A PROCESS FOR THE SYNTHESIS OF MONO AND BIMETALLIC NANOPARTICLES USING PLANT EXTRACT

Field of the Invention The present invention relates to a new biological method for the synthesis of mono and bimetallic nanoparticles. More particularly it relates to a method for synthesizing mono and bimetallic nanoparticles by an environment friendly and convenient method that relies on the use of plant extracts for reduction of metal ions in aqueous solution phase. The mono and bimetallic nanoparticles synthesized by this method can be used in numerous technological and medical applications, e.g., as, catalysts, sensors, nonlinear optics, electron microscopy marker, gene therapy and drug delivery. Background of the Invention An important area of research in nanotechnology concerns the synthesis of nanoparticles of different sizes, shapes and controlled polydispersity. Currently, there is a growing need to develop environmentally benign nanoparticle synthesis processes that do not use toxic chemicals in the synthesis protocol. Biological methods are a good competent for the chemical procedures, which are both environment friendly and economic. Plant extracts are very cost effective and environment friendly and thus can be an economic and efficient alternative for the large-scale synthesis of nanoparticles. Many applications require high concentration of nanoparticles in aqueous phase, which is not possible by existing protocols, but this method is very useful for synthesis of nanoparticles in high concentration in aqueous medium. The nanoparticles synthesized by this method are very stable and there is no need to use any external stabilizing agent as in other protocols. Compared to other biological methods this is very simple and there

, is no need to grow the pure culture of microorganisms, which is very cumbersome and also does not require the specific condition. Moreover, many of the chemical and physical based protocols used for synthesis of inorganic materials require non-ambient temperatures and/or non-ambient pressures that require capital-intensive equipment. Methods that can produce useful chemicals and materials at conditions closer to ambient conditions and use simple equipment are economically, ecologically and environmentally more desirable. Nanoparticles are extremely important materials with utility in different areas ranging from nano-technology, non-linear optics, diode lasers, smart sensors, markers in drugs, gene sequencing to catalysts (G. Schmid, Chem. Rev. 1992, 92, 1709). In the art, nano materials are obtained by different chemical and physical methods. Chemical methods for the preparation of nano-materials include borohydride and citrate reduction methods for the preparation of colloidal metal such as gold and silver. Physical methods for the preparation of nano materials include vapour deposition, lithographic processes and molecular beam epitaxy (MBE). Reduction of metal ions by radiolysis is also frequently used for the preparation of nano-sized metal particles.

Very little efforts have been devoted towards using biological species and more specifically plants for synthesis of metal nanoparticles. There have been few reports showing microorganisms such as bacteria and fungus being capable of synthesizing metal nanoparticles both intracellularly and extracellularly. Beveridge and co-workers have demonstrated that gold particles of nanoscale dimensions may be readily precipitated within bacterial cells by incubation of the cells with Au³⁺ ions (G. Southam and T. J. Beveridge, Geochim .Cosmochim.Acta, 1996, 60, 4369; T. J. Beveridge and R. G. E. Murray, J.Bacteriol., 1980, 141, 876). Klaus-Joerger et al have shown that the bacterium Pseudomonas stutzeri AG259 isolated from a silver mine when placed in a concentrated aqueous solution of AgNO₃, resulted in the reduction of the Ag⁺ ions and formation of silver nanoparticles of well-defined size and distinct morphology within the periplasmic space of the bacteria (T. Klaus, et al Proc.Nat.Acad.Sci., 1999, 96, 13611 ; T. Klaus-Joerger, et al Trends Biotech. 2001 , 19, 15; R. Joerger, et al Adv.Mater., 2000, 12 407). Nair and Pradeep have synthesized nanocrystals of gold, silver and their alloys by reaction of the corresponding metal ions within cells of lactic acid bacteria present in buttermilk (B. Nair and T. Pradeep, Crystal Growth & Design, 2002, 2, 293). Sastry's group have reported that the alkalothermophilic (extremophilic) actinomycete, Thermomonospora sp. can synthesize extra-cellularly high concentration of gold nanoparticles of 8 nm average size with good monodispersity (A. Ahmad, S. Senapati, M. I. Khan, R. Kumar and M. Sastry, Langmuir 2003, 19, 3550). Sastry's group has also shown that even fungi are capable of synthesizing gold nanoparticles (P. Mukherjee, et al Angew.Chem.lntEd., 2001 , 40, 3585).

To make possible use of biological species for large-scale synthesis of metal nanoparticles, Sastry's group have demonstrated the extracellular synthesis of gold nanoparticles (P. Mukherjee, et al ChemBioChem, 2002, 3, 461; P. Mukherjee, et al, Nano Lett, 2001 , 1, 515; A. Ahmad, et al, Coll.Surf.B. 2003, 28, 313).

Gold and silver nanoparticles have been synthesized within live alfalfa plants by gold/silver ion uptake from solid media (J. L. Gardea-Torresdey, et al Nano Lett., 2002, 2, 397; J. L. Gardea-Torresdey, et al Langmuir, 2003, 19, 1357). U.S. Pat. No. 6,537,344 discloses a biological process for the preparation of nano-sized colloidal metal particles by treating wet fungus or fungus extract with a metal ion solution of the desired metal and separating the biomass to obtain the nano-sized colloidal metal particles. A micellar liquid is formed when stabilizing agent / surfactant is added in sufficient quantity such that the stabilizing agent / surfactant molecules aggregate to form micelles. In a micellar liquid, micelles do not exhibit a significant degree of order, therefore the viscosity of the liquid is usually much less than that of more ordered liquid crystal phases, which are commonly gel-like. The mount of surfactant / stabilizing agent mixed with the solution is sufficient to produce a micellar liquid in which the micelles are closely spaced. The conditions under which the micellar liquid is formed will depend upon the particular surfactant being used. In practice, the main variables that needs to be controlled are the amount of surfactant added and the temperature. For some surfactants, the temperature should be elevated, whilst for other room temperature or below is necessary. As in the claimed invention, the inventors are not using any surfactant / stabilizing agent, the temperature and pressure are predetermined and is not a function of any other reagent.

Furthermore, many different procedures have been developed to prepare highly monodisperse nanoparticles. A common synthesis involves the reduction of a metal salt in the presence of capping agent molecules such as thiols, citrates or phosphines. The functionalities of these capping agents can be altered to yield various chemical properties. Most of them utilize the ability of capping agents to prepare the uniformly size distributed particles influencing the superlattice morphology of these nanoparticles. Although several different ligands like carboxylates have been used as capping ligands, in a majority of the cases leading to polydisperse particles. Here, we present a very effective way to prepare uniform size distributed mono and bimetallic nanoparticles using metal ion solution selected from the group consisting of halide, sulfate and nitrate without the use of any capping agent to control the agglomeration of nanoparticles. Such agglomeration of nanoparticels leads to big particles of poor monodispersity. Therefore, another novelty of the present invention clearly shows preparation of mono and bimetallic nanoparticles without using any capping agents.

In addition, plant extracts are very environmental friendly and as the reagents and reactants used in the preparation are less in terms of stabilizing agent and capping agent, the process is highly economic and cost effective and an efficient alternative to large-scale synthesis of nanoparticles. Unlike microorganism based biological processes, where growing of culture of microorganism in itself is cumbersome and time consuming, present invention utilizes plant extract in the preparation process.

However, the prior art methods described above suffer from several drawbacks. The chemical methods are environmentally hazardous and result in quick agglomeration of nano-particles leading to big particles of poor monodispersity. While specific capping agents are used in some of the above methods to restrict the size of the colloidal metal particles and to stabilize the particle size distribution, use of such capping agents makes the system complicated and user-unfriendly. The radiolysis method is quite complicated and gamma ray sources are not readily available. Many of the above biological processes are intracellular making the isolation of nanoparticles dificult. Growing pure strains of microorganism requires high skill, is tedious and requires careful maintenance of sterile conditions. Above all they are not cost effective.

Objects of the invention The main object of this invention is to produce biologically, mono and bimetallic nanoparticles.

Another object of is to produce mono and bimetallic nanoparticles of various metals on a large scale.

Yet another objective is, to produce mono and bimetallic nanoparticles using plant extracts from different parts of various plants.

Still another object of this invention is to produce metal nanoparticles of Au, Ag, Pt, Pd and Cu.

A further object of this invention is to produce bimetallic nanoparticles with composition

Au/Ag, Au/Pt, Au/Pd, Au/Cu, Ag/Pt, Ag/Pd, Ag/Cu, Pt/Pd, Pt/Cu and Pd/Cu. Yet another object of this invention is to produce mono and bimetallic nanoparticles by adding said plant extract to a corresponding aqueous solution of metal ions.

Still another object of this invention is to produce nanoparticles of various metals with different shapes.

A further another object of this invention is to produce mono and bimetallic nanoparticles, which does not require expensive and toxic chemicals.

Yet, another object of this invention is to provide a process for preparing mono and bimetallic nanoparticles, in which the conditions for preparation are not critical to the resulting particles of various metals.

Still another object of this invention is to produce mono and bimetallic nanoparticles, which are free of agglomerates. Yet another object of this invention is to provide a generic, low-cost process for producing mono and bimetallic nanoparticles. Summary of the Invention

The invention relates to a process for synthesis of mono and bimetallic nanoparticles, comprising reacting suitable metal salts and reducing extracts from different parts of various plants at a temperature in the range 15 to 100°C, such that the under the reducing action of the said extract the metal ions are reduced into metal nanoparticles with different size and shape in aqueous medium. Detailed Description of the Invention The present invention provides a process for preparing mono and bimetallic nanoparticles, comprising, reacting an aqueous metal ion solution or mixture of any two metal ion solutions with an aqueous extract from plant in water for a time of minimum 30 minutes in a temperature range of 15 to 100 °C to obtain the mono or bimetallic nanoparticles, separating the nanoparticles by conventional methods such as centrifugation.

In one of the embodiments of the present invention, the nanoparticles may be of different size and shapes such as spherical, triangular, rod shaped and/or cubic or of uniform size and shape depending on the extract of the plant used, ranging in size from 1 nanometer to 100 nanometers for spherical particles and from 1 nm to 5 microns for non-spherical particles.

In another embodiment of the present invention, the plants from whose extracts are obtained for the synthesis of mono and bimetallic nanoparticles are from different families exemplified herein below but not restricted to: Meliaceae: Azadirachta indica ^■ Geraniaceae: Pelargonium graveolens Poaceae: Cymbopogon flexuosus, Cymbopogon • winterianus, Cymbopogon martinii, Oryza sativa, T ticum aestivum, Saccharum officinarum. Lamiaceae : Mentha arvensis, Mentha citrata, Ocimum basilicum, Apocynaceae: Catharanthus roseus, Rauvolfia serpentina ^■ Myrtaceae: Eucalyptus globulus, Syzygium cumin i Euphorbiaceae: Phyllanthus amarus, Phyllanthus emblica Pinaceae/Coniferae: Pinus roxburghii Myrtaceae: Psidium guajava Rosaceae: Rosa damascena ^■ Santalaceae: Santalum album Caesalpiniaceae: Tamarindus indica, Cassia fistula, Cassia tora

Fabaceae: Trigonella foenum-graecum, Pisum sativum

Oleaceae: Jasminum grandiflorum

Labiatae: Rosmarinus officinalis, Pogostemon sp.

Solanaceae : Datura metel, Lycopersicon esculentum, Withania somnifera,

Atropa belladonna, Duboisia sp., Hyoscyamus sp.

Apiaceae : Daucus carota, Coriandrum sativum, Centella asiatica, Anethum graveolens

Rutaceae : Citrus limon

Fabaceae : Cicer arietinum

Rubiaceae : Cinchona officinalis

Musaceae : Musa paradisiaca

Cycadaceae : Cycas circinalis

Zingiberaceae : Costus speciosus

Araceae : Colocasia esculenta

Vitaceae : Cissus quadrangula s

Moraceae : Ficus benghalensis

Rhamnaceae : Zizipus mau tiana

Liliaceae : Gloriosa superba

Papaveraceae : Papaver somniferum

Piperaceae : Piper betle

Brassicaceae : Raphanus sativa

Cannabinaceae : Cannabis sativa

Caricaceae : Carica papaya

Marsileaceae : Marsilea quadrifolia

Scrophulariaceae: Bacopa monnieri, Digitalis sp.

Papilionaceae: Glycyrrhiza glabra

Dioscoreaceae: Dioscorea sp.

Plumbaginaceae: Plantago ovata

Compositae: Artemisia sp.

Lauraceae: Cinnamomum sp.

Umbelliferae: Foeniculum vulgare

Chenopodiaceae: Spinacia oleracea

Compositae: Chamomilla recutita In still another embodiment of the present invention, the extracts from different parts of plant or plants are used for synthesis of mono and bimetallic nanoparticles.

In still another embodiment of the invention, the different part of the plants is selected from the group consisting of leaf, flower, stem and root. In still another embodiment of the present invention, the metal nanoparticles that may be synthesized using the said process are of Au, Ag, Pt, Pd and Cu.

In still another embodiment of the present invention, the bimetallic nanoparticles that may be synthesized by the said process are of composition Au/Ag, Au/Pt, Au/Pd, Au/Cu,

Ag/Pt, Ag/Pd, Ag/Cu, Pt Pd, Pt/Cu and Pd/Cu. In still another embodiment of the present invention, the said metal ion solution is prepared by dissolving salts or acids of said metal ion in water.

In still another embodiment of the present invention, the said metal ion solution is selected from the group consisting of halide, sulfate and nitrate.

In another embodiment of the present invention, the concentration of metal salts may be varied from 10^"6 to 10^"2 M.

In still another embodiment of the present invention, the primary control over the particle size and shape is determined by the concentration of the metal ions and also by the plant extract and the type of the plant extract.

In still another embodiment of the present invention, various mono and bimetallic nanoparticles produced in accordance with one embodiment of the process of this invention have generally uniform particle sizes and shapes ranging from 1 nm to 500 nm.

The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the invention: EXAMPLE 1

This example illustrates the synthesis of gold nanoparticles with leaf extract of Pelargonium Graveolens. In a typical experiment, 20 g of thoroughly washed and finely cut Pelargonium Graveolens leaves were boiled in a 500 mL Erlenmeyer flask with 100 mL sterile distilled water for 2 min. After boiling, the solution was decanted and filtered. 5 mL of the broth thus obtained was added to 50 mL of 2 X 10^"3 M HAuCI₄ aqueous solution and kept in dark for 5 hour. After 5 hour the nanoparticles were separated out by centrifugation and redispersed in double distilled water. EXAMPLE 2 This example illustrates the synthesis of silver nanoparticles with leaf extract of Pelargonium Graveolens. In a typical experiment, 20 g of thoroughly washed and finely cut Pelargonium Graveolens leaves were boiled in a 500 mL Erlenmeyer flask with 100 mL sterile distilled water for 2 min. After boiling, the solution was decanted and filtered. 5 mL of the broth thus obtained was added to 2 X 10^"3 M AgNO₃ aqueous solution and kept in dark for 5 hour. After 5 hour the nanoparticles were separated out by centrifugation and redispersed in double distilled water. EXAMPLE 3

This example illustrates the synthesis of platinum nanoparticles with stem extract of Pelargonium Graveolens. In a typical experiment, 20 g of thoroughly washed and finely cut Pelargonium Graveolens stem pieces were boiled in a 500 mL Erlenmeyer flask with 100 mL sterile distilled water for 2 min. After boiling, the solution was decanted and filtered. 5 mL of the broth thus obtained was added to 50 mL of 2 X 10^"3 M H₂PtCI₆ aqueous solution and kept in dark for 5 hour. After 5 hour the nanoparticles were separated out by centrifugation and redispersed in double distilled water. EXAMPLE 4

This example illustrates the synthesis of palladium nanoparticles with root extract of Pelargonium Graveolens. In a typical experiment, 20 g of thoroughly washed and finely cut Pelargonium Graveolens root pieces were boiled in a 500 mL Erlenmeyer flask with 100 mL sterile distilled water for 2 min. After boiling, the solution was decanted and filtered. 5 mL of the broth thus obtained was added to 50 mL of 2 X 10^"3 M Pd(NO₃)₂ aqueous solution and kept in dark for 5 hour. After 5 hour the nanoparticles were separated out by centrifugation and redispersed in double distilled water. EXAMPLE 5 This example illustrates the synthesis of gold nanoparticles with leaf extract of Cymbopogon flexuosus. In a typical experiment, 20 g of thoroughly washed and finely cut Cymbopogon flexuosus leaves were boiled in a 500 mL Erlenmeyer flask with 100 mL sterile distilled water for 2 min. After boiling, the solution was decanted and filtered. 5 mL of the broth thus obtained was added to 50 mL of 2 X 10^"3 M HAuCI₄ aqueous solution and kept in dark for 5 hour. After 5 hour the nanoparticles were separated out by centrifugation and redispersed in double distilled water. EXAMPLE 6

This example illustrates the synthesis of gold-silver bimetallic nanoparticles with leaf extract of Azadirachta indica. In a typical experiment, 20 g of thoroughly washed and finely cut Azadirachta indica leaves were boiled in a 500 mL Erlenmeyer flask with 100 mL sterile distilled water for 2 min. After boiling, the solution was decanted and filtered. 10 mL of the broth thus obtained was added to 50 mL of 2 X 10^"3 M HAuCI₄ and 50 mL of 2 X 10^"3 M AgNO₃ aqueous solution and kept in dark for 5 hour. After 5 hour the nanoparticles were separated out by centrifugation and redispersed in double distilled water. Advantages of the process claimed in the present invention are:

1. Large scale synthesis is possible

2. Require less maneuvering.

3. Ambient experimental conditions.

4. Fast biological process. 5. Nanoparticles are synthesized extracellularly.

6. Isolation of the extracellularly synthesized nanoparticles is simple.

7. Cost effective/Economical system for the industry

8. Highly stable colloidal mono and bimetallic nanoparticles can be formed.

9. Nanoparticles of different shapes can be formed. 9. Environment friendly process.

10. Plant used for synthesis of nanoparticles can be easily grown anywhere.

11. Preparation of plant extract is very simple

12. Possibility of the reusability of nanoparticles.