Title of Invention

A PROCESS FOR MANUFACTURING SILVER METAL NANO PARTICLES

Abstract

ABSTRACT A process for making silver metal nano particles consisting the steps of preparing an aqueous solution of the silver metal salt in a sub-lethal concentration typically ranging between 0,1 to 5 mmoles, at temperatures ranging between 15 degrees Celsius to 45 degrees Celsius in deionized chemical free water and in an inert flask; adding yeast to the solution to obtain a turbidity ranging from 0.02 to 0.05 at a wavelength of 550 nm; allowing the yeast containing solution to stand on a shaker for 12 to 24 hours until the turbidity of the solution ranges between 1 to 8 at a wavelength of 550 nm; centrifugation between 1000 to 20000 rpm for 5 to 30 minutes of the turbid yeast containing solution until majority of the yeast settles down at the bottom of the flask; collecting the cell free supernatant liquid in a flask, extracting moisture from the supernatant liquid by freezing and thawing, obtaining metal silver nano particles ranging from 1 to lOOnanometer in diameter.

Full Text

FORM - 2 THE PATENTS ACT, 1970 COMPLETE SECTION-10 A PROCESS FOR MANUFACTURING SILVER METAL NANO PARTICLES AGARKAR RESEARCH INSTITUTE OF MAHARASHTRA ASSOCIATION FOR THE CULTIVATION OF SCIENCE of G.G.Agarkar Road, Pune 411 004, Maharashtra, India, A Society registered under the Societies Act ORIGINAL 292/MUM/2003 20/03/2003 THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED:- This invention relates to a process for manufacturing silver metal nano particles. In particular this invention relates to a process of manufacturing silver metal nano particles using yeast. Nanoparticles are part of an emerging science called 'nanotechnology'. The word nanotechnology comes from the Greek prefix 'nano'. In modern scientific parlance, a nanometer is one billionth of a meter, about the diameter often atoms placed side by side. Nanotechnology is about building things one atom at a time, and in doing so constructing particles and devices with unique capabilities. Nanoparticles of substances exhibit properties unlike the properties of their macro counterparts often with stunning new results. Physicist Richard Feynman first described the possibility of molecular engineering. In 1959 Feynman gave a lecture at the California Institute of Technology called "There's Plenty of Room at the Bottom" where he observed that the principles of physics do not deny the possibility of manipulating things atom by atom. He suggested using small machines to make even tinier machines, and so on down to the atomic level itself. Nanotechnology as it is understood now though, is the brainchild of Feynman's one-time student K. Eric Drexler. Drexler presented his key ideas in a paper on molecular engineering published in 1981, and expanded these in his books Engines of Creation, and Nanosystems: Molecular Machinery, Manufacturing and Computation, which describes the principles and mechanisms of molecular nanotechnology. 2 In 1981 the invention of the Scanning Tunneling Microscope or STM, by Gerd Binnig and Heinrich Rohrer at IBM's Zurich Research Labs, and the Atomic Force Microscope (AFM) five years later, made it possible to not only take photos of individual atoms, but to actual move a single atom around. Soon after, John Foster of IBM Almaden labs was able to spell "IBM" out of 35 xenon atoms on a nickel surface, using a scanning tunneling microscope to push the atoms into place. Nanoparticles are particles smaller than 100 nanometers in diameter and generally spherical in shape. The synthesis and characterization of nanoparticles has received attention in recent years for their use in industry and chemistry. A range of nanoparticles has been produced by physical, chemical and biological methods. There have been various attempts made to produce nanoparticles of metal Using physical and chemical methods. Researchers are developing a variety of techniques for building structures smaller than 100 nm. Two approaches have been adopted for nanofabrication - The Top down processes, which include the methods of synthesis that carve out or add aggregates of molecules to a surface. The second is the bottom up approach, which assembles atoms or molecules into nanostructures. Chemical and biological methods of synthesis come under this category. Physical methods include Electron beam lithography, Scanning probe method, Soft lithography, Microcontact printing, Micromoulding , Chemical methods include Wet chemical preparation, Surface passivation, Core shell synthesis, Organometallic precursor, Sol get method,. Langmuir- lodgett method, Precipitation in structured media, Zeolites, Micelles and inverse micelles, 3 Biological methods include Biomineralization using Bacteria, Yeast, Fungi, Plants and . Biotemplating using Ferritin, Lumazine synthase, Virus Surface layers DNA, PHYSICAL METHODS 1. Electron Beam Lithography The technology used to fabricate circuits on microchips can be modified to produce nanometer scale structures. In this technique an electron beam scans the surface of a semiconductor containing a buried layer of quantum well material. The resist gets removed where the beam has drawn a pattern. A metal layer is deposited on the resulting surface and then the solvent used to remove the remaining resist. Reactive ions etch away the chip except where it is protected by metal leaving metal in the form of quantum dots only where the electron beam exposed the resist. 2. Soft lithography This technique is an extension of the previous technique and overcomes the impracticability of applying electron beam lithography to large scale manufacturing by making a mould or a stamp, which can be used repeatedly. A bas-relief master is made using electron beam lithography to produce a pattern in a photoresist, which is on the surface of a Si wafer. A precursor to polydimethyl siloxane (PDMS) is poured over the bas-relief master and cured into the rubbery solid that matches the original pattern. The PDMS stamp is peeled of the master. This stamp can be used in various ways to make nanostructures. a Micro contact printing 4 The PDMS stamp is inked with a solution consisting of organic molecules called thiols and then pressed against a thin film of gold on a silicon plate. The thiols form a self-assembled monolayer on the gold surface that reproduces the stamp pattern; features in the pattern can be as small as 50 ran. Micromoulding in capillaries The PDMS stamp is placed on a hard surface, and a liquid polymer flows into the recesses between the surface and the stamp. The polymer solidifies into the desired pattern, which may contain features smaller than 10 nm. 3. Scanning probe methods A scanning probe microscope can image the surface of conducting materials with atomic scale detail. Hence single atoms can be placed at selected positions and structures can be built to a particular pattern atom by atom. It can also be used to make scratches on a surface and if the current flowing from the tip of the STM is increased the microscope becomes a very small source for an electron beam which can be use to write nanometer scale patterns. The STM tip can also push individual atoms around on a surface to build rings and wires that are only one atom wide. The main disadvantages of top down method are that they are expensive and technically difficult and too slow for mass production. Therefore there is a growing interest in bottom up methods. These methods can easily make the smallest nanostructures, with dimensions between 2 and 10 nm, and do so inexpensively. 5 4. Sonochemical method In this method an acoustic cavitation process is used to generate a transient localized hot zone with extremely high temperature gradient and pressure (Suslick et al. 1996). Such sudden changes in temperature and pressure bring about the destruction of the sonochemical precursor (e.g., organometallic solution) and the formation of nanoparticles. The technique can be used to produce a large volume of material for industrial applications. 5. Hydrodynamic cavitation Nanoparticles are synthesized by creation and release of gas bubbles inside sol-gel solutions (Sunstrom et al. 1996). Rapid pressurizing in a supercritical drying chamber and exposing to cavitational disturbance at high temperature bring about the mixing of the sol-gel solution. The erupted hydrodynamic bubbles are responsible for nucleation, growth, and quenching of the nanoparticles. The particle size can be controlled by adjusting the pressure and the solution retention time in the cavitation chamber. Microemulsions have been used for synthesis of metallic (Kishida et al. 1995), semiconductor (Kortan et al. 1990; Pileni et al. 1992), silica (Arriagada and Osseo-Assave 1995), barium sulfate (Hopwood and Mann 1997), magnetic, and superconductor (Pillai et al. 1995) nanoparticles. By controlling the very low interfacial tension (-10-3 mN/m) through the addition of a co-surfactant (e.g., an alcohol of intermediate chain length), these micro-emulsions are produced spontaneously without the need for significant mechanical agitation. The technique is useful for large-scale production of nanoparticles using relatively simple and inexpensive hardware (Higgins 1997). 6. High energy ball milling 6 This approach for nanoparticle synthesis, has been used for the generation of magnetic (Leslie- Pelecky and Reike 1996), catalytic (Ying and Sun 1997), and structural (Koch 1989) nanoparticles. The technique, which is already a commercial technology, has been considered dirty because of contamination problems from ball-milling processes. However, the availability of tungsten carbide components and the use of inert atmosphere and/or high vacuum processes have reduced impurities to acceptable levels for many industrial applications. Common drawbacks include the low surface area, the highly polydisperse size distributions, and the partially amorphous state of the as prepared powders. CHEMICAL METHODS A number of chemical strategies are now available for the construction of higher order structures. Organic molecules can be linked together by molecular recognition. For example, synergistic noncovalent donor acceptor interactions can give rise to intertwined rings (catenanes) (Ashton 1989). Liquid crystal polymers having self-organized structures can be formed from organic molecules containing head groups capable of complementary hydrogen bonding interactions. Organic molecules can be assembled around metal ions such as Cu (I) that provide stereo chemical constricts in the construction of double helices (Dietrich, 1991). The synthesis of inorganic clusters, by contrast, is usually dependent on passivating the surface of a growing aggregate by capping the surface sites with stabilizing ligands. Wet Chemical Preparation. 7 This method involves the reaction between a metal ion and the desired anion under controlled conditions to generate nanocrystals of desired size. Nanoparticles are extremely reactive as the coordination of surface atoms in nanoparticle is incomplete, and can lead to particle agglomeration. This problem is overcome by passivating the bare surface atoms with protecting groups. Capping or passivating the particle not only prevents agglomeration, it also protects the particles from its surrounding environment, and provides electronic stabilization to the surface. The capping agent usually takes the form of a Lewis base compound covalently bound to surface metal atoms. A few attempts have been made to synthesize metal nanoparticles using microorganisms. It was shown that CdS nanoparticles can be synthesized in the yeasts Candida glabrata and Schizosaccharomyces pombe (Dameron et al., 1989). These nanoparticles are coated with short peptides known as phytochelatins (Grill et al., 1986), which have the general structure (y-Glu-Cys)n-Gly where n varies from 2-6. The nanoparticles are size reproducible, more monodisperse, and have greater stability than synthetically produced nanoparticles (Williams et al., 1996b). Physical and chemical methods have been attempted in the manufacture of silver nano particles. These techniques involve controlling crystallite size by restraining the reaction environment. However, problems occur with general instability of the product and in achieving monodisperse size. This invention envisages a process using organic materials for the manufacture of silver metal nano particles. Some of the attractive features of organic materials are their flexibility, easy processing, and large quantum 8 efficiency for light emission. Efforts are being made to improve their stability, efficiency, and color tenability for diverse applications. For this purpose, composites of organic materials with nanoparticles, porous silicon, etc., have been probed. Some earlier attempts show that there are many advantages of using nanoparticles as an active material. This is because size-dependent properties enable, in case of nanoparticles, to tune their properties to a desired value. In the present invention, it is demonstrated that a silver-tolerant yeast species could precipitate a majority of silver(>99%) extracellularly as elemental nanoparticles when the cells are challenged with soluble silver in the log phase of growth. The microbial synthesis of single crystals of silver with well-defined composition and shapes such as equilateral triangles and hexagons by the culture Pseudomonas stutzeri has been reported earlier (Klaus, et al 1999). In this case the crystallites accumulated within the cell and were up to 200 nm in size. Most of the silver is found to be in elemental form and a small number as silver sulfide. However, when yeast cells are challenged by the Ag+ions, it is observed that silver is reduced extracellularly to metallic silver. The biologically synthesized silver nanoparticles could have many applications, such as spectrally selective coatings for solar energy absorption and intercalation material for electrical batteries (Klaus et al 2001), as optical receptors (Schultz et al, 2000), catalysts in chemical reactions, biolabeling (Hayat M A, 1989) etc. 9 According to this invention there is provided a process for making silver metal nano particles comprising the steps of: preparing an aqueous solution of the silver metal salt in a sub-lethal concentration typically ranging between 0.1 to 5 mMoles, at temperatures ranging between 15 degrees Celsius to 45 degrees Celsius in deionized chemical free water and in an inert vessel; adding yeast to the solution to obtain a turbidity ranging from 0.02 to 0.05 at a wavelength of 550 nm; allowing the yeast containing solution to stand on a shaker for 12 to 24 hours in the absence of light until the turbidity of the solution ranges between 1 to 8 at a wavelength of 550 nm; a first centrifugation between 1000 to 3000 rpm for 5 to 30 minutes of the turbid yeast containing solution in a centrifugation tube until majority of the yeast settles down at the bottom of the tube; to obtain metal silver nano particles ranging from 1 to 100 nanometer in diameter. The silver metal is passivated with organic material. Each nano particle is enveloped witfean organic passivating shell. The invention will now be described with reference to the accompanying examples: Example 1 Synthesis of silver nano particles using the yeast MKY3 10 The yeast species (strain MKY3) was isolated from garden soil during a screening program undertaken to isolate microorganisms capable of synthesizing metal-based nanoparticles. The culture could tolerate 0.8 mM silver and intracellular silver accumulation was negligible ( Production of silver nanoparticles MKY3 was inoculated at 0.5% level in 2 L Erlenmeyer flasks containing 1 L growth medium (2% tryptone, 1% yeast extract and 2% glucose, pH 5.6). The flasks were incubated at 30°C on a rotary shaker set at 100 rpm. Upon attaining the mid-log phase (between 9-10 h, O.D. 6oo = 2), the culture was challenged with 2.0 mM silver nitrate and incubated further in dark for 24 h. The cells were separated from the culture medium by centrifugation (5000 X g) and the cell-free medium was used for the recovery of precipitated silver nanoparticles. Appropriate controls (uninoculated medium + silver nitrate and MKY3 culture supernatant + silver nitrate) were run simultaneously. Recovery of silver nanoparticles The recovery of silver nanoparticles synthesized extracellularly by MKY3 was carried out in an apparatus, which could separate the silver particles from the medium on the basis of differences in their thawing temperatures (-8 C and 1°C, for silver nanoparticles and medium, respectively). The apparatus was a 1 L polycarbonate bottle fitted with a sampling cup. The 11 cell-free medium with suspended silver particles was filled in the bottle up . to the brim and kept in a freezer adjusted to -20°C in an upright position. During freezing, silver nanoparticles being denser than the medium settled at the bottom (as distinctly visible black layer). The bottle was then transferred to another freezer adjusted to 0°C and the contents were allowed to thaw. The thawing of frozen layer containing silver nanoparticles began at -8°C, and due to increase in volume the thawed suspension was pushed upwards (through microchannels formed along the walls of the bottle) under the weight of frozen block of the medium and got collected in the sampling cup. The concentrated colloidal suspension in the sampling cup was subjected to silver estimation by atomic absorption spectrophotometry (Unicam, England, Solar 929). The suspension was then centrifuged at 23000 X g for 1 h and the particles were resuspended in distilled water. The procedure was repeated twice before drying the particles in vacuo to obtain a powder. Example 2 ( Synthesis of silver nano particles using Issatchenkia orientalij). Issatchenkia orientalis culture was inoculated at 0.5 % level into growth medium [1 % yeast extract 2% tryptone and 2 % glucose ] of pH 5.6 in Erlenmeyer flasks. The flasks were incubated at 30 degrees Celsius on rotary shaker at 100 rpm. After 9 to 10 hours of growth, 2.0 mM of silver nitrate was added in the flasks and incubated further for 24 hours in the dark. After exposure to silver the culture exhibited characteristic color black of reduced silver. The cultures were harvested by centrifugation at 5000 x g for 10 minutes and the cell free supernatant solution was used for recovery of silver 12 nano particles. The recovery of silver nanoparticles synthesized extracellularly by Issatchenkia orientalis was carried out in an apparatus which could separate the silver particles from the medium on the basis of differences in their thawing temperatures (-8°C and 1°C, for silver nanoparticles and medium, respectively). The apparatus was a 1 L polycarbonate bottle fitted with a sampling cup. The cell-free medium with suspended silver particles was filled in the bottle up to the brim and kept in a freezer adjusted to -20 C in an upright position. During freezing, silver nanoparticles being denser than the medium settled at the bottom (as distinctly visible black layer). The bottle was then transferred to another freezer adjusted to 0°C and the contents were allowed to thaw. The thawing of frozen layer containing silver nanoparticles began at -8 C, and due to increase in volume the thawed suspension was pushed upwards (through microchannels formed along the walls of the bottle) under the weight of frozen block of the medium and got collected in the sampling cup. The concentrated colloidal suspension in the sampling cup was subjected to silver estimation by atomic absorption spectrophotometry (Unicam, England, Solar 929). The suspension was then centrifuged at 23000 X g for 1 h and the particles were resuspended in distilled water. The procedure was repeated twice before drying the particles in vacuo to obtain a powder. The size of the silver particles was found to be in the range of 2 to 5nm. Example 3 Synthesis of silver nano particles using pichia sp. Pichia sp culture was inoculated at 0.5 % level into growth medium [1 % yeast extract 2% tryptone and 2 % glucose ] of pH 5.6 in Erlenmeyer flasks. 13 The flasks were incubated at 30 degrees Celsius on rotary shaker at 100 rpm. After 9 to 10 hours of growth, 2.0 mM of silver nitrate was added in the flasks and incubated further for 24 hours in the dark. After exposure to silver the culture exhibited characteristic color black of reduced silver. The cultures were harvested by centrifugation at 5000 x g for 10 minutes and the cell free supernatant solution was used for recovery of silver nano particles. The recovery of silver nanoparticles synthesized extracellularly by pichia sp was carried out in an apparatus which could separate the silver particles from the medium on the basis of differences in their thawing temperatures (-8°C and 1°C, for silver nanoparticles and medium, respectively). The apparatus was a 1 L polycarbonate bottle fitted with a sampling cup. The cell-free medium with suspended silver particles was filled in the bottle up to the brim and kept in a freezer adjusted to -20°C in an upright position. During freezing, silver nanoparticles being denser than the medium settled at the bottom (as distinctly visible black layer). The bottle was then transferred to another freezer adjusted to 0°C and the contents were allowed to thaw. The thawing of frozen layer containing silver nanoparticles began at -8 C, and due to increase in volume the thawed suspension was pushed upwards (through microchannels formed along the walls of the bottle) under the weight of frozen block of the medium and got collected in the sampling cup. The concentrated colloidal suspension in the sampling cup was subjected to silver estimation by atomic absorption spectrophotometry (Unicam, England, Solar 929). The suspension was then centrifuged at 23000 X g for 1 h and the particles were resuspended in distilled water. The procedure was repeated twice before drying the particles in vacuo to obtain a powder. The size of the silver particles was found to be in the range of 2 to 5nm. 14 Although the invention and particular the system and the process, has been described in terms of particular embodiments and applications, one of ordinary skill in the art, in light of this teaching, can generate additional embodiments and modifications without departing from the spirit of or exceeding the scope of the invention. Accordingly, it is to be understood that the description herein is proffered by way of example to facilitate comprehension of the invention and should not be construed to limit the scope thereof. .. 15 We Claim: [1]A process for making silver metal nano particles comprising the steps of: (i) preparing an aqueous solution of the silver metal salt in a sub-lethal concentration typically ranging between 0.1 to 5 m Moles, at temperatures ranging between 15 degrees Celsius to 45 degrees Celsius in deionized chemical free water and in an inert flask; adding yeast to the solution to obtain a turbidity ranging from 0.02 to 0.05 at a wavelength of 550 nm; (ii)allowing the yeast containing solution to stand on a shaker for 12 to 24 hours until the turbidity of the solution ranges between 1 to 8 at a wavelength of 550 nm; (iii)centrifugation between 1000 to 20000 rpm for 5 to 30 minutes of the turbid yeast containing solution until majority of the yeast settles down at the bottom of the flask; (iv)collecting the cell free supernatant liquid in a flask, extracting moisture from the supernatant liquid by freezing and thawing, obtaining metal silver nano particles ranging from 1 to 100 nanometer in diameter. 2. A process for making silver metal nano particles as claimed in claim 1, in which the yeast is at least one yeast selected from a group of yeasts containing Issatchenkia orientalis, Hansenula sp., Torulopsis sp. , Schizosaccharomyces pombe, Candida glabrata, Trichosporon sp, Issatchenkia orientalis, and Pichia sp. 16 3. A process for making silver nano particles as decried herein with reference to the accompanying examples 1 to 3. Dated this 19th day of March 2003. Mohan Dewan ofR. K. Dewan&Co., Applicants' Patent Attorney 17

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