NANOTECHNOLOGYThe development of nanotechnology as a field was started since 1958. In 1959, Richard Feynman, the father of nanotechnology, initiated thought process by his statement “there is plenty of room at the bottom”. He suggested that the key to the future of technology would be scaling down to nanolevel and initiating from the bottom. Nanotechnology is manipulation of matter which have at least one dimension in the nano range. Briefly, nanotechnology is the study of extremely small structures. The prefix “nano” is a Greek word which means “dwarf”. The word “nano” means very small or miniature size. Nanotechnology uses individual atoms or molecules to produce materials and devices with special properties. There are two approaches to produce these nanomaterials such as top-down approach and bottom-up approach. Nanotechnology involves work from top down i.e. reducing the size of large structures to smallest structures (e.g. photonics applications in nano-electronics and nano-engineering) or the bottom up, which involves changing individual atoms and molecules into nanostructures and more closely resembles chemistry biology.Nanotechnology deals with materials in the nano-scale range (1-100 nm). Due to their smaller size, these materials display different properties from bulk counterparts, such as electrical conductance, optical effects, magnetism, chemical reactivity, physical strength, biological properties, etc.There are different fields which find potential applications of nanotechnology such as Health and Medicine, Electronics, Transportation, Energy and Environment, Space exploration.In the 20th century, the field of medicine and pharmaceuticals have been revolutionized with the advancement of nanotechnology. Nanomedicine is the latest advancement in the field of science. Nanoparticles can interact with biological molecules which are in nano-range, thus broadening the applications in the field of medicine.METALLIC NANOPARTICLESHISTORYMetallic nanoparticles have attracted people since the middle ages. Before the ancient Roman times silver and gold nanoparticles were used to stain glasses as “colouring agents”. The Lycurgus cup, manufactured in 4th – 5th century B.C., contains gold colloids. Hence, it shows ruby red colour in transmitted light and green colour in reflected light. In 15th – 16th centuries (Renaissance), pottery of Deruta was manufactured in Umbria, Italy. It contains glazes of copper and silver nanoparticles.The existence of metallic nanoparticles in solution was first recognized by Michael Faradayin 1857. Gold sols were prepared and studied by Faraday and it was the foundation of modern colloid science. He mentioned that individual gold (metal) nanoparticles (i.e. colloidal gold) had to be kinetically stabilized to avoid their aggregation, as colloidal metal nanoparticles were thermodynamically unstable. A quantitative explanation of their colour was given by Mie in 1908. In 1925, Richard Zsigmondy studied modern colloid chemistry and invented an ultra-microscope. He won the Nobel Prize in chemistry for his work. Due to the specific properties of colloidal metal nanoparticles, which cannot be seen in bulk counter metals, are very much of importance in research and technology. STABILIZATION OF NANOPARTICLESNanoparticles have large surface energy, therefore they always try to aggregate to form thermodynamically stable bulk particles. This agglomeration formation is affected by nanoparticle concentration, size, shape and surface charge. Hence, it is very important to stabilize these particles. This can be achieved by either steric or electrostatic stabilization. Steric stabilization is reached by using a capping agent such as surfactant, polymer, ligand or solid support. Electrostatic stabilization is reached by an electrical double layer arising from the attraction of negatively charged ions to the metal nanoparticles.PREPARATION OF NOBLE METAL NANOPARTICLESVarious methods are used to synthesize metal nanoparticles which are cheap, environmentally friendly and produce high yield. The size and shape of the nanoparticle should able to be controlled when using a particular method. Following preparation methods are discussed in reference of Ag nanoparticles, as an example.Oxidation – reduction methodAg+ salt precursor is mixed with a reducing agent in the presence of a stabilizing agent, which helps to control the morphology of metal nanoparticles. Silver nitrate is the commonly used precursor due to its cheap price and easy availability compared with other salts. Sodium citrate, sodium borohydride, hydroxylamine hydrochloride and alcohols are generally used as reducing agents to reduce Ag+ ions present in solution into Ag atoms which combine initially to make aggregates and finally convert to nanoparticles. Stabilizing agents are also introduced to maintain the size and shape and to stabilize the nanoparticles. This process can be controlled by controlling the concentration of reactants, concentration of stabilizer and the mixing rate.Electrolysis and PyrolysisElectrochemical approach can be used to synthesize metal nanoparticles. Spherical shaped Ag nanoparticles are synthesized using electrochemical method by reducing Ag+ ions in the presence of poly vinyl pyrrolidone (PVP), where titanium (Ti) electrode works as the cathode and platinum (Pt) electrode works as the anode. Ag nanoparticles are also synthesized by spray pyrolysis method in which an average grain size of 100 nm of Ag nanopowder is synthesized. Among other chemical synthesis methods, electrolysis and pyrolysis are considered environmental friendly as no harmful or toxic reducing agents are used to produce nanostructures.Radiation reduction methodSilver ions are extremely sensitive to light. Therefore, silver ions can absorb light energy and get reduced to silver atoms. For this purpose, high energy radiation can be used to prepare silver nanoparticles by reducing silver ions in solution. Ultraviolet rays and gamma rays can be used here.Hydrothermal Process Hydrothermal process is simple and has been widely used for the synthesis of different kinds of nanostructures. High temperature is required for this. For example, Yan et al., synthesized Ru nanoplates of triangular shape with thickness of about 3 nm using RuCl3?H2O and HCHO in the presence of PVP for 4h at 160?C. It was also worth noticing that only change in the concentration of Ru salt and PVP, irregular shaped Ru nanoplates were formed with thickness of about 1.5 nm. On the other hand, when Ru salt was substituted with silver nitrate, triangular shaped Ag nanoplates were formed in the presence of PVP for 6h at 160?C.Self-Assembly of Metal NanoparticlesSelf-assembly of metal nanoparticles can be distinguished on the basis of shape of individual nanoparticles, method used for self-assembly and the nature of forces involved for self-assembly of nanoparticles. Nanoparticles are the basic building blocks for self-assembly process. This process can generate larger products such as nanosheets. Self-assembly can be distinguished by the shape of individual nanoparticles, nature of forces involved and method used for preparation. Direct interaction of nanoparticles in solutions such as main driving forces, attractive and repulsive forces such as hydrophobic, electrostatic, hydrogen bonding influence this process.Biological Approach Microorganisms such as bacteria, fungi and plants have potential to synthesize metal nanoparticles such as cadmium sulfide (CdS), Ti/Ni, titanate, zirconia, Au and Ag. The use of living microorganisms for the synthesis is environmental friendly and it maintains the size distribution of nanostructures. For example, Ag nanostructures of size less than 200 nm were synthesized using bacteria.CHARACTERIZATION OF NOBLE METAL NANOPARTICLESVarious techniques/ instruments are available to characterize noble metal nanoparticles. Those can be divided as follows1. Size and shape of nanomaterial : Transmittance Electron Microscopy (TEM) Scanning Electron Microscopy (SEM)Atomic Force Microscopy (AFM)2. Optical and electrical properties: UV- Visible Absorption SpectraSurface-Enhanced Raman Scattering Spectroscopy (SERS)3. Crystalline structure and crystallinity: X-ray Diffraction (XRD)4. Surface modification: Fourier transform infrared spectroscopy (FTIR)UV-Visible Absorption SpectroscopyThe UV-visible absorption spectroscopy is a useful technique to characterize noble metal nanoparticles, as they possess bright colors which are visible to naked eye. Nanoparticles possess high extinction coefficient and their surface plasmon property depends on size and shape. Therefore, qualitative information about colloidal nanoparticles can be obtained by this technique.Metallic nanoparticles have free electrons at their natural frequency for co-oscillation. When the frequency of light and natural frequency of free electrons of metal nanoparticles and inherent oscillation frequency rates are equal, the motion of electrons can resonate with the irradiated light resulting in surface plasmon resonance. The appearance and location of the surface plasmon band depends on a few factors such as particle size, shape, extent of aggregation, nature of stabilizer, nature of surrounding medium and presence of any adsorbate on the surface of nanoparticles. Metal nanoparticles absorb light in the UV-visible range due to the metal surface plasmon resonance excitation. This technique has become one of the most simple and accurate methods to characterize and study metal nanoparticles. It is also the primary means of studying the optical properties of gold and silver colloids.Electron MicroscopySize and shape of nanoparticles can be determined using electron microscopy. TEM is the most commonly used method to characterize metal nanoparticles. TEM machine equipped with other accessories can provide information on morphology, topography, composition, dispersity and crystallography of the sample. High Resolution Transmission Electron Microscopy (HRTEM) can go upto a magnification of about 10,000,000 with a resolution of about 1 A°. SEM is developed in the 20th century. In SEM, the image of specimen surface is obtained by scanning the surface with an electron beam. SEM gives information about the morphology and topography of the sample and has a magnification of about 1,00,000 with a resolution of about 1.5 nm.FTIRIdentification of specific types of chemical bonds or functional groups is possible depending on their unique absorption signatures. Stretching and bending of chemical bonds take place due to absorption of energy. This energy is in the infra-red (IR) range of electromagnetic spectrum. Functional groups attached to the metal nanoparticle surface show different FTIR patterns than those of free groups. Therefore, FTIR gives information about the surface chemistry of nanomaterials.PROPERTIES OF NOBLE METAL NANOPARTICLESMetallic nanoparticles have unique morphologies, surface plasmon characteristics, quantum confinement, short range ordering, large surface energies and physicochemical properties. The characteristics of metallic nanoparticles depend on their size, shape, crystallinity and structure. The color of nanoparticle depends on size and shape of the nanoparticle and the dielectric constant of medium, leading to many studies and applications. These parameters can be changed or controlled to achieve desired properties of nanoparticles. Due to this, they make exceptional candidates for biomedical applications as a wide range of biological processes occur at nano level. Other applications of metallic nanoparticles range from catalysts, sensing, optics, antibacterial activity and storage.Metallic nanoparticles, especially silver and gold nanoparticles possess excellent optical properties such as Surface Plasmon Resonance (SPR), Surface Enhanced Raman Scattering (SERS), fluorescence, etc. Due to their efficient scattering properties, metallic nanoparticles are also used in various near field optical microscopy applications where they increase the signal output. The plasmon resonance, the large effective scattering cross section and nonbleaching properties of individual silver nanoparticles possess significant potential for single molecule labeling based biological assays.Surface Plasmon ResonanceThe characteristic plasmon absorption peak of silver nanoparticles is in the range of 380-440 nm, while that of gold nanoparticles is in the range of 510-550 nm.The free electrons in the metal (d electrons in silver and gold) can travel through the material. The mean free path in silver and gold is about 50 nm, thus no scattering is observed from the bulk in particles smaller than this. Therefore, all interactions are taking place with the surface. Light in resonance with the surface plasmon oscillation results in oscillation of free-electrons in the metal. As the wave-front of the light passes, the electron density in the particle is polarized to one surface and oscillates in resonance with the frequency of light resulting in a standing oscillation. The resonance condition is determined from absorption and scattering spectroscopy and it depends on shape, size and dielectric constants of metal and surrounding material. This phenomenon is located at the surface, therefore it is called surface plasmon resonance. The surface geometry of nanoparticle changes with its change in size and shape. This causes a shift in the electric field density on the surface. This causes a change in the oscillation frequency of the electrons, generating different cross-sections for the optical properties including absorption and scattering. Figure : Origin of SPR due to coherent interaction of the electrons in the conduction band with LightBy solving Maxwell’s equations for small spheres interacting with an electromagnetic field, Mie calculated the surface plasmon resonance. Gan was then able to extend this theory to apply to ellipsoidal geometries.Surface-Enhanced Raman Scattering (SERS)This phenomenon was observed in the early 70’s. Highly enhanced Raman scattering was observed when molecules deposited on rough noble metal surfaces. Rough surfaces are decorated with nanoparticle shapes with surface plasmon oscillations. This effect is known as surface enhanced Raman scattering (SERS). The aggregates of nanoparticles give the largest enhancement, due to the generation of large Raman signals with the interaction of multiple particles, while nanoparticles generate small or moderate signals. Fundamental theories point out the origin of the SERS signal as a ”hot spot” at the junction of two or more particles. Nanorods have higher SERS signals than spheres, due to the higher electric field generated at the edges of the nanorod. SERS is being developed as a powerful diagnostic tool. Large enhancement is accessible along with chemical information. There are two properties which affect the enhancement of the Raman signal, which are chemical enhancement and electromagnetic field enhancement. The chemical effect indicates that the enhancement factor is determined by the nature of the molecule. Different signal strengths are observed with different chemicals on the same substrate.Electromagnetic field increases at the junction of two nanoparticles or as the radius of curvature increases, or at sharp points of a triangle. Rough surfaces have many random geometries, hence enhance Raman signals. But nanoparticles give the opportunity to study the Raman enhancement in accurately designed systems.SILVER NANOPARTICLESSilver (Ag) has been used for medical applications apart from their uses as jewelry, coins, foils, photography, metalcraft, vessels or containers, water treatment throughout the history. Silver nanoparticles are also been used in many fields such as chemical and biological sensors (due to unique plasmon-resonance optical scattering), electronics and optoelectronics applications (due to electrical and thermal conductivity), energy harvesting, biomedicine, antimicrobial agents. Surprisingly at low concentrations, Ag nanoparticles are non-toxic to human and animal cells as the toxicity of Ag nanoparticles to the environment is considered extremely low when compared with other materials. Due to this reason, Ag nanoparticles have accomplished the highest commercialization level and account for >50% of nanomaterial-based products in the market. Ag nanostructures show plasmon resonance in the range of visible spectrum and therefore are used in various potential technologies.Compared with gold nanoparticles, silver nanoparticles have better catalytic activity and are more sensitive to electrons and protons. In last few decades, Au and Ag nanostructures have been prepared chemically on a large scale for plasmonic applications. Ag is 50 times cheaper than Au and became a good candidate in plasmonics due to its unique and convenient physicochemical properties for the next generation plasmonic technologies.BINDING OF BIOMOLECULES TO THE NANOPARTICLE SURFACEDue to larger surface-to-volume ratio, nanoparticles can interact with biomolecules such as nucleic acids, proteins, lipids and biological metabolites. Proteins are polypeptides which possess a definite conformation. The net surface charge of a protein depends on the pH of the medium. Proteins are about 10’s of nm in size. Owing to similar size scales, proteins can be incorporated with nanoparticles. Due to forces such as hydrogen bonds, solvation forces, Van der Waals interactions, etc proteins can get adsorbed onto nanoparticle surface. The “nanoparticle-protein complex” is referred to as the “nanoparticle-protein corona” (NP-PC). Biological reactivity of NP can be influenced by the NP-PC. Therefore, the overall NP-PC formation depends on the functional groups in the interacting protein, attraction of the protein towards NP surface, characteristics of the NP, NP composition, protein’s ability to completely occupy the NP surface, and the medium (temperature, pH). This binding of protein can be reversible or irreversible. The stability of this complex is determined by the rates of association and dissociation of protein with the NP surface. Colloidal solutions of NPs can often get agglomerated. NP agglomerates show different properties compared to monodispersed NPs. Hence, uneven surface of agglomerated NPs can induce conformational changes of proteins.Metallic nanoparticles can be modified using various chemical functional groups which allow them to conjugate with ligands, antibodies and drugs of interest. This opens a variety of potential applications in biotechnology and medicine such as magnetic separation, biosensors, targeted drug delivery, vehicles for gene and drug delivery, drug encapsulation and diagnostic imaging.Amongst various metals, nanoparticles such as magnetic nanoparticles (iron oxide), gold and silver nanoparticles, nanoshells and nanocages have been used in the medical field over the past years. They have also been modified to expand their use as diagnostic and therapeutic agents.APPLICATION OF METAL NANOPARTICLES FOR THE DETECTION OF LEPTOSPIROSISUse of Gold Nanoparticles for the detection of LeptospirosisGold nanoparticles were synthesized according to Turkevich citrate reduction method. Antibody specific to Leptospira interrogans serovar Bratislava was incubated with gold nanoparticle suspension at room temperature for 30 minutes to prepare antibody-coated gold nanoparticles. 1:10 antobody dilution was selected as the optimum dilution. Urine samples containing Leptospira was used for further testing. Urine containing Leptospira was mixed with antibody-coated nanoparticles. Positive result was indicated by the agglutination of red colour gold nanoparticles and it could be observed with the naked eye. When antibody-coated gold nanoparticles were tested with urine which did not contain Leptospira, no agglutination was observed. This assay is rapid and simple to perform. A positive result could be observed immediately or within 60 minutes if the amount of cells are low. Figure : Optimum antibody dilution for preparing antibody-coated gold particles. The suspension with antibody dilution 1:2, 1:10, 1:20 gave red colour (Tubes 1-3 respectively). The blue colour of suspension in Tube 4 (dilution 1:100) indicated that the amount of antibody dilution was insufficient.