2 nm (micro-cell mode)
0 to 10000 %T
-2 to 4 Abs
±0.002 Abs (0 to 0.5 Abs)
±0.003 Abs (0.5 to 1 Abs)
±0.3 %T (Tested with NIST SRM 930D)
Photometric repeatability ±0.001 Abs (0 to 0.5 Abs)
±0.001 Abs (0.5 to 1 Abs)
1 % (198 nm KCL 12 g/L aqueous solution)
0.005 % (220 nm NaI 10 g/L aqueous solution)
0.005 % (340 nm NaNO2 50 g/L aqueous solution)
0.005 % (370 nm NaNO2 50 g/L aqueous solution)
(spectral bandwidth: L2 nm, 10 mm cell)
(value obtained greater than two hours after turning on the light source;stabilized room temperature ; wavelength: 250 nm; response: slow; spectral bandwidth: 2nm
±0.0003 Abs (Value obtained after instrument
baseline correction with a temperature variation
of less than 5oC; wavelength range: 200 to 850 nm;response: medium; spectral bandwidth: 2 nm;
scanning speed: 400 nm/min based on JAIMA Standard JAIMAS-0001 )
0.00003 Abs (0 Abs; wavelength: 500 nm;
measurement time: 60 sec; response: medium; bandwidth: 2 nm)
Dimensions and weight 460(L) x 602(W) x 270(H) mm (excluding accessories)
2.2.3 Fourier Transform Infrared Spectroscopy
Fourier Transform Infrared (FTIR) spectroscopy first developed by astronomers in the early 1950s to study the infrared spectra of distant stars has now been developed into a very powerful technique for the detection of very weak signals from the environmental noise. It is a simple mathematical technique to resolve a complex wave into its frequency components. The conventional IR spectrometers are not of much use for the far IR region (20-400 cm-1) as the sources are weak and detectors insensitive. FTIR has made this energy limited region more accessible. It has made the middle infrared (400-4, 000 cm-1) also more useful. The basic components of a FTIR spectrometer are given below (Figure 2.19).
The conventional spectroscopy, called the frequency domain spectroscopy, records the radiant power G(?) as a function of frequency. In the time domain spectroscopy, the changes in radiant power f(t) is recorded as a function of time t. In a Fourier transform spectrometer, a time domain plot is converted into a frequency domain spectrum. In mathematics, the Fourier transform of the function f(t) is defined by
G(?) = 1/2? ?_(-?)^???f(t)e^i?t ? dt (2.6)
Then the inverse relation is
f(x)=1/2? ?_(-?)^???G(?)e^(-i?x) ? d? (2.7)
Equation (2.6) and (2.7) are said to form a Fourier transform pair.
To illustrate the use of Fourier transform, consider the superposition of two sine waves, Figures 2.20 (a) and (b) of the same amplitude but of slightly different frequencies. Figure 2.20 (c) represents the superposed wave. The Fourier transform of the individual sine waves and the superposed wave train gives the frequencies in the frequency domain and are represented in Figures 2.20 (d), (e) & (f).
In the same way, complicated time domain spectra could be transformed into frequency domain spectra. The actual calculation of the Fourier transform of such systems is done by means of high speed computers.
The source is the usual glower operated at very high temperatures. The Michelson interferometer consists of a source S, a beam splitter B and two plane mirrors M1 and M2 (Figure 2.21). Mirror M1 is fixed and M2 is capable of to and fro movements. The beam splitter allows 50% of the radiation to mirror M1 and the other 50% to mirror M2. The two beams are reflected back to B where they recombine with 50% going to the source and the other 50% going to the sample. For monochromatic source, if the path lengths BMIB and BMZB differ by an integral number including zero of wavelengths, one gets constructive interference of the two beams at B (bright beam). Destructive interference results when the difference in path lengths is half odd integral number of wavelengths.
Thus, if mirror M2 is moved towards or away from B, the sample and detector will see an alternation in intensity. If two different monochromatic frequencies v1 and v2 are used instead of one, a more complicated interference pattern would follow when M2 is moved. A Fourier transform of the resultant signal would give the two originals with the appropriate intensities. Extending this, a white light produces an extremely complicated interference pattern which can be transformed back to the original frequency distribution. The recombined beam if directed through a sample, the sample absorption will show up as gaps in the frequency distribution which on transformation gives a normal absorption spectrum. In the experiment, the detector signal is collected into a multichannel computer while mirror M2 is moved. The computer then carries out the Fourier transform of the stored data and plots it on a paper.
FTIR techniques have made significant impact with regard to (i) rapid scanning, (ii) signal to noise (S/N) ratio, (iii) high sensitivity, (iv) high resolution, (v) data processing. The simultaneous data collection helps one to investigate the spectrum of transient species such as unstable molecules or intermediaries in a chemical reaction and for the analysis of environmental samples, etc. The rapid scanning property of FTIR spectrometers is having its greatest impact in the field of gas chromatography-FTIR.
Signal to noise ratio of 3 to 4 is necessary for an unambiguous recognition of a signal in conventional spectrophotometers. The incorporation of powerful computers enables one to do computer averaging techniques. In this technique, the spectrum is recorded stepwise into the computer and stored in separate locations in the memory. For example, the region 400 to 4,000 cm-1 is recorded as 1,200 steps with each step covering a frequency range of 3 cm-1. This process is repeated number of times and the signals are added electronically. Thus a weak signal which may not be visible above the noise in the ordinary case will produce fairly intense signal as we have summed over n scans. It can be shown that in n scans the S/N ratio increases by ?(n ). The FTIR spectrum of sulphamic acid recorded for 10 scans is given in figure 2.22. For comparison, the normal IR spectrum is also given. . The resolution of the FTIR spectrum is really impressive.
The high sensitivity helps one to investigate species absorbed on metal oxide and supported metal catalysts, pharmaceuticals, proteins, etc. Its potential for the detection of photoacoustic signal and for biochemical research is enormous. The possibility for high resolution in the infrared permits a more detailed knowledge of molecular vibrations and energetics. High resolution FTIR spectra has enabled one to measure the presence of components in the atmosphere to the level of a few parts per billion.
The incorporation of a computer in the instrument allows rapid spectral searches as high as 10,000 in about 6 to 7 seconds at the end of a run. The subtraction of the spectrum of a reference sample from the spectrum of a mixture, the determination of the number of components in a mixture, curve routines etc., are some of the other popular operations possible with a FTIR spectrometer. 2.13
The FTIR Spectrometer used was PerkinElmer Spectrum 100 (Figure 2.23) installed at Department of Chemistry, Christian College, Chengannur. Spectrum 100 FTIR instrument was designed and manufactured to the highest quality standards to ensure quick and consistently superior results.Available for both the mid-infraredrange and near infrared range, the Spectrum 100 Series is engineered by the experienced and knowledgeable FTIR experts at PerkinElmer
Atmospheric Vapor Compensation (AVC) features an advanced digital filtering algorithm designed to subtract out CO2 and H2O absorptions in real time. AVC effectively eliminates interference from these atmospheric constituents, allowing to achieve the most accurate and reproducible results
Absolute Virtual Instrument (AVI) approach to calibration allows the same standardization used on individual measurements to be applied to measurements collected over time. AVI standardizes the instrument’s wavenumber scale to a far higher accuracy than can be achieved with conventional calibration methods, resulting in transmission spectra that can be related easily and directly to theory. AVI’s internal, traceable protocol allows data to be transferred quickly and accurately between instruments whether they are side by side or across the world.
Sigma-Delta modulators in the digitization of the FTIR interferogram with the Spectrum 100 improves dynamic range,reduces spectral artifacts and increases ordinate linearity producing more accurate, reproducible results. Inner components of PerkinElmer Spectrum 100 is shown in Figure 2.24
Electronically, temperature stabilized detector
Source doubling mirror
Sigma-Delta Conversion technology
User replaceable, electronically stabilized source
Second detector expandability
Upgrade to microscopy and imaging capabilities
2.3.4 Antimicrobial activity studies
Silver ions work against bacteria in a number of ways; silver ions interact with the thiol groups of enzyme and proteins that are important for the bacterial respiration and the transport of important substance across the cell membrane and within the cell, and silver ions are bound to the bacterial cell wall and outer bacterial cell, altering the function of the bacterial cell membrane, thus silver metal and its compounds were the effective preventing infection of the wound Silver can inhibit enzymatic systems of the respiratory chain and alter DNA synthesis Metal nanoparticles, which have a high specific surface area and a high fraction of surface atoms, have been studied extensively due to their unique physicochemical characteristics such as catalytic activity, optical properties, electronic properties, antimicrobial activity, and magnetic properties.
It can be expected that the high specific surface area and high fraction of surface atoms of nanosilver shapes will lead to high antimicrobial activity compared to bulk Ag metal. Recent, microbiological and chemical experiments implied that interaction of silver ion with thiol groups played an essential role in bacterial inactivation. Surface area involves the increase of contact surface, which is an important condition for the effects of silver nanoparticles. 2.15
Antibacterial activity of Ag NPs against Staphylococcus aureus (S aureus) and Escherichia coli (E coli) are tested here.
As per CLSI (Clinical laboratory standards institute) guidelines
Test organisms were collected from institute of Microbial Technology, Microbial Type Culture Collection Centre (IMTECH), Chandigarh.
The bacterial strains were maintained on their respective medium in slants at 2-8 0c.
Preparation of Muller Hinton Agar (MHA)
MHA medium was used for bacterial culture. MHA was prepared and sterilized at 1210c for 15 minutes. After sterilization required volume of the medium (20 ml) was poured in the sterile Petri dishes and allowed to solidify.
Use pure culture as inoculum. 3 to 4 similar colonies were selected and transferred them into 5 ml of suitable broth such as Trypton Soya Broth (TSB).Incubate at 2 to 8 hours until light to moderate turbidity develops.
Method of inoculation
Filter paper disc diffusion technique was applied for determining antibacterial activity.
Dipped a sterile nontoxic swab on a wooden applicator in to the standardized inoculum and rotated the soaked swab firmly against the upper side wall of the tube to express the excess fluid. Streaked the entire agar surface of the plate with the swab three times, turning the plates at 60 0 angle between each streaking. Allowed the inoculum to dry for 5to 15 minutes with lid in place.
Apply the disc (Hi media sterile 6mm disc) impregnated with the sample approximately 30 µl, using aseptic technique. Then place the disc with centers atleast 24mm apart.
Incubated immediately at 37 0c and examined after 16 to 18 hours or later if necessary. Measure the zone showing complete inhibition and record the diameters of the zones to the nearest millimeter.
Disc soaked in pure solvent Dimethyl sulfoxide (DMS) were used as control.
2.1. Schipper, M. A. A. 1984. A revision of the genus Rhizopus. I. The Rh.Stolonifergroup
and Rh. oryzae. CBS Studies in Mycology 25:119
2.3. C. Kittel., Introduction to solid state physics, John Wiley & Sons Inc. (1996).
2.4. H. P Kulg and L. E Alexander., X-ray diffraction procedures, 2nd Edition, John Wiley
& Son Inc. (1974).
2.5. W. L Bragg, Proc. Comb Phil. Soc. 17 (1912) 43
2.6. International Tables for X-ray crystallography, Kynoch Press, Birmingham U. K.
(1952) Vol. 1, (1959) Vol. II and (1962) Vol. III.
2.7. H. G. J. Mosely., Phil. Mag. 26(6) (1913) 1024.
2.8. B. D. Cullity, “Elements of X-ray diffraction”, Addition-Wesley Publishing
Company, Inc. Reading, Mass (1956).
2.9. 2nd edition PW 1840/01, N. V Philips Gloeilampenfabrieken-Eindhoven, Netherlands
2.10. H. P. Kulg and L. E. Alexander, X-ray diffraction procedures, 2nd edition, John Wiley
and Sons (1974)
2.13. G.Aruldas,Molecular Structure and Spectrocopy,PHI learning Pvt Ltd ( 2007)
2.15. B. Sadeghi, M. Jamali 2, Sh. Kia3, A. Amini nia3, S. Ghafari3, Synthesis and
characterization of silver nanoparticles for antibacterial activity, Int.J.Nano.Dim
1(2): 119-124, autumn 2010, ISSN: 2008-8868
RESULTS AND DISCUSSION
Green methods have now become the routine tool for nanoparticle synthesis 3.1. In this chapter, the detailed discussion of characteristic results obtained for the sample synthesized in chapter 2 (viz, Ag-NP-BM) is scripted. For the synthesis, 0.05M AgNO3 was used as precursor salt with Bread Mold extract as reductant. Black Bread Mold is a threadlike mold and a heterotrophic species; it is dependent on sugar or starch for its source of carbon substances for food. It uses food matter, generally breads or soft fruits, like grapes or strawberries, as a food source for growth nutrition and reproduction. Black Bread Mold is a mass of mycelium, the vegetative filaments of the fungus, and a fruiting structure. Most of the mycelium is composed of multi-nucleate, rapidly growing hyphae. When the mold’s spores are released they produce more mycelium through germination. As the mold matures it begins to turn black. It is an agent of plant disease; it breaks down organic matter through decomposition. When kept in a moist environment, such as a piece of bread, the parasite can quickly spread within a few days. Its spores are commonly found in the air. The spores grow most rapidly at temperatures between 15°C and 30°C where they are able to germinate to their full potential. Black Bread Molds are commercially used for manufacturing alcohol and organic acids. In kingdom fungi it comes under phylum zygomycota and class zygomycetes. The order and family is mucoraceae. In the rhizopus genus, it comes under the species rhizopus stolonifer, and hence the scientific name is Rhizopus stolnifer
X-ray diffraction Analysis
Figure 3.1 shows the x-ray diffraction pattern recorded for Ag-NP-BM sample, using XPERT-PRO Diffractometer system having type 0000000083005153 with continuous scan mode of step size, 2?°= 0.0170 in the gonio axis in the 2? range 10° to 89.9°, installed at National Centre for Earth Science Studies (NCESS), Thiruvananthapuram. X-rays from the Cu anode material include K-aplha1, K-alpha2 and K-beta radiations with respective wavelengths, 1.54060 Å, 1.54443 Å, and 1.39225 Å with K-alpha2 to K-alpha1 ratio equal to 0.50000. The 240 mm goniometer radius having 100 mm dist. focus-diverge-slit was used with generator settings 30 mA, 40kV. The indexed Bragg reflection peaks with respective d-values and width are listed in table 3.1, which is in accordance with JCPDS: ICDD PCPDF WIN #PDF 893722 corresponding to the face centered cubic lattice of Silver. Also, traces of silver oxide phases are evident from XRD patter due to the presence of an intense peak with matching d value at 32.5o.
Figure 3.1: X-ray diffraction pattern recorded for Ag-NP-BM
Table 3.1: List of peaks and corresponding d values
Pos. °2? Height cts FWHM °2? d-spacing Å Rel. Int. %
20.7673 5.19 0.4015 4.27732 4.39
28.1230 35.74 0.2007 3.17306 30.18
28.9134 12.28 0.1004 3.08809 10.37
29.6340 13.77 0.1673 3.01462 11.63
30.1508 15.26 0.1171 2.96412 12.89
32.5527 117.97 0.2342 2.75069 99.63
34.0406 7.28 0.2007 2.63379 6.15
35.9149 8.31 0.2007 2.50052 7.02
38.4836 18.94 0.4015 2.33932 16.00
39.1283 17.40 0.2007 2.30225 14.69
41.4248 9.47 0.2007 2.17978 8.00
41.9489 13.64 0.2007 2.15375 11.52
44.6073 8.15 0.5353 2.03137 6.89
46.5370 118.41 0.2676 1.95154 100.00
55.1318 29.41 0.2342 1.66592 24.84
57.7520 26.33 0.3346 1.59642 22.23
64.8066 3.70 0.4015 1.43865 3.12
67.7200 6.90 0.3346 1.38368 5.83
76.9920 3.97 0.5353 1.23853 3.36
85.6912 0.17 0.8029 1.13369 0.15
Considerably broadened X-ray reflection peaks from various crystallographic plane surfaces indicate the distribution of crystallite size in nanometric regime. The average crystallite size was evaluated using Debye-Scherer equation 3.2:
where D is the thickness (diameter) of the particle, l is the X-ray wavelength (1.5406 Å), ? is the full width at half maximum (FWHM) of the main peak under consideration, k is the shape factor and qB is the Bragg angle of reflection. The main intense peak at 46.5370° was taken for analysis to obtain the size estimation using Scherer formula. Accordingly, the estimated crystallite size is 37 nm, which is consistent with our other relate works 3.3 -3.8.
UV-Visible absorption spectrum of the sample was recorded using JASCO V-650 B118561150 spectrometer installed at Department of Physics, Sree Narayana College, Kollam. The data were obtained in absorbance photometric mode using a bandwidth of 5nm in 1 nm interval at a san speed of 400 nm/min. Figure 3.2 represents the spectrum obtained the prepared Ag-NP-BM sample.
Figure 3.2: UV-Visible Spectrum recorded for Ag-NP-BM
The peaks obtained at wavelength 228 nm, 273 nm and 373 nm are corresponding to the elemental absorption of Ag in Ag-NP-BM sample. In our previous observations on the green reduction of FeSO4.7H2O using the leaf extract of locally available kiriyath plant in Punalur region, it has been found that the UV-visible –NIR absorption at wavelengths 242 nm and 305 nm with absorbance 2.241 and 2.094 respectively, were corresponding to the elemental absorption of iron, which were further reported and presented 3.7 in National Seminar on Modern Trends in Physics Research (NSMPTR 2015) organized by Post Graduate Department of Physics, St. Stephens’s College, Pathanapuram. In another study 3.3 wherein FeSO4.7H2O is reduced by leaf extract of Garlic Vine, the absorption peaks were obtained at wavelengths, 241 nm, 305 nm and 378 nm respectively. In both cases, the spectra were obtained from UV-Visible spectrometer (Model: Lamda-45, Perkin Elmer make) installed at CEPCI lab Kollam. At present, the study on green synthesis of silver nanoparticles also give results on UV-visible data consistent with the above observations corresponding to the elemental absorption of silver.
FTIR spectral measurement was performed on PerkinElmer Spectrum Version 10.5.2 installed at Department of Chemistry, Christian College, Chengannur. Figure 3.3 shows the room temperature FTIR spectrum recorded in the wavelength range 4000 cm-1 -400 cm-1 for the sample, Ag-NP-BM. Five bands centered at 3206.68 cm-1, 1645.26 cm-1, 1403.00 cm-1, 1047.00 cm-1 and 607.33 cm-1 were observed.
Figure 3.3: FTIR spectrum recorded for Ag-NP-BM
The band at 607.33 cm-1 corresponds to the stretching vibrations at the metallic site of silver in the crystal. The intense broad band at 3206.68 cm-1 is due to the asymmetric and/or symmetric stretching modes of water molecules (H-O-H bonding) in the crystal. The band at 1645.26 cm-1 is attributed to the C=C ring stretching in flavonoids (organic capping) 3.9. The band at 1403.00 cm-1corresponds to the in-plane bending vibrations of –OH group in flavonoid 3.10 and that at 1047.00 cm-1 relates to the stretching vibration of C-O-C. The weak absorptions at the inorganic band positions against the comparatively larger absorptions at the capped polymer rings again infer that samples formed are of much reduced crystallites, which agrees well with the XRD result.
Antimicrobial activity studies
In this study, the antibacterial activity of Ag-NP-BM for Escherichia coli (E coli), Gram negative and Staphylococcus aureus (S aureus), Gram positive was measured by CLSI Standard M02-A10 method. The Ag-NP-BM (1817) shows moderate antibacterial activity as evidenced from the growth inhibition ring (8 mm dia) depicted in figure 3.4(a) and (b) for both E coli and S aureus).
Figure 3.4 (a): Antibacterial activity of Ag-NP-BM solution against E. Coli (1817)
Figure 3.4 (b): Antibacterial activity of Ag-NP-BM solution against S. aureus (1817)
The mechanism for antibacterial action of silver nanoparticles is bacterial membrane disruption by the ions silver released from the solution 3.11. The Ag ions form insoluble compounds with sulphydryl groups in the cellular wall of the microorganism that are responsible for the inhibition halo in the seeded culture media as observed in figure 3.4(a) and (b). The diminution in nanocrystallite site has also a great impact in antibacterial activities. All antibacterial activity tests were performed in triplicate and were done at least two different times to ensure reproducibility.
Silver based Nanocrystalline sample was successfully synthesized by the chemical reduction of AgNO3 using yeast Bread Mold extract. The crystalline behavior and size were estimated using X-ray diffraction analysis. From UV-visible spectrum the absorption by elemental silver could be identified. Using FTIR- the presence of silver nanoparticle in the sample Ag-NP-BM is confirmed and which agrees with the XRD result. The antibacterial activity of Ag-NP-BM for Escherichia coli (E coli), Gram negative and Staphylococcus aureus (S aureus), Gram positive was measured by CLSI Standard M02-A10 method.
Sangeetha Gunalan, Rajeshwari Sivaraj, rajendran Venckatesh, Aloe barbadensis Miller mediated green synthesis of mono-disperse copper oxide nanoparticles: Optical properties, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 97 (2012) 1140-1144.
B.D. Cullity, Elements of X-ray Diffraction, Addison-Wesley, Reading, 1978
Arun S. Prasad, S.N.Dolia, P.Predeep, Optical band gap study of nanocrystalline NiCr0.8Fe1.2O4, AIP Conf.Proc.1004 (2008) 112–116
Arun S. Prasad Iron oxide nanoparticles synthesized by controlled bio-precipitation using leaf extract of Garlic Vine (Mansoa alliacea) Materials Science in Semiconductor Processing 53 (2016) 79–83.
Athira Rajendran and Arun S. Prasad Green Synthesis and Spectroscopic Investigation of Copper Oxide Nanoparticles Proceedings of National Seminar on Nanomaterials for Energy Applications (NSNME) @ SN College, Punalur (2016)
Athira Rajendran, Pooja Dileep S., Jeena N. and Arun S. Prasad Characteristic and Thermo gravimetric Analysis of Biogenic reduction in Copper Sulphate Pentahydrate using the Leaf Extract of Eclipta alba Proceedings of National Seminar on Green Analytical Techniques (NSGAT-2015) at DB College Sasthamcotta, PP 13, pages 154-159
Pooja Dileep S. and Arun S. Prasad Green Reduction of Metallic Precursor Salts using Plant Extracts: a Novel route for Nanoparticle Synthesis Proceedings of National Seminar on Nanomaterials for Energy Applications (NSNME) @ SN College, Punalur (2016)
Pooja Dileep S., Athira Rajendran, Jeena N. and Arun S. Prasad Green Reduction and Spectroscopic Investigation of Iron (II) Sulphate Heptahydrate Precursor using Leaf Extract of Kiriyath (Andrographis paniculata) Proceedings of National Seminar on Modern Trent in Physics Research (NSMTPR) at St. Stephen’s College, Pathanapuram (2015)
Silverstein R., Bassler G., Morrill T., (1991) Spectrometric Identification of Organic Compounds, 5th ed., Courier Companies Inc., Canada
Shahwan T., Abu Sirriah S., Nairat M., Boyac? E., Ero?glub A.E., Scott T.B., Hallam K.R. (2011) Green synthesis of iron nanoparticles and their application as a Fenton-like catalyst for the degradation of aqueous cationic and anionic dyes. Chemical Engineering Journal. 172:258– 266.
B. Sadeghi , M. Jamali, Sh. Kia, A. Amini nia, S. Ghafari Synthesis and characterization of silver nanoparticles for antibacterial activity Int.J.Nano.Dim 1(2): 119-124, autumn 2010.
SUMMARY AND SCOPE FOR FUTURE WORKS
The purpose of my thesis is to synthesize silver nanoparticles through bioreduction of AgNO3. The bioreductant used is the extract of Black Bread Mold (Rhizopus stolonifera). The thesis is composed of three chapters. Chapter one is introductory and it gives the basic ideas behind the venture. The chapter is subdivided into five parts. Part one give the basic idea about nanoscience and nanotechnology. Part two describe the properties, classification and traditional method of synthesis of nanoparticles. In part three synthesis of nanoparticle using plant and bio-organism and factors affecting bio-reduction are mentioned. Part four contain some important application of nanoparticle and part five is an introduction about silver nanoparticle. It contains the characterization, antimicrobial activity and application of silver nanoparticles
Chapter two gives a relevant description of the materials used in the synthesis of silver nanoparticles and the various characterization techniques employed to study these particles. It consists of three parts. Part one and two deals with the preparation of the sample, describing in detail the materials used, procedure for the preparation of the yeast extract and the synthesis of nanoparticles. Part three deals with the description of the characterization techniques employed in the analysis namely, X-ray diffraction studies, UV- Visible spectroscopy, FTIR spectroscopy and Antimicrobial activity study with the respective instrument specifications.
Chapter three deals with the analysis of results and discussion. From the analysis of the X-ray Diffraction peaks, the average crystallite size is estimated to be 37 nm. The UV-Visible spectroscopic analysis peaks obtained at wavelength 228 nm, 273 nm and 373 nm are corresponding to the elemental absorption of Ag in Ag-NP-BM sample. The FTIR analysis band at 607.33 cm-1 corresponds to the stretching vibrations at the metallic site of silver in the crystal which agrees well with the XRD result and the antibacterial activity of Ag-NP-BM for Escherichia coli (E coli), Gram negative and Staphylococcus aureus (S aureus), Gram positive was measured by CLSI Standard M02-A10 method
The main aim of the thesis is to investigate on the formation of silver nanoparticles using an eco-friendly and cost less reductants which are commonly available. The fungal extract (Black Bread Mold) has been used as a reducing and capping agent. The biogenic reduction of metal ions to base metal is quite rapid, readily conducted at room temperature and pressure and easily scaled up. Synthesis mediated by fungal extracts is environmentally benign. It is a low-cost approach. Silver nanoparticles, owing to their antimicrobial activity to bacterial fish pathogens, could be used in fish treatment. They can also be used for the treatment of human diseases. So we can say that in general, the nanoparticles synthesized via fungal extracts have found a variety of applications and compare with another synthesis method it is easy, cost less and eco-friendly. Here 0.05 Molar AgNO3 solution is used for the preparation of silver nanoparticle and obtained 37nm nm silver nanoparticles. We can also reduce the size by reducing Molar concentration of AgNO3 because silver nanoparticle of small size have more useful properties like antibacterial activity and many application in electronics, optics, etc. Also we can use some other type fungi like bracket fungi, white root fungi, etc. as bioreductant because fungi are act as a good bioreductant for the synthesis of silver nanoparticle. Kerala is a cauldron of immense varieties of fungi or mushrooms. Many important varieties could be found in the Punalur region (around our research faculty). Thus there is a great scope of research in the green synthesis domain of nanoparticle synthesis.