Field of Invention
The present invention relates to rapid synthesis of mono-disperse platinum nanoparticle using fungal extracellular secrets of enzymatic protein. To obtain cell free filtrate, fungal biomass developed in form of balls which were incubated in Milli- Q- Water for 36 hours followed by separation using membrane filtration. Present invention is a reliable protocol for the synthesis of platinum nanoparticle over a range of biological composition, size and monodispersity.
Background of Invention
Conventional synthesis of nanoparticles involves a number of chemical and physical methods including chemical reduction in aqueous or non-aqueous solution, micro emulsion, template, sonochemical, and microwave-assisted methods. However, all these methods are energy and assets demanding, employ noxious chemicals, and often yield particles in non-polar organic solutions and preventing their biomedical applications.
WO/2012/095863 discloses a process to prepare novel platinum nanoparticls and
nanoparticles thereof. The modified platinum nanoparticle comprises of
a platinum nanoparticle having at least one mercapto alkyl acid selected from the group consisting of mercatopropyl sulfonic acid, mercapto propionic acid and mercapto succinic acid, attached thereon.
JP2011042863 discloses the method for manufacturing the metal nanoparticles comprises: a step of preparing a solution by adding a surfactant to a non-aqueous solvent; a step of preparing a platinum seed solution by adding a platinum salt to the solution; and a step of adding a metal salt to the platinum seed solution to induce a reaction.
KR 1020110019224 discloses A gold nanoparticle is formed with a platinum seed formed inside a non-water solvent, and contains 0.001~50mol% of platinum seed. The gold nanoparticle is produced by the following steps: producing a solution by adding a monomer surfactant to the non-water solvent; adding platinum salt to the solution for forming a platinum seed solution; and adding gold salt to the platinum seed solution for reacting.
JP 2010077526 discloses the method for preparing metal nanoparticles includes: a step of preparing a solution by adding a polymer surfactant to an alcohol solvent; heating the solution; forming a metal seed by adding a first metal salt of at least one metal salt selected from the group consisting of platinum palladium and indium in the heated solution; and adding a second metal salt into the solution including the metal seed.
Nelson Duran et.al. discloses the various mechanisms provided for the biological synthesis of metal nanoparticles by peptides, bacteria, fungi, and plants. Huaming Yang et al. discloses Anatase Ti02 nanoparticles of about 16nm in crystal size have been successfully
synthesized via a sol-gel method. Thermal treatment of the precursor at 500-600 °C results in the formation of different Ti02 phase compositions.
None of the prior art indicate above either alone or in combination with one another disclose what the present invention has disclosed. The present invention provides a rapid, efficient and simple method for synthesis of ecofriendly platinum nanoparticles, which can be easily scaled up, for the mass production in industry. There is a need for the development of clean, reliable, biocompatible and benign processes to synthesize nanoparticles leads to turning of more and more researchers to exploit biological systems as possible eco-friendly "nano-factories".
The main advantage of the present invention is the use of extracellular enzymatic protein secrets of fungus for the synthesis of shape, size, polymorph and poly-disperse controlled platinum nanoparticles, which is simple, efficient, ecofriendly and easy to handle. The invention uses naturally occurring fungus of arid zone soil. Synthesized nanoparticles were uniform in size and monodisperse with 100% yield. The nanoparticles formed are stable in aqueous solution for longer period of time as they are naturally encapsulated by fungal protein. More scope in scale up industrial production. The process is cost effective and easy downstream processing, therefore, useful for industry. The method is environmentally benign and there is no toxic by­products. Fungus biomass can be used for multiple times to collect extracellular secrets,
therefore, it is a rapid and cost effective process, and a large number of nanoparticles can be produced by the industry with no time.
Summary of Invention
The invention provide a method of producing platinum nanoparticle which may used in biomedical and engineering using fungus comprising (a) Isolation of fungal isolate from arid zone soil (b) Providing a culture of fungal isolate (c) Incubation time period, and (d) Precursor metal salt solution. The nanoparticle produced may not be limited to platinum. The fungal isolate was Ascomycota; more particularly Aspergillus flavus TFR12 (NCBI GenBank Accession No. JQ675295).
To isolate of fungi, sampling was done from the rhizosphere of clusterbean (Cyamopsis tetragonoloba L.) in arid soil. Fungus was isolated by serial dilution method followed by identification based on morphological and molecular level. Platinum oxide hydrated (Pt02.H20) used as precursor salt solution for platinum nanoparticles synthesis.
1. A mycogenic process for synthesis of platinum metal nanoparticles 100% yield which comprising the steps of: isolating fungal isolate from arid soil zone; providing a culture for inoculation soil fungal isolate; separating a fungal ball from soil culture; washing soil fungal ball with sterile distilled water; harvesting a fungal biomass from the culture; separating the biomass by filtration using membrane filter so as to obtain cell free filtrate; using the cell free filtrate in a salt solution; shaking the solution; synthesizing the solution so as to obtained metal nanoparticles. 2. The process of claim 1, wherein arid zone soils from Central Arid Zone Research Institute, Jodhpur. 3. The process of claim 1, wherein the fungal isolate comprises Aspergillus flavus TFR12 (GenBank NCBI Accession No. JQ675295). 4. The process of claim 1, wherein the fungus is used in whole cell form, wet solid mass and fungal extracellular secrets and the biomass of fungal isolate is incubated in ratio of 2:10 Milli-Q-water. 5. The process of claim 1, wherein the fungal extracellular secrets obtained at temperature 28°C and pH 6.4 with shaking (150 rpm). 6. The process of claim 1, wherein the said precursor salts is platinum oxide hydrated (PtCh.FbO). 7. The process of claim 1, wherein the concentration of precursor metal
salt in the solution is not less than 1(T3M. 8. The process of claim 1, wherein extracellular secrets is reacted with precursor salt for 12 hours with shacking at temperature 28°C and pH 6.4.; 9. The process of claim 1, wherein the reaction occurs in aqueous medium containing fungal extracellular enzymatic protein secrets. 10. The process of claim 1, wherein the average size of 100% biosynthesized nanoparticles are below 100 nm.
Brief Description of Drawings
The following drawings demonstrate the further aspects to present invention. The invention may be better understood by reference of more than one drawing in combination with detailed description of specific embodiments are presented herewith-
Figure 1 showing graph of dynamic light scattering (DLS) of platinum nanoparticles particularly number distribution with respect to size of particle using particle size analyzer. The results clearly demonstrated 100% particles are in nanoscale.
Figure 2 showing graph of Fourier Transformed Infrared (FTIR) spectrum of platinum nanoparticles recorded from bio-transformed products at the end of reaction.
Figure 3 showing Transmission Electron Microscope (TEM) micrograph of platinum nanoparticles respectively, which is recorded from drop coating method. The results clearly demonstrated 100% particles are in nanoscale at least at one dimension.
Figure 4 showing Energy Dispersive Spectroscopy (EDS) spectrum of platinum nanoparticles. The data confirmed that the nanoparticles are constituted by platinum metal only.
Detail Description of the Invention
Nanotechnology is a novel and innovative science that attracts researchers and scientists from different disciplines, including physicists, chemists, engineers, and biologists across the globe. Owing to its inimitable properties, nanotechnology provides the basic tools and subsequently the technology for gathering information and designing innovative devices to probe questions related to biological importance of metal availability, and the application of this knowledge in diverse sector particularly biomedical and engineering.
Various chemical (1-3) and physical (4,5) methods are available for the synthesis of platinum nanoparticles. The available processes for platinum nanosynthesis employ toxic chemical such as sodium borohydrate (NaBIHU) and require polymer (6), electrolyte (7,8), surfactant (9) and thiol group (10) to maintain the stability of synthesized nanoparticles. Chemical and physical methods for nanomaterial synthesis are usually energy intensive, employ toxic chemicals and require higher temperatures. Therefore, a clean, nontoxic and environment friendly process for the synthesis of nanoparticles is in great demand. Researchers have turned toward biological systems for inspiration, which could provide a possible route for the development of nanoparticle synthesis.
Synthesis of nanoparticles using fungi has several advantage over prokaryotic mediated approach regarding reproducibility of nano-size materials, these also includes ease to multiplication, grow, handling and rest of downstream process for this top down approach of nanobiosynthesis through nanofactories. Kingdom fungi encompasses an enormous diversity, has been estimated at around 1.52 million species. Fungi play crucial role in web of ecosystem in sense of biogeochemical and nutrient cycling. It is well known fact that fungi are saprophytes, which degrade organic matter in surroundings by extracellular enzymatic secretion. In present invention involved fungi, belongs to phylum Ascomycota, which develop Asci, a sac like structure, in which non motile ascospores are formed.
EXAMPLES
The present invention is described herein below with reference to the following examples, which however, should be understood not to limit the invention in any manner.
EXAMPLE 1
ISOLATION AND CULTIVATION OF FUNGAL BIOMASS
This example illustrates the isolation and cultivation of fungi, involving the method of isolation
and cultivation of fungi, including collection of soil sample from arid zone agricultural field
(26° 18' N 73° 01'E) of Central Arid Zone Research Institute (CAZRI) located in Jodhpur,
Rajasthan of India. The method employed for isolation of culture according to standard
microbiological research steps such as serial dilution technique followed by spread plate method
using fungal isolating Rose Bengal Agar medium which is especially for cultivation of fungi. Inoculated plates were incubated at 28°C for 72 hours in BOD incubator. Cultures were identified on the basis of morphological and molecular parameter. Stock culture was maintained by sub culturing at regular monthly interval. After growing at pH 5.3 and 28°C for 72 hours, the slants were preserved at 4°C. From an actively growing stock culture, subculture was made on the fresh slant and after 72 hours of incubation at 28°C, was used as starting material for nanoparticle synthesis.
EXAMPLE 2
CHARACTERIZATION AND IDENTIFICATION OF FUNGUS USED FOR EFFICIENT
NANOPARTICLE PRODUCTION
A number of fungal strains were isolated from soil and screened for platinum nanoparticle production. The Aspergillus flavus TFR12 found most efficient fungal strain for production of platinum nanoparticles. The molecular identification of fungal isolate was carried out on the basis of DNA nucleotide sequencing of nuclear 5.8S rRNA gene using universal primers viz., Internal Transcribed Spacer ITS1 (5'- TCCGTAGGTGAACCTGCG-3') and ITS 4 (5'-TCCTCCGCTTATTGATATGC-3'). The DNA sequence was submitted to gene bank of National Centre for Biotechnological Information (NCBI) and got accession no. JQ675295.
EXAMPLE 3
BIOSYNTHESIS OF PLATINUM NANOPARTICLES
This example illustrates the synthesis of platinum nanoparticles using fungus Aspergillus flavus
TFR12 (GenBank NCBI Accession No. JQ675295). The fungus was grown in 250 mL
Erlenmeyer flask containing 100 mL potato dextrose broth medium. After adjusting the pH of
medium to 6.4, the culture was grown with continuous shaking on a rotary shaker (150 rpm) at
28°C for 72 hours. After 72 hours, fungal balls of mycelia were separated from the culture broth
by filtration and then the fungal mycelia were washed with sterile distilled water under optimum
conditions. The harvested fungal biomass (20g wet weight) was re-suspend in 100 mL sterile
Milli-Q-water in 250 mL Erlenmeyer flask and again kept on a shaker (150 rpm) at 28°C for 36
hours. After incubation, the cell free filtrate was obtained by seperating the fungal biomass.
Using cell free filtrate, salt solution of platinum oxide hydrated (Pt02.H20) prepared in concentration of 1(T3M in Erlenmeyer flasks which was found to be optimum salt concentration. The entire mixture was kept on shaker (150 rpm) at 28°C and the reaction carried out for a period of 12 hours. The biotransformation were collected periodically and monitored for characterization.
EXAMPLE 4
CHARACTERIZATION OF PLATINUM NANOPARTICLES
Sampling of bio-transformed product was characterized by Dynamic Light Scattering (DLS) using particle size analyzer, Fourier Transform Infrared (FTIR) spectroscopy, Transmission Electron Microscope (TEM) and Energy Dispersive Spectroscopy (EDS).
Particle Size Analyzer uses Dynamic Light Scattering (DLS), determines particle size by measuring the rate of fluctuations in the laser light intensity scattered by particles as they diffuses through fluid. As shown in the figure 1 the average size of nanoparticles were in the range of 6.8 and 22.5 nm on the basis of number distribution. The graphs also had shown mono-dispersity of nanoparticle. Beckman DelsaNano C series model instrument was used for size measurement and confirmation of nanoparticles.
Fourier Transform Infrared (FTIR) spectroscopy is an effective analytical tool for identification of unknown samples on the basis of IR energy radiation absorb by the chemical bonds showing specific frequency at particular wavelength. Peaks in the figure 2, showed the presence of metal nanoparticles of platinum metal. FTIR spectra of samples were recorded on ABB FTLA 2000-100 model of IR spectroscopy and all measurements were carried out at a resolution of 16 cm-1.
Transmission Electron Microscope (TEM) is capable to evaluating morphology and nanoparticle size. Samples for TEM analysis were prepared by drop coating method using copper grid. Micrograph of TEM was taken from JEM-2100F TEM model with an accelerating voltage ranges of 200 kV for better resolution. Figure 3 showing TEM micrograph of platinum nanoparticles that further confirmed the shape and size of the nanoparticles.
Electron Dispersive Spectroscopy (EDS) is used to analysis the elemental composition of the nanoparticles sample based on the X-ray analysis. Figure 4 confirms that biotransformed product was of platinum metal only. EDS of the samples were carried out by Thermo Noran System SIX with high resolution LN2 cooled Si (Li) detector.
Advantage / Industrial Capabilities of Invention
Advantage of the process claimed in the present invention are-
The main advantage of the present invention is the use of extracellular enzymatic protein secrets of fungus for the synthesis of shape, size, polymorph and poly-disperse controlled platinum nanoparticles, which is simple, efficient, ecofriendly and easy to handle.
The invention uses naturally occurring fungus of arid zone soil.
Synthesized nanoparticles were uniform in size and monodisperse with 100% yield.
The nanoparticles formed are stable in aqueous solution for longer period of time as they are naturally encapsulated by fungal protein.
More scope in scale up industrial production.
The process is cost effective and easy downstream processing, therefore, useful for industry.
The method is environmentally benign and there is no toxic by-products.
8. Fungus biomass can be use for multiple times to collect extracellular secrets, therefore, it is a rapid and cost effective process, and a large number of nanoparticles can be produced by the industry with no time.
Flow chart method for platinum nanoparticle synthesis
The fungus, Aspergillus flavus TFR12 (GenBank NCBI Accession No. JQ675295) was grown in 250 mL Erlenmeyer flask containing 100 mL potato dextrose broth medium (pH 6.4)
The culture was grown with continuous shaking on a rotary shaker (150 rpm) at 28°C for 72
hours
Fungal balls of mycelia were separated from the culture broth by filtration
Fungal mycelia were washed with sterile distilled water
The harvested fungal biomass (20g wet weight) was re-suspended in 100 mL sterile Milli-Q-
water in 250 mL Erlenmeyer flask
↓
Kept on a shaker (150 rpm) at 28°C for 36 hours
↓
The cell free filtrate was obtained by separating the fungal biomass
Using cell free filtrate, salt solution of platinum oxide hydrated (PtOa.HaO) prepared in concentration of 10~3M in Erlenmeyer flasks
The whole mixture was kept on shaker (150 rpm) at 28°C and the reaction carried out for a
period of 12 hours
The biotransformed product (platinum nanoparticles) were collected periodically and monitored
for characterization
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We claim:
1. A mycogenic process for synthesis of platinum metal nanoparticles 100% yield comprising the
steps of:
isolating fungal isolate from arid soil zone;
providing a culture for inoculation soil fungal isolate;
separating a fungal ball from soil culture;
washing soil fungal ball with sterile distilled water;
harvesting a fungal biomass from the culture;
separating the biomass by filtration using membrane filter so as to obtain cell free filtrate; using the cell free filtrate in a salt solution; shaking the solution; synthesizing the solution so as to obtained metal nanoparticles.
The process of claim 1, wherein arid zone soils from Central Arid Zone Research Institute, Jodhpur.
The process of claim 1, wherein the fungal isolate comprises Aspergillus flavus TFR12 (GenBank NCBI Accession No. JQ675295).
The process of claim 1, wherein the fungus is used in whole cell form, wet solid mass and fungal extracellular secrets and the biomass of fungal isolate is incubated in ratio of 2:10 Milli-Q-water.
The process of claim 1, wherein the fungal extracellular secrets obtained at temperature 28°C and pH 6.4 with shaking (150 rpm).
6. The process of claim 1, wherein the said precursor salts is platinum oxide hydrated (PtO2.H2O).
7. The process of claim 1, wherein the concentration of precursor metal salt in the solution is not
less than 10"3M.
The process of claim 1, wherein extracellular secrets is reacted with precursor salt for 12 hours with shacking at temperature 28°C and pH 6.4
The process of claim 1, wherein the reaction occurs in aqueous medium containing fungal extracellular enzymatic protein secrets.
10. The process of claim 1, wherein the average size of 100% biosynthesized nanoparticles are below 100 nm.