Title of Invention

A PROCESS FOR MANUFACTURING GOLD METAL NANOPARTICLES

Abstract A process for making gold metal nanoparticles comprising the steps of (i) preparing an aqueous solution of the gold 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; (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) 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; (iv) discarding the supernatant liquid in the tube and thoroughly washing the yeast; (v) breaking the yeast using a breaking method selected from a group of breaking methods consisting of freeze-thawing, sonication, abrasion, zymolase treatment, treating with an alkali, treating with microwaves, heating, electroporation, protoplasting, and grinding; (vi) a second centrifugation at 5000 to 20000 rpm for 10 to 30 minutes until the yeast particles settle down; (vii) collecting the supernatant liquid; extracting moisture from the supernatant liquid by freezing and thawing, obtaining gold nanoparticles ranging from 1 to 100 nanometer in diameter.
Full Text FORM-2 THE PATENTS ACT, 1970
(39 of 1970)
COMPLETE Specification
(Section 10; rule 13)
A PROCESS FOR MANUFACTURING GOLD METAL NANOPARTICLES
AGHARKAR 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
GRANTED



THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED:-
2 JUL 2004
2-7-2004

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This invention relates to a process for manufacturing gold metal nanoparticles.
In particular this invention relates to a process of manufacturing gold metal nanoparticles 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.
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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
Sulfides 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
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micelles, 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.
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a Micro contact printing
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
nm.
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 used 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
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smallest nanostructures, with dimensions between 2 and 10 nm, and do so inexpensively.
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/ra) 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
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agitation. The technique is useful for large-scale production of nanoparticles using relatively simple and inexpensive hardware (Higgins 1997).
6. High energy ball milling
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
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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. 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 sulfides, typically cadmium sulfide (CdS) 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). Further work on microbial synthesis of CdS nanoparticles is scant and is limited to studies on characterization (Dameron and Winge, 1990a,b) and efficient production in batch cultivation (Williams et al., 1996a).
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Physical and chemical methods have been attempted in the manufacture of gold nanoparticles. 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 gold metal nanoparticles,. Some of the attractive features of organic materials are their flexibility, easy processing, and large quantum 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.
Metal nanoparticles have optical, electronic, magnetic and catalytic properties that are quite different from the bulk metals or atoms. Hence, these nanoparticles find novel applications. For example, nanosized gold has extensive use in nanoscale materials, devices fabrication (Schrnid, 1997, Dirix 1999), for biolabelling in electron microscopy techniques (Hayat 1989), in gene gun technology as carriers for the delivery of double-stranded DNA (dsDNA) and in sensors for biomedical diagnostics (Dubertret 2001, Storhoff 1999). The size of the gold particles seems to dictate their use. For example, in biolabelling it has been observed that with size reduction the sensitivity and resolution increase significantly. Very high labeling efficiency is achieved with gold particles in the size range of 3-8nm as these
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particles contribute less steric hindrance as compared to particles in 10-20 nm range (Yokota 1988). Conventionally gold nanoparticles are synthesized by chemical methods that use different reducing agents such as thiocyanate (Baschong et al 1985), white phosphorous (Roth 1982), sodium borohydride (Birrell et al 1987), a mixture of sodium citrate and tannic acid (Muhlpfordt 1982) etc. These chemical agents reduce chloroauric acid to colloidal gold nanoparticles. The use of chemical processes may become environmentally unacceptable, particularly when the nanomaterials will be required in large quantities for commercial use. Hence, there is a need to develop 'green' technologies for the synthesis of nanomaterials. Of late, the synthesis of nanoparticles using biological route is gaining prominence. Biological procedures are considered to be mild and environment friendly, as they do not make use of toxic/corrosive chemicals, very high temperatures or extreme pH.
Biosynthesis of gold nanoparticles using two fungal strains, viz. Fusarium oxysporum and Verticillium sp. was reported recently (Mukherjee et al 2002). In the former case, gold nanoparticles were synthesized extracellularly and were polydisperse (diameter 8-40 nm). In the latter case, the particles were synthesized intracellularly and were found to be 20+8 nm in size (after excluding gold particles larger than -100 nm). Thus, considerable polydispersity seems to be present in the gold nanoparticles synthesized by these two fungal strains. Very recently gold nanoparticles are reported to be synthesized within Alfa Alfa plants when grown on gold rich media (Gardea-Torresdey et al, 2002). This plant has the ability to take up gold from the media and transport it in the form of nanoparticles (2-20 nm sizes) throughout the plant.
10

According to this invention there is provided a process for making gold
metal nanoparticles consisting the steps of
Preparing an aqueous solution of the gold 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 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;
discarding the supernatant liquid in the tube and thoroughly washing the
yeast;
breaking the yeast using a breaking method selected from a group of
breaking methods consisting of freeze-thawing, sonication, abrasion,
zymolase treatment, treating with an alkali, treating with microwaves,
heating, electroporation, protoplasting, and grinding;
a second centrifugation at 5000 to 20000 rpm for 10 to 30 minutes until the
yeast particles settle down;
collecting the supernatant liquid; extracting moisture from the supernatant
liquid by freezing and thawing, obtaining gold nanoparticles ranging from 1
to 1 OOnanometer in diameter.
The gold metal is passivated with organic material. Each nanoparticle is enveioped with an organic passivating shefi.
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The invention will now be described with reference to the accompanying examples:
Example 1
SYNTHESIS OF MONODISPERSE GOLD NANOPARTICLES BY THE YEAST
ISSATCHENKIA ORIENTALIS
Gold nanoparticles in the size range of - 5-8 nm were synthesized by challenging the yeast Issatchenkia orientalis culture to AuCI4"ions. At low concentrations of auric acid in aqueous medium gold was found to accumulate in the form of nanocrystallites intracellularly. However with increase in the auric acid concentration, nanocrystallites were formed extracellulaly. Optical absorption, X-ray diffraction, X-ray photoelectron spectroscopy, transmission electron microscopy and energy dispersive analysis of X-rays of these gold nanoparticles was performed.
The yeast species used in this work was isolated from garden soil using a medium of pH 4 containing 0.5% yeast extract, 1% glucose and 0.35% sodium propionate. It was identified as Issatchenkia orientalis using the routine biochemical tests and sequence analysis of the internal transcribed spacer (ITS) region.
Reduction of AuCT4
The culture, Issatchenkia orientalis was inoculated in medium containing 0.5%) yeast extract and 1% glucose (pH 5.6) and allowed to grow for 8-10 h. Varying concentrations of gold (0.5mM and 1.25mM) in the form of HauCI4 3H20 were added and the cells incubated for further 12 h to get reduction of gold. Harvesting of cells was done by centrifugation at 6000 X g for 10
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minutes followed by two washes with distilled water. The cells were freeze dried in a lyophilizer (VirTis, USA) and the dry powders (Samples Al, A2, and A3 corresponding to gold concentrations of 0.5mM, l.0mM and 1.25mM, respectively) were used for further characterization studies.
The cell-free supematants were concentrated at room temperature under vacuum (Savant, USA Speedvac) and lyophilized to obtain dry powders (Samples Bl, B2 and B3 corresponding to gold concentrations of 0.5mM, 0.75mM and 1.25mM, respectively) that were used in further analysis. Absorbance measurements
The optical absorbance spectra of the samples were obtained by suspending the powders in distilled water. Spectra could be recorded in the range of 200 to 800 nm using a UV visible spectrophotometer (Shimadzu, Japan, UV 2501 PC).
Example 2
Synthesis of gold nanoparticles using Saccharomyces sp. Saccharomyces culture was inoculated into YES medium [0.5 % yeast extract and 1 % glucose ] of pH 5.2. The tubes were incubated at 25 degrees Celsius on a shaker at 100 rpm. After 12 hours of growth, 0.5 mM of gold chloride was added in the tubes and incubated further for 24 hours. After exposure to gold the culture exhibited characteristic colour blue of reduced gold. The cultures were harvested by centrifugation at 8000 x g for 10 minutes and the pellets obtained were washed with physiological saline followed by 0.01 M EDTA. TEM of the whole cells were performed to confirm the intracellular synthesis of gold nanoparticles using a Philips
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technai-12 electron microscope which operated at an accelerating voltage of 60 kV. The size of the gold particles was found to be below 5nm.
Example 3
Synthesis of gold nanoparticles using Trichosporon sp.
Trichosporon culture was inoculated into YES medium [0.5 % yeast extract and 1 % glucose ] of pH 5.2. The tubes were incubated at 25 degrees Celsius on a shaker at 100 rpm. After 12 hours of growth, 0.5 mM of gold chloride was added in the tubes and incubated further for 24 hours. After exposure to gold the culture exhibited characteristic colour blue of reduced gold. The cultures were harvested by centrifugation at 8000 x g for 10 minutes and the pellets obtained were washed with physiological saline followed by 0.01 M EDTA. TEM of the whole cells were performed to confirm the intracellular synthesis of gold nanoparticles using a Philips technai-12 electron microscope which operated at an accelerating voltage of 60 kV. The size of the gold particles was found to be below 5nm.
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.

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We Claim:
1. A process for making gold metal nanoparticles comprising the steps of
(i) preparing an aqueous solution of the gold 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;
(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) 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;
(iv) discarding the supernatant liquid in the tube and thoroughly washing the
yeast;
(v) breaking the yeast using a breaking method selected from a group of
breaking methods consisting of freeze-thawing, sonication, abrasion,
zymolase treatment, treating with an alkali, treating with microwaves,
heating, electroporation, protoplasting, and grinding;
(vi) a second centrifugation at 5000 to 20000 rpm for 10 to 30 minutes until
the yeast particles settle down;
(vii) collecting the supernatant liquid; extracting moisture from the
supernatant liquid by freezing and thawing, obtaining gold nanoparticles
ranging from 1 to 100 nanometer in diameter.
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2. A process for making gold metal nanoparticles 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.
3. A process for making gold metal nanoparticles as described herein with
reference to the accompanying examples 1 to 3.
Dated this 19th day of March 2003.

Documents:

290-mum-2003-cancelled pages(02-07-2004).pdf

290-mum-2003-claims(granted)-(02-07-2004).doc

290-mum-2003-claims(granted)-(02-07-2004).pdf

290-mum-2003-correspondence(12-07-2004).pdf

290-mum-2003-correspondence(ipo)-(29-03-2007).pdf

290-mum-2003-form 1(20-03-2003).pdf

290-mum-2003-form 19(02-06-2003).pdf

290-mum-2003-form 2(granted)-(02-07-2004).doc

290-mum-2003-form 2(granted)-(02-07-2004).pdf

290-mum-2003-form 3(20-03-2003).pdf

290-mum-2003-petition under rule 138(02-07-2004).pdf

290-mum-2003-power of attoreny(26-03-2003).pdf


Patent Number 205346
Indian Patent Application Number 290/MUM/2003
PG Journal Number 42/2008
Publication Date 17-Oct-2008
Grant Date 29-Mar-2007
Date of Filing 20-Mar-2003
Name of Patentee AGHARKAR RESEARCH INSTITUTE OF MAHARASHTRA ASSOCIATION FOR THE CULTIVATION OF SCIENCE
Applicant Address G.G.AGARKAR ROAD, PUNE 411 004, Maharashtra, India.
Inventors:
# Inventor's Name Inventor's Address
1 KISHORE MADHURKAR PAKNIKAR AGHARKAR RESEARCH INSTITUTE OF MAHARASHTRA ASSOCIATION FOR THE CULTIVATION OF SCIENCE, G.G.AGARKAR ROAD, PUNE 411 004, MAHARASHTRA, INDIA.
2 MEENAL KOWSHIK AGHARKAR RESEARCH INSTITUTE, G.G.AGARKAR ROAD, PUNE 411 004.
PCT International Classification Number B82B 3/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA